myelin-basic-protein and myelin-oligodendrocyte-glycoprotein-(35-55)

Antigen-specific therapy targeting selective inhibition of autoreactive responses holds promise for controlling multiple sclerosis (MS) without disturbing homeostasis of the whole immune system. Key autoantigens in MS include myelin proteins, such as myelin basic protein (MBP), proteolipid protein (PLP), and myelin oligodendrocyte glycoprotein (MOG). In this study, we examined the effect of transdermal therapy with myelin peptides on immune responses in the skin, lymph nodes, and peripheral blood immune cells of MS patients.. In a 1-year placebo-controlled study, 30 patients with relapsing-remitting MS were treated transdermally with a mixture of 3 myelin peptides: MBP85-99, PLP139-151, and MOG35-55, or placebo. The phenotype of immune cells in the skin was assessed using immunohistochemistry. Cell populations in lymph nodes were analyzed using flow cytometry. In peripheral blood immune cells, cytokine production was measured by enzyme-linked immunosorbent assay, and myelin-specific proliferation was examined by carboxyfluorescein succinimidyl ester-based assay.. We found that myelin peptides applied transdermally to MS patients activated dendritic Langerhans cells in the skin at the site of immunization and induced a unique population of granular dendritic cells in local lymph nodes. In the periphery, transdermal immunization with myelin peptides resulted in the generation of type 1, interleukin-10-producing regulatory T cells, suppression of specific autoreactive proliferative responses, and suppression of interferon-γ and transforming growth factor-β production.. We demonstrate for the first time the immunoregulatory potential of transdermal immunization with myelin peptides in MS patients.

Topics: Administration, Cutaneous; Adolescent; Adult; Autoimmunity; Cell Proliferation; Cytokines; Drug Combinations; Female; Glycoproteins; Humans; Lymph Nodes; Male; Middle Aged; Multiple Sclerosis, Relapsing-Remitting; Myelin Basic Protein; Myelin Proteins; Myelin Proteolipid Protein; Myelin-Oligodendrocyte Glycoprotein; Peptide Fragments; Skin

Experimental autoimmune encephalomyelitis, originally experimental allergic encephalomyelitis, is the well-known animal model of multiple sclerosis, an immune- mediated, demyelinating, inflammatory chronic disease of the central nervous system. The experimental disease is widely utilized to test new therapies in preclinical studies, to investigate new hypothesis on the possible pathogenic mechanisms of autoimmune reaction directed against the central nervous system or more generally to investigate the interactions between the immune system and the central nervous system that lead to neuroinflammation. The experimental autoimmune encephalomyelitis may be induced following different protocols in mammals, including nonhuman primates, and autoreactive CD4+ T-lymphocytes directed against myelin antigens are the main factors. Here, after introducing the model, we describe the protocol to induce active EAE in inbred mice, we report on a table the different clinical courses of EAE depending on the combination of antigen /mouse strain and we provide indications on how to evaluate the clinics and pathology of this induced disease.

Topics: Animals; Antigens; Encephalomyelitis, Autoimmune, Experimental; Female; Freund's Adjuvant; Lipids; Male; Mice, Inbred Strains; Mycobacterium tuberculosis; Myelin Basic Protein; Myelin Proteolipid Protein; Myelin-Oligodendrocyte Glycoprotein; Peptide Fragments; Phenotype; Research Design; Sex Factors; Species Specificity; T-Lymphocytes; Workflow

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 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

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

The protective and therapeutic mechanism of bee venom acupuncture (BVA) in neurodegenerative disorders is not clear. We investigated whether treatment with BVA (0.25 and 0.8 mg/kg) at the Zusanli (ST36) acupoints, located lateral from the anterior border of the tibia, has a beneficial effect in a myelin basic protein (MBP)(68-82)-induced acute experimental autoimmune encephalomyelitis (EAE) rat model. Pretreatment (every 3 days from 1 h before immunization) with BVA was more effective than posttreatment (daily after immunization) with BVA with respect to clinical signs (neurological impairment and loss of body weight) of acute EAE rats. Treatment with BVA at the ST36 acupoint in normal rats did not induce the clinical signs. Pretreatment with BVA suppressed demyelination, glial activation, expression of cytokines [interferon (IFN)-γ, IL-17, IL-17A, tumor necrosis factor-alpha (TNF-α), and IL-1β], chemokines [RANTES, monocyte chemotactic protein-1 (MCP-1), and macrophage inflammatory protein (MIP)-1α], and inducible nitric oxide synthase (iNOS), and activation of p38 mitogen-activated protein kinase (MAPK) and nuclear factor (NF)-κB (p65 and phospho-IκBα) signaling pathways in the spinal cord of acute EAE rats. Pretreatment with BVA decreased the number of CD4(+), CD4(+)/IFN-γ(+), and CD4(+)/IL-17(+) T cells, but increased the number of CD4(+)/Foxp3(+) T cells in the spinal cord and lymph nodes of acute EAE rats. Treatment with BVA at six placebo acupoints (SP9, GB39, and four non-acupoints) did not have a positive effect in acute EAE rats. Interestingly, onset and posttreatment with BVA at the ST36 acupoint markedly attenuated neurological impairment in myelin oligodendrocyte glycoprotein (MOG)(35-55)-induced chronic EAE mice compared to treatment with BVA at six placebo acupoints. Our findings strongly suggest that treatment with BVA with ST36 acupoint could delay or attenuate the development and progression of EAE by upregulating regulatory T cells and suppressing T-helper (Th) 17 and Th1 responses. These results warrant further investigation of BVA as a treatment for autoimmune disorders of the central nervous system.

Topics: Acupuncture Therapy; Animals; Bee Venoms; Encephalomyelitis, Autoimmune, Experimental; Female; Immunity, Cellular; Lymphocyte Depletion; Macrophages; MAP Kinase Signaling System; Mice; Mice, Inbred C57BL; Microglia; Myelin Basic Protein; Myelin-Oligodendrocyte Glycoprotein; Paraparesis; Peptide Fragments; Rats; Rats, Inbred Lew; T-Lymphocytes, Regulatory; Th1 Cells; Th17 Cells

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

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

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

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

The injection of antigens into the Anterior Chamber (AC) of the eye induces Anterior Chamber Associated Immune Deviation (ACAID), which is a potent form of immune deviation that is largely attributed to the effect of TGFβ2 in the aqueous humor on ocular antigen-presenting cells (APCs). ACAID antigen presentation via APCs and B cells leads to the generation of antigen-specific T regulatory cells. The encephalitogenic antigens Myelin oligodendrocyte glycoprotein (MOG) and Myelin basic protein (MBP) have an obvious clinical relevance. We hypothesized that the intravenous injection of in vitro-generated ACAID APCs or in vitro-generated ACAID B cells specific to the encephalitogenic antigens MOG35-55/MBP induces specific peripheral tolerance in recipient BALB/c mice. We examined the suppression of MOG35-55-specific/MBP-specific inflammatory responses using delayed-type hypersensitivity (DTH) assays and Local Adoptive Transfer (LAT) assays. Results indicated that MOG35-55-specific/MBP-specific tolerance was generated after the intravenous injections of MOG35-55-specific/MBP-specific ACAID APCs, MOG35-55-specific/MBP-specific ACAID B cells, and MOG35-55-specific/MBP-specific ACAID T regulatory cells. The specific immune deviation was in vitro-induced, cell-mediated, and specific to the encephalitogenic antigens MOG35-55/MBP. This in vitro-mediated approach for the generation of MOG35-55/MBP-specific tolerance opens up avenues for the application of ACAID as a tool for the therapy of Multiple Sclerosis, Schizophrenia, and other diseases.

Topics: Animals; Anterior Chamber; Antigen-Presenting Cells; B-Lymphocytes; Immune Tolerance; Mice; Mice, Inbred BALB C; Myelin Basic Protein; Myelin-Oligodendrocyte Glycoprotein; Peptide Fragments; T-Lymphocytes, Regulatory

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

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

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

Studies of MS histopathology are largely dependent on suitable animal models. While light microscopic analysis gives an overview of tissue pathology, it falls short in evaluating detailed changes in nerve fiber morphology. The ultrastructural data presented here and obtained from studies of myelin oligodendrocyte glycoprotein (MOG):35-55-induced experimental autoimmune encephalomyelitis (EAE) in C57BL/6 mice delineate that axonal damage and myelin pathology follow different kinetics in the disease course. While myelin pathology accumulated with disease progression, axonal damage coincided with the initial clinical disease symptoms and remained stable over time. This pattern applied both to irreversible axolysis and early axonal pathology. Notably, these histopathological patterns were reflected by the normal-appearing white matter (NAWM), suggesting that the NAWM is also in an active neurodegenerative state. The data underline the need for neuroprotection in MS and suggest the MOG model as a highly valuable tool for the assessment of different therapeutic strategies.

Topics: Animals; Axons; Encephalomyelitis, Autoimmune, Experimental; Female; Kinetics; Lumbar Vertebrae; Mice; Mice, Inbred C57BL; Multiple Sclerosis; Myelin Basic Protein; Myelin Proteolipid Protein; Myelin Sheath; Myelin-Oligodendrocyte Glycoprotein; Peptide Fragments; Recombinant Fusion Proteins; Spinal Cord

Induction of experimental autoimmune encephalomyelitis (EAE) in susceptible animals requires reactivation of encephalitogenic CD4(+) T cells by APCs in the CNS. However, it has remained unresolved from where APCs in the CNS acquire myelin Ag for T cell activation and under which conditions, that is, whether only during EAE or also in the naive CNS. In this study, we investigated the kinetics of myelin Ag uptake by CNS APCs during EAE and in the naive CNS. Our results show that during EAE CX3CR1(+)CD11b(+) microglia were the first APCs in the CNS to contain myelin Ag upon induction of disease, albeit in very small numbers. Dendritic cells (DCs) arrived in the CNS in sizable numbers significantly later (day 5 postimmunization), without detectable myelin Ag, but acquired it by day 7 postimmunization. Furthermore, a sharp increase in neuroantigen-containing DCs coincided with the onset of EAE symptoms. Importantly, in naive mice a low but consistent number of microglia contained myelin Ag, suggesting release by oligodendrocytes under steady state conditions. Although microglia isolated from naive brain and spinal cord did not elicit a strong CD4(+) T cell response in vitro, myelin Ag-containing microglia may still play a local role in modulating encephalitogenic CD4(+) T cell responses in early EAE prior to the arrival of other professional APCs, such as DCs. Finally, newly arriving DCs in the CNS not yet loaded with myelin Ag before the onset of EAE may be a potential therapeutic target.

Topics: Adoptive Transfer; Animals; Antigen Presentation; Antigens; Autoantibodies; Brain; Cell Movement; Dendritic Cells; Encephalomyelitis, Autoimmune, Experimental; Female; Freund's Adjuvant; Imaging, Three-Dimensional; Immunization; Injections, Intraperitoneal; Injections, Subcutaneous; Lymphocyte Activation; Male; Mice; Mice, Inbred C57BL; Mice, Inbred Strains; Mice, Neurologic Mutants; Mice, Transgenic; Microglia; Microscopy, Confocal; Myelin Basic Protein; Myelin Proteolipid Protein; Myelin-Oligodendrocyte Glycoprotein; Myeloid Cells; Peptide Fragments; Pertussis Toxin; Spinal Cord

Mice lacking IL-6 are resistant to autoimmune diseases, such as experimental autoimmune encephalomyelitis (EAE), which is driven by CNS-reactive CD4(+) T cells. There are multiple cellular sources of IL-6, but the critical source in EAE has been uncertain. Using cell-specific IL-6 deficiency in models of EAE induced by active immunization, passive transfer, T cell transfer, and dendritic cell transfer, we show that neither the pathogenic T cells nor CNS-resident cells are required to produce IL-6. Instead, the requirement for IL-6 was restricted to the early stages of T cell activation and was entirely controlled by dendritic cell-derived IL-6. This reflected the loss of IL-6R expression by T cells over time. These data explain why blockade of IL-6R only achieves protection against EAE if used at the time of T cell priming. The implications for therapeutic manipulation of IL-6 signaling in human T cell-driven autoimmune conditions are considered.

Topics: Adoptive Transfer; Animals; Autoantigens; CD4-Positive T-Lymphocytes; Crosses, Genetic; Dendritic Cells; Encephalomyelitis, Autoimmune, Experimental; Histocompatibility Antigens Class II; Immunization, Passive; Interleukin-6; Lymphocyte Activation; Lymphokines; Mice; Mice, Inbred C57BL; Mice, Knockout; Myelin Basic Protein; Myelin-Oligodendrocyte Glycoprotein; Peptide Fragments; Receptors, Interleukin-6; Specific Pathogen-Free Organisms; T-Cell Antigen Receptor Specificity

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

To elucidate the correlation between visual threshold of optokinetic tracking (OKT), visual evoked potential (VEP), and histopathology at different time points after induction of experimental autoimmune optic neuritis (EAON).. EAON was induced in C57BL/6 mice by subcutaneous immunization with an emulsified mixture of myelin oligodendrocyte glycoprotein (MOG)(35-55) peptide. OKT and VEP were measured on days 7, 14, 21, 28, and 42 postimmunization. After VEP measurements, the mice were killed and their eyes were enucleated for histopathological studies. Immunohistochemical staining was performed using cell-specific markers for characterization of cells in the optic nerve: CD3 (T cells), Iba-1 (microglia), MBP (myelin basic protein), and neurofilament (axons).. Functionally, OKT threshold decreased as early as day 7, and VEP latency was significantly prolonged on day 21. Axon degeneration was observed as early as day 14. Activated microglia infiltration was also observed on day 14, before T cell infiltration, which peaked on day 21. Demyelination, confirmed by MBP staining, was observed on day 21.. Microglial infiltration in the optic nerve coincided with decline in OKT threshold and preceded VEP latency prolongation, while VEP latency prolongation coincided with T cell infiltration and demyelination of the optic nerve. These findings may contribute to understanding of the pathophysiology of optic neuritis and future development of more effective therapeutic strategy for refractory optic neuritis.

Topics: Animals; Axons; Calcium-Binding Proteins; CD3 Complex; Evoked Potentials, Visual; Immunoenzyme Techniques; Mice; Mice, Inbred C57BL; Microfilament Proteins; Microglia; Myelin Basic Protein; Myelin-Oligodendrocyte Glycoprotein; Neuritis, Autoimmune, Experimental; Nystagmus, Optokinetic; Optic Nerve; Optic Neuritis; Peptide Fragments; T-Lymphocytes

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

IL-15 initially identified as a T proliferating cytokine has several structural and biological similarities with IL-2 and has been associated with a number of autoimmune diseases. Because of the scarcity of information available on the role of IL-15 in MS pathogenesis, we have investigated how the absence of IL-15 affected the development of experimental autoimmune encephalomyelitis, a mouse model of MS. Following immunization of IL-15(-/-) and C57BL/6 mice with MOG(35-55), we observed a more severe neurological impairment in the IL-15 knockout mice than in the wild-type group. The enhanced disease severity in IL-15(-/-) mice was associated with greater demyelination in the spinal cord, increased immune cell infiltration and inflammation. These events may be related to the higher CD4/CD8 ratio and the almost absent NK cell activity, congenital immune features of IL-15KO mice. Moreover, we found that the fractalkine receptor CX3CR1 was overexpressed in the spinal cord of IL-15(-/-) mice, mainly localized on infiltrating CD8(+) T cells. How these findings are contributing to the aggravated EAE development in IL-15 KO mice remain unclear and need to be further investigated.

Topics: Analysis of Variance; Animals; CD4 Antigens; CD8 Antigens; CX3C Chemokine Receptor 1; Cytokines; Demyelinating Diseases; Eliminative Behavior, Animal; Encephalomyelitis, Autoimmune, Experimental; Flow Cytometry; Gene Expression Regulation; Glycoproteins; Interleukin-15; Killer Cells, Natural; Mice; Mice, Inbred C57BL; Mice, Knockout; Myelin Basic Protein; Myelin-Oligodendrocyte Glycoprotein; Neutrophil Infiltration; Peptide Fragments; Receptors, Chemokine; Spinal Cord

Regulatory T cells (Tregs) play a vital role in autoimmune disorders. Among several markers, forkhead box p3 (Foxp3) is the most specific with regard to Treg activity. Therefore, understanding mechanisms that regulate Foxp3 expression is a critical step for unraveling the complicacy of autoimmune pathophysiology. The present study was undertaken to investigate the crosstalk between NO and Tregs. Interestingly, after myelin basic protein (MBP) priming, the expression of Foxp3 decreased in MBP-primed T cells. However, blocking NO either by inhibiting inducible NO synthase with l-N(6)-(1-iminoethyl)-lysine hydrochloride or through scavenging with PTIO or by pharmacological drugs, such as pravastatin, sodium benzoate, or gemfibrozil, restored the expression of Foxp3 in MBP-primed T cells. However, this restoration of Foxp3 by pharmacological drugs was reversed by S-nitrosoglutathione, an NO donor. Similarly, NO also decreased the populations of Tregs characterized by CD4(+)CD25(+) and CD25(+)FoxP3(+) phenotypes. We have further confirmed this inverse relationship between NO and Foxp3 by analyzing the mRNA expression of Foxp3 and characterizing CD25(+)FoxP3(+) or CD4(+)Foxp3(+) phenotypes from inducible NO synthase knockout mice. Moreover, this inverse relation between NO and Foxp3 also was observed during priming with myelin oligodendrocyte glycoprotein, another target neuroantigen in multiple sclerosis, as well as collagen, a target autoantigen in rheumatoid arthritis. Finally, we demonstrate that NO inhibited the expression of Foxp3 in MBP-primed T cells via soluble guanylyl cyclase-mediated production of cGMP. Taken together, our data imply a novel role of NO in suppressing Foxp3(+) Tregs via the soluble guanylyl cyclase pathway.

Topics: Animals; Cattle; Cells, Cultured; Down-Regulation; Female; Forkhead Transcription Factors; Glycoproteins; Mice; Mice, Inbred C57BL; Mice, Inbred Strains; Mice, Knockout; Myelin Basic Protein; Myelin-Oligodendrocyte Glycoprotein; Nitric Oxide; Nitric Oxide Synthase Type II; Peptide Fragments; T-Lymphocyte Subsets; 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

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

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

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

Oligodendrocyte death and demyelination are hallmarks of multiple sclerosis (MS). Here we show that ATP signaling can trigger oligodendrocyte excitotoxicity via activation of calcium-permeable P2X(7) purinergic receptors expressed by these cells. Sustained activation of P2X(7) receptors in vivo causes lesions that are reminiscent of the major features of MS plaques, i.e., demyelination, oligodendrocyte death, and axonal damage. In addition, treatment with P2X(7) antagonists of chronic experimental autoimmune encephalomyelitis (EAE), a model of MS, reduces demyelination and ameliorates the associated neurological symptoms. Together, these results indicate that ATP can kill oligodendrocytes via P2X(7) activation and that this cell death process contributes to EAE. Importantly, P2X(7) expression is elevated in normal-appearing axon tracts in MS patients, suggesting that signaling through this receptor in oligodendrocytes may be enhanced in this disease. Thus, P2X(7) receptor antagonists may be beneficial for the treatment of MS.

Topics: Adenosine Triphosphate; Animals; Animals, Newborn; Calcium; Cell Survival; Cells, Cultured; Encephalomyelitis, Autoimmune, Experimental; Glial Fibrillary Acidic Protein; Glycoproteins; Humans; Membrane Potentials; Mice; Microscopy, Immunoelectron; Myelin Basic Protein; Myelin-Oligodendrocyte Glycoprotein; Oligodendroglia; Optic Nerve; Patch-Clamp Techniques; Peptide Fragments; Platelet Aggregation Inhibitors; Purinergic P2 Receptor Antagonists; Rats; Rats, Sprague-Dawley; Receptors, Purinergic P2; Receptors, Purinergic P2X7

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

Multiple sclerosis is an inflammatory, neurodegenerative disease for which experimental autoimmune encephalomyelitis (EAE) is a model. Treatments with estrogens have been shown to decrease the severity of EAE through anti-inflammatory mechanisms. Here we investigated whether treatment with an estrogen receptor alpha (ERalpha) ligand could recapitulate the estrogen-mediated protection in clinical EAE. We then went on to examine both anti-inflammatory and neuroprotective mechanisms. EAE was induced in wild-type, ERalpha-, or ERbeta-deficient mice, and each was treated with the highly selective ERalpha agonist, propyl pyrazole triol, to determine the effect on clinical outcomes, as well as on inflammatory and neurodegenerative changes. ERalpha ligand treatment ameliorated clinical disease in both wild-type and ERbeta knock-out mice, but not in ERalpha knock-out mice, thereby demonstrating that the ERalpha ligand maintained ERalpha selectivity in vivo during disease. ERalpha ligand treatment also induced favorable changes in autoantigen-specific cytokine production in the peripheral immune system [decreased TNFalpha, interferon-gamma, and interleukin-6, with increased interleukin-5] and decreased CNS white matter inflammation and demyelination. Interestingly, decreased neuronal staining [NeuN+ (neuronal-specific nuclear protein)/beta3-tubulin+/Nissl], accompanied by increased immunolabeling of microglial/monocyte (Mac 3+) cells surrounding these abnormal neurons, was observed in gray matter of spinal cords of EAE mice at the earliest stage of clinical disease, 1-2 d after the onset of clinical signs. Treatment with either estradiol or the ERalpha ligand significantly reduced this gray matter pathology. In conclusion, treatment with an ERalpha ligand is highly selective in vivo, mediating both anti-inflammatory and neuroprotective effects in EAE.

Topics: Analysis of Variance; Animals; Antigens, Differentiation; Cytokines; Encephalomyelitis, Autoimmune, Experimental; Estrogen Receptor alpha; Female; Glycoproteins; Immunohistochemistry; Leukocyte Common Antigens; Lysosomal-Associated Membrane Protein 2; Mice; Mice, Inbred C57BL; Myelin Basic Protein; Myelin-Oligodendrocyte Glycoprotein; Neurons; Neuroprotective Agents; Ovariectomy; Peptide Fragments; Phosphopyruvate Hydratase; Selective Estrogen Receptor Modulators; Spinal Cord

In this study, we demonstrate for the first time the immunohistochemical expression of citrullinated proteins in the central nervous system (CNS) of mice with myelin oligodendrocyte glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis (EAE). By using an established monoclonal antibody (F95) against natural and synthetic citrullinated proteins (Nicholas and Whitaker [2002] Glia 37:328-336), numerous, small, previously unrecognized "patches" of citrullinated proteins were discovered throughout EAE brains, whereas EAE spinal cords showed similar but much larger lesions. On dual color immunofluorescence, these lesions were found to contain citrullinated myelin basic protein (MBP) and were surrounded by astrocytes immunoreactive for both glial fibrillary acidic protein (GFAP) and F95. These lesions became evident about the time when EAE mice became symptomatic and increased in size and number with increasing disease severity. In some sections of spinal cord but not brains of severely debilitated EAE mice, a widespread gliotic response was seen, with astrocytes containing citrullinated GFAP spread throughout the gray and white matter. Western blot analysis of acidic proteins from the brains and spinal cords of EAE mice had higher levels of multiple citrullinated GFAP isoforms compared with controls, with more F95-positive bands in the EAE brains vs. spinal cords. These results raise the possibility that citrullination of both GFAP and MBP may contribute to the pathophysiology of EAE and that the brains of EAE mice may contain more pathology than previously realized.

Topics: Animals; Antibodies, Monoclonal; Blotting, Western; Cerebral Cortex; Citrulline; Encephalomyelitis, Autoimmune, Experimental; Female; Fluorescent Antibody Technique; Glial Fibrillary Acidic Protein; Glycoproteins; Mice; Mice, Inbred C57BL; Myelin Basic Protein; Myelin-Oligodendrocyte Glycoprotein; Peptide Fragments; Spinal Cord; Time Factors

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

Serotonin (5-hydroxytryptamine, 5-HT) is one of the most extensively studied neurotransmitters of the central nervous system. It also has been identified in constituents of the immune system. Therefore serotonin has been suggested to serve as a mediator of bidirectional interactions between the nervous system and the immune system. We investigated this interaction in experimental autoimmune encephalomyelitis (EAE), a well-defined animal model of autoimmune disease of the central nervous system (CNS) mimicking features of the human disease multiple sclerosis. EAE was induced by immunization with the autoantigens myelin basic protein (MBP) or the immunodominant peptide of myelin oligodendrocyte glycoprotein (MOG) spanning amino acids 35-55 (MOGp 35-55). We studied EAE in knockout (KO) mice lacking the 5-HT transporter (5-HTT) on a C57.BL/6 background, in comparison with wild-type C57.BL/6 animals. After immunization with MOGp 35-55, or with rat MBP, the disease courses of the 5-HTT knockout mice were attenuated as compared to wildtype control mice. This difference was more pronounced in female animals. To dissect potential immune mechanisms underlying this phenomenon, histological studies of the CNS and cytokine measurements in mononuclear cells from the spleens of 5-HTT KO mice and wild-type controls were performed. We found a reduction of the inflammatory infiltrate in the CNS and of the neuroantigen-specific production of IFN-gamma in splenocytes, again accompanied by a gender difference. These findings suggest a potential role of extracellular 5-HT homeostasis in the fine-tuning of neuroantigen-specific immune responses.

Topics: Acute Disease; Animals; Autoantigens; Cell Division; Central Nervous System; Disease Susceptibility; Encephalomyelitis, Autoimmune, Experimental; Epitopes; Female; Glycoproteins; Immunohistochemistry; Interferon-gamma; Interleukins; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Myelin Basic Protein; Myelin-Oligodendrocyte Glycoprotein; Peptide Fragments; Rats; Serotonin; Sex Factors; T-Lymphocytes

FTY720, a sphingosine 1-phosphate receptor modulator, induces a marked decrease in the number of peripheral blood lymphocytes and exerts immunomodulating activity in various experimental allograft and autoimmune disease models. In this study, we evaluated the effect of FTY720 and its active metabolite, (S)-enantiomer of FTY720-phosphate [(S)-FTY720-P] on experimental autoimmune encephalomyelitis (EAE) in rats and mice. Prophylactic administration of FTY720 at 0.1 to 1 mg/kg almost completely prevented the development of EAE, and therapeutic treatment with FTY720 significantly inhibited the progression of EAE and EAE-associated histological change in the spinal cords of LEW rats induced by immunization with myelin basic protein. Consistent with rat EAE, the development of proteolipid protein-induced EAE in SJL/J mice was almost completely prevented and infiltration of CD4(+) T cells into spinal cord was decreased by prophylactic treatment with FTY720 and (S)-FTY720-P. When FTY720 or (S)-FTY720-P was given after establishment of EAE in SJL/J mice, the relapse of EAE was markedly inhibited as compared with interferon-beta, and the area of demyelination and the infiltration of CD4(+) T cells were decreased in spinal cords of EAE mice. Similar therapeutic effect by FTY720 was obtained in myelin oligodendrocyte glycoprotein-induced EAE in C57BL/6 mice. These results indicate that FTY720 exhibits not only a prophylactic but also a therapeutic effect on EAE in rats and mice, and that the effect of FTY720 on EAE appears to be due to a reduction of the infiltration of myelin antigen-specific CD4(+) T cells into the inflammation site.

Topics: Animals; Encephalomyelitis, Autoimmune, Experimental; Fingolimod Hydrochloride; Glycoproteins; Male; Mice; Myelin Basic Protein; Myelin Proteolipid Protein; Myelin-Oligodendrocyte Glycoprotein; Peptide Fragments; Propylene Glycols; Rats; Receptors, Lysosphingolipid; Sphingosine; 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