myelin-oligodendrocyte-glycoprotein-(35-55) and Disease-Models--Animal

Topics: Animals; Brain-Derived Neurotrophic Factor; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Fibroblast Growth Factor 2; Gene Expression Regulation; Humans; Immunologic Factors; Mice, Knockout; Microglia; Multiple Sclerosis; Myelin Sheath; Myelin-Oligodendrocyte Glycoprotein; Oligodendroglia; Peptide Fragments; Proto-Oncogene Proteins c-akt; Receptor, Fibroblast Growth Factor, Type 2; Remyelination; Signal Transduction

Modelling complex disorders presents considerable challenges, and multiple sclerosis (MS) is no exception to this rule. The aetiology of MS is unknown, and its pathophysiology is poorly understood. Moreover, the last two decades have witnessed a dramatic revision of the long-held view of MS as an inflammatory demyelinating white matter disease. Instead, it is now regarded as a global central nervous system (CNS) disorder with a neurodegenerative component. Currently, there is no animal model recapitulating MS immunopathogenesis. Available models are based on autoimmune-mediated demyelination, denoted experimental autoimmune encephalomyelitis (EAE) or virally or chemically induced demyelination. Of these, the EAE model has been the most commonly used. It has been extensively improved since its first description and now exists as a number of variants, including genetically modified and humanized versions. Nonetheless, EAE is a distinct disease, and each variant models only certain facets of MS. Whilst the search for more refined MS models must continue, it is important to further explore where mechanisms underlying EAE provide proof-of-principle for those driving MS pathogenesis. EAE variants generated with the myelin component myelin oligodendrocyte glycoprotein (MOG) have emerged as the preferred ones, because in this particular variant disease is associated with both T- and B-cell effector mechanisms, together with demyelination. MOG-induced EAE in the non-obese diabetic (NOD) mouse strain exhibits a chronic-relapsing EAE clinical profile and high disease incidence. We describe the generation of this variant, its contribution to the understanding of MS immune and pathogenetic mechanisms and potential for evaluation of candidate therapies.

Topics: Animals; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Mice; Mice, Inbred NOD; Multiple Sclerosis; Myelin-Oligodendrocyte Glycoprotein; Peptide Fragments

Astroglia, the most abundant cells in the human CNS, and even more prominent in multiple sclerosis patients, participate in CNS innate and adaptive immunity, and have been hypothesized to play an important role in multiple sclerosis progression. Experimental autoimmune encephalomyelitis elicited in mice by immunization with myelin oligodendrocyte glycoprotein peptide 35-55 provides a means by which to explore the genesis and disease significance of astrogliosis during a chronic immune-mediated CNS inflammatory/demyelinative disorder that, in its' pathological features, strongly resembles multiple sclerosis.

Topics: Animals; Astrocytes; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Gliosis; Humans; Models, Biological; Multiple Sclerosis; Myelin-Oligodendrocyte Glycoprotein; Peptide Fragments

The phylogenetic proximity between non-human primate species and humans is reflected by a high degree of immunological similarity. Non-human primates therefore provide important experimental models for disorders in the human population that are caused by the immune system, such as autoimmune diseases. In this paper we describe non-human primate models of multiple sclerosis, a chronic inflammatory and demyelinating disease of the human central nervous system. While reviewing data from the literature and our own research we will discuss the unique role of such models in the research of basic disease mechanisms and the development of new therapies.

Topics: Animals; Brain; Callithrix; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Female; Glycoproteins; Macaca; Male; Multiple Sclerosis; Myelin-Oligodendrocyte Glycoprotein; Peptide Fragments

Dietary interventions have not been effective in the treatment of multiple sclerosis (MS). Here, we show that periodic 3-day cycles of a fasting mimicking diet (FMD) are effective in ameliorating demyelination and symptoms in a murine experimental autoimmune encephalomyelitis (EAE) model. The FMD reduced clinical severity in all mice and completely reversed symptoms in 20% of animals. These improvements were associated with increased corticosterone levels and regulatory T (Treg) cell numbers and reduced levels of pro-inflammatory cytokines, TH1 and TH17 cells, and antigen-presenting cells (APCs). Moreover, the FMD promoted oligodendrocyte precursor cell regeneration and remyelination in axons in both EAE and cuprizone MS models, supporting its effects on both suppression of autoimmunity and remyelination. We also report preliminary data suggesting that an FMD or a chronic ketogenic diet are safe, feasible, and potentially effective in the treatment of relapsing-remitting multiple sclerosis (RRMS) patients (NCT01538355).

Topics: Animals; Antigens; Apoptosis; Autoimmunity; Diet; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Fasting; Female; Humans; Lymphocyte Count; Lymphocytes; Mice, Inbred C57BL; Models, Biological; Multiple Sclerosis; Myelin Sheath; Myelin-Oligodendrocyte Glycoprotein; Oligodendroglia; Peptide Fragments; Regeneration; Spinal Cord; Spleen

Fibrotic scar tissue limits central nervous system regeneration in adult mammals. The extent of fibrotic tissue generation and distribution of stromal cells across different lesions in the brain and spinal cord has not been systematically investigated in mice and humans. Furthermore, it is unknown whether scar-forming stromal cells have the same origin throughout the central nervous system and in different types of lesions. In the current study, we compared fibrotic scarring in human pathological tissue and corresponding mouse models of penetrating and non-penetrating spinal cord injury, traumatic brain injury, ischemic stroke, multiple sclerosis and glioblastoma. We show that the extent and distribution of stromal cells are specific to the type of lesion and, in most cases, similar between mice and humans. Employing in vivo lineage tracing, we report that in all mouse models that develop fibrotic tissue, the primary source of scar-forming fibroblasts is a discrete subset of perivascular cells, termed type A pericytes. Perivascular cells with a type A pericyte marker profile also exist in the human brain and spinal cord. We uncover type A pericyte-derived fibrosis as a conserved mechanism that may be explored as a therapeutic target to improve recovery after central nervous system lesions.

Topics: Aging; Animals; Astrocytes; Brain Injuries, Traumatic; Brain Ischemia; Brain Neoplasms; Central Nervous System; Cerebral Cortex; Cicatrix; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Extracellular Matrix; Fibroblasts; Fibrosis; Glioblastoma; Humans; Ischemic Stroke; Mice, Inbred C57BL; Mice, Transgenic; Myelin-Oligodendrocyte Glycoprotein; Peptide Fragments; Pericytes; Receptor, Platelet-Derived Growth Factor beta; Spinal Cord; Spinal Cord Injuries; Stromal Cells

Topics: Animals; Astrocytes; Body Weight; Cytokines; Disease Models, Animal; Dose-Response Relationship, Drug; Drug Evaluation, Preclinical; Encephalomyelitis, Autoimmune, Experimental; Female; Inflammasomes; Mice; Mice, Inbred C57BL; Microglia; Morphinans; Myelin-Oligodendrocyte Glycoprotein; NLR Family, Pyrin Domain-Containing 3 Protein; Peptide Fragments; Pyroptosis; Random Allocation; Specific Pathogen-Free Organisms; Spinal Cord

Topics: Animals; Anti-Inflammatory Agents; Cell Differentiation; Cells, Cultured; Cytokines; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Inflammation Mediators; Lymph Nodes; Male; Mevalonic Acid; Mice, Inbred C57BL; Myelin-Oligodendrocyte Glycoprotein; Peptide Fragments; Quinolines; Spinal Cord; T-Lymphocytes, Regulatory; Th17 Cells

The significance of monocytes has been demonstrated in multiple sclerosis (MS). One of the therapeutic challenges is developing medications that induce mild immunomodulation that is solely targeting specific monocyte subsets without affecting microglia. Muramyl dipeptide (MDP) activates the NOD2 receptor, and systemic MDP administrations convert Ly6C

Topics: Acetylmuramyl-Alanyl-Isoglutamine; Adjuvants, Immunologic; Animals; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Freund's Adjuvant; Immunomodulating Agents; Male; Mice; Mice, Inbred C57BL; Monocytes; Multiple Sclerosis; Myelin-Oligodendrocyte Glycoprotein; Neuroprotective Agents; Nod2 Signaling Adaptor Protein; Peptide Fragments

Pathologic roles of innate immunity in neurologic disorders are well described, but their beneficial aspects are less understood. Dectin-1, a C-type lectin receptor (CLR), is largely known to induce inflammation. Here, we report that Dectin-1 limited experimental autoimmune encephalomyelitis (EAE), while its downstream signaling molecule, Card9, promoted the disease. Myeloid cells mediated the pro-resolution function of Dectin-1 in EAE with enhanced gene expression of the neuroprotective molecule, Oncostatin M (Osm), through a Card9-independent pathway, mediated by the transcription factor NFAT. Furthermore, we find that the Osm receptor (OsmR) functioned specifically in astrocytes to reduce EAE severity. Notably, Dectin-1 did not respond to heat-killed Mycobacteria, an adjuvant to induce EAE. Instead, endogenous Dectin-1 ligands, including galectin-9, in the central nervous system (CNS) were involved to limit EAE. Our study reveals a mechanism of beneficial myeloid cell-astrocyte crosstalk regulated by a Dectin-1 pathway and identifies potential therapeutic targets for autoimmune neuroinflammation.

Topics: Animals; Astrocytes; Brain; CARD Signaling Adaptor Proteins; Cell Communication; Cells, Cultured; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Galectins; Gene Expression Regulation; Lectins, C-Type; Mice, Inbred C57BL; Mice, Knockout; Multiple Sclerosis; Myelin-Oligodendrocyte Glycoprotein; Myeloid Cells; Neurogenic Inflammation; Oncostatin M; Oncostatin M Receptor beta Subunit; Peptide Fragments; Receptors, Mitogen; Signal Transduction

Topics: Adjuvants, Immunologic; Animals; Capsules; Cell Wall; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Female; Freund's Adjuvant; Humans; Lymphocyte Activation; Mice; Mice, Inbred C57BL; Multiple Sclerosis; Myelin Proteolipid Protein; Myelin-Oligodendrocyte Glycoprotein; Peptide Fragments; Proteoglycans; Saccharomyces cerevisiae; Th17 Cells

Induction of T cell apoptosis constitutes a major mechanism by which therapeutically administered glucocorticoids (GCs) suppress inflammation and associated clinical symptoms, for instance in multiple sclerosis (MS) patients suffering from an acute relapse. The sensitivity of T cells to GC action depends on their maturation and activation status, but the precise effect of antigen-priming in a pathological setting has not been explored. Here we used transgenic and congenic mouse models to compare GC-induced apoptosis between naïve and antigen-specific effector T cells from mice immunized with a myelin peptide. Antigen-primed effector T cells were protected from the pro-apoptotic activity of the synthetic GC dexamethasone in a dose-dependent manner, which resulted in their accumulation relative to naïve T cells

Topics: Animals; Anti-Inflammatory Agents; Apoptosis; Cells, Cultured; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Glucocorticoids; Humans; Lymphocyte Activation; Mice; Mice, Inbred C57BL; Mice, Transgenic; Multiple Sclerosis; Myelin-Oligodendrocyte Glycoprotein; Peptide Fragments; Proto-Oncogene Proteins c-bcl-2; T-Lymphocytes

Experimental autoimmune encephalomyelitis (EAE) is an animal disease model of multiple sclerosis (MS) that involves the immune system and central nervous system (CNS). However, it is unclear how genetic predispositions promote neuroinflammation in MS and EAE. Here, we investigated how partial loss-of-function of suppressor of MEK1 (SMEK1), a regulatory subunit of protein phosphatase 4, facilitates the onset of MS and EAE.. C57BL/6 mice were immunized with myelin oligodendrocyte glycoprotein 35-55 (MOG. Here, we showed that partial Smek1 deficiency caused more severe symptoms in the EAE model than in controls by activating myeloid cells and that Smek1 was required for maintaining immunosuppressive function by modulating the indoleamine 2,3-dioxygenase (IDO1)-aryl hydrocarbon receptor (AhR) pathway. Single-cell sequencing and an in vitro study showed that Smek1-deficient microglia and macrophages were preactivated at steady state. After MOG. The present study suggests a protective role of Smek1 in autoimmune demyelination pathogenesis via immune suppression and inflammation regulation in both the immune system and the central nervous system. Our findings provide an instructive basis for the roles of Smek1 in EAE and broaden the understanding of the genetic factors involved in the pathogenesis of autoimmune demyelination.

Topics: Animals; Central Nervous System; Cytokines; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Gene Knockout Techniques; Indoleamine-Pyrrole 2,3,-Dioxygenase; Inflammation; Interferon-gamma; Mice; Mice, Inbred C57BL; Microglia; Multiple Sclerosis; Myelin-Oligodendrocyte Glycoprotein; Myeloid Cells; Peptide Fragments; Phosphoprotein Phosphatases; Receptors, Aryl Hydrocarbon; Signal Transduction; Spleen

The B-cell CLL/lymphoma 6 (Bcl6) oncogenic repressor is a master regulator of humoral immunity and B-cell lymphomagenesis. Although much research has focused on its regulation and function of GC B cells and T cells, the role of Bcl6 in regulating the functions of innate immunity is not well defined. Here, we demonstrated that EAE is exacerbated in LysM Cre

Topics: Animals; Cells, Cultured; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Immunity, Innate; Immunomodulation; Interleukin-6; Macrophage Activation; Macrophages; Mice; Mice, Inbred C57BL; Mice, Knockout; Multiple Sclerosis; Myelin-Oligodendrocyte Glycoprotein; Neutrophils; Peptide Fragments; Proto-Oncogene Proteins c-bcl-6; Sepsis; Th17 Cells

Multiple sclerosis (MS) is known as an autoimmune disease in the central nervous system (CNS) characterized by motor deficits, pain, fatigue, cognitive impairment, and sensory and visual dysfunction. MS is considered to be resulted from significant inflammatory response. Paricalcitol (Pari) is a vitamin D2 analogue, which has been indicated to show anti-inflammatory activities in kidney and heart diseases. In the present study, if Pari could ameliorate the experimental autoimmune encephalomyelitis (EAE) was investigated. Here, the C57BL/6 mice were immunized using myelin oligodendrocyte glycoprotein 35-55 (MOG

Topics: Animals; Anti-Inflammatory Agents; Apoptosis; Calcium-Binding Proteins; Caspase 3; Cell Line; Cell Proliferation; Cyclooxygenase 2; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Ergocalciferols; Glial Fibrillary Acidic Protein; Humans; I-kappa B Proteins; Inflammation; Jurkat Cells; Lipopolysaccharides; Macrophages; Male; Mice; Mice, Inbred C57BL; Microfilament Proteins; Microglia; Multiple Sclerosis; Myelin-Oligodendrocyte Glycoprotein; NF-kappa B; Nitric Oxide Synthase Type II; Peptide Fragments; Poly (ADP-Ribose) Polymerase-1; Primary Cell Culture; RAW 264.7 Cells; Signal Transduction; Spinal Cord

Multiple sclerosis (MS) is an inflammatory demyelination disease in the central nervous system (CNS) characterized by incomplete endogenous remyelination in the chronic phase. A shift of the balance between pro and anti-inflammatory cytokines is one of the important markers in the pathogenesis of MS. This study aimed to evaluate the effects of human adipose derived stem cells (hADSCs) overexpressing interleukin 11 and interleukin 13 (IL-11, 13-hADSCs) on the experimental autoimmune encephalomyelitis (EAE), an animal model of MS.12 days after immunization of C57Bl/6 female mice with MOG35-55 and initial clinical symptoms appearance, the IL-11, 13-hADSCs were injected via the tail vein into the EAE mice. Then, the mice were sacrificed at 30 days post-immunization (DPI) and the spinal cords of experimental groups were extracted for histopathological and real-time RT-PCR studies.The results indicated that the clinical scores and mononuclear cells infiltration into the spinal cords of EAE mice were significantly reduced in mice treated with IL-11, 13-hADSCs. Likewise, the remyelination and oligodendrogenesis were significantly enhanced in the mentioned treatment group. Real-time results demonstrated that pro/anti-inflammatory cytokine genes expression was reversed in IL-11, 13-hADSCs treatment group in comparison to the untreated EAE group.Expression of IL-11 as a neurotrophic cytokine and IL-13 as an anti-inflammatory cytokine by hADSCs could increase the immunomodulatory and neuroprotective effects of hADSCs and be a powerful candidate in stem cell therapy for future treatment of MS.

Topics: Adipose Tissue; Adult; Adult Stem Cells; Animals; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Female; Humans; Immunomodulation; Interleukin-11; Interleukin-13; Mice; Mice, Inbred C57BL; Multiple Sclerosis; Myelin-Oligodendrocyte Glycoprotein; Neuroprotective Agents; Peptide Fragments; Stem Cell Transplantation; Young Adult

Recent genome-wide association studies have identified over 230 genetic risk loci for multiple sclerosis. Current experimental autoimmune encephalomyelitis (EAE) models requiring active induction of disease may not be optimally suited for the characterization of the function of these genes. We have thus used gene expression profiling to study whether spontaneous opticospinal EAE (OSE) or MOG-induced EAE mirrors the genetic contribution to the pathogenesis of multiple sclerosis more faithfully. To this end, we compared gene expression in OSE and MOG EAE models and analyzed the relationship of both models to human multiple sclerosis risk genes and T helper cell biology. We observed stronger gene expression changes and an involvement of more pathways of the adaptive immune system in OSE than MOG EAE. Furthermore, we demonstrated a more extensive enrichment of human MS risk genes among transcripts differentially expressed in OSE than was the case for MOG EAE. Transcripts differentially expressed only in diseased OSE mice but not in MOG EAE were significantly enriched for T helper cell-specific transcripts. These transcripts are part of immune-regulatory pathways. The activation of the adaptive immune system and the enrichment of both human multiple sclerosis risk genes and T helper cell-specific transcripts were also observed in OSE mice showing only mild disease signs. These expression changes may, therefore, be indicative of processes at disease onset. In summary, more human multiple sclerosis risk genes were differentially expressed in OSE than was observed for MOG EAE, especially in T

Topics: Adaptive Immunity; Animals; Cytokines; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Humans; Immunomodulation; Mice; Mice, Inbred C57BL; Mice, Transgenic; Multiple Sclerosis; Myelin-Oligodendrocyte Glycoprotein; Peptide Fragments; Risk; Th1 Cells; Transcriptome

Multiple sclerosis (MS) is a potentially disabling disease of the central nervous system. Approximately half of the patients with MS experience severe pain; however, currently available therapeutics provide only insufficient relief. The mechanisms underlying the generation of neuropathic pain in patients with MS are not fully understood. Recently, we found that neutrophil elastase from accumulated neutrophils in the dorsal root ganglion (DRG) sensitizes DRG neurons and induces mechanical allodynia in a mouse model of experimental autoimmune encephalomyelitis (EAE). However, the mechanism underlying neutrophil accumulation in the DRG after myelin oligodendrocyte glycoprotein (MOG

Topics: Animals; Chemokine CXCL1; Disease Models, Animal; Disease Susceptibility; Encephalomyelitis, Autoimmune, Experimental; Ganglia, Spinal; Gene Expression Regulation; Hyperalgesia; Mice; Myelin-Oligodendrocyte Glycoprotein; Neutrophils; Peptide Fragments; Sensory Receptor Cells; Signal Transduction; Toll-Like Receptors

Oral tolerance is defined as the specific suppression of cellular and/or humoral immune responses to an Ag by prior administration of the Ag through the oral route. Although the investigation of oral tolerance has classically involved Ag feeding, we have found that oral administration of anti-CD3 mAb induced tolerance through regulatory T (Treg) cell generation. However, the mechanisms underlying this effect remain unknown. In this study, we show that conventional but not plasmacytoid dendritic cells (DCs) are required for anti-CD3-induced oral tolerance. Moreover, oral anti-CD3 promotes XCL1 secretion by small intestine lamina propria γδ T cells that, in turn, induces tolerogenic XCR1

Topics: Administration, Oral; Animals; CD3 Complex; Cell Movement; Chemokines, C; Dendritic Cells; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Female; Gene Knockout Techniques; Genes, T-Cell Receptor delta; Immune Tolerance; Intestinal Mucosa; Intraepithelial Lymphocytes; Lymph Nodes; Male; Mesentery; Mice; Mice, Inbred C57BL; Mice, Knockout; Muromonab-CD3; Myelin-Oligodendrocyte Glycoprotein; Peptide Fragments; T-Lymphocytes, Regulatory

Leukocyte trafficking is a key event during autoimmune and inflammatory responses. The subarachnoid space (SAS) and cerebrospinal fluid are major routes for the migration of encephalitogenic T cells into the central nervous system (CNS) during experimental autoimmune encephalomyelitis (EAE), the animal model of multiple sclerosis, and are sites of T cell activation before the invasion of CNS parenchyma. In particular, autoreactive Th1 and Th17 cell trafficking and reactivation in the CNS are required for the pathogenesis of EAE. However, the molecular mechanisms controlling T cell dynamics during EAE are unclear. We used two-photon laser microscopy to show that autoreactive Th1 and Th17 cells display distinct motility behavior within the SAS in the spinal cords of mice immunized with the myelin oligodendrocyte glycoprotein peptide MOG

Topics: Animals; Cell Movement; Central Nervous System; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Female; Gene Expression Profiling; Humans; Inflammation; Lymphocyte Activation; Lymphocyte Function-Associated Antigen-1; Mice, Inbred C57BL; Mice, Transgenic; Microscopy, Confocal; Multiple Sclerosis; Myelin-Oligodendrocyte Glycoprotein; Peptide Fragments; Spinal Cord; Th1 Cells; Th17 Cells

The pathogenesis of multiple sclerosis (MS) is mediated primarily by T cells, but most studies of MS and its animal model, experimental autoimmune encephalomyelitis (EAE), have focused on CD4 T cells. The aims of the current study were to determine the pathological interrelationship between CD4 and CD8 autoreactive T cells in MS/EAE.. Female C57BL/6 mice (n = 20) were induced by myelin oligodendrocyte glycoprotein (MOG)35-55 peptide. At 14 days after immunization, T cells were isolated from the spleen and purified as CD4 and CD8 T cells by using CD4 and CD8 isolation kits, and then the purity was determined by flow cytometric analysis. These cells were stimulated by MOG35-55 peptide and applied to proliferation assays. The interferon-gamma (IFN-γ) and interleukin (IL)-4 secretion of supernatant of cultured CD4 and CD8 T cells were measured by enzyme-linked immunosorbent assays (ELISA). For adoptive transfer, recipient mice were injected with MOG35-55-specific CD8 or CD4 T cells. EAE clinical course was measured by EAE score at 0-5 scale and spinal cord was examined by staining with hematoxylin and eosin and Luxol fast blue staining.. CD8CD3 and CD4CD3 cells were 86% and 94% pure of total CD3 cells after CD8/CD4 bead enrichment, respectively. These cells were stimulated by MOG35-55 peptide and applied to proliferation assays. Although the CD8 T cells had a generally lower response to MOG35-55 than CD4 T cells, the response of CD8 T cells was not always dependent on CD4. CD8 T cell secreted less IFN-γ and IL-4 compared with CD4 T cells. EAE was induced in wildtype B6 naïve mice by adoptive transfer of MOG35-55-specific T cells from B6 active-induced EAE (aEAE) mice. A similar EAE score and slight inflammation and demyelination were found in naive B6 mice after transferring of CD8 T cells from immunized B6 mice compared with transfer of CD4 T cells.. Our data suggest that CD8 autoreactive T cells in EAE have a lower encephalitogenic function but are unique and independent on pathogenic of EAE rather than their CD4 counterparts.

Topics: Adoptive Transfer; Animals; CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Female; Mice; Mice, Inbred C57BL; Myelin-Oligodendrocyte Glycoprotein; Peptide Fragments

In the injured central nervous system, myeloid cells, including macrophages and microglia, are key contributors to both myelin injury and repair. This immense plasticity emphasizes the need to further understand the precise molecular mechanisms that contribute to the dynamic regulation of myeloid cell polarization and function. Herein, we demonstrate that miR-223 is upregulated in multiple sclerosis (MS) patient monocytes and the alternatively-activated and tissue-regenerating M2-polarized human macrophages and microglia. Using miR-223 knock-out mice, we observed that miR-223 is dispensable for maximal pro-inflammatory responses, but is required for efficient M2-associated phenotype and function, including phagocytosis. Using the lysolecithin animal model, we further demonstrate that miR-223 is required to efficiently clear myelin debris and promote remyelination. These results suggest miR-223 constrains neuroinflammation while also promoting repair, a finding of important pathophysiological relevance to MS as well as other neurodegenerative diseases.

Topics: Animals; Case-Control Studies; Cells, Cultured; Corpus Callosum; Demyelinating Autoimmune Diseases, CNS; Disease Models, Animal; Freund's Adjuvant; Glial Fibrillary Acidic Protein; Humans; Lipopolysaccharides; Lysophosphatidylcholines; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Microglia; MicroRNAs; Monocytes; Myelin-Oligodendrocyte Glycoprotein; Myeloid Cells; Peptide Fragments; Phagocytosis; Reactive Oxygen Species

The developmental programs that generate a broad repertoire of regulatory T cells (T

Topics: Animals; Autoantigens; Cell Differentiation; Colitis; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Forkhead Transcription Factors; Freund's Adjuvant; Humans; Immune Tolerance; Interleukin-2 Receptor alpha Subunit; Lymphoid Progenitor Cells; Mice; Mice, Transgenic; Mycobacterium tuberculosis; Myelin-Oligodendrocyte Glycoprotein; Peptide Fragments; Signal Transduction; Specific Pathogen-Free Organisms; T-Lymphocytes, Regulatory; Thymus Gland

Multiple sclerosis (MS) is a neurodegenerative autoimmune disease with many known structural and functional changes in the central nervous system. A well-recognized, but poorly understood, complication of MS is chronic pain. Little is known regarding the influence of sex on the development and maintenance of MS-related pain. This is important to consider, as MS is a predominantly female disease. Using the experimental autoimmune encephalomyelitis (EAE) mouse model of MS, we demonstrate sex differences in measures of spinal cord inflammation and plasticity that accompany tactile hypersensitivity. Although we observed substantial inflammatory activity in both sexes, only male EAE mice exhibit robust staining of axonal injury markers and increased dendritic arborisation in morphology of deep dorsal horn neurons. We propose that tactile hypersensitivity in female EAE mice may be more immune-driven, whereas pain in male mice with EAE may rely more heavily on neurodegenerative and plasticity-related mechanisms. Morphological and inflammatory differences in the spinal cord associated with pain early in EAE progression supports the idea of differentially regulated pain pathways between the sexes. Results from this study may indicate future sex-specific targets that are worth investigating for their functional role in pain circuitry.

Topics: Animals; Axons; Calcium-Binding Proteins; Central Nervous System; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Estrous Cycle; Female; Freund's Adjuvant; Hyperalgesia; Male; Mice; Mice, Inbred C57BL; Microfilament Proteins; Motor Activity; Myelin-Oligodendrocyte Glycoprotein; Neuronal Plasticity; Pain; Pain Threshold; Peptide Fragments; Pertussis Toxin; Physical Stimulation; Sex Factors

Myeloid-derived suppressor cells (MDSCs) are a diverse group of cells that are recognized for their remarkable suppressive effects on pro-inflammatory T cells. The pleiotropic nature of these cells, however, has been demonstrated by their differential effects on immune responses in different settings. Our and others' work has demonstrated suppressive effects of these cells. We previously demonstrated that these cells were mobilized to the lungs during experimental autoimmune encephalomyelitis (EAE), which is a murine model of multiple sclerosis, and potently inhibited CD8

Topics: Animals; Autoimmunity; Cell Communication; Cell Lineage; Cell Movement; Central Nervous System; Coculture Techniques; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Female; Gene Expression; Humans; Interleukin-17; Lung; Mice, Inbred C57BL; Multiple Sclerosis; Myelin-Oligodendrocyte Glycoprotein; Myeloid-Derived Suppressor Cells; Peptide Fragments; Pertussis Toxin; Signal Transduction; Th17 Cells; Transforming Growth Factor beta

Neuromyelitis optica (NMO) is recognized as a different CNS autoimmune disease from multiple sclerosis (MS). Whether NMO-IgG contributes directly to the pathogenesis of NMO or is just a serologic marker of autoimmune responses of the disease needs to be clarified. We created MOG-induced experimental autoimmune encephalomyelitis (EAE) mice by passively transferring NMO-IgG to model the pathogenic findings in NMO patients. The mice were divided into three groups and administered intrathecal PBS, human complement with IgG from normal subjects, or IgG from AQP4(+) patients on days 8 and 11 after immunization. The EAE scores of EAE mice with intrathecal NMO-IgG injection were significantly elevated 14 days post-immunization. All of the mice were sacrificed for brain and spinal cord pathology analysis on day 21 post-immunization. Compared to mice given normal human IgG, EAE mice injected with NMO-IgG had markedly decreased AQP4 and glial fibrillary acidic protein (GFAP) expression and fluorescent intensity in the brain and spinal cord but more scattered deposition of complement (C9neo). Thus, our studies not only support the pathogenic role of NMO-IgG with complement in NMO disease but also provide a platform for the development of future therapeutics.

Topics: Analysis of Variance; Animals; Aquaporin 4; Brain; Complement System Proteins; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Female; Freund's Adjuvant; Glial Fibrillary Acidic Protein; HEK293 Cells; Humans; Immunoglobulin G; Injections, Spinal; Mice; Mice, Inbred C57BL; Myelin-Oligodendrocyte Glycoprotein; Peptide Fragments; Spinal Cord; Time Factors

Dysregulation of the JAK/STAT signaling pathway is associated with Multiple Sclerosis (MS) and its mouse model, Experimental Autoimmune Encephalomyelitis (EAE). Suppressors Of Cytokine Signaling (SOCS) negatively regulate the JAK/STAT pathway. We previously reported a severe, brain-targeted, atypical form of EAE in mice lacking Socs3 in myeloid cells (Socs3ΔLysM), which is associated with cerebellar neutrophil infiltration. There is emerging evidence that neutrophils are detrimental in the pathology of MS/EAE, however, their exact function is unclear. Here we demonstrate that neutrophils from the cerebellum of Socs3ΔLysM mice show a hyper-activated phenotype with excessive production of reactive oxygen species (ROS) at the peak of EAE. Neutralization of ROS in vivo delayed the onset and reduced severity of atypical EAE. Mechanistically, Socs3-deficient neutrophils exhibit enhanced STAT3 activation, a hyper-activated phenotype in response to G-CSF, and upon G-CSF priming, increased ROS production. Neutralization of G-CSF in vivo significantly reduced the incidence and severity of the atypical EAE phenotype. Overall, our work elucidates that hypersensitivity of G-CSF/STAT3 signaling in Socs3ΔLysM mice leads to atypical EAE by enhanced neutrophil activation and increased oxidative stress, which may explain the detrimental role of G-CSF in MS patients.

Topics: Animals; Cerebellum; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Flow Cytometry; Granulocyte Colony-Stimulating Factor; Mice; Myelin-Oligodendrocyte Glycoprotein; Neutrophil Activation; Neutrophils; Oxidative Stress; Peptide Fragments; Reactive Oxygen Species; Real-Time Polymerase Chain Reaction; RNA-Seq; Severity of Illness Index; Signal Transduction; STAT3 Transcription Factor; Suppressor of Cytokine Signaling 3 Protein

Contrasting studies are reported on the induction of IL-10 and IFN-γ via chitin microparticles (CMPs) during immune stimulation. Our previous studies have shown marked protection among CMP treated Leishmania-infected mice via regulated IL-10/IFN-γ response, at the present study, once more, examined the inconsistent responses regarding the immunologic response of CMPS. To verify whether CMPs could indeed up-regulate IL-10/IFN-γ axis, isolated spleen cells from the myelin oligodendrocyte glycoprotein (MOG) induced experimental autoimmune encephalomyelitis (EAE) mice were cultured in the presence of MOG peptide and/or CMPs. The effects of CMPs on IL-10, IFN-γ and IL-17 production were evaluated by Enzyme-linked Immunosorbent Assay (ELISA). Moreover, GATA binding protein 3 (Gata3), T-box transcription factor TBX21 (Tbx21), and RAR-related orphan receptor gamma (RORγT) expressions (real-time PCR) were investigated. MOG alone stimulated the production of IFN-γ (p≤0.004) but not, IL-10 (p≤0.140). MOG/chitin stimulation resulted in a significant increase in IFN-γ and IL-10 levels, respectively; (p≤0.004 and p≤0.003) rather than MOG. Additionally, the expression of Tbx21 (p≤0.001), but not Gata3 (p≤0.08), was increased in the MOG/chitin-treated spleen cells. All in all, CMP supports Gata3 independent IL-10 production and promotes Tbx21 dependent IFN-γ induction. These results, alongside our previous data, indicate that CMPs has particular adjuvant effects.

Topics: Adjuvants, Immunologic; Animals; Cell-Derived Microparticles; Cells, Cultured; Chitin; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Female; Humans; Immunomodulation; Interferon-gamma; Interleukin-10; Mice; Mice, Inbred C57BL; Multiple Sclerosis; Myelin-Oligodendrocyte Glycoprotein; Peptide Fragments; Spleen; T-Box Domain Proteins

Emerging evidence indicates that gut microbiota interacts with immune and nervous systems in the host and plays a critical role in the pathogenesis of multiple sclerosis (MS) and many psychiatric disorders such as depression and anxiety. The aim of this study was to explore the influence of gut bacterial depletion from early adolescence on adult immunological and neurobehavioral responses in mice with experimental-autoimmune-encephalomyelitis (EAE). We used an animal model of gut microbiota depletion induced by antibiotics from weaning to adulthood to assess clinical signs, cognitive function and depression-and anxiety-related symptoms in non-EAE and EAE-induced mice. We measured levels of interferon (IFN)-γ, interleukin (IL)-17A and IL-10 in serum, and BDNF, IL-1β and tumor necrosis factor (TNF)-α) in the hippocampus. Antibiotic-treated mice displayed a significant delay in the onset of clinical symptoms of EAE. However, a higher severity of EAE was found between days 19-22 post-immunization in antibiotics-treated mice, while a reduction in the clinical signs of MS was observed at days 24-25 post-immunization. Antibiotic administration decreased IFN-γ and IL-17A levels and increased IL-10 in serum of EAE-induced mice. Antibiotic treatment significantly decreased hippocampal BDNF and enhanced learning and memory impairments in EAE-induced mice. However, no significant changes were found in non-EAE mice. Non-EAE and EAE mice treated with antibiotics exhibited increased anxiety-related behaviors, whereas depression-related symptoms and increased hippocampal TNF-α and IL-1β were only observed in EAE-induced mice treated with antibiotics. This study supports the view that depletion of gut microbiota by antibiotics from weaning profoundly impacts adult immunological and neurobehavioral responses.

Topics: Animals; Anti-Bacterial Agents; Anxiety; Behavior, Animal; Brain-Derived Neurotrophic Factor; Depression; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Female; Gastrointestinal Microbiome; Hippocampus; Inflammation Mediators; Memory Disorders; Mice; Multiple Sclerosis; Myelin-Oligodendrocyte Glycoprotein; Peptide Fragments; Time Factors

Mouse experimental autoimmune encephalomyelitis (EAE) is widely used model of multiple sclerosis (MS). The role of autoreactive CD4. In this study, an EAE model with a clinical course containing acute onset, peak and chronic remission stages was established in C57BL/6J mice by myelin oligodendrocyte protein (MOG). The dynamic changes of inflammatory infiltration, myelin loss, and astrocyte proliferation in brain and spinal cord were highly consistent with clinical severity observed in EAE course. However, the frequencies of both MOG-specific CD4. Both CD4

Topics: Animals; Astrocytes; Autoantigens; Brain; CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; Cell Movement; Cell Proliferation; Cells, Cultured; Disease Models, Animal; Disease Progression; Encephalomyelitis, Autoimmune, Experimental; Female; Humans; Lymphocyte Activation; Mice; Mice, Inbred C57BL; Multiple Sclerosis; Myelin-Oligodendrocyte Glycoprotein; Peptide Fragments; Spinal Cord

Multiple sclerosis (MS) is an autoimmune-inflammatory neurodegenerative disease that is often accompanied by a debilitating neuropathic pain. Disease-modifying agents slow down the progression of multiple sclerosis and prevent relapses, yet it remains unclear if they yield analgesia. We explored the analgesic potential of fingolimod (FTY720), an agonist and/or functional antagonist at the sphingosine-1-phosphate receptor 1 (S1PR1), because it reduces hyperalgesia in models of peripheral inflammatory and neuropathic pain. We used a myelin oligodendrocyte glycoprotein 35 to 55 (MOG35-55) mouse model of experimental autoimmune encephalomyelitis, modified to avoid frank paralysis, and thus, allow for assessment of withdrawal behaviors to somatosensory stimuli. Daily intraperitoneal fingolimod reduced behavioral signs of central neuropathic pain (mechanical and cold hypersensitivity) in a dose-dependent and reversible manner. Both autoimmune encephalomyelitis and fingolimod changed hyperalgesia before modifying motor function, suggesting that pain-related effects and clinical neurological deficits were modulated independently. Fingolimod also reduced cellular markers of central sensitization of neurons in the dorsal horn of the spinal cord: glutamate-evoked Ca signaling and stimulus-evoked phospho-extracellular signal-related kinase ERK (pERK) expression, as well as upregulation of astrocytes (GFAP) and macrophage/microglia (Iba1) immunoreactivity. The antihyperalgesic effects of fingolimod were prevented or reversed by the S1PR1 antagonist W146 (1 mg/kg daily, i.p.) and could be mimicked by either repeated or single injection of the S1PR1-selective agonist SEW2871. Fingolimod did not change spinal membrane S1PR1 content, arguing against a functional antagonist mechanism. We conclude that fingolimod behaves as an S1PR1 agonist to reduce pain in multiple sclerosis by reversing central sensitization of spinal nociceptive neurons.

Topics: Anilides; Animals; Central Nervous System Sensitization; Disease Models, Animal; eIF-2 Kinase; Female; Fingolimod Hydrochloride; Immunosuppressive Agents; Male; Mice; Mice, Inbred C57BL; Motor Activity; Multiple Sclerosis; Myelin-Oligodendrocyte Glycoprotein; Neuralgia; Organophosphonates; Oxadiazoles; Pain Threshold; Peptide Fragments; Receptors, Lysosphingolipid; Sphingosine-1-Phosphate Receptors; Spinal Cord; Spinal Nerve Roots; Subcellular Fractions; Thiophenes

Oestrogen treatments are neuroprotective in a variety of neurodegenerative disease models. Selective oestrogen receptor modifiers are needed to optimize beneficial effects while minimizing adverse effects to achieve neuroprotection in chronic diseases. Oestrogen receptor beta (ERβ) ligands are potential candidates. In the multiple sclerosis model chronic experimental autoimmune encephalomyelitis, ERβ-ligand treatment is neuroprotective, but mechanisms underlying this neuroprotection remain unclear. Specifically, whether there are direct effects of ERβ-ligand on CD11c+ microglia, myeloid dendritic cells or macrophages in vivo during disease is unknown. Here, we generated mice with ERβ deleted from CD11c+ cells to show direct effects of ERβ-ligand treatment in vivo on these cells to mediate neuroprotection during experimental autoimmune encephalomyelitis. Further, we use bone marrow chimeras to show that ERβ in peripherally derived myeloid cells, not resident microglia, are the CD11c+ cells mediating this protection. CD11c+ dendritic cell and macrophages isolated from the central nervous system of wild-type experimental autoimmune encephalomyelitis mice treated with ERβ-ligand expressed less iNOS and T-bet, but more IL-10, and this treatment effect was lost in mice with specific deletion of ERβ in CD11c+ cells. Also, we extend previous reports of ERβ-ligand’s ability to enhance remyelination through a direct effect on oligodendrocytes by showing that the immunomodulatory effect of ERβ-ligand acting on CD11c+ cells is necessary to permit the maturation of oligodendrocytes. Together these results demonstrate that targeting ERβ signalling pathways in CD11c+ myeloid cells is a novel strategy for regulation of the innate immune system in neurodegenerative diseases. To our knowledge, this is the first report showing how direct effects of a candidate neuroprotective treatment on two distinct cell lineages (bone marrow derived myeloid cells and oligodendrocytes) can have complementary neuroprotective effects in vivo.awx315media15688130498001.

Topics: Animals; Basic Helix-Loop-Helix Transcription Factors; Bone Marrow Transplantation; Calcium-Binding Proteins; CD11 Antigens; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Estrogen Receptor beta; Female; Green Fluorescent Proteins; Histocompatibility Antigens Class II; Ligands; Macrophages; Mice; Mice, Transgenic; Microfilament Proteins; Myelin Basic Protein; Myelin-Oligodendrocyte Glycoprotein; Neuroprotective Agents; Nitric Oxide Synthase Type II; Ovariectomy; Peptide Fragments

The G protein-coupled receptor 65 (GPR65) gene has been genetically associated with several autoimmune diseases, including multiple sclerosis (MS). GPR65 is predominantly expressed in lymphoid organs and is activated by extracellular protons. In this study, we tested whether GPR65 plays a functional role in demyelinating autoimmune disease. Using a murine model of MS, experimental autoimmune encephalomyelitis (EAE), we found that Gpr65-deficient mice develop exacerbated disease. CD4

Topics: Adoptive Transfer; Animals; CD4-Positive T-Lymphocytes; Disease Models, Animal; Disease Progression; Encephalomyelitis, Autoimmune, Experimental; Mice, Inbred C57BL; Myelin-Oligodendrocyte Glycoprotein; Natural Killer T-Cells; Peptide Fragments; Receptors, G-Protein-Coupled

The genome organizer special AT-rich sequence binding protein 1 (SATB1) regulates specific functions through chromatin remodeling in T helper cells. It was recently reported by our team that T cells from SATB1 conditional knockout (SATB1cKO) mice, in which the Satb1 gene is deleted from hematopoietic cells, impair phosphorylation of signaling molecules in response to T cell receptor (TCR) crosslinking. However, in vivo T cell responses upon antigen presentation in the absence of SATB1 remain unclear. In the current study, it was shown that SATB1 modulates T cell antigen responses during the induction and effector phases. Expression of SATB1 was upregulated in response to TCR stimulation, suggesting that SATB1 is important for this antigen response. The role of SATB1 in TCR responses and induced experimental autoimmune encephalomyelitis (EAE) was therefore examined using the myelin oligodendrocyte glycoprotein peptide 35-55 (MOG35-55) and pertussis toxin. SATB1cKO mice were found to be resistant to EAE and had defects in IL-17- and IFN-γ-producing pathogenic T cells. Thus, SATB1 expression appears necessary for T cell function in the induction phase. To examine SATB1 function during the effector phase, a tamoxifen-inducible SATB1 deletion system, SATB1cKO-ER-Cre mice, was used. Encephalitogenic T cells from MOG35-55-immunized SATB1cKO-ER-Cre mice were transferred into healthy mice. Mice that received tamoxifen before the onset of paralysis were resistant to EAE. Furthermore, no disease progression occurred in recipient mice treated with tamoxifen after the onset of EAE. Thus, SATB1 is essential for maintaining TCR responsiveness during the induction and effector phases and may provide a novel therapeutic target for T cell-mediated autoimmune diseases.

Topics: Animals; Disease Models, Animal; Disease Progression; Encephalomyelitis, Autoimmune, Experimental; GPI-Linked Proteins; Interferon-gamma; Interleukin-17; Matrix Attachment Region Binding Proteins; Mice; Mice, Inbred C57BL; Mice, Knockout; Myelin Proteins; Myelin-Oligodendrocyte Glycoprotein; Peptide Fragments; Pertussis Toxin; Receptors, Antigen, T-Cell; T-Lymphocytes; Tamoxifen

Promoting remyelination is recognized as a novel strategy to foster repair in neurodegenerative demyelinating diseases, such as multiple sclerosis. In this respect, the receptor GPR17, recently emerged as a new target for remyelination, is expressed by early oligodendrocyte precursors (OPCs) and after a certain differentiation stage it has to be downregulated to allow progression to mature myelinating oligodendrocytes. Here, we took advantage of the first inducible GPR17 reporter mouse line (GPR17-iCreER

Topics: Animals; Cuprizone; Demyelinating Diseases; Disease Models, Animal; Female; Green Fluorescent Proteins; Male; Mice, Inbred C57BL; Mice, Transgenic; Myelin-Oligodendrocyte Glycoprotein; Nerve Tissue Proteins; Oligodendrocyte Precursor Cells; Peptide Fragments; Receptors, G-Protein-Coupled; Remyelination; Spinal Cord

Multiple sclerosis presents with profound changes in the network of molecules involved in maintaining central nervous system architecture, the extracellular matrix. The extracellular matrix components, particularly the chondroitin sulfate proteoglycans, have functions beyond structural support including their potential interaction with, and regulation of, inflammatory molecules. To investigate the roles of chondroitin sulfate proteoglycans in multiple sclerosis, we used the experimental autoimmune encephalomyelitis model in a time course study. We found that the 4-sulfated glycosaminoglycan side chains of chondroitin sulfate proteoglycans, and the core protein of a particular family member, versican V1, were upregulated in the spinal cord of mice at peak clinical severity, correspondent with areas of inflammation. Versican V1 expression in the spinal cord rose progressively over the course of experimental autoimmune encephalomyelitis. A particular structure in the spinal cord and cerebellum that presented with intense upregulation of chondroitin sulfate proteoglycans is the leucocyte-containing perivascular cuff, an important portal of entry of immune cells into the central nervous system parenchyma. In these inflammatory perivascular cuffs, versican V1 and the glycosaminoglycan side chains of chondroitin sulfate proteoglycans were observed by immunohistochemistry within and in proximity to lymphocytes and macrophages as they migrated across the basement membrane into the central nervous system. Expression of versican V1 transcript was also documented in infiltrating CD45+ leucocytes and F4/80+ macrophages by in situ hybridization. To test the hypothesis that the chondroitin sulfate proteoglycans regulate leucocyte mobility, we used macrophages in tissue culture studies. Chondroitin sulfate proteoglycans significantly upregulated pro-inflammatory cytokines and chemokines in macrophages. Strikingly, and more potently than the toll-like receptor-4 ligand lipopolysaccharide, chondroitin sulfate proteoglycans increased the levels of several members of the matrix metalloproteinase family, which are implicated in the capacity of leucocytes to cross barriers. In support, the migratory capacity of macrophages in vitro in a Boyden chamber transwell assay was enhanced by chondroitin sulfate proteoglycans. Finally, using brain specimens from four subjects with multiple sclerosis with active lesions, we found chondroitin sulfate proteoglycans to be associated with le

Topics: Animals; Brain; Cell Movement; Chondroitin Sulfate Proteoglycans; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Female; Freund's Adjuvant; Laminin; Lipopolysaccharides; Macrophages; Matrix Metalloproteinases; Mice; Mice, Inbred C57BL; Myelin-Oligodendrocyte Glycoprotein; Neutrophil Infiltration; Peptide Fragments; RNA, Messenger; Spinal Cord; Time Factors; Tumor Necrosis Factor-alpha; Up-Regulation; Versicans

MS and EAE are T cell-driven autoimmune diseases of the CNS where IL-17-producing Th17 cells promote damage and are pathogenic. Conversely, tolerogenic DCs induce Treg cells and suppress Th17 cells. Chloroquine (CQ) suppresses EAE through the modulation of DCs by unknown mechanisms. Here, we show that STAT 1 is necessary for CQ-induced tolerogenic DCs (tolDCs) to efficiently suppress EAE. We observed that CQ induces phosphorylation of STAT1 in DCs in vivo and in vitro. Genetic blockage of STAT1 abrogated the suppressive activity of CQ-treated DCs. Opposed to its WT counterparts, CQ-treated STAT1

Topics: Animals; Autoantigens; Cells, Cultured; Chloroquine; Dendritic Cells; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Gene Expression Regulation; Humans; Mice; Mice, Inbred C57BL; Mice, Knockout; Multiple Sclerosis; Myelin-Oligodendrocyte Glycoprotein; Neutrophils; Peptide Fragments; Signal Transduction; STAT1 Transcription Factor; Th17 Cells

Voltage-gated calcium channels (VGCCs) play a critical role in neuroinflammatory diseases, such as multiple sclerosis (MS). CTK 01512-2 is a recombinant version of the peptide Phα1β derived from the spider Phoneutria nigriventer, which inhibits N-type VGCC/TRPA1-mediated calcium influx. We investigated the effects of this molecule in the mouse model of experimental autoimmune encephalomyelitis (EAE). The effects of CTK 01512-2 were compared to those displayed by ziconotide-a selective N-type VGCC blocker clinically used for chronic pain-and fingolimod-a drug employed for MS treatment. The intrathecal (i.t.) treatment with CTK 01512-2 displayed beneficial effects, by preventing nociception, body weight loss, splenomegaly, MS-like clinical and neurological scores, impaired motor coordination, and memory deficits, with an efficacy comparable to that observed for ziconotide and fingolimod. This molecule displayed a favorable profile on EAE-induced neuroinflammatory changes, including inflammatory infiltrate, demyelination, pro-inflammatory cytokine production, glial activation, and glucose metabolism in the brain and spinal cord. The recovery of spatial memory, besides a reduction of serum leptin levels, allied to central and peripheral elevation of the anti-inflammatory cytokine IL-10, was solely modulated by CTK 01512-2, dosed intrathecally. The intravenous (i.v.) administration of CTK 01512-2 also reduced the EAE-elicited MS-like symptoms, similarly to that seen in animals that received fingolimod orally. Ziconotide lacked any significant effect when dosed by i.v. route. Our results indicate that CTK 01512-2 greatly improved the neuroinflammatory responses in a mouse model of MS, with a higher efficacy when compared to ziconotide, pointing out this molecule as a promising adjuvant for MS management.

Topics: Animals; Anti-Inflammatory Agents; Calcium Channel Blockers; Chemokines; Cognition Disorders; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Female; Fingolimod Hydrochloride; Hyperalgesia; Inflammation; Inflammation Mediators; Injections, Spinal; Mice, Inbred C57BL; Motor Activity; Multiple Sclerosis; Myelin Sheath; Myelin-Oligodendrocyte Glycoprotein; Nociception; omega-Conotoxins; Peptide Fragments

Increasing evidence points to an important role for neutrophils in participating in the pathogenesis of the human demyelinating disease MS and the animal model EAE. Therefore, a better understanding of the signals controlling migration of neutrophils as well as evaluating the role of these cells in demyelination is important to define cellular components that contribute to disease in MS patients. In this study, we examined the functional role of the chemokine CXCL1 in contributing to neuroinflammation and demyelination in EAE. Using transgenic mice in which expression of CXCL1 is under the control of a tetracycline-inducible promoter active within glial fibrillary acidic protein-positive cells, we have shown that sustained CXCL1 expression within the CNS increased the severity of clinical and histologic disease that was independent of an increase in the frequency of encephalitogenic Th1 and Th17 cells. Rather, disease was associated with enhanced recruitment of CD11b

Topics: Animals; Autoantigens; CD11b Antigen; Cells, Cultured; Central Nervous System; Chemokine CXCL1; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Gene Expression Regulation; Humans; Mice; Mice, Inbred C57BL; Mice, Transgenic; Molecular Targeted Therapy; Multiple Sclerosis; Myelin-Oligodendrocyte Glycoprotein; Neurogenic Inflammation; Neutrophil Infiltration; Neutrophils; Peptide Fragments; Signal Transduction; Spinal Cord

Topics: Adult; Animals; Autoantibodies; Autoimmunity; B-Lymphocytes; Brain; CD4-Positive T-Lymphocytes; Cytokines; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Female; Germinal Center; Humans; Lymphocyte Activation; Lymphocyte Count; Male; Mice; Middle Aged; Multiple Sclerosis, Relapsing-Remitting; Myelin-Oligodendrocyte Glycoprotein; Peptide Fragments; Spinal Cord; T-Lymphocytes, Helper-Inducer; Young Adult

The oral immunomodulatory agent laquinimod is currently evaluated for multiple sclerosis (MS) treatment. Phase II and III studies demonstrated a reduction of degenerative processes. In addition to anti-inflammatory effects, laquinimod might have neuroprotective properties, but its impact on the visual system, which is often affected by MS, is unknown. The aim of our study was to investigate potential protective effects of laquinimod on the optic nerve and retina in an experimental autoimmune encephalomyelitis (EAE) model.. Laquinimod reduced neurological EAE symptoms and improved the neuronal electrical output of the inner nuclear layer compared to untreated EAE mice. Furthermore, cellular infiltration, especially recruited phagocytes, and demyelination in the optic nerve were reduced. Microglia were diminished in optic nerve and retina. Retinal macroglial signal was reduced under treatment, whereas in the optic nerve macroglia were not affected. Additionally, laquinimod preserved retinal ganglion cells and reduced apoptosis. A later treatment with laquinimod in a therapeutic approach led to a reduction of clinical signs and to an improved b-wave amplitude. However, no changes in cellular infiltration and demyelination of the optic nerves were observed. Also, the number of retinal ganglion cells remained unaltered.. From our study, we deduce neuroprotective and anti-inflammatory effects of laquinimod on the optic nerve and retina in EAE mice, when animals were treated before any clinical signs were noted. Given the fact that the visual system is frequently affected by MS, the agent might be an interesting subject of further neuro-ophthalmic investigations.

Topics: Animals; Antigens, Differentiation; Calcium-Binding Proteins; Disease Models, Animal; Dose-Response Relationship, Drug; Electroretinography; Encephalomyelitis, Autoimmune, Experimental; Gene Expression Regulation; Mice; Mice, Inbred C57BL; Microfilament Proteins; Myelin Basic Protein; Myelin-Oligodendrocyte Glycoprotein; Nerve Tissue Proteins; Optic Nerve; Peptide Fragments; Phagocytes; Quinolones; Retina; RNA, Messenger

Myelin oligodendrocyte glycoprotein (MOG)-reactive T-helper (Th)1 cells induce conventional experimental autoimmune encephalomyelitis (cEAE), characterized by ascending paralysis and monocyte-predominant spinal cord infiltrates, in C57BL/6 wildtype (WT) hosts. The same T cells induce an atypical form of EAE (aEAE), characterized by ataxia and neutrophil-predominant brainstem infiltrates, in syngeneic IFNγ receptor (IFNγR)-deficient hosts. Production of ELR+ CXC chemokines within the CNS is required for the development of aEAE, but not cEAE. The cellular source(s) and localization of ELR+ CXC chemokines in the CNS and the IFNγ-dependent pathways that regulate their production remain to be elucidated.. The spatial distribution of inflammatory lesions and CNS expression of the ELR+ CXC chemokines, CXCL1 and CXCL2, were determined via immunohistochemistry and/or in situ hybridization. Levels of CXCL1 and CXCL2, and their cognate receptor CXCR2, were measured in/on leukocyte subsets by flow cytometric and quantitative PCR (qPCR) analysis. Bone marrow neutrophils and macrophages were cultured with inflammatory stimuli in vitro prior to measurement of CXCL2 and CXCR2 by qPCR or flow cytometry.. CNS-infiltrating neutrophils and monocytes, and resident microglia, are a prominent source of CXCL2 in the brainstem of IFNγRKO adoptive transfer recipients during aEAE. In WT transfer recipients, IFNγ directly suppresses CXCL2 transcription in microglia and myeloid cells, and CXCR2 transcription in CNS-infiltrating neutrophils. Consequently, infiltration of the brainstem parenchyma from the adjacent meninges is blocked during cEAE. CXCL2 directly stimulates its own expression in cultured neutrophils, which is enhanced by IL-1 and suppressed by IFNγ.. We provide evidence for an IFNγ-regulated CXCR2/CXCL2 autocrine/paracrine feedback loop in innate immune cells that determines the location of CNS infiltrates during Th1-mediated EAE. When IFNγ signaling is impaired, myeloid cell production of CXCL2 increases, which promotes brainstem inflammation and results in clinical ataxia. IFNγ, produced within the CNS of WT recipients, suppresses myeloid cell CXCR2 and CXCL2 production, thereby skewing the location of neuroinflammatory infiltrates to the spinal cord and the clinical phenotype to an ascending paralysis. These data reveal a novel mechanism by which IFNγ and CXCL2 interact to direct regional recruitment of leukocytes in the CNS, resulting in distinct clinical presentations.

Topics: Animals; Brain; Chemokine CXCL2; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Glial Fibrillary Acidic Protein; Interferon-gamma; Leukocyte Common Antigens; Mice; Mice, Inbred C57BL; Mice, Transgenic; Monocytes; Myelin-Oligodendrocyte Glycoprotein; Myeloid Cells; Neutrophil Infiltration; Neutrophils; Parenchymal Tissue; Peptide Fragments; RNA, Messenger; Signal Transduction

Multiple sclerosis (MS) is a chronic inflammatory autoimmune disease of the central nervous system (CNS). Currently, there is still lack of curative treatment for MS. Mesenchymal stem cell- (MSC-) based therapy is recently the subject of intense interest in autoimmune diseases. Here, we investigated the therapeutic effect and potential mechanism of human amnion mesenchymal cells (hAMC) on inflammation and remyelination in experimental autoimmune encephalomyelitis (EAE) mice. C57BL/6 mice were immunized with myelin oligodendrocyte glycoprotein (MOG) 35-55 peptide. hAMC were injected intraperitoneal when EAE was successfully established. The results demonstrated that application of hAMC significantly ameliorated the disease severity and histopathological changes in EAE mice. The production of proinflammatory cytokines such as IFN-

Topics: Animals; Cells, Cultured; Chronic Disease; Cytokines; Disease Models, Animal; Disease Progression; Encephalomyelitis, Autoimmune, Experimental; Female; Humans; Inflammation; Inflammation Mediators; Mesenchymal Stem Cells; Mice; Mice, Inbred C57BL; Multiple Sclerosis; Myelin-Oligodendrocyte Glycoprotein; Neurons; Peptide Fragments; Wound Healing

Very late antigen 4 (VLA-4; integrin α4β1) is critical for transmigration of T helper (T. Antibody blockade of VLA-4 and MCAM is assessed in murine models of CNS inflammation in conjunction with conditional ablation of α4-integrin expression in T cells. Effects of VLA-4 and MCAM blockade on lymphocyte migration are further investigated in the human system via in vitro T cell transmigration assays.. Compared to the broad effects of VLA-4 blockade on encephalitogenic T cell migration over endothelial barriers, MCAM blockade impeded encephalitogenic T cell migration in murine models of MS that especially depend on CNS migration across the choroid plexus (CP). In transgenic mice lacking T cell α4-integrin expression (CD4::Itga4. Our findings suggest that MCAM-laminin 411 interactions facilitate trans-endothelial migration of MCAM-expressing T cells into the CNS, which seems to be highly relevant to migration via the CP and to potential future clinical applications in neuroinflammatory disorders.

Topics: Animals; Antibodies; CD146 Antigen; Cell Movement; Cells, Cultured; Central Nervous System; Choroid Plexus; Cytokines; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Endothelial Cells; Freund's Adjuvant; Humans; Integrin alpha4beta1; Mice; Mice, Inbred C57BL; Mice, Transgenic; Myelin-Oligodendrocyte Glycoprotein; Peptide Fragments; Protein Kinases; T-Lymphocytes

Neuroinflammatory diseases, such as multiple sclerosis, are characterized by invasion of the brain by autoreactive T cells. The mechanism for how T cells acquire their encephalitogenic phenotype and trigger disease remains, however, unclear. The existence of lymphatic vessels in the meninges indicates a relevant link between the CNS and peripheral immune system, perhaps affecting autoimmunity. Here we demonstrate that meningeal lymphatics fulfill two critical criteria: they assist in the drainage of cerebrospinal fluid components and enable immune cells to enter draining lymph nodes in a CCR7-dependent manner. Unlike other tissues, meningeal lymphatic endothelial cells do not undergo expansion during inflammation, and they express a unique transcriptional signature. Notably, the ablation of meningeal lymphatics diminishes pathology and reduces the inflammatory response of brain-reactive T cells during an animal model of multiple sclerosis. Our findings demonstrate that meningeal lymphatics govern inflammatory processes and immune surveillance of the CNS and pose a valuable target for therapeutic intervention.

Topics: Animals; Antigens, CD; Central Nervous System; Dendritic Cells; Disease Models, Animal; Encephalitis; Green Fluorescent Proteins; Homeodomain Proteins; Lymph Nodes; Lymphatic Vessels; Male; Meninges; Mice; Mice, Inbred C57BL; Mice, Transgenic; MicroRNAs; Myelin-Oligodendrocyte Glycoprotein; Peptide Fragments; Photosensitizing Agents; Receptors, CCR7; Spleen; T-Lymphocytes; Tumor Suppressor Proteins

Axonal degeneration and neuronal loss have been described as the major causes of irreversible clinical disability in multiple sclerosis (MS). The aryl-hydrocarbon receptor nuclear translocator 2 (ARNT2) protein has been associated with neuroprotection in models of ischemia and neuronal responses to stressors.. To characterize its potential to influence inflammatory neurodegeneration, we examined ARNT2 expression in the experimental autoimmune encephalomyelitis (EAE) model of MS and characterized mediators that influence ARNT2 expression as well as plausible partners and targets.. Arnt2 message and protein levels dropped significantly in EAE spinal cords as disease developed and were lowest at peak disability. ARNT2 expression is prominent in neuronal cell bodies within the gray matter with some staining in glial fibrillary acidic protein (GFAP). Our data support ARNT2 as a neuronal transcription factor whose sustained expression is linked to neuronal and axonal health, protection that may primarily be driven through its partnering with Npas4 to influence BDNF expression.

Topics: Animals; Aryl Hydrocarbon Receptor Nuclear Translocator; Astrocytes; Axons; Basic Helix-Loop-Helix Transcription Factors; Cells, Cultured; Cerebral Cortex; Disease Models, Animal; Disease Progression; Embryo, Mammalian; Female; Freund's Adjuvant; Gene Expression Regulation; Hydrogen Peroxide; Male; Mice; Mice, Inbred C57BL; Multiple Sclerosis; Myelin-Oligodendrocyte Glycoprotein; Nerve Tissue Proteins; Neurons; Oxidative Stress; Peptide Fragments; Pertussis Toxin

V-type immunoglobulin domain-containing suppressor of T-cell activation (VISTA) is a negative checkpoint regulator (NCR) involved in inhibition of T cell-mediated immunity. Expression changes of other NCRs (PD-1, PD-L1/L2, CTLA-4) during inflammation of the central nervous system (CNS) were previously demonstrated, but VISTA expression in the CNS has not yet been explored. Here, we report that in the human and mouse CNS, VISTA is most abundantly expressed by microglia, and to lower levels by endothelial cells. Upon TLR stimulation, VISTA expression was reduced in primary neonatal mouse and adult rhesus macaque microglia in vitro. In mice, microglial VISTA expression was reduced after lipopolysaccharide (LPS) injection, during experimental autoimmune encephalomyelitis (EAE), and in the accelerated aging Ercc1

Topics: Animals; Animals, Newborn; Brain; Calcium-Binding Proteins; Cells, Cultured; Central Nervous System Diseases; Disease Models, Animal; DNA-Binding Proteins; Endonucleases; Female; Freund's Adjuvant; Gene Expression; Humans; Inflammation; Lipopolysaccharides; Macaca mulatta; Male; Membrane Proteins; Mice; Mice, Inbred C57BL; Mice, Transgenic; Microfilament Proteins; Microglia; Myelin-Oligodendrocyte Glycoprotein; Nerve Tissue Proteins; Peptide Fragments

Gap junctions (GJs) coupling oligodendrocytes to astrocytes and to other oligodendrocytes are formed mainly by connexin47 (Cx47) and a smaller portion by connexin32 (Cx32). Mutations in both connexins cause inherited demyelinating disorders, but their expression is also disrupted in multiple sclerosis (MS). To clarify whether the loss of either Cx47 or Cx32 could modify the outcome of inflammation and myelin loss, we induced experimental autoimmune encephalomyelitis (EAE) in fully backcrossed Cx32 knockout (KO) and Cx47KO mice and compared their outcome with wild type (WT, C57BI/6 N) mice. Cx47KO EAE mice developed the most severe phenotype assessed by clinical scores and behavioral testing, followed by Cx32KO and WT mice. Cx47KO more than Cx32KO EAE mice developed more microglial activation, myelin, and axonal loss than did WT mice. Oligodendrocyte apoptosis and precursor proliferation was also higher in Cx47KO than in Cx32KO or WT EAE mice. Similarly, blood-spinal cord barrier (BSCB) disruption and inflammatory infiltrates of macrophages, T- and B-cells were more severe in Cx47KO than either Cx32KO or WT EAE groups. Finally, expression profiling revealed that several proinflammatory cytokines were higher at the peak of inflammation in the Cx47KO mice and persisted at later stages of EAE in contrast to reduction of their levels in WT EAE mice. Thus, loss of oligodendrocyte GJs aggravates BSCB disruption and inflammatory myelin loss, likely due to dysregulation of proinflammatory cytokines. This mechanism may play an important role in MS brain with reduced connexin expression, as well as in patients with inherited mutations in oligodendrocyte connexins and secondary inflammation.

Topics: Animals; Apoptosis; Astrocytes; Blood-Brain Barrier; Calcium-Binding Proteins; Cell Proliferation; Connexins; Cytokines; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Freund's Adjuvant; Gap Junction beta-1 Protein; Gap Junctions; Gene Expression Regulation; Hand Strength; Macrophages; Mice; Mice, Inbred C57BL; Microfilament Proteins; Motor Activity; Myelin-Oligodendrocyte Glycoprotein; Oligodendroglia; Peptide Fragments

Recent studies in experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis (MS), suggest an involvement of the histone methyltransferase enhancer of zeste 2 polycomb repressive complex 2 subunit (EZH2) in important processes such as cell adhesion and migration.. Here, we aimed to expand these initial observations by investigating the role of EZH2 in MS. mRNA expression levels for EZH2 were measured by real-time PCR in peripheral blood mononuclear cells (PBMC) from 121 MS patients (62 untreated and 59 receiving treatment) and 24 healthy controls.. EZH2 expression levels were decreased in PBMC from untreated patients compared to that from controls, and treatment significantly upregulated EZH2 expression. Expression of miR-124 was increased in MS patients compared to controls. Blood immunophenotyping revealed EZH2 expression mostly restricted to CD4+ and CD8+ T cells, and circulating EZH2+ CD4+ and CD8+ T cells were decreased in untreated MS patients compared to controls. CD8+ T cells expressing EZH2 exhibited a predominant central memory phenotype, whereas EZH2+ CD4+ T cells were of effector memory nature, and both T cell subsets produced TNF-α. EZH2+ T cells were enriched in the cerebrospinal fluid compartment compared to blood and were found in chronic active lesions from MS patients. EZH2 inhibition and microarray analysis in PBMC was associated with significant downregulation of key T cell adhesion molecules.. These findings suggest a role of EZH2 in the migration of T cells in MS patients. The observation of TNF-α expression by CD4+ and CD8+ T cells expressing EZH2 warrants additional studies to explore more in depth the pathogenic potential of EZH2+-positive cells in MS.

Topics: Adult; Animals; Cohort Studies; Cytokines; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Enhancer of Zeste Homolog 2 Protein; Female; Freund's Adjuvant; Humans; Leukocytes, Mononuclear; Male; Mice; Mice, Inbred C57BL; MicroRNAs; Middle Aged; Multiple Sclerosis, Chronic Progressive; Multiple Sclerosis, Relapsing-Remitting; Myelin-Oligodendrocyte Glycoprotein; Peptide Fragments; Proto-Oncogene Proteins c-vav; T-Lymphocyte Subsets; Talin; Young Adult

Glial connexins (Cxs) form gap junction channels through which a pan-glial network plays key roles in maintaining homeostasis of the central nervous system (CNS). In multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE), expression of astrocytic Cx43 is lost in acute lesions but upregulated in chronic plaques, while astrocytic Cx30 is very low in normal white matter and changes in its expression have not been convincingly shown. In Cx30 or Cx43 single knockout (KO) mice and even in Cx30/Cx43 double KO mice, acute EAE is unaltered. However, the effects of Cx30/Cx43 deficiency on chronic EAE remains to be elucidated. We aimed to clarify the roles of Cx30 in chronic neuroinflammation by studying EAE induced by myelin oligodendrocyte glycoprotein peptide 35-55 in Cx30 KO mice. We found that Cx30 deficiency improved the clinical symptoms and demyelination of chronic but not acute EAE without influencing CD3

Topics: Acute Disease; Animals; Arginase; Brain-Derived Neurotrophic Factor; Chronic Disease; Connexin 30; Connexin 43; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Female; Humans; Mice; Mice, Inbred C57BL; Mice, Knockout; Microglia; Multiple Sclerosis; Myelin-Oligodendrocyte Glycoprotein; Neuroprotection; Peptide Fragments

Multiple sclerosis (MS) is a chronic autoimmune disease with demyelination and neurodegeneration of the central nervous system. It has been shown that the regulatory T (Treg) cells are responsible for maintaining tolerance to self-antigens and can suppress the autoimmune process in several animal models such as experimental autoimmune encephalomyelitis (EAE), a mouse model of MS. Recent basic studies have demonstrated that forkhead box P (FOXP3) and BTB domain and CNC homolog 2 (BACH2) are the master transcription factors of these cells playing a pivotal role in the polarization of naïve T cells into Treg cells. In the current study, the expression of FOXP3 and BACH2 genes and FOXP3 promoter methylation were evaluated in T cells of the EAE-induced mice. The results of this study showed a prominent and significant hypermethylation of the FOXP3 gene promoter in the EAE-induced mice compared to the sham and control groups. The expression of FOXP3 and BACH2 genes was significantly decreased in the EAE group in comparison with the sham and control groups. This study suggests that the epigenetic modification of FOXP3 gene is involved in the pathogenesis of EAE and this could be important in therapy in an appropriate and logical statement.

Topics: Animals; Antigens, CD; Basic-Leucine Zipper Transcription Factors; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Flow Cytometry; Forkhead Transcription Factors; Freund's Adjuvant; Gene Expression Regulation; Mice; Mice, Inbred C57BL; Multiple Sclerosis; Myelin-Oligodendrocyte Glycoprotein; Organophosphates; Peptide Fragments; RNA, Messenger; Severity of Illness Index; T-Lymphocytes, Regulatory

IL-23/STAT3 signaling pathway is a key process in Th17 cell differentiation, and Th17 cells are closely related to the development of autoimmune diseases. We previously designed and prepared rhIL23R-CHR protein to antagonize endogenous IL-23, showing effectiveness in the treatment of experimental autoimmune encephalomyelitis (EAE) in mice. To further elucidate the mechanism of action, mouse lncRNA microarray was used to screen expression profiles of lncRNAs, and a particular lncRNA, 1700040D17Rik was found to down-regulate in EAE model and its expression was significantly increased after the treatment by rhIL23R-CHR. The function of 1700040D17Rik was revealed to associate with the differentiation of Th17 cells through the regulation of the key transcription factor RORγt. Together, regulation of Th17 cells through lncRNA is responsible for the effects of rhIL23R-CHR to balance the immune responses, and 1700040D17Rik has the potential to serve as a therapeutic target or a biomarker for autoimmune diseases.

Topics: Animals; Cell Differentiation; Cells, Cultured; Disease Models, Animal; Down-Regulation; Encephalomyelitis, Autoimmune, Experimental; Female; Gene Expression Profiling; Humans; Interleukin-23; Mice; Mice, Inbred C57BL; Multiple Sclerosis; Myelin-Oligodendrocyte Glycoprotein; Nuclear Receptor Subfamily 1, Group F, Member 3; Oligonucleotide Array Sequence Analysis; Peptide Fragments; RNA, Long Noncoding; Signal Transduction; STAT3 Transcription Factor; Th17 Cells

Hypomethylation of the cathepsin Z locus has been proposed as an epigenetic risk factor for multiple sclerosis (MS). Cathepsin Z is a unique lysosomal cysteine cathepsin expressed primarily by antigen presenting cells. While cathepsin Z expression has been associated with neuroinflammatory disorders, a role for cathepsin Z in mediating neuroinflammation has not been previously established.. Experimental autoimmune encephalomyelitis (EAE) was induced in both wildtype mice and mice deficient in cathepsin Z. The effects of cathepsin Z-deficiency on the processing and presentation of the autoantigen myelin oligodendrocyte glycoprotein, and on the production of IL-1β and IL-18 were determined in vitro from cells derived from wildtype and cathepsin Z-deficient mice. The effects of cathepsin Z-deficiency on CD4+ T cell activation, migration, and infiltration to the CNS were determined in vivo. Statistical analyses of parametric data were performed by one-way ANOVA followed by Tukey post-hoc tests, or by an unpaired Student's t test. EAE clinical scoring was analyzed using the Mann-Whitney U test.. We showed that mice deficient in cathepsin Z have reduced neuroinflammation and dramatically lowered circulating levels of IL-1β during EAE. Deficiency in cathepsin Z did not impact either the processing or the presentation of MOG, or MOG- specific CD4+ T cell activation and trafficking. Consistently, we found that cathepsin Z-deficiency reduced the efficiency of antigen presenting cells to secrete IL-1β, which in turn reduced the ability of mice to generate Th17 responses-critical steps in the pathogenesis of EAE and MS.. Together, these data support a novel role for cathepsin Z in the propagation of IL-1β-driven neuroinflammation.

Topics: Animals; Antigen-Presenting Cells; Antigens, CD; Cathepsin Z; CD4-Positive T-Lymphocytes; Chemokine CXCL9; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Epilepsy; Interleukin-18; Interleukin-1beta; Leukocytes; Macrophages; Mice; Mice, Inbred C57BL; Mice, Transgenic; Myelin-Oligodendrocyte Glycoprotein; Peptide Fragments; Phagosomes; Spinal Cord

The influx of leukocytes into the central nervous system (CNS) is a key hallmark of the chronic neuro-inflammatory disease multiple sclerosis (MS). Strategies that aim to inhibit leukocyte migration across the blood-brain barrier (BBB) are therefore regarded as promising therapeutic approaches to combat MS. As the CD40L-CD40 dyad signals via TNF receptor-associated factor 6 (TRAF6) in myeloid cells to induce inflammation and leukocyte trafficking, we explored the hypothesis that specific inhibition of CD40-TRAF6 interactions can ameliorate neuro-inflammation.. Human monocytes were treated with a small molecule inhibitor (SMI) of CD40-TRAF6 interactions (6877002), and migration capacity across human brain endothelial cells was measured. To test the therapeutic potential of the CD40-TRAF6-blocking SMI under neuro-inflammatory conditions in vivo, Lewis rats and C57BL/6J mice were subjected to acute experimental autoimmune encephalomyelitis (EAE) and treated with SMI 6877002 for 6 days (rats) or 3 weeks (mice).. We here show that a SMI of CD40-TRAF6 interactions (6877002) strongly and dose-dependently reduces trans-endothelial migration of human monocytes. Moreover, upon SMI treatment, monocytes displayed a decreased production of ROS, tumor necrosis factor (TNF), and interleukin (IL)-6, whereas the production of the anti-inflammatory cytokine IL-10 was increased. Disease severity of EAE was reduced upon SMI treatment in rats, but not in mice. However, a significant reduction in monocyte-derived macrophages, but not in T cells, that had infiltrated the CNS was eminent in both models.. Together, our results indicate that SMI-mediated inhibition of the CD40-TRAF6 pathway skews human monocytes towards anti-inflammatory cells with reduced trans-endothelial migration capacity, and is able to reduce CNS-infiltrated monocyte-derived macrophages during neuro-inflammation, but minimally ameliorates EAE disease severity. We therefore conclude that SMI-mediated inhibition of the CD40-TRAF6 pathway may represent a beneficial treatment strategy to reduce monocyte recruitment and macrophage activation in the CNS and has the potential to be used as a co-treatment to combat MS.

Topics: Animals; Anti-Inflammatory Agents; CD40 Antigens; Cell Movement; Cells, Cultured; Cerebellum; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Gene Expression Regulation; Humans; Matrix Metalloproteinase 9; Mice; Monocytes; Myelin-Oligodendrocyte Glycoprotein; Nitric Oxide Synthase Type I; Peptide Fragments; Rats; Rats, Inbred Lew; Reactive Oxygen Species; Spinal Cord; TNF Receptor-Associated Factor 6; Tumor Necrosis Factor-alpha

Topics: Animals; Antigens; Antigens, CD; Bacterial Proteins; Central Nervous System; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Luminescent Proteins; Mice; Mice, Transgenic; Myelin-Oligodendrocyte Glycoprotein; Peptide Fragments; Pertussis Toxin; Proteoglycans; Receptor, Platelet-Derived Growth Factor beta; Th1 Cells; Th17 Cells

Sex hormones promote immunoregulatory effects on multiple sclerosis. In the current study we evaluated the composition of the gut microbiota and the mucosal-associated regulatory cells in estrogen or sham treated female mice before and after autoimmune encephalomyelitis (EAE) induction. Treatment with pregnancy levels of estrogen induces changes in the composition and diversity of gut microbiota. Additionally, estrogen prevents EAE-associated changes in the gut microbiota and might promote the enrichment of bacteria that are associated with immune regulation. Our results point to a possible cross-talk between the sex hormones and the gut microbiota, which could promote neuroprotection.

Topics: Animals; Antigens, CD; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Estrogens; Feces; Female; Interleukin-10; Intestines; Leukocytes; Lymph Nodes; Mice; Mice, Inbred C57BL; Mice, Transgenic; Microbiota; Mucous Membrane; Myelin-Oligodendrocyte Glycoprotein; Peptide Fragments; RNA, Ribosomal, 16S; Spinal Cord; Time Factors

Brain-intrinsic degenerative cascades are a proposed factor driving inflammatory lesion formation in multiple sclerosis (MS) patients. We recently described a model combining noninflammatory cytodegeneration (via cuprizone) with the classic active experimental autoimmune encephalomyelitis (Cup/EAE model), which exhibits inflammatory forebrain lesions. Here, we describe the histopathological characteristics and progression of these Cup/EAE lesions. We show that inflammatory lesions develop at various topographical sites in the forebrain, including white matter tracts and cortical and subcortical grey matter areas. The lesions are characterized by focal demyelination, discontinuation of the perivascular glia limitans, focal axonal damage, and neutrophil granulocyte extravasation. Transgenic mice with enhanced green fluorescent protein-expressing microglia and red fluorescent protein-expressing monocytes reveal that both myeloid cell populations contribute to forebrain inflammatory infiltrates. EAE-triggered inflammatory cerebellar lesions were augmented in mice pre-intoxicated with cuprizone. Gene expression studies suggest roles of the chemokines Cxcl10, Ccl2, and Ccl3 in inflammatory lesion formation. Finally, follow-up experiments in Cup/EAE mice with chronic disease revealed that forebrain, but not spinal cord, lesions undergo spontaneous reorganization and repair. This study underpins the significance of brain-intrinsic degenerative cascades for immune cell recruitment and, in consequence, MS lesion formation.

Topics: Amyloid beta-Protein Precursor; Animals; Disease Models, Animal; Disease Progression; Encephalitis; Encephalomyelitis, Autoimmune, Experimental; Female; Freund's Adjuvant; Gene Expression; Glial Fibrillary Acidic Protein; Intercellular Adhesion Molecule-1; Luminescent Proteins; Mice; Mice, Inbred C57BL; Mice, Transgenic; Microglia; Monocytes; Myelin-Oligodendrocyte Glycoprotein; Peptide Fragments; Receptors, CCR2; Receptors, Interleukin-8A; Sesquiterpenes

Several types of myeloid cell are resident in the CNS. In the steady state, microglia are present in the CNS parenchyma, whereas macrophages reside in boundary regions of the CNS, such as perivascular spaces, the meninges and choroid plexus. In addition, monocytes infiltrate into the CNS parenchyma from circulation upon blood-brain barrier breakdown after CNS injury and inflammation. Although several markers, such as CD11b and ionized calcium-binding adapter molecule 1 (Iba1), are frequently used as microglial markers, they are also expressed by other types of myeloid cell and microglia-specific markers were not defined until recently. Previous transcriptome analyses of isolated microglia identified a transmembrane lectin, sialic acid-binding immunoglobulin-like lectin H (Siglec-H), as a molecular signature for microglia; however, this was not confirmed by histological studies in the nervous system and the reliability of Siglec-H as a microglial marker remained unclear. Here, we demonstrate that Siglec-H is an authentic marker for microglia in mice by immunohistochemistry using a Siglec-H-specific antibody. Siglec-H was expressed by parenchymal microglia from developmental stages to adulthood, and the expression was maintained in activated microglia under injury or inflammatory condition. However, Siglec-H expression was absent from CNS-associated macrophages and CNS-infiltrating monocytes, except for a minor subset of cells. We also show that the Siglech gene locus is a feasible site for specific targeting of microglia in the nervous system. In conclusion, Siglec-H is a reliable marker for microglia that will allow histological identification of microglia and microglia-specific gene manipulation in the nervous system.

Topics: Animals; Animals, Newborn; Central Nervous System; Disease Models, Animal; Embryo, Mammalian; Encephalomyelitis, Autoimmune, Experimental; Gene Expression Regulation; Lectins; Macrophages; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Microglia; Myelin-Oligodendrocyte Glycoprotein; Myeloid Cells; Neuralgia; Neutrophil Infiltration; Peptide Fragments; Pertussis Toxin; Receptors, CCR2; Receptors, Cell Surface

Increased relapse rates in multiple sclerosis (MS) as a consequence of peripheral immune system activation, owing to infection for example, have been widely reported, but the mechanism remains unclear. Acute brain injury models can be exacerbated by augmenting the hepatic acute phase response (APR). Here, we explored the contribution of the hepatic APR to relapse in two rodent models of MS.. Mice with MOG-CFA-induced chronic relapsing experimental autoimmune encephalitis (CR-EAE) were killed before, during and after the first phase of disease, and the brain and liver chemokine, cytokine and acute phase protein (APP) mRNA expression profile was determined. During remission, the APR was reactivated with an intraperitoneal lipopolysaccharide (LPS) and clinical score was monitored throughout. To explore the downstream mediators, CXCL-1, which is induced as part of the APR, was injected into animals with a focal, cytokine/MOG-induced EAE lesion (fEAE) and the cellularity of the lesions was assessed.. Compared to CFA control, in a rodent CR-EAE model, an hepatic APR preceded clinical signs and central cytokine production in the initial phase of disease. Compared to administration in naïve animals, an LPS challenge during the asymptomatic remission phase of CR-EAE rodents provoked relapse and resulted in the increased and extended expression of specific peripheral hepatic chemokines. CXCL-1 and several other APPs were markedly elevated. A single intravenous administration of the highly induced chemokine, CXCL-1, was found to be sufficient to reactivate the lesions by increasing microglial activation and the recruitment of T cells in fEAE lesions.. The APR plays a contributing role to the pathology seen in models of chronic brain injury and in translating the effects of peripheral immune system stimulation secondary to trauma or infection into central pathology and behavioural signs. Further elucidation of the exact mechanisms in this process will inform development of more effective, selective therapies in MS that, by suppressing the hepatic chemokine response, may prevent relapse.

Topics: Acute-Phase Reaction; Amyloid beta-Protein Precursor; Animals; Brain; Chemokine CXCL1; Cytokines; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Female; Freund's Adjuvant; Gene Expression Regulation; Lipopolysaccharides; Liver; Mice; Muscle Strength; Myelin-Oligodendrocyte Glycoprotein; Peptide Fragments; RNA, Messenger; Serum Amyloid A Protein; Time Factors

Treatment with erythropoietin (Epo) in experimental autoimmune encephalomyelitis (EAE), the rodent model of multiple sclerosis (MS), has consistently been shown to ameliorate disease progression and improve overall outcome. The effect has been attributed to modulation of the immune response and/or preservation of the central nervous system (CNS) tissue integrity. It remains unclear, however, if (a) Epo acts primarily in the CNS or the periphery and if (b) Epo's beneficial effect in EAE is mainly due to maintaining CNS tissue integrity or to modulation of the immune response. If Epo acts primarily by modulating the immune system, where is this modulation required? In the periphery, the CNS or both?. To address these questions, we used two well-characterized transgenic mouse strains that constitutively overexpress recombinant human Epo (rhEpo) either systemically (tg6) or in CNS only (tg21) in a MOG-induced EAE model. We assessed clinical severity, disease progression, immunomodulation, and CNS tissue integrity, including neuronal survival.. Although disease onset remained unaffected, EAE progression was alleviated in transgenic animals compared to controls with both lines performing equally well showing that expression of Epo in the periphery is not required; Epo expression in the CNS is sufficient. Immunomodulation was observed in both strains but surprisingly the profile of modulation differed substantially between strains. Modulation in the tg21 strain was limited to a reduction in macrophages in the CNS, with no peripheral immunomodulatory effects observed. In contrast, in the tg6 strain, macrophages were upregulated in the CNS, and, in the periphery of this strain, T cells and macrophages were downregulated. The lack of a consistent immunomodulatory profile across both transgenic species suggests that immunomodulation by Epo is unlikely to be the primary mechanism driving amelioration of EAE. Finally, CNS tissue integrity was affected in all strains. Although myelin appeared equally damaged in all strains, neuronal survival was significantly improved in the spinal cord of tg21 mice, indicating that Epo may ameliorate EAE predominantly by protecting neurons.. Our data suggests that moderate elevated brain Epo levels provide clinically significant neuroprotection in EAE without modulation of the immune response making a significant contribution.

Topics: Animals; Central Nervous System; Cytokines; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Erythropoietin; Female; Gene Expression Regulation; Humans; Lymphocytes; Macrophages; Mice; Mice, Inbred C57BL; Mice, Transgenic; Monocytes; Myelin-Oligodendrocyte Glycoprotein; Neurons; Neuroprotection; Peptide Fragments; Proto-Oncogene Proteins c-sis; Spleen

Brain aging and neurodegeneration are associated with prominent microglial reactivity and activation of innate immune response pathways, commonly referred to as neuroinflammation. One such pathway, the type I interferon response, recognizes viral or mitochondrial DNA in the cytoplasm via activation of the recently discovered cyclic dinucleotide synthetase cGAS and the cyclic dinucleotide receptor STING. Here we show that the FDA-approved antiviral drug ganciclovir (GCV) induces a type I interferon response independent of its canonical thymidine kinase target. Inhibition of components of the STING pathway, including STING, IRF3, Tbk1, extracellular IFNβ, and the Jak-Stat pathway resulted in reduced activity of GCV and its derivatives. Importantly, functional STING was necessary for GCV to inhibit inflammation in cultured myeloid cells and in a mouse model of multiple sclerosis. Collectively, our findings uncover an unexpected new activity of GCV and identify the STING pathway as a regulator of microglial reactivity and neuroinflammation.

Topics: Animals; Animals, Newborn; Antiviral Agents; Cells, Cultured; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Female; Freund's Adjuvant; Ganciclovir; Gene Expression Regulation; Humans; Interferon Type I; Male; Membrane Proteins; Mice; Mice, Inbred C57BL; Mice, Transgenic; Microglia; Monocytes; Myelin-Oligodendrocyte Glycoprotein; Peptide Fragments; Pertussis Toxin; Signal Transduction

Topics: Animals; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Epitopes; Mice; Molecular Conformation; Molecular Dynamics Simulation; Myelin-Oligodendrocyte Glycoprotein; Nuclear Magnetic Resonance, Biomolecular; Oligodendroglia; Peptide Fragments; Rats

The preclinical selection of therapeutic candidates for progressive multiple sclerosis (MS) would be aided by the development of sensitive behavioural measures that accurately reflect the impact of autoimmune-mediated spinal cord damage on locomotion. Neurological deficits in mice subjected to experimental autoimmune encephalomyelitis (EAE) are typically scored using a clinical scale with 5-10 levels of increased disease severity. This ordinal scale represents a general impression of paralysis and impaired gait. By contrast, kinematic gait analyses generate ratio level data that have frequently been used to characterize walking deficits for MS patients and test the efficacy of treatments designed to improve them. Despite these advantages, kinematic gait analyses have not been systematically applied to the study of walking deficits for EAE mice. We have therefore used high speed video recordings (250 frames/s) of EAE mice walking on a treadmill to measure 8 kinematic parameters in the sagittal plane: average hip height (1), average toe height during swing (2), and average angle and range of motion for the hip (3-4), knee (5-6) and ankle (7-8). Kinematic measures of hip, knee and ankle movements were found to be early detectors of impaired locomotion for mice with mild EAE (median clinical score=1.0 at day post-immunization 26; DPI 26). These deficits occurred in the absence of reduced rotarod performance with impaired hip and knee movements observed 3days before disease onset as determined by clinical scores. Gait deficits for mild EAE mice were minor and often recovered fully by DPI 30. By contrast, severe EAE mice (median clinical score=2.5 at DPI 26) displayed much larger movement impairments for the knee and ankle that failed to completely recover by DPI 44. Moreover, impaired ankle movement was highly correlated with white matter loss in the spinal cords of EAE mice (r=0.96). Kinematic analyses therefore yield highly sensitive measures of motor deficits that predict spinal cord injury in EAE mice. These behavioural techniques should assist the selection of promising therapeutic candidates for clinical testing in progressive MS.

Topics: Animals; Ankle; Biomechanical Phenomena; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Female; Freund's Adjuvant; Gait; Hip; Locomotion; Mice; Mice, Inbred C57BL; Motor Disorders; Myelin-Oligodendrocyte Glycoprotein; Peptide Fragments; Pertussis Toxin; Range of Motion, Articular; Rotarod Performance Test; Spinal Cord Injuries

Topics: Animals; Chloride-Bicarbonate Antiporters; Disease Models, Animal; Infarction, Middle Cerebral Artery; Macrophages; Mice, Inbred C57BL; Microglia; Myelin-Oligodendrocyte Glycoprotein; Peptide Fragments; Stroke; Sulfate Transporters

Autoimmune diseases like multiple sclerosis (MS) develop from the activation and complex interactions of a wide network of immune cells, which penetrate the central nervous system (CNS) and cause tissue damage and neurological deficits. Experimental autoimmune encephalomyelitis (EAE) is a model used to study various aspects of MS, including the infiltration of autoaggressive T cells and pathogenic, inflammatory myeloid cells into the CNS. Various signature landscapes of immune cell infiltrates have proven useful in shedding light on the causes of specific EAE symptoms in transgenic mice. However, single cell analysis of these infiltrates has thus far been limited in conventional fluorescent flow cytometry methods by 14-16 parameter staining panels. With the advent of mass cytometry and metal-tagged antibodies, a staining panel of 35-45 parameters is now possible. With the aid of dimensionality reducing and clustering algorithms to visualize and analyze this high dimensional data, this allows for a more comprehensive picture of the different cell populations in an inflamed CNS, at a single cell resolution level. Here, we describe the induction of active EAE in C56BL/6 mice and, in particular, the staining of microglia and CNS invading immune cells for mass cytometry with subsequent data visualization and analysis.

Topics: Algorithms; Animals; Biomarkers; Cell Movement; Central Nervous System; Disease Models, Animal; Disease Progression; Encephalomyelitis, Autoimmune, Experimental; Female; Freund's Adjuvant; Image Cytometry; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Microglia; Multifactor Dimensionality Reduction; Multiple Sclerosis; Myelin-Oligodendrocyte Glycoprotein; Peptide Fragments; Single-Cell Analysis; Staining and Labeling; T-Lymphocytes

In April 2015, the US Food and Drug Administration approved the first generic glatiramer acetate, Glatopa® (M356), as fully substitutable for Copaxone® 20 mg/mL for relapsing forms of multiple sclerosis (MS). This approval was accomplished through an Abbreviated New Drug Application that demonstrated equivalence to Copaxone.. This article will provide an overview of the methods used to establish the biological and immunological equivalence of the two glatiramer acetate products, including methods evaluating antigenpresenting cell (APC) biology, T-cell biology, and other immunomodulatory effects.. In vitro and in vivo experiments from multiple redundant orthogonal assays within four biological processes (aggregate biology, APC biology, T-cell biology, and B-cell biology) modulated by glatiramer acetate in MS established the biological and immunological equivalence of Glatopa and Copaxone and are described. The following were observed when comparing Glatopa and Copaxone in these experiments: equivalent delays in symptom onset and reductions in "disease" intensity in experimental autoimmune encephalomyelitis; equivalent dose-dependent increases in Glatopa- and Copaxone- induced monokine-induced interferon-gamma release from THP-1 cells; a shift to a T helper 2 phenotype resulting in the secretion of interleukin (IL)-4 and downregulation of IL-17 release; no differences in immunogenicity and the presence of equivalent "immunofingerprints" between both versions of glatiramer acetate; and no stimulation of histamine release with either glatiramer acetate in basophilic leukemia 2H3 cell lines.. In summary, this comprehensive approach across different biological and immunological pathways modulated by glatiramer acetate consistently supported the biological and immunological equivalence of Glatopa and Copaxone.

Topics: Animals; Antigen-Presenting Cells; B-Lymphocytes; Cytokines; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Glatiramer Acetate; Histamine; Immunosuppressive Agents; Mice; Myelin Proteolipid Protein; Myelin-Oligodendrocyte Glycoprotein; Peptide Fragments; T-Lymphocytes; Therapeutic Equivalency

Multiple sclerosis (MS) is an inflammatory neurodegenerative disease of the central nervous system. The primary symptoms of MS include the loss of sensory and motor function. Exercise has been shown to modulate disease parameters in experimental autoimmune encephalomyelitis (EAE), a mouse model of MS, by reducing immune cell infiltration and oxidative stress. However, these initial studies were carried out exclusively in female mice. The present study compared the effects of daily voluntary wheel running on several disease parameters in male and female mice with EAE. Male and female mice were given access to a running wheel for 1h a day for 30 consecutive days. Daily wheel running significantly improved clinical scores in males with EAE but had little effect on clinical signs in females with the disease. Direct comparison of inflammation, axonal injury, and oxidative stress in male and female mice with EAE revealed significant differences in the amount of T-cell infiltration, microglia reactivity, demyelination and axon integrity. Male mice with EAE given daily access to running wheels also had significantly less ongoing oxidative stress compared to all other groups. Taken together, our results indicate that the inflammatory response generated in EAE is distinct between the sexes and its modulation by daily exercise can have sex-specific effects on disease-related outcomes.

Topics: Animals; Calcium-Binding Proteins; CD4 Antigens; Chromatography, High Pressure Liquid; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Female; Freund's Adjuvant; Gait Disorders, Neurologic; Glutathione Disulfide; Male; Mice; Mice, Inbred C57BL; Microfilament Proteins; Myelin-Oligodendrocyte Glycoprotein; Oligopeptides; Peptide Fragments; Running; Sex Characteristics; Spinal Cord; Statistics, Nonparametric; Superoxide Dismutase

Immunoregulatory sex hormones, including estrogen and estriol, may prevent relapses in multiple sclerosis during pregnancy. Our previous studies have demonstrated that regulatory B cells are crucial for estrogen-mediated protection against experimental autoimmune encephalomyelitis (EAE). Herein, we demonstrate an estrogen-dependent induction of alternatively activated (M2) macrophages/microglia that results in an increased frequency of regulatory B cells in the spinal cord of estrogen treated mice with EAE. We further demonstrate that cultured M2-polarized microglia promote the induction of regulatory B cells. Our study suggests that estrogen neuroprotection induces a regulatory feedback loop between M2 macrophages/microglia and regulatory B cells.

Topics: Animals; Arginase; B-Lymphocytes, Regulatory; Cells, Cultured; Coculture Techniques; Cytokines; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Estrogens; Female; Gene Expression Regulation; Interleukin-10; Macrophages; Mice; Mice, Inbred C57BL; Mice, Transgenic; Microglia; Myelin-Oligodendrocyte Glycoprotein; Nitric Oxide Synthase Type II; Peptide Fragments; Spinal Cord; Spleen; Time Factors

Month-season of birth (M-SOB) is a risk factor in multiple chronic diseases, including multiple sclerosis (MS), where the lowest and greatest risk of developing MS coincide with the lowest and highest birth rates, respectively. To determine whether M-SOB effects in such chronic diseases as MS can be experimentally modeled, we examined the effect of M-SOB on susceptibility of C57BL/6J mice to experimental autoimmune encephalomyelitis (EAE). As in MS, mice that were born during the M-SOB with the lowest birth rate were less susceptible to EAE than mice born during the M-SOB with the highest birth rate. We also show that the M-SOB effect on EAE susceptibility is associated with differential production of multiple cytokines/chemokines by neuroantigen-specific T cells that are known to play a role in EAE pathogenesis. Taken together, these results support the existence of an M-SOB effect that may reflect seasonally dependent developmental differences in adaptive immune responses to self-antigens independent of external stimuli, including exposure to sunlight and vitamin D. Moreover, our documentation of an M-SOB effect on EAE susceptibility in mice allows for modeling and detailed analysis of mechanisms that underlie the M-SOB effect in not only MS but in numerous other diseases in which M-SOB impacts susceptibility.-Reynolds, J. D., Case, L. K., Krementsov, D. N., Raza, A., Bartiss, R., Teuscher, C. Modeling month-season of birth as a risk factor in mouse models of chronic disease: from multiple sclerosis to autoimmune encephalomyelitis.

Topics: Animals; Birth Rate; Disease Models, Animal; Disease Susceptibility; Encephalitis; Hashimoto Disease; Mice; Mice, Inbred Strains; Multiple Sclerosis; Myelin-Oligodendrocyte Glycoprotein; Peptide Fragments; Retrospective Studies; Risk Factors; Seasons

MicroRNAs have emerged as an important class of modulators of gene expression. These molecules influence protein synthesis through translational repression or degradation of mRNA transcripts. Herein, we investigated the potential role of miR-142a isoforms, miR-142a-3p and miR-142a-5p, in the context of autoimmune neuroinflammation.. Expression of miR-142-5p was significantly increased in the frontal white matter from MS patients compared with white matter from non-MS controls. Likewise, expression levels of miR-142a-5p and miR-142a-3p showed significant upregulation in the spinal cords of EAE mice at days 15 and 25 post disease induction. Splenocytes stimulated with myelin oligodendrocyte glycoprotein (MOG) peptide or anti-CD3/anti-CD28 antibodies showed upregulation of miR-142a-5p and miR-142a-3p isoforms, whereas stimulated bone marrow-derived macrophages and primary astrocytes did not show any significant changes in miRNA expression levels. miR-142a-5p overexpression in activated lymphocytes shifted the pattern of T cell differentiation towards Th1 cells. Luciferase assays revealed SOCS1 and TGFBR1 as direct targets of miR-142a-5p and miR-142a-3p, respectively, and overexpression of miRNA mimic sequences suppressed the expression of these target transcripts in lymphocytes. SOCS1 levels were also diminished in MS white matter and EAE spinal cords.. Our findings suggest that increased expression of miR-142 isoforms might be involved in the pathogenesis of autoimmune neuroinflammation by influencing T cell differentiation, and this effect could be mediated by interaction of miR-142 isoforms with SOCS1 and TGFBR-1 transcripts.

Topics: Aged; Animals; Antigens, CD; Astrocytes; Cell Differentiation; Cells, Cultured; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Female; Freund's Adjuvant; Humans; Macrophages; Male; Mice; Mice, Inbred C57BL; MicroRNAs; Middle Aged; Multiple Sclerosis; Myelin Basic Protein; Myelin-Oligodendrocyte Glycoprotein; Peptide Fragments; Signal Transduction; T-Lymphocytes; Up-Regulation

CD4(+) memory T cells play an important role in induction of autoimmunity and chronic inflammatory responses; however, regulatory mechanisms of CD4(+) memory T cell-mediated inflammatory responses are poorly understood. Here we show that apoptotic cell-treated dendritic cells inhibit development and differentiation of CD4(+) effector memory T cells in vitro and in vivo. Simultaneously, intravenous transfer of apoptotic T cell-induced tolerogenic dendritic cells can block development of experimental autoimmune encephalomyelitis (EAE), an inflammatory disease of the central nervous system in C57 BL/6J mouse. Our results imply that it is effector memory CD4(+) T cells, not central memory CD4(+) T cells, which play a major role in chronic inflammatory responses in mice with EAE. Intravenous transfer of tolerogenic dendritic cells induced by apoptotic T cells leads to immune tolerance by specifically blocking development of CD4(+) effector memory T cells compared with results of EAE control mice. These results reveal a new mechanism of apoptotic cell-treated dendritic cell-mediated immune tolerance in vivo.

Topics: Animals; Apoptosis; CD4-Positive T-Lymphocytes; Cells, Cultured; Dendritic Cells; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Humans; Immunologic Memory; Immunotherapy; Mice; Mice, Inbred C57BL; Mice, Transgenic; Myelin-Oligodendrocyte Glycoprotein; Peptide Fragments; Peripheral Tolerance

Although interferon-β is used as first-line therapy for multiple sclerosis, the cell type-specific activity of type I interferons in multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis, remains obscure. In this study, we have elucidated the in vivo immunomodulatory role of type I interferon signaling in T cells during experimental autoimmune encephalomyelitis by use of a novel transgenic mouse, carrying a cd2-ifnar1 transgene on a interferon-α/β receptor 1 null genetic background, thus allowing expression of the interferon-α/β receptor 1 and hence, a functional type I interferon receptor exclusively on T cells. These transgenic mice exhibited milder experimental autoimmune encephalomyelitis with reduced T cell infiltration, demyelination, and axonal damage in the central nervous system. It is noteworthy that interferon-β administration in transgenic mice generated a more pronounced, protective effect against experimental autoimmune encephalomyelitis compared with untreated littermates. In vivo studies demonstrated that before experimental autoimmune encephalomyelitis onset, endogenous type I interferon receptor signaling in T cells led to impaired T-helper 17 responses, with a reduced fraction of CCR6(+) CD4(+) T cells in the periphery. At the acute phase, an increased proportion of interleukin-10- and interferon-γ-producing CD4(+) T cells was detected in the periphery of the transgenic mice, accompanied by up-regulation of the interferon-γ-induced gene Irgm1 in peripheral T cells. Together, these results reveal a hitherto unknown T cell-associated protective role of type I interferon in experimental autoimmune encephalomyelitis that may provide valuable clues for designing novel therapeutic strategies for multiple sclerosis.

Topics: Animals; CD4-Positive T-Lymphocytes; Central Nervous System; Cluster Analysis; Cytokines; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Gene Expression; Gene Expression Profiling; Interferon Type I; Lymphocyte Activation; Mice; Mice, Knockout; Mice, Transgenic; Myelin-Oligodendrocyte Glycoprotein; Organ Specificity; Peptide Fragments; Receptor, Interferon alpha-beta; Signal Transduction; T-Lymphocyte Subsets

Multiple sclerosis (MS) is an autoimmune demyelinating disease of the central nervous system (CNS) caused by the infiltration of TH1 and TH17 cells into the CNS. Ribosomal S6 kinase 2 (RSK2; RPS6KA3) regulates TH17 differentiation by attenuating RORγt transcriptional activities and IL-17A production. The pan-RSK inhibitor BI-D1870 also inhibits TH17 differentiation, but the effect of BI-D1870 in vivo remains unclear. Here, we generated mice with experimental autoimmune encephalomyelitis (EAE) and treated them with BI-D1870. BI-D1870 administration protected mice from EAE by reducing the infiltration of TH1 and TH17 cells into the CNS and decreasing mRNA levels of Ccr6 in TH17 cells. These results suggest that RSK inhibition is a promising strategy for the treatment of MS.

Topics: Animals; Cell Movement; Central Nervous System; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Gene Expression Regulation; Mice; Mice, Inbred C57BL; Multiple Sclerosis; Myelin-Oligodendrocyte Glycoprotein; Peptide Fragments; Protein Kinase Inhibitors; Pteridines; Receptors, CCR6; Ribosomal Protein S6 Kinases, 90-kDa; RNA, Messenger; Signal Transduction; Th1 Cells; Th17 Cells

Multiple sclerosis (MS) is a central nervous system disorder mainly characterized by inflammation, demyelination and axonal injury. Anti-inflammatory agents can be used to ameliorate the disease process. Hypericum perforatum L or St. John's wort is widely used as an anti-depressant and anti-inflammatory remedy in traditional and herbal medicine. Based on St. John's wort properties, the therapeutic potentials of an H. perforatum extract (HPE) and a single component, hyperforin were evaluated for effectiveness against MOG35-55-induced experimental autoimmune encephalomyelitis (EAE), an animal model for human multiple sclerosis. Female C57BL/6 mice were immunized with specific antigen MOG35-55 and then administered different doses of hyperforin or HPE post-immunization. Clinical symptoms/other relevant parameters were assessed daily. Histological analysis of the spinal cord was performed. T-cell proliferative activity was also evaluated using a BrdU assay. The effect of hyperforin on regulatory T-cells (Treg cells) was assessed using flow cytometry. The results indicate hyperforin and HPE reduced the incidence and severity of EAE, an outcome that closely correlated with an inhibition of pathological features (leukocyte infiltration and demyelination) and antigen-specific T-cell proliferation. The study also showed that hyperforin caused increased Treg cell levels in the spleen. These results indicated that hyperforin and HPE could attenuate EAE autoimmune responses by inhibiting immune cell infiltration and expansion of Treg cell and could eventually be considered as a potential candidate for use in the treatment of MS.

Topics: Animals; Cell Proliferation; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Female; Humans; Hypericum; Mice; Mice, Inbred C57BL; Multiple Sclerosis; Myelin-Oligodendrocyte Glycoprotein; Peptide Fragments; Phloroglucinol; Phytotherapy; Spleen; T-Lymphocytes, Regulatory; Terpenes

In the brain, chronic inflammatory activity may lead to compromised delivery of oxygen and glucose suggesting that therapeutic approaches aimed at restoring metabolic balance may be useful. In vivo exposure to chronic mild normobaric hypoxia (10 % oxygen) leads to a number of endogenous adaptations that includes vascular remodeling (angioplasticity). Angioplasticity promotes tissue survival. We have previously shown that induction of adaptive angioplasticity modulates the disease pattern in myelin oligodendrocyte glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis (EAE). In the present study, we define mechanisms by which adaptation to low oxygen functionally ameliorates the signs and symptoms of EAE and for the first time show that tissue hypoxia may fundamentally alter neurodegenerative disease.. C57BL/6 mice were immunized with MOG, and some of them were kept in the hypoxia chambers (day 0) and exposed to 10 % oxygen for 3 weeks, while the others were kept at normoxic environment. Sham-immunized controls were included in both hypoxic and normoxic groups. Animals were sacrificed at pre-clinical and peak disease periods for tissue collection and analysis.. Exposure to mild hypoxia decreased histological evidence of inflammation. Decreased numbers of cluster of differentiation (CD)4+ T cells were found in the hypoxic spinal cords associated with a delayed Th17-specific cytokine response. Hypoxia-induced changes did not alter the sensitization of peripheral T cells to the MOG peptide. Exposure to mild hypoxia induced significant increases in anti-inflammatory IL-10 levels and an increase in the number of spinal cord CD25+FoxP3+ T-regulatory cells.. Acclimatization to mild hypoxia incites a number of endogenous adaptations that induces an anti-inflammatory milieu. Further understanding of these mechanisms system may pinpoint possible new therapeutic targets to treat neurodegenerative disease.

Topics: Adaptation, Biological; Animals; Antigens, CD; Central Nervous System; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Female; Freund's Adjuvant; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Interferon-gamma; Interleukin-17; Mice; Mice, Inbred C57BL; Microglia; Myelin-Oligodendrocyte Glycoprotein; Neutrophil Infiltration; Oxygen; Peptide Fragments; Spinal Cord; T-Lymphocytes, Regulatory; Time Factors

Gray matter degeneration contributes to progressive disability in multiple sclerosis (MS) and can occur out of proportion to measures of white matter disease. Although white matter pathology, including demyelination and axon injury, can lead to secondary gray matter changes, we hypothesized that neurons can undergo direct excitatory injury within the gray matter independent of these. We tested this using a model of experimental autoimmune encephalomyelitis (EAE) with hippocampal degeneration in C57BL/6 mice, in which immunofluorescent staining showed a 28% loss of PSD95-positive excitatory postsynaptic puncta in hippocampal area CA1 compared with sham-immunized controls, despite preservation of myelin and VGLUT1-positive excitatory axon terminals. Loss of postsynaptic structures was accompanied by appearance of PSD95-positive debris that colocalized with the processes of activated microglia at 25 d after immunization, and clearance of debris was followed by persistently reduced synaptic density at 55 d. In vitro, addition of activated BV2 microglial cells to hippocampal cultures increased neuronal vulnerability to excitotoxic dendritic damage following a burst of synaptic activity in a manner dependent on platelet-activating factor receptor (PAFR) signaling. In vivo treatment with PAFR antagonist BN52021 prevented PSD95-positive synapse loss in hippocampi of mice with EAE but did not affect development of EAE or local microglial activation. These results demonstrate that postsynaptic structures can be a primary target of injury within the gray matter in autoimmune neuroinflammatory disease, and suggest that this may occur via PAFR-mediated modulation of activity-dependent synaptic physiology downstream of microglial activation.. Unraveling gray matter degeneration is critical for developing treatments for progressive disability and cognitive impairment in multiple sclerosis (MS). In a mouse model of MS, we show that neurons can undergo injury at their synaptic connections within the gray matter, independent of the white matter pathology, demyelination, and axon injury that have been the focus of most current and emerging treatments. Damage to excitatory synapses in the hippocampus occurs in association with activated microglia, which can promote excitotoxic injury via activation of receptors for platelet-activating factor, a proinflammatory signaling molecule elevated in the brain in MS. Platelet-activating factor receptor blockade protected synapses in the mouse model, identifying a potential target for neuroprotective treatments in MS.

Topics: Animals; Calcium-Binding Proteins; Cell Line, Tumor; Chromosome Pairing; Coculture Techniques; Disease Models, Animal; Disks Large Homolog 4 Protein; Encephalomyelitis, Autoimmune, Experimental; Female; Fibrinolytic Agents; Ginkgolides; Gray Matter; Guanylate Kinases; Hippocampus; Lactones; Male; Membrane Proteins; Mice; Mice, Inbred C57BL; Microfilament Proteins; Microglia; Myelin-Oligodendrocyte Glycoprotein; Peptide Fragments; Platelet Membrane Glycoproteins; Receptors, G-Protein-Coupled; Vesicular Glutamate Transport Protein 1

Brain-intrinsic degenerative cascades have been proposed to be an initial factor driving lesion formation in multiple sclerosis (MS). Here, we identify neurodegeneration as a potent trigger for peripheral immune cell recruitment into the mouse forebrain. Female C57BL/6 mice were fed cuprizone for 3 weeks, followed by a period of 2 weeks on normal chow to induce the formation of lesion foci in the forebrain. Subsequent immunization with myelin oligodendrocyte glycoprotein 35-55 peptide, which induces myelin autoreactive T cells in the periphery, resulted in massive immune cell recruitment into the affected forebrain. Additional adoptive transfer experiments together with flow cytometry analysis underline the importance of brain-derived signals for immune cell recruitment. This study clearly illustrates the significance of brain-intrinsic degenerative cascades for immune cell recruitment and MS lesion formation. Additional studies have to address the signaling cascades and mechanistic processes that form the top-down communication between the affected brain area, neurovascular unit, and peripheral immune cells.. We identify neurodegeneration as a potent trigger for peripheral immune cell recruitment into the forebrain. Thus, immune cell recruitment might be a second step during the formation of new inflammatory lesions in multiple sclerosis. A better understanding of factors regulating neurodegeneration-induced immune cell recruitment will pave the way for the development of novel therapeutic treatment strategies.

Topics: Adoptive Transfer; Animals; Calcium-Binding Proteins; CD3 Complex; Chelating Agents; Cuprizone; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Female; Freund's Adjuvant; Lymph Nodes; Lymphocytes; Mice; Mice, Inbred C57BL; Microfilament Proteins; Monocytes; Myelin-Oligodendrocyte Glycoprotein; Neurodegenerative Diseases; Peptide Fragments; Pertussis Toxin; Prosencephalon

Experimental autoimmune encephalitis (EAE) is an inflammatory demyelinating disease, which served as a useful model providing considerable insights into the pathogenesis of multiple sclerosis (MS). Mouse bone marrow mesenchymal stem cells (mBM-MSC) were shown to have neuroprotection capabilities in EAE. Resveratrol is a small polyphenolic compound and possess therapeutic activity in various immune-mediated diseases. The sensitivity of mBM-MSCs to resveratrol was determined by an established cell-viability assay. Resveratrol-treated mBM-MSCs were also characterized with flow cytometry using MSC-specific surface markers and analyzed for their multiple differentiation capacities. EAE was induced in C57BL/6 mice by immunization with MOG35-55. Interferon gamma (IFN-γ)/tumor necrosis factor alpha (TNF-α) and interleukin-4 (IL-4)/interleukin-10 (IL-10), the hallmark cytokines that direct T helper type 1 (Th1) and Th2 development, were detected with enzyme-linked immunosorbent assay (ELISA). In vivo efficacy experiments showed that mBM-MSCs or resveratrol alone led to a significant reduction in clinical scores, and combined treatment resulted in even more prominent reduction. The combined treatment with mBM-MSCs and resveratrol enhanced the immunomodulatory effects, showing suppressed proinflammatory cytokines (IFN-γ, TNF-α) and increased anti-inflammatory cytokines (IL-4, IL-10). The combination of mBM-MSCs and resveratrol provides a novel potential experimental protocol for alleviating EAE symptoms.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Cell Proliferation; Cell Survival; Cytokines; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Female; Freund's Adjuvant; L-Lactate Dehydrogenase; Mesenchymal Stem Cell Transplantation; Mesenchymal Stem Cells; Mice; Mice, Inbred C57BL; Myelin-Oligodendrocyte Glycoprotein; Peptide Fragments; Resveratrol; Severity of Illness Index; Stilbenes; Time Factors

Regulator of G-protein signaling (RGS) family proteins, which are GTPase accelerating proteins (GAPs) that negatively regulate G-protein-coupled receptors (GPCRs), are known to be important modulators of immune cell activation and function. Various single-nucleotide polymorphisms in RGS proteins highly correlate with increased risk for multiple sclerosis (MS), an autoimmune, neurodegenerative disorder. An in-depth search of the gene expression omnibus profile database revealed higher levels of RGS10 and RGS1 transcripts in peripheral blood mononuclear cells (PBMCs) in MS patients, suggesting potential functional roles for RGS proteins in MS etiology and/or progression.. To define potential roles for RGS10 in regulating autoimmune responses, we evaluated RGS10-null and wild-type (WT) mice for susceptibility to experimental autoimmune encephalomyelitis (EAE), a widely studied model of MS. Leukocyte distribution and functional responses were assessed using biochemical, immunohistological, and flow cytometry approaches.. RGS10-null mice displayed significantly milder clinical symptoms of EAE with reduced disease incidence and severity, as well as delayed onset. We observed fewer CD3+ T lymphocytes and CD11b+ myeloid cells in the central nervous system (CNS) tissues of RGS10-null mice with myelin oligodendrocyte protein (MOG)35-55-induced EAE. Lymph node cells and splenocytes of immunized RGS10-null mice demonstrated decreased proliferative and cytokine responses in response to in vitro MOG memory recall challenge. In adoptive recipients, transferred myelin-reactive RGS10-null Th1 cells (but not Th17 cells) induced EAE that was less severe than their WT counterparts.. These data demonstrate a critical role for RGS10 in mediating autoimmune disease through regulation of T lymphocyte function. This is the first study ever conducted to elucidate the function of RGS10 in effector lymphocytes in the context of EAE. The identification of RGS10 as an important regulator of inflammation might open possibilities for the development of more specific therapies for MS.

Topics: Animals; Antigen Presentation; Antigens, CD; Cell Proliferation; Cytokines; Dendritic Cells; Disease Models, Animal; Dose-Response Relationship, Drug; Encephalomyelitis, Autoimmune, Experimental; Freund's Adjuvant; Gene Expression Regulation; Immunization, Passive; Leukocytes, Mononuclear; Mice; Mice, Inbred C57BL; Mice, Transgenic; Myelin-Oligodendrocyte Glycoprotein; Peptide Fragments; RGS Proteins; T-Lymphocytes

CD30, a member of the tumor necrosis factor receptor superfamily, is expressed preferentially by effector or memory helper T cells. Here we show that experimental autoimmune encephalomyelitis (EAE) is ameliorated with markedly reduced induction of antigen-specific Th1 and Th17 cells in CD30 knockout mice. Passive EAE indicated that CD30 on non-hematopoietic parenchymal cell is not required and mixed bone marrow chimera experiments revealed that CD30 signaling on CD4 T cells amplified the development of antigen-specific and encephalitogenic CD4 T cells. Thus, CD30 expression on CD4 T cells is critically involved in the pathogenesis of central nervous system autoimmunity.

Topics: Animals; CD4-Positive T-Lymphocytes; Cytokines; Disease Models, Animal; Dose-Response Relationship, Drug; Encephalomyelitis, Autoimmune, Experimental; Flow Cytometry; Ki-1 Antigen; Mice; Mice, Inbred C57BL; Mice, Knockout; Myelin-Oligodendrocyte Glycoprotein; Peptide Fragments; Spinal Cord; Th1 Cells; Th17 Cells; Thymidine; Time Factors; Tritium

The aim of this study was to evaluate the effects of orexin-A (OX-A) on behavioral and pathological parameters and on gene expression of some multiple sclerosis-related peptides in a model of experimental autoimmune encephalomyelitis (EAE). EAE was induced by subcutaneous administration of MOG 35-55. Following immunization, the treatment was initiated by using SB.334867 (orexin-1 receptor antagonist) and/or OX-A. Locomotor activity and exploratory behaviors were monitored using open field and T-maze continuous alternation task (T-CAT) respectively. Pain sensitivity was assessed by hot-plate test. Histopathological assessments were performed by H&E staining. The expression of TGF-β, MBP, MMP-9, IL-12, iNOS and MCP-1 were measured using real-time PCR method in lumbar spinal cord. OX-A administration in EAE mice remarkably attenuated the clinical symptoms, increased latency response in hot plate test, inhibited infiltration of inflammatory cells, up-regulated mRNA expression of TGF-β as well as MBP and down-regulated mRNA expression of iNOS, MMP-9 and IL-12. In contrast SB.334867 administration in EAE mice deteriorated the clinical symptoms, decreased the alternation in T-CAT, increased infiltration of inflammatory cells, down-regulated mRNA expression of TGF-β and MBP and up-regulated mRNA expression of iNOS. Results of this study suggest that the orexinergic system might be involved in pathological development of EAE. These findings suggest orexinergic system as a potential target for treatment of multiple sclerosis.

Topics: Animals; Attention; Avoidance Learning; Benzoxazoles; Body Weight; Cytokines; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Exploratory Behavior; Female; Matrix Metalloproteinase 9; Maze Learning; Mice; Mice, Inbred C57BL; Myelin Basic Protein; Myelin-Oligodendrocyte Glycoprotein; Naphthyridines; Nitric Oxide Synthase Type II; Orexin Receptor Antagonists; Orexins; Peptide Fragments; Severity of Illness Index; Spinal Cord; Time Factors; Urea

Intravenous immunoglobulin (IVIG) proved to be an efficient anti-inflammatory treatment for a growing number of neuroinflammatory diseases and protects against the development of experimental autoimmune encephalomyelitis (EAE), a widely used animal model for multiple sclerosis (MS).. The clinical efficacy of IVIG and IVIG-derived F(ab')2 fragments, generated using the streptococcal cysteine proteinase Ide-S, was evaluated in EAE induced by active immunization and by adoptive transfer of myelin-specific T cells. Frequency, phenotype, and functional characteristics of T cell subsets and myeloid cells were determined by flow cytometry. Antibody binding to microbial antigen and cytokine production by innate immune cells was assessed by ELISA.. We report that the protective effect of IVIG is lost in the adoptive transfer model of EAE and requires prophylactic administration during disease induction. IVIG-derived Fc fragments are not required for protection against EAE, since administration of F(ab')2 fragments fully recapitulated the clinical efficacy of IVIG. F(ab')2-treated mice showed a substantial decrease in splenic effector T cell expansion and cytokine production (GM-CSF, IFN-γ, IL-17A) 9 days after immunization. Inhibition of effector T cell responses was not associated with an increase in total numbers of Tregs but with decreased activation of innate myeloid cells such as neutrophils, monocytes, and dendritic cells. Therapeutically effective IVIG-derived F(ab')2 fragments inhibited adjuvant-induced innate immune cell activation as determined by IL-12/23 p40 production and recognized mycobacterial antigens contained in Freund's complete adjuvant which is required for induction of active EAE.. Our data indicate that F(ab')2-mediated neutralization of adjuvant contributes to the therapeutic efficacy of anti-inflammatory IgG. These findings might partly explain the discrepancy of IVIG efficacy in EAE and MS.

Topics: Adoptive Transfer; Animals; Antigen Presentation; Antigens, CD; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Enzyme-Linked Immunosorbent Assay; Flow Cytometry; Freund's Adjuvant; Immunoglobulins, Intravenous; Immunologic Factors; Leukocytes; Mice; Mice, Inbred C57BL; Mice, Transgenic; Myelin-Oligodendrocyte Glycoprotein; Peptide Fragments; Receptors, Antigen, T-Cell; Spleen; Time Factors

Tumor necrosis factor (TNF) has pleiotropic functions during both the demyelinating autoimmune disease multiple sclerosis (MS) and its murine model experimental autoimmune encephalomyelitis (EAE). How TNF regulates disability during progressive disease remains unresolved. Using a progressive EAE model characterized by sustained TNF and increasing morbidity, this study evaluates the role of unregulated TNF in exacerbating central nervous system (CNS) pathology and inflammation.. Progressive MS was mimicked by myelin oligodendrocyte glycoprotein (MOG) peptide immunization of mice expressing a dominant negative IFN-γ receptor alpha chain under the human glial fibrillary acidic protein promoter (GFAPγR1∆). Diseased GFAPγR1∆ mice were treated with anti-TNF or control monoclonal antibody during acute disease to monitor therapeutic effects on sustained disability, demyelination, CNS inflammation, and blood brain barrier (BBB) permeability.. TNF was specifically sustained in infiltrating macrophages. Anti-TNF treatment decreased established clinical disability and mortality rate within 7 days. Control of disease progression was associated with a decline in myelin loss and leukocyte infiltration, as well as macrophage activation. In addition to mitigating CNS inflammation, TNF neutralization restored BBB integrity and enhanced CNS anti-inflammatory responses.. Sustained TNF production by infiltrating macrophages associated with progressive EAE exacerbates disease severity by promoting inflammation and disruption of BBB integrity, thereby counteracting establishment of an anti-inflammatory environment required for disease remission.

Topics: Animals; Antibodies; Antigens, CD; Blood-Brain Barrier; Calcium-Binding Proteins; Capillary Permeability; Central Nervous System; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Gene Expression Regulation; Glial Fibrillary Acidic Protein; Humans; Interferon-gamma; Macrophages; Mice; Mice, Transgenic; Microfilament Proteins; Myelin-Oligodendrocyte Glycoprotein; Neuroglia; Neutrophil Infiltration; Peptide Fragments; Tumor Necrosis Factor-alpha

Conditions of inflammatory tissue distress are associated with high extracellular levels of adenosine, due to increased adenosine triphosphate (ATP) degradation upon cellular stress or the release of extracellular ATP upon cell death, which can be degraded to adenosine by membrane-bound ecto-enzymes like CD39 and CD73. Adenosine is recognised to mediate anti-inflammatory effects via the adenosine A2a receptor (A2aR), as shown in experimental models of arthritis. Here, using pharmacological interventions and genetic inactivation, we investigated the roles of A2aR in experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS).. We used two independent mouse EAE variants, i.e. active immunization in C57BL/6 with myelin oligodendrocyte glycoprotein (MOG)35-55 or transfer-EAE by proteolipid protein (PLP)139-155-stimulated T lymphocytes and EAE in mice treated with A2aR-agonist CGS21680 at different stages of disease course and in mice lacking A2aR (A2aR(-/-)) compared to direct wild-type littermates. In EAE, we analysed myelin-specific proliferation and cytokine synthesis ex vivo, as well as inflammation and demyelination by immunohistochemistry. In vitro, we investigated the effect of A2aR on migration of CD4(+) T cells, macrophages and microglia, as well as the impact of A2aR on phagocytosis of macrophages and microglia. Statistical tests were Mann-Whitney U and Student's t test.. We found an upregulation of A2aR in the central nervous system (CNS) in EAE, predominantly detected on T cells and macrophages/microglia within the inflamed tissue. Preventive EAE treatment with A2aR-specific agonist inhibited myelin-specific T cell proliferation ex vivo and ameliorated disease, while application of the same agonist after disease onset exacerbated non-remitting EAE progression and resulted in more severe tissue destruction. Accordingly, A2aR-deficient mice showed accelerated and exacerbated disease manifestation with increased frequencies of IFN-γ-, IL-17- and GM-CSF-producing CD4(+) T helper cells and higher numbers of inflammatory lesions in the early stage. However, EAE quickly ameliorated and myelin debris accumulation was lower in A2aR(-/-) mice. In vitro, activation of A2aR inhibited phagocytosis of myelin by macrophages and primary microglia as well as migration of CD4(+) T cells, macrophages and primary microglia.. A2aR activation exerts a complex pattern in chronic autoimmune neurodegeneration: while providing anti-inflammatory effects on T cells and thus protection at early stages, A2aR seems to play a detrimental role during later stages of disease and may thus contribute to sustained tissue damage within the inflamed CNS.

Topics: Adenosine; Animals; Anti-Inflammatory Agents; CD4-Positive T-Lymphocytes; Cell Movement; Cell Proliferation; Cells, Cultured; Cytokines; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Macrophages; Mice; Mice, Inbred C57BL; Mice, Transgenic; Microglia; Myelin Proteolipid Protein; Myelin-Oligodendrocyte Glycoprotein; Peptide Fragments; Phagocytosis; Phenethylamines; Receptor, Adenosine A2A; Time Factors; Up-Regulation

7,8-Dihydroxyflavone (DHF), is a recently described TrkB agonist that readily crosses the blood brain barrier. We treated C57Bl/6 mice with MOG--induced EAE daily with DHF starting on the day of disease induction. Clinical severity of impairment was reduced throughout the course of disease. Pathological examination of brains and spinal cords on day 28 showed that DHF treatment increased the phosphorylation of TrkB and activated downstream signaling pathways including AKT and STAT3 and reduced inflammation, demyelination and axonal loss compared to EAE controls. DHF treatment duplicated the central nervous system effects of brain derived neurotrophic factor in the EAE.

Topics: 2',3'-Cyclic-Nucleotide Phosphodiesterases; Animals; Apoptosis; bcl-2-Associated X Protein; Brain; Cytokines; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Female; Flavones; Humans; Mice; Mice, Inbred C57BL; Multiple Sclerosis; Myelin Basic Protein; Myelin-Oligodendrocyte Glycoprotein; Peptide Fragments; Severity of Illness Index; Signal Transduction; Spinal Cord; Time Factors

Immoderate immunoreaction of antigen-specific Th17 and Treg cell dysfunction play critical roles in the pathogenesis of multiple sclerosis. We examined Th17/Treg immune responses and the underlying mechanisms in response to metformin in C57BL/6 mice with experimental autoimmune encephalomyelitis (EAE). Metformin reduced Th17 and increased Treg cell percentages along with the levels of associated cytokines. Molecules involved in cellular metabolism were altered in mice with EAE. Suppressed activation of mTOR and its downstream target, HIF-1α, likely mediated the protective effects of metformin. Our findings demonstrate that regulation of T cell metabolism represents a new therapeutic target for CNS autoimmune disorders.

Topics: Analysis of Variance; Animals; Cytokines; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Enzyme-Linked Immunosorbent Assay; Female; Hypoglycemic Agents; Hypoxia-Inducible Factor 1, alpha Subunit; Metformin; Mice; Mice, Inbred C57BL; Myelin-Oligodendrocyte Glycoprotein; Peptide Fragments; RNA, Messenger; T-Lymphocytes, Regulatory; Th17 Cells

Daphnetin, a coumarin derivative extracted from Daphne odora var. marginata, has been reported to have antiinflammatory and immunosuppressive properties. Our previous study indicated that it was able to remarkably suppress the neuroinflammation and suggested its potential application in treating neuroinflammatory diseases. Multiple sclerosis (MS), a Th cell-mediated autoimmune disease, is the most common inflammatory demyelinating disease of the central nervous system (CNS). We examined whether daphnetin treatment can protect mice against experimental autoimmune encephalomyelitis (EAE), an animal model for MS.. To assess the effect of daphnetin in neuroinflammatory diseases, the EAE mice were established and treated with daphnetin at 8 mg/kg for 28 days. The severity of neuroinflammation and demyelination in the spinal cords was examined histopathologically. Infiltration of CD4(+) T cells into the CNS was assessed by immunohistochemistry, and the cytokine production was determined by ELISA. Meanwhile, the effect of daphnetin on the activity of dendritic cells (DCs) was evaluated, as assessed by DCs' capability to express surface markers, secrete cytokines, and activate naïve CD4(+) T cells. Furthermore, we explored the molecular mechanisms whereby DAPH regulated DCs' activity and thereby CD4(+) T cell responses.. The administration of daphnetin markedly alleviated the clinical symptoms of EAE and reduced the CNS inflammation and demyelination in experimental mice. Th1 and Th17 cell responses were profoundly repressed in daphnetin-treated EAE mice. Mechanistically, daphnetin treatment significantly repressed the activation, maturation, and antigen-presenting capability of DCs. NF-κB signaling was significantly reduced in daphnetin-treated DCs, along with a concomitant induction of heme oxygenase-1, a negative regulator of inflammatory signaling.. Our findings for the first time demonstrate the property of daphnetin in regulating DCs' function and subsequently Th development. Given the low or absent toxicity associated with daphnetin, our data may suggest a novel safe and effective approach to control autoimmune neuroinflammation.

Topics: Animals; Central Nervous System; Cytokines; Dendritic Cells; Disease Models, Animal; Dose-Response Relationship, Drug; Encephalomyelitis, Autoimmune, Experimental; Enzyme Activation; Female; Heme Oxygenase-1; Lipopolysaccharides; Lymphocytes; Membrane Proteins; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Myelin-Oligodendrocyte Glycoprotein; NF-kappa B; Peptide Fragments; Signal Transduction; Spleen; Time Factors; Umbelliferones

Sex hormones promote immunoregulatory effects on multiple sclerosis. The current study evaluated estrogen effects on regulatory B cells and resident CNS microglia during experimental autoimmune encephalomyelitis (EAE). Herein, we demonstrate an estrogen-dependent induction of multiple regulatory B cell markers indicative of IL-10 dependent as well as IFN-γ dependent pathways. Moreover, although estrogen pretreatment of EAE mice inhibited the infiltration of pro-inflammatory cells into the CNS, it enhanced the frequency of regulatory B cells and M2 microglia. Our study suggests that estrogen has a broad effect on the development of regulatory B cells during EAE, which in turn could promote neuroprotection.

Topics: Animals; B-Lymphocytes, Regulatory; Brain; Cell Proliferation; Cytokines; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Estradiol; Freund's Adjuvant; Leukocytes; Mice; Mice, Inbred C57BL; Microglia; Myelin-Oligodendrocyte Glycoprotein; Peptide Fragments; Pertussis Toxin; RNA, Messenger; Spinal Cord; Spleen; Statistics, Nonparametric; Time Factors

Multiple sclerosis (MS) is a major neuroinflammatory demyelinating disease of the CNS. Current MS treatments, including immunomodulators and immunosuppressants, do not result in complete remission. Stem cells from human exfoliated deciduous teeth (SHEDs) are mesenchymal stem cells derived from dental pulp. Both SHED and SHED-conditioned medium (SHED-CM) exhibit immunomodulatory and regenerative activities and have the potential to treat various diseases. In this study, we investigated the efficacy of SHED-CM in treating experimental autoimmune encephalomyelitis (EAE), a mouse model of MS. EAE mice treated with a single injection of SHED-CM exhibited significantly improved disease scores, reduced demyelination and axonal injury, and reduced inflammatory cell infiltration and proinflammatory cytokine expression in the spinal cord, which was associated with a shift in the microglia/macrophage phenotype from M1 to M2. SHED-CM also inhibited the proliferation of myelin oligodendrocyte glycoprotein-specific CD4(+) T cells, as well as their production of proinflammatory cytokines in vitro. Treatment of EAE mice with the secreted ectodomain of sialic acid-binding Ig-like lectin-9, a major component of SHED-CM, recapitulated the effects of SHED-CM treatment. Our data suggest that SHED-CM and secreted ectodomain of sialic acid-binding Ig-like lectin-9 may be novel therapeutic treatments for autoimmune diseases, such as MS.

Topics: Animals; Antigens, CD; CD4-Positive T-Lymphocytes; Cells, Cultured; Culture Media, Conditioned; Cytokines; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Female; Humans; Lymphocyte Activation; Macrophages; Mesenchymal Stem Cells; Mice; Mice, Inbred C57BL; Microglia; Multiple Sclerosis; Myelin-Oligodendrocyte Glycoprotein; Peptide Fragments; Sialic Acid Binding Immunoglobulin-like Lectins; Tooth, Deciduous

Multiple sclerosis (MS) is a chronic autoimmune demyelinating disease of the central nervous system (CNS). The high costs, inconvenient administration, and side effects of current Food and Drug Administration (FDA)-approved drugs often lead to poor adherence to the long-term treatment of MS. Molecular hydrogen (H2) has been reported to exhibit anti-oxidant, anti-apoptotic, anti-inflammatory, anti-allergy, and anti-cancer effects. In the present study, we explored the prophylactic and therapeutic effects of hydrogen-rich water (HRW) on the progress of experimental autoimmune encephalomyelitis (EAE), the animal model for MS. We found that prophylactic administration of both 0.36mM and 0.89mM HRW was able to delay EAE onset and reduce maximum clinical scores. Moreover, 0.89mM HRW also reduced disease severity, CNS infiltration, and demyelination when administered after the onset of disease. Furthermore, HRW treatment prevented infiltration of CD4(+) T lymphocytes into the CNS and inhibited Th17 cell development without affecting Th1 cell populations. Because HRW is non-toxic, inexpensive, easily administered, and can readily cross the blood-brain barrier, our experiments suggest that HRW may have great potential in the treatment of MS.

Topics: Analysis of Variance; Animals; Cytokines; Disease Models, Animal; Dose-Response Relationship, Drug; Encephalomyelitis, Autoimmune, Experimental; Female; Flow Cytometry; Hydrogen; Leukocytes, Mononuclear; Mice; Mice, Inbred C57BL; Myelin-Oligodendrocyte Glycoprotein; Nervous System Diseases; Peptide Fragments; Recovery of Function; Statistics, Nonparametric; Water

The development of multiple sclerosis, a major neurodegenerative disease, is due to both genetic and environmental factors that might trigger aberrant epigenetic changes of the genome. In this study, we analysed global DNA methylation in the brain of mice upon induction of experimental autoimmune encephalomyelitis (EAE), and the effect of environmental enrichment (EE). We demonstrate that global DNA methylation decreased in the striatum, but not in the cortex, of EAE mice compared to healthy controls, in particular in neuronal nitric oxide synthase (nNOS)-positive interneurons of this brain area. Also, in the striatum but again not in the cortex, decreased DNA methylation of the nNOS downstream effector, dexamethasone-induced Ras protein 1 (Dexras 1), was observed in EAE mice, and was paralleled by an increase in its mRNA. Interestingly, EE was able to revert EAE effects on mRNA expression and DNA methylation levels of Dexras 1 and reduced gene expression of nNOS and 5-lipoxygenase (Alox5). Conversely, interleukin-1β (IL-1β) gene expression was found up-regulated in EAE mice compared to controls and was not affected by EE. Taken together, these data demonstrate an unprecedented epigenetic modulation of nNOS-signaling in the pathogenesis of multiple sclerosis, and show that EE can specifically revert EAE effects on Dexras 1 along this pathway.

Topics: 5-Methylcytosine; Animals; Anti-Inflammatory Agents; Arachidonate 5-Lipoxygenase; Brain; Cytokines; Dexamethasone; Disease Models, Animal; Dopamine and cAMP-Regulated Phosphoprotein 32; Encephalomyelitis, Autoimmune, Experimental; Epigenesis, Genetic; Female; Mice; Mice, Inbred C57BL; Myelin-Oligodendrocyte Glycoprotein; Neurons; Nitric Oxide Synthase Type I; Peptide Fragments; ras Proteins; Signal Transduction

Experimental autoimmune encephalomyelitis (EAE) is T-cell-dependent disease of the central nervous system (CNS) of mice. This model resembles multiple sclerosis (MS) in many aspects. Therapies that focus in the modulation of the immune response and cellular infiltration in the CNS present best effects in the clinics. Artesunate (Art) is a semi-synthetic sesquiterpene derivative from artemisinin and has been shown to reduce the clinical signs of autoimmune disease models through mechanisms not yet understood. In this study, we aimed to evaluate whether administration of Art would ameliorate EAE.. C57BL6 mice were immunized with MOG35-55 peptide to induce EAE. At the same time, Art treatment started (3 mg/kg/day via i.p.) for five consecutive days. We found that Art treatment reduced the clinical signs of EAE and that correlated with a reduced infiltration of cells in the CNS. Disease amelioration did not correlate with immunomodulation as recall responses, leukocyte subpopulations, and gene expression analysis were similar among treated and untreated mice. Ultimately, further analysis provided data indicating that a possible mechanism of action for Art is dependent on the cellular migration to the CNS.. Artesunate reduces the severity of EAE by inhibiting migration of pathogenic T cells to the CNS.

Topics: Analysis of Variance; Animals; Anti-Inflammatory Agents; Artemisinins; Artesunate; Brefeldin A; Cell Movement; Central Nervous System; Cytokines; Disease Models, Animal; DNA-Binding Proteins; Encephalomyelitis, Autoimmune, Experimental; Enzyme Inhibitors; Female; Flow Cytometry; Gene Expression Regulation; Ionomycin; Leukocytes; Mice; Mice, Inbred C57BL; Mice, Knockout; Myelin-Oligodendrocyte Glycoprotein; Peptide Fragments; Phorbol Esters

Tolerogenic dendritic cells (tolDC) have been postulated as a potent immunoregulatory therapy for autoimmune diseases such as multiple sclerosis (MS). In a previous study, we demonstrated that the administration of antigen-specific vitamin D3 (vitD3) tolDC in mice showing clinical signs of experimental autoimmune encephalomyelitis (EAE; the animal model of MS) resulted in abrogation of disease progression. With the purpose to translate this beneficial therapy to the clinics, we have investigated the effectivity of vitD3-frozen antigen-specific tolDC pulsed with myelin oligodendrocyte glycoprotein 40-55 peptide (f-tolDC-MOG) since it would reduce the cost, functional variability and number of leukapheresis to perform to the patients.. Mice showing EAE clinical signs were treated with repetitive doses of f-tolDC-MOG. Tolerogenic mechanisms induced by the therapy were analysed by flow cytometry and T cell proliferation assays.. Treatment with f-tolDC-MOG was effective in ameliorating clinical signs of mice with EAE, inhibiting antigen-specific reactivity and inducing Treg. In addition, the long-term treatment was well tolerated and leading to a prolonged maintenance of tolerogenicity mediated by induction of Breg, reduction of NK cells and activation of immunoregulatory NKT cells.. The outcomes of this study show that the use of antigen-specific f-tolDC promotes multiple and potent tolerogenic mechanisms. Moreover, these cells can be kept frozen maintaining their tolerogenic properties, which is a relevant step for their translation to the clinic. Altogether, vitD3 f-tolDC-MOG is a potential strategy to arrest the autoimmune destruction in MS patients.

Topics: Animals; Autoantigens; Cell Transplantation; Cholecalciferol; Cryopreservation; Cytokines; Dendritic Cells; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Female; Follow-Up Studies; Killer Cells, Natural; Mice; Mice, Inbred C57BL; Myelin-Oligodendrocyte Glycoprotein; Peptide Fragments; Polysaccharides; Time Factors

Multiple Sclerosis, a chronic inflammatory demyelinating disease of the central nervous system, involves an increased expression of monocyte chemotactic protein 1 MCP1-/CCL2. For exerting its chemotactic effects, chemokine binding to glycosaminoglycans (GAGs) is required and therefore this interaction represents a potential target for therapeutic intervention. We have designed an anti-inflammatory decoy variant, Met-CCL2 (Y13A S21K Q23R), embodying increased affinity for GAGs as well as knocked-out GPCR activation properties. This non-signalling dominant-negative mutant is shown here to be able to displace wild type CCL2 from GAGs by which it is supposed to interfere with the chemokine-related inflammatory response. In vivo, the anti-inflammatory properties were successfully demonstrated in a murine model of zymosan-induced peritonitis as well as in an experimental autoimmune encephalomyelitis, a model relevant for multiple sclerosis, where the compound lead to significantly reduced clinical scores due to reduction of cellular infiltrates and demyelination in spinal cord and cerebellum. These findings indicate a promising potential for future therapeutic development.

Topics: Animals; Anti-Inflammatory Agents; Cerebellum; Chemokine CCL2; Dexamethasone; Disease Models, Animal; Encephalitis; Encephalomyelitis, Autoimmune, Experimental; Female; Glycosaminoglycans; Inhibitory Concentration 50; Male; Mice; Mice, Inbred C57BL; Monocytes; Myelin-Oligodendrocyte Glycoprotein; Peptide Fragments; Peritonitis; Spinal Cord; Zymosan

Our previous studies showed that the non-psychoactive cannabinoid, cannabidiol (CBD), ameliorates the clinical symptoms in mouse myelin oligodendrocyte glycoprotein (MOG)35-55-induced experimental autoimmune encephalomyelitis model of multiple sclerosis (MS) as well as decreases the memory MOG35-55-specific T cell (TMOG) proliferation and cytokine secretion including IL-17, a key autoimmune factor. The mechanisms of these activities are currently poorly understood.. Herein, using microarray-based gene expression profiling, we describe gene networks and intracellular pathways involved in CBD-induced suppression of these activated memory TMOG cells. Encephalitogenic TMOG cells were stimulated with MOG35-55 in the presence of spleen-derived antigen presenting cells (APC) with or without CBD. mRNA of purified TMOG was then subjected to Illumina microarray analysis followed by ingenuity pathway analysis (IPA), weighted gene co-expression network analysis (WGCNA) and gene ontology (GO) elucidation of gene interactions. Results were validated using qPCR and ELISA assays.. Gene profiling showed that the CBD treatment suppresses the transcription of a large number of proinflammatory genes in activated TMOG. These include cytokines (Xcl1, Il3, Il12a, Il1b), cytokine receptors (Cxcr1, Ifngr1), transcription factors (Ier3, Atf3, Nr4a3, Crem), and TNF superfamily signaling molecules (Tnfsf11, Tnfsf14, Tnfrsf9, Tnfrsf18). "IL-17 differentiation" and "IL-6 and IL-10-signaling" were identified among the top processes affected by CBD. CBD increases a number of IFN-dependent transcripts (Rgs16, Mx2, Rsad2, Irf4, Ifit2, Ephx1, Ets2) known to execute anti-proliferative activities in T cells. Interestingly, certain MOG35-55 up-regulated transcripts were maintained at high levels in the presence of CBD, including transcription factors (Egr2, Egr1, Tbx21), cytokines (Csf2, Tnf, Ifng), and chemokines (Ccl3, Ccl4, Cxcl10) suggesting that CBD may promote exhaustion of memory TMOG cells. In addition, CBD enhanced the transcription of T cell co-inhibitory molecules (Btla, Lag3, Trat1, and CD69) known to interfere with T/APC interactions. Furthermore, CBD enhanced the transcription of oxidative stress modulators with potent anti-inflammatory activity that are controlled by Nfe2l2/Nrf2 (Mt1, Mt2a, Slc30a1, Hmox1).. Microarray-based gene expression profiling demonstrated that CBD exerts its immunoregulatory effects in activated memory TMOG cells via (a) suppressing proinflammatory Th17-related transcription, (b) by promoting T cell exhaustion/tolerance, (c) enhancing IFN-dependent anti-proliferative program, (d) hampering antigen presentation, and (d) inducing antioxidant milieu resolving inflammation. These findings put forward mechanism by which CBD exerts its anti-inflammatory effects as well as explain the beneficial role of CBD in pathological memory T cells and in autoimmune diseases.

Topics: Analysis of Variance; Animals; Antigen-Presenting Cells; Cannabidiol; Coculture Techniques; Cytokines; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Enzyme-Linked Immunosorbent Assay; Gene Expression Regulation; Gene Regulatory Networks; Glycoproteins; Lymphocyte Activation; Male; Mice; Mice, Inbred C57BL; Myelin-Oligodendrocyte Glycoprotein; Peptide Fragments; RNA, Messenger; Spleen; T-Lymphocytes

Chronic neuropathic pain is a common symptom of multiple sclerosis (MS). MOG35-55-induced experimental autoimmune encephalomyelitis (EAE) has been used as an animal model to investigate the mechanisms of pain in MS. Previous studies have implicated sensitization of spinal nociceptive networks in the pathogenesis of pain in EAE. However, the involvement of supraspinal sites of nociceptive integration, such as the primary somatosensory cortex (S1), has not been defined. We therefore examined functional, structural, and immunological alterations in S1 during the early stages of EAE, when pain behaviors first appear. We also assessed the effects of the antidepressant phenelzine (PLZ) on S1 alterations and nociceptive (mechanical) sensitivity in early EAE. PLZ has been shown to restore central nervous system (CNS) tissue concentrations of GABA and the monoamines (5-HT, NA) in EAE. We hypothesized that PLZ treatment would also normalize nociceptive sensitivity in EAE by restoring the balance of excitation and inhibition (E-I) in the CNS.. We used in vivo flavoprotein autofluorescence imaging (FAI) to assess neural ensemble responses in S1 to vibrotactile stimulation of the limbs in early EAE. We also used immunohistochemistry (IHC), and Golgi-Cox staining, to examine synaptic changes and neuroinflammation in S1. Mechanical sensitivity was assessed at the clinical onset of EAE with Von Frey hairs.. Mice with early EAE exhibited significantly intensified and expanded FAI responses in S1 compared to controls. IHC revealed increased vesicular glutamate transporter (VGLUT1) expression and disrupted parvalbumin+ (PV+) interneuron connectivity in S1 of EAE mice. Furthermore, peri-neuronal nets (PNNs) were significantly reduced in S1. Morphological analysis of excitatory neurons in S1 revealed increased dendritic spine densities. Iba-1+ cortical microglia were significantly elevated early in the disease. Chronic PLZ treatment was found to normalize mechanical thresholds in EAE. PLZ also normalized S1 FAI responses, neuronal morphologies, and cortical microglia numbers and attenuated VGLUT1 reactivity-but did not significantly attenuate the loss of PNNs.. These findings implicate a pro-excitatory shift in the E-I balance of the somatosensory CNS, arising early in the pathogenesis EAE and leading to large-scale functional and structural plasticity in S1. They also suggest a novel antinociceptive effect of PLZ treatment.

Topics: Animals; Antidepressive Agents; Calcium-Binding Proteins; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Female; Freund's Adjuvant; Hyperalgesia; Mice; Mice, Inbred C57BL; Microfilament Proteins; Myelin-Oligodendrocyte Glycoprotein; Neuralgia; Neurons; Pain Measurement; Pain Threshold; Parvalbumins; Peptide Fragments; Phenelzine; Plant Lectins; Receptors, N-Acetylglucosamine; Somatosensory Cortex; Synapses

Experimental allergic encephalomyelitis (EAE), an animal model of multiple sclerosis produced by immunization with myelin oligodendrocyte glycoprotein (MOG) and adjuvants, results from profound T-cell mediated CNS demyelination. EAE is characterized by progressive, ascending motor dysfunction and symptoms of ongoing pain and hypersensitivity, in some cases preceding or concomitant with the motor deficits. In this regard, the EAE model mimics major features of multiple sclerosis, where a central neuropathic pain state is common. Although the latter condition is presumed to arise from a CNS loss of inhibitory controls secondary to the demyelination, dysfunction of sensory neurons may also contribute. Based on our previous studies that demonstrated the utility of monitoring expression of activating transcription factor 3 (ATF3), a sensitive marker of injured sensory neurons, here we followed both ATF3 and CD4+ T cells invasion of sensory ganglia (as well as the CNS) at different stages of the EAE model. We found that ATF3 is induced in peripheral sensory ganglia and brainstem well before the appearance of motor deficits. Unexpectedly, the ATF3 induction always preceded T cell infiltration, typically in adjacent, but non-overlapping regions. Surprisingly, control administration of the pertussis toxin and/or Complete Freund's adjuvants, without MOG, induced ATF3 in sensory neurons. In contrast, T cell infiltration only occurred with MOG. Taken together, our results suggest that the clinical manifestations in the EAE result not only from central demyelination but also from neuronal stress and subsequent pathophysiology of sensory neurons.

Topics: Activating Transcription Factor 3; Animals; Brain; CD4-Positive T-Lymphocytes; Disease Models, Animal; Disease Progression; Encephalomyelitis, Autoimmune, Experimental; Female; Freund's Adjuvant; Ganglia; Macrophage Colony-Stimulating Factor; Mice; Mice, Inbred C57BL; Myelin-Oligodendrocyte Glycoprotein; Nerve Tissue Proteins; Neurons; Neurotransmitter Agents; Neutrophil Infiltration; Peptide Fragments; Pertussis Toxin; TRPV Cation Channels

Vasoactive intestinal peptide (VIP) and pituitary adenylyl cyclase-activating polypeptide (PACAP) are two highly homologous neuropeptides. In vitro and ex vivo experiments repeatedly demonstrate that these peptides exert pronounced immunomodulatory (primarily anti-inflammatory) actions which are mediated by common VPAC1 and VPAC2 G protein-coupled receptors. In agreement, we have shown that mice deficient in PACAP ligand or VPAC2 receptors exhibit exacerbated experimental autoimmune encephalomyelitis (EAE). However, we observed that VIP-deficient mice are unexpectedly resistant to EAE, suggesting a requirement for this peptide at some stage of disease development. Here, we investigated the involvement of VPAC1 in the development of EAE using a VPAC1-deficient mouse model.. EAE was induced in wild-type (WT) and VPAC1 knockout (KO) mice using myelin oligodendrocyte glycoprotein 35-55 (MOG35-55), and clinical scores were assessed continuously over 30 days. Immune responses in the spinal cords were determined by histology, real-time PCR and immunofluorescence, and in the draining lymph nodes by antigen-recall assays. The contribution of VPAC1 expression in the immune system to the development of EAE was evaluated by means of adoptive transfer and bone marrow chimera experiments. In other experiments, VPAC1 receptor analogs were given to WT mice.. MOG35-55-induced EAE was ameliorated in VPAC1 KO mice compared to WT mice. The EAE-resistant phenotype of VPAC1 KO mice correlated with reduced central nervous system (CNS) histopathology and cytokine expression in the spinal cord. The immunization phase of EAE appeared to be unimpaired because lymph node cells from EAE-induced VPAC1 KO mice stimulated in vitro with MOG exhibited robust proliferative and Th1/Th17 responses. Moreover, lymph node and spleen cells from KO mice were fully capable of inducing EAE upon transfer to WT recipients. In contrast, WT cells from MOG-immunized mice did not transfer the disease when administered to VPAC1 KO recipients, implicating a defect in the effector phase of the disease. Bone marrow chimera studies suggested that the resistance of VPAC1-deficient mice was only minimally dependent on the expression of this receptor in the immunogenic/hematopoietic compartment. Consistent with this, impaired spinal cord inductions of several chemokine mRNAs were observed in VPAC1 KO mice. Finally, treatment of WT mice with the VPAC1 receptor antagonist PG97-269 before, but not after, EAE induction mimicked the clinical phenotype of VPAC1 KO mice.. VPAC1 gene loss impairs the development of EAE in part by preventing an upregulation of CNS chemokines and invasion of inflammatory cells into the CNS. Use of VPAC1 antagonists in WT mice prior to EAE induction also support a critical role for VPAC1 signaling for the development of EAE.

Topics: Adoptive Transfer; Animals; Central Nervous System; Cytokines; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Female; Freund's Adjuvant; Laminin; Lymph Nodes; Mice; Mice, Inbred C57BL; Mice, Knockout; Myelin-Oligodendrocyte Glycoprotein; Peptide Fragments; Receptors, Vasoactive Intestinal Polypeptide, Type I; RNA, Messenger; Spinal Cord; Th1 Cells; Th17 Cells; Time Factors

The present study was designed to investigate the beneficial effects of cornel iridoid glycoside (CIG), a main component extract from Cornus officinalis, on neurotrophin expression in mouse experimental autoimmune encephalomyelitis (EAE), a classical model of multiple sclerosis (MS). After EAE initiation, CIG was intragastrically administered daily for 32 days and reduced disease severity. Histopathological staining and western blotting both showed that CIG could prevent brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) loss in the spinal cord of EAE mice. In conclusion, our findings indicated that CIG treatment suppressed disease severity of EAE partially through blocking downregulation of neurotrophic factor expression such as BDNF and NGF, suggesting that CIG may have beneficial effects for the treatment of demyelinating diseases such as MS.

Topics: Animals; Brain-Derived Neurotrophic Factor; Disease Models, Animal; Dose-Response Relationship, Drug; Encephalomyelitis, Autoimmune, Experimental; Female; Freund's Adjuvant; Gene Expression Regulation; Iridoid Glucosides; Mice; Mice, Inbred C57BL; Microtubule-Associated Proteins; Multiple Sclerosis; Myelin-Oligodendrocyte Glycoprotein; Nerve Growth Factor; Nervous System Diseases; Peptide Fragments; Phosphopyruvate Hydratase; Spinal Cord; Statistics, Nonparametric; Time Factors

Relapsing-remitting multiple sclerosis (MS)(2) is characterized by phases of acute neuroinflammation followed by spontaneous remission. Termination of inflammation is accompanied by an influx of regulatory T cells (Tregs).(3) The molecular mechanisms responsible for directing Tregs into the inflamed CNS tissue, however, are incompletely understood. In an MS mouse model we show that the stress kinase general control non-derepressible 2 (GCN2),(4) expressed in T cells, contributes to the resolution of autoimmune neuroinflammation. Failure to recover from acute inflammation was associated with reduced frequencies of CNS-infiltrating Tregs. GCN2 deficient Tregs displayed impaired migration to a CCL2 gradient. These data suggest an important contribution of the T cell stress response to the resolution of autoimmune neuroinflammation.

Topics: Animals; Annexin A5; Astrocytes; Brain; Cell Movement; Cytokines; Disease Models, Animal; Disease Progression; Encephalomyelitis, Autoimmune, Experimental; Endothelial Cells; Female; Flow Cytometry; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Myelin-Oligodendrocyte Glycoprotein; Peptide Fragments; Protein Serine-Threonine Kinases; Statistics, Nonparametric; T-Lymphocytes, Regulatory; Time Factors

Increased α-synuclein immunoreactivity has been associated with inflammatory activity in multiple sclerosis (MS) lesions, but the function of α-synuclein in neuroinflammation remains unknown. The aim of this study was to examine the role of α-synuclein in immunological processes in murine experimental autoimmune encephalomyelitis (EAE) as a model of MS.. We studied EAE in wildtype (aSyn(+/+)) and α-synuclein knockout (aSyn(-/-)) mice on a C57BL/6N background. In the spleen and spinal cord of aSyn(+/+) mice, we observed a gradual reduction of α-synuclein expression during EAE, starting already in the pre-symptomatic disease phase. Compared to aSyn(+/+) mice, aSyn(-/-) mice showed an earlier onset of symptoms but no differences in symptom severity at the peak of disease. Earlier symptom onset was accompanied by increased spinal cord infiltration of CD4(+) T cells, predominantly of interferon-γ-producing T helper 1 (Th1) cells, and reduced infiltration of regulatory T cells, whereas antigen-presenting cells were unaltered. Pre-symptomatically, aSyn(-/-) mice exhibited hyperproliferative CD4(+) splenocytes consistent with increased splenic interleukin-2 mRNA expression, resulting in increased numbers of Th1 cells in the spleen at the onset of symptoms.. Our findings indicate a functional role of α-synuclein in early EAE by increasing Th1 cell-mediated immune response.

Topics: alpha-Synuclein; Analysis of Variance; Animals; Antigen-Presenting Cells; Cell Proliferation; Cytokines; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Gene Expression Regulation; Mice; Mice, Inbred C57BL; Myelin-Oligodendrocyte Glycoprotein; Peptide Fragments; RNA, Messenger; Spinal Cord; Th1 Cells

Data from multiple sclerosis (MS) and the MS rodent model, experimental autoimmune encephalomyelitis (EAE), highlighted an inflammation-dependent synaptopathy at the basis of the neurodegenerative damage causing irreversible disability in these disorders. This synaptopathy is characterized by an imbalance between glutamatergic and GABAergic transmission and has been proposed to be a potential therapeutic target. Siponimod (BAF312), a selective sphingosine 1-phosphate1,5 receptor modulator, is currently under investigation in a clinical trial in secondary progressive MS patients. We investigated whether siponimod, in addition to its peripheral immune modulation, may exert direct neuroprotective effects in the central nervous system (CNS) of mice with chronic progressive EAE.. Minipumps allowing continuous intracerebroventricular (icv) infusion of siponimod for 4 weeks were implanted into C57BL/6 mice subjected to MOG35-55-induced EAE. Electrophysiology, immunohistochemistry, western blot, qPCR experiments, and peripheral lymphocyte counts were performed. In addition, the effect of siponimod on activated microglia was assessed in vitro to confirm the direct effect of the drug on CNS-resident immune cells.. Siponimod administration (0.45 μg/day) induced a significant beneficial effect on EAE clinical scores with minimal effect on peripheral lymphocyte counts. Siponimod rescued defective GABAergic transmission in the striatum of EAE, without correcting the EAE-induced alterations of glutamatergic transmission. We observed a significant attenuation of astrogliosis and microgliosis together with reduced lymphocyte infiltration in the striatum of EAE mice treated with siponimod. Interestingly, siponimod reduced the release of IL-6 and RANTES from activated microglial cells in vitro, which might explain the reduced lymphocyte infiltration. Furthermore, the loss of parvalbumin-positive (PV+) GABAergic interneurons typical of EAE brains was rescued by siponimod treatment, providing a plausible explanation of the selective effects of this drug on inhibitory synaptic transmission.. Altogether, our results show that siponimod has neuroprotective effects in the CNS of EAE mice, which are likely independent of its peripheral immune effect, suggesting that this drug could be effective in limiting neurodegenerative pathological processes in MS.

Topics: Animals; Antigens, CD; Azetidines; Benzyl Compounds; Calcium-Binding Proteins; Cell Line, Transformed; Cerebral Cortex; Cytokines; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Freund's Adjuvant; Mice; Microfilament Proteins; Myelin-Oligodendrocyte Glycoprotein; Nerve Degeneration; Neural Conduction; Neuroglia; Neurons; Neuroprotective Agents; Peptide Fragments; Synapses; Synaptic Potentials; T-Lymphocytes; White Matter

T-helper 17 (Th17) cells play an important role in the pathogenesis of multiple sclerosis (MS), an autoimmune demyelinating disease that affects the CNS. In the present study, MicroRNA sequencing (miRNA-seq) was performed in mouse Th0 and Th17 cells to determine the critical miRNAs that are related to Th17 differentiation. We found that miR-30a was significantly downregulated during mouse Th17 differentiation. In addition, the level of miR-30a in CD4(+) T cells from peripheral blood of MS patients and experimental autoimmune encephalomyelitis (EAE) animal models was also decreased and inversely correlated with the expression of interleukin 17a, the canonical cytokine of Th17 cells. Moreover, overexpression of miR-30a inhibited Th17 differentiation and prevented the full development of EAE, whereas interference of miR-30a promoted Th17 differentiation. Mechanism studies showed that miR-30a reduced IRF4 expression by specifically binding with the 3'-untranslated region. Through screening of 640 different Food and Drug Administration (FDA)-approved drugs, we found that disulfiram and diphenhydramine hydrochloride were effective candidates for inhibiting Th17 differentiation and ameliorating EAE development through upregulating miR-30a. To our knowledge, the present work is not only the first miRNA-seq study focusing on Th17 differentiation, but also the first chemical screening for FDA-approved drugs that inhibit Th17 differentiation through regulating miRNA expression.. The present work is the first miRNA sequencing (miRNA-seq) study focusing on T-helper 17 (Th17) differentiation. By miRNA deep sequencing, we found that miR-30a was downregulated during Th17 differentiation. miR-30a was also decreased in CD4(+) T cells from multiple sclerosis patients and experimental autoimmune encephalomyelitis (EAE) mice. miR-30a reduced IRF4 expression by specific binding with the 3'-untranslated region and thus suppressed Th17 differentiation and prevented the full development of EAE. Interestingly, by performing a chemical screen with Food and Drug Administration-approved small-molecule drugs, we found that disulfiram and diphenhydramine upregulated miR-30a and suppressed Th17-associated autoimmune demyelination.

Topics: Animals; Case-Control Studies; Cell Differentiation; Cells, Cultured; Diphenhydramine; Disease Models, Animal; Disulfiram; Encephalomyelitis, Autoimmune, Experimental; Female; HEK293 Cells; Humans; Interferon Regulatory Factors; Interleukin-17; Male; Mice; Mice, Inbred C57BL; MicroRNAs; Myelin Proteolipid Protein; Myelin-Oligodendrocyte Glycoprotein; Peptide Fragments; Statistics, Nonparametric; Transfection; Up-Regulation

Mood disorders, including anxiety and depression, are frequently diagnosed in multiple sclerosis (MS) patients, even independently of the disabling symptoms associated with the disease. Anatomical, biochemical, and pharmacological evidence indicates that type-1 cannabinoid receptor (CB1R) is implicated in the control of emotional behavior and is modulated during inflammatory neurodegenerative diseases such as MS and experimental autoimmune encephalomyelitis (EAE).. We investigated whether CB1R could exert a role in anxiety-like behavior in mice with EAE. We performed behavioral, pharmacological, and electrophysiological experiments to explore the link between central inflammation, mood, and CB1R function in EAE.. We observed that EAE-induced anxiety was associated with the downregulation of CB1R-mediated control of striatal GABA synaptic transmission and was exacerbated in mice lacking CB1R (CB1R-KO mice). Central blockade of interleukin-1β (IL-1β) reversed the anxiety-like phenotype of EAE mice, an effect associated with the concomitant rescue of dopamine (DA)-regulated spontaneous behavior, and DA-CB1R neurotransmission, leading to the rescue of striatal CB1R sensitivity.. Overall, results of the present investigation indicate that synaptic dysfunction linked to CB1R is involved in EAE-related anxiety and motivation-based behavior and contribute to clarify the complex neurobiological mechanisms underlying mood disorders associated to MS.

Topics: Amphetamine; Animals; Antirheumatic Agents; Anxiety; Central Nervous System Stimulants; Cerebral Cortex; Corpus Striatum; Dark Adaptation; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Excitatory Amino Acid Antagonists; Exploratory Behavior; Female; Interleukin 1 Receptor Antagonist Protein; Interleukin-1beta; Mice; Mice, Inbred C57BL; Mice, Knockout; Myelin-Oligodendrocyte Glycoprotein; Peptide Fragments; Receptor, Cannabinoid, CB1; Signal Transduction

Though several functional properties of laquinimod have been identified, our understanding of the underlying mechanisms is still incomplete. Since the compound elicits similar immunomodulatory effects to ligands of the aryl hydrocarbon receptor (AhR), we compared the efficacy of laquinimod in experimental autoimmune encephalomyelitis (EAE)-afflicted wild-type and AhR-deficient mice. Laquinimod failed to ameliorate clinical symptoms and leukocyte infiltration in AhR-deficient mice; however, treatment exerted neuroprotection by elevation of brain-derived neurotrophic factor (BDNF) independent of genetic profile. Thus, our data identify the AhR pathway in these mutant mice as crucial for the immunomodulatory, but not neuroprotective, efficacy of laquinimod in EAE.

Topics: Analysis of Variance; Animals; Axons; Brain-Derived Neurotrophic Factor; CD3 Complex; Demyelinating Autoimmune Diseases, CNS; Disease Models, Animal; Gene Expression Regulation; Immunologic Factors; Leukemic Infiltration; Mice, Inbred C57BL; Mice, Knockout; Myelin-Oligodendrocyte Glycoprotein; Peptide Fragments; Quinolones; Rats; Receptors, Aryl Hydrocarbon; RNA, Messenger; Spinal Cord; T-Lymphocytes; White Matter

Experimental autoimmune encephalomyelitis (EAE) is a common rodent model for multiple sclerosis (MS). Yet, the long-term consequences for retina and optic nerve (ON) are unknown. C57BL/6 mice were immunized with an encephalitogenic peptide (MOG35-55) and the controls received the carriers or PBS. Clinical symptoms started at day 8, peaked at day 14, and were prevalent until day 60. They correlated with infiltration and demyelination of the ON. In MOG-immunized animals more microglia cells in the ONs and retinas were detected at day 60. Additionally, retinal ganglion cell (RGC) loss was combined with an increased macroglia response. At this late stage, an increased number of microglia was associated with axonal damage in the ON and in the retina with RGC loss. Whether glial activation contributes to repair mechanisms or adversely affects the number of RGCs is currently unclear.

Topics: Analysis of Variance; Animals; Axons; Calcium-Binding Proteins; Central Nervous System Stimulants; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Freund's Adjuvant; Glial Fibrillary Acidic Protein; Mice; Mice, Inbred C57BL; Microfilament Proteins; Microglia; Myelin-Oligodendrocyte Glycoprotein; Optic Nerve; Peptide Fragments; Picrotoxin; Protein Kinase C-alpha; Retina; Transcription Factor Brn-3A; Vimentin

Amyloid hexapeptide molecules are effective in the treatment of the murine model of neuroinflammation, known as experimental autoimmune encephalomyelitis (EAE). Efficacy however differs between two inbred mouse strains, C57BL/6J (B6) and C57BL/10SnJ (B10). Amyloid hexapeptide treatments improved the clinical outcomes of B6, but not B10 mice, indicating that genetic background influences therapeutic efficacy. Moreover, although previous studies indicated that prion protein deficiency results in more severe EAE in B6 mice, we observed no such effect in B10 mice. In addition, we found that amyloid hexapeptide treatments of B10 and B6 mice elicited differential IL4 responses. Thus, the modulatory potential of prion protein and related treatments with other amyloid hexapeptides in EAE depends on mouse strain.

Topics: Amyloid beta-Peptides; Animals; Anti-Inflammatory Agents; Cells, Cultured; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Eosinophils; Female; Gene Expression Regulation; Genetic Background; Interleukin-4; Lymphocytes; Mice; Mice, Inbred Strains; Mice, Transgenic; Monocytes; Myelin-Oligodendrocyte Glycoprotein; Peptide Fragments; Prion Proteins; Species Specificity; tau Proteins

Since ancient times, Capparis species have been widely used in traditional medicine to treat various diseases. Our recent investigations have suggested Capparis ovata's potential anti-neuroinflammatory application for the treatment of multiple sclerosis (MS). The present study was designed to precisely determine the underlying mechanism of its anti-neuroinflammatory effect in a mouse model of MS. C. ovata water extract (COWE) was prepared using the plant's fruit, buds, and flower parts (Turkish Patent Institute, PT 2012/04,093). We immunized female C57BL/6J mice with MOG35-55/CFA. COWE was administered at a daily dose of 500mg/kg by oral gavage either from the day of immunization (T1) or at disease onset (T2) for 21days. Gene expression analysis was performed using a Mouse Multiple Sclerosis RT² Profiler PCR Array, and further determinations and validations of the identified genes were performed using qPCR. Whole-genome transcriptome profiling was analyzed using Agilent SurePrint G3 Mouse GE 8X60K microarrays. Immunohistochemical staining was applied to brain sections of the control and treated mice to examine the degree of degeneration. COWE was further fractionated and analyzed phytochemically using the Zivak Tandem Gold Triple Quadrupole LC/MS-MS system. COWE remarkably suppressed the development of EAE in T1, and the disease activity was completely inhibited. In the T2 group, the maximal score was significantly reduced compared with that of the parallel EAE group. The COWE suppression of EAE was associated with a significantly decreased expression of genes that are important in inflammatory signaling, such as TNFα, IL6, NF-κB, CCL5, CXCL9, and CXCK10. On the other hand, the expression of genes involved in myelination/remyelination was significantly increased. Immunohistochemical analysis further supported these effects, showing that the number of infiltrating immune cells was decreased in the brains of COWE-treated animals. In addition, differential expression profiling of the transcriptome revealed that COWE treatment caused the down regulation of a group of genes involved in the immune response, inflammatory response, antigen processing and presentation, B-cell-mediated immunity and innate immune response. Collectively, these results suggest anti-neuroinflammatory mechanisms by which COWE treatment delayed and suppressed the development of EAE and ameliorated the disease in mice with persistent clinical signs.

Topics: Analysis of Variance; Animals; Anti-Inflammatory Agents; Brain; Capparis; Cytokines; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Female; Gene Expression Profiling; Gene Expression Regulation; Mice; Mice, Inbred C57BL; Myelin Proteins; Myelin-Oligodendrocyte Glycoprotein; Peptide Fragments; Phytotherapy; Plant Extracts; Signal Transduction

The mechanism of leukocyte transendothelial migration (TEM) across the highly restrictive blood-brain barrier (BBB) remains enigmatic, with paracellular TEM thought to require leukocytes to somehow navigate the obstructive endothelial tight junctions (TJs). Transient interactions between TJ proteins on the respective leukocyte and endothelial surfaces have been proposed as one mechanism for TEM. Given the expanding role of extracellular vesicles (EVs) in intercellular communication, we investigated whether EVs derived from brain microvascular endothelial cells (BMEC) of the BBB may play a role in transferring a major TJ protein, claudin-5 (CLN-5), to leukocytes as a possible basis for such a mechanism during neuroinflammation.. High-resolution 3D confocal imaging was used to highlight CLN-5 immunoreactivity in the central nervous system (CNS) and on leukocytes of mice with the neuroinflammatory condition experimental autoimmune encephalomyelitis (EAE). Both Western blotting of circulating leukocytes from wild-type mice and fluorescence imaging of leukocyte-associated eGFP-CLN-5 in the blood and CNS of endothelial-targeted, Tie-2-eGFP-CLN-5 transgenic mice were used to confirm the presence of CLN-5 protein on these cells. EVs were isolated from TNF-α-stimulated BMEC cultures and blood plasma of Tie-2-eGFP-CLN-5 mice with EAE and evaluated for CLN-5 protein by Western blotting and fluorescence-activated cell sorting (FACS), respectively. Confocal imaging and FACS were used to detect binding of endothelial-derived EVs from these two sources to leukocytes in vitro. Serial electron microscopy (serial EM) and 3D contour-based surface reconstruction were employed to view EV-like structures at the leukocyte:BBB interface in situ in inflamed CNS microvessels.. A subpopulation of leukocytes immunoreactive for CLN-5 on their surface was seen to infiltrate the CNS of mice with EAE and reside in close apposition to inflamed vessels. Confocal imaging of immunostained samples and Western blotting established the presence of CLN-5. During neuroinflammation, CLN-5

Topics: Animals; Cells, Cultured; Central Nervous System; Cytokines; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Endothelial Cells; Endothelium, Vascular; Extracellular Vesicles; Female; Leukocytes; Lysosomal Membrane Proteins; Membrane Glycoproteins; Mice; Mice, Inbred C57BL; Mice, Transgenic; Myelin-Oligodendrocyte Glycoprotein; Peptide Fragments

Multiple sclerosis (MS) is a chronic CNS autoimmune disease characterized by inflammation, demyelination, and neuronal degeneration, where myelin-specific CD4 T cells play critical roles in the formation of acute MS lesions and disease progression. The suppression of IL-7Rα expression and the upregulation of inhibitory receptors (PD-1, etc.) are essential parts of the cell-intrinsic immunosuppressive program regulating T effector functions to prevent autoimmunity. However, little is known on the factors regulating IL-7Rα/PD-1 balance in myelin-specific CD4 T effector/memory cells during the development of CNS autoimmunity.. We analyzed the roles of the transcription factor T-bet in regulating the expression of IL-7Rα and inhibitory receptors in myelin-specific CD4 T cells. Furthermore, we compared the effects of different inflammatory cytokines that are crucial for Th1 and Th17 development in regulating the IL-7Rα/PD-1 balance.. We discovered that T-bet suppresses the expression of inhibitory receptors (PD-1 and LAG-3) and promotes IL-7Rα expression in myelin-specific CD4 T cells in vitro and in vivo. As a result, T-bet skews IL-7Rα/PD-1 balance towards IL-7Rα and promotes enhanced effector function. Furthermore, IL-12 enhances IL-7Rα expression in a T-bet independent manner in myelin-specific Th1 cells. Meanwhile, IL-6, the cytokine inducing highly encephalitogenic Th17 differentiation, suppresses PD-1 while upregulating IL-7Rα, skewing IL-7Rα/PD-1 balance towards IL-7Rα, and promoting enhanced effector function. Moreover, blocking IL-7 signaling in myelin-specific CD4 T cells by αIL-7Rα significantly delays experimental autoimmune encephalomyelitis (EAE) onset and reduces disease severity.. T-bet is a major transcription factor regulating IL-7Rα/PD-1 balance in myelin-specific CD4 T cells during EAE development, and there is a positive correlation between several major determinants promoting T cell encephalitogenicity (T-bet, IL-6, IL-12) and an IL-7Rα/PD-1 balance skewed towards IL-7Rα. Furthermore, IL-7 signaling inhibits PD-1 expression in myelin-specific CD4 T cells and blocking IL-7 signaling suppresses T cell encephalitogenicity. Therefore, interference with inhibitory pathways and IL-7Rα expression may suppress the encephalitogenic potential of myelin-specific CD4 T cells and have therapeutic benefits for MS patients.

Topics: Animals; CD4-Positive T-Lymphocytes; Central Nervous System; Cytokines; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Gene Expression Regulation; Mice; Mice, Transgenic; Myelin-Oligodendrocyte Glycoprotein; Nerve Tissue Proteins; Peptide Fragments; Programmed Cell Death 1 Receptor; Receptors, Cell Surface; Receptors, Interleukin-17; Th1 Cells

Antigen presenting cells (APC) are critical for regulating immune responses. We tested mannan-peptide conjugates for targeting myelin peptides to APC to induce T cell tolerance and resistance to experimental autoimmune encephalomyelitis (EAE). Myelin peptides conjugated to mannan in oxidized (OM) or reduced (RM) forms protected mice against EAE in prophylactic and therapeutic protocols, with OM-conjugated peptides giving best results. Protection was peptide-specific and associated with reduced antigen-specific T cell proliferation, but not alterations in Th1, Th17 and Treg cell differentiation or T cell apoptosis compared to EAE controls. Bone marrow-derived dendritic cells (DC) loaded with OM-MOG showed up-regulated expression of co-stimulatory molecules, reduced PD-L1 expression and enhanced CD40-inducible IL-12 and IL-23 production compared to MOG DC, features consistent with immunogenic DC. OM-MOG induced active T cell tolerance because i.d. administration or passive transfer of OM-MOG DC suppressed ongoing EAE, while OM-MOG-vaccinated mice did not reduce the proliferation of transferred MOG-specific T cells. As in vivo, MOG-specific T cells cultured with OM-MOG DC showed reduced proliferation and equal Th1 and Th17 cell differentiation compared to those with MOG DC, but surprisingly cytokine production was unresponsive to CD40 engagement. Impaired effector T cell function was further evidenced in spinal cord sections from OM-MOG-vaccinated EAE mice, where markedly reduced numbers of CD3(+) T cells were present, restricted to leptomeninges and exceptional parenchymal lesions. Our results show that mannan-conjugated myelin peptides protect mice against EAE through the expansion of antigen-specific Th1 and Th17 cells with impaired proliferation responses and APC-induced co-stimulatory signals that are required for licensing them to become fully pathogenic T cells.

Topics: Animals; Apoptosis; Cell Differentiation; Cell Movement; Cell Proliferation; Cytokines; Dendritic Cells; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Ki-67 Antigen; Lymphocyte Activation; Mannans; Mice; Mice, Inbred C57BL; Mice, Transgenic; Myelin Basic Protein; Myelin-Oligodendrocyte Glycoprotein; Peptide Fragments; Peptides; Th1 Cells; Th17 Cells; Time Factors

Inflammatory signals present in demyelinated multiple sclerosis lesions affect the reparative remyelination process conducted by oligodendrocyte progenitor cells (OPCs). Interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), and interleukin (IL)-6 have differing effects on the viability and growth of OPCs, however the effects of IL-17A are largely unknown. Primary murine OPCs were stimulated with IL-17A and their viability, proliferation, and maturation were assessed in culture. IL-17A-stimulated OPCs exited the cell cycle and differentiated with no loss in viability. Expression of the myelin-specific protein, proteolipid protein, increased in a cerebellar slice culture assay in the presence of IL-17A. Downstream, IL-17A activated ERK1/2 within 15 min and induced chemokine expression in 2 days. These results demonstrate that IL-17A exposure stimulates OPCs to mature and participate in the inflammatory response.

Topics: Animals; Cell Differentiation; Cells, Cultured; Cerebellum; Cytokines; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Flow Cytometry; Freund's Adjuvant; Gene Expression Regulation; Green Fluorescent Proteins; In Vitro Techniques; MAP Kinase Signaling System; Mice; Mice, Inbred C57BL; Mice, Transgenic; Myelin Proteolipid Protein; Myelin-Oligodendrocyte Glycoprotein; Oligodendroglia; Organ Culture Techniques; Peptide Fragments; Receptors, Interleukin-17; Stem Cells

Recently, parasite infections or parasite-derived products have been suggested as a therapeutic strategy with suppression of immunopathology, which involves the induction of regulatory T cells or/and T helper type 2 (Th2) responses. In a recent study, researchers reported that constructed recombinant galectin (rTl-gal) isolated from an adult worm of the gastrointestinal nematode parasite Toxascaris leonina attenuated clinical symptoms of inflammatory bowel disease in mice treated with dextran sulphate sodium. Noting the role of rTl-gal in inflammatory disease, we attempted to investigate the effect of the parasite via its rTl-gal on neuronal autoimmune disease using experimental autoimmune encephalomyelitis (EAE), a mouse inflammatory and demyelinating autoimmune disease model of human multiple sclerosis. In this model, rTl-gal-treated experimental autoimmune encephalomyelitis (EAE) mice failed to recover after the peak of the disease, leading to persistent central nervous system (CNS) damage, such as demyelination, gliosis and axonal damage. Further, rTl-gal-treated EAE mice markedly increased the number of CD45R/B220(+) B cells in both infiltrated inflammation and the periphery, along with the increased production of autoantibody [anti-myelin oligodendrocyte glycoprotein (MOG)35-55 ] in serum at chronic stage. Upon antigen restimulation, rTl-gal treatment affected the release of overall cytokines, especially interferon (IFN)-γ and tumour necrosis factor (TNF)-α. Our results suggest that galectin isolated from a gastrointestinal parasite can deliver a harmful effect to EAE contrary to its beneficial effect on inflammatory bowel disease.

Topics: Animals; Autoantibodies; Axons; B-Lymphocyte Subsets; Cytokines; Demyelinating Diseases; Disease Models, Animal; Disease Progression; Encephalomyelitis, Autoimmune, Experimental; Female; Galectins; Gliosis; Immunomodulation; Leukocyte Common Antigens; Mice; Myelin-Oligodendrocyte Glycoprotein; Parasites; Peptide Fragments; Severity of Illness Index; Spinal Cord

Iron accumulation occurs in the CNS in multiple sclerosis (MS) and in experimental autoimmune encephalomyelitis (EAE). However, the mechanisms underlying such iron accumulation are not fully understood. We studied the expression and cellular localization of molecules involved in cellular iron influx, storage, and efflux. This was assessed in two mouse models of EAE: relapsing-remitting (RR-EAE) and chronic (CH-EAE). The expression of molecules involved in iron homeostasis was assessed at the onset, peak, remission/progressive and late stages of the disease. We provide several lines of evidence for iron accumulation in the EAE spinal cord which increases with disease progression and duration, is worse in CH-EAE, and is localized in macrophages and microglia. We also provide evidence that there is a disruption of the iron efflux mechanism in macrophages/microglia that underlie the iron accumulation seen in these cells. Macrophages/microglia also lack expression of the ferroxidases (ceruloplasmin and hephaestin) which have antioxidant effects. In contrast, astrocytes which do not accumulate iron, show robust expression of several iron influx and efflux proteins and the ferroxidase ceruloplasmin which detoxifies ferrous iron. Astrocytes therefore are capable of efficiently recycling iron from sites of EAE lesions likely into the circulation. We also provide evidence of marked dysregulation of mitochondrial function and energy metabolism genes, as well as of NADPH oxidase genes in the EAE spinal cord. This data provides the basis for the selective iron accumulation in macrophage/microglia and further evidence of severe mitochondrial dysfunction in EAE. It may provide insights into processes underling iron accumulation in MS and other neurodegenerative diseases in which iron accumulation occurs.

Topics: Animals; Cation Transport Proteins; CD11b Antigen; Ceruloplasmin; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Female; Ferritins; Freund's Adjuvant; Glial Fibrillary Acidic Protein; Hepcidins; Iron; Iron Metabolism Disorders; Membrane Proteins; Mice; Mice, Inbred C57BL; Mitochondria; Myelin-Oligodendrocyte Glycoprotein; Nerve Tissue Proteins; Peptide Fragments; Receptors, Transferrin; Spinal Cord; Time Factors

Multiple sclerosis (MS) has been associated with a history of sub-optimal exposure to ultraviolet light, implicating vitamin D3 as a possible protective agent. We evaluated whether 1,25(OH)2D3 attenuates the progression of experimental autoimmune encephalomyelitis (EAE), and explored its potential mechanisms. EAE was induced in C57BL/6 mice via immunization with MOG35-55, and some mice received 1,25(OH)2D3. 1,25(OH)2D3 inhibited EAE progression. Additionally, 1,25(OH)2D3 reduced inflammation, demyelination, and neuron loss in the spinal cord. The protective effect of 1,25(OH)2D3 was associated with significantly elevated expression of Beclin1, increased Bcl-2/Bax ratio, and decreased LC3-II accumulation. Thus, 1,25(OH)2D3 may represent a promising new MS treatment.

Topics: Animals; Apoptosis Regulatory Proteins; Autophagy; bcl-2-Associated X Protein; Beclin-1; Calcitriol; Calcium Channel Agonists; Cyclin D1; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Extremities; Female; Gene Expression Regulation; Mice; Microtubule-Associated Proteins; Myelin-Oligodendrocyte Glycoprotein; Neurodegenerative Diseases; Peptide Fragments; Phosphopyruvate Hydratase; Spinal Cord

High mobility group box 1 (HMGB1), a nonhistone chromatin associated protein, plays different roles according to the expression pattern such as the amount, cell location and sub-cellular location. It has been recently demonstrated that the systemic HMGB1 is associated with autoimmune encephalomyelitis. However, the dynamic change of HMGB1 expression pattern in spinal cords that may be involved in the progression of disease is not fully understood. In this study, the amount, cell location and subcellular location of HMGB1 in adult mice spinal cords during various stages of experimental autoimmune encephalomyelitis (EAE) are investigated. HMGB1 is expressed in the nuclei of spinal cord resident cells such as some astrocytes, microglia and a few neurons in normal situation. During EAE progression, the total and extracellular HMGB1 in the spinal cord are increased, more HMGB1 positive astrocytes and microglia are observed, and the intra-neurons HMGB1 in the ventral horn and around the central canal localize majorly in the cytoplasm accompanied by the increasing extracellular HMGB1. Blockade of HMGB1 in central nervous system (CNS) locally attenuates the severity of EAE significantly. Our findings indicate that the HMGB1 expression pattern in the spinal cord is associated with the progression of EAE. HMGB1 may be a potential target for autoimmune encephalomyelitis (multiple sclerosis in human) therapy.

Topics: Animals; Antibodies; Astrocytes; CD11b Antigen; Disease Models, Animal; Disease Progression; Female; Freund's Adjuvant; Gene Expression Profiling; Gene Expression Regulation; Glial Fibrillary Acidic Protein; HMGB1 Protein; Mice; Mice, Inbred C57BL; Multiple Sclerosis; Myelin-Oligodendrocyte Glycoprotein; Peptide Fragments; Pertussis Toxin; Phosphopyruvate Hydratase; Spinal Cord; Time Factors

Previous work has demonstrated that the hormone prolactin promotes oligodendrocyte precursor proliferation and remyelination following lysolecithin-induced demyelination of the mouse spinal cord. Prolactin, however, can elicit pro-inflammatory responses, and its use in the prototypical demyelinating and inflammatory condition, multiple sclerosis (MS), should thus be approached cautiously. Here, we sought to determine whether recombinant prolactin could alter the course of experimental autoimmune encephalomyelitis (EAE), an inflammatory animal model of MS. Consistent with previous literature, we found that prolactin activated leukocytes in vitro. Daily treatment with prolactin from around the time of onset of clinical signs, for 9 (days 9 to 17) or 25 (days 9 to 33) days did not increase clinical or histological signs of EAE over that of vehicle-treated mice. Instead, the combination of prolactin and a suboptimal dose of recombinant murine interferon-β resulted in (days 9 to 17 group) or trended towards (days 9 to 33 group), a greater amelioration of clinical signs of EAE, compared to either treatment alone or to vehicle controls. Histological analyses corroborated the clinical EAE data. These results suggest that prolactin may be beneficial when administered in combination with interferon-β in MS.

Topics: Animals; Antigen-Presenting Cells; Cell Proliferation; Disease Models, Animal; Drug Therapy, Combination; Female; Immunologic Factors; Interferon-beta; Mice; Mice, Inbred C57BL; Multiple Sclerosis; Myelin-Oligodendrocyte Glycoprotein; Peptide Fragments; Prolactin; Spinal Cord; Time Factors

Myo9b regulates leukocyte migration by controlling RhoA signaling. Here we assessed its role in active experimental autoimmune encephalomyelitis (EAE). Myo9b(-/-) mice show a delay in the onset of EAE symptoms. The delay in disease onset was accompanied by reduced numbers of Th1 and Th17 cells in the CNS. Myo9b(-/-) mice showed no recovery from disease symptoms and exhibited elevated numbers of both Th17 cells and CD11b+ macrophages. Bone marrow chimeric mice demonstrated that the absence of a leukocyte source of Myo9b was responsible for the delayed leukocyte infiltration into the CNS, delayed EAE onset and lack of recovery.

Topics: Animals; Antigens, CD; Cell Proliferation; Central Nervous System; Cytokines; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Flow Cytometry; Gene Expression Regulation; Image Cytometry; Leukocyte Common Antigens; Leukocytes; Mice; Mice, Inbred C57BL; Mice, Transgenic; Myelin-Oligodendrocyte Glycoprotein; Myosins; Peptide Fragments; Recovery of Function; Time Factors

Exogenous transplanted neural precursor cells (NPCs) exhibit miscellaneous immune-modulatory effects in models of autoimmune demyelination. However, the regional interactions of NPCs with the host brain tissue in remissive inflammatory events have not been adequately studied. In this study we used the chronic MOG-induced Experimental Autoimmune Encephalomyelitis (EAE) model in C57BL/six mice. Based on previous data, we focused on neuropathology at Day 50 post-induction (D50) and studied the expression of connexin43 (Cx43) and Cx47, two of the main glial gap junction (GJ) proteins, in relation to the intraventricular transplantation of GFP(+) NPCs and their integration with the host tissue. By D50, NPCs had migrated intraparenchymally and were found in the corpus callosum at the level of the lateral ventricles and hippocampus. The majority of GFP(+) cells differentiated with simple or ramified processes expressing mainly markers of mature GLIA (GFAP and NogoA) and significantly less of precursor glial cells. GFP(+) NPCs expressed connexins and formed GJs around the hippocampus more than lateral ventricles. The presence of NPCs did not alter the increase in Cx43 GJ plaques at D50 EAE, but prevented the reduction of oligodendrocytic Cx47, increased the number of oligodendrocytes, local Cx47 levels and Cx47 GJ plaques per cell. These findings suggest that transplanted NPCs may have multiple effects in demyelinating pathology, including differentiation and direct integration into the panglial syncytium, as well as amelioration of oligodendrocyte GJ loss, increasing the supply of potent myelinating cells to the demyelinated tissue.

Topics: Age Factors; Animals; Brain; Cell Differentiation; Connexin 43; Connexins; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Female; Gene Expression Regulation; Mice; Mice, Inbred C57BL; Mice, Transgenic; Microscopy, Electron, Transmission; Myelin Basic Protein; Myelin-Oligodendrocyte Glycoprotein; Nerve Tissue Proteins; Neural Stem Cells; Neuroglia; Peptide Fragments

Foxp3(+) regulatory T cell (Treg)-based immunotherapy holds promise for autoimmune diseases. However, this effort has been hampered by major caveats, including the low frequency of autoantigen-specific Foxp3(+) Tregs and lack of understanding of their molecular and cellular targets, in an unmanipulated wild-type (WT) immune repertoire. In this study, we demonstrate that infusion of myelin in WT mice results in the de novo induction of myelin-specific Foxp3(+) Tregs in WT mice and amelioration of experimental autoimmune encephalomyelitis. Myelin-specific Foxp3(+) Tregs exerted their effect both by diminishing Ag-bearing inflammatory dendritic cell (iDC) recruitment to lymph nodes and by impairing their function. Transcriptome analysis of ex vivo-isolated Treg-exposed iDCs showed significant enrichment of transcripts involved in functional properties of iDCs, including chemotaxis-related genes. To this end, CCR7 expression by iDCs was significantly downregulated in tolerant mice and this was tightly regulated by the presence of IL-10. Collectively, our data demonstrate a novel model for deciphering the Ag-specific Foxp3(+) Treg-mediated mechanisms of tolerance and delineate iDCs as a Foxp3(+) Treg cellular target in unmanipulated mice.

Topics: Animals; Autoantigens; Autoimmunity; Chemotaxis; Cluster Analysis; Dendritic Cells; Disease Models, Animal; Disease Progression; Encephalomyelitis, Autoimmune, Experimental; Female; Forkhead Transcription Factors; Gene Expression Profiling; Immune Tolerance; Inflammation; Interleukin-10; Lymph Nodes; Lymphocyte Depletion; Mice; Mice, Knockout; Myelin-Oligodendrocyte Glycoprotein; Peptide Fragments; Receptors, CCR7; Signal Transduction; T-Cell Antigen Receptor Specificity; T-Lymphocytes, Regulatory

MicroRNAs (miRNAs) associate with Argonaute (Ago), GW182, and FXR1 proteins to form RNA-induced silencing complexes (RISCs). RISCs represent a critical checkpoint in the regulation and bioavailability of miRNAs. Recent studies have revealed dysregulation of miRNAs in multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE); however, the function of RISCs in EAE and MS is largely unknown. Here, we examined the expression of Ago, GW182, and FXR1 in CNS tissue, oligodendrocytes (OLs), brain-infiltrating T lymphocytes, and CD3(+)splenocytes (SCs) of EAE mic, and found that global RISC protein levels were significantly dysregulated. Specifically, Ago2 and FXR1 levels were decreased in OLs and brain-infiltrating T cells in EAE mice. Accordingly, assembly of Ago2/GW182/FXR1 complexes in EAE brain tissues was disrupted, as confirmed by immunoprecipitation experiments. In parallel with alterations in RISC complex content in OLs, we found downregulation of miRNAs essential for differentiation and survival of OLs and myelin synthesis. In brain-infiltrating T lymphocytes, aberrant RISC formation contributed to miRNA-dependent proinflammatory helper T-cell polarization. In CD3(+) SCs, we found increased expression of both Ago2 and FXR1 in EAE compared with nonimmunized mice. Therefore, our results demonstrate a gradient in expression of miRNA between primary activated T cells in the periphery and polarized CNS-infiltrating T cells. These results suggest that, in polarized autoreactive effector T cells, miRNA synthesis is inhibited in response to dysregulated RISC assembly, allowing these cells to maintain a highly specific proinflammatory program. Therefore, our findings may provide a mechanism that leads to miRNA dysregulation in EAE/MS.

Topics: Animals; Argonaute Proteins; Autoantigens; Carboxypeptidases; CD11b Antigen; Central Nervous System; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Female; Gene Expression Regulation; Glial Fibrillary Acidic Protein; Leukocyte Common Antigens; Mice; Mice, Inbred C57BL; MicroRNAs; Myelin-Oligodendrocyte Glycoprotein; Neurons; Peptide Fragments; Receptors, Antigen, T-Cell, alpha-beta; RNA-Binding Proteins; RNA, Small Nuclear

To evaluate the role of NADPH oxidase-mediated reactive oxygen species (ROS) production in multiple sclerosis pathogenesis, we examined the effects of apocynin, an NADPH oxidase assembly inhibitor, on experimental autoimmune encephalomyelitis (EAE).. EAE was induced by immunization with myelin oligodendrocyte glycoprotein (MOG (35-55)) in C57BL/6 female mice. Three weeks after initial immunization, the mice were analyzed for demyelination, immune cell infiltration, and ROS production. Apocynin (30 mg/kg) was given orally once daily for the entire experimental course or after the typical onset of clinical symptom (15 days after first MOG injection).. Clinical signs of EAE first appeared on day 11 and reached a peak level on day 19 after the initial immunization. The daily clinical symptoms of EAE mice were profoundly reduced by apocynin. The apocynin-mediated inhibition of the clinical course of EAE was accompanied by suppression of demyelination, reduced infiltration by encephalitogenic immune cells including CD4, CD8, CD20, and F4/80-positive cells. Apocynin reduced MOG-induced pro-inflammatory cytokines in cultured microglia. Apocynin also remarkably inhibited EAE-associated ROS production and blood-brain barrier (BBB) disruption. Furthermore, the present study found that post-treatment with apocynin also reduced the clinical course of EAE and spinal cord demyelination.. These results demonstrate that apocynin inhibits the clinical features and neuropathological changes associated with EAE. Therefore, the present study suggests that inhibition of NADPH oxidase activation by apocynin may have a high therapeutic potential for treatment of multiple sclerosis pathogenesis.

Topics: Acetophenones; Animals; Animals, Newborn; Blood-Testis Barrier; Brain; Cells, Cultured; Cytokines; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Enzyme Inhibitors; Female; Leukoencephalopathies; Male; Mice; Mice, Inbred C57BL; Myelin-Oligodendrocyte Glycoprotein; NADPH Oxidases; Peptide Fragments; Reactive Oxygen Species; Severity of Illness Index; Spinal Cord

Total glucosides of peony (TGP), an active compound extracted from the roots of Paeonia lactiflora Pall, has wide pharmacological effects on nervous system. Here we examined the effects of TGP on experimental autoimmune encephalomyelitis (EAE), an established model of multiple sclerosis (MS). The results showed that TGP can reduce the severity and progression of EAE in C57 BL/6 mice. In addition, TGP also down-regulated the Th1/Th17 inflammatory response and prevented the reduced expression of brain-derived neurotrophic factor and 2',3'-cyclic nucleotide 3'-phosphodiesterase of EAE. These findings suggest that TGP could be a potential therapeutic agent for MS.

Topics: Analysis of Variance; Animals; Brain-Derived Neurotrophic Factor; Cytokines; Disease Models, Animal; Dose-Response Relationship, Drug; Dose-Response Relationship, Immunologic; Encephalomyelitis, Autoimmune, Experimental; Enzyme-Linked Immunosorbent Assay; Female; Freund's Adjuvant; Glucosides; Mice; Mice, Inbred C57BL; Myelin-Oligodendrocyte Glycoprotein; Paeonia; Peptide Fragments; Pertussis Toxin; Phytotherapy; RNA, Messenger; Spinal Cord; Transcription Factors

Multiple sclerosis (MS) is a demyelinating disease of the CNS characterized by inflammation and neurodegeneration. Animal models that enable the study of remyelination in the context of ongoing inflammation are greatly needed for the development of novel therapies that target the pathological inhibitory cues inherent to the MS plaque microenvironment. We report the development of an innovative animal model combining cuprizone-mediated demyelination with transfer of myelin-reactive CD4(+) T cells. Characterization of this model reveals both Th1 and Th17 CD4(+) T cells infiltrate the CNS of cuprizone-fed mice, with infiltration of Th17 cells being more efficient. Infiltration correlates with impaired spontaneous remyelination as evidenced by myelin protein expression, immunostaining, and ultrastructural analysis. Electron microscopic analysis further reveals that demyelinated axons are preserved but reduced in caliber. Examination of the immune response contributing to impaired remyelination highlights a role for peripheral monocytes with an M1 phenotype. This study demonstrates the development of a novel animal model that recapitulates elements of the microenvironment of the MS plaque and reveals an important role for T cells and peripheral monocytes in impairing endogenous remyelination in vivo. This model could be useful for testing putative MS therapies designed to enhance remyelination in the setting of active inflammation, and may also facilitate modeling the pathophysiology of denuded axons, which has been a challenge in rodents because they typically remyelinate very quickly.

Topics: Adoptive Transfer; Animals; Cell Polarity; Cells, Cultured; Central Nervous System; Cuprizone; Demyelinating Diseases; Disease Models, Animal; Freund's Adjuvant; Interleukin-17; Leukocyte Common Antigens; Mice; Mice, Inbred C57BL; Mice, Transgenic; Monoamine Oxidase Inhibitors; Monocytes; Myelin Proteins; Myelin Sheath; Myelin-Oligodendrocyte Glycoprotein; Neutrophil Infiltration; Peptide Fragments; Regeneration; Th17 Cells; Time Factors

DRα1-mouse(m)MOG-35-55, a novel construct developed in our laboratory as a simpler and potentially less immunogenic alternative to two-domain class II constructs, was shown previously to target the MIF/CD74 pathway and to reverse clinical and histological signs of experimental autoimmune encephalomyelitis (EAE) in DR*1501-Tg mice in a manner similar to the parent DR2β1-containing construct.. In order to determine whether DRα1-mMOG-35-55 could treat EAE in major histocompatibility complex (MHC)-mismatched mice and to evaluate the treatment effect on central nervous system (CNS) inflammation, C57BL/6 mice were treated with DRα1-mMOG-35-55. In addition, gene expression profile was analyzed in spinal cords of EAE DR*1501-Tg mice that were treated with DRα1-mMOG-35-55.. We here demonstrate that DRα1-mMOG-35-55 could effectively treat EAE in MHC-mismatched C57BL/6 mice by reducing CNS inflammation, potentially mediated in part through an increased frequency of M2 monocytes in the spinal cord. Microarray analysis of spinal cord tissue from DRα1-mMOG-35-55-treated vs. vehicle control mice with EAE revealed decreased expression of a large number of pro-inflammatory genes including CD74, NLRP3, and IL-1β and increased expression of genes involved in myelin repair (MBP) and neuroregeneration (HUWE1).. These findings indicate that the DRα1-mMOG-35-55 construct retains therapeutic, anti-inflammatory, and neuroprotective activities during treatment of EAE across MHC disparate barriers.

Topics: Animals; CD11 Antigens; Cell Count; Cell Survival; Central Nervous System Diseases; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; HLA-DR alpha-Chains; Inflammation; Lectins, C-Type; Macrophages; Male; Mannose Receptor; Mannose-Binding Lectins; Mice; Mice, Inbred C57BL; Myelin-Oligodendrocyte Glycoprotein; Nerve Regeneration; Neuroprotection; Neuroprotective Agents; Peptide Fragments; Receptors, Cell Surface

In experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis, mice genetically deficient in the transcription factor signal transducer and activator of transcription 4 (STAT4) are resistant to disease. In contrast, deletion or inhibition of the Th1-associated cytokines IL-12 or IFNγ which act upstream and downstream of STAT4, respectively, does not ameliorate disease. These discordant findings imply that STAT4 may act in a non-canonical role during EAE. Recently, STAT4 has been shown to regulate GM-CSF production by CD4 T cells and this cytokine is necessary for the induction of EAE. However, it is not known if STAT4 controls GM-CSF production by both Th1 and Th17 effector CD4 T cells.. This study utilized the MOG(35-55) peptide immunization model of EAE. Intracellular cytokine staining and novel mixed bone marrow chimeric mice were used to study the CD4 T cell-intrinsic role of STAT4 during disease. STAT4 chromatin-immunoprecipitation (ChIP-PCR) experiments were performed to show STAT4 directly interacts with the Csf2 gene loci.. Herein, we demonstrate that STAT4 controls CD4 T cell-intrinsic GM-CSF production by both Th1 and Th17 CD4 T cells during EAE as well as in vitro. Importantly, we show that STAT4 interacts with the Csf2 locus in MOG(35-55)-activated effector CD4 T cells demonstrating direct modulation of GM-CSF.. Overall, these studies illustrate a previously unrecognized role of STAT4 to regulate GM-CSF production by not only Th1 cells, but also Th17 effector CD4 T cell subsets during EAE pathogenesis. Critically, these data highlight for the first time that STAT4 is able to modulate the effector profile of Th17 CD4 T cell subsets, which redefines our current understanding of STAT4 as a Th1-centric factor.

Topics: Animals; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Female; Granulocyte-Macrophage Colony-Stimulating Factor; Homeodomain Proteins; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Multiple Sclerosis; Myelin-Oligodendrocyte Glycoprotein; Peptide Fragments; STAT4 Transcription Factor; Th1 Cells; Th17 Cells

Astrocytes, the most abundant cell population in mammal central nervous system (CNS), contribute to a variety of functions including homeostasis, metabolism, synapse formation, and myelin maintenance. White matter (WM) reactive astrocytes are important players in amplifying autoimmune demyelination and may exhibit different changes in transcriptome profiles and cell function in a disease-context dependent manner. However, their transcriptomic profile has not yet been defined because they are difficult to purify, compared to gray matter astrocytes. Here, we isolated WM astrocytes by laser capture microdissection (LCM) in a murine model of multiple sclerosis to better define their molecular profile focusing on selected genes related to inflammation. Based on previous data indicating anti-inflammatory effects of estrogen only at high nanomolar doses, we also examined mRNA expression for enzymes involved in steroid inactivation.. Experimental autoimmune encephalomyelitis (EAE) was induced in female C57BL6 mice with MOG35-55 immunization. Fluorescence activated cell sorting (FACS) analysis of a portion of individual spinal cords at peak disease was used to assess the composition of immune cell infiltrates. Using custom Taqman low-density-array (TLDA), we analyzed mRNA expression of 40 selected genes from immuno-labeled laser-microdissected WM astrocytes from lumbar spinal cord sections of EAE and control mice. Immunohistochemistry and double immunofluorescence on control and EAE mouse spinal cord sections were used to confirm protein expression in astrocytes.. The spinal cords of EAE mice were infiltrated mostly by effector/memory T CD4+ cells and macrophages. TLDA-based profiling of LCM-astrocytes identified EAE-induced gene expression of cytokines and chemokines as well as inflammatory mediators recently described in gray matter reactive astrocytes in other murine CNS disease models. Strikingly, SULT1A1, but not other members of the sulfotransferase family, was expressed in WM spinal cord astrocytes. Moreover, its expression was further increased in EAE. Immunohistochemistry on spinal cord tissues confirmed preferential expression of this enzyme in WM astrocytic processes but not in gray matter astrocytes.. We described here for the first time the mRNA expression of several genes in WM astrocytes in a mouse model of multiple sclerosis. Besides expected pro-inflammatory chemokines and specific inflammatory mediators increased during EAE, we evidenced relative high astrocytic expression of the cytoplasmic enzyme SULT1A1. As the sulfonation activity of SULT1A1 inactivates estradiol among other phenolic substrates, its high astrocytic expression may account for the relative resistance of this cell population to the anti-neuroinflammatory effects of estradiol. Blocking the activity of this enzyme during neuroinflammation may thus help the injured CNS to maintain the anti-inflammatory activity of endogenous estrogens or limit the dose of estrogen co-regimens for therapeutical purposes.

Topics: Animals; Arylsulfotransferase; Astrocytes; Biomarkers; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Female; Gene Expression Profiling; Glucuronidase; Laser Capture Microdissection; Mice; Mice, Inbred C57BL; Multiple Sclerosis; Myelin-Oligodendrocyte Glycoprotein; Peptide Fragments; RNA, Messenger; White Matter

Multiple sclerosis (MS) is a neurodegenerative disease resulting from an autoimmune attack on the axon-myelin unit. A female MS bias becomes evident after puberty and female incidence has tripled in the last half-century, implicating a female sex hormone interacting with a modifiable environmental factor. These aspects of MS suggest that many female MS cases may be preventable. Mechanistic knowledge of this hormone-environment interaction is needed to devise strategies to reduce female MS risk. We previously demonstrated that vitamin D3 (D3) deficiency increases and D3 supplementation decreases experimental autoimmune encephalomyelitis (EAE) risk in a female-biased manner. We also showed that D3 acts in an estrogen (E2)-dependent manner, since ovariectomy eliminated and E2 restored D3-mediated EAE protection. Here we probed the hypothesis that E2 and D3 interact synergistically within CD4(+) T cells to control T cell fate and prevent demyelinating disease. The E2 increased EAE resistance in wild-type (WT) but not T-Vdr(0) mice lacking Vdr gene function in CD4(+) T cells, so E2 action depended entirely on Vdr(+)CD4(+) T cells. The E2 levels were higher in WT than T-Vdr(0) mice, suggesting the Vdr(+)CD4(+) T cells produced E2 or stimulated its production. The E2 decreased Cyp24a1 and increased Vdr transcripts in T cells, prolonging the calcitriol half-life and increasing calcitriol responsiveness. The E2 also increased CD4(+)Helios(+)FoxP3(+) T regulatory (Treg) cells in a Vdr-dependent manner. Thus, CD4(+) T cells have a cooperative amplification loop involving E2 and calcitriol that promotes CD4(+)Helios(+)FoxP3(+) Treg cell development and is disrupted when the D3 pathway is impaired. The global decline in population D3 status may be undermining a similar cooperative E2-D3 interaction controlling Treg cell differentiation in women, causing a breakdown in T cell self tolerance and a rise in MS incidence.

Topics: Animals; Autoimmune Diseases of the Nervous System; CD4-Positive T-Lymphocytes; Disease Models, Animal; DNA-Binding Proteins; Drug Synergism; Estrogens; Female; Forkhead Transcription Factors; Gene Expression Regulation, Developmental; In Vitro Techniques; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Myelin-Oligodendrocyte Glycoprotein; Ovariectomy; Peptide Fragments; Pregnancy; Receptors, Calcitriol; T-Lymphocytes, Regulatory; Time Factors; Transcription Factors; Uterus; Vitamin D

Multiple sclerosis (MS) is an autoimmune inflammatory and neurodegenerative disease of the central nervous system (CNS). It is widely accepted that inflammatory cells play major roles in the pathogenesis of MS, possibly through the use of serine protease granzyme B (GrB) secreted from the granules of cytotoxic T cells. We have previously identified GrB as a mediator of axonal injury and neuronal death. In this study, our goal was to evaluate the effect of GrB inhibition in the human system in vitro, and in vivo in EAE using the newly isolated GrB-inhibitor serpina3n.. We used a well-established in vitro model of neuroinflammation characterized by a co-culture system between human fetal neurons and lymphocytes. In vivo, we induced EAE in 10- to 12-week-old female C57/BL6 mice and treated them intravenously with serpina3n.. In the in vitro co-culture system, pre-treatment of lymphocytes with serpina3n prevented neuronal killing and cleavage of the cytoskeletal protein alpha-tubulin, a known substrate for GrB. Moreover, in EAE, 50 μg serpina3n substantially reduced the severity of the disease. This dose was administered intravenously twice at days 7 and 20 post EAE induction. serpina3n treatment reduced axonal and neuronal injury compared to the vehicle-treated control group and maintained the integrity of myelin. Interestingly, serpina3n treatment did not seem to reduce the infiltration of immune cells (CD4(+) and CD8(+) T cells) into the CNS.. Our data suggest further studies on serpina3n as a potentially novel therapeutic strategy for the treatment of inflammatory-mediated neurodegenerative diseases such as MS.

Topics: Acute-Phase Proteins; Animals; Antigens, CD; Cells, Cultured; Coculture Techniques; Disease Models, Animal; Dose-Response Relationship, Drug; Encephalomyelitis, Autoimmune, Experimental; Female; Fetus; Freund's Adjuvant; Humans; Mice; Mice, Inbred C57BL; Myelin Sheath; Myelin-Oligodendrocyte Glycoprotein; Neurofilament Proteins; Neurons; Neuroprotective Agents; Peptide Fragments; Serpins; T-Lymphocytes; Tubulin

Proteoglycans (PGs) are the components of extracellular matrices in the central nervous system (CNS). Keratan sulfate (KS) is a glycosaminoglycan that is included in the KSPG that acts as an inhibitory factor in nerve regeneration after CNS injury. To investigate the role of KS in immune diseases, we induced experimental autoimmune encephalomyelitis (EAE) in mice that were deficient in the N-acetylglucosamine (GlcNAc)-6-O-sulfotransferase 1 (GlcNAc6ST1) gene (KS-KO). KS-KO mice developed less severe EAE and showed repressed recall response in the induction phase. Furthermore, GlcNAc6ST1 might have roles in the passage of the pathogenic lymphocytes through the blood-brain barrier via adhesion molecules. Thus, modulation of KS may become a treatment for neuroimmunological diseases.

Topics: Adoptive Transfer; Animals; Carbohydrate Sulfotransferases; Cell Proliferation; Cytokines; Disease Models, Animal; Down-Regulation; Encephalomyelitis, Autoimmune, Experimental; Freund's Adjuvant; Keratan Sulfate; Mice; Mice, Inbred C57BL; Mice, Knockout; Myelin-Oligodendrocyte Glycoprotein; Peptide Fragments; Sulfotransferases; T-Lymphocytes

Experimental autoimmune encephalomyelitis (EAE) is a model of inflammatory demyelinating diseases mediated by different types of leukocytes. How these cells communicate with each other to orchestrate autoimmune attacks is not fully understood, especially in the case of neutrophils, whose importance in EAE is newly established. The present study aimed to determine the expression pattern and role of different components of the IL-36 signaling pathway (IL-36α, IL-36β, IL-36γ, IL-36R) in EAE.. EAE was induced by either active immunization with myelin peptide, passive transfer of myelin-reactive T cells or injection of pertussis toxin to transgenic 2D2 mice. The molecules of interest were analyzed using a combination of techniques, including quantitative real-time PCR (qRT-PCR), flow cytometry, Western blotting, in situ hybridization, and immunohistochemistry. Microglial cultures were treated with recombinant IL-36γ and analyzed using DNA microarrays. Different mouse strains were subjected to clinical evaluation and flow cytometric analysis in order to compare their susceptibility to EAE.. Our observations indicate that both IL-36γ and IL-36R are strongly upregulated in nervous and hematopoietic tissues in different forms of EAE. IL-36γ is specifically expressed by neutrophils, while IL-36R is expressed by different immune cells, including microglia and other myeloid cells. In culture, microglia respond to recombinant IL-36γ by expressing molecules involved in neutrophil recruitment, such as Csf3, IL-1β, and Cxcl2. However, mice deficient in either IL-36γ or IL-36R develop similar clinical and histopathological signs of EAE compared to wild-type controls.. This study identifies IL-36γ as a neutrophil-related cytokine that can potentially activate microglia, but that is only correlative and not contributory in EAE.

Topics: Adoptive Transfer; Animals; Animals, Newborn; Antigens, CD; Brain; Cells, Cultured; Central Nervous System; Cytokines; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Interleukin-1; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Microglia; Myelin-Oligodendrocyte Glycoprotein; Neutrophils; Peptide Fragments; Receptors, Interleukin-1; Signal Transduction

The family of 14-3-3 proteins plays an important role in the regulation of cell survival and death. Here, we investigate the role of the 14-3-3 gamma (14-3-3 γ) subunit for glial responses in autoimmune demyelination.. Expression of 14-3-3 γ in glial cell culture was investigated by reverse transcription polymerase chain reaction (RT-PCR) and immunocytochemistry. 14-3-3 γ knockout mice were subjected to murine myelin oligodendrocyte-induced experimental autoimmune encephalomyelitis (MOG-EAE), an animal model mimicking inflammatory features and neurodegenerative aspects of multiple sclerosis (MS).. Expression studies in cell culture confined expression of 14-3-3 γ to both, oligodendrocytes (OL) and astrocytes. RT-PCR analysis revealed an increased expression of 14-3-3 γ mRNA in the spinal cord during the late chronic phase of MOG-EAE. At that stage, EAE was more severe in 14-3-3 γ knockout mice as compared to age- and gender-matched controls. Histopathological analyses on day 56 post immunization (p.i.) revealed significantly enhanced myelin damage as well as OL injury and secondary, an increase in axonal injury and gliosis in 14-3-3 γ -/- mice. At the same time, deficiency in 14-3-3 γ protein did not influence the immune response. Further histological studies revealed an increased susceptibility towards apoptosis in 14-3-3 γ-deficient OL in the inflamed spinal cord.. These data argue for a pivotal role of 14-3-3 γ-mediated signalling pathways for OL protection in neuroinflammation.

Topics: 14-3-3 Proteins; Animals; Apoptosis; Axons; Cells, Cultured; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Glial Fibrillary Acidic Protein; Mice; Mice, Inbred C57BL; Mice, Knockout; Myelin Proteins; Myelin-Oligodendrocyte Glycoprotein; Neuroglia; Nogo Proteins; Oligodendroglia; Peptide Fragments; Phosphopyruvate Hydratase; RNA, Messenger; Time Factors

The aim of the present study was to investigate effect of two different ages (6 weeks [6 W] vs. 6 months [6 M]) on blood-brain barrier (BBB) disruption in EAE and evaluate the expression and correlations of NADPH oxidase, MMP-9, ICAM-1, and VCAM-1 following increased age and EAE induction. Higher disease severity was observed in 6 M-EAE than 6 W-EAE. The four factors were significantly elevated and correlated in 6 M-EAE. BBB permeability increased with statistically significant interaction between age and EAE effects. We suggest strong correlations between NADPH oxidase and the other factors play important roles in increased BBB disruption and EAE susceptibility in middle-aged mice.

Topics: Aging; Animals; Blood-Brain Barrier; Body Weight; Cell Adhesion Molecules; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Female; Freund's Adjuvant; Male; Matrix Metalloproteinase 9; Mice; Mice, Inbred C57BL; Mycobacterium tuberculosis; Myelin-Oligodendrocyte Glycoprotein; NADPH Oxidases; Peptide Fragments; Permeability; Pertussis Toxin; Time Factors

Cortical lesions are a crucial part of MS pathology and it is critical to determine that new MS therapies have the ability to alter cortical inflammatory lesions given the differences between white and gray matter lesions. We tested lipoic acid (LA) in a mouse focal cortical EAE model. Brain sections were stained with antibodies against CD4, CD11b and galectin-3. Compared with vehicle, treatment with LA significantly decreased CD4+ and galectin-3+ immune cells in the brain. LA treated mice had fewer galectin-3+ cells with no projections indicating decrease in the number of infiltrating monocytes. LA significantly reduces inflammation in a focal cortical model of MS.

Topics: Animals; Antioxidants; CD11b Antigen; Cerebral Cortex; Cytokines; Disease Models, Animal; Encephalitis; Encephalomyelitis, Autoimmune, Experimental; Female; Galectin 3; Mice; Mice, Inbred C57BL; Myelin-Oligodendrocyte Glycoprotein; Peptide Fragments; Statistics, Nonparametric; Thioctic Acid; Time Factors

Myelinating cells synthesize large amounts of membrane protein through the secretory pathway, which makes these cells particularly sensitive to perturbations of the endoplasmic reticulum (ER). Ig binding protein (BiP), also known as glucose-regulated protein 78 (GRP78), is a critical ER chaperone that also plays a pivotal role in controlling the cellular response to ER stress. To examine the potential importance of BiP to myelinating cells, we used a conditional knock-out approach to BiP gene inactivation in oligodendrocytes during development, in adulthood, and in response to experimental autoimmune encephalomyelitis (EAE), an animal model of the inflammatory demyelinating disorder multiple sclerosis (MS). During development, mice lacking functional BiP gene expression in oligodendrocytes developed tremors and ataxia and died before reaching maturity. When BiP gene inactivation in oligodendrocytes was initiated in adulthood, the mice displayed severe neurological symptoms including tremors and hind-limb paralysis. The inactivation of BiP in oligodendrocytes during development or in adulthood resulted in oligodendrocyte loss and corresponding severe myelin abnormalities. Mice heterozygous for the oligodendrocyte-specific inactivation of BiP, which were phenotypically normal without evidence of neuropathology, displayed an exacerbated response to EAE that correlated with an increased loss of oligodendrocytes. Furthermore, mice in which the BiP gene was specifically inactivated in developing Schwann cells displayed tremor that progressed to hindlimb paralysis, which correlated with diminished numbers of myelinating Schwann cells and severe PNS hypomyelination. These studies demonstrate that BiP is critical for myelinating cell survival and contributes to the protective response of oligodendrocyte against inflammatory demyelination.

Topics: 2',3'-Cyclic Nucleotide 3'-Phosphodiesterase; Animals; Animals, Newborn; Cell Survival; Cytokines; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Endoplasmic Reticulum; Endoplasmic Reticulum Chaperone BiP; Female; Freund's Adjuvant; Gene Expression Regulation, Developmental; Mice; Mice, Inbred C57BL; Myelin Proteins; Myelin Sheath; Myelin-Oligodendrocyte Glycoprotein; Oligodendroglia; Oligopeptides; Peptide Fragments; Peripheral Nerves; Transcription Factor CHOP

F1 (SJL/J×C57BL/6) mice with MOG35-55-induced EAE recover from disease when treated with Ig-MOG carrying MOG35-55 peptide. However, Ig-PLP1, carrying PLP139-151, induced reduction of anti-MOG antibodies and exacerbated EAE. Herein, we show that Ig-PLP1 specifically reduces the frequency of B cells producing protective IgG2a/b anti-MOG antibodies. Surprisingly, these cells were marginal zone (MZ), rather than follicular (FO) or newly formed (NF), B cells and transfer of MZ B cells into sick mice nullified disease exacerbation by Ig-PLP1 in a complement dependent manner. These findings reveal a potential self-limiting regulatory mechanism involving auto-antibodies in MOG EAE.

Topics: Animals; Antibodies; B-Lymphocytes; Cytokines; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Enzyme-Linked Immunosorbent Assay; Female; Flow Cytometry; Immune Tolerance; Lymphocyte Activation; Mice; Mice, Inbred C57BL; Myelin-Oligodendrocyte Glycoprotein; Peptide Fragments; Statistics, Nonparametric; T-Lymphocytes

Astrocytes respond to insult with a process of cellular activation known as reactive astrogliosis. One of the key signals regulating this phenomenon is the transcription factor nuclear factor-kappa B (NF-κB), which is responsible for modulating inflammation, cell survival, and cell death. In astrocytes, following trauma or disease, the expression of NF-κB-dependent genes is highly activated. We previously demonstrated that inactivation of astroglial NF-κB in vivo (GFAP-IκBα-dn mice) leads to improved functional outcome in experimental autoimmune encephalomyelitis (EAE), and this is accompanied by reduction of pro-inflammatory gene expression in the CNS. Here we extend our studies to show that recovery from EAE in GFAP-IκBα-dn mice is associated with reduction of peripheral immune cell infiltration into the CNS at the chronic phase of EAE. This is not dependent on a less permeable blood-brain barrier, but rather on a reduced immune cell mobilization from the periphery. Furthermore, once inside the CNS, the ability of T cells to produce pro-inflammatory cytokines is diminished during acute disease. In parallel, we found that the number of total and activated microglial cells is reduced, suggesting that functional improvement in GFAP-IκBα-dn mice is dependent upon reduction of the overall inflammatory response within the CNS sustained by both resident and infiltrating cells. This results in preservation of myelin compaction and enhanced remyelination, as shown by electron microscopy analysis of the spinal cord. Collectively our data indicate that astrocytes are key players in driving CNS inflammation and are directly implicated in the pathophysiology of EAE, since blocking their pro-inflammatory capability results in protection from the disease.

Topics: Animals; Astrocytes; Central Nervous System; Claudin-5; Cytokines; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Gene Expression Regulation; Glial Fibrillary Acidic Protein; I-kappa B Proteins; Immunoglobulin G; Inflammation; Leukocytes; Mice; Mice, Transgenic; Microscopy, Electron; Myelin Sheath; Myelin-Oligodendrocyte Glycoprotein; NF-KappaB Inhibitor alpha; Peptide Fragments; Spinal Cord; T-Lymphocytes

Down-regulation of soluble or membrane-bound co-stimulatory molecules by RNAi in dendritic cells can prevent the activation of immune responses. Therefore, this study was designed to evaluate the therapeutic efficacy of bone marrow-derived DCs (BMDCs) transduced with lentiviral vectors to permanently expressed shRNA specific for CD40 (CD40LV-DCs) and/or p19 subunit of interleukin (IL)-23 (p19LV-DCs) mRNAs in experimental autoimmune encephalomyelitis (EAE). In-vitro studies showed that double-transduced BMDCs (CD40(+) p19LV-DCs) resemble tolerogenic DCs due to profound down-regulation of CD40, lower expression of proinflammatory cytokines (IL-6 and IL-12), increased IL-10 production and stronger stimulation of myelin oligodendrocyte glycoprotein (MOG)35-55 -specific T cells for production of IL-10 compared with CD40LV-DCs, p19LV-DCs and BMDCs transduced with control lentiviral vector (CoLV-DCs). Moreover, injection of transduced CD40(+) p19LV- BMDCs in EAE mice resulted in more reduction in clinical score, significant reduction in IL-17 or increased production of IL-10 by mononuclear cells derived from the lymph nodes or spinal cord compared with CoLV-DCs-treated EAE mice. In conclusion, simultaneous knock-down of CD40 and IL-23 production by BMDCs may represent a promising therapeutic tool for the treatment of IL-17-dependent autoimmune diseases, including multiple sclerosis.

Topics: Animals; CD4-Positive T-Lymphocytes; CD40 Antigens; Cell Proliferation; Coculture Techniques; Cytokines; Dendritic Cells; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Flow Cytometry; Humans; Interleukin-10; Interleukin-17; Interleukin-23 Subunit p19; Lentivirus; Mice; Mice, Inbred C57BL; Multiple Sclerosis; Myelin-Oligodendrocyte Glycoprotein; Peptide Fragments; RNA Interference; RNA, Small Interfering; Th17 Cells; Treatment Outcome

Multiple sclerosis (MS) is a chronic demyelinating immune mediated disease of the central nervous system. The immunoproteasome is a distinct class of proteasomes found predominantly in monocytes and lymphocytes. Recently, we demonstrated a novel function of immunoproteasomes in cytokine production and T cell differentiation. In this study, we investigated the therapeutic efficacy of an inhibitor of the immunoproteasome (ONX 0914) in two different mouse models of MS. ONX 0914 attenuated disease progression after active and passive induction of experimental autoimmune encephalomyelitis (EAE), both in MOG₃₅-₅₅ and PLP₁₃₉₋₁₅₁-induced EAE. Isolation of lymphocytes from the brain or spinal cord revealed a strong reduction of cytokine-producing CD4(+) cells in ONX 0914 treated mice. Additionally, ONX 0914 treatment prevented disease exacerbation in a relapsing-remitting model. An analysis of draining lymph nodes after induction of EAE revealed that the differentiation to Th17 or Th1 cells was strongly impaired in ONX 0914 treated mice. These results implicate the immunoproteasome in the development of EAE and suggest that immunoproteasome inhibitors are promising drugs for the treatment of MS.

Topics: Animals; Brain; Cell Differentiation; Disease Models, Animal; Disease Progression; Encephalomyelitis, Autoimmune, Experimental; Female; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Inflammation; Mice; Mice, Inbred C57BL; Myelin-Oligodendrocyte Glycoprotein; Oligopeptides; Peptide Fragments; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Spinal Cord; T-Lymphocytes

Expression of chemokine CCL2 in the normal central nervous system (CNS) is nearly undetectable, but is significantly upregulated and drives neuroinflammation during experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis which is considered a contributing factor in the human disease. As astrocytes and brain microvascular endothelial cells (BMEC) forming the blood-brain barrier (BBB) are sources of CCL2 in EAE and other neuroinflammatory conditions, it is unclear if one or both CCL2 pools are critical to disease and by what mechanism(s).. Mice with selective CCL2 gene knockout (KO) in astrocytes (Astro KO) or endothelial cells (Endo KO) were used to evaluate the respective contributions of these sources to neuroinflammation, i.e., clinical disease progression, BBB damage, and parenchymal leukocyte invasion in a myelin oligodendrocyte glycoprotein peptide (MOG35-55)-induced EAE model. High-resolution 3-dimensional (3D) immunofluorescence confocal microscopy and colloidal gold immuno-electron microscopy were employed to confirm sites of CCL2 expression, and 3D immunofluorescence confocal microscopy utilized to assess inflammatory responses along the CNS microvasculature.. Cell-selective loss of CCL2 immunoreactivity was demonstrated in the respective KO mice. Compared to wild-type (WT) mice, Astro KO mice showed reduced EAE severity but similar onset, while Endo KO mice displayed near normal severity but significantly delayed onset. Neither of the KO mice showed deficits in T cell proliferation, or IL-17 and IFN-γ production, following MOG35-55 exposure in vitro, or altered MOG-major histocompatibility complex class II tetramer binding. 3D confocal imaging further revealed distinct actions of the two CCL2 pools in the CNS. Astro KOs lacked the CNS leukocyte penetration and disrupted immunostaining of CLN-5 at the BBB seen during early EAE in WT mice, while Endo KOs uniquely displayed leukocytes stalled in the microvascular lumen.. These results point to astrocyte and endothelial pools of CCL2 each regulating different stages of neuroinflammation in EAE, and carry implications for drug delivery in neuroinflammatory disease.

Topics: Animals; Astrocytes; Central Nervous System; Chemokine CCL2; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Endothelium; Imaging, Three-Dimensional; Mice; Mice, Knockout; Microscopy, Confocal; Microvessels; Myelin-Oligodendrocyte Glycoprotein; Peptide Fragments

Pertussis toxin (PTx) has various effects in experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS). This study was designed to explore the protective effects of PTx of different doses and subunits. EAE model was induced with myelin oligodendrocyte glycoprotein (MOG35-55, 200 ug) plus complete Freund's adjuvant in 6-7 week-old female C57BL/6 mice. PTx reduced clinical deficits of EAE by 91.3%. This reduction in clinical deficits was achieved by attenuating demyelination by 75.5%. Furthermore, PTx reduced the lymphocyte infiltration, deactivated microglia activation and changed T cell profile by increasing T helper (type 1 and 2) and T regulatory cells.

Topics: Analysis of Variance; Animals; Antigen Presentation; Calcium-Binding Proteins; Cell Proliferation; Cytokines; Disease Models, Animal; Dose-Response Relationship, Drug; Encephalomyelitis, Autoimmune, Experimental; Flow Cytometry; Forkhead Transcription Factors; Mice; Mice, Inbred C57BL; Microfilament Proteins; Microglia; Myelin-Oligodendrocyte Glycoprotein; Neurologic Examination; Peptide Fragments; Pertussis Toxin; Spleen; T-Lymphocytes; Time Factors

Cinnabarinic acid (CA) is an endogenous metabolite of the kynurenine pathway which acts as an orthosteric agonist of type-4 metabotropic glutamate receptor (mGlu4). We now report that systemic administration of CA (0.1-10 mg/kg, i.p.) was highly protective against experimental autoimmune encephalomyelitis (EAE) induced by the myelin oligodendrocyte glycoprotein (MOG35-55) peptide, which models multiple sclerosis in mice. Full protection against EAE required daily injections of CA since the time of immunization, similarly to what reported for the mGlu4 enhancer N-phenyl-7-(hydroxyimino)cyclopropa[b]chromen-1acarboxamide (PHCCC). CA treatment boosted an immune response dominated by regulatory T (Treg) cells at the expenses of Th17 cells. In addition, exogenous CA enhanced endogenous CA formation in lymphocytes, suggesting the occurrence of a positive feedback loop sustaining immune tolerance. To examine whether activation of mGlu4 could account for the protective activity of CA against EAE, we used mGlu4 knockout mice. As expected, these mice displayed a more severe form of EAE in response to immunization. CA was still protective against EAE in mGlu4-deficient mice, although its action was significantly reduced both at high and low CA doses. This suggests that the action of CA against neuroinflammation involves multiple mechanisms including the activation of mGlu4. These data further suggest that CA is one possible bridge between activation of the kynurenine pathway and immune tolerance aimed at restraining neuroinflammation.

Topics: Animals; Benzopyrans; Central Nervous System; Cytokines; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Excitatory Amino Acid Agonists; Male; Mice; Mice, Inbred C57BL; Myelin Basic Protein; Myelin-Oligodendrocyte Glycoprotein; Myosin Type II; Nuclear Receptor Subfamily 1, Group F, Member 3; Oxazines; Peptide Fragments; Receptors, Metabotropic Glutamate; T-Box Domain Proteins; Th17 Cells; Time Factors; Transcription Factors

We provide evidence of cortical neuronopathy in myelin oligodendrocyte glycoprotein peptide-induced experimental autoimmune encephalomyelitis, an established model of chronic multiple sclerosis. To investigate phenotypic perturbations in neurons in this model, we used apoptotic markers and immunohistochemistry with antibodies to NeuN and other surrogate markers known to be expressed by adult pyramidal Layer V somas, including annexin V, encephalopsin, and Emx1. We found no consistent evidence of chronic loss of Layer V neurons but detected both reversible and chronic decreases in the expression of these markers in conjunction with evidence of cortical demyelination and presynaptic loss. These phenotypic perturbations were present in, but not restricted to, the neocortical Layer V. We also investigated inflammatory responses in the cortex and subcortical white matter of the corpus callosum and spinal dorsal funiculus and found that those in the cortex and corpus callosum were delayed compared with those in the spinal cord. Inflammatory infiltrates initially included T cells, neutrophils, and Iba1-positive microglia/macrophages in the corpus callosum, whereas only Iba1-positive cells were present in the cortex. These data indicate that we have identified a new temporal pattern of subtle phenotypic perturbations in neocortical neurons in this chronic multiple sclerosis model.

Topics: Animals; Caspase 3; Cell Death; Disease Models, Animal; Encephalitis; Freund's Adjuvant; Humans; In Situ Nick-End Labeling; Male; Mice; Mice, Inbred C57BL; Motor Neurons; Multiple Sclerosis; Myelin Basic Protein; Myelin-Oligodendrocyte Glycoprotein; Neocortex; Peptide Fragments; Phosphopyruvate Hydratase; Synaptophysin; Time Factors

Recent data suggest that oxidative injury may play an important role in demyelination and neurodegeneration in multiple sclerosis (MS). We compared the extent of oxidative injury in MS lesions with that in experimental models driven by different inflammatory mechanisms. It was only in a model of coronavirus-induced demyelinating encephalomyelitis that we detected an accumulation of oxidised phospholipids, which was comparable in extent to that in MS. In both, MS and coronavirus-induced encephalomyelitis, this was associated with massive microglial and macrophage activation, accompanied by the expression of the NADPH oxidase subunit p22phox but only sparse expression of inducible nitric oxide synthase (iNOS). Acute and chronic CD4(+) T cell-mediated experimental autoimmune encephalomyelitis lesions showed transient expression of p22phox and iNOS associated with inflammation. Macrophages in chronic lesions of antibody-mediated demyelinating encephalomyelitis showed lysosomal activity but very little p22phox or iNOS expressions. Active inflammatory demyelinating lesions induced by CD8(+) T cells or by innate immunity showed macrophage and microglial activation together with the expression of p22phox, but low or absent iNOS reactivity. We corroborated the differences between acute CD4(+) T cell-mediated experimental autoimmune encephalomyelitis and acute MS lesions via gene expression studies. Furthermore, age-dependent iron accumulation and lesion-associated iron liberation, as occurring in the human brain, were only minor in rodent brains. Our study shows that oxidative injury and its triggering mechanisms diverge in different models of rodent central nervous system inflammation. The amplification of oxidative injury, which has been suggested in MS, is only reflected to a limited degree in the studied rodent models.

Topics: Aging; Animals; CD4 Antigens; CD8 Antigens; Coronavirus Infections; Cuprizone; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Gene Expression; Iron; Lipopolysaccharides; Macrophages; Mice, Inbred C57BL; Microglia; Multiple Sclerosis; Myelin-Oligodendrocyte Glycoprotein; Oxidative Stress; Peptide Fragments; Rats; Rats, Inbred Lew; Respiratory Burst; T-Lymphocytes

Multiple sclerosis (MS) is a chronic demyelinating disease often displaying a relapsing-remitting course of neurological manifestations that is mimicked by experimental autoimmune encephalomyelitis (EAE) in animal models of MS. In particular, NOD mice immunized with myelin oligodendrocyte glycoprotein peptide 35-55 develop chronic relapsing-remitting EAE (CREAE). To elucidate the mechanisms that cause MS relapse, we investigated the histopathology and cytokine production of spleen cells and mRNA expression levels in the central nervous system (CNS) of CREAE mice. During the first attack, inflammatory cell infiltration around small vessels and in the subarachnoid space was observed in the spinal cord. Spleen cell production and mRNA expression in the CNS of several cytokines, including IFN-γ, TNF-α, IL-6, IL-17, and CC chemokine ligand 2 (CCL2), were higher in CREAE mice than in controls. Afterwards, parenchymal infiltration and demyelination were observed histologically in the spinal cord and corresponded with the more severe clinical symptoms of the first and second relapses. IL-17 and CCL2, but not IFN-γ, TNF-α, or IL-6, were also produced by spleen cells during recurrences. Our results suggested that the immune mechanisms in relapses were different from those in the first attack for CREAE. Further investigation of CREAE mechanisms may provide important insights into successful therapies for human relapsing-remitting MS.

Topics: Animals; Cytokines; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Female; Freund's Adjuvant; Mice; Myelin-Oligodendrocyte Glycoprotein; Peptide Fragments; RNA, Messenger; Severity of Illness Index; Spinal Cord; Time Factors

Axon degeneration has been identified as a major contributor to non-remitting neurological deficits in patients with multiple sclerosis (MS), which has elicited substantial interest in the development of neuroprotective therapies. Sodium channel blockers, including phenytoin, carbamazepine, flecainide and lamotrigine, have been shown to protect axons from degeneration, attenuate immune cell infiltrates and slow the acquisition of neurological deficits in mice with experimental autoimmune encephalomyelitis (EAE), a model of MS. However, the sudden withdrawal of sodium channel blockers, phenytoin and carbamazepine, is associated with severe exacerbation of EAE characterized by massive inflammatory infiltrates and high mortality. In the present study, we asked whether a slow, tapered withdrawal of phenytoin treatment from mice with EAE produced sudden worsening similar to that of sudden withdrawal. Our results demonstrate that gradual withdrawal of phenytoin treatment from mice with EAE is associated with worsening of clinical scores which approach non-treated levels, but was not associated with increased immune cell infiltrates or deaths as have been observed with abrupt withdrawal. These observations support sodium channel blockers as a potential therapeutic agent in the treatment of MS, but indicate caution if treatment is ceased.

Topics: Animals; Disease Models, Animal; Drug Administration Schedule; Encephalomyelitis, Autoimmune, Experimental; Mice; Mice, Inbred C57BL; Myelin-Oligodendrocyte Glycoprotein; Neurologic Examination; Neuroprotective Agents; Peptide Fragments; Phenytoin; Time Factors

Despite recent advances in development of treatments for multiple sclerosis, there is still an unmet need for more effective and also safe therapies. Based on the modes of action of interferon-beta (IFN-β) and dimethyl fumarate (DMF), we hypothesized that anti-inflammatory and neuroprotective effects may synergize in experimental autoimmune encephalomyelitis (EAE).. EAE was induced in C57BL/6 mice by immunization with MOG35-55-peptide. Murine IFN-β was injected s.c. every other day at 10.000IU, and DMF was provided at 15mg/kg by oral gavage twice daily. Control mice received PBS injections and were treated by oral gavage with the vehicle methylcellulose. Mice were scored daily by blinded observers and histological, FACS and cytokine studies were performed to further elucidate the underlying mechanism of action.. Combination therapy significantly ameliorated EAE disease course in comparison to controls and monotherapy with IFN-β. Histological analyses showed a significant effect on axon preservation with almost twice as much axons present in inflamed lesions as compared to control. Remarkably, the effect on axonal preservation was more pronounced under combination therapy than with both monotherapies. Neither monotherapy nor combination therapy demonstrated modulation of cytokines and frequency of antigen presenting cells.. Combination of IFN-β and DMF resulted in greater beneficial effects with improved tissue protection as compared to the respective monotherapies. Further combination studies of these safe therapies in human disease are warranted.

Topics: 2',3'-Cyclic-Nucleotide Phosphodiesterases; Animals; Antigens, Differentiation; Axons; CD3 Complex; Cell Proliferation; Dimethyl Fumarate; Disease Models, Animal; Drug Synergism; Encephalomyelitis, Autoimmune, Experimental; Enzyme-Linked Immunosorbent Assay; Fumarates; Immunologic Factors; Interferon-beta; Lymphocytes; Mice; Mice, Inbred C57BL; Myelin-Oligodendrocyte Glycoprotein; Neurofilament Proteins; Peptide Fragments; Time Factors

Although multiple sclerosis (MS) has been associated with the coagulation system, the temporal and spatial regulation of coagulation activity in neuroinflammatory lesions is unknown. Using a novel molecular probe, we characterized the activity pattern of thrombin, the central protease of the coagulation cascade, in experimental autoimmune encephalomyelitis. Thrombin activity preceded onset of neurological signs, increased at disease peak, and correlated with fibrin deposition, microglial activation, demyelination, axonal damage, and clinical severity. Mice with a genetic deficit in prothrombin confirmed the specificity of the thrombin probe. Thrombin activity might be exploited for developing sensitive probes for preclinical detection and monitoring of neuroinflammation and MS progression.

Topics: Animals; Axons; Blood Coagulation Factors; Connexin 30; Connexins; Demyelinating Diseases; Disease Models, Animal; Disease Progression; Encephalomyelitis, Autoimmune, Experimental; Fibrin; Green Fluorescent Proteins; Mice; Mice, Inbred C57BL; Mice, Transgenic; Myelin Basic Protein; Myelin-Oligodendrocyte Glycoprotein; Peptide Fragments; Poly I-C; Thrombin

Calcitonin Gene-Related Peptide (CGRP) inhibits microglia inflammatory activation in vitro. We here analyzed the involvement of CGRP and Receptor Component Protein (RCP) in experimental autoimmune encephalomyelitis (EAE). Alpha-CGRP deficiency increased EAE scores which followed the scale alpha-CGRP null>heterozygote>wild type. In wild type mice, CGRP delivery into the cerebrospinal fluid (CSF) 1) reduced chronic EAE (C-EAE) signs, 2) inhibited microglia activation (revealed by quantitative shape analysis), and 3) did not alter GFAP expression, cell density, lymphocyte infiltration, and peripheral lymphocyte production of IFN-gamma, TNF-alpha, IL-17, IL-2, and IL-4. RCP (probe for receptor involvement) was expressed in white matter microglia, astrocytes, oligodendrocytes, and vascular-endothelial cells: in EAE, also in infiltrating lymphocytes. In relapsing-remitting EAE (R-EAE) RCP increased during relapse, without correlation with lymphocyte density. RCP nuclear localization (stimulated by CGRP in vitro) was I) increased in microglia and decreased in astrocytes (R-EAE), and II) increased in microglia by CGRP CSF delivery (C-EAE). Calcitonin like receptor was rarely localized in nuclei of control and relapse mice. CGRP increased in motoneurons. In conclusion, CGRP can inhibit microglia activation in vivo in EAE. CGRP and its receptor may represent novel protective factors in EAE, apparently acting through the differential cell-specific intracellular translocation of RCP.

Topics: Adrenomedullin; Animals; Calcitonin Gene-Related Peptide; Cells, Cultured; Cytokines; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Enzyme Inhibitors; Freund's Adjuvant; Gene Expression Regulation; Lymphocytes; Mice; Mice, Inbred C57BL; Mice, Knockout; Microglia; Myelin-Oligodendrocyte Glycoprotein; Nerve Tissue Proteins; Peptide Fragments; Receptors, Calcitonin Gene-Related Peptide

In multiple sclerosis (MS), inflammation leads to damage of central nervous system myelin and axons. Previous studies have postulated impaired GABA transmission in MS, and recent postmortem analysis has shown that GABAergic parvalbumin (PV)-positive interneurons are decreased in the primary motor cortex (M1) of patients with MS. In this report, we present evidence for the loss of a specific population of GABAergic interneurons in the experimental autoimmune encephalomyelitis mouse model of MS. Using experimental autoimmune encephalomyelitis, we evaluated the distribution of both PV-positive interneurons and of the inhibitory presynaptic input in the M1 of experimental autoimmune encephalomyelitis and control mice. Our results demonstrate a specific decrease in the number of PV-positive interneurons in the M1 of mice with experimental autoimmune encephalomyelitis. We detected a significant reduction in the number of PV-positive interneurons in the layers II and III of the M1 of diseased mice, while there was no difference in the number of calretinin (CR)-positive cells between animals with experimental autoimmune encephalomyelitis and control animals. Moreover, we observed a significant reduction in the inhibitory presynaptic input in the M1 of treated mice. These changes were specific for the mice with elevated clinical score, while they were not detectable in the mice with low clinical score. Our results support the hypothesis that reinforcing the action of the GABAergic network may represent a therapeutic alternative to limit the progression of the neuronal damage in MS patients.

Topics: Animals; Apoptosis; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Female; Glial Fibrillary Acidic Protein; In Situ Nick-End Labeling; Interneurons; Leukocyte Common Antigens; Mice; Mice, Inbred C57BL; Motor Cortex; Myelin-Oligodendrocyte Glycoprotein; Neural Inhibition; Parvalbumins; Peptide Fragments; Time Factors; Vesicular Inhibitory Amino Acid Transport Proteins

Multiple sclerosis (MS) is a common disabling autoimmune disease without an effective treatment in young adults. Ginsenoside Rd, extracted from Panax notoginseng, has multiple pharmacological effects and potential therapeutic applications in diseases of the central nervous system. In this study, we explore the efficacy of ginsenoside Rd in experimental autoimmune encephalomyelitis (EAE), an established model of MS. EAE was induced by myelin oligodendrocyte glycoprotein 35-55-amino-acid peptide. Ginsenoside Rd (10-80 mg/kg/day) or vehicle was intraperitoneally administered on the disease onset day, and the therapy persisted throughout the experiments. The dose of 40 mg/kg/day of ginsenoside Rd was selected as optimal. Ginsenoside Rd effectively ameliorated the clinical severity in EAE mice, reduced the permeability of the blood-brain barrier, regulated the secretion of interferon-gamma and interleukin-4, promoted the Th2 shift in vivo (cerebral cortex) and in vitro (splenocytes culture supernatants), and prevented the reduction in expression of brain-derived neurotrophic factor and nerve growth factor in both cerebral cortex and lumbar spinal cord of EAE mice. This study establishes the potency of ginsenoside Rd in inhibiting the clinical course of EAE. These findings suggest that ginsenoside Rd could be a promising agent for amelioration of neuroimmune dysfunction diseases such as MS.

Topics: Analysis of Variance; Animals; Blood-Brain Barrier; Brain; Brain-Derived Neurotrophic Factor; Capillary Permeability; Disease Models, Animal; Dose-Response Relationship, Drug; Encephalomyelitis, Autoimmune, Experimental; Enzyme-Linked Immunosorbent Assay; Female; Ginsenosides; Interferon-gamma; Interleukin-4; Lymphocytes; Mice; Mice, Inbred C57BL; Myelin-Oligodendrocyte Glycoprotein; Peptide Fragments; Spinal Cord

Transmigration of peripheral leukocytes to the brain is a major contributor to cerebral ischemic cell death mechanisms. Humanized partial major histocompatibility complex class II constructs (pMHC), covalently linked to myelin peptides, are effective for treating experimental stroke in males, but new evidence suggests that some inflammatory cell death mechanisms after brain injury are sex-specific. We here demonstrate that treatment with pMHC constructs also improves outcomes in female mice with middle cerebral artery occlusion (MCAO). HLA-DR2 transgenic female mice with MCAO were treated with RTL1000 (HLA-DR2 moiety linked to human MOG-35-55 peptide), HLA-DRa1-MOG-35-55, or vehicle (VEH) at 3, 24, 48, and 72 h after reperfusion and were recovered for 96 h or 2 weeks post-injury for measurement of histology (TTC staining) or behavioral testing. RTL1000- and DRa1-MOG-treated mice had profoundly reduced infarct volumes as compared to the VEH group, although higher doses of DRa1-MOG were needed for females vs. males evaluated previously. RTL1000-treated females also exhibited strongly improved functional recovery in a standard cylinder test. In novel studies of post-ischemic ultrasonic vocalization (USV), as measured by animal calls to their cage mates, we modeled in mice the post-stroke speech deficits common in human stroke survivors. The number of calls was reduced in injured animals relative to pre-MCAO baseline regardless of RTL1000 treatment status. However, call duration was significantly improved by RTL1000 treatment, suggesting benefit to the animal's recovery of vocalization capability. We conclude that both the parent RTL1000 molecule and the novel non-polymorphic DRα1-MOG-35-55 construct were highly effective immunotherapies for treatment of transient cerebral ischemia in females.

Topics: Animals; Brain; Brain Ischemia; Disease Models, Animal; Female; HLA-DR Antigens; Humans; Ligands; Mice; Myelin-Oligodendrocyte Glycoprotein; Neuroprotective Agents; Peptide Fragments; Receptors, Antigen, T-Cell; Recombinant Fusion Proteins; Recovery of Function; Sex Factors; Stroke; Vocalization, Animal

Multiple sclerosis (MS) is characterized by central nervous system (CNS) inflammation, demyelination, and axonal degeneration. CXCL10 (IP-10), a chemokine for CXCR3+ T cells, is known to regulate T cell differentiation and migration in the periphery, but effects of CXCL10 produced endogenously in the CNS on immune cell trafficking are unknown. We created floxed cxcl10 mice and crossed them with mice carrying an astrocyte-specific Cre transgene (mGFAPcre) to ablate astroglial CXCL10 synthesis. These mice, and littermate controls, were immunized with myelin oligodendrocyte glycoprotein peptide 35-55 (MOG peptide) to induce experimental autoimmune encephalomyelitis (EAE). In comparison to the control mice, spinal cord CXCL10 mRNA and protein were sharply diminished in the mGFAPcre/CXCL10fl/fl EAE mice, confirming that astroglia are chiefly responsible for EAE-induced CNS CXCL10 synthesis. Astroglial CXCL10 deletion did not significantly alter the overall composition of CD4+ lymphocytes and CD11b+ cells in the acutely inflamed CNS, but did diminish accumulation of CD4+ lymphocytes in the spinal cord perivascular spaces. Furthermore, IBA1+ microglia/macrophage accumulation within the lesions was not affected by CXCL10 deletion. Clinical deficits were milder and acute demyelination was substantially reduced in the astroglial CXCL10-deleted EAE mice, but long-term axon loss was equally severe in the two groups. We concluded that astroglial CXCL10 enhances spinal cord perivascular CD4+ lymphocyte accumulation and acute spinal cord demyelination in MOG peptide EAE, but does not play an important role in progressive axon loss in this MS model.

Topics: Animals; Astrocytes; Axons; Calcium-Binding Proteins; Central Nervous System; Chemokine CXCL10; Disease Models, Animal; Disease Progression; Encephalomyelitis, Autoimmune, Experimental; Glial Fibrillary Acidic Protein; Leukocytes; Lymph Nodes; Mice; Mice, Inbred C57BL; Mice, Knockout; Microfilament Proteins; Myelin Basic Protein; Myelin-Oligodendrocyte Glycoprotein; Peptide Fragments; Spinal Cord; Spleen; Time Factors

Topics: Adoptive Transfer; Animals; B-Lymphocytes; CD3 Complex; Dendritic Cells; Dinucleoside Phosphates; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Flow Cytometry; Forkhead Transcription Factors; Mice; Mice, Inbred C57BL; Myelin-Oligodendrocyte Glycoprotein; Peptide Fragments; Toll-Like Receptor 9

Multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE), are autoimmune diseases characterized by the immune-mediated demyelination and neurodegeneration of the CNS. Our previous studies showed that Rho kinase inhibitor Fasudil can delay onset, and ameliorate severity of EAE, accompanied by the improvement in myelination and the inhibition of inflammatory responses in the CNS. In this study, we found that Fasudil inhibited the migration of T cells indirectly by affecting the production of inflammatory factors and the expression of chemokines in astrocytes functions, indicating that Fasudil treatment reduced inflammatory cytokines such as TNF-α and IL-6, reactive oxygen species (NO) and chemokines like MIP-3α (CCL-20), RANTES (CCL5), MIP-1α (CCL-3) and MCP-1 (CCL2) in vitro, and blocked the chemotaxis of reactive mononuclear cells in EAE mice. Further studies found that Fasudil treatment reduced the infiltration and accumulation of pathogenic T cells into the CNS. Astrocytes expressing GFAP and CCL-20 were inhibited in Fasudil-treated EAE compared with control mice. These results demonstrate that Fasudil alleviates the pathogenesis of EAE possibly by blocking astrocyte-derived chemokine-mediated migration of inflammatory macrophages and pathogenic T cells, and might be used to treat MS.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Animals; Animals, Newborn; Astrocytes; CD4-Positive T-Lymphocytes; Cell Movement; Cells, Cultured; Cytokines; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Female; Gene Expression Regulation; Glial Fibrillary Acidic Protein; Leukocytes, Mononuclear; Lipopolysaccharides; Mice; Mice, Inbred C57BL; Myelin-Oligodendrocyte Glycoprotein; Nitrites; Peptide Fragments; Protein Kinase Inhibitors; Reactive Oxygen Species; rho-Associated Kinases

We reported that AS101 (organotellurium compound, trichloro(dioxoethylene-O,O') tellurate) inhibited the differentiation of Th17 cells and reduced the production of IL-17 and GM-CSF. In addition, AS101 promoted the production of IL-2 in activated T cells. Flow cytometric analysis showed that AS101 inhibited Th17 cell proliferation. AS101 blocked the activation of transcriptional factor NFAT, Stat3, and RORγt, and increased activation of Erk1/2, suggesting a mechanism of action of AS101. We further demonstrated that AS101 was effective in amelioration of experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis. Finally, by real-time PCR analysis we showed that AS101 reduces the IL-17, IFN-γ, GM-CSF, and IL-6 mRNA expression in inflammatory cells of spinal cords. Additionally, flow cytometry analysis also indicated that the CD4+ T cells and IL-17 and GM-CSF-producing cells were reduced in the spinal cords of AS101 treated mice compared to those treated with PBS.

Topics: Animals; CD4-Positive T-Lymphocytes; Cell Polarity; Cell Proliferation; Cytokines; Disease Models, Animal; Dose-Response Relationship, Drug; Encephalomyelitis, Autoimmune, Experimental; Ethylenes; Female; Freund's Adjuvant; Immunologic Factors; Mice; Mice, Inbred C57BL; Myelin-Oligodendrocyte Glycoprotein; Oncogene Protein v-akt; Peptide Fragments; Signal Transduction; Spinal Cord; Th17 Cells

The paracaspase mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1) is crucial for lymphocyte activation through signaling to the transcription factor NF-κB. Besides functioning as a scaffold signaling protein, MALT1 also acts as a cysteine protease that specifically cleaves a number of substrates and contributes to specific T cell receptor-induced gene expression. Recently, small molecule inhibitors of MALT1 proteolytic activity were identified and shown to have promising anticancer properties in subtypes of B cell lymphoma. However, information on the therapeutic potential of small compound inhibitors that target MALT1 protease activity in autoimmunity is still lacking.. The present study aimed to elucidate whether MALT1 protease inhibitors are also useful in the treatment of lymphocyte-mediated autoimmune pathologies such as multiple sclerosis (MS). For this, we studied the therapeutic potential of a recently identified inhibitor of MALT1 protease activity, the phenothiazine derivative mepazine, in the context of experimental autoimmune encephalomyelitis (EAE), the main animal model for MS.. We demonstrate that administration of mepazine prophylactically or after disease onset, can attenuate EAE. Importantly, while complete absence of MALT1 affects the differentiation of regulatory T (Treg) cells in vivo, the MALT1 protease inhibitor mepazine did not affect Treg development.. Altogether, these data indicate that small molecule inhibitors of MALT1 not only hold great promise for the treatment of B cell lymphomas but also for autoimmune disorders such as MS.

Topics: Animals; Antigens, CD; Caspases; Cell Differentiation; Cells, Cultured; Cytokines; Disease Models, Animal; Encephalitis; Encephalomyelitis, Autoimmune, Experimental; Follow-Up Studies; Lymphocyte Activation; Mice; Mucosa-Associated Lymphoid Tissue Lymphoma Translocation 1 Protein; Multiple Sclerosis; Myelin-Oligodendrocyte Glycoprotein; Neoplasm Proteins; NF-kappa B; Peptide Fragments; Phenothiazines; Spinal Cord; T-Lymphocytes

Experimental autoimmune encephalomyelitis (EAE) is an animal model of autoimmune inflammatory demyelination that is mediated by Th1 and Th17 cells. The transcription factor interferon regulatory factor 3 (IRF3) is activated by pathogen recognition receptors and induces interferon-β production.. To determine the role of IRF3 in autoimmune inflammation, we immunised wild-type (WT) and irf3(-/-) mice to induce EAE. Splenocytes from WT and irf3(-/-) mice were also activated in vitro in Th17-polarising conditions.. Clinical signs of disease were significantly lower in mice lacking IRF3, with reduced Th1 and Th17 cells in the central nervous system. Peripheral T-cell responses were also diminished, including impaired proliferation and Th17 development in irf3(-/-) mice. Myelin-reactive CD4+ cells lacking IRF3 completely failed to transfer EAE in Th17-polarised models as did WT cells transferred into irf3(-/-) recipients. Furthermore, IRF3 deficiency in non-CD4+ cells conferred impairment of Th17 development in antigen-activated cultures.. These data show that IRF3 plays a crucial role in development of Th17 responses and EAE and warrants investigation in human multiple sclerosis.

Topics: Animals; CD4-Positive T-Lymphocytes; Cell Proliferation; Cells, Cultured; Cytokines; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Female; Flow Cytometry; Interferon Regulatory Factor-3; Mice; Mice, Inbred C57BL; Mice, Transgenic; Myelin-Oligodendrocyte Glycoprotein; Peptide Fragments; Phorbol Esters; Spinal Cord; T-Lymphocytes; Th17 Cells; Transfection

Monocyte-derived macrophages (mo-MΦs) and T cells have been shown to contribute to spinal cord repair. Recently, the remote brain choroid plexus epithelium (CP) was identified as a portal for monocyte recruitment, and its activation for leukocyte trafficking was found to be IFN-γ-dependent. Here, we addressed how the need for effector T cells can be reconciled with the role of inflammation-resolving immune cells in the repair process. Using an acute spinal cord injury model, we show that in mice deficient in IFN-γ-producing T cells, the CP was not activated, and recruitment of inflammation-resolving mo-MΦ to the spinal cord parenchyma was limited. We further demonstrate that mo-MΦ locally regulated recruitment of thymic-derived Foxp3(+) regulatory T (Treg) cells to the injured spinal cord parenchyma at the subacute/chronic phase. Importantly, an ablation protocol that resulted in reduced Tregs at this stage interfered with tissue remodeling, in contrast to Treg transient ablation, restricted to the 4 d period before the injury, which favored repair. The enhanced functional recovery observed following such a controlled decrease of Tregs suggests that reduced systemic immunosuppression at the time of the insult can enhance CNS repair. Overall, our data highlight a dynamic immune cell network needed for repair, acting in discrete compartments and stages, and involving effector and regulatory T cells, interconnected by mo-MΦ. Any of these populations may be detrimental to the repair process if their level or activity become dysregulated. Accordingly, therapeutic interventions must be both temporally and spatially controlled.

Topics: Animals; Antigens, CD; CD11c Antigen; CX3C Chemokine Receptor 1; Diphtheria Toxin; Disease Models, Animal; Forkhead Transcription Factors; Green Fluorescent Proteins; Humans; Macrophages; Mice; Mice, Inbred C57BL; Mice, Transgenic; Myelin-Oligodendrocyte Glycoprotein; Nerve Regeneration; Peptide Fragments; Receptors, Chemokine; Recovery of Function; Spinal Cord Injuries; T-Box Domain Proteins; T-Lymphocytes, Regulatory; Vaccination

Recent studies have demonstrated that atypical antipsychotic agents, which are known to antagonize dopamine D2 and serotonin 5-HT2a receptors, have immunomodulatory properties. Given the potential of these drugs to modulate the immune system both peripherally and within the central nervous system, we investigated the ability of the atypical anti-psychotic agent, risperidone, to modify disease in the animal model of multiple sclerosis (MS)4, experimental autoimune encephalomyelitis (EAE). We found that chronic oral administration of risperidone dose-dependently reduced the severity of disease and decreased both the size and number of spinal cord lesions. Furthermore, risperidone treatment substantially reduced antigen-specific interleukin (IL)-17a, IL-2, and IL-4 but not interferon (IFN)-γ production by splenocytes at peak disease and using an in vitro model, we show that treatment of macrophages with risperidone alters their ability to bias naïve T cells. Another atypical antipsychotic agent, clozapine, showed a similar ability to modify macrophages in vitro and to reduce disease in the EAE model but this effect was not due to antagonism of the type 1 or type 2 dopamine receptors alone. Finally, we found that while risperidone treatment had little effect on the in vivo activation of splenic macrophages during EAE, it significantly reduced the activation of microglia and macrophages in the central nervous system. Together these studies indicate that atypical antipsychotic agents like risperidone are effective immunomodulatory agents with the potential to treat immune-mediated diseases such as MS.

Topics: Animals; Antigens; Antipsychotic Agents; Cell Survival; Cytokines; Disease Models, Animal; Dose-Response Relationship, Drug; Encephalomyelitis, Autoimmune, Experimental; Epitopes, T-Lymphocyte; Female; Macrophage Activation; Macrophages; Mice; Microglia; Multiple Sclerosis; Myelin-Oligodendrocyte Glycoprotein; Nitric Oxide; Peptide Fragments; Risperidone; Severity of Illness Index; Spinal Cord; T-Lymphocytes

The immunomodulatory effects of the IL-27 and IL-33 and the anti-inflammatory effects of ginger have been reported in some studies. The aim was to evaluate the effects of the ginger extract on the expression of IL-27 and IL-33 in a model of experimental autoimmune encephalomyelitis (EAE). In PBS-treated EAE mice the expression of IL-27 P28 was significantly lower whereas the expression of IL-33 was significantly higher than unimmunized control mice. In 200 and 300 mg/kg ginger-treated EAE groups the expression of IL-27 P28 and IL-27 EBI3 was significantly higher whereas the expression of IL-33 was significantly lower than PBS-treated EAE mice. The EAE clinical symptoms and the pathological scores were significantly lower in ginger-treated EAE groups. These results showed that the ginger extract modulates the expression of the IL-27 and IL-33 in the spinal cord of EAE mice and ameliorates the clinical symptoms of disease.

Topics: Animals; Body Weight; Central Nervous System; Chemotaxis, Leukocyte; Disease Models, Animal; Dose-Response Relationship, Drug; Encephalomyelitis, Autoimmune, Experimental; Female; Freund's Adjuvant; Interferon-gamma; Interleukin-27; Interleukin-33; Interleukin-7; Interleukins; Mice; Mice, Inbred C57BL; Myelin-Oligodendrocyte Glycoprotein; Peptide Fragments; Phytotherapy; Plant Extracts; Time Factors; Zingiber officinale

Multiple sclerosis (MS) is a CNS inflammatory demyelinating disorder. T helper 1 (Th1) and T helper 17 (Th17) cells are important in MS immunopathogenesis. Level of endothelin-1 (ET-1), a potent vasoconstrictor, is increased in sera of MS patients. We studied the role of ET-1 in experimental allergic encephalomyelitis (EAE), a MS animal model.. EAE is induced in transgenic mice overexpressing endothelial ET-1 (TET-1), transgenic mice overexpressing astrocytic ET-1 (GET-1) and non-transgenic (NTg) mice by immunization with myelin oligodendrocyte glycoprotein (MOG)35-55 peptide. EAE scores, spinal cord histology, serum proinflammatory cytokines levels, and proinflammatory cytokines production from splenocytes of ET-1 transgenic and NTg mice with EAE were studied.. ET-1 transgenic mice developed more severe EAE than NTg with increased inflammation and demyelination in spinal cord. The mean maximum EAE scores for GET-1, TET-1 and NTg mice with EAE were 4.84, 4.31 and 4.05 respectively (p<0.05). Serum levels of IL-6, IL-17A, IFN-γ and TNF-α were higher in ET-1 transgenic than NTg mice with EAE (p<0.05) while serum IL-4 levels were similar. mRNA levels of IL-6, IL-17A, IFN-γ and TNF-α from cultured splenocytes were higher in ET-1-transgenic than NTg mice with EAE (p<0.05) while IL-4 mRNA levels were similar. Consistently, levels of IL-6, IL-17A, IFN-γ and TNF-α in culture media of splenocytes were higher in ET-1 transgenic than NTg mice with EAE (p<0.05) while IL-4 levels were similar.. Mice with endothelial or astrocytic ET-1 overexpression developed more severe EAE with increased splenic lymphocyte production of Th1 and Th17 proinflammatory cytokines.

Topics: Analysis of Variance; Animals; Cytokines; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Endothelin-1; Enzyme-Linked Immunosorbent Assay; Gene Expression Regulation; Lymphocytes; Mice; Mice, Inbred C57BL; Myelin-Oligodendrocyte Glycoprotein; Peptide Fragments; Spinal Cord; Spleen

Reduced levels of noradrenaline (NA) in CNS of multiple sclerosis patients could be due to metabolism by catechol-O-methyltransferase (COMT). In mice immunized with myelin oligodendrocyte glycoprotein peptide, the BBB-permeable COMT inhibitor dinitrocatechol (DNC) reduced clinical signs, while entacapone, a non-BBB-permeable inhibitor, had no effect. Spinal cord NA levels were slightly increased by DNC, and there was an inverse correlation between NA levels and average clinical signs. Spinal cord COMT mRNA levels were not increased during EAE, but were found increased in the frontal cortex of MS patients. These results suggest that COMT inhibitors could provide benefit to MS patients.

Topics: Aged; Aged, 80 and over; Animals; Blood-Brain Barrier; Catechol O-Methyltransferase; Catechol O-Methyltransferase Inhibitors; Catechols; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Female; Freund's Adjuvant; Humans; Male; Mice; Mice, Inbred C57BL; Middle Aged; Multiple Sclerosis; Myelin-Oligodendrocyte Glycoprotein; Nitriles; Peptide Fragments; Spinal Cord; T-Lymphocytes; Time Factors

In this study, we investigate the basis of T cell recognition of myelin that governs the progression from acute symptoms into disease remission, relapse, and chronic progression in a secondary progressive model of demyelinating disease. Until now, the frequency and affinity of myelin-reactive CD4 T cells that elicit relapsing-remitting disease have not been quantified. The micropipette adhesion frequency assay was used to obtain a sensitive and physiologically relevant two-dimensional measurement of frequency and TCR affinity for myelin, as the inherent low affinity does not allow the use of specific peptide:MHC-II tetramers for this purpose. We found the highest affinity and frequency of polyclonal myelin oligodendrocyte glycoprotein-reactive cells infiltrate the CNS during acute disease, whereas affinities during remission, relapse, and chronic disease are not significantly different from each other. Frequency analysis revealed that the vast majority of CNS-infiltrating CD4 T cells are myelin oligodendrocyte glycoprotein reactive at all time points, demonstrating epitope spread is not a predominant factor for disease progression. Furthermore, time points at which mice were symptomatic were characterized by an infiltration of Th17 cells in the CNS, whereas symptom remission showed an enrichment of cells producing IFN-γ. Also, the ratio of regulatory T cells to Foxp3(-) CD4 T cells was significantly higher in the CNS at remission than during acute disease. The results of this study indicate that a high frequency of T cells specific for a single myelin Ag, rather than increased TCR affinity or epitope spread, governs the transition from acute symptoms through remission, relapse, and chronic disease states.

Topics: Animals; CD4-Positive T-Lymphocytes; Demyelinating Autoimmune Diseases, CNS; Disease Models, Animal; Disease Progression; Encephalomyelitis, Autoimmune, Experimental; Epitopes; Female; Interleukin-17; Lymphocyte Activation; Male; Mice; Mice, Inbred NOD; Myelin-Oligodendrocyte Glycoprotein; Peptide Fragments; Receptors, Antigen, T-Cell; T-Lymphocyte Subsets

The role of microglia and monocyte-derived macrophages in experimental autoimmune encephalomyelitis pathogenesis has been controversial. To gain insight into their respective roles, we developed a method for differentiating between microglia and monocyte-derived macrophages in the CNS by flow cytometry utilizing anti-CD44 antibodies. We used this system to monitor changes in cell number, activation status, and gene expression by RNA sequencing over the course of disease. This in vivo characterization and RNA-Seq dataset improves our understanding of macrophage biology in the brain under inflammatory conditions and may lead to strategies to identify therapies for neuroinflammatory diseases.

Topics: Animals; Antigens, CD; Base Sequence; Cell Proliferation; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Female; Flow Cytometry; Macrophages, Peritoneal; Mice; Mice, Inbred C57BL; Microglia; Myelin-Oligodendrocyte Glycoprotein; Peptide Fragments; Signal Transduction; Time Factors

Evidence from experimental autoimmune encephalomyelitis (EAE) suggests that CNS-infiltrating dendritic cells (DCs) are crucial for restimulation of coinfiltrating T cells. Here we systematically quantified and visualized the distribution and interaction of CNS DCs and T cells during EAE. We report marked periventricular accumulation of DCs and myelin-specific T cells during EAE disease onset prior to accumulation in the spinal cord, indicating that the choroid plexus-CSF axis is a CNS entry portal. Moreover, despite emphasis on spinal cord inflammation in EAE and in correspondence with MS pathology, inflammatory lesions containing interacting DCs and T cells are present in specific brain regions.

Topics: Adoptive Transfer; Analysis of Variance; Animals; Bone Marrow Cells; CD11c Antigen; Central Nervous System; Dendritic Cells; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Flow Cytometry; Luminescent Proteins; Mice; Mice, Inbred C57BL; Mice, Transgenic; Mutation; Myelin-Oligodendrocyte Glycoprotein; Peptide Fragments; T-Lymphocytes; Time Factors

Topics: Analysis of Variance; Animals; Anti-Inflammatory Agents; Calcium-Binding Proteins; Central Nervous System; Cytokines; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Forkhead Transcription Factors; Freund's Adjuvant; Glial Fibrillary Acidic Protein; Mice; Mice, Inbred C57BL; Microfilament Proteins; Myelin-Oligodendrocyte Glycoprotein; Nerve Tissue Proteins; Peptide Fragments; Primaquine; Severity of Illness Index; T-Lymphocytes, Regulatory

CD4(+) T-cells play a key role in the pathogenesis of multiple sclerosis (MS). Altered peptide ligands capable of modulating T-cell autoreactivity are considered a promising strategy for development of antigen-specific therapies for MS. Since peptides are inherently unstable, the current study explored single β-amino acid substitution as a means of stabilizing an epitope of myelin oligodendrocyte glycoprotein. β-Amino acid substitution at position 44, the major T-cell receptor contact residue, increased the half-life of active metabolites. Vaccination with one altered peptide, MOG44βF, conferred protection from EAE, decreased T-cell autoreactivity and pro-inflammatory cytokine production. Additional studies using MOG44βF in an oral treatment regimen, administered after EAE induction, also attenuated disease severity. Thus, altered peptides such as those reported here may lead to the development of novel and more specific treatments for MS.

Topics: Amino Acid Substitution; Analysis of Variance; Animals; Cell Proliferation; Central Nervous System; Cytokines; Disease Models, Animal; Dose-Response Relationship, Drug; Encephalomyelitis, Autoimmune, Experimental; Female; Forkhead Transcription Factors; Freund's Adjuvant; Gene Expression Regulation; Mice; Mice, Inbred C57BL; Myelin-Oligodendrocyte Glycoprotein; Peptide Fragments; T-Lymphocytes; Time Factors

Apolipoprotein A1 (Apo A-I), the most abundant component of high-density lipoprotein (HDL), is an anti-inflammatory molecule, yet its potential role in the pathogenesis of multiple sclerosis (MS) has not been fully investigated. In this study, Western blot analyses of human plasma showed differential Apo A-I expression in healthy controls compared to MS patients. Further, primary progressive MS patients had less plasma Apo A-I than other forms of MS. Using experimental allergic encephalomyelitis (EAE) as a model for MS, Apo A-I deficient mice exhibited worse clinical disease and more neurodegeneration concurrent with increased levels of pro-inflammatory cytokines compared to wild-type animals. These data suggest that Apo A-I plays a role in the pathogenesis of EAE, a model for MS, creating the possibility for agents that increase Apo A-I levels as potential therapies for MS.

Topics: Adult; Animals; Apolipoprotein A-I; Case-Control Studies; Cytokines; Disease Models, Animal; Electrophoresis, Gel, Two-Dimensional; Encephalomyelitis, Autoimmune, Experimental; Evoked Potentials, Visual; Female; Fluoresceins; Freund's Adjuvant; Gene Expression Regulation; Humans; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Multiple Sclerosis; Myelin-Oligodendrocyte Glycoprotein; Peptide Fragments

Cellular therapies are becoming a major focus for the treatment of demyelinating diseases such as multiple sclerosis (MS), therefore it is important to identify the most effective cell types that promote myelin repair. Several components contribute to the relative benefits of specific cell types including the overall efficacy of the cell therapy, the reproducibility of treatment, the mechanisms of action of distinct cell types and the ease of isolation and generation of therapeutic populations. A range of distinct cell populations promote functional recovery in animal models of MS including neural stem cells and mesenchymal stem cells derived from different tissues. Each of these cell populations has advantages and disadvantages and likely works through distinct mechanisms. The relevance of such mechanisms to myelin repair in the adult central nervous system is unclear since the therapeutic cells are generally derived from developing animals. Here we describe the isolation and characterization of a population of neural cells from the adult spinal cord that are characterized by the expression of the cell surface glycoprotein NG2. In functional studies, injection of adult NG2(+) cells into mice with ongoing MOG35-55-induced experimental autoimmune encephalomyelitis (EAE) enhanced remyelination in the CNS while the number of CD3(+) T cells in areas of spinal cord demyelination was reduced approximately three-fold. In vivo studies indicated that in EAE, NG2(+) cells stimulated endogenous repair while in vitro they responded to signals in areas of induced inflammation by differentiating into oligodendrocytes. These results suggested that adult NG2(+) cells represent a useful cell population for promoting neural repair in a variety of different conditions including demyelinating diseases such as MS.

Topics: Animals; Animals, Newborn; Antigens; Bromodeoxyuridine; CD3 Complex; Cell Transplantation; Cells, Cultured; Culture Media, Conditioned; Disease Models, Animal; Female; Glial Fibrillary Acidic Protein; Green Fluorescent Proteins; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Multiple Sclerosis; Myelin Proteolipid Protein; Myelin-Oligodendrocyte Glycoprotein; Neurons; Oligodendroglia; Organ Culture Techniques; Peptide Fragments; Proteoglycans; Proto-Oncogene Proteins c-sis; Recovery of Function; Spinal Cord; Time Factors

Previous work from our laboratory implicated protein carbonylation in the pathophysiology of both MS (multiple sclerosis) and its animal model EAE (experimental autoimmune encephalomyelitis). Subsequent in vitro studies revealed that the accumulation of protein carbonyls, triggered by glutathione deficiency or proteasome inhibition, leads to protein aggregation and neuronal cell death. These findings prompted us to investigate whether their association can be also established in vivo. In the present study, we characterized protein carbonylation, protein aggregation and apoptosis along the spinal cord during the course of MOG (myelin-oligodendrocyte glycoprotein)(35-55) peptide-induced EAE in C57BL/6 mice. The results show that protein carbonyls accumulate throughout the course of the disease, albeit by different mechanisms: increased oxidative stress in acute EAE and decreased proteasomal activity in chronic EAE. We also show a temporal correlation between protein carbonylation (but not oxidative stress) and apoptosis. Furthermore, carbonyl levels are significantly higher in apoptotic cells than in live cells. A high number of juxta-nuclear and cytoplasmic protein aggregates containing the majority of the oxidized proteins are present during the course of EAE. The LC3 (microtubule-associated protein light chain 3)-II/LC3-I ratio is significantly reduced in both acute and chronic EAE indicating reduced autophagy and explaining why aggresomes accumulate in this disorder. Taken together, the results of the present study suggest a link between protein oxidation and neuronal/glial cell death in vivo, and also demonstrate impaired proteostasis in this widely used murine model of MS.

Topics: Animals; Apoptosis; Autophagy; Calpain; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Female; Freund's Adjuvant; Glutathione; In Situ Nick-End Labeling; Mice; Mice, Inbred C57BL; Myelin-Oligodendrocyte Glycoprotein; Peptide Fragments; Proteasome Endopeptidase Complex; Protein Carbonylation; Spinal Cord; Time Factors

Neural precursor cells (NPCs) located in the subgranular zone (SGZ) of the dentate gyrus (DG) give rise to thousands of new cells every day, mainly hippocampal neurons, which are integrated into existing neuronal circuits. Aging and chronic degenerative disorders have been shown to impair hippocampal neurogenesis, but the consequence of inflammation is somewhat controversial. The present study demonstrates that the inflammatory environment prevailing in the brain of experimental autoimmune encephalomyelitis (EAE) mice enhances the proliferation of NPCs in SGZ of the dorsal DG and alters the proportion between radial glial cells and newborn neuroblasts. The injection protocol of the cell cycle marker bromodeoxyuridine and the immunohistochemical techniques that were employed revealed that the proliferation of NPCs is increased approximately twofold in the SGZ of the dorsal DG of EAE mice, at the acute phase of the disease. However, although EAE animals exhibited significant higher percentage of newborn radial-glia-like NPCs, the mean percentage of newborn neuroblasts rather was decreased, indicating that the robust NPCs proliferation is not followed by a proportional production of newborn neurons. Significant positive correlations were detected between the number of proliferating cells in the SGZ and the clinical score or degree of brain inflammation of diseased animals. Finally, enhanced neuroproliferation in the acute phase of EAE was not found to trigger compensatory apoptotic mechanisms. The possible causes of altered neurogenesis observed in this study emphasize the need to understand more precisely the mechanisms regulating adult neurogenesis under both normal and pathological conditions.

Topics: Animals; Bromodeoxyuridine; Cell Count; Cell Cycle; Cell Proliferation; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Female; Hippocampus; Mice; Mice, Inbred C57BL; Myelin-Oligodendrocyte Glycoprotein; Neural Stem Cells; Neurogenesis; Peptide Fragments; Time Factors

Fingolimod is an immunomodulating agent that has been approved for the treatment of multiple sclerosis. Fingolimod-phosphate is an antagonist of sphingosine-1-phosphate receptor and known to act by preventing infiltration of autoreactive lymphocytes into the central nervous system. In this study, we investigated whether fingolimod prevents experimental autoimmune optic neuritis (EAON).. EAON was induced by immunizing C57BL/6 mice with myelin oligodendrocyte glycoprotein-derived peptide 35-55 (MOG-p). After MOG-p immunization, fingolimod was administered intragastrically from day 1 (entire phase study) or from day 9 (effector phase study) until day 35. Visual acuity of the mice was measured using OptoMotry on days 7, 14, 21, 28, and 35 after immunization. On day 35 after immunization, the mice were killed and eyes and entire length of the optic nerves were submitted for histopathologic evaluation.. In the positive control group, visual acuity decreased markedly from approximately day 14 after immunization, reaching a nadir on day 21. In the fingolimod-treated groups in both entire phase and effector phase studies, there was only minimal decline in visual acuity on day 14 after immunization, and mild deterioration on day 21, followed by recovery. Histopathologic study showed that fingolimod given throughout the entire phase or only from the effector phase suppressed murine EAON. Immunohistochemical study for neurofilament demonstrated no irregularity of the linear structure of the optic nerve in the fingolimod-treated mice compared with the positive control group.. Fingolimod ameliorated EAON even when started after optic neuritis had developed. Further study is warranted to examine whether these findings are applicable to human disease.

Topics: Animals; Disease Models, Animal; Female; Fingolimod Hydrochloride; Freund's Adjuvant; Immunosuppressive Agents; Mice; Mice, Inbred C57BL; Myelin-Oligodendrocyte Glycoprotein; Neuritis, Autoimmune, Experimental; Optic Nerve; Peptide Fragments; Pertussis Toxin; Propylene Glycols; Sphingosine; Statistics, Nonparametric; Visual Acuity

Multiple sclerosis (MS) is an autoimmune disease characterized by inflammation and neurodegeneration. Current MS treatments were designed to reduce inflammation in MS rather than directly to prevent neurodegeneration. Estrogen has well-documented neuroprotective effects in a variety of disorders of the CNS, including experimental autoimmune encephalomyelitis (EAE), the most widely used mouse model of MS. Treatment with an estrogen receptor-β (ERβ) ligand is known to ameliorate clinical disease effectively and provide neuroprotection in EAE. However, the protective effects of this ERβ ligand have been demonstrated only when administered prior to disease (prophylactically). Here we tested whether ERβ ligand treatment could provide clinical protection when treatment was initiated after onset of disease (therapeutically). We found that therapeutic treatment effectively ameliorated clinical disease in EAE. Specifically, ERβ ligand-treated animals exhibited preserved axons and myelin compared with vehicle-treated animals. We observed no difference in the number of T lymphocytes, macrophages, or microglia in the CNS of vehicle- vs. ERβ ligand-treated animals. Our findings show that therapeutically administered ERβ ligand successfully treats clinical EAE, bearing translational relevance to MS as a candidate neuroprotective agent.

Topics: Animals; Axons; Demyelinating Diseases; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Female; Freund's Adjuvant; Mice; Mice, Inbred C57BL; Myelin-Oligodendrocyte Glycoprotein; Neuroprotective Agents; Nitriles; Peptide Fragments; Propionates; Receptors, Estrogen; Severity of Illness Index

We have reported earlier that pertussis toxin (PTx) attenuates the motor deficits in experimental autoimmune encephalomyelitis (EAE), an animal model for human multiple sclerosis. PTx protects neurons from inflammatory insults. Vascular endothelial growth factor (VEGF) is also neuroprotective. However, the effect of PTx on VEGF has never been studied. We investigated whether PTx modulates neuronal VEGF expression and how it affects the pathogenesis of EAE. EAE was induced by injecting myelin oligodendrocyte glycoprotein 35-55 peptides with adjuvants into C57BL/6 mice. Clinical scores of EAE were evaluated daily for 19 days. Brain and spinal cord samples were collected and assessed for inflammation and demyelination. VEGF, NeuN for neurons, and Caspase-3 for apoptosis were stained for localization using immunohistochemistry techniques, followed by western blot analysis for quantification. Primary neurons were cultured to assess the direct effect of PTx on neuronal VEGF expression. PTx treatment increases neuronal VEGF expression by up to ∼75% in vitro and ∼60% in vivo, preventing neurons from apoptosis. This leads to resolution in inflammation and remyelination and amendment in motor deficits. Our findings suggest that upregulation of endogenous neuronal VEGF by PTx protects motor deficits in EAE and it is a potential therapeutic option for multiple sclerosis.

Topics: Animals; Animals, Newborn; Cells, Cultured; Cerebral Cortex; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Mice; Mice, Inbred C57BL; Myelin-Oligodendrocyte Glycoprotein; Neurons; Peptide Fragments; Pertussis Toxin; Up-Regulation; Vascular Endothelial Growth Factor A

Many symptoms in multiple sclerosis (MS) can be related to changes in the levels of key neurotransmitters. These neurotransmitters have a direct role in the maintenance of neurons and also have immunomodulatory properties. Previously we have shown that when treatment began prior to the onset of clinical signs, daily treatment with the monoamine oxidase (MAO) inhibitor phenelzine (PLZ), which also elevates CNS levels of GABA, lead to substantial behavioral improvements in the experimental autoimmune encephalomyelitis (EAE), the animal model for MS. To determine whether PLZ could have beneficial effects in an already established disease state, we conducted experiments in which PLZ treatment only began when mice with EAE exhibited the first clinical signs of the disease. Using this more clinically relevant treatment approach, we find that PLZ treatment can reduce the severity of clinical signs and improve exploratory behaviors for the duration of the experiment in mice with EAE. Treatment with PLZ did not affect the infiltration of CD4+ T-cells into the spinal cord nor did it reduce the degree of reactive gliosis as measured by Iba1 immunostaining. Beginning PLZ treatment after the start of clinical signs did however lead to significantly better 5-HT innervation density in the ventral horn of the spinal cord and also resulted in higher levels of GABA, dopamine and norepinephrine in the brain and spinal cord. These results indicate that even in an established EAE disease state, PLZ can have clinical benefits. These benefits likely derive from PLZ's ability to normalize the innervation to ventral horn motor neuron pools as well as the elevations in GABA and biogenic amines that have been shown to have anti-inflammatory properties.

Topics: Analysis of Variance; Animals; CD4 Antigens; Central Nervous System; Chromatography, High Pressure Liquid; Disease Models, Animal; Dopamine; Encephalomyelitis, Autoimmune, Experimental; Exploratory Behavior; Female; Freund's Adjuvant; gamma-Aminobutyric Acid; Mice; Mice, Inbred C57BL; Monoamine Oxidase; Monoamine Oxidase Inhibitors; Myelin-Oligodendrocyte Glycoprotein; Norepinephrine; Peptide Fragments; Phenelzine; Rotarod Performance Test; Serotonin; T-Lymphocytes; Time Factors; Treatment Outcome

Multiple sclerosis (MS) causes significant neurological disability. Experimental autoimmune encephalomyelitis (EAE) is an animal model of MS. Human bone marrow mesenchymal stem cells (hMSCs) possess anti-inflammatory and immunosuppressive effects. We studied whether hMSCs affect CD1d(high)CD5(+) regulatory B-cell activity in EAE.. EAE was induced in C57BL/6N mice by immunization with MOG35-55 peptide. hMSCs were injected intravenously into EAE mice on day 3 and day 12 after first immunization. Mice were sacrificed on day 26. Immunohistochemistry of the spinal cord, serum cytokines levels, production of cytokines by cultured splenic cells, and flow cytometry for splenic Th17 and CD1d(high)CD5(+) regulatory B cells were studied.. EAE mice with hMSC treatment on day 3 and day 12 had reduced EAE scores from day 14 to day 26 compared to EAE mice without hMSC treatment, and reduced infiltration of inflammatory cells and demyelination in the spinal cord. EAE mice with hMSC treatment on day 3 and day 12 had: (1) lower serum levels of IL-6, TNF-α (p < 0.0005), and IL-17 (p < 0.005 for day 3, p < 0.0005 for day 12); (2) reduced splenic cell production and secretion of IL-6, TNF-α (p < 0.05), and IL-17 (p < 0.05), and increased splenic production of IL-10; (3) reduced splenic Th17 cells (p < 0.05 for day 3, p < 0.005 for day 12), and (4) increased CD1d(high)CD5(+) regulatory B cells (p < 0.005) compared to EAE mice without hMSC treatment.. hMSC treatment on day 3 and day 12 suppresses EAE severity. The underlying mechanisms involve downregulation of Th17 cells and upregulation of CD1d(high)CD5(+) regulatory B-cell activity.

Topics: Analysis of Variance; Animals; Antigens, CD1d; B-Lymphocytes, Regulatory; CD5 Antigens; Cytokines; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Female; Flow Cytometry; Humans; Mesenchymal Stem Cell Transplantation; Mesenchymal Stem Cells; Mice; Mice, Inbred C57BL; Myelin-Oligodendrocyte Glycoprotein; Peptide Fragments; RNA, Messenger; Up-Regulation

Neuromyelitis optica (NMO) is a devastating inflammatory disorder of the optic nerves and spinal cord characterized by frequently recurring exacerbations of humoral inflammation. NMO is associated with the highly specific NMO-IgG biomarker, an antibody that binds the aquaporin-4 water channel. Aquaporin-4 is present on glial endfeet in the central nervous system (CNS). In humans, the NMO-IgG portends more frequent exacerbations and a worse long-term clinical outcome.. We tested the longer-term outcome of mice with EAE injected with NMO-IgG and followed them for 60 days. Clinical exams and pathology of the spinal cord and optic nerves were compared to mice that received control human IgG.. Passively transferred human NMO-IgG leads to more severe neurology disability over two months after onset of disease. Clinical worsening is associated with an increased concentration of large demyelinating lesions primarily to subpial AQP4-rich regions of the spinal cord.. NMO-IgG is pathogenic in the context of EAE in mice.

Topics: Animals; Antigens, CD; Antigens, Ly; Aquaporin 4; Demyelinating Diseases; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Female; Humans; Immunoglobulin G; Mice; Mice, Inbred C57BL; Myelin Sheath; Myelin-Oligodendrocyte Glycoprotein; Neuromyelitis Optica; Optic Nerve; Peptide Fragments; Spinal Cord; Time Factors

We aimed at validating a plasma biomarker for neuronal damage that can be used in acute and chronic models of neurological diseases.. We investigated two different models, middle cerebral artery occlusion followed by reperfusion and MOG35-55-induced experimental autoimmune encephalomyelitis (EAE). In stroke experiments we measured infarct sizes by magnetic resonance imaging and vital stainings and correlated them with plasma levels of neuron specific enolase (NSE) at different time points after reperfusion. Equally, in EAE experiments, we correlated NSE levels with neurological scores and histopathological damage of axons at different time points. We detected plasma NSE levels by ELISA.. Plasma NSE levels correlated significantly with stroke size, EAE score and histopathological damage in EAE. Investigations into the dynamics of neuronal loss over time correlated well with the dynamics of NSE levels. NSE even predicted the onset of EAE, before clinical signs were recordable.. Plasma NSE is a valid and simple experimental biomarker that allows quantifying the degree of neuronal injury in a non-invasive approach.

Topics: Amyloid beta-Protein Precursor; Animals; Brain; Brain Infarction; Cells, Cultured; Disease Models, Animal; Embryo, Mammalian; Glutamic Acid; Infarction, Middle Cerebral Artery; L-Lactate Dehydrogenase; Magnetic Resonance Imaging; Mice; Mice, Inbred C57BL; Microtubule-Associated Proteins; Multiple Sclerosis; Myelin-Oligodendrocyte Glycoprotein; Neurons; Peptide Fragments; Phosphopyruvate Hydratase; Time Factors

We investigated the CCL5-glutamate interaction in the cortex and in the spinal cord from mice with Experimental Autoimmune Encephalomyelitis (EAE) at 13 and 21/30 days post immunization (d.p.i.), representing the onset and the peak of the disease, respectively. An early reduction of the KCl-evoked glutamate release was observed in cortical terminals from EAE mice at 13 d.p.i., persisting until 21/30 d.p.i. A concomitant reduction of the depolarization-evoked cyclic adenosine monophosphate (cAMP), but not of the inositol 1,4,5-trisphosphate (IP3) cortical production also occurred at 13 d.p.i, that still was detectable at the acute stage of disease (21 dp.i.). Inasmuch, the CCL5-mediated inhibition of glutamate exocytosis observed in control mice turned to facilitation in EAE mouse cortex at 13 d.p.i., then becoming undetectable at 21/30 d.p.i. Differently, glutamate exocytosis, as well as IP3 and cAMP productions were unaltered in spinal cord synaptosomes from EAE mice at 13 d.p.i., but significantly increased at 21/30 d.p.i., while the presynaptic CCL5-mediated facilitation of glutamate exocytosis observed in control mice remained unchanged. In both CNS regions, the presynaptic defects were parallelled by increased CCL5 availability. Inasmuch, the presynaptic defects so far described in EAE mice were reminiscent of the effects acute CCL5 exerts in control conditions. Based on these observations we propose that increased CCL5 bioavailability could have a role in determining the abovedescribed impaired presynaptic impairments in both CNS regions. These presynaptic defects could be relevant to the onset of early cognitive impairments and acute neuroinflammation and demyelinating processes observed in multiple sclerosis patients.

Topics: Age Factors; Animals; Animals, Newborn; Central Nervous System; Chemokine CCL5; Colforsin; D-Aspartic Acid; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Exocytosis; Female; Glutamic Acid; In Vitro Techniques; Mice; Mice, Inbred C57BL; Myelin-Oligodendrocyte Glycoprotein; Peptide Fragments; Potassium Chloride; Second Messenger Systems; Synaptosomes; Time Factors; Tritium

The interactions between a prior program of regular exercise and the development of experimental autoimmune encephalomyelitis (EAE)-mediated responses were evaluated. In the exercised EAE mice, although there was no effect on infiltrated cells, the cytokine and derived neurotrophic factor (BDNF) levels were altered, and the clinical score was attenuated. Although, the cytokine levels were decreased in the brain and increased in the spinal cord, BDNF was elevated in both compartments with a tendency of lesser demyelization volume in the spinal cord of the exercised EAE group compared with the unexercised.

Topics: Analysis of Variance; Animals; Brain; Brain-Derived Neurotrophic Factor; Cytokines; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Enzyme-Linked Immunosorbent Assay; Exercise Therapy; Female; Freund's Adjuvant; Leukocyte Count; Mice; Mice, Inbred C57BL; Myelin-Oligodendrocyte Glycoprotein; Peptide Fragments; Physical Conditioning, Animal; Spinal Cord; Swimming; Time Factors

Optical neuritis (ON) is characterized by inflammation of the optic nerve, and is one of the first clinical signs of multiple sclerosis (MS). Experimental autoimmune encephalomyelitis (EAE) is the animal model used to study MS and ON. The present study evaluated the induction, development and progression of ON using an EAE model induced by 100 μg or 300 μg of MOG35-55. An EAE model was induced in C57BL/6 mice by tail base injection of 100 μg or 300 μg of MOG35-55 in complete Freund's adjuvant, supplemented with Mycobacterium tuberculosis. On the day of injection and 48 h later, animals received intraperitoneally 300 ng of pertussis toxin. On days 7, 10, 14, 21 and 58 the optic nerve was dissected for histological analysis, production of CCL5 and immunohistochemical detection of CD4 and CD8. The histological changes observed in the optic nerves consisted of inflammatory cell infiltrates showing varying degrees of ON in the two groups. The onset of ON in the 300 μg of MOG35-55 group was coincident with higher production of CCL5, on day 10 after induction. However, the 100 μg MOG35-55 group showed more intense inflammatory infiltrate on day 14 after induction, with higher amounts of CD4 and CD8, reaching an excessive demyelination process on days 21 and 58 after induction. The results suggest that two different concentrations of MOG35-55 lead to different forms of evolution of optic neuritis.

Topics: Animals; CD4 Antigens; CD4-Positive T-Lymphocytes; CD8 Antigens; CD8-Positive T-Lymphocytes; Chemokine CCL5; Disease Models, Animal; Dose-Response Relationship, Drug; Encephalomyelitis, Autoimmune, Experimental; Female; Humans; Inflammation; Inflammation Mediators; Mice; Mice, Inbred C57BL; Multiple Sclerosis; Myelin-Oligodendrocyte Glycoprotein; Optic Neuritis; Peptide Fragments

Multiple sclerosis (MS) is a complex multifactorial disease of the central nervous system (CNS) for which animal models have mainly addressed downstream immunopathology but not potential inducers of autoimmunity. In the absence of a pathogen known to cause neuroinflammation in MS, Mycobacterial lysate is commonly used in the form of complete Freund's adjuvant to induce autoimmunity to myelin proteins in Experimental Allergic Encephalomyelitis (EAE), an animal model for MS. The present study demonstrates that a protein from the human endogenous retrovirus HERV-W family (MSRV-Env) can be used instead of mycobacterial lysate to induce autoimmunity and EAE in mice injected with MOG, with typical anti-myelin response and CNS lesions normally seen in this model. MSRV-Env was shown to induce proinflammatory response in human macrophage cells through TLR4 activation pathway. The present results demonstrate a similar activation of murine dendritic cells and show the ability of MSRV-Env to trigger EAE in mice. In previous studies, MSRV-Env protein was reproducibly detected in MS brain lesions within microglia and perivascular macrophages. The present results are therefore likely to provide a model for MS, in which the upstream adjuvant triggering neuroinflammation is the one detected in MS active lesions. This model now allows pre-clinical studies with therapeutic agents targeting this endogenous retroviral protein in MS.

Topics: Adjuvants, Immunologic; Animals; Cells, Cultured; Central Nervous System; Dendritic Cells; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Gene Expression; Gene Products, env; Humans; Immunity, Innate; Lipopolysaccharide Receptors; Mice; Mice, Inbred C57BL; Mice, Knockout; Multiple Sclerosis; Myelin-Oligodendrocyte Glycoprotein; Peptide Fragments; Pregnancy Proteins; Recombinant Proteins; Toll-Like Receptor 4

Fetal neural stem/precursor cells (NPCs) possess powerful immunomodulatory properties which enable them to protect the brain from immune-mediated injury. A major issue in developing neural stem/precursor cell (NPC) therapy for chronic neuroinflammatory disorders such as multiple sclerosis is whether cells maintain their immune-regulatory properties for prolonged periods of time. Therefore, we studied time-associated changes in NPC immunomodulatory properties. We examined whether intracerebrally-transplanted NPCs are able to inhibit early versus delayed induction of autoimmune brain inflammation and whether allogeneic NPC grafts continuously inhibit host rejection responses. In two experimental designs, intraventricular fetal NPC grafts attenuated clinically and pathologically brain inflammation during early EAE relapse but failed to inhibit the disease relapse if induced at a delayed time point. In correlation, long-term cultured neural precursors lost their capacity to inhibit immune cell proliferation in vitro. Loss of NPC immune functions was associated with transition into a quiescent undifferentiated state. Also, allogeneic fetal NPC grafts elicited a strong immune reaction of T cell and microglial infiltration and were rejected from the host brain. We conclude that long-term functional changes in transplanted neural precursor cells lead to loss of their therapeutic immune-regulatory properties, and render allogeneic grafts vulnerable to immunologic rejection. Thus, the immunomodulatory effects of neural precursor cell transplantation are limited in time.

Topics: Adrenergic Agents; Animals; Cell Proliferation; Corpus Striatum; Cytokines; Disease Models, Animal; Embryo, Mammalian; Encephalitis; Encephalomyelitis, Autoimmune, Experimental; Female; Glial Fibrillary Acidic Protein; Graft Rejection; Graft Survival; Green Fluorescent Proteins; Interferon-gamma; Lymphocytes; Macrophages; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Transgenic; Microglia; Myelin-Oligodendrocyte Glycoprotein; Neural Stem Cells; Neurotoxicity Syndromes; Oxidopamine; Peptide Fragments; Pregnancy; Stem Cell Transplantation; Time Factors

β-Lapachone is a naturally occurring quinine, originally isolated from the bark of the lapacho tree (Tabebuia avellanedae) which is currently being evaluated in clinical trials for the treatment of cancer. In addition, recent investigations suggest its potential application for treatment of inflammatory diseases. Multiple sclerosis (MS) is an autoimmune disorder characterized by CNS inflammation and demyelination. Reactive T cells including IL-17 and IFN-γ-secreting T cells are believed to initiate MS and the associated animal model system experimental autoimmune encephalomyelitis (EAE). IL-12 family cytokines secreted by peripheral dendritic cells (DCs) and CNS microglia are capable of modulating T-cell phenotypes. The present studies demonstrated that β-lapachone selectively inhibited the expression of IL-12 family cytokines including IL-12 and IL-23 by DCs and microglia, and reduced IL-17 production by CD4(+) T-cells indirectly through suppressing IL-23 expression by microglia. Importantly, our studies also demonstrated that β-lapachone ameliorated the development on EAE. β-Lapachone suppression of EAE was associated with decreased expression of mRNAs encoding IL-12 family cytokines, IL-23R and IL-17RA, and molecules important in Toll-like receptor signaling. Collectively, these studies suggest mechanisms by which β-lapachone suppresses EAE and suggest that β-lapachone may be effective in the treatment of inflammatory diseases such as MS.

Topics: Analysis of Variance; Animals; Animals, Newborn; Anti-Inflammatory Agents, Non-Steroidal; Bone Marrow Cells; CD4-Positive T-Lymphocytes; Cell Survival; Cells, Cultured; Cerebral Cortex; Cytokines; Dendritic Cells; Disease Models, Animal; Dose-Response Relationship, Drug; Drug Interactions; Encephalomyelitis, Autoimmune, Experimental; Enzyme-Linked Immunosorbent Assay; Freund's Adjuvant; Mice; Mice, Inbred C57BL; Microglia; Myelin-Oligodendrocyte Glycoprotein; Naphthoquinones; Peptide Fragments; Polysaccharides; Severity of Illness Index; Spleen; Statistics, Nonparametric; Time Factors

Vorinostat, a histone deacetylase inhibitor, has been used clinically as an anticancer drug and also has immunosuppressive properties. However, the underlying mechanisms of effects of vorinostat on central nervous system (CNS) inflammatory diseases remain incomplete. Here, this study investigates the effects of vorinostat on human CD14(+) monocyte-derived dendritic cells (DCs) and mouse immature DC in vitro. Furthermore, we explore the therapeutic effects and cellular mechanisms of vorinostat on animal model of multiple sclerosis, experimental autoimmune encephalomyelitis (EAE) in vivo. Our findings demonstrate that vorinostat inhibited human CD14(+) monocyte-derived DCs differentiation, maturation, endocytosis, and further inhibited mDCs' stimulation of allogeneic T-cell proliferation. In addition, vorinostat inhibited DC-directed Th1- (Type 1T helper) and Th17-polarizing cytokine production. Furthermore, vorinostat ameliorated Th1- and Th17-mediated EAE by reducing CNS inflammation and demyelination. What's more, Th1 and Th17 cell functions were suppressed in vorinostat-treated EAE mice. Finally, vorinostat suppressed expression of costimulatory molecules of DC in EAE mice. These suggest therapeutic effects of vorinostat on EAE which may by suppress DCs and DCs-mediated Th1 and Th17 cell functions. Our findings warrant further investigation in the potential of vorinostat for the treatment of human multiple sclerosis.

Topics: Animals; Antigens, CD; Cell Count; Cell Differentiation; Cell Polarity; Cell Proliferation; Cells, Cultured; Cytokines; Dendritic Cells; Disease Models, Animal; Dose-Response Relationship, Drug; Encephalomyelitis, Autoimmune, Experimental; Female; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Lipopolysaccharides; Lymphocyte Activation; Lymphocytes; Mice; Mice, Inbred C57BL; Myelin-Oligodendrocyte Glycoprotein; Peptide Fragments; RNA, Messenger; Vorinostat

Multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis (EAE), are autoimmune diseases characterized by the immune-mediated demyelination and neurodegeneration of the CNS. Overactivation of CD4(+) T cells, especially the Th1 and Th17 subpopulations, is thought to be the direct cause of this disease. Aurintricarboxylic acid (ATA), an inhibitor of protein-nucleic acid interaction, has been reported to block with the JAK/STAT signaling pathway that is critical for Th cell differentiation. In this study, we discovered that ATA treatment significantly reduces the clinical score of EAE, but it does not directly inhibit the differentiation of Th1 and Th17 cells in vitro. ATA was found to block the chemotaxis and accumulation of dendritic cells in the spleen of EAE mice before the onset of the disease and to reduce the percentage of Th1 and Th17 cells in the spleen. Further study revealed that ATA also blocks the infiltration of pathogenic T cells into the CNS and blocks the onset of passive EAE. ATA was found to inhibit the functions of many chemokine receptors. By blocking chemokine-mediated migration of dendritic cells and pathogenic T cells, ATA alleviates the pathogenesis of EAE and might be used to treat autoimmune diseases, including multiple sclerosis.

Topics: Adoptive Transfer; Amino Acid Sequence; Animals; Apoptosis; Aurintricarboxylic Acid; CD4-Positive T-Lymphocytes; Cells, Cultured; Central Nervous System; Chemotaxis, Leukocyte; Dendritic Cells; Disease Models, Animal; Drug Evaluation, Preclinical; Encephalomyelitis, Autoimmune, Experimental; Female; Lymphopoiesis; Mice; Mice, Inbred C57BL; Models, Immunological; Molecular Sequence Data; Myelin-Oligodendrocyte Glycoprotein; Peptide Fragments; Receptors, Chemokine; Spleen; Th17 Cells

Multiple sclerosis is associated with a high incidence of depression, cognitive impairments and neuropathic pain. Previously, we demonstrated that tactile allodynia is present at disease onset in an animal model of MS, experimental autoimmune encephalomyelitis (EAE). We have now monitored changes in object recognition in mice with EAE to determine if altered nociceptive sensitivity is also associated with behavioral signs indicative of cognitive impairment in this model. At the onset of clinical signs, mice with EAE showed impairments in the novel object recognition (NOR) assay, indicative of deficits in cognitive functioning early in the disease course. At the spinal level, we found increased gene expression for the cytokines IL-1β, IL-6 and the glutamate transporter EAAT-2 that coincide with increased nociceptive sensitivity and deficits in object recognition. Increased levels of EAAT-2 mRNA appear to be a response to perturbed protein levels of the transporter as we found a loss of EAAT-2 protein levels in the spinal cord of EAE mice. To determine if changes in the levels of EAAT-2 were responsible for the observed changes in nociceptive sensitivity and cognitive deficits, we treated EAE mice with the β-lactam antibiotic ceftriaxone, an agent known to increase glutamate transporter levels in vivo. Ceftriaxone prevented tactile hypersensitivity and normalized performance in the NOR assay in EAE mice. These findings highlight the important interrelationship between pain and cognitive function in the disease and suggest that targeting spinally mediated pain hypersensitivity is a novel therapeutic avenue to treat impairments in other higher order cortical processes.

Topics: Analysis of Variance; Animals; Brain; Ceftriaxone; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Excitatory Amino Acid Transporter 2; Exploratory Behavior; Female; Freund's Adjuvant; Gene Expression Regulation; Hyperalgesia; Interleukin-1beta; Interleukin-6; Mice; Mice, Inbred C57BL; Motor Activity; Myelin-Oligodendrocyte Glycoprotein; Pain Measurement; Pain Threshold; Peptide Fragments; Receptors, N-Methyl-D-Aspartate; Recognition, Psychology; RNA, Messenger; Rotarod Performance Test; Spinal Cord; Time Factors

The present study aimed to evaluate the therapeutic potential of clioquinol (CQ), a metal chelator, on multiple sclerosis pathogenesis. Experimental autoimmune encephalomyelitis was induced by immunization with myelin oligodendrocyte glycoprotein (MOG(35-55)) in female mice. Three weeks after the initial immunization, demyelination and immune cell infiltration in the spinal cord were analyzed. CQ (30mg/kg) was given by gavage once per day for the entire experimental course. CQ profoundly reduced the daily clinical score and incidence rate of EAE mice. The CQ-mediated inhibition of the clinical course of EAE was accompanied by suppression of demyelination and reduced infiltration by encephalitogenic immune cells including CD4, CD8, CD20 and F4/80 positive cells. CQ also remarkably inhibited EAE-associated BBB disruption and MMP-9 activation. Autophagy contributes to clearance of aggregated proteins in astrocytes and neurons. The present study found that EAE increased the induction of autophagy and CQ further increased this expression. Furthermore, the present study found that post-treatment with CQ also reduced the clinical score of EAE and spinal cord demyelination. These results demonstrate that CQ inhibits the clinical features and neuropathological changes associated with EAE. The present study suggests that transition metals may be involved in several steps of multiple sclerosis pathogenesis.

Topics: Animals; Axons; Behavior, Animal; Blotting, Western; Chelating Agents; Clioquinol; Copper; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Female; Mice; Mice, Inbred C57BL; Multiple Sclerosis; Myelin-Oligodendrocyte Glycoprotein; Peptide Fragments; Spinal Cord; Zinc

During immune-mediated demyelinating lesions, the endocannabinoid system is involved in the pathogenesis of both neuroinflammation and neurodegeneration through different mechanisms. Here we explored the cellular distribution of the CB1 receptor (CB1R) in the central nervous system (CNS) and detected the level of CB1R expression during experimental autoimmune encephalomyelitis (EAE) by RT-qPCR, Western blotting, and immunostaining. Expression of CB1R was observed in neurons and microglia/macrophages but was barely detected in astrocytes. During EAE, the expression of CB1R in spinal cords was reduced at days 9, 17, and 28 postimmunization (p.i.), but the level of CB1R expression in spleens did not show a significant difference compared with complete Freund's adjuvant (CFA)-immunized mice. A selective CB1R antagonist (SR141716A) increased EAE clinical score, accompanied by weight loss. Unexpectedly, SR141716A inhibited the expression of CB1R but increased the expression of CB2R in brains, spinal cords, and spleens simultaneously. The administration of SR141716A increased interferon-γ, interleukin-17 (IL-17), and inflammatory cytokines such as IL-1β, IL-6, and tumor necrosis factor-α in brains and/or spinal cords. A similar increase was observed in spontaneous and specific antigen-stimulated splenic mononuclear cells compared with vehicle controls. Interestingly, the expression of CX3CL1 was increased in brains and spinal cords but declined in spleens of EAE mice treated with SR141716A. These results indicate that manipulation of the CB1R may have therapeutic value in MS, but its complexity remains to be carefully considered and studied in further clinical application.

Topics: Analysis of Variance; Animals; Cells, Cultured; Cytokines; Disease Models, Animal; Embryo, Mammalian; Encephalomyelitis, Autoimmune, Experimental; Enzyme-Linked Immunosorbent Assay; Female; Glycoproteins; Hippocampus; Mice; Mice, Inbred C57BL; Myelin-Oligodendrocyte Glycoprotein; Neuroglia; Neurons; Peptide Fragments; Piperidines; Pyrazoles; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2; Rimonabant; RNA, Messenger; Spinal Cord; Spleen

Our earlier studies described a disruption of heart rate and blood pressure diurnal rhythms in mice with experimental autoimmune encephalomyelitis (EAE). The present study investigates whether these observations could be extended to additional clock-regulated rhythms in mice with EAE. Analysis of clock gene expression in the liver of EAE mice demonstrated significant variability associated with Per2 rhythmic expression. Corticosterone and leptin hormone rhythms were also altered in EAE mice. The results presented here indicate that disturbances in clock-regulated rhythms are associated with EAE and present a suitable model for investigating the relationship between circadian disruption and autoimmune inflammatory disease.

Topics: Animals; Chronobiology Disorders; Circadian Rhythm; CLOCK Proteins; Corticosterone; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Gene Expression Regulation; Glycoproteins; Hypoxanthine Phosphoribosyltransferase; Leptin; Liver; Male; Mice; Mice, Inbred C57BL; Myelin-Oligodendrocyte Glycoprotein; Peptide Fragments; Period Circadian Proteins; Pertussis Toxin; T-Lymphocytes; Time Factors

Murine experimental autoimmune encephalomyelitis (EAE), a model for multiple sclerosis, presents typically as ascending paralysis. However, in mice in which interferon-gamma (IFNγ) signaling is disrupted by genetic deletion, limb paralysis is accompanied by atypical deficits, including head tilt, postural imbalance, and circling, consistent with cerebellar/vestibular dysfunction. This was previously attributed to intense cerebellar and brainstem infiltration by peripheral immune cells and formation of neutrophil-rich foci within the CNS. However, the exact mechanism by which IFNγ signaling prohibits the development of vestibular deficits, and whether the distribution and composition of inflammatory foci within the CNS affects the course of atypical EAE remains elusive.. We induced EAE in IFNγ-/- mice and bone marrow chimeric mice in which IFNγR is not expressed in the CNS but is intact in the periphery (IFNγRCNSKO) and vice versa (IFNγRperiKO). Blood-brain barrier permeability was determined by Evans blue intravenous administration at disease onset. Populations of immune cell subsets in the periphery and the CNS were quantified by flow cytometry. CNS tissues isolated at various time points after EAE induction, were analyzed by immunohistochemistry for composition of inflammatory foci and patterns of axonal degeneration.. Incidence and severity of atypical EAE were more pronounced in IFNγRCNSKO as compared to IFNγRperiKO mice. Contrary to what we anticipated, cerebella/brainstems of IFNγRCNSKO mice were only minimally infiltrated, while the same areas of IFNγRperiKO mice were extensively populated by peripheral immune cells. Furthermore, the CNS of IFNγRperiKO mice was characterized by persistent neutrophil-rich foci as compared to IFNγRCNSKO. Immunohistochemical analysis of the CNS of IFNγ-/- and IFNγR chimeric mice revealed that IFNγ protective actions are exerted through microglial STAT1.. Alterations in distribution and composition of CNS inflammatory foci are not sufficient for the onset of atypical EAE. IFNγ dictates the course of neuroinflammatory disorders mainly through actions exerted within the CNS. This study provides strong evidence that link microglial STAT1 inactivation to vestibular dysfunction.

Topics: Animals; Antigens, CD; Blood-Brain Barrier; Central Nervous System; Cytokines; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Flow Cytometry; Glycoproteins; Interferon-gamma; Leukocyte Common Antigens; Leukocytes; Lymph Nodes; Mice; Mice, Inbred C57BL; Mice, Knockout; Myelin-Oligodendrocyte Glycoprotein; Nervous System Diseases; Neutrophil Infiltration; Peptide Fragments; Signal Transduction; Spleen

The NF-κB/REL-family of transcription factors plays a central role in coordinating the expression of a wide variety of genes controlling immune responses including autoimmunity of the central nervous system (CNS). The inactive form of NF-κB consists of a heterodimer which is complexed with its inhibitor, IκB. Conditional knockout-mice for IκBα in myeloid cells (lysMCreIκBα(fl/fl)) have been generated and are characterized by a constitutive activation of NF-κB proteins allowing the study of this transcription factor in myelin-oligodendrocyte-glycoprotein induced experimental autoimmune encephalomyelitis (MOG-EAE), a well established experimental model for autoimmune demyelination of the CNS.In comparison to controls, lysMCreIκBα(fl/fl) mice developed a more severe clinical course of EAE. Upon histological analysis on day 15 p.i., there was an over two fold increased infiltration of T-cells and macrophages/microglia. In addition, lysMCreIκBα(fl/fl) mice displayed an increased expression of the NF-κB dependent factor inducible nitric oxide synthase in inflamed lesions. These changes in the CNS are associated with increased numbers of CD11b positive splenocytes and a higher expression of Ly6c on monocytes in the periphery. Well in accordance with these changes in the myeloid cell compartment, there was an increased production of the monocyte cytokines interleukin(IL)-12 p70, IL-6 and IL-1beta in splenocytes. In contrast, production of the T-cell associated cytokines interferon gamma (IFN-gamma) and IL-17 was not influenced.In summary, myeloid cell derived NF-κB plays a crucial role in autoimmune inflammation of the CNS and drives a pathogenic role of monocytes and macrophages independently from T-cells.

Topics: Animals; CD11b Antigen; Cell Proliferation; Cells, Cultured; Cytokines; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Enzyme-Linked Immunosorbent Assay; Flow Cytometry; Glycoproteins; I-kappa B Proteins; Macrophages; Mice; Mice, Inbred C57BL; Mice, Transgenic; Monocytes; Myelin-Oligodendrocyte Glycoprotein; Neutrophil Infiltration; Nitric Oxide Synthase Type II; Peptide Fragments; Spleen

Current treatments used in Multiple Sclerosis (MS) are partly effective in the early stages of the disease but display very limited benefits in patients affected by progressive MS. One possible explanation is that these therapies are unable to target the inflammatory component most active during the progressive phase of the disease, and compartmentalized behind the blood-brain barrier. Our findings show that Rapamycin ameliorates clinical and histological signs of chronic EAE when administered during ongoing disease. Moreover, Rapamycin significantly reduced the hyperalgesia observed before clinical development of EAE which, in turn, is completely abolished by the administration of the drug.

Topics: Analysis of Variance; Animals; Cytokines; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Female; Gene Expression Regulation; Glial Fibrillary Acidic Protein; Glycoproteins; Hyperalgesia; Immunosuppressive Agents; Mice; Mice, Inbred C57BL; Myelin Basic Protein; Myelin Sheath; Myelin-Oligodendrocyte Glycoprotein; Neuralgia; Pain Threshold; Peptide Fragments; Pertussis Toxin; RNA, Messenger; Sirolimus; T-Lymphocytes; Time Factors

Although multiple sclerosis (MS) has traditionally been considered to be an inflammatory disease, recent evidence has brought neurodegeneration into the spotlight, suggesting that accumulated damage and loss of axons is critical to disease progression and the associated irreversible disability. Proposed mechanisms of axonal degeneration in MS posit cytosolic and subsequent mitochondrial Ca(2+) overload, accumulation of pathologic reactive oxygen species (ROS), and mitochondrial dysfunction leading to cell death. In this context, the role of the p66 isoform of ShcA protein (p66) may be significant. The ShcA isoform is uniquely targeted to the mitochondrial intermembrane space in response to elevated oxidative stress, and serves as a redox enzyme amplifying ROS generation in a positive feedforward loop that eventually mediates cell death by activation of the mitochondrial permeability transition pore. Consequently, we tested the hypothesis that genetic inactivation of p66 would reduce axonal injury in a murine model of MS, experimental autoimmune encephalomyelitis (EAE). As predicted, the p66-knockout (p66-KO) mice developed typical signs of EAE, but had less severe clinical impairment and paralysis than wild-type (WT) mice. Histologic examination of spinal cords and optic nerves showed significant axonal protection in the p66-KO tissue, despite similar levels of inflammation. Furthermore, cultured p66-KO neurons treated with agents implicated in MS neurodegenerative pathways showed greater viability than WT neurons. These results confirm the critical role of ROS-mediated mitochondrial dysfunction in the axonal loss that accompanies EAE, and identify p66 as a new pharmacologic target for MS neuroprotective therapeutics.

Topics: Animals; Axons; Cell Proliferation; Cells, Cultured; Cerebral Cortex; Cyclophilins; Cytokines; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Freund's Adjuvant; Glycoproteins; Hydrogen Peroxide; Leukemic Infiltration; Mice; Mice, Inbred C57BL; Mice, Knockout; Microscopy, Electron, Transmission; Myelin-Oligodendrocyte Glycoprotein; Nerve Fibers, Myelinated; Neurons; Optic Nerve; Peptide Fragments; Peptidyl-Prolyl Isomerase F; Shc Signaling Adaptor Proteins; Spinal Cord; Src Homology 2 Domain-Containing, Transforming Protein 1; T-Lymphocytes

Experimental autoimmune encephalomyelitis (EAE) is a widely used model of multiple sclerosis. In NOD mice, EAE develops as a relapsing-remitting disease that transitions to a chronic progressive disease, making the NOD model the only mouse model that recapitulates the full clinical disease course observed in most multiple sclerosis patients. We have generated a TCR transgenic mouse that expresses the α- and β-chains of a myelin oligodendrocyte glycoprotein (MOG) 35-55-reactive TCR (1C6) on the NOD background. 1C6 TCR transgenic mice spontaneously generate both CD4(+) and CD8(+) T cells that recognize MOG and produce proinflammatory cytokines, allowing for the first time to our knowledge the simultaneous examination of myelin-reactive CD4(+) and CD8(+) T cells in the same host. 1C6 CD8(+) T cells alone can induce optic neuritis and mild EAE with delayed onset; however, 1C6 CD4(+) T cells alone induce severe EAE and predominate in driving disease when both cell types are present. When 1C6 mice are crossed with mice bearing an IgH specific for MOG, the mice develop spontaneous EAE with high incidence, but surprisingly the disease pattern does not resemble the neuromyelitis optica-like disease observed in mice bearing CD4(+) T cells and B cells reactive to MOG on the C57BL/6 background. Collectively, our data show that although myelin-reactive CD8(+) T cells contribute to disease, disease is primarily driven by myelin-reactive CD4(+) T cells and that the coexistence of myelin-reactive T and B cells does not necessarily result in a distinct pathological phenotype.

Topics: Amino Acid Sequence; Animals; B-Lymphocyte Subsets; CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Female; Gene Knock-In Techniques; Glycoproteins; Humans; Male; Mice; Mice, Inbred C57BL; Mice, Inbred NOD; Mice, Transgenic; Molecular Sequence Data; Myelin-Oligodendrocyte Glycoprotein; Peptide Fragments

Prions, composed of a misfolded protein designated PrP(Sc), are infectious agents causing fatal neurodegenerative diseases. We have shown previously that, following induction of experimental autoimmune encephalomyelitis, prion-infected mice succumb to disease significantly earlier than controls, concomitant with the deposition of PrP(Sc) aggregates in inflamed white matter areas. In the present work, we asked whether prion disease acceleration by experimental autoimmune encephalomyelitis results from infiltration of viable prion-infected immune cells into the central nervous system.. C57Bl/6 J mice underwent intraperitoneal inoculation with scrapie brain homogenates and were later induced with experimental autoimmune encephalomyelitis by inoculation of MOG(35-55) in complete Freund's adjuvant supplemented with pertussis toxin. Spleen and lymph node cells from the co-induced animals were reactivated and subsequently injected into naïve mice as viable cells or as cell homogenates. Control groups were infected with viable and homogenized scrapie immune cells only with complete Freund's adjuvant. Prion disease incubation times as well as levels and sites of PrP(Sc) deposition were next evaluated.. We first show that acceleration of prion disease by experimental autoimmune encephalomyelitis requires the presence of high levels of spleen PrP(Sc). Next, we present evidence that mice infected with activated prion-experimental autoimmune encephalomyelitis viable cells succumb to prion disease considerably faster than do mice infected with equivalent cell extracts or other controls, concomitant with the deposition of PrP(Sc) aggregates in white matter areas in brains and spinal cords.. Our results indicate that inflammatory targeting of viable prion-infected immune cells to the central nervous system accelerates prion disease propagation. We also show that in the absence of such targeting it is the load of PrP(Sc) in the inoculum that determines the infectivity titers for subsequent transmissions. Both of these conclusions have important clinical implications as related to the risk of prion disease contamination of blood products.

Topics: Animals; Central Nervous System; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Glycoproteins; Humans; Lymphocytes; Mice; Mice, Inbred C57BL; Myelin-Oligodendrocyte Glycoprotein; Peptide Fragments; Prion Diseases; Prions

During central nervous system autoimmunity, interactions between infiltrating immune cells and brain-resident cells are critical for disease progression and ultimately organ damage. Here, we demonstrate that local cross-talk between invading autoreactive T cells and auto-antigen-presenting myeloid cells within the central nervous system results in myeloid cell activation, which is crucial for disease progression during experimental autoimmune encephalomyelitis, the animal model of multiple sclerosis. This T cell-mediated licensing of central nervous system myeloid cells triggered astrocytic CCL2-release and promoted recruitment of inflammatory CCR2(+)-monocytes, which are the main effectors of disease progression. By employing a cell-specific knockout model, we identify the nuclear receptor peroxisome proliferator-activated receptor γ (PPARγ) in myeloid cells as key regulator of their disease-determining interactions with autoreactive T cells and brain-resident cells, respectively. LysM-PPARγ(KO) mice exhibited disease exacerbation during the effector phase of experimental autoimmune encephalomyelitis characterized by enhanced activation of central nervous system myeloid cells accompanied by pronounced local CCL2 production and inflammatory monocyte invasion, which finally resulted in increased demyelination and neuronal damage. Pharmacological PPARγ activation decreased antigen-specific T cell-mediated licensing of central nervous system myeloid cells, reduced myeloid cell-mediated neurotoxicity and hence dampened central nervous system autoimmunity. Importantly, human monocytes derived from patients with multiple sclerosis clearly responded to PPARγ-mediated control of proinflammatory activation and production of neurotoxic mediators. Furthermore, PPARγ in human monocytes restricted their capacity to activate human astrocytes leading to dampened astrocytic CCL2 production. Together, interference with the disease-promoting cross-talk between central nervous system myeloid cells, autoreactive T cells and brain-resident cells represents a novel therapeutic approach that limits disease progression and lesion development during ongoing central nervous system autoimmunity.

Topics: Analysis of Variance; Animals; Animals, Newborn; Antigens, CD; Autoimmunity; CD4-Positive T-Lymphocytes; Cell Differentiation; Cells, Cultured; Central Nervous System; Cerebellum; Coculture Techniques; Cytokines; Disease Models, Animal; Embryo, Mammalian; Encephalomyelitis, Autoimmune, Experimental; Flow Cytometry; Freund's Adjuvant; Gene Knockdown Techniques; Glycoproteins; Green Fluorescent Proteins; Hippocampus; Humans; Hypoglycemic Agents; Mice; Mice, Congenic; Mice, Inbred C57BL; Mice, Knockout; Monocytes; Myelin-Oligodendrocyte Glycoprotein; Myeloid Cells; Neuroglia; Neurons; Peptide Fragments; Pioglitazone; PPAR gamma; Receptors, CCR2; RNA, Small Interfering; T-Lymphocytes; Thiazolidinediones

The abundant axonal microtubule-associated protein tau regulates microtubule and actin dynamics, thereby contributing to normal neuronal function. We examined whether mice deficient in tau (Tau(-/-)) or with high levels of human tau differ from wild-type (WT) mice in their susceptibility to neuroaxonal injury in experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis. After sensitization with MOG35-55, there was no difference in clinical disease course between human tau and WT mice, but Tau mice had more severe clinical disease and significantly more axonal damage in spinal cord white matter than those in WT mice. Axonal damage in gray matter correlated with clinical severity in individual mice. By immunoblot analysis, the early microtubule-associated protein-1b was increased 2-fold in the spinal cords of Tau mice with chronic experimental autoimmune encephalomyelitis versus naive Tau mice. This difference was not detected in comparable WT animals, which suggests that there was compensation for the loss of tau in the deficient mice. In addition, levels of the growth arrest-specific protein 7b, a tau-binding protein that is stabilized when bound to tau, were higher in WT than those in Tau(-/-) spinal cord samples. These data indicate that loss of tau exacerbates experimental autoimmune encephalomyelitis and suggest that maintaining tau integrity might reduce the axonal damage that occurs in inflammatory neurodegenerative diseases such as multiple sclerosis.

Topics: Age Factors; Animals; Axons; Cytoskeletal Proteins; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Freund's Adjuvant; Gene Expression Regulation; Genetic Predisposition to Disease; Glycoproteins; Humans; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Myelin-Oligodendrocyte Glycoprotein; Nerve Fibers, Myelinated; Nerve Tissue Proteins; Neurons; Paralysis; Peptide Fragments; Phosphorylation; Spinal Cord; tau Proteins

Natalizumab inhibits the influx of leukocytes into the central nervous system (CNS) via blockade of alpha-4 subunit of very late activation antigen (VLA)-4. The association of natalizumab therapy with progressive multifocal leukoencephalopathy (PML) suggests a disturbance of CNS immune surveillance in a small percentage of Multiple Sclerosis (MS) patients exposed to the medication. Natural killer (NK) cells are known to play an important role in modulating the evolution of different phases of this lymphocyte mediated disease, and we investigated the effects of natalizumab on the NK cell phenotype and infiltration in the CNS in experimental autoimmune encephalomyelitis (EAE), a murine model of MS. Our data show that both resting (from naïve mice) and activated (from EAE mice) NK cells express high levels of VLA-4, and anti-VLA-4 antibody treatment significantly decreases NK cells frequency in the CNS of EAE mice. Moreover, we find that anti-VLA-4 possibly impairs NK cells migratory potential, since unblocked VLA-4 expression levels were downregulated on those NK cells that penetrate the CNS. These data suggest that treatment with antibody to VLA-4 may alter immune surveillance of the CNS by impacting NK cell functions and might contribute to the understanding of the mechanisms leading to the development of PML in some MS patients.

Topics: Animals; Antibodies; Antigens, CD; Cell Movement; Central Nervous System; Disease Models, Animal; Down-Regulation; Encephalomyelitis, Autoimmune, Experimental; Female; Flow Cytometry; Glycoproteins; Integrin alpha4; Killer Cells, Natural; Leukocytes, Mononuclear; Mice; Mice, Inbred C57BL; Mice, SCID; Myelin-Oligodendrocyte Glycoprotein; Neutrophil Infiltration; Peptide Fragments

IL-33 is a recently described member of the IL-1 family that has been reported to have a pathogenic role in several inflammatory diseases. In this study, we evaluated the role of IL-33 in a murine model of multiple sclerosis, experimental autoimmune encephalomyelitis (EAE). We showed that the expression of IL-33 and its receptor, ST2, was markedly elevated in the spinal cord of mice during myelin oligodendrocyte glycoprotein (MOG)(35-55) peptide-induced EAE. Administration of a blocking anti-IL-33 antibody in mice of EAE during the induction phase significantly inhibited the onset and severity of EAE and reduced MOG(35-55)-induced IFN-γ and IL-17 production. In contrast, treatment with recombinant IL-33 worsened the disease course of EAE in association with increased induction of both IFN-γ and IL-17. Furthermore, anti-IL-33 treatment caused a remarkable decrease in expression of IL-17, IFN-γ, T-bet and RORγt, and an upregulation of IL-10 and TGF-β in the spinal cord of EAE mice. These results demonstrate that endogenous IL-33 plays a pivotal role in the pathogenesis of EAE and indicate that blockade of IL-33 has a significant protective effect against EAE.

Topics: Analysis of Variance; Animals; Antibodies; Cytokines; Disease Models, Animal; Dose-Response Relationship, Immunologic; Encephalomyelitis, Autoimmune, Experimental; Enzyme-Linked Immunosorbent Assay; Female; Gene Expression Regulation; Glycoproteins; Interleukin-1 Receptor-Like 1 Protein; Interleukin-33; Interleukins; Mice; Mice, Inbred C57BL; Myelin-Oligodendrocyte Glycoprotein; Peptide Fragments; Receptors, Interleukin; RNA, Messenger; Spinal Cord; T-Lymphocytes, Helper-Inducer; Time Factors

Intravenous (i.v.) administration of autoantigen effectively induces Ag-specific tolerance against experimental autoimmune encephalomyelitis (EAE). We and others have shown enhanced EAE severity in mice lacking IL-12 or its receptor, strongly suggesting an immunoregulatory effect of IL-12 signaling. To examine the role of IL-12 responsiveness in autoantigen-induced tolerance in EAE, we administered autoantigen i.v. in two distinct treatment regimes to wildtype and IL-12Rβ2(-/-) mice, immunized to develop EAE. Administration at the induction phase suppressed EAE in wildtype and IL-12Rβ2(-/-) mice however the effect was somewhat less potent in the absence of IL-12Rβ2. Expression of pro-inflammatory cytokines such as IFN-γ, IL-17 and IL-2, was inhibited in wild-type tolerized mice but less so in IL-12Rβ2(-/-) mice. I.v. antigen was also effective in suppressing disease in both genotypes when given during the clinical phase of disease with similar CNS inflammation, demyelination and peripheral inflammatory cytokine profiles observed in both genotypes. There was however a mild impact of a lack of IL-12 signaling on Treg induction during tolerance induction compared to WT mice in this treatment regime. These findings show that the enhanced severity of EAE that occurs in the absence of IL-12 signaling can be effectively overcome by i.v. autoantigen, indicating that this therapeutic effect is not primarily mediated by IL-12 and that i.v. tolerance could be a powerful approach in suppressing severe and aggressive MS.

Topics: Animals; Cell Proliferation; Central Nervous System; Cytokines; Disease Models, Animal; Dose-Response Relationship, Immunologic; Encephalomyelitis, Autoimmune, Experimental; Female; Flow Cytometry; Glycoproteins; Immune Tolerance; Injections, Intravenous; Interleukin-2 Receptor beta Subunit; Leukocytes, Mononuclear; Mice; Mice, Inbred C57BL; Mice, Knockout; Myelin-Oligodendrocyte Glycoprotein; Peptide Fragments; Signal Transduction; Statistics, Nonparametric; Time Factors

Multiple sclerosis involves demyelination and axonal degeneration of the central nervous system. The molecular mechanisms of axonal degeneration are relatively unexplored in both multiple sclerosis and its mouse model, experimental autoimmune encephalomyelitis. We previously reported that targeting the axonal growth inhibitor, Nogo-A, may protect against neurodegeneration in experimental autoimmune encephalomyelitis; however, the mechanism by which this occurs is unclear. We now show that the collapsin response mediator protein 2 (CRMP-2), an important tubulin-associated protein that regulates axonal growth, is phosphorylated and hence inhibited during the progression of experimental autoimmune encephalomyelitis in degenerating axons. The phosphorylated form of CRMP-2 (pThr555CRMP-2) is localized to spinal cord neurons and axons in chronic-active multiple sclerosis lesions. Specifically, pThr555CRMP-2 is implicated to be Nogo-66 receptor 1 (NgR1)-dependent, since myelin oligodendrocyte glycoprotein (MOG)(35-55)-induced NgR1 knock-out (ngr1(-)(/)(-)) mice display a reduced experimental autoimmune encephalomyelitis disease progression, without a deregulation of ngr1(-)(/)(-) MOG(35-55)-reactive lymphocytes and monocytes. The limitation of axonal degeneration/loss in experimental autoimmune encephalomyelitis-induced ngr1(-)(/)(-) mice is associated with lower levels of pThr555CRMP-2 in the spinal cord and optic nerve during experimental autoimmune encephalomyelitis. Furthermore, transduction of retinal ganglion cells with an adeno-associated viral vector encoding a site-specific mutant T555ACRMP-2 construct, limits optic nerve axonal degeneration occurring at peak stage of experimental autoimmune encephalomyelitis. Therapeutic administration of the anti-Nogo(623-640) antibody during the course of experimental autoimmune encephalomyelitis, associated with an improved clinical outcome, is demonstrated to abrogate the protein levels of pThr555CRMP-2 in the spinal cord and improve pathological outcome. We conclude that phosphorylation of CRMP-2 may be downstream of NgR1 activation and play a role in axonal degeneration in experimental autoimmune encephalomyelitis and multiple sclerosis. Blockade of Nogo-A/NgR1 interaction may serve as a viable therapeutic target in multiple sclerosis.

Topics: Adult; Analysis of Variance; Animals; Antibodies; Axons; CD3 Complex; Cell Line, Tumor; Demyelinating Diseases; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Female; Gene Expression Regulation; Glycoproteins; GPI-Linked Proteins; Green Fluorescent Proteins; Humans; Immunoprecipitation; Intercellular Signaling Peptides and Proteins; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Middle Aged; Multiple Sclerosis; Mutation; Myelin Proteins; Myelin-Oligodendrocyte Glycoprotein; Nerve Degeneration; Nerve Tissue Proteins; Neuroblastoma; Neurofilament Proteins; Nogo Receptor 1; Optic Nerve; Peptide Fragments; Phosphorylation; Receptors, Cell Surface; Retinal Ganglion Cells; Severity of Illness Index; Silver Staining; Spinal Cord; tau Proteins; Time Factors; Transduction, Genetic; Tubulin

The development of experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis, has been studied in mice that were (i) vitamin D-deficient, (ii) minus the vitamin D receptor, (iii) minus a vitamin D 25-hydroxylase, and (iv) minus the vitamin D 25-hydroxyvitamin D-1α-hydroxylase. EAE development was markedly suppressed in mice lacking the vitamin D receptor and partially suppressed in vitamin D-insufficient mice. However, the absence of either of the two key hydroxylases (i.e., 25-hydroxylase and 1α-hydroxylase) neither inhibits nor enhances the development of EAE. These results indicate that vitamin D and the vitamin D receptor are required for the development of EAE. The results also suggest that 1,25-dihydroxyvitamin D(3) may not play a role in this autoimmune response.

Topics: 25-Hydroxyvitamin D3 1-alpha-Hydroxylase; Animals; Body Weight; Calcium; Cholestanetriol 26-Monooxygenase; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Female; Glycoproteins; Humans; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Myelin-Oligodendrocyte Glycoprotein; Peptide Fragments; Receptors, Calcitriol; Vitamin D; Vitamin D Deficiency

Studies with multiple sclerosis patients and animal models of experimental autoimmune encephalomyelitis (EAE) implicate adenosine and adenosine receptors in modulation of neuroinflammation and brain injury. Although the involvement of the A(1) receptor has been recently demonstrated, the role of the adenosine A(2A) receptor (A(2A)R) in development of EAE pathology is largely unknown. Using mice with genetic inactivation of the A(2A) receptor, we provide direct evidence that loss of the A(2A)R exacerbates EAE pathology in mice. Compared with wild-type mice, A(2A)R knockout mice injected with myelin oligodendroglia glycoprotein peptide had a higher incidence of EAE and exhibited higher neurological deficit scores and greater decrease in body weight. A(2A)R knockout mice displayed increased inflammatory cell infiltration and enhanced microglial cell activation in cortex, brainstem, and spinal cord. In addition, demyelination and axonal damage in brainstem were exacerbated, levels of Th1 cytokines increased, and Th2 cytokines decreased. Collectively, these findings suggest that extracellular adenosine acting at A(2A)Rs triggers an important neuroprotective mechanism. Thus, the A(2A) receptor is a potential target for therapeutic approaches to multiple sclerosis.

Topics: Adenosine A1 Receptor Antagonists; Animals; Astrocytes; Axons; Brain Injuries; Cell Proliferation; Cells, Cultured; Cerebral Cortex; Cytokines; Demyelinating Diseases; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Enzyme-Linked Immunosorbent Assay; Female; Filtration; Flow Cytometry; Freund's Adjuvant; Gene Expression Regulation; Mice; Mice, Inbred C57BL; Mice, Knockout; Microglia; Myelin-Oligodendrocyte Glycoprotein; Peptide Fragments; Protein Binding; Receptor, Adenosine A2A; RNA, Messenger; Spinal Cord; Spleen; Statistics, Nonparametric; Tritium; Xanthines

There is a growing evidence to suggest that the antecedents of some adult diseases can be traced back to their fetal origins. Despite extensive research on such diseases, to our knowledge, there has been no research investigating the relationship between the origins of multiple sclerosis (MS) disease and maternal infections. The aim of this study was to examine the role of prenatal exposure to endotoxin in fetal programming with respect to induction of susceptibility/vulnerability to MS.. The pregnant dams were administered a single intraperitoneal injection of lipopolysaccharide in gestational day 10. The male offspring were weighed and examined for clinical signs of experimental autoimmune encephalomyelitis in a blinded fashion within 36 days after immunization (postnatal day 63-98).. Our data provide the evidence showing prenatal exposures to higher doses of Lipopolysaccharide resulted in an earlier onset of the disease, an augmentation of its clinical signs, and lower body weight in the prenatally Lipopolysaccharide -treated C57BL/6 mice after the immunization.. Therefore, the present research can provide evidence that prenatal stress may play a role in enhancing the clinical symptoms of experimental autoimmune encephalomyelitis/MS.

Topics: Animals; Body Weight; Disease Models, Animal; Dose-Response Relationship, Drug; Encephalomyelitis, Autoimmune, Experimental; Female; Fetal Development; Male; Mice; Mice, Inbred C57BL; Myelin-Oligodendrocyte Glycoprotein; Peptide Fragments; Polysaccharides; Pregnancy; Prenatal Exposure Delayed Effects; Severity of Illness Index; Time Factors

Levetiracetam (LEV) is an established anticonvulsant with numerous mechanisms of action. Apart from its anti-epileptic effects, recent experimental studies suggest anti-inflammatory properties via modulation of interleukin (IL)-1β and transforming-growth-factor (TGF)-β1. However, its anti-inflammatory properties have not yet been examined in an autoimmune inflammatory disease of the central nervous system (CNS). We investigated LEV anti-inflammatory properties in experimental autoimmune encephalomyelitis, an established mouse model of multiple sclerosis. FACS analyses, ELISA, histology and rt-PCR experiments were done to explore potential anti-inflammatory effects. In line with prior studies, we demonstrate that LEV modulates both the relative gene expression and secretion of IL-1β and TGF-1β. However, these changes were not sufficient to alter the disease course or histological parameters. Additionally, LEV showed no effects on the absolute number of different immune cell subsets. In summary, LEV showed only minor anti-inflammatory effects not sufficient to ameliorate disease course in an autoimmune inflammatory disease of CNS.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Cells, Cultured; Disease Models, Animal; Disease Progression; Encephalomyelitis, Autoimmune, Experimental; Female; Gene Expression Regulation; Immunomodulation; Interleukin-1beta; Levetiracetam; Mice; Mice, Inbred C57BL; Multiple Sclerosis; Myelin-Oligodendrocyte Glycoprotein; Neuroglia; Peptide Fragments; Piracetam; Transforming Growth Factor beta1

Optic neuritis is an acute, demyelinating neuropathy of the optic nerve often representing the first appreciable symptom of multiple sclerosis. Wallerian degeneration of irreversibly damaged optic nerve axons leads to death of retinal ganglion cells, which is the cause of permanent visual impairment. Although the specific mechanisms responsible for triggering these events are unknown, it has been suggested that a key pathological factor is the activation of immune-inflammatory processes secondary to leukocyte infiltration. However, to date, there is no conclusive evidence to support such a causal role for infiltrating peripheral immune cells in the etiopathology of optic neuritis.. To dissect the contribution of the peripheral immune-inflammatory response versus the CNS-specific inflammatory response in the development of optic neuritis, we analyzed optic nerve and retinal ganglion cells pathology in wild-type and GFAP-IκBα-dn transgenic mice, where NF-κB is selectively inactivated in astrocytes, following induction of EAE.. We found that, in wild-type mice, axonal demyelination in the optic nerve occurred as early as 8 days post induction of EAE, prior to the earliest signs of leukocyte infiltration (20 days post induction). On the contrary, GFAP-IκBα-dn mice were significantly protected and showed a nearly complete prevention of axonal demyelination, as well as a drastic attenuation in retinal ganglion cell death. This correlated with a decrease in the expression of pro-inflammatory cytokines, chemokines, adhesion molecules, as well as a prevention of NAD(P)H oxidase subunit upregulation.. Our results provide evidence that astrocytes, not infiltrating immune cells, play a key role in the development of optic neuritis and that astrocyte-mediated neurotoxicity is dependent on activation of a transcriptional program regulated by NF-κB. Hence, interventions targeting the NF-κB transcription factor in astroglia may be of therapeutic value in the treatment of optic neuritis associated with multiple sclerosis.

Topics: Analysis of Variance; Animals; Astrocytes; Cell Count; Cytokines; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Glial Fibrillary Acidic Protein; Membrane Glycoproteins; Mice; Mice, Transgenic; Myelin-Oligodendrocyte Glycoprotein; NADPH Oxidase 2; NADPH Oxidases; Neutrophil Infiltration; NF-kappa B; Optic Nerve; Optic Nerve Diseases; Oxidative Stress; Peptide Fragments; Phenylenediamines; Phosphopyruvate Hydratase; Retinal Ganglion Cells

Our previous studies showed that ligands to type 2 imidazoline receptors (I₂R), including 2-(2-Benzofuranyl)-2-imidazoline (2-BFI) and Idazoxan, were effective in reducing spinal cord inflammation caused by experimental autoimmune encephalomyelitis (EAE). In the present study, we determined the effective therapeutic time window of 2-BFI and found that administration of 2-BFI in mice before the appearance of ascending flaccid paralysis (1-10 days post immunization), but not during the period when neurological deficits occurred (11-20 days post immunization), significantly ameliorated EAE-induced neurobehavioral deficits, reduced the infiltration of inflammatory cells into the spinal cord, and reduced the level of demyelination. More interestingly, giving 2-BFI during 1-10 days post immunization selectively suppressed IL-17 levels in the peripheral blood, which strongly suggests that IL-17 may be a good early marker to indicate EAE progression and that 2-BFI may target CD4⁺ T lymphocytes, especially Th17 cells to reduce IL-17 expression. Collectively, these studies led us to envisage that 2-BFI can be a useful drug to treat multiple sclerosis (MS) when used in combination with an early indicator of MS progression, such as IL-17.

Topics: Analysis of Variance; Animals; Benzofurans; Calcium-Binding Proteins; Cytokines; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Enzyme-Linked Immunosorbent Assay; Freund's Adjuvant; Imidazoles; Indoles; Mice; Mice, Inbred C57BL; Microfilament Proteins; Myelin Sheath; Myelin-Oligodendrocyte Glycoprotein; Nervous System Diseases; Peptide Fragments; Spinal Cord Injuries; Time Factors

Pain can be one of the most severe symptoms associated with multiple sclerosis (MS) and develops with varying levels and time courses. MS-related pain is difficult to treat, since very little is known about the mechanisms underlying its development. Animal models of experimental autoimmune encephalomyelitis (EAE) mimic many aspects of MS and are well-suited to study underlying pathophysiological mechanisms. Yet, to date very little is known about the sensory abnormalities in different EAE models. We therefore aimed to thoroughly characterize pain behavior of the hindpaw in SJL and C57BL/6 mice immunized with PLP139-151 peptide or MOG35-55 peptide respectively. Moreover, we studied the activity of pain-related molecules and plasticity-related genes in the spinal cord and investigated functional changes in the peripheral nerves using electrophysiology.. We analyzed thermal and mechanical sensitivity of the hindpaw in both EAE models during the whole disease course. Qualitative and quantitative immunohistochemical analysis of pain-related molecules and plasticity-related genes was performed on spinal cord sections at different timepoints during the disease course. Moreover, we investigated functional changes in the peripheral nerves using electrophysiology.. Mice in both EAE models developed thermal hyperalgesia during the chronic phase of the disease. However, whereas SJL mice developed marked mechanical allodynia over the chronic phase of the disease, C57BL/6 mice developed only minor mechanical allodynia over the onset and peak phase of the disease. Interestingly, the magnitude of glial changes in the spinal cord was stronger in SJL mice than in C57BL/6 mice and their time course matched the temporal profile of mechanical hypersensitivity.. Diverse EAE models bearing genetic, clinical and histopathological heterogeneity, show different profiles of sensory and pathological changes and thereby enable studying the mechanistic basis and the diversity of changes in pain perception that are associated with distinct types of MS.

Topics: Animals; Body Weight; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Exploratory Behavior; Female; Freund's Adjuvant; Hyperalgesia; Mice; Mice, Inbred C57BL; Motor Activity; Myelin Proteolipid Protein; Myelin-Oligodendrocyte Glycoprotein; Pain; Pain Measurement; Pain Threshold; Peptide Fragments; Peripheral Nerves

Neutrophil influx is an important sign of hyperacute neuroinflammation, whereas the entry of activated lymphocytes into the brain parenchyma is a hallmark of chronic inflammatory processes, as observed in multiple sclerosis (MS) and its animal models of experimental autoimmune encephalomyelitis (EAE). Clinically approved or experimental therapies for neuroinflammation act by blocking leukocyte penetration of the blood brain barrier. However, in view of unsatisfactory results and severe side effects, complementary therapies are needed. We have examined the effect of chlorite-oxidized oxyamylose (COAM), a potent antiviral polycarboxylic acid on EAE.. EAE was induced in SJL/J mice by immunization with spinal cord homogenate (SCH) or in IFN-γ-deficient BALB/c (KO) mice with myelin oligodendrocyte glycoprotein peptide (MOG₃₅₋₅₅). Mice were treated intraperitoneally (i.p.) with COAM or saline at different time points after immunization. Clinical disease and histopathology were compared between both groups. IFN expression was analyzed in COAM-treated MEF cell cultures and in sera and peritoneal fluids of COAM-treated animals by quantitative PCR, ELISA and a bioassay on L929 cells. Populations of immune cell subsets in the periphery and the central nervous system (CNS) were quantified at different stages of disease development by flow cytometry and differential cell count analysis. Expression levels of selected chemokine genes in the CNS were determined by quantitative PCR.. We discovered that COAM (2 mg i.p. per mouse on days 0 and 7) protects significantly against hyperacute SCH-induced EAE in SJL/J mice and MOG₃₅₋₅₅-induced EAE in IFN-γ KO mice. COAM deviated leukocyte trafficking from the CNS into the periphery. In the CNS, COAM reduced four-fold the expression levels of the neutrophil CXC chemokines KC/CXCL1 and MIP-2/CXCL2. Whereas the effects of COAM on circulating blood and splenic leukocytes were limited, significant alterations were observed at the COAM injection site.. These results demonstrate novel actions of COAM as an anti-inflammatory agent with beneficial effects on EAE through cell deviation. Sequestration of leukocytes in the non-CNS periphery or draining of leukocytes out of the CNS with the use of the chemokine system may thus complement existing treatment options for acute and chronic neuroinflammatory diseases.

Topics: Amylose; Animals; Ascitic Fluid; Cell Differentiation; Cells, Cultured; Central Nervous System; Cytokines; Disease Models, Animal; Embryo, Mammalian; Encephalomyelitis, Autoimmune, Experimental; Enzyme-Linked Immunosorbent Assay; Fibroblasts; Flow Cytometry; Freund's Adjuvant; Gene Expression Regulation; Interferon-gamma; Leukocytes; Mice; Mice, Inbred BALB C; Mice, Knockout; Myelin-Oligodendrocyte Glycoprotein; Neutrophil Infiltration; Peptide Fragments; Receptors, Chemokine; RNA, Messenger; Spleen; Statistics, Nonparametric; Time Factors

The nature of pathogenic mechanisms associated with the development of multiple sclerosis (MS) have long been debated. However, limited research was conducted to define the interplay between infiltrating lymphocytes and resident cells of the central nervous system (CNS). Data presented in this report describe a novel role for astrocyte-mediated alterations to myelin oligodendrocyte glycoprotein (MOG)(35-55) -specific lymphocyte responses, elicited during the development of experimental autoimmune encephalitomyelitis (EAE). In-vitro studies demonstrated that astrocytes inhibited the proliferation and interferon (IFN)-γ, interleukin (IL)-4, IL-17 and transforming growth factor (TGF)-β secretion levels of MOG(35-55) -specific lymphocytes, an effect that could be ameliorated by astrocyte IL-27 neutralization. However, when astrocytes were pretreated with IFN-γ, they could promote the proliferation and secretion levels of MOG(35-55) -specific lymphocytes, coinciding with apparent expression of major histocompatibility complex (MHC)-II on astrocytes themselves. Quantitative polymerase chain reaction (qPCR) demonstrated that production of IL-27 in the spinal cord was at its highest during the initial phases. Conversely, production of IFN-γ in the spinal cord was highest during the peak phase. Quantitative analysis of MHC-II expression in the spinal cord showed that there was a positive correlation between MHC-II expression and IFN-γ production. In addition, astrocyte MHC-II expression levels correlated positively with IFN-γ production in the spinal cord. These findings suggested that astrocytes might function as both inhibitors and promoters of EAE. Astrocytes prevented MOG(35-55) -specific lymphocyte function by secreting IL-27 during the initial phases of EAE. Then, in the presence of higher IFN-γ levels in the spinal cord, astrocytes were converted into antigen-presenting cells. This conversion might promote the progression of pathological damage and result in a peak of EAE severity.

Topics: Animals; Astrocytes; Cell Communication; Coculture Techniques; Cytokines; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Epitopes; Female; Gene Expression Regulation; Genes, MHC Class II; Interferon-gamma; Interleukins; Lymphocyte Activation; Lymphocytes; Mice; Mice, Inbred C57BL; Myelin-Oligodendrocyte Glycoprotein; Peptide Fragments; Spinal Cord

Alkaline phosphatase (AP) is a ubiquitously expressed enzyme which can neutralize endotoxin as well as adenosine triphosphate (ATP), an endogenous danger signal released during brain injury. In this study we assessed a potential therapeutic role for AP in inhibiting neuroinflammation using three complementary approaches.. Mice were immunized to induce experimental autoimmune encephalomyelitis (EAE) and treated with AP for seven days during different phases of disease. In addition, serological assays to determine AP activity, endotoxin levels and endotoxin-reactive antibodies were performed in a cohort of multiple sclerosis (MS) patients and controls. Finally, the expression of AP and related enzymes CD39 and CD73 was investigated in brain tissue from MS patients and control subjects.. AP administration during the priming phase, but not during later stages, of EAE significantly reduced neurological signs. This was accompanied by reduced proliferation of splenocytes to the immunogen, myelin oligodendrocyte glycoprotein peptide. In MS patients, AP activity and isoenzyme distribution were similar to controls. Although endotoxin-reactive IgM was reduced in primary-progressive MS patients, plasma endotoxin levels were not different between groups. Finally, unlike AP and CD73, CD39 was highly upregulated on microglia in white matter lesions of patients with MS.. Our findings demonstrate that: 1) pre-symptomatic AP treatment reduces neurological signs of EAE; 2) MS patients do not have altered circulating levels of AP or endotoxin; and 3) the expression of the AP-like enzyme CD39 is increased on microglia in white matter lesions of MS patients.

Topics: Adenosine Triphosphate; Adult; Alkaline Phosphatase; Animals; Antigens, CD; Blood Vessels; Brain; Cell Proliferation; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Endotoxins; Enzyme-Linked Immunosorbent Assay; Female; HLA-DR Antigens; Humans; Male; Mice; Mice, Inbred C57BL; Middle Aged; Multiple Sclerosis; Myelin-Oligodendrocyte Glycoprotein; Peptide Fragments; Postmortem Changes; Statistics, Nonparametric; T-Lymphocytes; Thymidine; Tritium; Tumor Necrosis Factor-alpha; Young Adult

Transplanted Neural Precursor Cells (NPCs) are capable of long-distance migration inside the inflamed CNS, but exhibit limited myelinating capacities in animal models of Multiple Sclerosis (MS). Inflammation seems to be both beneficial for the recruitment and migration of NPCs and restrictive for their terminal differentiation. In the present study, a set of transplantation experiments was applied in order to investigate the migratory potential, the differentiation pattern and long-term survival of NPCs in Experimental Autoimmune Encephalomyelitis (EAE) mice, the animal model of MS. The in vitro differentiation potential of NPCs in the presence of either pro- (TNFa, INFγ) or anti- (TGFb) inflammatory cytokines was also analyzed. According to the in vivo results obtained, at the acute phase of EAE only a small fraction of transplanted NPCs succeed to differentiate, whereas at chronic phase most of them followed a differentiation process to glial cell lineage along white matter tracts. However, this differentiation was not fully completed, since 8 months after their transplantation a number of NPCs remained as pre-oligodendrocytes. Glial differentiation of NPCs was also found to be inhibited or promoted following their treatment with TNFa or TGFb respectively, in vitro. Our findings suggest that inflammation triggers migration whereas the anti-inflammatory component is a prerequisite for NPCs to follow glial differentiation thereby providing myelinating oligodendrocytes. It is speculated that the fine balance between the pro- and anti-inflammatory determinants in the CNS may be a key factor for transplanted NPCs to exhibit a better therapeutic effect in EAE and MS. This article is part of a Special Issue entitled "Interaction between repair, disease, & inflammation."

Topics: Animals; Animals, Newborn; Antigens; Cell Differentiation; Cell Movement; Cytokines; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Female; Gene Expression Regulation; Glial Fibrillary Acidic Protein; Glycoproteins; Green Fluorescent Proteins; Intermediate Filament Proteins; Mice; Mice, Inbred C57BL; Mice, Transgenic; Myelin-Oligodendrocyte Glycoprotein; Nerve Tissue Proteins; Nestin; Neural Stem Cells; Neurons; Peptide Fragments; Proteoglycans; Severity of Illness Index; Statistics, Nonparametric; Time Factors

Multiple sclerosis (MS) is a severely debilitating neurodegenerative diseases marked by progressive demyelination and axonal degeneration in the CNS. Although inflammation is the major pathology of MS, the mechanism by which it occurs is not completely clear. The primary symptoms of MS are movement difficulties caused by conduction block resulting from the demyelination of axons. The possible mechanism of functional loss is believed to be the exposure of potassium channels and increase of outward current leading to conduction failure. 4-Aminopyridine (4-AP), a well-known potassium channel blocker, has been shown to enhance conduction in injured and demyelinated axons. However, 4-AP has a narrow therapeutic range in clinical application. Recently, we developed a new fast potassium channel blocker, 4-aminopyridine-3-methanol (4-AP-3-MeOH). This novel 4-AP derivative is capable of restoring impulse conduction in ex vivo injured spinal cord without compromising the ability of axons to follow multiple stimuli. In the current study, we investigated whether 4-AP-3-MeOH can enhance impulse conduction in an animal model of MS. Our results showed that 4-AP-3-MeOH can significantly increase axonal conduction in ex vivo experimental autoimmune encephalomyelitis mouse spinal cord.

Topics: Action Potentials; Aminopyridines; Animals; Axons; Disease Models, Animal; Glycoproteins; Mice; Mice, Inbred C57BL; Multiple Sclerosis; Myelin Sheath; Myelin-Oligodendrocyte Glycoprotein; Neural Conduction; Neurofilament Proteins; Peptide Fragments; Potassium Channel Blockers; Reaction Time; Spinal Cord

Cyclic GMP (cGMP)-mediated pathways regulate inflammatory responses in immune and CNS cells. Recently, cGMP phosphodiesterase inhibitors such as sildenafil, commonly used to treat sexual dysfunction in humans including multiple sclerosis (MS) patients, have been reported to be neuroprotective in animal models of stroke, Alzheimer's disease, and focal brain lesion. In this work, we have examined if sildenafil ameliorates myelin oligodendrocyte glycoprotein peptide (MOG₃₅₋₅₅)-induced experimental autoimmune encephalomyelitis (EAE), a mouse model of MS. We show for the first time that treatment with sildenafil after disease onset markedly reduces the clinical signs of EAE by preventing axonal loss and promoting remyelination. Furthermore, sildenafil decreases CD3+ leukocyte infiltration and microglial/macrophage activation in the spinal cord, while increasing forkhead box transcription factor 3-expressing T regulatory cells (Foxp3 Tregs). However, sildenafil treatment did not significantly affect MOG₃₅₋₅₅-stimulated proliferation or release of Th1/Th2 cytokines in splenocytes but decreased ICAM-1 in spinal cord infiltrated cells. The presence of reactive astrocytes forming scar-like structures around infiltrates was enhanced by sildenafil suggesting a possible mechanism for restriction of leukocyte spread into healthy parenchyma. These results highlight novel actions of sildenafil that may contribute to its beneficial effects in EAE and suggest that treatment with this widely used and well-tolerated drug may be a useful therapeutic intervention to ameliorate MS neuropathology.

Topics: Animals; CD3 Complex; Demyelinating Diseases; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Female; Forkhead Transcription Factors; Gene Expression Regulation; Glial Fibrillary Acidic Protein; Gliosis; Glycoproteins; Intercellular Adhesion Molecule-1; Mice; Mice, Inbred C57BL; Myelin-Oligodendrocyte Glycoprotein; Neurofilament Proteins; Peptide Fragments; Phosphodiesterase 5 Inhibitors; Piperazines; Purines; Sildenafil Citrate; Spinal Cord; Sulfones; Time Factors

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS) characterized by inflammation, but also degenerative changes. Besides neurological deficits, the rate of affective disorders such as depression and anxiety is at least six fold increased. Many aspects of MS can be mimicked in the animal model of myelin oligodendrocyte glycoprotein experimental autoimmune encephalomyelitis (MOG-EAE). Here we investigate behavioral changes in C57BL/6 mice suffering from mild MOG-EAE. In the later phase of the disease, mice were subjected to behavioral tests including the light-dark-box (LD Box), the acoustic startle response (SR) with a pre-pulse inhibition protocol as well as the learned helplessness (LH) paradigm. Behavioral data were correlated with the motor performance in an open field and rotarod test (RR). In the RR and open field, there was no significant difference in the motor performance between controls and mice suffering from mild MOG-EAE. Yet EAE mice displayed an increased anxiety-like behavior with a 23% reduction of the time spent in the bright compartment of the LD Box as well as an increased SR. In the LH paradigm, mice suffering from MOG-EAE were twice as much prone to depressive-like behavior. These changes correlate with an increase of hippocampal tissue tumor necrosis factor alpha levels and neuronal loss in the hippocampus. Modulation of monoaminergic transmission by chronic application of the antidepressant amitriptyline resulted in a decreased startle reaction and increased hippocampal norepinephrine levels. These data imply that chronic inflammation in the CNS may impact on emotional responses in rodent models of anxiety.

Topics: Acoustic Stimulation; Amitriptyline; Analysis of Variance; Animals; Antidepressive Agents, Tricyclic; Anxiety; Central Nervous System; Chromatography, High Pressure Liquid; Cytokines; Dark Adaptation; Demyelinating Diseases; Depression; Disease Models, Animal; Dose-Response Relationship, Drug; Exploratory Behavior; Female; Gene Expression Regulation; Glycoproteins; Helplessness, Learned; Inflammation; Mice; Mice, Inbred C57BL; Motor Activity; Multiple Sclerosis; Myelin-Oligodendrocyte Glycoprotein; Peptide Fragments; Pertussis Toxin; Phosphopyruvate Hydratase; Psychoacoustics; Reflex, Startle; Rotarod Performance Test; Stereotaxic Techniques; Time Factors

The endogenous neurotransmitter noradrenaline exerts anti-inflammatory and neuroprotective effects in vitro and in vivo. Several studies report that noradrenaline levels are altered in the central nervous system of patients with multiple sclerosis and rodents with experimental autoimmune encephalomyelitis, which could contribute to pathology. Since the major source of noradrenaline are neurons in the locus coeruleus, we hypothesized that alterations in noradrenaline levels are a consequence of stress or damage to locus coeruleus neurons. In C57BL/6 mice immunized with myelin oligodendrocyte glycoprotein peptide 35-55 to develop chronic disease, cortical and spinal cord levels of noradrenaline were significantly reduced versus control mice. Immunohistochemical staining revealed increased astrocyte activation in the ventral portion of the locus coeruleus in immunized mice. The immunized mice showed neuronal damage in the locus coeruleus detected by a reduction of average cell size of tyrosine hydroxylase stained neurons. Analysis of the locus coeruleus of multiple sclerosis and control brains showed a significant increase in astrocyte activation, a reduction in noradrenaline levels, and neuronal stress indicated by hypertrophy of tyrosine hydroxylase stained cell bodies. However, the magnitude of these changes was not correlated with extent of demyelination or of cellular infiltrates. Together these findings demonstrate the presence of inflammation and neuronal stress in multiple sclerosis as well as in experimental autoimmune encephalomyelitis. Since reduced noradrenaline levels could be permissive for increased inflammation and neuronal damage, these results suggest that methods to raise noradrenaline levels or increase locus coeruleus function may be of benefit in treating multiple sclerosis.

Topics: Aged; Aged, 80 and over; Analysis of Variance; Animals; Brain-Derived Neurotrophic Factor; Cerebral Cortex; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Enzyme-Linked Immunosorbent Assay; Eosinophil-Derived Neurotoxin; Female; Glial Fibrillary Acidic Protein; Glycoproteins; Humans; Locus Coeruleus; Male; Mice; Mice, Inbred C57BL; Middle Aged; Multiple Sclerosis; Myelin-Oligodendrocyte Glycoprotein; Neurons; Norepinephrine; Peptide Fragments; RNA, Messenger; Spinal Cord; Tyrosine 3-Monooxygenase

Inflammation and oxidative stress are thought to promote tissue damage in multiple sclerosis. Thus, novel therapeutics enhancing cellular resistance to free radicals could prove useful for multiple sclerosis treatment. BG00012 is an oral formulation of dimethylfumarate. In a phase II multiple sclerosis trial, BG00012 demonstrated beneficial effects on relapse rate and magnetic resonance imaging markers indicative of inflammation as well as axonal destruction. First we have studied effects of dimethylfumarate on the disease course, central nervous system, tissue integrity and the molecular mechanism of action in an animal model of chronic multiple sclerosis: myelin oligodendrocyte glycoprotein induced experimental autoimmune encephalomyelitis in C57BL/6 mice. In the chronic phase of experimental autoimmune encephalomyelitis, preventive or therapeutic application of dimethylfumarate ameliorated the disease course and improved preservation of myelin, axons and neurons. In vitro, the application of fumarates increased murine neuronal survival and protected human or rodent astrocytes against oxidative stress. Application of dimethylfumarate led to stabilization of the transcription factor nuclear factor (erythroid-derived 2)-related factor 2, activation of nuclear factor (erythroid-derived 2)-related factor 2-dependent transcriptional activity and accumulation of NADP(H) quinoline oxidoreductase-1 as a prototypical target gene. Furthermore, the immediate metabolite of dimethylfumarate, monomethylfumarate, leads to direct modification of the inhibitor of nuclear factor (erythroid-derived 2)-related factor 2, Kelch-like ECH-associated protein 1, at cysteine residue 151. In turn, increased levels of nuclear factor (erythroid-derived 2)-related factor 2 and reduced protein nitrosylation were detected in the central nervous sytem of dimethylfumarate-treated mice. Nuclear factor (erythroid-derived 2)-related factor 2 was also upregulated in the spinal cord of autopsy specimens from untreated patients with multiple sclerosis. In dimethylfumarate-treated mice suffering from experimental autoimmune encephalomyelitis, increased immunoreactivity for nuclear factor (erythroid-derived 2)-related factor 2 was detected by confocal microscopy in neurons of the motor cortex and the brainstem as well as in oligodendrocytes and astrocytes. In mice deficient for nuclear factor (erythroid-derived 2)-related factor 2 on the same genetic background, the dimethylfumarate mediated ben

Topics: 2',3'-Cyclic-Nucleotide Phosphodiesterases; Aldehyde Reductase; Animals; Antigens, Differentiation; Antioxidants; Astrocytes; Axons; CD3 Complex; Cell Proliferation; Cells, Cultured; Chromatography, High Pressure Liquid; Cytokines; Disease Models, Animal; Embryo, Mammalian; Encephalomyelitis, Autoimmune, Experimental; Female; Fumarates; Gene Expression Regulation; Glycoproteins; Green Fluorescent Proteins; Humans; Hydrogen Peroxide; Mass Spectrometry; Mice; Mice, Inbred C57BL; Motor Neurons; Multiple Sclerosis; Myelin Proteins; Myelin-Oligodendrocyte Glycoprotein; NAD(P)H Dehydrogenase (Quinone); Neuroprotective Agents; NF-E2-Related Factor 2; Nogo Proteins; Oxidative Stress; Peptide Fragments; RNA, Small Interfering; Signal Transduction; Sleep; Spinal Cord; Statistics, Nonparametric; Tandem Mass Spectrometry; Time Factors; Transfection

In the present study, we demonstrate that the histopathologic features of myelin oligodendrocyte glycoprotein (MOG) peptide 35-55-induced experimental autoimmune encephalomyelitis (EAE) in C57BL/6 mice closely mirror the hallmarks of MS pathology. On the one hand, we depict a time-dependent transition from acute inflammation to chronic neurodegeneration in spinal cord histopathology and provide distinct criteria (i.e. parenchymal edema, cellular infiltration and perivascular inflammatory infiltrates) by which acute and chronic stages of the disease can be distinguished. On the other hand, we assessed the extent of spinal cord plaque formation in relation to the total white matter area and we demonstrate a strong correlation with the clinical disease severity. Additionally, we report on the involvement of different spinal cord regions, focusing on the anterolateral, posterior and pyramidal tract. Our results help to further characterize histopathology of MOG peptide 35-55-induced EAE and reinforce the importance of this model for structural and functional studies of MS features.

Topics: Animals; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Female; Glycoproteins; Mice; Mice, Inbred C57BL; Multiple Sclerosis; Myelin-Oligodendrocyte Glycoprotein; Nerve Degeneration; Peptide Fragments; Spinal Cord

Multiple sclerosis (MS) is an inflammatory disease of the CNS mediated by CD4(+) T cells directed against myelin antigens. Experimental autoimmune encephalomyelitis (EAE) is induced by immunization with myelin antigens like myelin oligodendrocyte glycoprotein (MOG). We have explored the transfer of EAE using MOG(35-55)-specific TCR transgenic (2D2) T cells. Unsorted 2D2 Th1 cells reliably transferred EAE. Further, we found that CD44(hi)CD62L(lo) effector/memory CD4(+) T cells are likely responsible for the disease transfer due to the up-regulation of CD44. Given the importance of MOG in MS pathogenesis, mechanistic insights into adoptively transferred EAE by MOG-specific Th1 cells could prove valuable in MS research.

Topics: Adoptive Transfer; Animals; Antigens, CD; Brain; Cell Differentiation; Cells, Cultured; Cytokines; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Enzyme-Linked Immunosorbent Assay; Female; Flow Cytometry; Glycoproteins; Lymphocyte Activation; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Myelin-Oligodendrocyte Glycoprotein; Peptide Fragments; Receptors, Antigen, T-Cell, alpha-beta; Sex Factors; Spinal Cord; T-Lymphocytes; Time Factors

B cell activating factor (BAFF) is critical for B cell survival, a function that is mediated by BAFF receptor, (BAFF-R). The role of BAFF (or BAFF-R) in the multiple sclerosis model, experimental autoimmune encephalomyelitis (EAE), was examined using BAFF-R-deficient mice. BAFF-R deficiency resulted in paradoxically increased severity of EAE induced by myelin-oligodendrocyte glycoprotein (MOG) peptide 35-55. Inflammatory foci in BAFF-R-deficient mice comprised increased numbers of activated macrophages expressing BAFF and correlated with increased BAFF secretion. Thus, BAFF-R may be important in EAE pathogenesis, possibly by influencing macrophage function through a mechanism that involves modulation of BAFF expression.

Topics: Animals; Autoimmunity; B-Cell Activation Factor Receptor; Central Nervous System; Cytokines; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Enzyme-Linked Immunosorbent Assay; Glycoproteins; Lymphocytes; Macrophages; Mice; Mice, Inbred C57BL; Mice, Knockout; Myelin-Oligodendrocyte Glycoprotein; Peptide Fragments; Spleen; Statistics, Nonparametric

Hematopoietic stem cell transplantation (HSCT) has been proposed as a novel therapy for multiple sclerosis (MS). CD4 + CD25 + regulatory T cells (Tregs) expressing Foxp3 play an important role in the maintenance of immune tolerance to self. Our study was conducted to confirm the efficiency of nonmyeloablative conditioning and syngeneic bone marrow transplantation (BMT) on experimental autoimmune encephalomyelitis (EAE) mice and to determine whether Tregs plays a role in the underlying mechanism.. EAE were induced in C57BL/6 mice and were randomly divided into 4 groups: the Conditioning group received the conditioning regimen, the Normal-EAE BMT group received conditioning and bone marrow (BM) grafts from normal mice, the EAE-EAE BMT group received conditioning and BM grafts from EAE mice and the EAE control group received no further therapy. The cumulative clinical score was used to assess the efficacy of the different treatments, and the proportion of Tregs in the spleen was measured by flow cytometry on day 40, 80 and 120 after BMT. Foxp3 mRNA expression was assessed by real-time PCR, and the expression of Foxp3 protein was tested by western blot on day 120 after BMT.. Conditioning and conditioning with BMT led to a significant clinical improvement on day 80 after BMT compared with EAE without further treatment. On day 120 after BMT, the clinical score of the Conditioning group showed no significant difference from that of the EAE control group, whereas BMT led to a further amelioration of the disease score. On day 80 and day 120 after BMT, the proportions of Tregs of the two BMT groups were significantly higher than that in EAE control group, whereas no statistically significant difference was found between the Conditioning group and the EAE control group. On day 120 after BMT, the Foxp3 mRNA level and Foxp3 protein expression was higher in the two BMT groups than in EAE control group or Conditioning group.. Nonmyeloablative conditioning could temporarily reverse already established EAE, but it was not sufficient for the induction of long-term EAE remission. Transplantation by BM cells from healthy or diseased donors was necessary and responsible for complete and long-time remission of EAE, and these beneficial effects may be the result of the induction of Tregs and the Treg-related factor Foxp3.

Topics: Analysis of Variance; Animals; Bone Marrow Transplantation; CD4 Antigens; Cyclophosphamide; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Female; Flow Cytometry; Gene Expression Regulation; Glycoproteins; Hepatocyte Nuclear Factor 3-gamma; Immunosuppressive Agents; Interleukin-2 Receptor alpha Subunit; Mice; Mice, Inbred C57BL; Myelin-Oligodendrocyte Glycoprotein; Peptide Fragments; Severity of Illness Index; T-Lymphocytes, Regulatory; Time Factors

It has been reported that autoimmune inflammatory processes in human multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE), may induce an alteration in neurogenesis. Studies with transgenic EAE mice have demonstrated an enhancement of neurogenesis in the subventricular zone (SVZ). In contrast, a reduction of stem cell proliferation in the same region has been observed by Pluchino et al. [Brain 2008;131:2564-2578] in myelin oligodendrocyte glycoprotein (MOG)-induced EAE mice. We immunized female C57BL/6 mice with MOG 35-55 peptide and successfully developed chronic/nonremitting EAE, which is believed to be analogous to the progressive form of MS. On day 21 postimmunization, coronal brain sections were collected and stained with anti-5-bromo-2'-deoxyuridine (BrdU) antibody. By counting the number of BrdU-labeled cells, we demonstrated that the neural stem/progenitor cell (NSC/NPC) proliferation decreased in the SVZ, which basically confirms the study of Pluchino et al. on the changes in the SVZ. A reduction of NSC/NPC proliferation also occurred in the hippocampal subgranular zone of the dentate gyrus. The hippocampus is well known to be an important region involved in learning and memory; thus, our finding may offer a possible explanation for the cognitive impairment in human chronic MS.

Topics: Animals; Brain; Bromodeoxyuridine; CD4 Antigens; Cell Proliferation; Cerebral Ventricles; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Female; Glycoproteins; Mice; Mice, Inbred C57BL; Myelin-Oligodendrocyte Glycoprotein; Neural Stem Cells; Peptide Fragments; Pertussis Toxin; Spinal Cord; T-Lymphocytes; Time Factors

Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS) thought to be primarily mediated by T cells. However, emerging evidence supports an important role for B cells in the pathogenesis and inhibition of MS. Glatiramer acetate (GA), a Food and Drug Administration-approved drug for the treatment of MS, has a good safety profile. But GA's mechanism of action in MS is still elusive. In this study, we showed that B cells from GA-treated mice increased production of IL-10 and reduced expression of co-stimulatory molecules viz.: CD80 and CD86. B cells from GA-treated mice also diminished proliferation of myelin oligodendrocyte glycoprotein (MOG(35-55)) specific T cells. Purified B cells transferred from GA-treated mice suppressed experimental autoimmune encephalomyelitis (EAE) in recipient mice compared with B cells transferred from mice treated with PBS or ovalbumin. The treatment effect of GA in EAE was abrogated in B cell-deficient mice. Transfer of B cells from GA-treated mice inhibited the proliferation of autoreactive T cells as well as the development of Th1 and Th17 cells but promoted IL-10 production in recipient mice. The number of peripheral CD11b(+) macrophages in recipient mice also decreased after transfer of B cells from GA-treated mice; however, the number of dendritic cells and regulatory T cells remained unaltered. These results suggest that B cells are important to the protective effects of GA in EAE.

Topics: Adoptive Transfer; Animals; Antigens, CD; B-Lymphocytes; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Flow Cytometry; Gene Expression Regulation; Glatiramer Acetate; Glycoproteins; Immunosuppressive Agents; Interleukin-10; Interleukin-4; Lymphocyte Activation; Mice; Mice, Inbred C57BL; Mice, Transgenic; Myelin Proteins; Myelin-Associated Glycoprotein; Myelin-Oligodendrocyte Glycoprotein; Peptide Fragments; Peptides; T-Lymphocytes; Transforming Growth Factor beta

Multiple sclerosis (MS) is an autoimmune disease characterized by peripheral activation of CD4(+) T cells that migrate into the central nervous system (CNS) and mount an autoimmune neuroinflammatory attack on myelin and oligodendrocytes. Secondary to these events, however equally destructive, is the generation of inflammatory-mediated reactive oxygen and nitrogen species generated by persistently activated microglia and astrocytes. Nuclear factor-erythroid 2-related factor 2 (Nrf2) is a basic leucine zipper transcription factor that regulates genetic expression of many protective antioxidant and detoxication enzymes. Here we describe the Nrf2 modulation of innate and adaptive immune responses in an acute autoimmune model of MS, experimental autoimmune encephalomyelitis (EAE). Wild-type (WT) mice and Nrf2 knockout mice were immunized with myelin oligodendrocyte glycoprotein (MOG 35-55) and monitored daily for clinical scores of disease. Disruption of Nrf2 resulted in a more severe clinical course, a more rapid onset, and a greater percentage of mice with the disease. Furthermore, increased immune cell infiltration and glial cell activation in spine was observed. In conjunction, we observed increased inflammatory enzyme (iNOS, phox-47, gp91-phox, and phox-67), cytokine (IFN-gamma, IL1-b, TNF-alpha, and IL-12), and chemokine (BLC and MIG) gene expression levels in the Nrf2-deficient mice compared to the WT mice, supporting the notion that Nrf2 can modulate an autoimmune neuroinflammatory response. Our results show that the absence of Nrf2 exacerbates the development of EAE and thus suggests that activation of Nrf2 may then attenuate pathogenesis of autoimmune diseases such as MS as well as other neurodegenerative diseases that present with neuroinflammation.

Topics: Animals; Chimera; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Enzymes; Female; Gene Expression; Glycoproteins; Male; Mice; Mice, Inbred Strains; Mice, Knockout; Multiple Sclerosis; Myelin-Oligodendrocyte Glycoprotein; NF-E2-Related Factor 2; Oxidative Stress; Peptide Fragments; Species Specificity; Spinal Cord

Naltrexone, an opioid antagonist, has been shown to modulate expression of experimental autoimmune encephalomyelitis (EAE), an animal model of MS, suggesting that endogenous opioids are inhibitory trophic factors in EAE. In the present study, we investigated the effects of one native opioid peptide, opioid growth factor ([Met(5)]-enkephalin), on the onset and progression of EAE. C57Bl/6 mice injected with myelin oligodendrocyte glycoprotein (MOG) received daily injections of 10 mg/kg OGF (MOG+OGF) or saline (MOG+Vehicle). Over 60% of the MOG+OGF animals did not exhibit behavioral signs of disease (EAE) in contrast to 100% of the mice in the MOG+Vehicle group. The severity and disease indices of EAE in the OGF-treated mice were markedly reduced from MOG+Vehicle cohorts. By day 30, 60% of MOG+OGF mice had a remission, relative to 4% in the MOG+Vehicle group. MOG-injected mice receiving OGF had significant reductions in activated astrocytes and damaged neurons compared to MOG+Vehicle animals. Unlike MOG+Vehicle and MOG+OGF mice with behavioral signs of disease, MOG+OGF animals without manifestation of disease had no lumbar spinal cord demyelination. Both OGF and OGF receptor were detected in splenic-derived T lymphocytes by immunohistochemistry. OGF treatment decreased both DNA synthesis and cell proliferation in comparison to vehicle-treated T cell lymphocyte cultures. These results indicate that an endogenous opioid, OGF, inhibits the onset and progression of EAE, and suggest that clinical studies on the use of OGF treatment for MS are merited.

Topics: Animals; Disease Models, Animal; Disease Progression; Encephalomyelitis, Autoimmune, Experimental; Enkephalin, Methionine; Female; Glycoproteins; Lymphocyte Activation; Mice; Mice, Inbred C57BL; Myelin-Oligodendrocyte Glycoprotein; Neurotransmitter Agents; Peptide Fragments; Receptors, Opioid; Spinal Cord; Time Factors

Hepatocyte growth factor (HGF) is a pleiotropic cytokine able to evoke a wide array of cellular responses including proliferation, migration, and survival through activation of its receptor c-met. Various types of leukocytes have been described to express c-met suggesting that HGF/c-met signaling may directly influence leukocyte responses in inflammation. We have investigated the HGF/c-met pathway in experimental autoimmune encephalomyelitis (EAE), a common mouse model of multiple sclerosis (MS), in which macrophages play a dual role, contributing directly to CNS damage at disease onset but promoting recovery during remission by removing myelin debris. Here we show that during EAE both HGF and c-met are expressed in the CNS and that c-met is activated. We subsequently demonstrate that c-met is primarily expressed in inflammatory lesions by macrophages and a small number of dendritic cells (DCs) and oligodendrocyte progenitor cells (OPCs) but not by microglia or T cells. Complementary in vitro experiments show that only LPS and TNFalpha, but not IL-6, IL-10, or IL-13, are able to induce c-met expression in macrophages. In addition, using TNF signaling deficient macrophages we demonstrate that LPS and TNFalpha induce c-met through distinct pathways. Furthermore, TNFalpha- and LPS-induced c-met is functional because treatment of macrophages with recombinant HGF results in rapid phosphorylation of c-met. Interestingly, HGF/c-met signaling does not modulate cytokine expression, phagocytosis, or antigen presentation but promotes proliferation of activated macrophages. Taken together, our data indicate a pro-inflammatory role for the HGF/c-met pathway in EAE rather than a role in the initiation of repair mechanisms.

Topics: Animals; Antigens, CD; Bone Marrow Cells; Cell Proliferation; Cells, Cultured; Central Nervous System; Cytokines; Dendritic Cells; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Female; Flow Cytometry; Gene Expression Regulation; Glycoproteins; Hepatocyte Growth Factor; Lipopolysaccharides; Macrophages; Mice; Mice, Inbred C57BL; Mice, Knockout; Microglia; Myelin-Oligodendrocyte Glycoprotein; Nitrates; Oligodendroglia; Peptide Fragments; Phagocytosis; Proto-Oncogene Proteins c-met; Stem Cells; Time Factors; Tumor Necrosis Factor-alpha

We studied the effects of combination treatment with an anti-inflammatory agent, interferon (IFN)-beta, and a putative neuroprotective agent, an estrogen receptor (ER)-beta ligand, during EAE. Combination treatment significantly attenuated EAE disease severity, preserved axonal densities in spinal cord, and reduced CNS inflammation. Combining ERbeta treatment with IFNbeta reduced IL-17, while it abrogated IFNbeta-mediated increases in Th1 and Th2 cytokines from splenocytes. Additionally, combination treatment reduced VLA-4 expression on CD4+ T cells, while it abrogated IFNbeta-mediated decreases in MMP-9. Our data demonstrate that combination treatments can result in complex effects that could not have been predicted based on monotherapy data alone.

Topics: Analysis of Variance; Animals; Antigens, CD; Antigens, Differentiation; Cytokines; Disease Models, Animal; Drug Therapy, Combination; Encephalomyelitis, Autoimmune, Experimental; Female; Glycoproteins; Immunologic Factors; Integrin alpha4beta1; Interferon-beta; Luminescent Proteins; Macrophages; Matrix Metalloproteinase 9; Mice; Mice, Transgenic; Myelin Basic Protein; Myelin-Oligodendrocyte Glycoprotein; Neuroprotective Agents; Neutrophil Infiltration; Nitriles; Peptide Fragments; Propionates; Severity of Illness Index; Spinal Cord

B cells are of increasing importance as a target for multiple sclerosis treatment. Here we show that GA treatment of mice with experimental autoimmune encephalomyelitis (EAE) biases cytokine production by B cells towards cytokines associated with regulation in MS including interleukin (IL)-4, -10 and -13 and reduces pro-inflammatory IL-6, IL-12, and TNF alpha levels. GA also down-regulates expression of B cell-activating factor (BAFF) of the TNF family and a proliferation-inducing ligand (APRIL), as well as the BAFF receptor in mice with EAE. Thus, GA impacts both B cell survival and B cell cytokine production during CNS inflammatory disease in an EAE model.

Topics: Animals; Antigens, CD19; B-Cell Activating Factor; B-Lymphocytes; Brain; CD5 Antigens; Cells, Cultured; Cytokines; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Female; Flow Cytometry; Gene Expression Regulation; Glatiramer Acetate; Glycoproteins; Immunosuppressive Agents; Lymph Nodes; Mice; Mice, Inbred C57BL; Myelin-Oligodendrocyte Glycoprotein; Peptide Fragments; Peptides; RNA, Messenger; Spleen; Tumor Necrosis Factor Ligand Superfamily Member 13

The migration of aberrant inflammatory cells into the central nervous system plays an important role in the pathogenesis of demyelinating diseases potentially through the Rho/Rho-kinase (Rock) pathway, but direct evidence from human and animal models remains inadequate. Here we further confirm that Fasudil, a selective Rock inhibitor, has therapeutic potential in a mouse model of myelin oligodendrocyte glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis (EAE). The results show that Fasudil decreased the development of EAE in C57BL/6 mice. Immunohistochemistry disclosed that expression of Rock-II in the perivascular spaces and vascular endothelial cells of spleens, spinal cords, and brains was elevated in EAE and was inhibited in the Fasudil-treated group. T-cell proliferation specific to MOG(35-55) was markedly reduced, together with a significant down-regulation of interleukin (IL)-17, IL-6, and MCP-1. In contrast, secretion of IL-4 was increased, and IL-10 was slightly elevated. There were no differences in the percentages of CD4(+)CD25(+), CD8(+)CD28(-), and CD8(+)CD122(+) in mononuclear cells. Histological staining disclosed a marked decrease of inflammatory cells in spinal cord and brain of Fasudil-treated mice. These results, together with previous studies showing the inhibitory effect of Fasudil on T-cell migration, might expand its clinical application as a new therapy for multiple sclerosis by decreasing cell migration and regulating immune balance.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Analysis of Variance; Animals; Cell Proliferation; Cells, Cultured; Cytokines; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Enzyme-Linked Immunosorbent Assay; Female; Flow Cytometry; Gene Expression Regulation, Enzymologic; Glycoproteins; Leukocytes, Mononuclear; Mice; Mice, Inbred C57BL; Myelin-Oligodendrocyte Glycoprotein; Peptide Fragments; Protein Kinase Inhibitors; rho-Associated Kinases; Severity of Illness Index; Spinal Cord; Spleen

Experimental autoimmune encephalomyelitis (EAE) is a mouse model for multiple sclerosis, where disease is mediated by autoantigen-specific T cells. Although there is evidence linking CD4(+) T cells that secrete IL-17, termed Th17 cells, and IFN-gamma-secreting Th1 cells with the pathogenesis of EAE, the precise contribution of these T cell subtypes or their associated cytokines is still unclear. We have investigated the infiltration of CD4(+) T cells that secrete IFN-gamma, IL-17 or both cytokines into CNS during development of EAE and have examined the role of T cells in microglial activation. Our findings demonstrate that Th17 cells and CD4(+) T cells that produce both IFN-gamma and IL-17, which we have called Th1/Th17 cells, infiltrate the brain prior to the development of clinical symptoms of EAE and that this coincides with activation of CD11b(+) microglia and local production of IL-1beta, TNF-alpha and IL-6 in the CNS. In contrast, significant infiltration of Th1 cells was only detected after the development of clinical disease. Co-culture experiments, using mixed glia and MOG-specific T cells, revealed that T cells that secreted IFN-gamma and IL-17 were potent activators of pro-inflammatory cytokines but T cells that secrete IFN-gamma, but not IL-17, were less effective. In contrast both Th1 and Th1/Th17 cells enhanced MHC-class II and co-stimulatory molecule expression on microglia. Our findings suggest that T cells which secrete IL-17 or IL-17 and IFN-gamma infiltrate the CNS prior to the onset of clinical symptoms of EAE, where they may mediate CNS inflammation, in part, through microglial activation.

Topics: Animals; CD11b Antigen; CD4-Positive T-Lymphocytes; Cells, Cultured; Central Nervous System; Coculture Techniques; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Enzyme-Linked Immunosorbent Assay; Glycoproteins; Injections, Subcutaneous; Interferon-gamma; Interleukin-17; Interleukin-1beta; Interleukin-6; Mice; Mice, Inbred C57BL; Microglia; Multiple Sclerosis; Myelin-Oligodendrocyte Glycoprotein; Peptide Fragments; Polymerase Chain Reaction; Th1 Cells; Time Factors; Tumor Necrosis Factor-alpha

Steroid receptor coactivator-3 (SRC-3) has been demonstrated to regulate lipid metabolism by inhibiting adipocyte differentiation. In this study, the potential role of SRC-3 in experimental autoimmune encephalomyelitis (EAE), which characterized by inflammatory demyelination in central nervous system (CNS), was examined by analyzing disease progression in SRC-3-deficient (SRC-3(-/-)) mice. We found that SRC-3 deficiency significantly attenuated the disease severity of EAE along with decreased inflammatory infiltration and demyelination. However, these effects are not caused by inhibition of peripheral T cell response, but by upregulated expression of peroxisome proliferator-activated receptor (PPAR)-beta in CNS, which induced an alternative activation state of microglia in SRC-3(-/-) mice. These alternatively activated microglia inhibited CNS inflammation through inhibition of proinflammatory cytokines and chemokines, such as TNF-alpha, IFN-gamma, CCL2, CCL3, CCL5, and CXCL10, as well as upregulation of anti-inflammatory cytokine IL-10 and opsonins, such as C1qa and C1qb. Moreover, microglia alternative activation promoted myelin regeneration through increased accumulation of oligodendrocyte precursors in white matter and elevated expression of myelin genes in the spinal cords of SRC-3(-/-) mice. Our results build up a link between lipid metabolic regulation and immune functions, and the modulation of the expression of SRC-3 or PPAR-beta may hopefully has therapeutic modality in MS and possibly other neurodegenerative diseases.

Topics: Analysis of Variance; Animals; Cell Proliferation; Central Nervous System; Cytokines; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Flow Cytometry; Gene Expression Regulation; Glycoproteins; Leukocytes, Mononuclear; Mice; Mice, Inbred C57BL; Mice, Knockout; Microglia; Myelin Sheath; Myelin-Oligodendrocyte Glycoprotein; Nuclear Receptor Coactivator 3; Peptide Fragments; PPAR-beta; RNA, Messenger

Immune-based self-recognition and failure to modulate this response are believed to contribute to the debilitating autoimmune pathology observed in multiple sclerosis (MS). Studies from its murine model, experimental autoimmune encephalomyelitis (EAE), have shown that neuroantigen-specific CD4+T cells are capable of inducing disease, while their immune sibling, the CD8+T cells, have largely been ignored. To understand their role in autoimmune demyelination, we first confirmed that, similar to our observations in human MS, there is robust induction of neuroantigen-reactive CD8+T cells in several models, including MOG(35-55)/CFA-induced EAE. However, MOG(35-55)-specific CD8+T-cells, when purified, were unable to adoptively transfer disease into naïve mice (in contrast to CD4+T-cells). In fact, we observed that the transfer of these neuroantigen-specific CD8+T cells was able to suppress the induction of EAE and to inhibit ongoing EAE. These regulatory CD8+T cells produced IFN-gamma and perforin and were able to kill MOG loaded CD4+T-cells as well as CD4-depleted APC, suggesting a cytotoxic/suppressor mechanism. Inhibition of EAE was associated with both the modulation of APC function as well as decreased MOG-specific CD4+T cell responses. Our studies reveal a novel and unexpected immune regulatory function for neuroantigen-specific CD8+T cells and have interesting biologic and therapeutic implications.

Topics: Animals; CD8-Positive T-Lymphocytes; Cells, Cultured; Central Nervous System; Cytotoxicity, Immunologic; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Female; Glycoproteins; Humans; Interferon-gamma; Mice; Mice, Inbred C57BL; Multiple Sclerosis; Myelin-Oligodendrocyte Glycoprotein; Peptide Fragments; T-Lymphocytes, Regulatory

Clinical symptoms in MOG-induced EAE mice significantly exacerbated following chondroitin sulfate A (CS-A) injection, whereas administration of a degraded product, CSPG-DS, caused dramatic inhibition of EAE development. Also, administration of CSPG-DS but not CS-A, after the onset of clinical symptoms of EAE, was able to suppress the disease. Further studies demonstrated that CS-A up-regulated STAT4 expression and thus, induced IFN-gamma production and Th1 CD4 T cell differentiation. CS-A also up-regulated STAT3 and IL-23 expression and thus increased IL-17 producing T cells. CSPG-DS treatment both in vivo and in vitro decreased TNFalpha production from splenocytes. In vitro and in vivo studies indicated that CSPG-DS treatment in EAE mice significantly blocked migration of lymphocytes, whereas CS-A treatment increased lymphocyte infiltration in the brain.

Topics: Adjuvants, Immunologic; Amino Acid Sequence; Animals; Anti-Inflammatory Agents, Non-Steroidal; Chondroitin Sulfate Proteoglycans; Disaccharides; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Female; Glycoproteins; Mice; Mice, Inbred C57BL; Molecular Sequence Data; Multiple Sclerosis; Myelin-Oligodendrocyte Glycoprotein; Peptide Fragments; Toxoids

The purpose of the present study was to develop a peptide for treatment of multiple sclerosis (MS). We have tested the effect of a novel anti-inflammatory peptide (KGHYAERVG, termed IIIM1) on experimental autoimmune encephalitis (EAE), an animal model of MS. Our findings demonstrate significant reduction in neurological score following oral administration of IIIM1. Structural studies revealed that the entire peptide is required for activity. The peptide caused significant reduction in IL17, interferon gamma, IL23 and IL12 production by isolated splenocytes and concomitant elevation of anti-inflammatory cytokines. IIIM1 elevated T regulatory cells (Tregs, CD4(+)CD25(+)FoxP3(+)) in brain and spleen of EAE mice. Similar proliferative effect was observed in isolated human and mouse Tregs in vitro. Stimulation of Tregs by IIIM1 caused production of a new peptide termed RA1 present in Oryza Sativa Japonica group. This Japanese rice peptide ameliorated neurological symptoms in the EAE model. Similar beneficial effect was observed upon oral administration of an extract of Japanese rice. In conclusion, oral treatment with IIIM1 ameliorates EAE symptoms via stimulation of Tregs to proliferate and produce RA1 which reduces EAE symptoms. RA1 might be involved in the relatively low prevalence of MS in Japan and other Japanese rice-eating populations.

Topics: Animals; Anti-Inflammatory Agents; Brain; CD4 Antigens; Cell Proliferation; Cells, Cultured; Cytokines; Disease Models, Animal; Disease Progression; Encephalomyelitis, Autoimmune, Experimental; Female; Forkhead Transcription Factors; Freund's Adjuvant; Glycoproteins; Histones; Humans; Interleukin-2 Receptor alpha Subunit; Mice; Mice, Inbred C57BL; Multiple Sclerosis; Myelin Proteolipid Protein; Myelin-Oligodendrocyte Glycoprotein; Oryza; Peptide Fragments; Plant Extracts; Plant Proteins; Rats; Rats, Inbred Lew; T-Lymphocytes, Regulatory

To determine if suppressing Nogo-A, an axonal inhibitory protein, will promote functional recovery in a murine model of multiple sclerosis (MS).. A small interfering RNA was developed to specifically suppress Nogo-A (siRNA-NogoA). The siRNA-NogoA silencing effect was evaluated in vitro and in vivo via immunohistochemistry. The siRNA was administered intravenously in 2 models of experimental autoimmune encephalomyelitis (EAE). Axonal repair was measured by upregulation of GAP43. Enzyme-linked immunosorbent assay, flow cytometry, and (3)H-thymidine incorporation were used to determine immunological changes in myelin-specific T cells in mice with EAE.. The siRNA-NogoA suppressed Nogo-A expression in vitro and in vivo. Systemic administration of siRNA-NogoA ameliorated EAE and promoted axonal repair, as demonstrated by enhanced GAP43+ axons in the lesions. Myelin-specific T-cell proliferation and cytokine production were unchanged in the siRNA-NogoA-treated mice.. Silencing Nogo-A in EAE promotes functional recovery. The therapeutic benefit appears to be mediated by axonal growth and repair, and is not attributable to changes in the encephalitogenic capacity of the myelin-specific T cells. Silencing Nogo-A may be a therapeutic option for MS patients to prevent permanent functional deficits caused by immune-mediated axonal damage.

Topics: Animals; Cell Line, Tumor; Cell Proliferation; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Enzyme-Linked Immunosorbent Assay; Flow Cytometry; GAP-43 Protein; Gene Expression Regulation; Glycoproteins; Interferon-gamma; Interleukin-10; Lymphocytes; Mice; Mice, Inbred C57BL; Mice, Transgenic; Myelin Basic Protein; Myelin Proteins; Myelin-Oligodendrocyte Glycoprotein; Neuroblastoma; Nogo Proteins; Peptide Fragments; RNA, Small Interfering; Spinal Cord; Tetradecanoylphorbol Acetate; Transfection

Combination therapies with existing or novel drugs for multiple sclerosis (MS) have great clinical potential to improve MS treatment outcomes. Our previous studies had confirmed that the combined treatment of minocycline and prednisone produced beneficial effects partially through preventing the reduction of brain-derived neurotrophic factor and nerve growth factor mRNA expression in the cerebral cortex of experimental autoimmune encephalomyelitis (EAE) mice. As high-dose methylprednisolone administered intravenously has more superior efficacy than oral prednisone and had been provided as a stable therapy for MS patients at the onset of an acute relapse, we further evaluated the effects of combined methylprednisolone and minocycline at suboptimal doses on EAE mice at the acute stage in this study. Interferon gamma (IFN-γ) and interleukin-4 (IL-4), the hallmark cytokines that direct Th1 and Th2 development and play an important role in the pathogenesis of MS as well as EAE, were also assayed. Obtained results showed that combined treatment could successfully attenuate severe clinical deficit and suppress histopathological events in EAE. In addition, reduced IFN-γ and increased IL-4 production/expression were found in the splenocytes culture supernatants and brains of EAE mice by the combined treatment. Our data indicate that the combination of methylprednisolone and minocycline may be a promising therapy for MS.

Topics: Analysis of Variance; Animals; Anti-Bacterial Agents; Brain-Derived Neurotrophic Factor; Disease Models, Animal; Dose-Response Relationship, Drug; Drug Synergism; Encephalomyelitis, Autoimmune, Experimental; Enzyme-Linked Immunosorbent Assay; Female; Freund's Adjuvant; Glycoproteins; Interferon-gamma; Interleukin-4; Methylprednisolone; Mice; Mice, Inbred C57BL; Minocycline; Myelin-Oligodendrocyte Glycoprotein; Nerve Growth Factor; Neuroprotective Agents; Peptide Fragments; RNA, Messenger

Multiple sclerosis (MS) is an inflammatory demyelinating disease of the CNS and remyelination in MS ultimately fails. Although strategies to promote myelin repair are eagerly sought, mechanisms underlying remyelination in vivo have been elusive. CXCR2 is expressed on neutrophils and oligodendrocyte lineage cells in the CNS. CXCR2-positive neutrophils facilitate inflammatory demyelination in demyelination models such as experimental autoimmune encephalomyelitis (EAE) and cuprizone intoxication. Systemic injection of a small molecule CXCR2 antagonist at the onset of EAE decreased demyelinated lesions. These results left the cellular target of the CXCR2 antagonist uncertain and did not clarify whether CXCR2 blockade prevented demyelination or promoted remyelination. Here, we show that the actions of CXCR2 on nonhematopoietic cells unexpectedly delay myelin repair. Bone marrow chimeric mice (Cxcr2(+/-)-->Cxcr2(-/-) and Cxcr2(+/-)-->Cxcr2(+/+)) were subjected to two distinct models of myelin injury. In all cases, myelin repair was more efficient in Cxcr2(+/-)-->Cxcr2(-/-) animals. Oligodendrocyte progenitor cells (OPCs) in demyelinated lesions of Cxcr2(+/-)-->Cxcr2(-/-) mice proliferated earlier and more vigorously than in tissues from Cxcr2(+/-)--> Cxcr2(+/+) animals. In vitro demyelinated CNS slice cultures also showed better myelin repair when CXCR2 was blocked with neutralizing antibodies or was genetically deleted. Our results suggest that CXCR2 inactivation permits optimal spatiotemporal positioning of OPCs in demyelinating lesions to receive local proliferative and differentiating signals. Given that CXCR2 exerts dual functions that promote demyelination and decrease remyelination by actions toward hematopoietic cells and nonhematopoietic cells, respectively, our findings identify CXCR2 as a promising drug target for clinical demyelinating disorders.

Topics: Animals; Animals, Newborn; Antibodies; Bone Marrow; Cell Differentiation; Central Nervous System Stimulants; Cerebellum; Cuprizone; Demyelinating Diseases; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Flow Cytometry; Freund's Adjuvant; Glycoproteins; In Vitro Techniques; Leukocyte Common Antigens; Lipopolysaccharides; Mice; Mice, Inbred C57BL; Mice, Knockout; Microscopy, Electron, Transmission; Myelin Basic Protein; Myelin Proteolipid Protein; Myelin Sheath; Myelin-Oligodendrocyte Glycoprotein; Nerve Regeneration; Neurologic Examination; Oligodendroglia; Peptide Fragments; Picrotoxin; Proliferating Cell Nuclear Antigen; Receptors, Interleukin-8B; Recovery of Function; Severity of Illness Index; Stem Cells; Time Factors

3G11, a sialylated carbohydrate epitope on the disialoganglioside molecule, is expressed predominantly on the surface of mouse CD4(+) T cells. Our previous studies suggested that lack of the 3G11 molecule could be a new cell surface marker for regulatory CD4(+) T cells. In the present study, we explore the relationship between 3G11(-) and CD25(+) T cells, a well-defined, naturally occurring regulatory T cell population. We found that a large proportion of CD25(+)CD4(+) T cells lack expression of 3G11 and that more 3G11(-)CD4(+) T cells express Foxp3 compared to the 3G11(+)CD4(+) population. Based on 3G11 and CD25 expression we sorted four CD4(+) T cell subpopulations and tested their phenotypes. Among four CD25/3G11-related CD4(+) T cell subpopulations, CD25(+)3G11(-) T cells expressed the highest levels of Foxp3 and IL-10 and most efficiently inhibited mitogenic and antigen-specific immune responses in vitro and clinical EAE in vivo, while CD25(-)3G11(+) T cells produced a higher level of proinflammatory cytokines and enhanced autoimmune responses. Thus, among CD4(+)CD25(+) T cells, CD25(+)3G11(-) T cells represent a more effective Treg subpopulation than CD25(+)3G11(+) T cells.

Topics: Animals; Antigens, CD; Antigens, Surface; Cell Proliferation; Coculture Techniques; Cytokines; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Epitopes; Female; Flow Cytometry; Fluoresceins; Forkhead Transcription Factors; Gene Expression Regulation; Glycoproteins; Mice; Mice, Inbred C57BL; Mice, Transgenic; Myelin-Oligodendrocyte Glycoprotein; Peptide Fragments; Receptors, Antigen, T-Cell; Statistics, Nonparametric; Succinimides; T-Lymphocytes, Regulatory

Clinical studies indicate that anti-CD20 B-cell depletion may be an effective multiple sclerosis (MS) therapy. We investigated mechanisms of anti-CD20-mediated immune modulation using 2 paradigms of experimental autoimmune encephalomyelitis (EAE).. Murine EAE was induced by recombinant myelin oligodendrocyte glycoprotein (rMOG), a model in which B cells are considered to contribute pathogenically, or MOG peptide (p)35-55, which does not require B cells.. In EAE induced by rMOG, B cells became activated and, when serving as antigen-presenting cells (APCs), promoted differentiation of proinflammatory MOG-specific Th1 and Th17 cells. B-cell depletion prevented or reversed established rMOG-induced EAE, which was associated with less central nervous system (CNS) inflammation, elimination of meningeal B cells, and reduction of MOG-specific Th1 and Th17 cells. In contrast, in MOG p35-55-induced EAE, B cells did not become activated or efficiently polarize proinflammatory MOG-specific T cells, similar to naive B cells. In this setting, anti-CD20 treatment exacerbated EAE, and did not impede development of Th1 or Th17 cells. Irrespective of the EAE model used, B-cell depletion reduced the frequency of CD4(+)CD25(+)Foxp3(+) regulatory T cells (Treg), and increased the proinflammatory polarizing capacity of remaining myeloid APCs.. Our study highlights distinct roles for B cells in CNS autoimmunity. Clinical benefit from anti-CD20 treatment may relate to inhibition of proinflammatory B cell APC function. In certain clinical settings, however, elimination of unactivated B cells, which participate in regulation of T cells and other APC, may be undesirable. Differences in immune responses to MOG protein and peptide may be important considerations when choosing an EAE model for testing novel B cell-targeting agents for MS.

Topics: Animals; Antibodies; Antigens, CD20; B-Lymphocytes; CD4-Positive T-Lymphocytes; Cytokines; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Female; Flow Cytometry; Forkhead Transcription Factors; Glycoproteins; Humans; Interleukin-2 Receptor alpha Subunit; Lymphocyte Activation; Mice; Mice, Inbred C57BL; Mice, Transgenic; Myelin-Oligodendrocyte Glycoprotein; Peptide Fragments; Statistics, Nonparametric; T-Lymphocytes, Regulatory

Experimental autoimmune encephalomyelitis (EAE) is a T cell-mediated neuroinflammatory disease that is often used as a model of multiple sclerosis. EAE can follow either relapsing-remitting (RR) or chronic (CH) courses, yet the factors responsible for differentially inducing these forms of disease remain largely unknown. Proinflammatory cytokines play an important role in EAE, and signaling by these cytokines can be negatively regulated by the suppressor of cytokine signaling 1 protein (SOCS1). We assessed if differential expression of SOCS1 could contribute to the clinical course of RR and CH forms of EAE induced in the same mouse strain (C57BL/6) using the same myelin antigen (myelin oligodendrocyte glycoprotein). We show that SOCS1 mRNA levels are significantly elevated in the spinal cord in early stages of both RR- and CH-EAE. SOCS1 protein is highly expressed in immune cells in EAE lesions in the spinal cord, with expression predominantly localized within macrophages. Importantly, the number of Mac-1(+) macrophages expressing SOCS1 at the peak stage of RR disease is three-fold greater than in CH-EAE. Furthermore, the macrophage effector molecule iNOS, whose expression is regulated by SOCS1, is significantly reduced at the peak of RR- versus CH-EAE. Finally, the administration of a SOCS1-mimetic peptide reduces disease severity in the CH-EAE model. Thus, the differential expression of SOCS1 may contribute to the development of RR and CH forms of EAE.

Topics: Animals; CD8 Antigens; Disease Models, Animal; Disease Progression; Encephalomyelitis, Autoimmune, Experimental; Female; Freund's Adjuvant; Galectin 3; Ganglia, Spinal; Gene Expression Regulation; Glycoproteins; Macrophages; Mice; Mice, Inbred C57BL; Myelin-Oligodendrocyte Glycoprotein; Nitric Oxide Synthase Type II; Peptide Fragments; Recurrence; RNA, Messenger; Severity of Illness Index; Spinal Cord; Suppressor of Cytokine Signaling 1 Protein; Suppressor of Cytokine Signaling Proteins

We have investigated the spontaneous and the depolarisation-induced release of [(3)H]D-aspartate ([(3)H]D-ASP), a non-metabolisable analogue of glutamate, in spinal cord slices, synaptosomes and gliosomes from mice with experimental autoimmune encephalomyelitis (EAE) at 13, 21 and 55 days post-immunisation (d.p.i.), representing onset, peak and chronic phases of the pathology. At 13 and 21 d.p.i., the KCl-evoked, calcium-dependent overflow of [(3)H]D-ASP in spinal cord slices was significantly lower (30-40%), whereas at 55 d.p.i. it was significantly higher (30%), than that elicited in matched controls. When the release was measured from spinal cord synaptosomes and gliosomes in superfusion, a different picture emerged. The spontaneous and the KCl(15 mM)-induced release of [(3)H]D-ASP were significantly increased both in synaptosomes (17% and 45%, respectively) and gliosomes (26% and 25%, respectively) at 21, but not at 13, d.p.i. At 55 d.p.i., the KCl-induced [(3)H]D-ASP release was significantly increased (40%) only in synaptosomes. Finally, uptake of [(3)H]D-ASP was markedly (50-60%) increased in spinal cord synaptosomes, but not in gliosomes, obtained from EAE mice at 21 d.p.i., whereas no differences could be detected at 13 d.p.i. Our data indicate that glutamatergic neurotransmission is altered in the spinal cord of EAE mice.

Topics: Animals; Aspartic Acid; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Female; Glutamic Acid; Glycoproteins; In Vitro Techniques; Mice; Mice, Inbred C57BL; Myelin-Oligodendrocyte Glycoprotein; Neuroglia; Neurons; Peptide Fragments; Potassium Chloride; Spinal Cord; Subcellular Fractions; Tritium

Oxidized phospholipids (Ox-PLs) are generated in abundance at sites of inflammation. Recent studies have indicated that Ox-PLs may also exhibit anti-inflammatory activities. In this study, we investigated the beneficial effect of VB-201, a pure synthetic Ox-PL analog that we synthesized, on the development of a central nervous system (CNS) autoimmune inflammatory disease, in vivo. Oral administration of VB-201 ameliorated the severity of experimental autoimmune encephalomyelitis (EAE) induced by myelin oligodendrocyte glycoprotein (MOG) peptide MOG35-55, and restrained the encephalogenicity of MOG35-55-specific T-cells. Our data presents a novel prospect for the role of Ox-PL analogs in CNS inflammatory diseases.

Topics: Animals; Bone Marrow Cells; Bromodeoxyuridine; CD4 Antigens; Cell Differentiation; Central Nervous System; Cytokines; Dendritic Cells; Disease Models, Animal; Drug Interactions; Encephalitis; Encephalomyelitis, Autoimmune, Experimental; Enzyme-Linked Immunosorbent Assay; Female; Forkhead Transcription Factors; Freund's Adjuvant; Glycerylphosphorylcholine; Glycoproteins; Ionomycin; Ionophores; Lymph Nodes; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Myelin-Oligodendrocyte Glycoprotein; Peptide Fragments; Pertussis Toxin; Phosphorylcholine; Polymethacrylic Acids; Severity of Illness Index; T-Lymphocytes; Time Factors

Multiple sclerosis (MS) is a demyelinating disease characterized by the breakdown of the blood-brain barrier (BBB), and accumulation of inflammatory infiltrates in the central nervous system. Tight junctions are specialized cell-cell adhesion structures and critical components of the BBB that have previously been shown to be abnormally distributed in MS tissue. To evaluate whether experimental autoimmune encephalomyelitis (EAE) provides a suitable model for this aspect of MS disease, we examined the expression and distribution of ZO-1 over the course of disease in EAE. We observed a dramatic relocalization of ZO-1 which precedes overt clinical disease and correlates with the sites of inflammatory cell accumulation. Treatment of in vitro cultures of murine brain endothelial cells with components of EAE induction provided similar findings, with relocalization of ZO-1 and increased permeability of endothelial monolayers. BBB disruption in the EAE model appears to parallel disease progression in MS, with direct effects on the cerebrovascular endothelium, making it an ideal tool for future evaluation of tight junction breakdown and repair in MS-like pathology.

Topics: Animals; Blood-Brain Barrier; Brain; Calcium-Binding Proteins; Capillary Permeability; Cell Adhesion Molecules; Cells, Cultured; Dextrans; Disease Models, Animal; Disease Progression; Encephalomyelitis, Autoimmune, Experimental; Endothelial Cells; Female; Gene Expression Regulation; Glycoproteins; Lipopolysaccharides; Membrane Proteins; Mice; Mice, Inbred C57BL; Microfilament Proteins; Myelin-Oligodendrocyte Glycoprotein; Nerve Fibers, Myelinated; Occludin; Peptide Fragments; Pertussis Toxin; Phosphoproteins; Receptors, Cell Surface; Spinal Cord; Tight Junctions; Zonula Occludens-1 Protein

Aquaporin-4 (AQP4) is a water channel protein that plays an important role in water movement in the central nervous system (CNS). Recently, presence of anti-AQP4 antibody has been reported in the sera from patients with neuromyelitis optica. AQP4 is therefore a possible target for inflammatory mechanisms in CNS. In the present investigation, we performed semi-quantitative analysis of AQP4-mRNA in brain and spinal cord from mice affected with experimental autoimmune encephalomyelitis (EAE) using real-time PCR. AQP4-mRNA expression was increased in EAE; reaching a peak in the spinal cord at 14 days, and in the brain at 21 days after first inoculation. Immunohistochemical analysis showed that AQP4 is expressed on astrocytes, indicating that the increase in AQP4 expression may correlate with astrocytic activation. This is the first study to demonstrate upregulation of AQP4 in EAE. The upregulation of AQP4 could be involved in the development of inflammation in the acute phase of EAE.

Topics: Animals; Aquaporin 4; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Glial Fibrillary Acidic Protein; Glycoproteins; Mice; Mice, Inbred C57BL; Myelin-Oligodendrocyte Glycoprotein; Peptide Fragments; RNA, Messenger; Up-Regulation

Acridine-iminodibenzyl chimeric compounds were previously introduced as a class of cholesterol-redistributing substances with antiprion effects. Here, we show that administration of the lead compound quinpramine to mice with experimental autoimmune encephalitis, an animal model of multiple sclerosis (MS), significantly ameliorates disease in preventive and therapeutic paradigms. Quinpramine treatment decreased the number of inflammatory CNS lesions, antigen-specific T-cell proliferation, and pro-inflammatory cytokines IFNgamma and IL-17. Quinpramine is thus an immunoregulatory drug that is a candidate pharmaceutical for MS.

Topics: Animals; Cell Proliferation; Cytokines; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Female; Glycoproteins; Mice; Mice, Inbred C57BL; Myelin-Oligodendrocyte Glycoprotein; Peptide Fragments; Quinolinium Compounds; T-Lymphocytes

LFA-1 (CD11a/CD18) is a member of the beta(2)-integrin family of adhesion molecules important in leukocyte trafficking and activation. Although LFA-1 is thought to contribute to the development of experimental autoimmune encephalomyelitis (EAE) primarily through its functions on effector T cells, its importance on other leukocyte populations remains unexplored. To address this question, we performed both adoptive transfer EAE experiments involving CD11a(-/-) mice and trafficking studies using bioluminescent T cells expressing luciferase under the control of a CD2 promoter (T-lux cells). Transfer of encephalitogenic CD11a(-/-) T cells to wild type mice resulted in a significant reduction in overall EAE severity compared to control transfers. We also observed, using in vivo imaging techniques, that CD11a(-/-) T-lux cells readily infiltrated lymph nodes and the CNS of wild type recipients with kinetics comparable to CD11a(+/+) transfers, although their overall numbers in these organs were reduced. Surprisingly, transfer of encephalitogenic wild type T cells to CD11a(-/-) mice induced a severe and sometimes fatal EAE disease course, associated with massive T cell infiltration and proliferation in the CNS. These data indicate that LFA-1 expression on leukocytes in recipient mice plays an important immunomodulatory role in EAE. Thus, LFA-1 acts as a key regulatory adhesion molecule during the development of EAE, serving both pro- and anti-inflammatory roles in disease pathogenesis.

Topics: Analysis of Variance; Animals; CD11a Antigen; Central Nervous System; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Glycoproteins; Luciferases; Lymphocyte Activation; Lymphocyte Function-Associated Antigen-1; Mice; Mice, Inbred C57BL; Mice, Transgenic; Myelin-Oligodendrocyte Glycoprotein; Peptide Fragments; Statistics, Nonparametric; T-Lymphocytes; Time Factors

Human Mesenchymal Stem Cells (MSCs) were previously reported to ameliorate neuronal functional deficits in the MOG35-55-induced experimental autoimmune encephalomyelitis (EAE) mice by inducing T cell anergy. Human Ciliary neurotrophic factor (CNTF) recently was found to promote myelogenesis and reduce inflammation in CNTF-deficient EAE mice. We ectopically overexpressed CNTF in human MSCs to investigate its potential role in promoting remyelination and improving functional recovery in EAE mice. MSCs transfected by Ad-CNTF-IRES-EGFP (MSC-CNTF) were injected intravenously into EAE mice 10 days after the immunization. Neurological functional tests were scored daily by grading clinical signs (score 0-6). Immunofluorescence microscopy was used to detect MSC-CNTF in spinal cord. Expression of NG2, CNTF, and cleaved caspase-3 was measured by immunohistochemistry. CNTF expression was also analyzed by Western blot. Myelin was detected by Solochrome Cyanin staining. Our results found that CNTF concentration in MSC-CNTF cells was 20-fold higher than that in either MSC or Ad-EGFP-transfected MSCs (MSC-EGFP) in vitro. Mice receiving MSC-CNTF cells showed remarkable neuronal functional recovery: the cumulative clinical scores were significantly decreased, and the disease onset was statistically delayed. Mice receiving MSC-CNTF cells showed reduced TNF-alpha, IFN-gamma and increased the level of cytokine IL-10 in peripheral blood and a large number of MSC-CNTF cells were detected in the spleen, but were not detected in other organs such as lung, liver and kidney. In the lesions of these mice, 1) the number of cleaved caspase3-positive cells was significantly reduced; 2) MSC-CNTF- and NG2-positive cells were significantly increased; and 3) the expression of CNTF was dramatically increased. In addition, demyelination was significantly reduced in MSC-CNTF mice. These data indicated that MSC-CNTF may improve functional recovery in EAE mice, possibly by exerting their immunoregulatory activity, inhibiting inflammation, homing MSC-CNTF cells to the lesions, elevating CNTF expression, reducing demyelination, and stimulating oligodendrogenesis.

Topics: Adenoviridae; Amino Acids; Analysis of Variance; Animals; Antigens; Caspase 3; Cell Count; Ciliary Neurotrophic Factor; Cytokines; Demyelinating Diseases; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Female; Genetic Vectors; Glycoproteins; Green Fluorescent Proteins; Humans; Mesenchymal Stem Cell Transplantation; Mesenchymal Stem Cells; Mice; Mice, Inbred C57BL; Mice, Knockout; Myelin-Oligodendrocyte Glycoprotein; Peptide Fragments; Proteoglycans; Recovery of Function; Statistics, Nonparametric; Time Factors

Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system (CNS). While the primary symptoms of MS are losses of sensory and motor functions, it is now recognized that chronic pain is also a major concern affecting between 50% and 80% of MS patients. To date, however, few studies have examined the underlying mechanisms of chronic pain in MS or in the animal model, experimental autoimmune encephalomyelitis (EAE), which shares many features of MS pathology. We, therefore, set out to characterize the changes in pain sensitivity that arises in a chronic-relapsing model of EAE. We show here that female C57BL/6 mice immunized with myelin oligodendrocyte glycoprotein (MOG(35-55)) develop a robust allodynia to both cold and tactile stimuli. Allodynia emerges early in the disease process, often before any signs of neurological deficit and is independent of the overall symptom severity in these mice. "Classical" cellular substrates for neuropathic pain and allodynia such as altered expression of sensory neuropeptides in the dorsal horn of the spinal do not appear to underlie these changes in sensory function. There is, however, a significant influx of CD3+ T cells and increased astrocyte and microglia/macrophage reactivity in the superficial dorsal horn of mice with MOG(35-55) EAE. This suggests that inflammation and reactive gliosis may be key mediators of allodynia in MOG(35-55) EAE similar to peripheral nerve and spinal cord injury models. Taken together, our results show that the MOG(35-55) EAE model is a useful tool to study neuropathic pain in MS.

Topics: Analysis of Variance; Animals; Antigens, CD; Antigens, Differentiation; Calcitonin Gene-Related Peptide; Disease Models, Animal; Disease Progression; Encephalomyelitis, Autoimmune, Experimental; Female; Gliosis; Glycoproteins; Hyperalgesia; Mice; Mice, Inbred C57BL; Motor Activity; Myelin-Oligodendrocyte Glycoprotein; Neuralgia; Pain Threshold; Peptide Fragments; Reaction Time; Spinal Cord

Dendritic cells (DCs) appear in higher numbers within the CNS as a consequence of inflammation associated with autoimmune disorders, such as multiple sclerosis, but the contribution of these cells to the outcome of disease is not yet clear. Here, we show that stimulatory or tolerogenic functional states of intracerebral DCs regulate the systemic activation of neuroantigen-specific T cells, the recruitment of these cells into the CNS and the onset and progression of experimental autoimmune encephalomyelitis (EAE). Intracerebral microinjection of stimulatory DCs exacerbated the onset and clinical course of EAE, accompanied with an early T-cell infiltration and a decreased proportion of regulatory FoxP3-expressing cells in the brain. In contrast, the intracerebral microinjection of DCs modified by tumor necrosis factor alpha induced their tolerogenic functional state and delayed or prevented EAE onset. This triggered the generation of interleukin 10 (IL-10)-producing neuroantigen-specific lymphocytes in the periphery and restricted IL-17 production in the CNS. Our findings suggest that DCs are a rate-limiting factor for neuroinflammation.

Topics: Animals; CD4-Positive T-Lymphocytes; Central Nervous System; Central Nervous System Stimulants; Dendritic Cells; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Female; Flow Cytometry; Forkhead Transcription Factors; Freund's Adjuvant; Glycoproteins; Immune System Phenomena; Interferon-alpha; Interferon-gamma; Interleukin-7; Lipopolysaccharides; Mice; Mice, Inbred C57BL; Mycobacterium tuberculosis; Myelin-Oligodendrocyte Glycoprotein; Peptide Fragments; Picrotoxin; T-Lymphocytes; Time Factors

An increased expression of vascular endothelial growth factor (VEGF) is associated with demyelinated lesions in both multiple sclerosis (MS) and its model (EAE), implicating changes in vasculature as a potential component of CNS plaque formation. The purpose of this study was to investigate the vascular changes in acute and chronic EAE in C57BL/6 mice induced with myelin oligodendrocyte glycoprotein (MOG ((35-55))) peptide. We investigated the functional contribution of VEGF to acute and chronic EAE by treating immunized mice with SU5416 (Semaxinib), a potent and selective inhibitor of VEGF receptor 2 (VEGFR2). Animals received seven daily injections of SU5416 (50 mg/kg) or vehicle beginning on the day after disease onset (acute study) or on day 45 post-immunization (chronic study). Spinal cord sections were collected on the day of sacrifice. Modulation of angiogenic gene expression was determined using RNA isolated from 4 acute and 4 non-immunized controls. MOG peptide induction produced extensive demyelination, immune cell infiltration, tissue laminin deposits, and axonal loss in lesions. VEGF expression was extensively increased in the acute mice, which correlated positively with clinical score. In the acute study, SU5416 treatment produced a significant clinical improvement versus vehicle controls (p<0.001), with less demyelination (-37%) and cellular infiltration (-23%) in the spinal cord (p<0.05). Treated animals also had significantly fewer blood vessels per section than controls (56.1+/-6.1 v. 81.6+/-11.5, p<0.05), and significantly reduced laminin abnormalities (28.9% of lesion area v. 46.8%, p<0.05). There was no improvement in clinical score or tissue pathology, and no difference in vessel number or lesion laminin expression, when SU5416 was administered during the chronic disease (all p>0.05). In the acute study only, VEGF staining correlated with demyelination and the extent of cellular infiltration in both control (r=0.723, r=0.665) and treated (r=0.681, r=0.487) animals (all p<0.05). Laminin staining in lesion areas was strongly correlated with tissue pathology for all animals in both the acute and chronic study (all p<0.001). Vascular alterations in MOG peptide-induced EAE in the mouse are accompanied by increased lesion-specific levels of VEGF, extensive laminin deposits in the tissue and altered transcription of numerous angiogenic factors. In the microarray studies, acute mice showed a significant increase in several angiogenic RNA tran

Topics: Angiogenesis Inhibitors; Angiogenic Proteins; Animals; Blood Vessels; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Female; Glycoproteins; Indoles; Laminin; Mice; Mice, Inbred C57BL; Myelin Sheath; Myelin-Oligodendrocyte Glycoprotein; Neovascularization, Pathologic; Peptide Fragments; Pyrroles; RNA, Messenger; Spinal Cord; Up-Regulation; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factor Receptor-2; Wallerian Degeneration

The response of the immune system during injury of the central nervous system may play a role in protecting neurons. We have previously reported that immunization with MOG 35-55 prior to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced injury of the dopaminergic system promotes less dopamine depletion and less dopaminergic damage of neurons in mice. In this study, we evaluate the influence of MOG immunization on the inflammatory reaction that occurs at the place of injury. C57Bl male mice, 2 and 12 months old, received i.p. injections of MPTP (40 mg/kg) and some groups animals also received an additional injection with myelin oligodendrocyte glycoprotein (MOG) 35-55 in CFA 6 days before MPTP administration. MPTP caused a common inflammatory reaction characterized by microglial activation, infiltration of T cells into the substantia nigra and striatum and increased expression of mRNA encoding pro-inflammatory cytokines (IL-1 beta, TNFalpha, INF gamma) and trophic factors (TGFbeta, GDNF). MOG immunization prior to MPTP administration significantly diminished the microglial reaction and reduced the levels of infiltrating CD8+ lymphocytes. The number of CD4+ T cells remained at the same level as in the MPTP group. Expression of pro-inflammatory cytokines was diminished. The mRNA expression of GDNF was significantly higher in the MOG pretreated mice relative to the MPTP group, both in the 2 month old and 12 month old groups. Since MOG immunization prior to MPTP intoxication appears to prevent nigrostriatal injury, the observed decrease of inflammation and increase of GDNF mRNA expression in the injured areas might represent one of the mechanisms of observed neuroprotection.

Topics: 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine; Animals; Brain; CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; Cytokines; Disease Models, Animal; Glycoproteins; Inflammation; Male; Mice; Mice, Inbred C57BL; MPTP Poisoning; Myelin-Oligodendrocyte Glycoprotein; Neurons; Peptide Fragments

Myelin reactive T cells are central in the development of the autoimmune response leading to CNS destruction in Multiple Sclerosis and Experimental Autoimmune Encephalomyelitis (EAE). Investigations on the mechanisms underlying the activation and expansion of myelin reactive T have stressed the importance of non-autoimmune conditions impinging the autoimmune repertoire potentially involved in the disease. Here, we show that CNS injury caused by the toxic cuprizone results in the generation of immunoreactivity towards several myelin components. Paradoxically, exposure to CNS injury does not increase the susceptibility to develop EAE, but render mice protected to the pathogenic autoimmune response against myelin antigens.

Topics: Animals; Antibody Specificity; Antigens; Chelating Agents; Cuprizone; Cytokines; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Female; Glycoproteins; Immune Tolerance; Lymphocyte Activation; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Myelin Proteins; Myelin Sheath; Myelin-Oligodendrocyte Glycoprotein; Peptide Fragments; T-Lymphocytes

We investigated the possible therapeutic effect of cilostazol, a specific inhibitor of phosphodiesterase-3, for experimental autoimmune encephalomyelitis (EAE). Mice affected with EAE induced by inoculation with MOG(35-55) were fed with cilostazol or vehicle control. The clinical EAE scores of the cilostazol-fed mice were lower than those of the controls. Serum level of soluble intercellular adhesion molecule-1 was significantly lower in the cilostazol-fed mice than in the controls. In the recall responses with MOG(35-55), proliferation and IFN-gamma production by lymphocytes from cilostazol-fed mice were significantly reduced. Cilostazol may exhibit repressive effects on EAE by reducing the antigen-specific T-cell response and decreasing the expression of the adhesion molecules. Cilostazol is a hopeful choice for the treatment of multiple sclerosis.

Topics: Animals; Cilostazol; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Glycoproteins; Intercellular Adhesion Molecule-1; Interferon-gamma; Interleukin-10; Interleukin-17; Lymph Nodes; Mice; Mice, Inbred C57BL; Multiple Sclerosis; Myelin-Oligodendrocyte Glycoprotein; P-Selectin; Peptide Fragments; Phosphodiesterase 3 Inhibitors; Phosphodiesterase Inhibitors; Severity of Illness Index; T-Lymphocytes; Tetrazoles; Tumor Necrosis Factor-alpha; Vascular Cell Adhesion Molecule-1

Voluntary exercise is beneficial in models of primarily neurodegenerative disorders. Whether exercise also affects inflammatory neurodegeneration is unknown. In the present study, we evaluated the clinical, synaptic and neuropathological effects of voluntary wheel running in mice with myelin oligodendrocyte glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis. Exercising EAE mice exhibited less severe neurological deficits compared to control EAE animals. The sensitivity of striatal GABA synapses to the stimulation of cannabinoid CB1 receptors was dramatically downregulated following EAE induction, and was rescued by exercise in EAE mice with access to a running wheel. Finally, we found that exercise was able to contrast dendritic spine loss induced by EAE in striatal neurons, although the degree of inflammatory response was similar in the two experimental groups. Our work suggests that life style and experiences can impact the clinical course of inflammatory neurodegenerative diseases by affecting their synaptic bases.

Topics: Animals; Antigens, CD; Corpus Striatum; Dendrites; Disease Models, Animal; Dronabinol; Encephalomyelitis, Autoimmune, Experimental; Female; Glutamic Acid; Glycoproteins; In Vitro Techniques; Mice; Mice, Inbred C57BL; Movement Disorders; Myelin-Oligodendrocyte Glycoprotein; Neurons; Neuroprotective Agents; Patch-Clamp Techniques; Peptide Fragments; Physical Conditioning, Animal; Silver Staining; Statistics, Nonparametric; Synapses; Synaptic Potentials

Multiple sclerosis (MS) is a neurodegenerative disease characterized by demyelination/remyelination episodes that ultimately fail. Chemokines and their receptors have been implicated in both myelination and remyelination failure. Chemokines regulate migration, proliferation and differentiation of immune and neural cells during development and pathology. Previous studies have demonstrated that the absence of the chemokine receptor CXCR2 results in both disruption of early oligodendrocyte development and long-term structural alterations in myelination. Histological studies suggest that CXCL1, the primary ligand for CXCR2, is upregulated around the peripheral areas of demyelination suggesting that this receptor/ligand combination modulates responses to injury. Here we show that in focal LPC-induced demyelinating lesions, localized inhibition of CXCR2 signaling reduced lesion size and enhanced remyelination while systemic treatments were relatively less effective. Treatment of spinal cord cultures with CXCR2 antagonists reduced CXCL1 induced A2B5+ cell proliferation and increased differentiation of myelin producing cells. More critically, treatment of myelin oligodendrocyte glycoprotein peptide 35-55-induced EAE mice, an animal model of multiple sclerosis, with small molecule antagonists against CXCR2 results in increased functionality, decreased lesion load, and enhanced remyelination. Our findings demonstrate the importance of antagonizing CXCR2 in enhancing myelin repair by reducing lesion load and functionality in models of multiple sclerosis and thus providing a therapeutic target for demyelinating diseases.

Topics: Animals; Antibodies; Axons; Cell Differentiation; Cell Proliferation; Cells, Cultured; Chemokine CXCL1; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Female; Glycoproteins; Mice; Mice, Inbred C57BL; Multiple Sclerosis; Myelin Sheath; Myelin-Oligodendrocyte Glycoprotein; Oligodendroglia; Peptide Fragments; Rats; Rats, Sprague-Dawley; Receptors, Interleukin-8B; Recovery of Function; Spinal Cord; Wallerian Degeneration

The involvement of astrocyte water channel aquaporin-4 (AQP4) in autoimmune diseases of the central nervous system has been suggested following the identification of AQP4 autoantibodies in neuromyelitis optica, an inflammatory demyelinating disease.. We investigated the involvement of AQP4 in disease severity in an established mouse model of experimental autoimmune encephalomyelitis (EAE) produced by immunization with myelin oligodendrocyte glycoprotein (MOG 35-55) peptide. EAE was remarkably attenuated in AQP4 null mice compared to identically treated wildtype mice. Whereas most wildtype mice developed progressive tail and hindlimb paralysis, clinical signs were virtually absent in AQP4 null mice. Brain and spinal cords from AQP1 null mice showed greatly reduced mononuclear cell infiltration compared to wildtype mice, with relatively little myelin loss and axonal degeneration.. The reduced severity of autoimmune encephalomyelitis in AQP4 deficiency suggests AQP4 as a novel determinant in autoimmune inflammatory diseases of the central nervous system and hence a potential drug target.

Topics: Animals; Aquaporin 4; Autoantibodies; Body Weight; Brain; Cell Proliferation; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Female; Fluorescent Antibody Technique; Glycoproteins; Immunohistochemistry; Lymphocyte Activation; Mice; Mice, Knockout; Myelin-Oligodendrocyte Glycoprotein; Peptide Fragments; Severity of Illness Index; Spinal Cord

Recent studies in experimental autoimmune encephalomyelitis (EAE) have found that CNS injury in Daf1(-/-) mice is much greater than in wild types (WTs), suggesting that upregulating DAF levels in vivo might ameliorate disease. To test this, we generated a Daf1 transgenic (Tg) mouse which had elevated DAF levels on its cell surfaces. In by-stand C3b uptake assays, Daf1 Tg mouse erythrocytes took up less C3b on their surfaces than WT erythrocytes. When co-cultured with OT-II CD4(+) T cells together with OVA(323-339) peptide, Daf1 Tg mouse bone marrow derived dendritic cells (BM-DCs) produced less C5a and C3a than WT BM-DCs and stimulated a lesser T cell response. In MOG(35-55) immunization induced EAE model, Daf1 Tg mice exhibited delayed disease onset and decreased clinical scores compared to WTs. Histological analyses showed that there were less inflammation and demyelination in spinal cords in Daf1 Tg mice than those in WTs. In accordance with these results, Daf1 Tg mice had decreased MOG(35-55) specific Th1 and Th17 responses. These data provide further evidence that DAF suppresses autoreactive T cell responses in EAE, and indicate that augmenting its expression levels could be effective therapeutically in treating multiple sclerosis as well as other T cell mediated diseases.

Topics: Animals; CD55 Antigens; Complement C3a; Complement C5a; Dendritic Cells; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Erythrocytes; Glycoproteins; Interferon-gamma; Interleukin-17; Lymphocyte Activation; Mice; Mice, Inbred C57BL; Mice, Knockout; Mice, Transgenic; Myelin-Oligodendrocyte Glycoprotein; Ovalbumin; Peptide Fragments; Spinal Cord; T-Lymphocyte Subsets; T-Lymphocytes, Helper-Inducer

Obesity is associated with numerous inflammatory conditions including atherosclerosis, autoimmune disease and cancer. Although the precise mechanisms are unknown, obesity-associated rises in TNF-alpha, IL-6 and TGF-beta are believed to contribute. Here we demonstrate that obesity selectively promotes an expansion of the Th17 T-cell sublineage, a subset with prominent pro-inflammatory roles. T-cells from diet-induced obese mice expand Th17 cell pools and produce progressively more IL-17 than lean littermates in an IL-6-dependent process. The increased Th17 bias was associated with more pronounced autoimmune disease as confirmed in two disease models, EAE and trinitrobenzene sulfonic acid colitis. In both, diet-induced obese mice developed more severe early disease and histopathology with increased IL-17(+) T-cell pools in target tissues. The well-described association of obesity with inflammatory and autoimmune disease is mechanistically linked to a Th17 bias.

Topics: Animals; Colitis; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Glycoproteins; Interleukin-17; Interleukin-6; Male; Mice; Mice, Inbred C57BL; Myelin-Oligodendrocyte Glycoprotein; Obesity; Peptide Fragments; T-Lymphocytes, Helper-Inducer; Trinitrobenzenesulfonic Acid

Immunization with an altered myelin-derived peptide (MOG45D) improves recovery from acute CNS insults, partially via recruitment of monocyte-derived macrophages that locally display a regulatory activity. Here, we investigated the local alterations in the cellular and molecular immunological milieu associated with attenuation of Alzheimer's disease-like pathology following immunotherapy. We found that immunization of amyloid precursor protein/presenilin 1 double-transgenic mice with MOG45D peptide, loaded on dendritic cells, led to a substantial reduction of parenchymal and perivascular amyloid beta (Abeta)-plaque burden and soluble Abeta((1-42)) peptide levels as well as reduced astrogliosis and levels of a key glial scar protein (chondroitin sulphate proteoglycan). These changes were associated with a shift in the local innate immune response, manifested by increased Iba1+/CD45(high) macrophages that engulfed Abeta, reduced pro-inflammatory (tumor necrosis factor-alpha) and increased anti-inflammatory (interleukin-10) cytokines, as well as a significant increase in growth factors (IGF-1 and TGFbeta) in the brain. Furthermore, the levels of matrix metalloproteinase-9, an enzyme shown to degrade Abeta and is associated with glial scar formation, were significantly elevated in the brain following immunization. Altogether, these results indicate that boosting systemic immune cells leads to a local immunomodulation manifested by elevated levels of anti-inflammatory cytokines and metalloproteinases that contribute to ameliorating Alzheimer's disease pathology.

Topics: Alzheimer Disease; Amyloid beta-Peptides; Amyloid beta-Protein Precursor; Animals; Brain; Chondroitin Sulfate Proteoglycans; Dendritic Cells; Disease Models, Animal; Enzyme-Linked Immunosorbent Assay; Female; Flow Cytometry; Gene Expression Regulation; Glycoproteins; Humans; Insulin-Like Growth Factor I; Interleukin-10; Male; Matrix Metalloproteinase 9; Mice; Mice, Transgenic; Mutation; Myelin-Oligodendrocyte Glycoprotein; Peptide Fragments; Presenilin-1; Transforming Growth Factor beta

The onset of neurological signs in experimental autoimmune encephalomyelitis is tightly associated with infiltration and reactivation of T cells in the central nervous system. The anatomic localization of the initial T cell-antigen-presenting cell (APC) interactions leading to reactivation of T cells in the central nervous system is, however, still unclear. We hypothesized that activated CD4(+) T cells gain direct access to the subarachnoid space and become reactivated on encounter with cognate antigen in this compartment.. C57Bl/6 mice were immunized with MOG35-55, and interactions between CD4(+) T cells and major histocompatibility class II+ APCs in the subarachnoid space were investigated using flow cytometry, confocal microscopy of leptomeningeal whole-mount preparations, time-lapse microscopy of leptomeningeal explants, and in vitro proliferation assays.. CD4(+) T cells, polarized to produce Th1/Th17 cytokines, accumulated in the subarachnoid space early during the course of experimental autoimmune encephalomyelitis, before CD4(+) T cells were detected in the spinal cord parenchyma. At this time point, leptomeningeal but not parenchymal CD4(+) T cells incorporated bromodeoxyuridine, indicating local proliferation of CD4(+) T cells in the subarachnoid space. Time-lapse microscopy indicated that these CD4(+) T cells actively scanned the tissue and interacted with local major histocompatibility class II+ APCs, resulting in long-lasting interactions between CD4(+) T cells and major histocompatibility class II+ APCs, suggestive of immunological synapses.. These results support the concept that immune surveillance of the central nervous system involves the subarachnoid space and indicate that the leptomeninges play an important role in experimental autoimmune encephalomyelitis initiation.

Topics: Animals; Antigen-Presenting Cells; Antigens, CD; Bromodeoxyuridine; CD4-Positive T-Lymphocytes; Cell Movement; Cell Proliferation; Central Nervous System; Cytokines; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Flow Cytometry; Glycoproteins; Histocompatibility Antigens Class I; Immunologic Surveillance; Meninges; Mice; Mice, Inbred C57BL; Microscopy, Confocal; Myelin-Oligodendrocyte Glycoprotein; Peptide Fragments; Spinal Cord; Subarachnoid Space; Time Factors

Multiple sclerosis (MS) is characterized by a dynamic inflammatory process in which CNS lesions of distinct cellular composition coexist. In particular the formation of B cell plaques has been ascribed an important role as predictor of disease progression. Here we show that the novel MBP-PLP fusion protein (MP4)-induced experimental autoimmune encephalomyelitis (EAE) of C57BL/6 mice fulfils these criteria inducing differential cellular infiltration of B cells, T cells, macrophages and granulocytes and permitting the quantification and staging of the disease. On the contrary, both key features - dynamic CNS inflammation and B cell infiltration - were absent in the classical MOG:35-55-induced EAE of C57BL/6 mice, which was characterized by a static CD4(+) T cell and macrophage-mediated CNS immunopathology throughout the disease. MP4-induced EAE may thus provide a unique opportunity for studying immune-pathomechanisms of the disease that have been previously neglected due to experimental shortcomings in murine EAE.

Topics: Animals; B-Lymphocytes; Brain; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Female; Glycoproteins; Mice; Mice, Inbred C57BL; Multiple Sclerosis; Myelin Basic Protein; Myelin Proteolipid Protein; Myelin-Oligodendrocyte Glycoprotein; Peptide Fragments; Recombinant Fusion Proteins; Spinal Cord

To explore the model of chronic experimental autoimmune encephalomyelitis (EAE)for the further study of multiple sclerosis.. A total of 72 female SPF C57BL/6J mice (inbred strain, aged 8 approximately 10 weeks), were randomly divided into an EAE group, a blank group and an adjuvant group, and each group was divided into 3 subgroups: an onset group, a peak group and a chronic phase group. The EAE group was immunized with mMOG35-55.. At the end of the study, and 83.3% of the mice in EAE group suffered the onset, and 8.3% of the mice died. The highest clinical score reached grade 5, namely paralysis of the whole body and then death. In the EAE group, after being immunized first, the mice were all anosis during the first 13 days. They got ill on the third week, and in about 20 approximately 24 days the clinical symptom reached the peak, and in 28 approximately 32 days the chronic phase arrived,when parts of the clinical symptoms got relieved. On the contrary, both the adjuvant group and the blank group were healthy all the time. Characteristic appearance was detected in the EAE group.. Antigen emulsion, mixture of artificially synthesized mMOG35-55 and complete Freundos adjuvant can successfully induce chronic EAE in the mice. The model of EAE duplicated in our study has the characteristics of high incidence, low death rate and stability, which can be used to carry out further research on multiple sclerosis.

Topics: Animals; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Female; Freund's Adjuvant; Glycoproteins; Mice; Mice, Inbred C57BL; Myelin-Oligodendrocyte Glycoprotein; Peptide Fragments; Random Allocation

T helper 17 (Th17) cells are pivotal in the immune pathogenesis of EAE. Glatiramer acetate (GA) can enhance Treg FOXp3 expression. We demonstrate that GA downregulates the expression of both IL-17 and IL-6 in two different EAE models. Increased mRNA expression in CNS for ROR gamma t, IL-17, IL-12/IL-23, IL-6, TNF-alpha, STAT4 and Th1 cytokines were significantly reduced by GA with a concomitant rise in SMAD3. The increased expression of TNF-alpha, IL-6, and IL-17 in CNS of CD25+ depleted animals was suppressed by GA treatment. This study demonstrates that both Th1 polarization and Th17 expression are modulated by GA.

Topics: Animals; CD4-Positive T-Lymphocytes; Central Nervous System; Cytokines; Disease Models, Animal; Down-Regulation; Encephalomyelitis, Autoimmune, Experimental; Female; Forkhead Transcription Factors; Glatiramer Acetate; Glycoproteins; Immunosuppressive Agents; Interleukin-17; Interleukin-6; Mice; Mice, Inbred C57BL; Mice, Transgenic; Myelin-Oligodendrocyte Glycoprotein; Peptide Fragments; Peptides; Receptors, Antigen, T-Cell; RNA, Messenger; Smad3 Protein; STAT4 Transcription Factor

Cytosolic phospholipase A2 alpha (cPLA2 alpha) is the rate-limiting enzyme for release of arachidonic acid, which is converted primarily to prostaglandins via the cyclooxygenase (COX) 1/2 pathways, and leukotrienes via the 5-lipoxygenase (LO) pathway. We utilized inhibitors of cPLA2 alpha, COX-1/2 and 5-LO to determine the potential roles of these enzymes in development of experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS). Blocking cPLA2 alpha prevented EAE development and greatly reduced antigen-induced production of Th1-type cytokines and IL-17. Blocking COX-1/2 delayed onset and reduced severity of EAE, and reduced production of Th1-type cytokines, but not IL-17. Blocking 5-LO delayed onset and reduced cumulative severity of EAE, but did not reduce production of Th1-type cytokines or IL-17. Finally, blockade of cPLA2 alpha from the onset of clinical EAE reduced duration of EAE relapses. Therefore, cPLA2 alpha represents a potential therapeutic target for treatment of MS.

Topics: Analysis of Variance; Animals; Benzoates; Cell Proliferation; Cells, Cultured; Cyclooxygenase Inhibitors; Cytokines; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Enzyme Inhibitors; Female; Glycoproteins; Group IV Phospholipases A2; Hydroxyurea; Lipoxygenase Inhibitors; Mice; Mice, Inbred C57BL; Mice, Inbred NOD; Myelin-Oligodendrocyte Glycoprotein; Naproxen; Peptide Fragments; Severity of Illness Index; Seveso Accidental Release; Sulfonamides; Th1 Cells; Time Factors

Multiple sclerosis is a currently incurable inflammatory demyelinating syndrome. Recent reports suggest that bone marrow derived mesenchymal stem cells may have therapeutic potential in experimental models of demyelinating disease, but various alternative mechanisms, ranging from systemic immune effects to local cell replacement, have been proposed. Here we used intraperitoneal delivery of human mesenchymal stem cells to help test (a) whether human cells can indeed suppress disease, and (b) whether CNS infiltration is required for any beneficial effect. We found pronounced amelioration of clinical disease but profoundly little CNS infiltration. Our findings therefore help confirm the therapeutic potential of mesenchymal stem cells, show that this does indeed extend to human cells, and are consistent with a peripheral or systemic immune effect of human MSCs in this model.

Topics: Animals; Antigens, Nuclear; Cell Transplantation; Central Nervous System; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Glycoproteins; Green Fluorescent Proteins; Humans; Injections, Intraperitoneal; Mesenchymal Stem Cell Transplantation; Mesenchymal Stem Cells; Mice; Mice, Inbred C57BL; Myelin-Oligodendrocyte Glycoprotein; Peptide Fragments; Severity of Illness Index; Transfection; Transplantation, Heterologous

Soluble egg antigen (SEA) is strongly antigenic and inherently induces Th2-biased immune responses. In this study, we tested whether SEA from Schistosoma japonicum is able to prevent experimental autoimmune encephalomyelitis (EAE) induced by MOG(35-55) peptide, an established animal model of multiple sclerosis (MS). Intraperitoneal administration with SEA before EAE induction and in the preclinical phase after EAE induction successfully ameliorated the severity and progression of EAE on mice compared with phosphate buffered saline (PBS) controls, while no protective effect was shown when SEA immunization began after disease onset. This effect was associated with reduced interferon gamma (IFN-gamma) production and/or increased interleukin 4 (IL-4) production in spleen and central nervous system (CNS) even at the chronic stage. Similarly, we observed reduced inflammation and demyelination in spinal cords of SEA pretreated EAE mice compared with controls. Our data indicate that immunization with SEA from S. japonicum induces a preestablished Th2-biased microenvironment that provides preventive immune-modulating effects on EAE progression. This study may have important implications for its promising therapeutic use in MS and other autoimmune diseases.

Topics: Amino Acid Sequence; Animals; Antigens, Helminth; Central Nervous System; Disease Models, Animal; Disease Progression; Drug Administration Schedule; Encephalomyelitis, Autoimmune, Experimental; Female; Glycoproteins; Immunologic Factors; Injections, Intraperitoneal; Interferon-gamma; Interleukin-4; Mice; Mice, Inbred C57BL; Molecular Sequence Data; Myelin-Oligodendrocyte Glycoprotein; Peptide Fragments; Schistosoma japonicum; Spleen; Th2 Cells

Experimental autoimmune encephalomyelitis is a widely used animal model to understand not only multiple sclerosis but also basic principles of immunity. The disease is scored typically by observing signs of paralysis, which do not always correspond with pathological changes.. Experimental autoimmune encephalomyelitis was induced in transgenic mice expressing an injury responsive luciferase reporter in astrocytes (GFAP-luc). Bioluminescence in the brain and spinal cord was measured non-invasively in living mice. Mice were sacrificed at different time points to evaluate clinical and pathological changes. The correlation between bioluminescence and clinical and pathological EAE was statistically analyzed by Pearson correlation analysis.. Bioluminescence from the brain and spinal cord correlates strongly with severity of clinical disease and a number of pathological changes in the brain in EAE. Bioluminescence at early time points also predicts severity of disease.. These results highlight the potential use of bioluminescence imaging to monitor neuroinflammation for rapid drug screening and immunological studies in EAE and suggest that similar approaches could be applied to other animal models of autoimmune and inflammatory disorders.

Topics: Animals; Antigens, CD; Antigens, Differentiation, Myelomonocytic; Brain; CD4 Antigens; Diagnostic Imaging; Disease Models, Animal; Disease Progression; Encephalomyelitis, Autoimmune, Experimental; Glial Fibrillary Acidic Protein; Glycoproteins; Luciferases; Mice; Mice, Inbred C57BL; Mice, Transgenic; Myelin-Oligodendrocyte Glycoprotein; Peptide Fragments; Severity of Illness Index; Spinal Cord; Time Factors

The co-inhibitory B7-homologue 1 (B7-H1/PD-L1) influences adaptive immune responses and has been proposed to contribute to the mechanisms maintaining peripheral tolerance and limiting inflammatory damage in parenchymal organs. To understand the B7-H1/PD1 pathway in CNS inflammation, we analyzed adaptive immune responses in myelin oligodendrocyte glycoprotein (MOG)(35-55)-induced EAE and assessed the expression of B7-H1 in human CNS tissue. B7-H1(-/-) mice exhibited an accelerated disease onset and significantly exacerbated EAE severity, although absence of B7-H1 had no influence on MOG antibody production. Peripheral MOG-specific IFN-gamma/IL-17 T cell responses occurred earlier and enhanced in B7-H1(-/-) mice, but ceased more rapidly. In the CNS, however, significantly higher numbers of activated neuroantigen-specific T cells persisted during all stages of EAE. Experiments showing a direct inhibitory role of APC-derived B7-H1 on the activation of MOG-specific effector cells support the assumption that parenchymal B7-H1 is pivotal for delineating T cell fate in the target organ. Compatible with this concept, our data investigating human brain tissue specimens show a strong up-regulation of B7-H1 in lesions of multiple sclerosis. Our findings demonstrate the critical importance of B7-H1 as an immune-inhibitory molecule capable of down-regulating T cell responses thus contributing to the confinement of immunopathological damage.

Topics: Animals; Antibody Formation; Antigen-Presenting Cells; Antigens, CD; Apoptosis; B7-1 Antigen; B7-H1 Antigen; Cell Count; Central Nervous System; Disease Models, Animal; Glycoproteins; Humans; Interferon-gamma; Interleukin-17; Kinetics; Lymphocyte Activation; Membrane Glycoproteins; Mice; Mice, Inbred C57BL; Mice, Knockout; Multiple Sclerosis; Myelin Proteins; Myelin-Associated Glycoprotein; Myelin-Oligodendrocyte Glycoprotein; Peptide Fragments; Peptides; Spleen; T-Lymphocyte Subsets; T-Lymphocytes; Vaccination

Experimental allergic encephalomyelitis (EAE) is a T cell-mediated autoimmune disease model for multiple sclerosis (MS). We have shown earlier that 15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)) and curcumin ameliorate EAE by modulating inflammatory signaling pathways in T lymphocytes. Toll-like receptors (TLRs), expressed primarily in innate immune cells, play critical roles in the pathogenesis of EAE. T lymphocytes also express TLRs and function as costimulatory receptors to upregulate proliferation and cytokine production in response to specific agonists.. In this study, we show that naïve CD4(+) and CD8(+) T cells express detectable levels of TLR4 and TLR9 and that increase after the induction of EAE in SJL/J and C57BL/6 mice by immunization with PLPp139-151 and MOGp35-55 antigen, respectively. It is interesting to note that in vivo treatment with 15d-PGJ2 or curcumin results in a significant decrease in TLR4 and TLR9 expression in CD4(+) and CD8(+) T cells in association with the amelioration of EAE.. Although the exact mechanisms are not known, the modulation of TLR expression in T lymphocytes by 15d-PGJ(2) and curcumin suggests new therapeutic targets in the treatment of T cell-mediated autoimmune diseases.

Topics: Animals; CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; Curcumin; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Female; Freund's Adjuvant; Glycoproteins; Humans; Immunization; Mice; Mice, Inbred C57BL; Multiple Sclerosis; Myelin Proteolipid Protein; Myelin-Oligodendrocyte Glycoprotein; Peptide Fragments; Prostaglandin D2; Toll-Like Receptor 4; Toll-Like Receptor 9

The enzyme Fatty Acid Amide Hydrolase (FAAH) is a key regulator of the endogenous levels of a family of biologically active lipid mediators, the fatty acid amides. These include anandamide, oleoyl ethanolamide and palmitoyl ethanolamide, and their effects are mediated by a variety of downstream targets including cannabinoid receptors and peroxisome proliferator-activated receptors (PPARs). Activation of both of these may have anti-inflammatory and neuroprotective effects. Levels of all three mediators are low in normal nervous tissue, but substantially elevated in mice lacking FAAH as a result of genetic deletion. There is a long anecdotal history of cannabis use by patients suffering from multiple sclerosis, and preclinical studies have indicated beneficial effects of cannabinoid receptor stimulation on both long-term outcome and acute muscle spasm in rodent models of multiple sclerosis (experimental autoimmune encephalitis; EAE). Thus far no report has appeared on the effect of inhibition of FAAH on the progression of EAE. Using a chronic mouse EAE model, we present data indicating that mice lacking FAAH experience an initial inflammatory phase of EAE similar in severity to wild type controls, but exhibited a more substantial clinical remission compared to wild type mice.

Topics: Amidohydrolases; Animals; CD3 Complex; Chronic Disease; Demyelinating Diseases; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Glycoproteins; Mice; Mice, Inbred C57BL; Mice, Knockout; Myelin-Oligodendrocyte Glycoprotein; Peptide Fragments; Spinal Cord; T-Lymphocytes; Time Factors

Recently we reported that antioxidant system in brain and spinal cord in experimental autoimmune encephalomyelitis (EAE) mice is mainly affected at early stages of the disease [M. Zargari, A. Allameh, M.H. Sanati, T. Tiraihi, S.H. Lavasani, O. Emadyan, Relationship between the clinical scoring and demyelination in central nervous system with total antioxidant capacity of plasma during experimental autoimmune encephalomyelitis development in mice, Neurosci. Lett. 412 (2007), 24-28]. The aim of the present study was to investigate the role of uric acid (UA) on antioxidant system in liver and plasma of EAE mice. EAE was induced in C57/BL6 mice (n=60), followed by i.p. administration of UA (10mg/kg BW) in 30 mice at three distinct clinical stages (A: prior to onset, B: after onset, C: after development of EAE). Livers were removed and processed for measurement of lipid peroxidation products, reduced glutathione (GSH), and glutathione S-transferase (GST) and total antioxidant capacity of plasma (FRAP). The results showed that lipid peroxidation products in liver of EAE mice was increased significantly ( approximately 85%) as compared to normal. UA administration to EAE mice caused a significant suppression of liver lipid peroxidation products ( approximately 45%) at early stages (A and B). There was an inverse relationship between lipid peroxidation and cellular GSH in liver. GSH was significantly depleted in mice liver during the EAE progression, but it was recovered ( approximately 29%) when UA was injected before the onset of the disease (groups A and B). Plasma total antioxidant capacity was significantly decreased during the development of EAE, however it was subsided in mice treated with UA as compared to the corresponding controls (21%) in groups A and B. Elevated liver GST as a result of EAE induction was reversed in mice treated with UA particularly in groups A and B. These results indicate that hepatic glutathione system, particularly GST plays a major role in modulation of oxidative damages to central nervous system (CNS) during EAE induction. The positive response of antioxidant system to UA administration in EAE mice was corroborated with improvement of clinical manifestation of the animals.

Topics: Animals; Antioxidants; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Glutathione; Glycoproteins; Liver; Mice; Mice, Inbred C57BL; Myelin-Oligodendrocyte Glycoprotein; Oxidation-Reduction; Oxidative Stress; Peptide Fragments; Severity of Illness Index; Time Factors; Uric Acid

Although demyelination is a cardinal feature in multiple sclerosis, axonal injury also occurs. We tested whether a delay in axonal degeneration could affect the disease severity in two models for multiple sclerosis: experimental autoimmune encephalomyelitis (EAE) and Theiler's murine encephalomyelitis virus (TMEV) infection. We compared wild-type C57BL/6 (B6) mice with C57BL/Wld(s) (Wld) mice, which carry a mutation that delays axonal degeneration. In EAE, both mouse strains were sensitized with myelin oligodendrocyte glycoprotein (MOG)(35-55) peptide and showed a similar disease onset, MOG-specific lymphoproliferative responses, and inflammation during the acute stage of EAE. However, during the chronic stage, B6 mice continued to show paralysis with a greater extent of axonal damage, demyelination, and MOG-specific lymphoproliferative responses compared with Wld mice, which showed complete recovery. In TMEV infection, only Wld mice were paralyzed and had increased inflammation, virus antigen-positive cells, and TMEV-specific lymphoproliferative responses versus infected B6 mice. Because TMEV can use axons to disseminate in the brain, axonal degeneration in B6 mice might be a beneficial mechanism that limits the virus spread, whereas slow axonal degeneration in Wld mice could favor virus spread. Therefore, axonal degeneration plays contrasting roles (beneficial versus detrimental) depending on the initiator driving the disease.

Topics: Animals; Antigens, Viral; Cardiovirus Infections; Demyelinating Diseases; Disease Models, Animal; Disease Progression; Encephalomyelitis, Autoimmune, Experimental; Female; Glycoproteins; Mice; Mice, Inbred C57BL; Multiple Sclerosis; Mutation; Myelin-Oligodendrocyte Glycoprotein; Nerve Degeneration; Peptide Fragments; Species Specificity; Theilovirus; Time Factors

ThIL-17 (IL-17+/IFN-gamma-) cell lines are significantly more encephalitogenic than Th1 (IL-17-/IFN-gamma+) cell lines in adoptive transfer EAE models. In actively induced EAE short ex vivo peptide stimulation identifies an IL-17+/IFN-gamma+ population of CD4+ CNS-infiltrating MOG35-55-specific T cells, which outnumber IL-17+/IFN-gamma- cells by approximately 3:1 as disease develops. A decrease in numbers of IL-17+/IFN-gamma+ cells following in vitro culture is accompanied by an increase in IL-17-/IFN-gamma+ cell numbers. Together these ex vivo and in vitro observations imply that the Th1 lineage is more encephalitogenic than is suggested by adoptive transfer of Th1 (IL-17-/IFN-gamma+) cell lines which have been terminally differentiated in vitro.

Topics: Animals; Cell Proliferation; Central Nervous System; Cytokines; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Flow Cytometry; Glycoproteins; Interferon-gamma; Interleukin-17; Mice; Mice, Inbred C57BL; Myelin-Oligodendrocyte Glycoprotein; Peptide Fragments; Th1 Cells; Thymidine; Tritium

Multiple sclerosis (MS), the most common nontraumatic cause of neurologic disability in young adults in economically developed countries, is characterized by inflammation, gliosis, demyelination, and neuronal degeneration in the CNS. Bone marrow transplantation (BMT) can suppress inflammatory disease in a majority of patients with MS but retards clinical progression only in patients treated in the early stages of the disease. Here, we applied BMT in a mouse model of neuroinflammation, experimental autoimmune encephalomyelitis (EAE), and investigated the kinetics of reconstitution of the immune system in the periphery and in the CNS using bone marrow cells isolated from syngeneic donors constitutively expressing green fluorescent protein. This approach allowed us to dissect the contribution of donor cells to the turnover of resident microglia and to the pathogenesis of observed disease relapses after BMT. BMT effectively blocked or delayed EAE development when mice were treated early in the course of the disease but was without effect in mice with chronic disease. We found that there is minimal overall replacement of host microglia with donor cells in the CNS and that newly transplanted cells do not appear to contribute to disease progression. In contrast, EAE relapses are accompanied by the robust activation of endogenous microglial and macroglial cells, which further involves the maturation of endogenous Olig2 glial progenitor cells into reactive astrocytes through the cytoplasmic translocation of Olig2 and the expression of CD44 on the cellular membrane. The observed maturation of large numbers of reactive astrocytes from glial progenitors and the chronic activation of host microglial cells have relevance for our understanding of the resident glial response to inflammatory injury in the CNS. Our data indicate that reactivation of a local inflammatory process after BMT is sustained predominantly by endogenous microglia/macrophages.

Topics: Animals; Antigens, CD; Basic Helix-Loop-Helix Transcription Factors; Bone Marrow Transplantation; Brain; Cell Proliferation; Disease Models, Animal; Disease Progression; Encephalomyelitis, Autoimmune, Experimental; Flow Cytometry; Glial Fibrillary Acidic Protein; Glycoproteins; Mice; Mice, Inbred C57BL; Microglia; Multiple Sclerosis; Myelin-Oligodendrocyte Glycoprotein; Nerve Tissue Proteins; Oligodendrocyte Transcription Factor 2; Peptide Fragments; Spinal Cord; Stem Cells

In vitro observations and studies in murine experimental autoimmune encephalomyelitis (EAE) have shown protective effects of sodium channel blockers on central nervous system axons and improved clinical status when treatment is continued throughout the period of observation. Several clinical studies of sodium channel blockers are under way in patients with multiple sclerosis. Here we asked whether a protective effect would persist after withdrawal of a sodium channel blocker.. We studied a mouse model of myelin oligodendrocyte glycoprotein-induced EAE treated with phenytoin or carbamazepine.. Both phenytoin and carbamazepine significantly improved the clinical course of the disease. Withdrawal of phenytoin resulted in acute exacerbation, accompanied by a significantly increased inflammatory infiltrate within the central nervous system and the death of nearly 60% of EAE mice. There were no clinical worsening or deaths in control mice after withdrawal of phenytoin. Withdrawal of carbamazepine led to acute worsening of EAE symptoms, increased inflammatory infiltrate, and was associated with the death of 8% of mice.. These results, together with results showing effects of sodium channel blockers in immune cells, raise questions about the long-term effects of sodium channel blockers in neuroinflammatory disorders, and suggest that clinical studies of sodium channel blockers in these disorders should be planned carefully.

Topics: Animals; Anticonvulsants; Antigens, CD; Axons; Carbamazepine; Cell Count; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Flow Cytometry; Gene Expression Regulation; Glycoproteins; Mice; Mice, Inbred C57BL; Myelin-Oligodendrocyte Glycoprotein; NAV1.6 Voltage-Gated Sodium Channel; Nerve Tissue Proteins; Peptide Fragments; Phenytoin; Pyramidal Tracts; Severity of Illness Index; Sodium Channels; Substance Withdrawal Syndrome

Mechanism-oriented studies of EAE rely mostly on gene-modified mice on the C57BL/6 background. Here we report that MP4-induced EAE displays characteristic differences in CNS pathology as compared to MOG peptide 35-55-elicited disease. While in the latter, the topology of CNS infiltration remained unchanged throughout the disease, in MP4-induced EAE it was dynamic and stage-dependent shifting from the brain to the spinal cord and finally to the cerebellum. Unlike in the MOG peptide model, the frequencies and sizes of CNS lesions in MP4-induced disease showed a clear correlation with clinical disease severity. These characteristic features of MP4-induced EAE may contribute to modelling the complex spectrum of disease manifestations seen in MS.

Topics: Animals; Central Nervous System; Disease Models, Animal; Drug Delivery Systems; Encephalomyelitis, Autoimmune, Experimental; Female; Glycoproteins; Mice; Mice, Inbred C57BL; Myelin Basic Protein; Myelin Proteolipid Protein; Myelin-Oligodendrocyte Glycoprotein; Peptide Fragments; Recombinant Fusion Proteins; Time Factors

Autoimmune optic neuritis is a common early manifestation of multiple sclerosis (MS), yet early therapeutic interventions for MS often have high ocular toxicity associated with increased risks for glaucoma, cataract, or retinopathy. This need to discover better early treatment options prompted our development of a sensitive and reliable means to quantify the broad range of pathologies that potentially develop very early in autoimmune optic neuritis. Tissue microfluorimetry was used to measure seven established markers for human MS pathology in normal and autoimmune optic nerves 13 days after antigen exposure, in a Brown Norway rat model of myelin oligodendrocyte glycoprotein (MOG) peptide (35-55)-induced autoimmune optic neuritis. Optic neuritis rats demonstrated early and significant pathologic changes in five established indices for neuroinflammation, immune infiltration, and demyelination that accurately modeled pathologies characteristic of MS. Two indices of MS-like axon damage advanced significantly within 13 days of antigen exposure. Fluorimetrically measured immunoreactivity (-ir) was significantly decreased for paranodin (PN, the requisite axonal paranodal junction protein) and significantly increased for amyloid precursor protein (APP), indicating loss of paranodal junctions and impaired fast axonal transport, respectively. Measurements showing decreased PN-ir with increased APP-ir quantitatively defined a pattern of early axonal damage in autoimmune optic neuritis.

Topics: Amyloid beta-Protein Precursor; Animals; Axons; Cell Adhesion Molecules, Neuronal; Disease Models, Animal; Endothelin-1; Female; Flow Cytometry; Gene Expression Regulation; Glial Fibrillary Acidic Protein; Glycoproteins; Mast Cells; Myelin-Oligodendrocyte Glycoprotein; Optic Neuritis; Peptide Fragments; Rats

Analysis of T regulatory cells (Treg) and T effector cells (Teff) in experimental autoimmune encephalomyelitis is complicated by the fact that both cell types express CD4 and CD25. We demonstrate that encephalitogenic T cells, following antigen recognition, up-regulate cell surface expression of CD4. The CD4(high) sub-population contains all of the antigen response as shown by proliferation and cytokine secretion, and only these cells are capable of transferring EAE to naive animals. On the other hand, a FACS separable CD25(+) sub-population of cells displayed consistent levels of CD4 prior to and after antigen stimulation. These cells displayed characteristics of Treg, such as expressing high levels of the Foxp3 gene and the ability to suppress mitogenic T cell responses.

Topics: Adoptive Transfer; Animals; CD4 Antigens; CD4-Positive T-Lymphocytes; Cell Proliferation; Disease Models, Animal; Dose-Response Relationship, Immunologic; Encephalomyelitis, Autoimmune, Experimental; Flow Cytometry; Forkhead Transcription Factors; Gene Expression Regulation; Glycoproteins; In Vitro Techniques; Interferon-gamma; Interleukin-2 Receptor alpha Subunit; Interleukin-7; Mice; Mice, Inbred C57BL; Myelin Basic Protein; Myelin-Oligodendrocyte Glycoprotein; Peptide Fragments; T-Lymphocyte Subsets; T-Lymphocytes, Regulatory

Autoimmune encephalomyelitis, a mouse model for multiple sclerosis, is characterized by the activation of immune cells, demyelination of axons in the CNS, and paralysis. We found that TGF-beta1 synthesis in glial cells and TGF-beta-induced signaling in the CNS were activated several days before the onset of paralysis in mice with autoimmune encephalomyelitis. While early production of TGF-beta1 was observed in glial cells TGF-beta signaling was activated in neurons and later in infiltrating T cells in inflammatory lesions. Systemic treatment with a pharmacological inhibitor of TGF-beta signaling ameliorated the paralytic disease and reduced the accumulation of pathogenic T cells and expression of IL-6 in the CNS. Priming of peripheral T cells was not altered, nor was the generation of TH17 cells, indicating that this effect was directed within the brain, yet affected the immune system. These results suggest that early production of TGF-beta1 in the CNS creates a permissive and dangerous environment for the initiation of autoimmune inflammation, providing a rare example of the brain modulating the immune system. Importantly, inhibition of TGF-beta signaling may have benefits in the treatment of the acute phase of autoimmune CNS inflammation.

Topics: Animals; Brain; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Genes, Reporter; Glycoproteins; Inflammation; Mice; Mice, Inbred C57BL; Mice, Transgenic; Multiple Sclerosis; Myelin-Oligodendrocyte Glycoprotein; Neuroglia; Neurons; Peptide Fragments; Signal Transduction; T-Lymphocyte Subsets; T-Lymphocytes, Helper-Inducer; Transforming Growth Factor beta1

Macrophage colony-stimulating factor (M-CSF) is a critical cytokine in the development of monocytic lineage and may have immunoregulatory properties. Here we show that peritoneal antigen presenting cells (APCs) treated with M-CSF produced decreased levels of proinflammatory cytokines IFN-gamma, TNF-alpha and IL-12. These APCs treated with M-CSF+autoantigen peptide significantly suppressed antigen-specific T cell proliferation, induced regulatory CD4(+) and CD8(+) T cells in vitro and in vivo, and significantly suppressed experimental autoimmune encephalomyelitis (EAE). Thus, in vitro treatment of APCs with M-CSF+autoantigen can be a novel therapeutic option for autoimmune diseases.

Topics: Animals; Antigen-Presenting Cells; Autoantigens; CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; Cell Differentiation; Cell Proliferation; Coculture Techniques; Cytokines; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Female; Forkhead Transcription Factors; Glycoproteins; Macrophage Colony-Stimulating Factor; Mice; Mice, Inbred C57BL; Mice, Transgenic; Myelin-Oligodendrocyte Glycoprotein; Peptide Fragments; Receptors, Antigen, T-Cell; Time Factors

Erythropoietin (EPO) mediates a wide range of neuroprotective activities, including amelioration of disease and neuroinflammation in rat models of EAE. However, optimum dosing parameters are currently unknown. In the present study, we used a chronic EAE model induced in mice by immunization with the myelin oligodendrocyte glycoprotein peptide (MOG35-55) to compare the effect of EPO given with different treatment schedules. EPO was administered intraperitoneally at 0.5, 5.0 or 50 microg/kg three times weekly starting from day 3 after immunization (preventive schedule), at the onset of clinical disease (therapeutic schedule) or 15 days after the onset of symptoms (late therapeutic schedule). The results show that EPO is effective even when given after the appearance of clinical signs of EAE, but with a reduced efficacy compared to the preventative schedule. To determine whether this effect requires the homodimeric EPO receptor (EPOR2)-mediated hematopoietic effect of EPO, we studied the effect of carbamylated EPO (CEPO) that does not bind EPOR2. CEPO, ameliorated EAE without changing the hemoglobin concentration. Another non-erythropoietic derivative, asialoEPO was also effective. Both EPO and CEPO equivalently decreased the EAE-associated production of TNF-alpha, IL-1beta and IL-1Ra in the spinal cord, and IFN-gamma by peripheral lymphocytes, indicating that their action involves targeting neuroinflammation. The lowest dosage tested appeared fully effective. The possibility to dissociate the anti-neuroinflammatory action of EPO from its hematopoietic action, which may cause undesired side effects in non-anemic patients, present new avenues to the therapy of multiple sclerosis.

Topics: Analysis of Variance; Animals; Body Weight; Chronic Disease; Cytokines; Disease Models, Animal; Dose-Response Relationship, Drug; Drug Administration Schedule; Encephalomyelitis, Autoimmune, Experimental; Erythropoietin; Female; Glycoproteins; Hematocrit; Humans; Immunohistochemistry; Mice; Mice, Inbred C57BL; Myelin-Oligodendrocyte Glycoprotein; Neuroprotective Agents; Peptide Fragments; Recombinant Proteins; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Severity of Illness Index; Spinal Cord; Spleen; Statistics, Nonparametric; Time Factors; Treatment Outcome; Tumor Necrosis Factor-alpha

Infections can trigger or exacerbate the course of Multiple Sclerosis, and both bacterial and viral agents have been implicated. These agents are recognized by host cells via pathogen-associated molecular patterns activating TLRs. We investigated the role that PAMPs play in the animal model Experimental Autoimmune Encephalomyelitis, and found various MyD88-dependent PAMPs can participate as the adjuvant to induce EAE. Studies with IRAK1-deficient mice suggest that signaling through TLRs is not required in the target organ to develop disease. This suggests that PAMPs play an important role in priming of autoreactive T cells in EAE and potentially MS.

Topics: Adaptor Proteins, Signal Transducing; Adjuvants, Pharmaceutic; Animals; Autoimmunity; Cell Proliferation; Cells, Cultured; Cytokines; Disease Models, Animal; Dose-Response Relationship, Drug; Drug Interactions; Encephalomyelitis, Autoimmune, Experimental; Glycoproteins; Interleukin-1 Receptor-Associated Kinases; Intracellular Signaling Peptides and Proteins; Lymph Nodes; Mice; Mice, Inbred C57BL; Mice, Transgenic; Myelin-Oligodendrocyte Glycoprotein; Myeloid Differentiation Factor 88; Peptide Fragments; Protein Serine-Threonine Kinases; Spleen; Thymidine; Time Factors; Toll-Like Receptors; Tritium

Alpha-fetoprotein (AFP) is a 65-kDa oncofetal glycoprotein found in fetal and maternal fluids during pregnancy. Clinical remissions during pregnancy have been observed in several autoimmune diseases, such as multiple sclerosis (MS), and have been attributed to the presence of pregnancy-associated natural immune-reactive substances, including AFP which can exert immunomodulatory effects on immune cells. In this study, we tested the effect of recombinant human AFP (rhAFP) isolated from transgenic goats, which contain the genomic DNA for hAFP, on experimental autoimmune encephalomyelitis (EAE), the animal model used for the study of MS. RhAFP treatment markedly improved the clinical manifestations of EAE, preventing central nervous system (CNS) inflammation and axonal degeneration. RhAFP exerted a broad immunomodulating activity, influencing the various populations of immune cells. T cells from treated mice had significantly reduced activity towards the encephalitogenic peptide of myelin oligodendrocyte glycoprotein (MOG), exhibiting less proliferation and reduced Th1 cytokine secretion. Moreover, AFP affected the humoral response, causing an inhibition in MOG-specific antibody production. The expression of CD11b, MHC class II and the chemokine receptor CCR5 was also down-regulated. This is the first study demonstrating reduced inflammation and axonal damage exerted by recombinant AFP. In light of our findings, rhAFP may serve as a potential candidate for treatment of MS and other autoimmune diseases.

Topics: alpha-Fetoproteins; Analysis of Variance; Animals; Antibodies; Axons; CD11b Antigen; Cytokines; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Enzyme-Linked Immunosorbent Assay; Female; Flow Cytometry; Glycoproteins; Humans; Immunization; Immunoglobulin G; Inflammation; Mice; Mice, Inbred C57BL; Myelin-Oligodendrocyte Glycoprotein; Peptide Fragments; Radioimmunoassay; Recombinant Proteins; T-Lymphocytes; Thymidine; Time Factors

Vasoactive intestinal peptide (VIP) has been found to act as a potent anti-inflammatory factor through regulating the production of both anti- and pro-inflammatory mediators and promoting Th2-type responses. In this study, we used myelin oligodendrocyte glycoprotein-induced experimental autoimmune encephalomyelitis (EAE) model in C57BL/6 mice to investigate the potential effects of VIP on multiple sclerosis. Our results showed that in vivo treatment of EAE-induced mice with VIP had great protective benefit at both clinical and histological levels. Disease suppression was associated with the inhibition of T cells proliferation, shifting of the immune response toward a Th2-type response and influencing the expression of pro-inflammatory cytokines including IFN-gamma, IL-6 and IL-2 as well as chemotactic factors such as RANTES. In conclusion, the study provides evidence that VIP had great protective effect on EAE through its inhibition actions on pathogenic T cells and through a specific effect on the Th1 response.

Topics: Animals; Cell Proliferation; Chemokine CCL5; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Glycoproteins; Interferon-gamma; Interleukin-2; Interleukin-6; Lymphocyte Activation; Male; Mice; Mice, Inbred C57BL; Multiple Sclerosis; Myelin-Oligodendrocyte Glycoprotein; Peptide Fragments; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Spinal Cord; Th1 Cells; Th2 Cells; Vasoactive Intestinal Peptide

Infiltration of the central nervous system (CNS) by CD4+ Th1 cells precedes onset and relapses of experimental autoimmune encephalomyelitis (EAE). We reported that (B6xSJL) F1 (H-2b/s) mice with severe relapsing-remitting disease had extensive infiltration by CD4+ T cells compared to that in C57BL/6 (B6) (H-2b) mice, which developed mild low-relapsing disease in response to myelin oligodendrocyte peptide 35-55 (MOG(35-55)). This observation led us to search for mechanisms that specifically regulate trafficking of CD4+ cells in relapsing H-2b/s mice. We show that the CD4+ cell chemoattractant cytokine interleukin (IL)-16 has an important role in regulation of relapsing EAE induced by MOG(35-55) in the (B6xSJL) F1 (H-2b/s) mice. We found production of IL-16 in the CNS of mice with EAE. IL-16 levels in the CNS correlated well with the extent of CD4+ T-cell and B-cell infiltration during acute and relapsing disease. Infiltrating CD4+ T cells, B cells, and to a lesser extent CD8+ T cells all contained IL-16 immunoreactivity. Treatment with neutralizing anti-IL-16 antibody successfully reversed paralysis and ameliorated relapsing disease. In treated mice, diminished infiltration by CD4+ T cells, less demyelination, and more sparing of axons was observed. Taken together, our results show an important role for IL-16 in regulation of relapsing EAE. We describe a novel therapeutic approach to specifically impede CD4+ T cell chemoattraction in EAE based on IL-16 neutralization. Our findings have high relevance for the development of new therapies for relapsing EAE and potentially MS.

Topics: Animals; Antibodies; B-Lymphocytes; Blotting, Western; CD4 Antigens; CD4-Positive T-Lymphocytes; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Female; Glycoproteins; Immunization; Immunohistochemistry; Immunotherapy; Indoles; Interleukin-16; Leukocyte Common Antigens; Lymphocyte Activation; Mice; Mice, Inbred C57BL; Microscopy, Electron, Transmission; Multiple Sclerosis, Relapsing-Remitting; Myelin-Oligodendrocyte Glycoprotein; Paralysis; Peptide Fragments; Phenotype; Reaction Time; Severity of Illness Index; Spinal Cord; Staining and Labeling; Time Factors

The central role of T cells in the induction of tolerance to autoantigens has been well documented. However, the role of antigen-presenting cells (APCs) in this process is not yet fully understood. To better understand the contribution of APCs in tolerance, we studied the interaction of purified APCs and CD4(+) T cells in a model of intravenous (i.v.) tolerance to experimental autoimmune encephalomyelitis (EAE). As expected, we found that T cells were tolerized to the autoantigen after i.v. injection. However, purified APCs obtained from MOG-i.v.-treated mice were paradoxically immuno-stimulatory, as they induced a non-specific Th1-type response both in vitro and in vivo. We conclude that blocking such APC activation would enhance the effectiveness of tolerance induction.

Topics: Animals; Antigen-Presenting Cells; CD4-Positive T-Lymphocytes; Cell Proliferation; Coculture Techniques; Cytokines; Disease Models, Animal; Dose-Response Relationship, Immunologic; Encephalomyelitis, Autoimmune, Experimental; Female; Flow Cytometry; Glycoproteins; Immune Tolerance; Immunization, Passive; Injections, Intravenous; Mice; Mice, Inbred C57BL; Myelin-Oligodendrocyte Glycoprotein; Peptide Fragments; Receptors, Cytokine; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Statistics, Nonparametric; Time Factors

We demonstrate that the histone deacetylase (HDAC) inhibitor drug trichostatin A (TSA) reduces spinal cord inflammation, demyelination, neuronal and axonal loss and ameliorates disability in the relapsing phase of experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis (MS). TSA up-regulates antioxidant, anti-excitotoxicity and pro-neuronal growth and differentiation mRNAs. TSA also inhibits caspase activation and down-regulates gene targets of the pro-apoptotic E2F transcription factor pathway. In splenocytes, TSA reduces chemotactic, pro-Th1 and pro-proliferative mRNAs. A transcriptional imbalance in MS may contribute to immune dysregulation and neurodegeneration, and we identify HDAC inhibition as a transcriptional intervention to ameliorate this imbalance.

Topics: Animals; Cell Death; Cells, Cultured; Cerebral Cortex; Cytokines; Disease Models, Animal; Drug Administration Schedule; Drug Interactions; Embryo, Mammalian; Encephalomyelitis, Autoimmune, Experimental; Female; Gene Expression Profiling; Gene Expression Regulation; Glycoproteins; Hydroxamic Acids; Immunohistochemistry; Mice; Mice, Inbred C57BL; Myelin-Oligodendrocyte Glycoprotein; Neurons; Oligonucleotide Array Sequence Analysis; Peptide Fragments; Protein Synthesis Inhibitors; Rats; Rats, Sprague-Dawley; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Severity of Illness Index; Spleen; Tetrazolium Salts; Thiazoles; Time Factors

Mucosal tolerance has been used successfully to treat animal models of autoimmune diseases and is being tested in human diseases. In this work we demonstrate the reduction of infarct size following mucosal tolerance by myelin oligodendrocyte glycoprotein (MOG) (35-55) peptide in mouse stroke model. Nasal MOG was most efficacious and reduced ischemic infarct size by 70% at 24 h as well as improving behavior score. Using immunohistological methods and IL-10 -/- mice, we demonstrate the importance of IL-10-producing CD4+ T cells in the reduction of the ischemic infarct volume following middle cerebral artery occlusion (MCAO). Furthermore, adoptive transfer of CD4+ T cells from nasally tolerized mice to untreated mice prior to MCAO surgery significantly decreased stroke size (p<0.001 vs. control), whereas CD4+ T cells from nasally tolerized IL-10-deficient mice had no significant effect. Based on these results, modulation of cerebral inflammation by mucosal tolerance to myelin antigens may have applicability both as prophylactic therapy and treatment following ischemia attacks.

Topics: Analysis of Variance; Animals; Antigens, CD; Behavior, Animal; Brain Infarction; CD4-Positive T-Lymphocytes; Cells, Cultured; Cytokines; Disease Models, Animal; Drug Administration Routes; Drug Administration Schedule; Enzyme-Linked Immunosorbent Assay; Female; Glycoproteins; Immune Tolerance; Immunohistochemistry; Infarction, Middle Cerebral Artery; Interleukin-10; Mice; Mice, Inbred C57BL; Mice, Knockout; Models, Immunological; Myelin-Oligodendrocyte Glycoprotein; Peptide Fragments; Severity of Illness Index; Stroke; Time Factors

We studied the immunoregulatory features of murine mesenchymal stem cells (MSCs) in vitro and in vivo. MSCs inhibited T-cell receptor (TCR)-dependent and -independent proliferation but did not induce apoptosis on T cells. Such inhibition was paired with a decreased interferon (IFN)-gamma and tumor necrosis factor (TNF)-alpha production and was partially reversed by interleukin-2 (IL-2). Thus, we used MSCs to treat myelin oligodendrocyte glycoprotein (MOG)35-55-induced experimental autoimmune encephalomyelitis (EAE) in C57BL/6J mice. We injected intravenously 1 x 10(6) MSCs before disease onset (preventive protocol) and at different time points after disease occurrence (therapeutic protocol). MSC administration before disease onset strikingly ameliorated EAE. The therapeutic scheme was effective when MSCs were administered at disease onset and at the peak of disease but not after disease stabilization. Central nervous system (CNS) pathology showed decreased inflammatory infiltrates and demyelination in mice that received transplants of MSCs. T-cell response to MOG and mitogens from MSC-treated mice was inhibited and restored by IL-2 administration. Upon MSC transfection with the enhanced green fluorescent protein (eGFP), eGFP(+) cells were detected in the lymphoid organs of treated mice. These data suggest that the immunoregulatory properties of MSCs effectively interfere with the autoimmune attack in the course of EAE inducing an in vivo state of T-cell unresponsiveness occurring within secondary lymphoid organs.

Topics: Animals; Cell Proliferation; Cells, Cultured; Clonal Anergy; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Glycoproteins; Green Fluorescent Proteins; Interferon-gamma; Interleukin-2; Mesenchymal Stem Cell Transplantation; Mesenchymal Stem Cells; Mice; Mice, Inbred C57BL; Myelin-Oligodendrocyte Glycoprotein; Peptide Fragments; Receptors, Antigen, T-Cell; Signal Transduction; T-Lymphocytes; Tumor Necrosis Factor-alpha

Agonists of the peroxisome proliferator-activated receptor gamma (PPARgamma) exert anti-inflammatory and anti-proliferative effects which led to testing of these drugs in experimental autoimmune encephalomyelitis (EAE), a model for multiple sclerosis. In contrast, the effect of PPARdelta (PPARdelta) agonists in EAE is not yet known. We show that oral administration of the selective PPARdelta agonist GW0742 reduced clinical symptoms in C57BL/6 mice that had been immunized with encephalitogenic myelin oligodendrocyte glycoprotein (MOG) peptide. In contrast to previous results with PPARgamma agonists, GW0742 only modestly attenuated clinical symptoms when the drug was provided simultaneously with immunization, but a greater reduction was observed if administered during disease progression. Reduced clinical symptoms were accompanied by a reduction in the appearance of new cortical lesions, however cerebellar lesion load was not reduced. Treatment of T-cells with GW0742 either in vivo or in vitro did not reduce IFNgamma production; however GW0742 reduced astroglial and microglial inflammatory activation and IL-1beta levels in EAE brain. RTPCR analysis showed that GW0742 increased expression of some myelin genes. These data demonstrate that PPARdelta agonists, like other PPAR ligands, can exert protective actions in an autoimmune model of demyelinating disease.

Topics: Animals; Brain; Concanavalin A; Disease Models, Animal; Dose-Response Relationship, Drug; Drug Interactions; Encephalomyelitis, Autoimmune, Experimental; Enzyme-Linked Immunosorbent Assay; Female; Gene Expression Regulation, Enzymologic; Glycoproteins; Immunohistochemistry; Interferon-gamma; Lymphocyte Activation; Lymphocytes; Mice; Mice, Inbred C57BL; Myelin Basic Protein; Myelin-Oligodendrocyte Glycoprotein; Neuroglia; Peptide Fragments; PPAR delta; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Severity of Illness Index; Thiazoles; Time Factors

Insulin-like growth factor 1 (IGF-1) has been identified as a critical molecule in the induction of myelination in the central nervous system (CNS). Systemic injection of IGF-1 has been shown to have a varied and transiently protective effect on the clinical course of experimental autoimmune encephalomyelitis (EAE). Since systemic IGF-1 can also modulate peripheral immune lymphocytes, we examined whether a sustained and local delivery of IGF-1 into the spinal cord would have any influence on the chronic course of EAE in C57/BL6 mice. The capability of adeno-associated virus (AAV) to be retrogradely transported efficiently from muscle to motor neurons of the spinal cord was used to overcome the difficulty routinely encountered when attempting chronic delivery of molecules into the CNS. We demonstrate that AAV-mediated delivery of IGF-1 in CNS did not have any beneficial effect on the clinical course of EAE. Injection of AAV-IGF1 after induction of the disease worsened the clinical symptoms. Furthermore, CNS expression of IGF-1 did not affect the pathogenic anti-MOG T cell response, as examined by proliferation and cytokine secretion. Thus, enhanced expression of IGF-1 in the CNS during inflammation does not have a significant effect on myelination. These data have important implications for the potential use of IGF-1 in the treatment of multiple sclerosis.

Topics: Animals; Blotting, Northern; Cell Line; Cell Proliferation; Central Nervous System; Cytokines; Disease Models, Animal; Dose-Response Relationship, Drug; Encephalomyelitis, Autoimmune, Experimental; Gene Expression; Gene Expression Regulation; Glycoproteins; Humans; Immunohistochemistry; Insulin-Like Growth Factor I; Lymphocyte Activation; Mice; Mice, Inbred C57BL; Muscle, Skeletal; Myelin-Oligodendrocyte Glycoprotein; Peptide Fragments; Reverse Transcriptase Polymerase Chain Reaction; Spinal Cord; Time Factors

Complement is implicated in the pathology of neurodegenerative and inflammatory disease in the central nervous system (CNS). Although studies demonstrate that inhibition of complement activation attenuates disease development in the CNS, the specific complement components that contribute to the pathogenesis of CNS diseases remain unclear. To dissect the role of C5a in CNS disease, we developed a transgenic mouse that produces C5a exclusively in the brain using the astrocyte-specific, murine glial fibrillary acidic protein (GFAP) promoter. C5a/GFAP mice develop normally and do not demonstrate any signs of spontaneous inflammation or neurodegeneration with age. Using C5a/GFAP mice, we examined the outcome of the animal model of multiple sclerosis, experimental autoimmune encephalomyelitis (EAE). To our surprise the onset and severity of myelin oligodendrocyte glycoprotein-induced EAE was essentially identical between C5a/GFAP and control mice. These results demonstrate that C5a, despite it is pro-inflammatory functions, is not critical to the development and progression of EAE.

Topics: Animals; Brain; Complement C5a; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Fluorescent Antibody Technique; Gene Expression Regulation; Glial Fibrillary Acidic Protein; Glycoproteins; Humans; Mice; Mice, Transgenic; Myelin-Oligodendrocyte Glycoprotein; Peptide Fragments; Severity of Illness Index

Once considered to be of sole importance in allergy and parasitic infections, the role of mast cells in other pathologic and protective immune responses is becoming increasingly evident. We previously demonstrated that mast cells contribute to the severity of EAE, the rodent model of multiple sclerosis. Here we show that one mode of mast cell action is through effects on the autoreactive T cell response. Early indices of both peripheral CD4 and CD8 T cell activation, including IFN-gamma production and increases in CD44 and CD11a expression, are attenuated in mast cell-deficient (W/Wv) mice after myelin oligodendrocyte glycoprotein(35-55) priming when compared to WT animals. Reduced infiltrates of activated T cells in the central nervous system are also observed. Importantly, selective repletion of the mast cell compartment restores most T cell responses in the lymph nodes and the central nervous system, correlating with reconstitution of severe disease. The adoptive transfer of WT-derived encephalitogenic T cells results in significantly less severe disease in W/Wv recipients, indicating that mast cells also exert potent effects after the initial T cell response is generated. Our data provide the first in vivo evidence that mast cells can significantly influence T cell responses and suggest that mast cells exacerbate disease during both the inductive and effector phases.

Topics: Animals; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Female; Glycoproteins; H-2 Antigens; Lymphocyte Activation; Mast Cells; Mice; Mice, Congenic; Mice, Inbred C57BL; Mice, Knockout; Multiple Sclerosis; Myelin-Oligodendrocyte Glycoprotein; Peptide Fragments; T-Lymphocytes

To characterize T cell and antibody responses in remitting-relapsing experimental autoimmune encephalomyelitis (RR-EAE), we compared myelin oligodendrocyte glycoprotein (MOG)-induced RR-EAE in C57BL/6 (B6) x SJL (F1) mice and chronic-progressive EAE (CP-EAE) in B6 mice at week 8 p.i. when clinical scores were comparable. Although these two strains exhibited similar inflammation/demyelination pattern and MOG-induced T cell responses, RR-EAE mice produced significantly higher levels of anti-MOG IgG1/IgG2a antibodies. Further, lymphocytes of RR-EAE mice proliferated vigorously to the secondary epitope myelin basic protein (MBP) 1-11. These results support a potential involvement of anti-MOG antibodies and epitope spreading in T cell responses in the development of MOG-induced RR-EAE model.

Topics: Analysis of Variance; Animals; Antibodies; Cells, Cultured; Cytokines; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Enzyme-Linked Immunosorbent Assay; Flow Cytometry; Glycoproteins; Histological Techniques; Immunization; Immunoglobulin G; Lymphocyte Activation; Mice; Mice, Inbred Strains; Multiple Sclerosis, Chronic Progressive; Multiple Sclerosis, Relapsing-Remitting; Myelin Basic Protein; Myelin Proteolipid Protein; Myelin-Oligodendrocyte Glycoprotein; Peptide Fragments; Spinal Cord; T-Lymphocytes; Time Factors

Given the abnormalities in B-cell activity occurring in the central nervous system (CNS) of patients with multiple sclerosis (MS), we have explored the possibility that CNS inflammation induced in mouse models of experimental autoimmune encephalomyelitis (EAE) triggers expression of molecules that control the development and functional organization of lymphoid follicles, the sites where B-cell responses are initiated. By reverse transcription-polymerase chain reaction (RT-PCR), we find that gene expression of CXCL13, a chemokine involved in B-cell recruitment into lymphoid follicles, and BAFF, a key regulator of B-cell survival, is markedly and persistently upregulated in the CNS of mice with relapsing-remitting and chronic-relapsing EAE. Using immunohistochemical techniques, we also show the presence of lymphoid follicle-like structures containing B cells and a reticulum of CXCL13+ and FDC-M1+ follicular dendritic cells within the meninges of several mice undergoing progressive relapsing EAE. These observations indicate that, under chronic inflammatory conditions, the less immunoprivileged meningeal compartment is the site where ectopic lymphoid follicles preferentially develop and where pathogenic B-cell responses could be sustained in autoimmune disorders of the CNS.

Topics: Animals; B-Cell Activating Factor; CD4 Antigens; Central Nervous System; Chemokine CXCL13; Chemokines, CXC; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Female; Glycoproteins; Immunization; Immunohistochemistry; Ki-67 Antigen; Leukocyte Common Antigens; Lymphocytes; Membrane Proteins; Meninges; Mice; Mice, Inbred Strains; Myelin Proteolipid Protein; Myelin-Oligodendrocyte Glycoprotein; Peptide Fragments; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Species Specificity; Spinal Cord; Time Factors; Tumor Necrosis Factor-alpha

We have investigated the role of p75NTR in inflammation in experimental allergic encephalomyelitis (EAE), a model for the human disease multiple sclerosis (MS). Induction of EAE in C57/BL6 wild-type mice resulted in expression of p75NTR in endothelial cells in the CNS. In contrast to the clinical manifestation of EAE observed in wild-type C57/BL6 mice, mice deficient for p75NTR (p75NTR knockout mice) developed severe or lethal disease and concomitant increased levels of inflammation in the CNS. Our findings suggest a physiological significant role for p75NTR in CNS endothelial cells during inflammation and involvement in preservation of blood-brain barrier integrity during a severe infiltrative attack.

Topics: Animals; Blood Vessels; CD11 Antigens; CD3 Complex; Central Nervous System; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Endothelial Cells; Gene Expression; Glycoproteins; Immunization; Immunohistochemistry; Mice; Mice, Inbred C57BL; Mice, Knockout; Microscopy, Electron; Multiple Sclerosis; Myelin-Oligodendrocyte Glycoprotein; Peptide Fragments; Probability; Receptor, Nerve Growth Factor; Receptors, Nerve Growth Factor; Time Factors

Myelin oligodendrocyte glycoprotein (MOG) is an important autoantigen in multiple sclerosis and in experimental autoimmune encephalomyelitis (EAE). We generated a T cell receptor (TCR) transgenic (Tg) mouse expressing a TCR derived from an encephalitogenic T cell clone specific for MOG(35-55). This mouse failed to develop EAE spontaneously and developed mild EAE at late onset when immunized with MOG(35-55). The Tg T cells produced large amounts of IL-4 when stimulated with MOG(35-55) and underwent FAS/FAS-L-mediated activation-induced cell death when stimulated with MOG(35-55) and IL-12. The unique phenotype of these autoantigen-specific T cells may represent an important mechanism of protection against autoimmune disease.

Topics: Animals; Animals, Newborn; CD4 Antigens; CD8 Antigens; Cells, Cultured; Cloning, Molecular; Disease Models, Animal; Dose-Response Relationship, Immunologic; Drug Interactions; Encephalomyelitis, Autoimmune, Experimental; Flow Cytometry; Glycoproteins; In Situ Nick-End Labeling; In Vitro Techniques; Interleukin-12; Interleukin-2; Ionophores; Lymph Nodes; Mice; Mice, Inbred C57BL; Mice, Transgenic; Myelin-Oligodendrocyte Glycoprotein; Peptide Fragments; Receptors, Antigen, T-Cell; Spleen; T-Lymphocytes; Thymus Gland; Time Factors

Estrogen treatment has been found to be protective in experimental autoimmune encephalomyelitis (EAE) and possibly multiple sclerosis (MS). We investigated whether the effect of estrogen treatment is gender-specific. Estrogen receptor (ER) expressions, ERalpha and ERbeta, were found to be equivalent in both genders. EAE disease severity in both females and males was decreased with estriol treatment as compared to placebo. Finally, proinflammatory cytokine production during autoantigen-specific immune responses was decreased with estriol treatment in both females and males. These data support a potential role for estriol treatment for men in addition to women with MS.

Topics: Analysis of Variance; Animals; Cell Culture Techniques; Cytokines; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Estriol; Estrogen Receptor alpha; Estrogen Receptor beta; Female; Glycoproteins; Lymphocytes; Male; Mice; Mice, Inbred C57BL; Multiple Sclerosis; Myelin-Oligodendrocyte Glycoprotein; Peptide Fragments; Receptors, Estrogen; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sex Characteristics; Time Factors

Kallikrein 6 (K6, MSP) is a newly identified member of the Kallikrein family of serine proteases that is preferentially expressed in the adult central nervous system (CNS). We have previously demonstrated that K6 is abundantly expressed by inflammatory cells at sites of CNS inflammation and demyelination in animal models of multiple sclerosis (MS) and in human MS lesions. To test the hypothesis that this novel enzyme is a mediator of pathogenesis in CNS inflammatory disease, we have evaluated whether autonomously generated K6 antibodies alter the clinicopathological course of disease in murine proteolipid protein139-151-induced experimental autoimmune encephalomyelitis (PLP139-151 EAE). We demonstrate that immunization of mice with recombinant K6 generates antibodies that block K6 enzymatic activity in vitro, including the breakdown of myelin basic protein (MBP), and that K6-immunized mice exhibit significantly delayed onset and severity of clinical deficits. Reduced clinical deficits were reflected in significantly less spinal cord pathology and meningeal inflammation and in reduced Th1 cellular responses in vivo and in vitro. These data demonstrate for the first time that K6 participates in enzymatic cascades mediating CNS inflammatory disease and that this unique enzyme may represent a novel therapeutic target for the treatment of progressive inflammatory disorders, including MS.

Topics: Animals; Autoantibodies; Chemotaxis, Leukocyte; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Female; Glycoproteins; Immunization; Immunoglobulin G; Immunotherapy; Kallikreins; Lymphocyte Activation; Meninges; Mice; Mice, Inbred BALB C; Multiple Sclerosis; Myelin Proteolipid Protein; Myelin-Oligodendrocyte Glycoprotein; Peptide Fragments; Recombinant Proteins; Signal Transduction; Spinal Cord; Th1 Cells

Recent studies have indicated that, in addition to demyelination and axonal degeneration, a third factor, dysregulated ion channel expression, contributes to the pathophysiology of experimental autoimmune encephalomyelitis (EAE) and multiple sclerosis (MS). Consistent with this suggestion, upregulated expression of sodium channel Na(v)1.8 is observed in Purkinje neurons in EAE and MS, and biophysical studies indicate that aberrant expression of Na(v)1.8 produces abnormal Purkinje cell firing which may contribute to the development of cerebellar ataxia. However, the molecular mechanisms that contribute to the upregulation of Na(v)1.8 in Purkinje cells in EAE and MS have not yet been determined. Previous studies have shown that neurotrophic factors can modulate sodium channel expression and that elevated levels of NGF are present in EAE and MS. Using immunocytochemical methods, we examined the relationship between the upregulation of Na(v)1.8 and the expression of the NGF receptors p75 and TrkA in EAE. Here we demonstrate that upregulation of Na(v)1.8 is associated with expression of p75 and low levels of TrkA in the majority of Purkinje cells in EAE. These findings, together with previous studies demonstrating a modulatory role of NGF on sodium channel expression, suggest that NGF acting via p75 contributes to the upregulation of Na(v)1.8 in Purkinje cells in EAE.

Topics: Adaptor Proteins, Signal Transducing; Animals; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Fluorescent Antibody Technique; Glycoproteins; Mice; Myelin-Oligodendrocyte Glycoprotein; NAV1.8 Voltage-Gated Sodium Channel; Peptide Fragments; Purkinje Cells; Receptor, trkA; Sodium Channels; Trans-Activators; Transcription Factors; Up-Regulation

IL-10 has been shown to be an important anti-inflammatory mediator that has both down-regulatory and immunomodulatory effects. Utilizing IL-10(-/-) mice we demonstrate the induction of low dose oral tolerance characterized by the up-regulation of TGF-beta and IL-4 and the suppression of Ag specific proliferation with little suppression of INF-gamma. More severe EAE was found in IL-10(-/-) mice than in wild type controls, however, feeding resulted in amelioration of disease severity in both groups. Orally tolerized IL-10(-/-) mice had greater disease severity compared to orally tolerized wild type mice. IL-4 was present in the GALT of IL-10(-/-) mice and up-regulation of TGF-beta was detected in the lamina propria of fed mice. These results demonstrate that IL-10 is not required for the induction of low dose oral tolerance but is required for the regulation of INF-gamma which affects severity of disease in tolerized mice.

Topics: Administration, Oral; Animals; Cell Proliferation; Cells, Cultured; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Gene Deletion; Glycoproteins; Immune Tolerance; Immunohistochemistry; Interleukin-10; Mice; Mice, Inbred C57BL; Mice, Knockout; Myelin-Oligodendrocyte Glycoprotein; Peptide Fragments; Spleen; Transforming Growth Factor beta

Pituitary adenylate cyclase-activating polypeptide (PACAP), a 38-amino acid neuropeptide belonging to the secretin-glucagon-vasoactive intestinal peptide (VIP) family, performs a variety of functions in both the nervous and immune systems. In this study, we examined the effects of PACAP on experimental autoimmune encephalomyelitis (EAE) in C57BL/6 mice. When administrated intraperitoneally every other day after immunization with myelin oligodendrocyte glycoprotein (MOG) peptide 35-55, PACAP ameliorated both the clinical and pathological manifestations of EAE Ex vivo examination revealed a significant inhibition of MOG35-55-specific Th1 response in mice treated with PACAP. In vitro analysis revealed that PACAP suppressed the production of inflammatory cytokines, including TNF-alpha, IL-1beta, and IL-12, and expression of the costimulatory factor B7-2 on macrophage and microglia, which may function as antigen presenting cells (APC) in the CNS. While PACAP suppressed the differentiation of MOG35-55-specific T cells into Th1 effectors upon restimulation with MOG35-55-expressing APC, it did not affect interferon (IFN)-gamma production by MOG35-55-specific T cells stimulated with anti-CD3 and anti-CD28. These observations suggested that PACAP suppressed induction of EAE primarily via suppression of APC function and inflammatory cytokine production. PACAP may be useful in the future treatment of Th1-mediated autoimmune diseases, such as multiple sclerosis.

Topics: Animals; Antigen-Presenting Cells; CD28 Antigens; CD3 Complex; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Glycoproteins; Histocompatibility Antigens Class II; Inflammation Mediators; Interferon-gamma; Interleukin-12; Macrophages; Mice; Mice, Inbred C57BL; Microglia; Multiple Sclerosis; Myelin-Oligodendrocyte Glycoprotein; Nerve Growth Factors; Neuropeptides; Neurotransmitter Agents; Peptide Fragments; Pituitary Adenylate Cyclase-Activating Polypeptide; Th1 Cells

Chemokines are low molecular weight cytokines which act as chemoattractants for infiltrating cells bearing appropriate receptors (CCR) to sites of inflammation. It has been proposed that CCR2 on monocytes is responsible for their recruitment into the central nervous system (CNS) in experimental autoimmune encephalomyelitis (EAE), a model for multiple sclerosis, and two previous reports have described resistance of CCR2(-/-) mice to EAE. The present study examined three different mouse strains with CCR2 deletions for susceptibility to EAE. Animals were studied up to 4 months post-sensitization and were examined by neuropathology, RNase protection assay, in situ hybridization, and in vitro assays. All three strains were found to be susceptible to EAE: C57BL/6 x J129 and Balb c strains, 100%; and C57BL/6, 67%. Unusual in CNS lesions of CCR2(-/-) mice was an overabundance of neutrophils versus monocytes in wild-type animals. An attempt of the immune system to develop compensatory mechanisms for the lack of CCR2 was evidenced by a corresponding increase in mRNA for other chemokines and CCR. Inasmuch as neutrophils replaced monocytes and led to demyelination, our findings support the concept that promiscuity of chemokines and CCR was able to surmount the deletion of CCR2, still resulting in full expression of this autoimmune disease.

Topics: Animals; Cell Division; Crosses, Genetic; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Genetic Predisposition to Disease; Glycoproteins; Immunity, Innate; Immunohistochemistry; In Situ Hybridization; Lymphocytes; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Inbred Strains; Mice, Knockout; Myelin Sheath; Myelin-Oligodendrocyte Glycoprotein; Nuclease Protection Assays; Peptide Fragments; Receptors, CCR2; Receptors, Chemokine; RNA, Messenger; Species Specificity

The severity of the experimental autoimmune encephalomyelitis (EAE) induced by peptide myelin oligodendrocyte glycoprotein(35-55)(pMOG(35-55)) is thought to be predominantly influenced by the major histocompatibility complex (MHC), so that C57BL6/J mice, on H2(b) strain, were only mildly sick. However, it remains unclear as to how non-MHC gene regions affect EAE. To determine whether the immunization protocol could have an influence on clinical signs, C57BL6/J mice were immunized with a multiple antigen peptide (MAP) containing eight pMOG(35-55)branches synthesized directly onto a lysine core, myelin oligodendrocyte glycoprotein (35-55)-multiple antigen peptide (MOG(35-55)-MAP), in complete Freund's adjuvant (CFA). In most of the mice, clinical onset (marked weakness) occurred approximately at day 15. All mice injected with MOG(35-55)-MAP had more severe symptoms than those injected with pMOG(35-55), which developed no leg paralysis. All MOG(35-55)-MAP-immunized mice developed EAE symptoms, but 50% had primary-progressive EAE, while the other 50% had relapsing-remitting disease. Leukocyte infiltrations, associated with increased glial fibrillary acidic protein (GFAP) expression by reactive astrocytes, were observed around the lateral ventricles and blood vessels in the brain. Significant positive correlations were established between anti-MOG(35-55)antibody levels and clinical scores or GFAP positivity in the spinal cord. The heterogeneity of EAE progression, observed in these genetically identical individuals, suggests that the environment rather than the genetics plays a role. This observation is highly pertinent as it corresponds to what is seen in clinical MS.

Topics: Animals; Antibodies; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Female; Glycoproteins; Mice; Mice, Inbred C57BL; Multiple Sclerosis; Myelin-Oligodendrocyte Glycoprotein; Peptide Fragments

To find immune mechanisms underlying relapse regulation, we developed a model of relapsing-remitting experimental autoimmune encephalomyelitis (EAE) in (B6xSJL) F1 (H-2(b/s)) mice by immunization with myelin oligodendrocyte glycoprotein peptide 35-55 (MOG(35-55)) and compared with low/non-relapsing B6 (H-2(b)) mice. In relapsing H-2(b/s) mice, inflammatory lesions scattered throughout the white matter with extensive demyelination, consisted of CD4(+) T and B220(+) B cells with fewer Mac3(+) macrophages. Memory T cell proliferation to MOG(35-55) was significantly enhanced. Switch of macrophage chemoattractant protein-1 (MCP-1) production from GFAP(+) astrocytes to CD3(+) T cells was observed. Distinct patterns of inflammation and demyelination, MOG(35-55) memory T cell response and regulation of MCP-1 are associated with relapsing H-2(b/s) phenotype.

Topics: Animals; Antigens, Differentiation; Astrocytes; B-Lymphocytes; CD4 Antigens; CD4-Positive T-Lymphocytes; Chemokine CCL2; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Epitopes; Female; Glycoproteins; H-2 Antigens; Lymphocyte Activation; Macrophages; Mice; Mice, Inbred C57BL; Multiple Sclerosis, Relapsing-Remitting; Myelin-Oligodendrocyte Glycoprotein; Nerve Fibers, Myelinated; Peptide Fragments; Receptors, CCR2; Receptors, Chemokine; T-Lymphocytes

Inflammation plays an important role in ischemic stroke and in humans IL-10 may have a beneficial effect in stroke. We mucosally administered myelin oligodendrocyte glycoprotein (MOG) 35-55 peptide to C57BL/6 mice before middle cerebral artery occlusion (MCAO) to induce an anti-inflammatory T cell response directed at CNS myelin. Nasal and oral administration of MOG(35-55) peptide decreased ischemic infarct size at 24 and 72 h after MCAO surgery. Nasal MOG(35-55) peptide was most efficacious and reduced infarct size by 70% at 24 h and by 50% at 72 h (p

Topics: Administration, Intranasal; Administration, Oral; Adoptive Transfer; Animals; Brain Ischemia; CD4-Positive T-Lymphocytes; Cytokines; Disease Models, Animal; Female; Glycoproteins; Immune Tolerance; Immunity, Mucosal; Immunohistochemistry; Infarction, Middle Cerebral Artery; Interleukin-10; Mice; Mice, Inbred C57BL; Mice, Knockout; Myelin-Oligodendrocyte Glycoprotein; Neuroprotective Agents; Peptide Fragments; Vaccines

Experimental autoimmune encephalomyelitis (EAE) is an autoimmune demyelinating disease commonly used to model the pathogenetic mechanisms involved in multiple sclerosis (MS). In this study, we examined the effects of immunization with the myelin oligodendrocyte glycoprotein MOG(35-55) on the expression of molecules of the innate immune system, namely toll-like receptor 2 (TLR2) and CD14. Expression of the mRNA encoding TLR2 increased in the choroid plexus, the leptomeninges and within few isolated cells in the CNS parenchyma 4 to 8 days after immunization with MOG. At day 10, the signal spread across the meninges, few perivascular regions and over isolated groups of parenchymal cells. Three weeks after the MOG treatment, at which time animals showed severe clinical symptoms, a robust expression of both TLR2 and CD14 transcripts occurred in barrier-associated structures, as well as parenchymal elements of the spinal cord, and within numerous regions of the brain including, the medulla, cerebellum and the cortex. Dual labeling provided the anatomical evidence that microglia/macrophages were positive for TLR2 in the brain of EAE mice. The regions that exhibited chronic expression of TLR2 and CD14 were also associated with an increase in NF-kappaB activity and transcriptional activation of genes encoding numerous proinflammatory molecules. The present data provide evidence that receptors of the pathogen-associated molecular patterns are strongly induced in the CNS of EAE mice, further reinforcing the concept that the innate immune system plays a determinant role in this autoimmune demyelinating disease.

Topics: Animals; Disease Models, Animal; Drosophila Proteins; Encephalomyelitis, Autoimmune, Experimental; Female; Glycoproteins; Immunohistochemistry; In Situ Hybridization; Lipopolysaccharide Receptors; Membrane Glycoproteins; Mice; Mice, Inbred C57BL; Microglia; Multiple Sclerosis; Myelin-Oligodendrocyte Glycoprotein; Peptide Fragments; Receptors, Cell Surface; RNA, Messenger; Toll-Like Receptor 2; Toll-Like Receptors; Transcriptional Activation; Up-Regulation

IL-12 plays a critical role in the priming of Th1 responses to bacterial/parasitic antigens and autoantigens. Several studies have demonstrated a dependency on CD40/CD40L interactions and IL-12 for maintenance of both antibacterial/parasitic and autoreactive Th1 cells in vivo. However, it is still unclear if fully differentiated Th1 effectors require continued stimulation by IL-12. We demonstrate that the proliferative response and IFN-gamma production by a fully differentiated T cell line specific for myelin oligodendrocyte glycoprotein are completely independent of IL-12 and CD40/CD40L interactions. The capacity of this line to adoptively transfer experimental autoimmune encephalomyelitis is also independent of IL-12 and CD40/CD40L. These results have important implications regarding the therapeutic usefulness of blockade of IL-12 or the CD40/CD40L pathway for treatment of autoimmune disease.

Topics: Adoptive Transfer; Animals; CD40 Antigens; CD40 Ligand; Cell Differentiation; Cell Division; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Female; Flow Cytometry; Glycoproteins; Interferon-gamma; Interleukin-12; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Myelin-Oligodendrocyte Glycoprotein; Peptide Fragments; Receptors, Interleukin; Receptors, Interleukin-12; Th1 Cells; Th2 Cells

Estrogen treatment has been found to have suppressive activity in several models of autoimmunity. To investigate the mechanism of 17 beta-estradiol (E2) suppression of experimental autoimmune encephalomyelitis, we evaluated E2 effects on TNF-alpha expression in the central nervous system (CNS) and spleen of C57BL/6 mice immunized with MOG 35-55/CFA. Kinetic analysis demonstrated that E2 treatment drastically decreased the recruitment of total inflammatory cells as well as TNF-alpha(+) macrophages and T cells into the CNS at disease onset. In contrast, E2 had only moderate effects on the relatively high constitutive TNF-alpha expression by resident CNS microglial cells. E2 treatment also had profound inhibitory effects on expression of TNF-alpha by splenic CD4(+) T cells, including those responsive to MOG 35-55 peptide. We propose that the mechanism of E2 protection may involve both systemic inhibition of TNF-alpha expression and local (CNS) recruitment of inflammatory cells, with modest effects on TNF-alpha expression by resident CNS microglial cells.

Topics: Animals; Central Nervous System; Disease Models, Animal; Encephalomyelitis; Estradiol; Female; Flow Cytometry; Glycoproteins; Kinetics; Macrophages; Mice; Mice, Inbred C57BL; Microglia; Myelin-Oligodendrocyte Glycoprotein; Nerve Tissue Proteins; Peptide Fragments; Spleen; T-Lymphocytes; Tumor Necrosis Factor-alpha