Mei Zong

Pathogenesis, classification and treatment of inflammatory myopathies.

The inflammatory myopathies—collectively, myositis—are a heterogeneous group of chronic muscle disorders that differ in response to immunosuppressive treatment. Insufficient knowledge of the molecular pathways that drive pathogenesis (and underlie the clinical differences between subtypes) has hindered accurate classification, which in turn has been detrimental for clinical research. Nevertheless, new insights into pathogenesis are paving the way for improvements in diagnosis, classification and treatment. Accumulating data suggest that both immune and nonimmune mechanisms cause muscle weakness. Phenotyping of the T cells that accumulate in muscle tissue has identified proinflammatory, apoptosis resistant and cytotoxic CD4+ and CD8+ CD28null populations. Several myositis-specific autoantibodies have been identified, associated with distinct clinical phenotypes. Thus, adaptive immunity is involved in pathogenesis, and both T and B cells are interesting targets for therapy. Furthermore, genotyping has revealed activation of the type I interferon pathway in patients with dermatomyositis or with expression of particular autoantibodies. Decreased release of Ca2+ from the sarcoplasmic reticulum, as a consequence of release of proinflammatory cytokines and high mobility group protein B1, might contribute to muscle weakness, and nonimmune mechanisms potentially include a role for endoplasmic reticulum stress, autophagy and hypoxia. Deeper understanding, careful phenotyping of patients—and new classification criteria—will expedite clinical research.

Anakinra treatment in patients with refractory inflammatory myopathies and possible predictive response biomarkers: a mechanistic study with 12 months follow-up

Objective To perform a mechanistic study on the effect of interleukin (IL)-1 blockade by anakinra in patients with refractory myositis and to explore possible predictive biomarkers. Methods Fifteen patients with refractory myositis were treated with anakinra for 12 months. Clinical response was assessed by the six-item core set measures of disease activity International Myositis Assessment and Clinical Studies (IMACS) and functional index (FI). Repeated muscle biopsies were investigated for cellular infiltrates, IL-1α, IL-1β, IL-1Ra and major histocompatibility complex-class I by immunohistochemistry. Serum levels of IL-1Ra and granulocyte colony-stimulating factor (G-CSF) were measured by ELISA. T cell phenotype and functional assays were investigated by multicolour flow cytometry. Results Seven patients had clinical response according to IMACS, four of them also showed improved FI. Responders had higher baseline extramuscular score compared with non-responders. In muscle biopsies, baseline CD163 macrophages and IL-1α expression were inversely correlated with muscle performance after 6 months treatment; all responders had IL-1Ra expression in the post-treatment biopsies but only 3/8 non-responders. In serum, IL-1Ra levels were increased and G-CSF was decreased after 6 months treatment, but their levels and changes were not related to clinical response. For T cells, an inverse correlation between baseline frequency of CD4 activated/memory T cells and decreased creatine kinase levels was observed. Five of six patients demonstrated less IL-17A and more IFN-γ secreting CD4 T cells after 6 months treatment. Moreover, anakinra reduced IL-17A secretion in vitro. Conclusions Patients with myositis may respond to anakinra. Extramuscular score, muscle CD163 macrophages and IL-1α expression, blood CD4 activated/memory T cells might associate with anakinra treatment response. Blocking the IL-1 receptor disfavoured Th17 cell differentiation both in vivo and in vitro.

TLR4 as receptor for HMGB1 induced muscle dysfunction in myositis

Objectives Polymyositis and dermatomyositis are characterised by muscle weakness and fatigue even in patients with normal muscle histology via unresolved pathogenic mechanisms. In this study, we investigated the mechanisms by which high mobility group box protein 1 (HMGB1) acts to accelerate muscle fatigue development. Methods Intact single fibres were dissociated from flexor digitorum brevis (FDB) of wild type, receptor for advanced glycation endproduct (RAGE) knockout and toll like receptor 4 (TLR4) knockout mice and cultured in the absence or presence of recombinant HMGB1. A decrease in sarcoplasmic reticulum Ca2+ release during a series of 300 tetanic contractions, which reflects the development of muscle fatigue, was determined by measuring myoplasmic free tetanic Ca2+. TLR4 and major histocompatibility complex (MHC)-class I expression in mouse FDB fibres were investigated by immunofluorescence and confocal microscopy. Immunohistochemistry was used to investigate TLR4, MHC-class I and myosin heavy chain expression in muscle fibres of patients. Results Our results demonstrate that TLR4 is expressed in human and mouse skeletal muscle fibres, and coexpressed with MHC-class I in muscle fibres of patients with myositis. Furthermore, we show that HMGB1 acts via TLR4 but not RAGE to accelerate muscle fatigue and to induce MHC-class I expression in vitro. In order to bind and signal via TLR4, HMGB1 must have a reduced cysteine 106 and a disulphide linkage between cysteine 23 and 45. Conclusions The HMGB1-TLR4 pathway may play an important role in causing muscle fatigue in patients with polymyositis or dermatomyositis and thus is a potential novel target for future therapy.

Expanded T cell receptor Vβ-restricted T cells from patients with sporadic inclusion body myositis are proinflammatory and cytotoxic CD28null T cells.

OBJECTIVE Sporadic inclusion body myositis (IBM) is characterized by T cell infiltrates in muscle tissue, but their functional role is unclear. Systemic signs of inflammation are lacking, and the absence of beneficial effects following immunosuppression has challenged the notion of a role for the immune system. This study was undertaken to investigate the phenotype and functionality of T cells, specifically a subset of proinflammatory, cytotoxic, and apoptosis-resistant T cells defined as CD28(null) T cells, in the pathogenesis of sporadic IBM. METHODS A cohort of 27 patients with sporadic IBM was analyzed for the frequency of circulating and muscle-infiltrating CD28(null) T cells. The T cell receptor (TCR) V(β) usage was determined using flow cytometry and immunohistochemistry. Anti-CD3-stimulated peripheral blood mononuclear cells were analyzed for intracellular interferon-γ and cytotoxic potential by flow cytometry. RESULTS We found striking accumulations of both CD8+CD28(null) and CD4+CD28(null) T cells, which represented the TCR V(β) -expanded T cells in sporadic IBM. Such CD28(null) T cells were abundant both in the inflamed muscle tissue and in the circulation. Although the specific TCR V(β) expansions varied between patients, both CD8+CD28(null) and CD4+CD28(null) T cells consistently displayed a highly proinflammatory and cytotoxic potential. CONCLUSION Our results suggest that CD28null T cell expansions represent the previously described expanded T cell subsets in sporadic IBM, and their proinflammatory capacity and presence in both muscle tissue and the circulation may imply a role of immune activation in sporadic IBM. In addition, CD4+CD28(null) T cells may exert cytotoxic effects directly on muscle fibers due to a cytotoxic potential similar to that in CD8+ T cells.

Effects of immunosuppressive treatment on interleukin-15 and interleukin-15 receptor α expression in muscle tissue of patients with polymyositis or dermatomyositis

Objectives To investigate the expression of interleukin (IL)-15 and IL-15 receptor α (IL-15Rα) in muscle tissue from patients with polymyositis or dermatomyositis before and after conventional immunosuppressive (IS) treatment. Methods Muscle biopsies from 17 patients before and after conventional IS treatment and seven healthy individuals were investigated by immunohistochemistry using antibodies against IL-15 and IL-15Rα. Quantification was performed by computerised image analysis. Cellular localisation of IL-15 was determined by double immunofluorescence. Clinical outcome was measured by the functional index and serum creatine kinase. Human myotubes were cultured and IL-15 staining was performed by immunocytochemistry. Results IL-15 was observed in mononuclear inflammatory cells of muscle tissue while IL-15Rα was localised to mononuclear inflammatory cells, capillaries and large vessels. Double staining showed localisation of IL-15 to CD163+ macrophages. A significantly larger number of IL-15 and IL-15Rα-positive cells were seen in muscle tissue of patients compared with healthy individuals. Baseline IL-15 expression correlated negatively with improvement in muscle function. After conventional IS treatment, a significantly lower number of IL-15 and IL-15Rα-positive cells was found. However, compared with controls, eight of 17 patients still had more IL-15-positive cells and less muscle function improvement was shown in this group of patients, both in short-term and long-term observations. Human differentiated myotubes were negative for IL-15 staining. Conclusions IL-15 and its receptor are expressed in the muscle tissue of patients with myositis and IL-15 expression is correlated with improvement in muscle function. IL-15 may play a role in the pathogenesis of myositis and could be a biological treatment target, at least in a subgroup of patients with polymyositis or dermatomyositis.

Activated LTB4 pathway in muscle tissue of patients with polymyositis or dermatomyositis

Objective To investigate the involvement of the leukotriene B4 (LTB4) pathway in polymyositis (PM) and dermatomyositis (DM) and the effect of immunosuppressive treatment on the LTB4 pathway. Methods 5-lipoxygenase (5-LO), 5-LO activating protein (FLAP) and LTB4 receptor-1 (BLT1) expression was analysed by immunohistochemistry in muscle tissue from patients with PM/DM before and after immunosuppressive treatment and from healthy individuals. In vivo LTB4 in thigh muscle was measured by microdialysis at rest and after acute exercise in another cohort of patients and healthy controls. Results The number of 5-LO-positive cells and BLT1-positive capillaries was higher in patients with PM/DM than in healthy individuals. The number of FLAP-expressing cells divided the patients into two groups (high/low expression). Treatment reduced the number of FLAP-positive cells in the group with initial high levels, however the expression remained high compared with healthy individuals. The number of BLT1-positive cells was also reduced while staining for 5-LO was unchanged. An inverse correlation was observed between the number of 5-LO or FLAP-positive cells in muscle tissue and muscle performance. LTB4 could be detected in dialysate of muscle tissue in vivo in both patients and healthy controls and was significantly increased after exercise in patients. Conclusion The LTB4 pathway is upregulated in muscle tissue from patients with PM/DM and this upregulation correlated negatively to muscle performance, suggesting a role for LTB4 in myositis muscle weakness. The immunosuppressive treatment was insufficient on the LTB4 pathway and, for patients with high expression of FLAP, FLAP inhibitors may be considered as possible therapy.

Anakinra effects on T cells in patients with refractory idiopathic inflammatory myopathies

Background and objectives Interleukin 1 (IL-1) is overexpressed in muscle tissue of inflammatory myopathies. A clinical response to IL-1 blockade with anakinra was seen in 7/15 patients with refractory myositis, but the biological explanation for improvement has not been clarified nor has predictors of response been identified (Dorph et al, Abstract ACR 2009). IL-1 is a multifunctional cytokine, and anakinra therapy could affect many cell types. The authors have here focused on its role in mediating T cell differentiation into Th17-like cells. The authors have here explored possible predictive biomarkers for anakinra therapy response, and investigated changes in effector T cell function and phenotype. Materials and methods 15 refractory myositis patients were included in a 12-month open-label study (100 mg anakinra per day). Serum creatine kinase (s-CK) was measured as a surrogate marker for clinical response. Peripheral blood samples were collected before and after 6 months. Serum levels of IL-1 receptor antagonist (IL-1Ra) were detected by a commercial ELISA, and T cells were studied by flow cytometry. To assess systemic immune activation, the proportions of naïve versus memory T cells were determined. The effector function of in vivo primed T cells was assessed by re-stimulating cells with anti-CD3 for 6 h and performing intracellular cytokine staining for interferon γ (IFNγ), tumour necrosis factor α and IL-17A. Results Before treatment the patients demonstrated elevated levels of serum IL-1Ra (n=14, mean=2559 pg/ml, range 467–6937 pg/ml) compared to healthy controls (mean=354 pg/ml, range 11–839 pg/ml, Son et al, Intern Med, 2000). 10/14 patients were still on anakinra therapy at 6 months and thus displayed significantly elevated IL-1Ra (mean=19 571 pg/ml, range 19 572–18 844 pg/ml, p=0.005). Viable cells from peripheral blood were available at baseline for eight patients and following 6 months of therapy for six subjects (three responders and three non-responders). When dissecting the pool of naïve versus activated/memory T cells, a strong negative correlation was seen between baseline level of CD4 CD45RO T cells and changes in CK levels after 6 months (R=−0.9, p=0.001). The levels of IFNγ-secreting CD4 T cells (mean=1.1%, range 0.08–4.96%) increased after 6 months, while IL-17 producing T cells (mean=0.16%, range 0.03–0.4%) decreased in 2/3 responders. Conclusions Patients with myositis may respond to anakinra treatment. This functional assay indicates that anakinra might favour T cell differentiation into Th1 rather than Th17 as indicated by more IFNγ and less IL-17A secretion. Furthermore, a high level of CD4 activated memory T cells might indicate a worse clinical outcome as demonstrated by less decreased CK levels.

A8.24 autophagy may contribute to glucocorticoid resistance in myositis patients by maitaining muscle T cells homeostasis

Background and Objectives Infiltrating T cells is a typical histopathologic feature in muscles of patients with myositis and they play important roles in the disease development. In contrast to macrophages which usually can be reduced extensively after glucocorticoid (GC) treatment, T cells often persist, and the reasons are still unclear. Autophagy helps cells to survive under variable cellular stresses. In this context the effects of endogenous, cytosolic high mobility group box 1 (HMGB1) protein is of interest as HMGB1 can induce autophagy by binding Beclin1 (an upstream protein initiating autophagy) and thereby contribute to cell survival. In this study, we will investigate whether autophagy initiated by HMGB1-Beclin1 binding can contribute to T cell survival in the muscles of patients with myositis, and whether these homeostatic T cells are related to GC treatment resistance. Materials and Methods Muscle biopsies were obtained from poly- and dermato- myositis patients with no or limited clinical response to GC and from patients with good response to GC. Biopsies were investigated by immunohistochemistry for macrophages (CD163, CD68), T cells (CD3), HMGB1 and Beclin1. Computer image analysis was performed for each marker. Co-localization of HMGB1, Beclin1 and T cells was done by consecutive section staining and was confirmed by double fluorescence staining. Results Both HMGB1 and Beclin1 expression was detected in muscle tissue of patients with myositis; furthermore, the expression co-localised to the infiltrating T cells as demonstrated by consecutive section staining and double fluorescence staining. Moreover, the expression of autophagy marker Beclin1 correlated with both HMGB1 and T cells (p<0.05). In nine patients who are good responders to GC, the number of T cells in the muscles was decreased after treatment, and simultaneously the HMGB1 and Beclin1 expression was decreased. Analyses are ongoing on the non-responders. According to our hypothesis in these patients T cells will not be reduced as much as good responders after treatment and HMGB1 and Beclin1 expression will maintain at high levels too. Conclusions Markers of autophagy are present in the invading T cells in muscle tissue of myositis patients. Autophagy initiated by HMGB1-Beclin1 binding may contribute to T cell survival in the muscles of patients with myositis. And this homeostasis in T cells could be a factor that contributes to the GC resistance.

The Role of Reactive Oxygen Species in β-Adrenergic Signaling in Cardiomyocytes from Mice with the Metabolic Syndrome

The metabolic syndrome is associated with prolonged stress and hyperactivity of the sympathetic nervous system and afflicted subjects are prone to develop cardiovascular disease. Under normal conditions, the cardiomyocyte response to acute β-adrenergic stimulation partly depends on increased production of reactive oxygen species (ROS). Here we investigated the interplay between beta-adrenergic signaling, ROS and cardiac contractility using freshly isolated cardiomyocytes and whole hearts from two mouse models with the metabolic syndrome (high-fat diet and ob/ob mice). We hypothesized that cardiomyocytes of mice with the metabolic syndrome would experience excessive ROS levels that trigger cellular dysfunctions. Fluorescent dyes and confocal microscopy were used to assess mitochondrial ROS production, cellular Ca2+ handling and contractile function in freshly isolated adult cardiomyocytes. Immunofluorescence, western blot and enzyme assay were used to study protein biochemistry. Unexpectedly, our results point towards decreased cardiac ROS signaling in a stable, chronic phase of the metabolic syndrome because: β-adrenergic-induced increases in the amplitude of intracellular Ca2+ signals were insensitive to antioxidant treatment; mitochondrial ROS production showed decreased basal rate and smaller response to β-adrenergic stimulation. Moreover, control hearts and hearts with the metabolic syndrome showed similar basal levels of ROS-mediated protein modification, but only control hearts showed increases after β-adrenergic stimulation. In conclusion, in contrast to the situation in control hearts, the cardiomyocyte response to acute β-adrenergic stimulation does not involve increased mitochondrial ROS production in a stable, chronic phase of the metabolic syndrome. This can be seen as a beneficial adaptation to prevent excessive ROS levels.

HMGB1 mediates muscle fatigue via TLR4 - a possible mechanism for muscle fatigue in patients with inflammatory myopathies

Backgroundand objectives Idiopathic inflammatory myopathies (IIMs) are chronic inflammatory diseases characterised by muscle weakness and the mechanisms are still unclear. High mobility group box protein 1 (HMGB1) is often found together with aberrant expression of major histocompatibility complex (MHC) class I in muscle fibres of patients with IIMs but not in healthy individuals. Exogenous HMGB1 can accelerate development of muscle fatigue and increase MHC-class I expression in adult mice skeletal muscle fibres. In other tissues it has been shown that HMGB1 could mediate functions via different receptors including the receptor for advanced glycation end products (RAGE) and Toll-like receptor 4 (TLR4). In this study, the authors set out to investigate whether HMGB1 contribute to increased muscle fatigue and MHC-class I expression in muscle fibres via RAGE or TLR4 in adult skeletal muscle fibres. Materials and methods Intact single fibres were dissociated from flexor digitorum brevis (FDB) of wild type (WT), RAGE knockout (RAGE-/-) and TLR4 knockout (TLR4-/-) mice and cultured in the absence or presence of 10 µg/ml recombinant HMGB1. Decrease in sarcoplasmic reticulum (SR) Ca2+ release, which reflects the development of muscle fatigue, was determined by measuring myoplasmic free tetanic Ca2+ ((Ca2+)i) during a series of 300 tetanic contractions. MHC-class I and TLR4 expression in FDB fibres was investigated by immunofluorescence and confocal microscopy. Immunohistochemistry was used to investigate TLR4, MHC-class I and myosin heavy chain expression in muscle fibres of patients with IIMs. Results Intact single muscle fibres from WT and RAGE-/- but not from TLR4-/- mice showed a greater decline in tetanic (Ca2+)i at the end of a series of 300 tetanic contractions when cultured with rHMGB1 compared to without rHMGB1; MHC-class I was detected on the membrane of single fibers from WT but not TLR4-/- mice when exposed to rHMGB1; TLR4 expression was detected on the sarcolemma of muscle fibres from WT mice. TLR4 and MHC-class I co-expression was observed in muscle fibres from patients with IIMs and this staining was essentially confined to skeletal type II fast twitch muscle fibers. Conclusions TLR4 mediates muscle fatigue and MHC-class I expression in differentiated skeletal muscle fibres after exposure to the alarmin HMGB1. TLR4, MHC-class I and HMGB1 co-localisation is frequently observed in muscle fibres of patients with IIMs, thus HMGB1-TLR4 pathway may play a role in causing muscle weakness in patients with IIMs and is thus a possible novel target for therapy in IIM patients.