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BiP (binding immunoglobulin protein) is a key chaperone mostly residing in the Endoplasmic / Sarcoplasmic Reticulum (ER/SR). It plays an important role in maintaining protein homeostasis by facilitating correct folding, assembly, and clearance of proteins across diverse cellular compartments. Despite its implication in diverse (neuro-)muscular disorders associated with ER stress, the precise involvement of BiP in muscle physiology and pathology, as in the context of IIM, remains poorly understood. To study the role of BiP in IIM, quadriceps muscle biopsy specimens obtained from IIM patients were subjected to unbiased proteomic profiling, and results were validated by immunostaining. Hereby, following diseases were studied: anti-synthetase syndrome (ASS), dermatomyositis (DM), inclusion body myositis (IBM), immune-mediated necrotizing myopathy (IMNM), and systemic sclerosis associated with myositis and vasculopathy (SSc/MMCP). In patients with idiopathic inflammatory myopathies (IIM), proteomics revealed the most substantial elevation in BiP levels in muscle biopsies derived from dermatomyositis (DM) patients, with a median increase of 3.49-fold. This was followed by patients with immune-mediated necrotizing myopathy (IMNM), showing a mean increase of 2.62-fold in SRP54-seropositive individuals and a mean increase of 2.23-fold in HMGCR-seropositive individuals. In patients with inclusion body myositis (IBM), a mean increase of 2.33-fold in BiP levels was identified. Among anti-synthetase syndrome patients, PL-7-seropositive individuals exhibited a median increase of 1.56-fold, while PL-12- and JO-1-seropositive patients showed elevations, with mean increases of 1.31-fold and 1.51-fold, respectively. Additionally, muscle protein extracts from patients with systemic sclerosis/mixed connective tissue disease (SSc/MMCP) showed increased BiP levels, with a mean increase of 1.40-fold. Immunostaining confirmed the elevation of BiP in the sarcoplasm of patients with IMNM, IBM, ASS and DM. Interestingly, muscle biopsy samples from systemic sclerosis (MMCP) patients showed an extracellular increase in BiP levels indicative for a function in immunomodulation. In conclusion, BiP is upregulated throughout all IIM subtypes, with DM showing the highest intracellular abundance suggesting a role in proteostasis. However, BiP was also found in the extracellular space in SSc patients, hinting at additional biological roles in the molecular aetiology of muscular diseases with an autoimmune background. [ABSTRACT FROM AUTHOR]

Systemic sclerosis (SSc) is a rare autoimmune disease belonging to the group of connective tissue disorders. It is characterized by obliterative vasculopathy, accumulation of extracellular matrix in terms of fibrosis and autoimmune-mediated inflammation; all of these three features can affect almost any organ, including skeletal muscle, leading to potentially life-threatening complications such as interstitial lung disease or pulmonary-arterial hypertension. There is therefore a great medical need for a better understanding of the pathophysiology and thus improved therapy. Recent studies unveiled involvement of ER-stress in white blood cells and muscle tissue, which lead to activation of the unfolded protein response (UPR). BiP, a chaperone, is a major player that binds nascent polypeptides as they translocate into the ER and misfolded/aberrant proteins destined for degradation. Since therapy in SSc is currently oriented towards individual symptoms rather than the causes of the disease, a deeper understanding of the pathophysiology is invaluable to provide improved therapies. Therefore, UPR modulation represents an attractive target, as it is therapeutically addressable by e.g. small molecules. Hence, we investigated the potential involvement of BiP in the etiology of SSc. For this we analyzed muscle tissue from 15 patients with SSc in comparison to five non-disease controls by immune histochemistry, unbiased proteomic profiling, and sera by ELISA. Proteomic profiling revealed clear increase of BiP in skeletal muscle from SSc patients. Immunostaining also revealed an increase of BiP. Interestingly, BiP-deposits were not identified within the muscle fibers, but rather within the extracellular space. Notably, previous studies have shown that extracellular BiP-localization impacts immune cell function and thus immune response. Especially T cells are attracted by high extracellular BiP concentrations. Our ELISA-based quantification showed lower serum concentrations. In contrast, anti-BiP in serum showed a significant increase, supporting the concept of cellular BiP-release and thus supports the interesting finding of BiP in the extracellular space seen in skeletal muscle. This migth hint to an inflammatory role of BiP in SSc. Our combined results unravel BiP as a tissue and blood biomarker with potential impact on immune responses in SSc. Further functional studies are crucial to address a potential therapeutic impact of this finding. [ABSTRACT FROM AUTHOR]

Immune-mediated necrotizing myopathy (IMNM) is a form of idiopathic inflammatory myopathy (IIM) characterized by severe, rapidly progressing muscle weakness, creatine kinase elevation and muscle fibre necrosis. IMNMs are divided into three subgroups depending on the presence of muscle specific autoantibodies: anti-SRP-positive, anti-HMGCR-positive and seronegative IMNM. Vascular abnormalities, particularly alterations in capillaries, have been mentioned in IMNM. However, the link between autoimmune muscle damage and abnormal capillary morphology has remained unclear. The type of capillary pathology is a relevant feature, since vascular damage is key e.g. in dermatomyositis and systemic sclerosis as highlighted recently. In this project, we aimed to characterize vascular/capillary abnormalities in muscle biopsies derived from HMGCR-Ab, SRP-Ab, and seronegative IMNM patients. Light and electron microscopy were used to assess structural changes in capillaries, while quantitative real-time PCR was used to study the expression of genes related to vascular function. Additionally, we analyzed the proteomic signature of IMNM in relation to vascularization. Distinct patterns of capillary abnormalities were identified in all IMNM subgroups. Capillaries showed a spectrum of alterations, including a broad range of basement membrane duplications, activation of endothelial subcellular organelles featuring vesicle formation, proliferation of pericytes and degeneration to string vessels, but they do not show endothelial tubuloreticular inclusions. By integrating these methods, this study sought to provide a comprehensive understanding of the vascular abnormalities observed in IMNM, shedding light on a previously neglected aspect of the disease's pathophysiology. [ABSTRACT FROM AUTHOR]

Anti-synthetase syndrome (ASyS) is an autoimmune condition, characterized by the presence of autoantibodies directed against an aminoacyl-tRNA synthetase (anti-ARS). Patients present clinical symptoms such as myositis, interstitial lung disease, Raynaud's phenomenon, and arthritis. Anti-Jo-1, anti-PL-7 and anti-PL-12 are the most frequent anti-ARS. However, their role in ASyS pathogenesis remains incompletely understood. Therefore, robust animal models are essential to gain a detailed insight into the underlying pathophysiology. Aiming to characterize these pathophysiological features, we established and studied a mouse model for Jo-1, PL-7 and PL-12 associated ASyS. ASyS was induced in NOD.Idd 3/5 mice by injection of 200 µg Jo-1, PL-7 or PL-12 recombinant protein emulsified in Complete Freund's Adjuvant (CFA) in combination with OX86. Controls received CFA and Phosphate Buffered Saline only. Muscle strength was assessed by rotarod tests and the effects on the peripheral immune system were investigated by flow cytometry in spleen and lymph nodes. Morphological characteristics of skeletal muscle and lung tissue of immunized mice and the tissue infiltrating immune cells were validated using histology and immunohistochemistry. Immunization of mice led to clinical symptoms including muscle weakness and demonstrated variations in the immune cell response between the ARS subtypes. Histological analysis of skeletal muscle tissues showed infiltration by immune cells in the epimysium, spreading into the adjacent perifascicular area with progressing disease. Analysis of lung specimens by immunohistological staining demonstrated peribronchial accentuated accumulation of lymphocyte aggregates. We present a mouse model, which recapitulates features of the human phenotype of ASyS, to study the molecular pathogenesis and provide new insights into the pathomechanisms. [ABSTRACT FROM AUTHOR]