C. Preusse

Anti-Jo-1 antibody-positive patients show a characteristic necrotizing perifascicular myositis

Idiopathic inflammatory myopathies can be classified as polymyositis, dermatomyositis, immune-mediated necrotizing myopathy, sporadic inclusion body myositis or non-specific myositis. Anti-Jo-1 antibody-positive patients are assigned to either polymyositis or dermatomyositis suggesting overlapping pathological features. We aimed to determine if anti-Jo-1 antibody-positive myopathy has a specific morphological phenotype. In a series of 53 muscle biopsies of anti-Jo-1 antibody-positive patients, relevant descriptive criteria defining a characteristic morphological pattern were identified. They were tested in a second series of anti-Jo-1 antibody-positive patients and compared to 63 biopsies from patients suffering from other idiopathic inflammatory myopathies. In anti-Jo-1 antibody-positive patients, necrotic fibres, which strongly clustered in perifascicular regions, were frequently observed. Sarcolemmal complement deposition was detected specifically in perifascicular areas. Inflammation was mainly located in the perimysium and around vessels in 90.6%. Perimysial fragmentation was observed in 90% of cases. Major histocompatibility complex class I staining was diffusely positive, with a perifascicular reinforcement. Multivariate analysis showed that criteria defining perifascicular pathology: perifascicular necrosis, atrophy, and perimysial fragmentation allow the distinction of anti-Jo-1 antibody-positive patients, among patients suffering from other idiopathic inflammatory myopathies. Anti-Jo-1 antibody-positive patients displayed perifascicular necrosis, whereas dermatomyositis patients exhibited perifascicular atrophy.

MIF Receptor CD74 is Restricted to Microglia/Macrophages, Associated with a M1‐Polarized Immune Milieu and Prolonged Patient Survival in Gliomas

The macrophage migration inhibitory factor (MIF) receptor CD74 is overexpressed in various neoplasms, mainly in hematologic tumors, and currently investigated in clinical studies. CD74 is quickly internalized and recycles after antibody binding, therefore it constitutes an attractive target for antibody‐based treatment strategies. CD74 has been further described as one of the most up‐regulated molecules in human glioblastomas. To assess the potential relevance for anti‐CD74 treatment, we determined the cellular source and clinicopathologic relevance of CD74 expression in human gliomas by immunohistochemistry, immunofluorescence, immunoblotting, cell sorting analysis and quantitative polymerase chain reaction (qPCR). Furthermore, we fractionated glioblastoma cells and glioma‐associated microglia/macrophages (GAMs) from primary tumors and compared CD74 expression in cellular fractions with whole tumor lysates. Our results show that CD74 is restricted to GAMs in vivo, while being absent in tumor cells, the latter strongly expressing its ligand MIF. Most interestingly, a higher amount of CD74‐positive GAMs was associated with beneficial patient survival constituting an independent prognostic parameter and with an anti‐tumoral M1 polarization. In summary, CD74 expression in human gliomas is restricted to GAMs and positively associated with patient survival. In conclusion, CD74 represents a positive prognostic marker most probably because of its association with an M1‐polarized immune milieu in high‐grade gliomas.

Pathogenic role of anti-signal recognition protein and anti-3-Hydroxy-3-methylglutaryl-CoA reductase antibodies in necrotizing myopathies: Myofiber atrophy and impairment of muscle regeneration in necrotizing autoimmune myopathies.

Immune‐mediated necrotizing myopathies (IMNM) may be associated with either anti–signal recognition protein (SRP) or anti–3‐hydroxy‐3‐methylglutaryl‐CoA reductase (HMGCR) antibodies (Abs), and the titer of these Abs is correlated with disease activity. We investigated whether anti‐SRP and anti‐HMGCR Abs could be involved in muscle damage.

Necrosis in anti-SRP+ and anti-HMGCR+myopathies: Role of autoantibodies and complement

Objective To characterize muscle fiber necrosis in immune-mediated necrotizing myopathies (IMNM) with anti–signal recognition particle (SRP) or anti–3-hydroxy-3-methylglutarylcoenzyme A reductase (HMGCR) antibodies and to explore its underlying molecular immune mechanisms. Methods Muscle biopsies from patients with IMNM were analyzed and compared to biopsies from control patients with myositis. In addition to immunostaining and reverse transcription PCR on muscle samples, in vitro immunostaining on primary muscle cells was performed. Results Creatine kinase levels and muscle regeneration correlated with the proportion of necrotic fibers (r = 0.6, p < 0.001). CD68+iNOS+ macrophages and a Th-1 immune environment were chiefly involved in ongoing myophagocytosis of necrotic fibers. T-cell densities correlated with necrosis but no signs of cytotoxicity were detected. Activation of the classical pathway of the complement cascade, accompanied by deposition of sarcolemmal immunoglobulins, featured involvement of humoral immunity. Presence of SRP and HMGCR proteins on altered myofibers was reproduced on myotubes exposed to purified patient-derived autoantibodies. Finally, a correlation between sarcolemmal complement deposits and fiber necrosis was observed (r = 0.4 and p = 0.004). Based on these observations, we propose to update the pathologic criteria of IMNM. Conclusion These data further corroborate the pathogenic role of anti-SRP and anti-HMGCR autoantibodies in IMNM, highlighting humoral mechanisms as key players in immunity and myofiber necrosis.

Th2-M2 immunity in lesions of muscular sarcoidosis and macrophagic myofasciitis.

To analyse the paradox of a lack of giant cell formation and fibrosis in chronic lesions of macrophagic myofasciitis (MMF) in comparison with muscular sarcoidosis (MuS).

JAK inhibitor improves type I interferon induced damage: proof of concept in dermatomyositis.

Dermatomyositis is an acquired auto-immune disease characterized by skin lesions and muscle-specific pathological features such as perifascicular muscle fibre atrophy and vasculopathy. Dermatomyositis patients display an upregulation of type I interferon-inducible genes in muscle fibres, endothelial cells, skin and peripheral blood. However, the effect of type I interferon on muscle tissue has not yet been determined. Our aim was to study the pathogenicity of type I interferon in vitro and to evaluate the efficacy of the type I interferon pathway blockade for therapeutic purposes. The activation of type I interferon in differentiating myoblasts abolished myotube formation with reduced myogenin expression while in differentiated myotubes, we observed a reduction in surface area and an upregulation of atrophy-associated genes. In vitro endothelial cells exposure to type I interferon disrupted vascular network organization. All the pathogenic effects observed in vitro were abolished by ruxolitinib. Finally, four refractory dermatomyositis patients were treated with ruxolitinib and improvement ensued in skin lesions, muscle weakness and a reduced serum type I interferon levels and interferon-inducbile genes scores. We propose JAK inhibition as a mechanism-based treatment for dermatomyositis, a finding that is relevant for the design of future clinical trials targeting dermatomyositis.

C5b-9 deposits on endomysial capillaries in non-dermatomyositis cases

Deposits of the terminal-membrane-attack-complex (MAC) C5b-9 on perfascicular endomysial capillaries are generally regarded as diagnostic hallmark of dermatomyositis (DM). Although the pathophysiology is not clear, C5b-9 deposits on capillaries seem to be associated with microinfarctions and vascular damage. Here, we report on a series of 19 patients presenting with C5b-9 accumulation on endomysial capillaries in the absence of features for DM. To decipher differences in the capillary C5b-9 accumulation pattern between DM and non-DM cases, we assessed the extent of endomysial capillary C5b-9 deposits related to capillary density and extent of myofiber necrosis by immunohistochemistry in 12 DM and 8 control patients. We found similar numbers of C5b-9-positive myofibers in both DM and non-DM C5b-9(+) cases. The distribution pattern differed as DM cases showed significantly more perifascicular capillary C5b-9 deposits as compared to non-DM cases, which presented stronger endomysial capillary C5b-9 deposits in a diffuse pattern. While total capillary density was not differing, DM patients displayed significantly more C5b-9(+) necrotic fibers as compared to non-DM C5b-9(+). In summary, endomysial capillary C5b-9 deposits are present in a variety of non-DM cases, however with differing distribution pattern. In conclusion, capillary C5b-9(+) deposits should be assessed critically, taking into consideration the distribution pattern.

P.21.3 Skeletal muscle provides a permissive environment for Th2-M2 polarisation in neuromuscular sarcoidosis

Neuromuscular involvement may affect more then 60% of patients suffering from sarcoidosis. We have recently described that macrophages and giant cells in skeletal muscle exhibit an unexpected status of alternative activation (M2). Objective: The intrinsic immune signature of the granulomas, was compared to the cytokine profile of adjacent non-inflamed muscle tissue and to healthy control muscle. Granulomas and contiguous muscle from 9 patients with biopsy-proven neuromuscular sarcoidosis were cut out by laser capture microdissection (LCM). Markers and activators of the T helper cell 1 (Th1) – classical macrophage activation (M1) and Th2 – alternatively activated (M2) immune response as well as molecules involved in giant cell development were assessed by real-time PCR. STAT-6-induced Th2 immunity leads to upregulated expression of CD206 and SOCS1 in the granuloma in comparison to adjacent tissue. DAP12 and RAC1, genes that regulate giant cell formation, are significantly induced in the granulomas. Conversely, STAT-1-induced Th1 immunity, IFN γ and CXCR3 are expressed in the granulomas and the surrounding tissue at elevated levels, however without statistical differences. While Th1-mediated immunity is upregulated in the whole inflamed muscle specimen, Th2-M2 markers are expressed at significantly higher levels in the granulomata. These results indicate that muscle tissue per se may provide a permissive environment for M2 polarisation in neuromuscular sarcoidosis.

P.21.2 New insights into eosinophilic fasciitis

Eosinophilic fasciitis (EF), first described by Shulman in 1974, is a rare disease characterized by fibrosis and inflammatory infiltration of the muscle fascia as well as scleroderma-like skin indurations and blood eosinophilia. In contrast to other inflammatory myopathies, patients generally show less muscle weakness and myalgia, and a frequent increase in body weight. Thus, we hypothesize a unique immune mechanism underlying Shulman syndrome. The immunohistochemical expression pattern of leucocytes and a comprehensive panel of cytokine and chemokine expression on RNA level of muscle specimen from EF patients were compared to healthy control muscle. In patients with biopsy-proven EF the immune phenotype of inflammatory cells was determined and RNA from fascia and adjacent muscle was isolated to assess activators of the T helper cell 1 (Th1) – classical macrophage activation (M1) and Th2–M2 immune response by real-time PCR. The inflammatory infiltrate located at the muscle-fascia interface is mainly composed of CD206+ M2 macrophages with focal accumulations of CD4+ and CD8+ T cells while B cells and plasma cells are rare. Eosinophils could be detected in all of the EF cases. MHC class I is upregulated diffusely while MHC class II expression is pronounced in the perifascicular region accompanied by a perifascicular atrophy of muscle fibers, comparable to Dermatomyositis. Activators of the Th2–M2 pathway like STAT6 and IL-4 are upregulated leading to higher expression levels of CD206. Activators of the Th1–M1 pathway like STAT1 and interferon-a are also upregulated, however, they do not translate into a significant upregulation of M1 markers. We could show a specific immune phenotype of leucocyte infiltrates in Shulman syndrome with a mixed Th1/Th2 phenotype that resulted in a dominant M2 immune response.

P.20.3 Targeting fibrosis and inflammation in Duchenne Muscular Dystrophy

Duchenne Muscular Dystrophy is the most frequent genetic muscle disease worldwide affecting ∼1:5000 male births. It is caused by a defective DMD gene, which leads to reduced and defective dystrophin protein expression. The constant breakdown of fibres leads to focal necrosis, myophagocytosis and a considerable influx of inflammatory cells into the muscle tissue, which is followed by increasing endomysial fibrosis. Both, inflammation and fibrosis as well as a putative relation are not yet understood immunologically. Fibrosis directly correlates with adverse outcome and early loss of ambulation. We have studied how inflammation is linked to fibrosis in DMD, with an emphasis on the communication between fibroblasts, macrophages and the immune response, especially Th2-immunity, at different time points of disease and identified target molecules involved in this process. Classically activated M1 macrophages exhibit a pro-inflammatory phenotype, while alternatively activated (M2) macrophages are profibrotic and are therefore thought to exhibit a ‘repair phenotype’ but may be deleterious at certain stages of the disease in DMD. We have analysed muscle biopsies derived from patients suffering from DMD, which were obtained at different time-points after onset of disease. The immune response was studied on the protein and on the mRNA levels. Depending on the time-point and disease activity, the immune response showed a Th1-M1 or Th2-M2 phenotype respectively. Increasing fibrosis was associated with an M2 polarized immune milieu. The results of this study may provide a basis for the development of a specifically targeted and putatively time-dependent immune intervention in DMD patients, based on the immune profile of their muscle biopsy specimen, which can be systematically and individually assessed. This approach may provide a useful additional therapeutic intervention in addition to modern gene-therapeutic approaches.