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Objectives EF is a rare disease characterized by fibrosis and inflammation of the fascia, scleroderma-like skin indurations and optional blood eosinophilia. We aimed to expand the knowledge about its aetiology and pathogenesis. Methods Biopsy specimens from 16 EF patients were assessed by histology, immunohistochemistry and quantitative reverse transcription PCR in comparison with anti-Mi-2+ DM patients and non-disease controls. Results Histologically, EF shows mild to severe inflammation at the muscle–fascia interface, with frequent involvement of the underlying muscle tissue, though varying in degree. CD206+ macrophages predominate and eosinophils are detected within the fascia in the majority of cases, however in quite small numbers, and seen infrequently within the muscle. Activators of the so-called Th2–M2 pathway like STAT6 and IL-4 are upregulated leading to high expression levels of CD206. Activators of the so-called Th1–M1 pathway like STAT1 and IFN-γ (IFNG) are also upregulated, though not translating into a significant upregulation of the effector molecule COX2. Interestingly, activators or chemoattractants of eosinophils show no significant upregulation in EF compared with DM. EF shows features of perifascicular pathology comparable to DM, with upregulation of MHC class I and II; however, this is not accompanied by perifascicular atrophy or any signs of a type I IFN response or hypoxia-mediated processes. Conclusions Our findings highlight a specific immune phenotype of leucocyte infiltrates in EF along features of perifascicular pathology similar to DM, while there is no evidence of hypoxia-mediated or type I IFN–associated processes with perifascicular fibre atrophy, indicating different pathomechanisms of muscle involvement.

The SARS-CoV-2 pandemic not only resulted in millions of acute infections worldwide, but also caused innumerable cases of post-infectious syndromes, colloquially referred to as “long COVID”. Due to the heterogeneous nature of symptoms and scarcity of available tissue samples, little is known about the underlying mechanisms. We present an in-depth analysis of skeletal muscle biopsies obtained from eleven patients suffering from enduring fatigue and post-exertional malaise after an infection with SARS-CoV-2. Compared to two independent historical control cohorts, patients with post-COVID exertion intolerance had fewer capillaries, thicker capillary basement membranes and increased numbers of CD169+macrophages. SARS-CoV-2 RNA could not be detected in the muscle tissues, but transcriptomic analysis revealed distinct gene signatures compared to the two control cohorts, indicating immune dysregulations and altered metabolic pathways. We hypothesize that the initial viral infection may have caused immune-mediated structural changes of the microvasculature, potentially explaining the exercise-dependent fatigue and muscle pain.

The original version of this article unfortunately contained mistake. One of the author name was missspelled during revision and finalization of the manuscript. Dr. Kiran Polavarapu's surname "Polaparapu" should be spelled "Polavarapu". The original article has been corrected.

Background: In juvenile idiopathic inflammatory myopathies (IIMs), morphological characteristic features of distinct subgroups are not well defined. New treatment strategies require a precise diagnosis of the subgroups in IIM, and, therefore, knowledge about the pathomorphology of juvenile IIMs is warranted. Methods: Muscle biopsies from 15 patients (median age 8 (range 3–17) years, 73% female) with IIM and seven controls were analyzed by standard methods, immunohistochemistry, and transmission electron microscopy (TEM). Detailed clinical and laboratory data were accessed retrospectively. Results: Proximal muscle weakness and skin symptoms were the main clinical symptoms. Dermatomyositis (DM) was diagnosed in 9/15, antisynthetase syndrome (ASyS) in 4/15, and overlap myositis (OM) in 2/15. Analysis of skeletal muscle tissues showed inflammatory cells and diffuse upregulation of MHC class I in all subtypes. Morphological key findings were COX-deficient fibers as a striking pathology in DM and perimysial alkaline phosphatase positivity in anti-Jo-1-ASyS. Vascular staining of the type 1 IFN-surrogate marker, MxA, correlated with endothelial tubuloreticular inclusions in both groups. None of these specific morphological findings were present in anti-PL7-ASyS or OM patients. Conclusions: Morphological characteristics discriminate IIM subtypes in juvenile patients, emphasizing differences in aetiopathogenesis and supporting the notion of individual and targeted therapeutic strategies.

Background There have been numerous classification systems to diagnose corresponding myositis subtypes and select appropriate therapeutic measures. However, the lack of a broad consensus on diagnostic criteria has led to clinical uncertainties. The objective of this study was to compare two commonly used dermatomyositis-classification systems regarding their clinical practicability and to point out their specific advantages and disadvantages. Methods This study included 30 patients diagnosed with dermatomyositis at the Charité university hospital, Berlin, Germany from 2010 to 2017. Patient files with complete data and defined historical classifications were enrolled and ENMC (2003) and EULAR/ACR (2017) criteria retrospectively applied. Results According to the ENMC approach, 14 patients were classified as "definite" and 12 as "probable" dermatomyositis. One patient exhibited an "amyopathic dermatomyositis" and three a "DM without dermatitis". Regarding the criteria probability of the EULAR/ACR set, 16 patients had a "high", 13 a "medium" and one a "low probability". There was a significant difference (p = 0.004) between the subclasses of the ENMC in relation to the EULAR/ACR score. The agreement between the classification probabilities of "definite/high" (κ = 0.400) and "possible/medium" (κ = 0.324) was fair. Conclusions It is important to find a consensus among the medical disciplines involved and to establish a structured procedure. Future studies with newer approaches are warranted to conclusively decide which system to use for the physician.

Systemic sclerosis represents a chronic connective tissue disease featuring fibrosis, vasculopathy and autoimmunity, affecting skin, multiple internal organs, and skeletal muscles. The vasculopathy is considered obliterative, but its pathogenesis is still poorly understood. This may partially be due to limitations of conventional transmission electron microscopy previously being conducted only in single patients. The aim of our study was therefore to precisely characterize immune inflammatory features and capillary morphology of systemic sclerosis patients suffering from muscle weakness. In this study, we identified 18 individuals who underwent muscle biopsy because of muscle weakness and myalgia in a cohort of 367 systemic sclerosis patients. We performed detailed conventional and immunohistochemical analysis and large-scale electron microscopy by digitizing entire sections for in-depth ultrastructural analysis. Muscle biopsies of 12 of these 18 patients (67%) presented minimal features of myositis but clear capillary alteration, which we termed minimal myositis with capillary pathology (MMCP). Our study provides novel findings in systemic sclerosis-associated myositis. First, we identified a characteristic and specific morphological pattern termed MMCP in 67% of the cases, while the other 33% feature alterations characteristic of other overlap syndromes. This is also reflected by a relatively homogeneous clinical picture among MMCP patients. They have milder disease with little muscle weakness and a low prevalence of interstitial lung disease (20%) and diffuse skin involvement (10%) and no cases of either pulmonary arterial hypertension or renal crisis. Second, large-scale electron microscopy, introducing a new level of precision in ultrastructural analysis, revealed a characteristic capillary morphology with basement membrane thickening and reduplications, endothelial activation and pericyte proliferation. We provide open-access pan-and-zoom analysis to our datasets, enabling critical discussion and data mining. We clearly highlight characteristic capillary pathology in skeletal muscles of systemic sclerosis patients.

Genes 13(5), 893 (2022). doi:10.3390/genes13050893 special issue: "Special Issue "Genetics of Motor Neuron Diseases" / Special Issue Editor: Christopher Grunseich, Guest Editor", Published by MDPI, Basel

Objective:To characterize morphological and molecular underpinnings of polymyositis with mitochondrial pathology (PM-Mito) in comparison to sporadic inclusion body myositis (IBM) and to define common and distinct pathophysiological features with a focus on interferon-associated inflammation and T-cell-response.Methods:In this cross-sectional study, skeletal muscle biopsy samples as well as clinical and laboratory data from PM-Mito and IBM patients were analyzed at Charité university hospital in Berlin, Germany. All available PM-Mito biopsy samples and an equal number of randomly selected IBM biopsy samples were included in the study as well as randomly selected non-diseased controls (NDC). Biopsy samples were studied by histopathology, immunohistochemistry, and quantitative PCR and compared to biopsies derived from NDC. Primary outcomes included cell counts for immunohistochemistry, and gene expression (fold-change values compared to NDCs) for quantitative PCR.Results:Twenty-five skeletal muscle biopsy samples of patients with PM-Mito and IBM were included in the study and compared to five biopsy samples from non-diseased controls. PM-Mito and IBM qualitatively harbored a strikingly similar molecular signature and shared important histopathological features. Expression of interferon-induced GBP6 and T-cell function-related KLRG1 distinguished IBM from PM-Mito biopsies with IBM patients showing significantly higher expression of GBP6 and KLRG1. Cryptic exon expression was detected in both patient groups with IBM patients showing higher expression levels. Skeletal muscle biopsies from IBM patients showed significantly more GBP6+ cells and KLRG1+ lymphocytes in comparison to biopsies from PM-Mito patients. CD45+, CD68+, CD57+, PD1+ and CD8+ cytotoxic T-cells were also significantly more abundant in IBM. Clinically, PM-Mito patients presented with a spectrum of muscle-related symptoms including myalgia, proximal paraparesis, proximal tetraparesis and incomplete IBM-like patterns. 13 out of 14 (93%) PM-Mito patients for whom clinical follow-up was available later developed clinically defined IBM. Notably, two follow-up biopsies obtained 5 and 7 years after the first ones were available in this cohort, both showing histopathological progress to net IBM including GBP6 and KLRG1 upregulation.Conclusions:Our combined data suggest that specific interferon-mediated inflammation plays a key role in both IBM and PM-Mito. GBP6 was identified as a new molecule of type II interferon-induced inflammation distinguishing IBM from PM-Mito. Skeletal muscles from both groups harbor dysfunctional T-cells of similar type, albeit in different quantity. T-cell senescence exemplified by KLRG1-positivity does not play a significant role in PM-Mito. Based on these findings, we propose to include PM-Mito in the spectrum of IBM (IBM-spectrum disease, IBM-SD) as a possible early form of this disease. The establishment of IBM-SD as a larger entity could potentially have a significant impact on the design of trials and therapeutic interventions.

Patients suffering from immune-mediated necrotizing myopathies (IMNM) harbor, the pathognomonic myositis-specific auto-antibodies anti-SRP54 or -HMGCR, while about one third of them do not. Activation of chaperone-assisted autophagy was described as being part of the molecular etiology of IMNM. Endoplasmic reticulum (ER)/sarcoplasmic reticulum (SR)-stress accompanied by activation of the unfolded protein response (UPR) often precedes activation of the protein clearance machinery and represents a cellular defense mechanism toward restoration of proteostasis. Here, we show that ER/SR-stress may be part of the molecular etiology of IMNM. To address this assumption, ER/SR-stress related key players covering the three known branches (PERK-mediated, IRE1-mediated, and ATF6-mediated) were investigated on both, the transcript and the protein levels utilizing 39 muscle biopsy specimens derived from IMNM-patients. Our results demonstrate an activation of all three UPR-branches in IMNM, which most likely precedes the activation of the protein clearance machinery. In detail, we identified increased phosphorylation of PERK and eIF2a along with increased expression and protein abundance of ATF4, all well-documented characteristics for the activation of the UPR. Further, we identified increased general XBP1-level, and elevated XBP1 protein levels. Additionally, our transcript studies revealed an increased ATF6-expression, which was confirmed by immunostaining studies indicating a myonuclear translocation of the cleaved ATF6-form toward the forced transcription of UPR-related chaperones. In accordance with that, our data demonstrate an increase of downstream factors including ER/SR co-chaperones and chaperones (e.g., SIL1) indicating an UPR-activation on a broader level with no significant differences between seropositive and seronegative patients. Taken together, one might assume that UPR-activation within muscle fibers might not only serve to restore protein homeostasis, but also enhance sarcolemmal presentation of proteins crucial for attracting immune cells. Since modulation of ER-stress and UPR via application of chemical chaperones became a promising therapeutic treatment approach, our findings might represent the starting point for new interventional concepts. CA extern

Anti-synthetase syndrome (ASyS)-associated myositis is a major subgroup of the idiopathic inflammatory myopathies (IIM) and is characterized by disease chronicity with musculoskeletal, dermatological and pulmonary manifestations. One of eight autoantibodies against the aminoacyl-transferase RNA synthetases (ARS) is detectable in the serum of affected patients. However, disease-specific therapeutic approaches have not yet been established.To obtain a deeper understanding of the underlying pathogenesis and to identify putative therapeutic targets, we comparatively investigated the most common forms of ASyS associated with anti-PL-7, anti-PL-12 and anti-Jo-1. Our cohort consisted of 80 ASyS patients as well as healthy controls (n = 40), diseased controls (n = 40) and non-diseased controls (n = 20). We detected a reduced extent of necrosis and regeneration in muscle biopsies from PL-12+ patients compared to Jo-1+ patients, while PL-7+ patients had higher capillary dropout in biopsies of skeletal muscle. Aside from these subtle alterations, no significant differences between ASyS subgroups were observed. Interestingly, a tissue-specific subpopulation of CD138+ plasma cells and CXCL12+/CXCL13+CD20+ B cells common to ASyS myositis were identified. These cells were localized in the endomysium associated with alkaline phosphatase+ activated mesenchymal fibroblasts and CD68+MHC-II+CD169+ macrophages. An MHC-I+ and MHC-II+ MxA negative type II interferon-driven milieu of myofiber activation, topographically restricted to the perifascicular area and the adjacent perimysium, as well as perimysial clusters of T follicular helper cells defined an extra-medullary immunological niche for plasma cells and activated B cells. Consistent with this, proteomic analyses of muscle tissues from ASyS patients demonstrated alterations in antigen processing and presentation. In-depth immunological analyses of peripheral blood supported a B-cell/plasma-cell-driven pathology with a shift towards immature B cells, an increase of B-cell-related cytokines and chemokines, and activation of the complement system. We hypothesize that a B-cell-driven pathology with the presence and persistence of a specific subtype of plasma cells in the skeletal muscle is crucially involved in the self-perpetuating chronicity of ASyS myositis. This work provides the conceptual framework for the application of plasma-cell-targeting therapies in ASyS myositis.

Background: Pulmonary affection (PA) is associated with a substantial increase in morbidity and mortality in patients with idiopathic inflammatory myopathies (IIM). However, the underlying immune mechanisms of PA remain enigmatic and prompt deeper immunological analyses. Importantly, the Janus-faced role of natural killer (NK) cells, capable of pro-inflammatory as well as regulatory effects, might be of interest for the pathophysiologic understanding of PA in IIM. Methods: To extend our understanding of immunological alterations in IIM patients with PA, we compared the signatures of NK cells in peripheral blood using multi-color flow cytometry in IIM patients with (n = 12, of which anti-synthetase syndrome = 8 and dermatomyositis = 4) or without PA (n = 12). Results: We did not observe any significant differences for B cells, CD4, and CD8 T cells, while total NK cell numbers in IIM patients with PA were reduced compared to non-PA patients. NK cell alterations were driven by a particular decrease of CD56dim NK cells, while CD56bright NK cells remained unchanged. Comparisons of the cell surface expression of a large panel of NK receptors revealed an increased mean fluorescence intensity of NKG2D+ on NK cells from patients with PA compared with non-PA patients, especially on the CD56dim subset. NKG2D+ and NKp46+ cell surface levels were associated with reduced vital capacity, serving as a surrogate marker for clinical severity of PA. Conclusion: Our data illustrate that PA in IIM is associated with alterations of the NK cell repertoire, suggesting a relevant contribution of NK cells in certain IIMs, which might pave the way for NK cell-targeted therapeutic approaches.

Aims Variable degrees of inflammation, necrosis, regeneration and fibrofatty replacement are part of the pathological spectrum of the dystrophic process in alpha dystroglycanopathy LGMDR9 (FKRP-related, OMIM #607155), one of the most prevailing types of LGMDs worldwide. Inflammatory processes and their complex interplay with vascular, myogenic and mesenchymal cells may have a major impact on disease development. The purpose of our study is to describe the specific immune morphological features in muscle tissue of patients with LGMDR9 in order to enable a better understanding of the phenotype of muscle damage leading to disease progression. Methods We have analysed skeletal muscle biopsies of 17 patients genetically confirmed as having LGMDR9 by histopathological and molecular techniques. Results We identified CD206+ MHC class II+ and STAT6+ immune-repressed macrophages dominating the endomysial infiltrate in areas of myofibre regeneration and fibrosis. Additionally, PDGFRβ+ pericytes were located around MHC class II+ activated capillaries residing in close proximity to areas of fibrosis and regenerating fibres. Expression of VEGF was found on many regenerating neonatal myosin+ fibres myofibres and CD206+ macrophages also co-expressed VEGF. Conclusion Our results show characteristic immune inflammatory features in LGMDR9 and more specifically shed light on the predominant role of macrophages and their function in vascular organization, fibrosis and myogenesis. Understanding disease-specific immune phenomena potentially inform about possibilities for anti-fibrotic and anti-inflammatory therapeutic strategies, which may complement Ribitol replacement and gene therapies for LGMDR9 that may be available in the future.

International audience; Recent data suggest the implication of T, B and NK cells in the anti-synthetase syndrome (ASyS); nevertheless their role and activation states are poorly described. We performed deep immune-profiling using 37 markers on peripheral blood cells from 10 ASyS patients versus 17 healthy donors (HD) and 26 myositis control patients. We show decreased percentages of memory B cells in ASyS patients (mean ± SEM: ASyS = 13 ± 3%, HD = 37 ± 4% and 'myositis controls' = 32 ± 3), counterbalanced by increased percentages of naïve B cells. Interestingly, perifascicular infiltrations of memory B cells within muscle biopsies of ASyS patients suggest that they niche within the muscle.

International audience; Diffuse myofiber necrosis in the context of inflammatory myopathy is the hallmark of immune-mediated necrotizing myopathy (IMNM). We have previously shown that skeletal muscle fibers of IMNM patients may display nonrimmed vacuoles and sarcoplasmic irregularities. The dysfunctional chaperone activity has been linked to the defective assembly of skeletal muscle proteins and their degradation via lysosomes, autophagy and the proteasomal machinery. This study was undertaken to highlight a chaperone-assisted selective autophagy (CASA) pathway, functionally involved in protein homeostasis, cell stress and the immune response in skeletal muscle of IMNM patients. Skeletal muscle biopsies from 54 IMNM patients were analyzed by immunostaining, as well as by qPCR. Eight biopsies of sIBM patients served as pathological controls, and eight biopsies of nondisease control subjects were included. Alteration of autophagy was detectable in all IMNM biopsy samples highlighted via a diffuse sarcoplasmic staining pattern by p62 and LC3 independent of vacuoles. This pattern was at variance with the coarse focal staining pattern mostly confined to rimmed vacuoles in sIBM. Colocalization of p62 with the chaperone proteins HSP70 and αB-crystalline points to the specific targeting of misfolded proteins to the CASA machinery. Bcl2-associated athanogene 3 (BAG3) positivity of these fibers emphasizes the selectivity of autophagy processes and these fibers also express MHC class I sarcolemma. Expression of genes involved in autophagy and endoplasmic reticulum (ER) stress pathways studied here is significantly upregulated in IMNM. We highlight that vacuoles without sarcolemmal features may arise in IMNM muscle biopsies, and they must not be confounded with sIBM-specific vacuoles. Further, we show the activation of selective autophagy and emphasize the role of chaperones in this context. CASA occurs in IMNM muscle, and specific molecular pathways of autophagy differ from the ones in sIBM, with p62 as a unique identifier of this process.

Female carriers of DMD gene mutations may be symptomatic and show variable skeletal as well as cardiac muscle symptoms. Skeletal muscle can exhibit morphological alterations. However, inflammatory, degenerative and fibrotic changes as seen in Duchenne boys have not been specifically analysed yet, so we addressed the question whether skeletal muscle of female carriers show such alterations. Thirteen carriers with an age range of 3 to 50 years were studied retrospectively. Five out of 13 women had clinically affected relatives. Clinically, most women showed mild muscle weakness, while the CK levels were increased in nine of them. Histomorphological analyses highlighted the typical mosaic pattern of dystrophin-positive and -negative fibres. Regenerating fibres were diffusely scattered and focally pronounced, while endo- and perimysial fibrosis was a variable but constant feature. Infiltration of CD206+TGFs+ macrophages and scattered T cells was noted in the endomysium. TGFb and CCL18, were significantly increased. However, gene expression of markers involved in Th1/Th2 immunity did not reach statistical significance compared to non-diseased controls. In summary, skeletal muscle of clinically manifest female DMD gene mutation carriers shows mild fibrosis and increased regeneration associated with endomysial CD206+TGFβ+ and STAT6+ macrophages, which are most likely involved in fibrotic remodelling.

// Tobias Ruck 1 , Stefan Bittner 2 , Ali Maisam Afzali 1 , Kerstin Gobel 1 , Sarah Glumm 1 , Peter Kraft 3 , Claudia Sommer 3 , Christoph Kleinschnitz 3 , Corinna Preuse 4 , Werner Stenzel 4 , Heinz Wiendl 1,* and Sven G. Meuth 1,* 1 Department of Neurology, University of Muenster, Muenster, Germany 2 Department of Neurology, University Medical Center of the Johannes Gutenberg-University Mainz, Mainz, Germany 3 Department of Neurology, University Hospital of Wuerzburg, Wuerzburg, Germany 4 Department of Neuropathology, Charite-Universitatsmedizin, Berlin, Germany * These authors have contributed equally to this work Correspondence to: Tobias Ruck, email: // Keywords : NKG2D, IL-15, polymyositis, idiopathic inflammatory myopathies, T cell activation, Pathology Section Received : June 08, 2015 Accepted : November 14, 2015 Published : December 04, 2015 Abstract NKG2D is an activating receptor on T cells, which has been implicated in the pathogenesis of autoimmune diseases. T cells are critically involved in idiopathic inflammatory myopathies (IIM) and have been proposed as specific therapeutic targets. However, the mechanisms underlying T cell-mediated progressive muscle destruction in IIM remain to be elucidated. We here determined the involvement of the NKG2D – IL-15 signaling pathway. Primary human myoblasts expressed NKG2D ligands, which were further upregulated upon inflammatory stimuli. In parallel, shedding of the soluble NKG2D ligand MICA (sMICA) decreased upon inflammation potentially diminishing inhibition of NKG2D signaling. Membrane-related expression of IL-15 by myoblasts induced differentiation of naive CD8 + T cells into highly activated, cytotoxic CD8 + NKG2D high T cells demonstrating NKG2D-dependent lysis of myoblasts in vitro . CD8 + NKG2D high T cell frequencies were increased in the peripheral blood of polymyositis (PM) patients and correlated with serum creatinine kinase concentrations, while serum sMICA levels were not significantly changed. In muscle biopsy specimens from PM patients expression of the NKG2D ligand MICA/B was upregulated, IL-15 was expressed by muscle cells, CD68 + macrophages as well as CD4 + T cells, and CD8 + NKG2D + cells were frequently detected within inflammatory infiltrates arguing for a local signaling circuit in the inflammatory muscle milieu. In conclusion, the NKG2D – IL-15 signaling pathway contributes to progressive muscle destruction in IIM potentially opening new therapeutic avenues.

ObjectiveTo study the B-cell content, organization, and existence of distinct B-cell subpopulations in relation to the expression of type 1 interferon signature related genes in dermatomyositis (DM).MethodsEvaluation of skeletal muscle biopsies from patients with adult DM (aDM) and juvenile DM (jDM) by histology, immunohistochemistry, electron microscopy, and quantitative reverse-transcription PCR.ResultsWe defined 3 aDM subgroups—classic (containing occasional B cells without clusters), B-cell–rich, and follicle-like aDM—further elucidating IM B-lymphocyte maturation and immunity. The quantity of B cells and formation of ectopic lymphoid structures in a subset of patients with aDM were associated with a specific profile of cytokines and chemokines involved in lymphoid neogenesis. Levels of type 1 interferon signature related gene expression paralleled B-cell content and architectural organization and link B-cell immunity to the interferon type I signature.ConclusionThese data corroborate the important role of B cells in DM, highlighting the direct link between humoral mechanisms as key players in B-cell immunity and the role of type I interferon–related immunity.

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.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.Creatine kinase levels and muscle regeneration correlated with the proportion of necrotic fibers (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.

We describe a patient with a clinically atypical presentation of inflammatory myopathy with abundant macrophages (IMAM) but with convincing muscle biopsy features of this subform of inflammatory myopathy. IMAM is characterized mainly by a conspicuous infiltration of muscle and connective tissue by numerous macrophages remote from necrotic and basophilic regenerating muscle fibers. Typically few, mostly CD4(+) T helper (Th) cells are also present. Here, we report a patient with IMAM and demonstrate, that most macrophages express the macrophage mannose receptor 1 (CD206) corresponding to alternatively activated (M2) polarization. Accordingly, signal transducer and activator of transcription 6 (STAT6), involved in Th2-M2 immunity, was expressed at high levels in skeletal muscle. However, TNFα, IFNγ and STAT1, mediators of the T helper 1-classically activated (M1) response were elevated in skeletal muscle and in blood, while expression of CD206 was elevated in skeletal muscle only. Our results argue that IMAM could be a distinct entity between the inflammatory myopathies rather than a subform of dermatomyositis.

Dermatomyositis (DM) can occur in both adults and juveniles with considerable clinical differences. The links between immune-mediated mechanisms and vasculopathy with respect to development of perifascicular pathology are incompletely understood. We investigated skeletal muscle from newly diagnosed, treatment-naïve juvenile (jDM) and adult dermatomyositis (aDM) patients focusing on hypoxia-related pathomechanisms, vessel pathology, and immune mechanisms especially in the perifascicular region. Therefore, we assessed the skeletal muscle biopsies from 21 aDM, and 15 jDM patients by immunohistochemistry and electron microscopy. Transcriptional analyses of genes involved in hypoxia, as well as in innate and adaptive immunity were performed by quantitative Polymerase chain reaction (qPCR) of whole tissue cross sections including perifascicular muscle fibers. Through these analysis, we found that basic features of DM, like perifascicular atrophy and inflammatory infiltrates, were present at similar levels in jDM and aDM patients. However, jDM was characterized by predominantly hypoxia-driven pathology in perifascicular small fibers and by macrophages expressing markers of hypoxia. A more pronounced regional loss of capillaries, but no relevant activation of type-1 Interferon (IFN)-associated pathways was noted. Conversely, in aDM, IFN-related genes were expressed at significantly elevated levels, and Interferon-stimulated gene (ISG)15 was strongly positive in small perifascicular fibers whereas hypoxia-related mechanisms did not play a significant role. In our study we could provide new molecular data suggesting a conspicuous pathophysiological ‘dichotomy’ between jDM and aDM: In jDM, perifascicular atrophy is tightly linked to hypoxia-related pathology, and poorly to innate immunity. In aDM, perifascicular atrophy is prominently associated with molecules driving innate immunity, while hypoxia-related mechanisms seem to be less relevant. Electronic supplementary material The online version of this article (doi:10.1186/s40478-016-0308-5) contains supplementary material, which is available to authorized users.

The anti-melanoma differentiation-associated gene 5 (MDA5) autoantibody is specifically associated with dermatomyositis (DM). Nevertheless, anti-MDA5(+)-patients experience characteristic symptoms distinct from classic DM, including severe signs of extramuscular involvement; however, the clinical signs of myopathy are mild or even absent. The morphological and immunological features are not yet described in adulthood. Data concerning the pathophysiology of anti-MDA5 DM are sparse; however, the importance of the interferon (IFN) type I pathway involved in DM has been shown. Our aim was to define morphological alterations of the skeletal muscle and the intrinsic immune response of anti-MDA5-positive DM patients. Immunohistological and RT-PCR analysis of muscle biopsy specimens from anti-MDA5 and classic DM were compared. Those with anti-MDA5 DM did not present the classic features of perifascicular fiber atrophy and major histocompatibility complex class I expression. They did not show significant signs of capillary loss; tubuloreticular formations were observed less frequently. Inflammation was focal, clustering around single vessels but significantly less intense. Expression of IFN-stimulated genes was up-regulated in anti-MDA5 DM; however, the IFN score was significantly lower. Characteristic features were observed in anti-MDA5 DM and not in classic DM patients. Only anti-MDA5 DM showed numerous nitric oxide synthase 2-positive muscle fibers with sarcoplasmic colocalization of markers of regeneration and cell stress. Anti-MDA5-positive patients demonstrate a morphological pattern distinct from classic DM.

Objective: To analyze antisynthetase syndrome–associated myositis by modern myopathologic methods and to define its place in the spectrum of idiopathic inflammatory myopathies (IIMs). Methods: Skeletal muscle biopsies from antisynthetase syndrome–associated myositis and other IIMs from different institutions worldwide were analyzed by histopathology, quantitative PCR, and electron microscopy. Results: Myonuclear actin filament inclusions were identified as a unique morphologic hallmark of antisynthetase syndrome–associated myositis. Nuclear actin inclusions were never found in dermatomyositis, polymyositis, sporadic inclusion body myositis, autoimmune necrotizing myopathy associated with signal recognition particle or 3-hydroxy-3-methylglutaryl-coenzyme A reductase autoantibodies, or nonspecific myositis associated with other systemic diseases, harboring myositis-associated autoantibodies, and presenting myofiber necrosis. We show that molecules involved in actin filament formation and actin shuttling mechanisms are altered in antisynthetase syndrome, and may thus be involved in pathologic myonuclear actin aggregation. In addition, we have identified a typical topographic distribution of necrotic myofibers predominantly located at the periphery of muscle fascicles accompanied by inflammation and destruction of the perimysial connective tissue. Conclusion: Antisynthetase syndrome–associated myositis is characterized by distinctive myonuclear actin filament inclusions, including rod formations and a typical necrotizing perimysial myositis. This supports the hypothesis that antisynthetase syndrome–associated myositis is unique and should not be grouped among dermatomyositis, polymyositis, sporadic inclusion body myositis, necrotizing autoimmune myositis, or nonspecific myositis. Classification of evidence: This study provides Class II evidence that for patients with IIMs, the presence of myonuclear actin filament inclusions accurately identifies patients with antisynthetase syndrome–associated myositis (sensitivity 81%, specificity 100%).

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).Inflammatory lesions and contiguous muscle regions from biopsy samples of 10 patients with MuS and 10 patients with MMF were cut out by laser microdissection. Mediators of the T helper cell (Th)1 inducing classical macrophage activation (e.g. STAT1, IFNγ and CXCR3), and Th2 inducing alternative activation of macrophages (e.g. CD206/MRC1, STAT6, SOCS1), molecules involved in development of fibrosis (e.g. TGFβ) and giant cells (e.g. TYROBP), were assessed by immunohistochemistry and real-time polymerase chain reaction (PCR).STAT6-induced Th2 immunity was associated with up-regulated gene expression of MRC1, SOCS1 and TGFB in inflammatory foci, in comparison with adjacent tissue. TYROBP and TREM2, genes regulating giant cell formation, were more strongly expressed in lesions of MuS patients than in those of MMF. TGFβ co-localized with CD206(+) macrophages in MuS but not in MMF. Conversely, Th1 immunity was illustrated by STAT1 staining both in macrophages and myofibres in MuS, but not in MMF. Also, STAT1-induced IFNG and CXCR3 expression in lesions and the surrounding tissue was elevated compared with normal controls, but without statistically significant differences.Giant cell and typical granuloma formations, including fibrogenesis, is dependent on two main mechanisms, both involving specific macrophage activation: a strong Th2-M2 polarization and a significant expression of TYROBP and TGFβ in macrophages. The low-grade alternative activation of macrophages in MMF lesions and poor TYROBP and TGFβco-expression are obviously insufficient to produce giant cells.

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.

Murine Staphylococcus aureus-mediated brain abscess comprises 2 major phases, an initial phase of cerebritis, followed by a healing phase characterized by capsule formation.C57BL/6 wild-type (WT) and IL-12p35(-/-) mice were intracerebrally infected with S. aureus to induce brain abscesses. Clinical disease activity and bacterial load were monitored. The cell populations that were involved, as well as their specific mediators, were analyzed by immunohistochemistry, quantitative real-time polymerase chain reaction, and flow cytometry.In the acute phase, IL-12p35(-/-) mice were protected from disease. This was associated with enhanced recruitment of granulocytes, accompanied by upregulated expression of Il17a, Csf2 (which encodes granulocyte-macrophage colony-stimulating factor), Cxcl1, and Cxcl5, as well as increased expression of proinflammatory mediators, including Nos2 (which encodes inducible nitric oxide synthase), Ptgs2 (which encodes cyclooxygenase 2), and Tnf, that were primarily produced by granulocytes and activated microglia/macrophages. Furthermore, mechanisms associated with beneficial wound healing, including an accelerated formation of a fibrous capsule, were demonstrated by prominent VEGF-A production and collagen deposition driven by an earlier onset of T-helper 2 immunity in the absence of interleukin 12 (IL-12).Brain abscess development is orchestrated by IL-12 at different stages of disease. Our data indicate that IL-12 has a nonprotective role in the acute phase and that IL-12 deficiency results in the accelerated formation of a protective capsule during the healing phase, which we consider crucial for early recovery from disease.

Immune-mediated necrotizing myopathy (IMNM) is considered one of the idiopathic inflammatory myopathies, comprising dermatomyositis, polymyositis, and inclusion body myositis. The heterogeneous group of necrotizing myopathies shows a varying amount of necrotic muscle fibers, myophagocytosis, and a sparse inflammatory infiltrate. The underlying immune response in necrotizing myopathy has not yet been addressed in detail. Affected muscle tissue, obtained from 16 patients with IMNM, was analyzed compared with eight non-IMNM (nIMNM) tissues. Inflammatory cells were characterized by IHC, and immune mediators were assessed by quantitative real-time PCR. We demonstrate that immune- and non–immune-mediated disease can be distinguished by a specific immune profile with significantly more prominent major histocompatibility complex class I expression and complement deposition and a conspicuous inflammatory infiltrate. In addition, patients with IMNM exhibit a strong type 1 helper T cell (T1)/classically activated macrophage M1 response, with detection of elevated interferon-γ, tumor necrosis factor-α, IL-12, and STAT1 levels in the muscle tissue, which may serve as biomarkers and aid in diagnostic decisions. Furthermore, B cells and high expression of the chemoattractant CXCL13 were identified in a subgroup of patients with defined autoantibodies. Taken together, we propose a diagnostic armamentarium that allows for clear differentiation between IMNM and nIMNM. In addition, we have characterized a Th1-driven, M1-mediated immune response in most of the autoimmune necrotizing myopathies, which may guide therapeutic options in the future.

Granulomatous inflammation of skeletal muscle is the hallmark of neuromuscular sarcoidosis (NMS) and macrophagic myofasciitis (MMF). We have recently described an M2-polarized immune response in neuromuscular sarcoidosis. Now, the intrinsic immune signature of granulomas from both entities, and the adjacent non-inflamed muscle tissue was analysed. Granulomas and contiguous muscle from ten patients with NMS and ten patients with MMF were cut out by laser microdissection. Mediators of the T helper cell 1 – classical macrophage activation and Th2 – alternatively activated macrophage immune response as well as molecules involved in development of fibrosis and giant cells were assessed by immunohistochemistry and real-time PCR. STAT6-induced Th2 immunity leads to up-regulated expression of CD206, SOCS1 and TGF- β in granulomas, in comparison to adjacent tissue. DAP12 and TREM2, molecules regulating giant cell formation, are more strongly expressed in granulomas of NMS patients than in those of MMF, while RAC1 is not up-regulated in both entities. TGF- β co-localizes with CD206 + macrophages in NMS but not in MMF. Conversely, Th1 immunity with STAT1-induced IFN- γ and CXCR3 expression in granulomas and the surrounding tissue was elevated, without statistically significant differences. While Th1-mediated immunity is up-regulated in the entire inflamed muscle specimen, Th2-M2 markers are expressed at significantly higher levels in granulomas. These results indicate that muscle tissue per se may provide a permissive environment for M2 polarization in NMS and MMF. Further, DAP12/TREM2-induced giant cell formation and expression of TGF- β in M2 macrophages are hallmarks of NMS and share common activation signalling.

As the most frequent genetic muscle disease worldwide, Duchenne Muscular Dystrophy affects ∼1:5 000 male births. Caused by a defective DMD gene, the dystrophin protein expression is severely hampered and the protein is only partially functional or completely dysfunctional. The constant breakdown of fibres leads to focal necrosis, myophagocytosis and a disease-stage dependent influx of inflammatory cells into the muscle tissue. A further hallmark of the disease is the development of endomysial fibrosis, whereby the amount of fibrosis directly correlates with an adverse outcome and early loss of ambulation. The molecular pathomechanisms underlying development and nature of fibrosis in DMD are not well understood. To study the link between inflammation and fibrosis we analysed macrophage and T cell immunology with regard to development of fibrosis at different time points and identified target molecules involved in this process. We have analysed muscle biopsies derived from DMD patients obtained at different time-points after onset of disease and studied the immune response at protein and mRNA level. Especially the polarisation of macrophages into pro-inflammatory, classically activated M1 phenotype and their switch to an alternatively activated (M2-‘repair phenotype’ during disease progression was investigated. We show a direct link between aaMph’s and fibrogenesis linked to a certain time frame in DMD. An automated whole slide image based quantification of fibrosis showed an increase of fibrosis over time, while the number of macrophages decreased. Polarisation of macrophages showed CD206 positivity. Comparison to patients suffering from Becker muscular dystrophy and female carriers of the defective DMD gene showed less development of fibrosis, and macrophages were only scant. Hopefully the results of this study may add to the development of a time-dependent immune intervention therapy, which can be ideally supplemental to other e.g. gene-therapeutic approaches.