C Protein-induced Myositis Research Articles

Pathogenicity of functionally activated PD-1+CD8+ cells and counterattacks by muscular PD-L1 through IFNγ in myositis

Programmed-cell-death 1 (PD-1) expression is associated not only with T-cell activation but with exhaustion. Specifically, PD-1+ T cells present an exhausted phenotype in conditions of chronic antigen exposure, such as tumor microenvironments and chronic viral infection. However, the immune status regarding exhaustion of PD-1+CD8+ T cells in chronic autoimmune diseases including idiopathic inflammatory myopathies (IIMs) remains unclear. We aimed to clarify the role of PD-1+CD8+ T cells and PD-1 ligand (PD-L1) in IIMs. We showed that PD-1+ cells infiltrated into PD-L1-expressing muscles in patients with IIMs and immune checkpoint inhibitor-related myopathy. According to the peripheral blood immunophenotyping, the PD-1+CD8+ cell proportions were comparable between the active and inactive patients. Of note, PD-1+CD8+ cells in the active patients highly expressed cytolytic molecules, indicating their activation, while PD-1−CD8+ cells expressed low levels of cytolytic molecules in the active and inactive patients. A part of PD-1+CD8+ cells expressed the HMG-box transcription factor TOX highly and presented the exhausted phenotype in the active patients. Among PD-1+CD4+ T cells, PD-1highCXCR5−CD45RO+CD4+ peripheral helper T cells were increased in the active patients. PD-L1-deficient mice developed severer C-protein-induced myositis (CIM), a model of polymyositis, with abundant infiltration of PD-1+CD8+ cells expressing cytolytic molecules than wild-type mice, indicating pathogenicity of the PD-1+CD8+ cells and the protective role of PD-L1. The deficiency of IFNγ, a general PD-L1-inducer, impaired muscular PD-L1 expression and exacerbated CIM, indicating IFNγ-dependent muscular PD-L1 regulation. IFNγ-induced PD-L1 on myotubes was protective in an established muscle injury model. In conclusion, PD-1+CD8+ T cells rather than PD-1−CD8+ T cells were a pathogenic subset of IIMs. Muscular PD-L1 was regulated by IFNγ and exerted protective properties in IIMs.

Effective therapy of polymyositis in mice via selective inhibition of the immunoproteasome.

Polymyositis (PM) is a chronic autoimmune inflammatory myopathy resulting in muscle weakness. The limited approved therapies and their poor efficacy contribute to its comorbidity. We investigated the therapeutic use of ONX 0914 and KZR-616, selective inhibitors of the immunoproteasome, in C protein-induced myositis (CIM), a mouse model of PM that closely resembles the human disease. Diseased mice (day 13 postimmunization) were treated with 10 mg/kg ONX 0914, KZR-616, or vehicle on alternate days until day 28. Endpoints included muscle strength assessed by a grip strength meter, serum creatine kinase activity, histology, and immunohistochemistry analysis. Treatment with ONX 0914 or KZR-616 prevented the loss of grip strength in mice after CIM induction, while vehicle-treated animals displayed progressive muscle weakness. Immunoproteasome inhibition lowered PM-associated leukocyte infiltration of the muscle and prevented increased serum creatine kinase levels. LMP7-deficient mice were resistant to CIM induction, as they showed no alterations in grip strength or creatine kinase (CK) levels or muscular alterations. In conclusion, selective inhibition of the immunoproteasome displays therapeutic efficacy in a preclinical mouse model of PM with suppression of muscle inflammation and preservation of muscle strength. Positive results from this study support the rationale for using KZR-616 in clinical studies.

Amelioration of inflammatory myopathies by glucagon‐like peptide‐1 receptor agonist via suppressing muscle fibre necroptosis

BackgroundAs glucocorticoids induce muscle atrophy during the treatment course of polymyositis (PM), novel therapeutic strategy is awaited that suppresses muscle inflammation but retains muscle strength. We recently found that injured muscle fibres in PM undergo FASLG‐mediated necroptosis, a form of regulated cell death accompanied by release of pro‐inflammatory mediators, contributes to accelerate muscle inflammation and muscle weakness. Glucagon‐like peptide‐1 receptor (GLP‐1R) agonists have pleiotropic actions including anti‐inflammatory effects, prevention of muscle atrophy, and inhibition of cell death, in addition to anti‐diabetic effect. We aimed in this study to examine the role of GLP‐1R in PM and the effect of a GLP‐1R agonist on in vivo and in vitro models of PM.MethodsMuscle specimens of PM patients and a murine model of PM, C protein‐induced myositis (CIM), were examined for the expression of GLP‐1R. The effect of PF1801, a GLP‐1R agonist, on CIM was evaluated in monotherapy or in combination with prednisolone (PSL). As an in vitro model of PM, C2C12‐derived myotubes were treated with FASLG to induce necroptosis. The effect of PF1801 on this model was analysed.ResultsGLP‐1R was expressed on the inflamed muscle fibres of PM and CIM. The treatment of CIM with PF1801 in monotherapy (PF) or in combination with PSL (PF + PSL) suppressed CIM‐induced muscle weakness (grip strength, mean ± SD (g); PF 227 ± 6.0 (P < 0.01), PF + PSL 224 ± 8.5 (P < 0.01), Vehicle 162 ± 6.0) and decrease in cross‐sectional area of muscle fibres (mean ± SD (μm2); PF 1896 ± 144 (P < 0.05), PF + PSL 2018 ± 445 (P < 0.01), Vehicle 1349 ± 199) as well as the severity of histological inflammation scores (median, interquartile range; PF 0.0, 0.0–0.5 (P < 0.05), PF + PSL 0.0, 0.0–0.0 (P < 0.01), Vehicle 1.9, 1.3–3.3). PF1801 decreased the levels of inflammatory mediators such as TNFα, IL‐6, and HMGB1 in the serum of CIM. PF1801 inhibited necroptosis of the myotubes in an AMP‐activated protein kinase (AMPK)‐dependent manner. PF1801 activated AMPK and decreased the expression of PGAM5, a mitochondrial protein, which was crucial for necroptosis of the myotubes. PF1801 promoted the degradation of PGAM5 through ubiquitin‐proteasome activity. Furthermore, PF1801 suppressed FASLG‐induced reactive oxygen species (ROS) accumulation in myotubes, also crucial for the execution of necroptosis, thorough up‐regulating the antioxidant molecules including Nfe2l2, Hmox1, Gclm, and Nqo1.ConclusionsGLP‐1R agonist could be a novel therapy for PM that recovers muscle weakness and suppresses muscle inflammation through inhi biting muscle fibre necroptosis.

Interleukin-23 as a therapeutic target for inflammatory myopathy

Current treatments of polymyositis and dermatomyositis (PM/DM) depend on non-specific immunosuppressants. This study was performed to elucidate the role of interleukin (IL)-23, as their possible therapeutic target. As was reported earlier in PM/DM patients, serum IL-23 levels were elevated in mice with C protein induced-myositis (CIM), a murine model of PM. IL-23 was expressed by macrophages in the PM/DM and CIM muscles and by dendritic cells and macrophages in the lymph nodes from the CIM mice. It was also expressed by macrophages in the chemically injured muscles, but not those recruited into the muscles by footpad injection of Freund’s complete adjuvant, demonstrating that IL-23 production should be associated with muscle damage. Genetic deletion of IL-23 as well as preventive and therapeutic administration of blocking antibodies against IL-23p19 subunit suppressed CIM. When lymph node cells from the CIM mice were transferred adoptively into naive wild type or IL-23p19 deficient recipient mice, both recipients developed myositis equally. Thus, elevated IL-23 should promote dendritic cells and macrophages to activate the autoaggressive T cells. Our findings suggest that IL-23 should mediate positive feedback loop from the muscle damage to the T cell activation and be a promising therapeutic target for autoimmune myositis.

Interferon‐γ, but Not Interleukin‐4, Suppresses Experimental Polymyositis

C protein-induced myositis (CIM) is a mouse model of polymyositis in which activated antigen-specific CD8+ T cells injure the muscles. Animal models of autoimmunity that have been examined in the past for the effects of the type 1 cytokine interferon-γ (IFNγ) and the type 2 cytokine interleukin-4 (IL-4) are all mediated by pathogenic CD4+ T cells. In those models, the disruption of IFNγ leads to up-regulation of IL-17A, exacerbating the disease with neutrophil infiltration into sites of inflammation. This study was undertaken to investigate the roles of IFNγ and IL-4, as well as IL-17A in the absence of IFNγ, in CD8+ T cell-mediated CIM. IFNγ(-/-) mice, anti-IL-17A antibody-treated IFNγ(-/-) mice, IFNγ(-/-) IL-17A(-/-) mice, IL-4(-/-) mice, and wild-type C57BL/6 mice were immunized with skeletal muscle C protein fragments to induce CIM. Muscle tissue specimens were examined histologically. IFNγ(-/-) mice developed myositis at a higher incidence and with greater severity than wild-type mice. Unlike wild-type mice, IFNγ(-/-) mice had infiltration of neutrophils into the endomysial sites of the affected muscles. IFNγ(-/-) mice treated with anti-IL-17A antibodies and IFNγ(-/-) IL-17A(-/-) mice developed myositis with an incidence and severity comparable to those in IFNγ(-/-) mice and showed neutrophil infiltration similar to that in IFNγ(-/-) mice. IL-4(-/-) mice developed CIM comparable to that in wild-type mice. Our findings indicate that IFNγ, but not IL-4, plays a suppressive role in the development of CIM. Unlike in CD4+ T cell-mediated autoimmune disease models, IFNγ prevents factors other than IL-17A from exacerbating myositis and neutrophil infiltration in CD8+ T cell-mediated CIM.

A Crucial Role of L‐Selectin in C Protein–Induced Experimental Polymyositis in Mice

To investigate the role of adhesion molecules in C protein-induced myositis (CIM), a murine model of polymyositis (PM). CIM was induced in wild-type mice, L-selectin-deficient (L-selectin(-/-) ) mice, intercellular adhesion molecule 1 (ICAM-1)-deficient (ICAM-1(-/-) ) mice, and mice deficient in both L-selectin and ICAM-1 (L-selectin(-/-) ICAM-1(-/-) mice). Myositis severity, inflammatory cell infiltration, and messenger RNA expression in the inflamed muscles were analyzed. The effect of dendritic polyglycerol sulfate, a synthetic inhibitor that suppresses the function of L-selectin and endothelial P-selectin, was also examined. L-selectin(-/-) mice and L-selectin(-/-) ICAM-1(-/-) mice developed significantly less severe myositis compared to wild-type mice, while ICAM-1 deficiency did not inhibit the development of myositis. L-selectin(-/-) mice that received wild-type T cells developed myositis. Treatment with dendritic polyglycerol sulfate significantly diminished the severity of myositis in wild-type mice compared to treatment with control. These data indicate that L-selectin plays a major role in the development of CIM, whereas ICAM-1 plays a lesser role, if any, in the development of CIM. L-selectin-targeted therapy may be a candidate for the treatment of PM.

Therapeutic effect of anti-C-X-C motif chemokine 10 (CXCL10) antibody on C protein-induced myositis mouse.

IntroductionC-X-C motif chemokine 10 (CXCL10) is a chemokine that plays a critical role in the infiltration of T cells in autoimmune diseases and is reported to be expressed in muscle tissue of polymyositis. To determine the therapeutic efficacy of CXCL10 blockade, we investigated the role of CXCL10 and the effect of anti-CXCL10 antibody treatment in C protein-induced myositis (CIM), an animal model of polymyositis.MethodsCIM was induced with human skeletal muscle C protein fragment in female C57BL/6 mice. Immunohistochemistry of CXCL10 and C-X-C motif chemokine receptor 3 (CXCR3) and measurement of serum CXCL10 were performed. Cell surface markers and interferon-gamma (IFN-γ) and tumor necrosis factor-alpha (TNF-α) in CIM lymph node cells was investigated by flow cytometry. Mice with CIM were treated with anti-CXCL10 antibody or control antibody (anti-RVG1) and the inflammation in muscle tissue was assessed.ResultsImmunohistochemistry showed increased expression of CXCL10 and CXCR3 in the inflammatory lesions of muscle in CIM. Especially, CD8+ T cells invading myofiber expressed CXCR3. Serum level of CXCL10 was increased in CIM compared to the level in normal mice (normal mouse, 14.3 ± 5.3 pg/ml vs. CIM, 368.5 ± 135.6 pg/ml, P < 0.001). CXCR3 positivity in CD8+ T cells was increased compared to that of CD4+ T cells in the lymph node cells of CIM (CXCR3+ among CD8+ T cell, 65.9 ± 2.1% vs. CXCR3+ among CD4+ T cell, 23.5 ± 4.7%, P <0.001). Moreover, IFN-γ+ cells were increased among CXCR3+CD8+ T cells compared to CXCR3–CD8+ T cells (CXCR3+CD8+ T cell, 28.0 ± 4.2% vs. CXCR3-CD8+ T cell, 9.5 ± 1.5%, P = 0.016). Migration of lymph node cells was increased in response to CXCL10 (chemotactic index was 1.91 ± 0.45). CIM mice treated with anti-CXCL10 antibody showed a lower inflammation score in muscles than those with anti-RVG1 (median, anti-CXCL10 treatment group, 0.625 vs. anti-RVG1 treatment group, 1.25, P = 0.007).ConclusionsCXCL10/CXCR3 expression was increased in the inflammation of CIM model and its blockade suppressed inflammation in muscle.

Roles of mast cells in the pathogenesis of inflammatory myopathy

IntroductionIn addition to the pivotal roles of mast cells in allergic diseases, recent data suggest that mast cells play crucial roles in a variety of autoimmune responses. However, their roles in the pathogenesis of autoimmune skeletal muscle diseases have not been clarified despite their distribution in skeletal muscle. Therefore, the objective of this study is to determine the roles of mast cells in the development of autoimmune skeletal muscle diseases.MethodsThe number of mast cells in the affected muscle was examined in patients with dermatomyositis (DM) or polymyositis (PM). The susceptibility of mast cell-deficient WBB6F1-KitW/KitWv mice (W/Wv mice) to a murine model of polymyositis, C protein-induced myositis (CIM), was compared with that of wild-type (WT) mice. The effect of mast cell reconstitution with bone marrow-derived mast cells (BMMCs) on the susceptibility of W/Wv mice to CIM was also evaluated.ResultsThe number of mast cells in the affected muscle increased in patients with PM as compared with patients with DM. W/Wv mice exhibited significantly reduced disease incidence and histological scores of CIM as compared with WT mice. The number of CD8+ T cells and macrophages in the skeletal muscles of CIM decreased in W/Wv mice compared with WT mice. Engraftment of BMMCs restored the incidence and histological scores of CIM in W/Wv mice. Vascular permeability in the skeletal muscle was elevated in WT mice but not in W/Wv mice upon CIM induction.ConclusionMast cells are involved in the pathogenesis of inflammatory myopathy.

T lymphocytes and muscle condition act like seeds and soil in a murine polymyositis model

It has been reported that polymyositis (PM) is driven by CD8+ cytotoxic T lymphocytes. The C protein-induced myositis (CIM) model we have established is similar to PM in pathology except that it undergoes spontaneous remission. We undertook the present study to delineate the roles of innate and acquired immunity in myositis. C57BL/6 mice were immunized with recombinant C protein fragments together with Freund's complete adjuvant (CFA) and Toll-like receptor (TLR) ligands at hind leg footpads and tail bases. CIM mediated by adoptive transfer of T cells to naive mice was treated with cytokine antagonists. Second immunization with C protein fragments revealed no induction of tolerance. Injection of CFA and TLR ligands at the hind leg footpads reinduced myositis in the same legs. Interestingly, initial myositis was observed only in the CFA-treated forelegs. Transfer of C protein fragment-specific T cells from mice with CIM induced myositis in CFA- and TLR ligand-treated legs of recipient mice. CFA treatment resulted in the recruitment of macrophages producing inflammatory cytokines. Induction of myositis was inhibited by blocking interleukin-1 receptor or tumor necrosis factor α. Myositis development requires activation of autoaggressive T cells and conditioning of muscle tissue. CIM regression is due to attenuation of local CFA-induced immune activation. These results are in accordance with a "seed and soil" model of disease development and might offer clues to decipher clinical aspects of PM.

Interleukin‐1 and tumor necrosis factor α blockade treatment of experimental polymyositis in mice

Histologic studies of the muscles suggest that cytokines are involved in inflammatory myopathy. The therapeutic effects of cytokine blockade are controversial, with anecdotal reports of clinical efficacy. The aim of this study was to discern the significance of interleukin-1 (IL-1) and tumor necrosis factor α (TNFα) as therapeutic targets in polymyositis (PM) by studying their involvement and the effects of their blockade in C protein-induced myositis (CIM), a murine model of PM. C57BL/6 mice were immunized with recombinant skeletal C protein fragments to induce CIM. The expression of IL-1 and TNFα in the muscles of mice with CIM was detected using immunohistochemical and real-time polymerase chain reaction analyses. After the onset of myositis, the mice with CIM were treated with recombinant IL-1 receptor antagonist (IL-1Ra), anti-IL-1R monoclonal antibody, recombinant TNF receptor (p75)-fusion protein (TNFR-Fc), or anti-TNFα monoclonal antibody. The muscles were examined histologically for the severity of myositis. IL-1α- and TNFα-positive macrophages were observed in the muscle tissue of mice with CIM as early as 7 days after immunization. IL-1α, IL-1β, and TNFα expression in the muscles increased as the severity of myositis peaked, at both the messenger RNA and protein levels. Continuous subcutaneous delivery of IL-1Ra resulted in suppression of established CIM. Intermittent delivery (1-day intervals) of anti-IL-1R monoclonal antibody suppressed myositis, while intermittent delivery of IL-1Ra did not suppress myositis. Treatment with anti-TNFα monoclonal antibody and with TNFR-Fc also reduced the severity of CIM. IL-1 and TNF blockade ameliorated CIM after disease onset and should potentially be a new strategy for the treatment of inflammatory myopathy. As IL-1 blockade, treatment with anti-IL-1R monoclonal antibody appeared more feasible than the other approaches.

Crucial roles of mast cells in the development of a murine model of polymyositis (171.20)

Abstract Objective: In addition to a pivotal role of mast cells in allergic diseases, recent data suggest that mast cells play a crucial role in a variety of immune responses. However, their role in the pathogenesis of autoimmune skeletal muscle diseases has not been clarified despite their distribution in skeletal muscle. Therefore, the objective of this study is to determine the role of mast cells in the development of a murine model of polymyositis, C protein-induced myositis (CIM). Methods: The susceptibility of mast cell-deficient WBB6F1-KitW/KitWv mice (W/Wv mice) to CIM was compared with that of wild-type (WT) mice. The effect of mast cell reconstitution with bone marrow-derived mast cells (BMMCs) on the susceptibility of W/Wv mice to CIM was also evaluated. Results: W/Wv mice exhibited significantly reduced disease incidence and histological scores of CIM as compared with WT mice. The infiltration of macrophages but not of CD8+ T cells in the skeletal muscle was attenuated in CIM of W/Wv mice. The expression of monocyte chemotactic protein-1 (MCP-1) in skeletal muscle was reduced in W/Wv mice. Vascular permeability in skeletal muscle was elevated in WT mice but not in W/Wv mice upon CIM induction. Engraftment of BMMCs restored the incidence and histological scores of CIM in W/Wv mice. Conclusion: Mast cells play critical roles in the development of CIM.

Definitive engagement of cytotoxic CD8 T cells in C protein–induced myositis, a murine model of polymyositis

To substantiate a pathogenic role of cytotoxic CD8 T cells in the development of a murine polymyositis model, C protein-induced myositis (CIM). Beta(2)-microglobulin-null mutant, perforin-null mutant, and wild-type (WT) C57BL/6 mice were immunized with skeletal muscle C protein fragments to provoke CIM. Regional lymph node CD8 or CD4 T cells stimulated with C protein-pulsed dendritic cells were transferred adoptively to naive mice. Inflammation and damage of the muscle tissues were evaluated histologically. The incidence of myositis development was significantly lower in β₂-microglobulin-null and perforin-null mutant mice compared with WT mice. Inflammation was less severe in mutant mice, and the incidence of muscle injury was reduced significantly. Adoptive transfer of lymph node T cells from mice with CIM induced myositis in naive recipient mice. The CD8 T cell-induced muscle injuries were significantly more severe than the CD4 T cell-induced muscle injuries. Perforin-mediated cytotoxicity by CD8 T cells is definitively responsible for muscle injury in CIM.

Therapeutic effects of interleukin‐6 blockade in a murine model of polymyositis that does not require interleukin‐17A

To explore new molecular targets in the treatment of polymyositis (PM) by examining a recently established murine model of PM, C protein-induced myositis (CIM), for involvement of an interleukin-6 (IL-6)/IL-17A pathway. CIM was induced by immunizing wild-type mice as well as IL-6-null and IL-17A-null C57BL/6 mice with recombinant mouse skeletal C protein fragments. Some mice were treated with anti-IL-6 receptor (anti-IL-6R) monoclonal antibodies or control antibodies. Muscle tissue samples were examined histologically and immunohistochemically. The syngeneic C protein fragments successfully induced inflammation in the skeletal muscles of wild-type mice. IL-6 was expressed by mononuclear cells, especially in macrophages, infiltrating in the muscles. IL-6-null mice developed myositis with significantly lower incidence and milder severity than wild-type mice. In contrast, IL-17A-null mice were as susceptible to CIM as wild-type mice. Intraperitoneal administration of anti-IL-6R monoclonal antibodies, but not of control monoclonal antibodies, ameliorated CIM both preventively and therapeutically. Our findings indicate that IL-6 is critically involved in the development of CIM. Although many other autoimmune models require IL-6 for differentiation of pathogenic T cells producing IL-17A, IL-17A was dispensable in CIM. Nevertheless, treatment with anti-IL-6R antibodies was effective. IL-6 blockade is potentially a new approach to the treatment of autoimmune myositis, via processes distinct from interference in the IL-6/IL-17A pathway.