pr-957 and Disease-Models--Animal

Topics: Animals; Anti-Inflammatory Agents; Cells, Cultured; Coxsackievirus Infections; Disease Models, Animal; Enterovirus B, Human; Host-Pathogen Interactions; Inflammation; Male; Mice, Knockout; Myeloid Cells; Myocarditis; Myocytes, Cardiac; Oligopeptides; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Proteolysis

The immunoproteasome, a special isoform of the 20S proteasome, is expressed when the cells receive an inflammatory signal. Immunoproteasome inhibition proved efficacy in the treatment of autoimmune diseases. However, the role of the immunoproteasome in the pathogenesis of immune thrombocytopenia (ITP) remains unknown. We found that the expression of the immunoproteasome catalytic subunit, large multifunctional protease 2 (LMP2), was significantly upregulated in peripheral blood mononuclear cells of active ITP patients compared to those of healthy controls. No significant differences in LMP7 expression were observed between patients and controls. ML604440, an specific LMP2 inhibitor, had no significant impact on the platelet count of ITP mice, while ONX-0914 (an inhibitor of both LMP2 and LMP7) increased the number of platelets.

Topics: Adolescent; Adult; Aged; Animals; Case-Control Studies; Cysteine Endopeptidases; Disease Models, Animal; Female; Humans; Lymphocyte Activation; Macrophages; Male; Mice, Inbred C57BL; Middle Aged; Oligopeptides; Phagocytosis; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Purpura, Thrombocytopenic, Idiopathic; Receptors, IgG; Signal Transduction; T-Lymphocytes; Young Adult

Chronic antibody-mediated rejection is the leading cause of allograft dysfunction and loss after kidney transplantation, and current immunosuppressive regimens fail to target the plasma cells that produce alloantibodies. We previously showed that treatment with the immunoproteasome inhibitor ONX 0914 prevented the expansion of plasma cells and prevented chronic allograft nephropathy and organ failure after kidney transplantation in rats, but the mechanism has remained elusive. In the current study, we confirmed a long-term reduction in alloantibody production and improvements in allograft histology in rats treated with ONX 0914 or with the broad-spectrum proteasome inhibitor bortezomib. Plasma cells from allotransplanted rats expressed immunoproteasomes at high levels. Immunoproteasome inhibition with ONX 0914 led to ubiquitin-conjugate accumulation, activation of the unfolded protein response, and induction of apoptosis in plasma cells. In addition, ONX 0914 suppressed the expression of adhesion molecules (VLA-4 and LFA-1), plasma cell survival factors (APRIL and IL-6), and IFN-γ-inducible chemokines in bone marrow, while the APRIL receptor BCMA, the IL-6 receptor, and the chemokine receptors CXCR4 and CXCR3 were down-regulated on plasma cells. Taken together, immunoproteasome inhibition blocked alloantibody production by inducing apoptosis of plasma cells through activating the unfolded protein response and suppressing plasma cell survival factors in the bone marrow.

Topics: Allografts; Animals; Apoptosis; Bortezomib; Cell Survival; Disease Models, Animal; Graft Rejection; Humans; Isoantibodies; Kidney; Kidney Transplantation; Male; Oligopeptides; Plasma Cells; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Rats; Rats, Inbred F344; Rats, Inbred Lew; Transplantation, Homologous; Unfolded Protein Response

Chronic antibody-mediated rejection is the major cause of fading allograft function and loss after renal transplantation. Currently, pharmacological agents for the suppression of chronic antibody-mediated rejection are lacking. Non-selective proteasome inhibitors suppress antibody-mediated allograft rejection. However, extensive adverse side effects of these inhibitors severely limit their application. In contrast, immunoproteasome inhibition is effective in preclinical models of autoimmune diseases and was applied over weeks without obvious adverse side effects. ONX 0914, an immunoproteasome subunit LMP7 (β5i)-selective inhibitor, impeded the chronic rejection of kidneys transplanted from Fischer to allogeneic Lewis rats. ONX 0914 inhibited immunoproteasome induction both in immune organs and renal allografts. Selective immunoproteasome inhibition reduced the numbers of B and plasma cells, and suppressed donor-specific alloantibody production. The infiltration of T cells, B cells and macrophages as well as interferon-γ, interleukin-17, IgG and complement deposition were reduced in renal allografts of ONX 0914-treated recipients. Chronic nephropathy was ameliorated and renal allograft function preserved, enabling long-term survival of recipients. Thus, our studies define a critical role of the immunoproteasome in chronic kidney allograft rejection and suggest immunoproteasome inhibition as a promising therapeutic approach to suppress chronic antibody-mediated rejection.

Topics: Allografts; Animals; Chronic Disease; Disease Models, Animal; Graft Rejection; Graft Survival; Immunosuppressive Agents; Isoantibodies; Kidney; Kidney Transplantation; Male; Oligopeptides; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Rats, Inbred F344; Rats, Inbred Lew; Time Factors

Acute ischaemic stroke can induce secondary brain injury by activating an inflammatory response that contributes to clinical impairment. As a specific inhibitor of the immunoproteasome subunit low molecular weight polypeptide 7 (LMP7), PR-957 may participate in regulating pathophysiological and inflammatory responses in multiple diseases of the central nervous system (CNS). We investigated the neuroprotective properties of PR-957 in a mouse model of stroke, induced by middle cerebral artery occlusion (MCAO). After MCAO and injections of PR-957 or vehicle, we evaluated mice behaviourally using modified Neurological Severity Scores (mNSS) and sensorimotor tests, including the adhesive-removal test, a foot-fault test and an inclined plane test. Infarct volume was measured 24 and 72 h after MCAO. Infiltration by different lymphocyte subpopulations was evaluated by flow cytometry and immunofluorescent staining of brain tissue from the penumbral area. Quantitative real-time polymerase chain reaction analysis and enzyme-linked immunosorbent assay were used to measure the expression of proinflammatory cytokines: interkeukin (IL)-1α, IL-1β, IL-2, IL-4, IL-6, IL-10, IL-12, IL-17A, interferon (IFN)-γ, tumour necrosis factor (TNF)-α, granulocyte colony-stimulating factor (GCSF) and granulocyte-macrophage colony-stimulating factor (GM-CSF). Expression of phosphorylated signal transducer and activator of transcription 3 (pSTAT-3) protein levels in brain was measured by immunoblot. MCAO mice treated with PR-957 showed a significant decrease in infarct volume and had mild neurological deficits compared to vehicle-treated mice. PR-957 administration also significantly decreased IL-1β, IL-6, IL-12, IL-17A and TNF-α. PR-957 provides neuroprotection via inhibiting T lymphocyte infiltration and decreasing T helper type 17 (Th17) cell differentiation in MCAO mice, which may result from the reduced expression of pSTAT-3. The neuroprotective effect of PR-957 indicates its potential utility as anti-inflammatory therapy for ischaemic stroke.

Topics: Acute Disease; Animals; Anti-Inflammatory Agents; Cell Differentiation; Cytokines; Disease Models, Animal; Humans; Inflammation Mediators; Ischemia; Male; Mice; Mice, Inbred C57BL; Neuroprotection; Neuroprotective Agents; Oligopeptides; STAT3 Transcription Factor; Stroke; Th17 Cells

Accumulating evidence shows that the immunoproteasome participates in the immune response, beyond its initial role in the protein degradation. Here, we tested the effects of the selective immunoproteasome inhibitor, ONX-0914, on experimental autoimmune myasthenia gravis (EAMG). We found that ONX-0914 ameliorated the severity of ongoing EAMG by reducing the autoantibody affinity, accompanied with decreased Tfh cells and antigen presenting cells. Also it reduced the percentage of Th17 cells and inhibited the secretion of IL-17. Our data indicated ONX-0914 may bring benefit for MG therapy.

Topics: Animals; Antigen-Presenting Cells; Antigens, CD; Disease Models, Animal; Enzyme-Linked Immunosorbent Assay; Female; Flow Cytometry; Immunity, Humoral; Immunoglobulin G; Interleukin-17; Lymph Nodes; Myasthenia Gravis, Autoimmune, Experimental; Oligopeptides; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Rats; Rats, Inbred Lew; Spleen; T-Lymphocytes, Helper-Inducer

Apart from its role in MHC class I antigen processing, the immunoproteasome has recently been implicated in the modulation of T helper cell differentiation under polarizing conditions in vitro and in the pathogenesis of autoimmune diseases in vivo. In this study, we investigated the influence of LMP7 on T helper cell differentiation in response to the fungus Candida albicans. We observed a strong effect of ONX 0914, an LMP7-selective inhibitor of the immunoproteasome, on IFN-γ and IL-17A production by murine splenocytes and human peripheral blood mononuclear cells (PBMCs) stimulated with C. albicans in vitro. Using a murine model of systemic candidiasis, we could confirm reduced generation of IFN-γ- and IL-17A-producing cells in ONX 0914 treated mice in vivo. Interestingly, ONX 0914 treatment resulted in increased susceptibility to systemic candidiasis, which manifested at very early stages of infection. Mice treated with ONX 0914 showed markedly increased kidney and brain fungal burden which resulted in enhanced neutrophil recruitment and immunopathology. Together, these results strongly suggest a role of the immunoproteasome in promoting proinflammatory T helper cells in response to C. albicans but also in affecting the innate antifungal immunity in a T helper cell-independent manner.

Topics: Animals; Antifungal Agents; Candida albicans; Candidiasis; Colony Count, Microbial; Cytokines; Disease Models, Animal; Humans; Immunity, Innate; Immunomodulation; Kidney; Leukocyte Count; Leukocytes, Mononuclear; Mice; Neutrophil Infiltration; Neutrophils; Oligopeptides; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Spleen; T-Lymphocyte Subsets

Duchenne muscular dystrophy is an inherited fatal genetic disease characterized by mutations in dystrophin gene, causing membrane fragility leading to myofiber necrosis and inflammatory cell recruitment in dystrophic muscles. The resulting environment enriched in proinflammatory cytokines, like IFN-γ and TNF-α, determines the transformation of myofiber constitutive proteasome into the immunoproteasome, a multisubunit complex involved in the activation of cell-mediate immunity. This event has a fundamental role in producing peptides for antigen presentation by MHC class I, for the immune response and also for cytokine production and T-cell differentiation. Here, we characterized for the first time the presence of T-lymphocytes activated against revertant dystrophin epitopes, in the animal model of Duchenne muscular dystrophy, the mdx mice. Moreover, we specifically blocked i-proteasome subunit LMP7, which was up-regulated in dystrophic skeletal muscles, and we demonstrated the rescue of the dystrophin expression and the amelioration of the dystrophic phenotype. The i-proteasome blocking lowered myofiber MHC class I expression and self-antigen presentation to T cells, thus reducing the specific antidystrophin T cell response, the muscular cell infiltrate, and proinflammatory cytokine production, together with muscle force recovery. We suggest that i-proteasome inhibition should be considered as new promising therapeutic approach for Duchenne muscular dystrophy pathology.

Topics: Animals; Cell Differentiation; Disease Models, Animal; Genetic Therapy; Immunoproteins; Mice; Mice, Inbred mdx; Muscular Dystrophy, Duchenne; Oligopeptides; Proteasome Endopeptidase Complex; Proteasome Inhibitors; T-Lymphocytes

The proteasome is the core of the ubiquitin-proteasome system and is involved in synaptic protein metabolism. The incorporation of three inducible immuno-subunits into the proteasome results in the generation of the so-called immunoproteasome, which is endowed of pathophysiological functions related to immunity and inflammation. In healthy human brain, the expression of the key catalytic β5i subunit of the immunoproteasome is almost absent, while it is induced in the epileptogenic foci surgically resected from patients with pharmaco-resistant seizures, including temporal lobe epilepsy. We show here that the β5i immuno-subunit is induced in experimental epilepsy, and its selective pharmacological inhibition significantly prevents, or delays, 4-aminopyridine-induced seizure-like events in acute rat hippocampal/entorhinal cortex slices. These effects are stronger in slices from epileptic vs normal rats, likely due to the more prominent β5i subunit expression in neurons and glia cells of diseased tissue. β5i subunit is transcriptionally induced in epileptogenic tissue likely by Toll-like receptor 4 signaling activation, and independently on promoter methylation. The recent availability of selective β5i subunit inhibitors opens up novel therapeutic opportunities for seizure inhibition in drug-resistant epilepsies.

Topics: Animals; Disease Models, Animal; Entorhinal Cortex; Epilepsy; Hippocampus; Male; Oligopeptides; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Protein Subunits; Rats; Rats, Wistar; RNA, Messenger

Aside from antimalarials, there is currently no treatment for cerebral malaria, a fulminant neurological complication of P. falciparum infection that is a leading cause of death in African children. In the mouse model of cerebral malaria, cross-presentation of parasite antigens by brain endothelial cells is thought to be a crucial late step in pathogenesis. We have investigated three proteasome inhibitors as potential adjunct therapies: bortezomib, carfilzomib and ONX-0914. Only carfilzomib, an irreversible inhibitor of both constitutive proteasomes and immunoproteasomes, was able to inhibit cross-presentation of malaria antigen by murine brain endothelial cells in vitro. To mimic the clinical setting, carfilzomib was co-administered with artesunate only when infected mice exhibited neurological defects. However, there was no improvement in survival compared to artesunate monotherapy. The treatment failure was explained by the inability of daily or twice daily bolus doses of carfilzomib to inhibit cross-presentation by brain endothelial cells in vivo. We also report here that bortezomib, which has been associated with neurological adverse events, accelerated death in ECM-infected mice. Future investigations of proteasome inhibitors for modulating cross-presentation during malaria infection should focus on sustained and targeted delivery to brain endothelial cells.

Topics: Animals; Antigen Presentation; Antimalarials; Bortezomib; Brain; Cells, Cultured; Disease Models, Animal; Endothelial Cells; In Vitro Techniques; Malaria, Cerebral; Malaria, Falciparum; Mice; Mice, Inbred C57BL; Oligopeptides; Plasmodium berghei; Proteasome Inhibitors

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

Major histocompatibility complex (MHC) class I-restricted T cell epitopes are generated mainly by the immunoproteasome in antigen-presenting cells. Therefore, inhibition of activity of this proteolytic complex molecule is thought to be a potential treatment for cell-mediated autoimmune diseases. We therefore studied the efficacy of an immunoproteasome inhibitor, ONX 0914 (formerly PR-957), for the treatment of autoimmune thyroid diseases, including cell-mediated Hashimoto's thyroiditis and autoantibody-mediated Graves' hyperthyroidism using mouse models. Our data show that ONX 0914 was effective prophylactically and therapeutically at suppressing the degree of intrathyroidal lymphocyte infiltration and, to a lesser degree, the titres of anti-thyroglobulin autoantibodies in non-obese diabetic (NOD)-H2(h4) mice, an iodine-induced autoimmune thyroiditis model. It also inhibited differentiation of T cells to T helper type 1 (Th1) and Th17 cells, effector T cell subsets critical for development of thyroiditis in this mouse strain. In contrast, its effect on the Graves' model was negligible. Although ONX 0914 exerts its immune-suppressive effect through not only suppression of immune proteasome but also other mechanism(s), such as inhibition of T cell differentiation, the present results suggest that the immunoproteasome is a novel drug target in treatment of Hashimoto's thyroiditis in particular and cell-mediated autoimmune diseases in general.

Topics: Animals; Antibody Formation; Autoantibodies; Cells, Cultured; Cysteine Proteinase Inhibitors; Disease Models, Animal; Graves Disease; Hashimoto Disease; Humans; Immunity, Cellular; Iodine; Mice; Mice, Inbred NOD; Oligopeptides; Proteasome Inhibitors; Th1 Cells; Th17 Cells; Thyroglobulin; Thyroid Gland

The proteasome, a multicatalytic protease, is responsible for the degradation of intracellular proteins. Stimulation of cells with inflammatory cytokines, such as IFN-gamma, leads to the replacement of the constitutive catalytic proteasome subunits by the inducible subunits low molecular mass polypeptide (LMP)2 (beta1i), multicatalytic endopeptidase complex-like-1 (beta2i), and LMP7 (beta5i), which are required for the production of certain MHC class I-restricted T cell epitopes. In this study, we investigated the effect of immunoproteasomes on the development of dextran sulfate sodium-induced colitis. Colitis induction in LMP2-, LMP7-, and multicatalytic endopeptidase complex-like-1-deficient mice caused reduced weight loss compared with wild-type mice. Although colon lengths were shortened in wild-type mice, no reduction was observed in immunoproteasome-deficient mice. In accordance with this, proinflammatory cytokines, such as TNF-alpha and IL-1beta, were not upregulated in these mice. Blockage of LMP7 by a novel LMP7-selective inhibitor (PR-957) strongly reduced pathological symptoms of dextran sulfate sodium-induced colitis. Production of numerous cytokines in PR-957-treated mice was suppressed, resulting in reduced inflammation and tissue destruction. Taken together, these results demonstrate that an immunoproteasome-specific inhibitor can be used to attenuate autoimmune diseases like colitis.

Topics: Animals; Autoimmune Diseases; Colitis, Ulcerative; Disease Models, Animal; Female; Mice; Mice, Inbred C57BL; Mice, Knockout; Oligopeptides; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Up-Regulation