Autoantibodies Produced at the Site of Tissue Damage Provide Evidence of Humoral Autoimmunity in Inclusion Body Myositis

Inclusion body myositis (IBM) belongs to a group of muscle diseases known as the inflammatory myopathies. The presence of antibody-secreting plasma cells in IBM muscle implicates the humoral immune response in this disease. However, whether the humoral immune response actively contributes to IBM pathology has not been established. We sought to investigate whether the humoral immune response in IBM both in the periphery and at the site of tissue damage was directed towards self-antigens. Peripheral autoantibodies present in IBM serum but not control serum recognized self-antigens in both muscle tissue and human-derived cell lines. To study the humoral immune response at the site of tissue damage in IBM patients, we isolated single plasma cells directly from IBM-derived muscle tissue sections and from these cells, reconstructed a series of recombinant immunoglobulins (rIgG). These rIgG, each representing a single muscle-associated plasma cell, were examined for reactivity to self-antigens. Both, flow cytometry and immunoblotting revealed that these rIgG recognized antigens expressed by cell lines and in muscle tissue homogenates. Using a mass spectrometry-based approach, Desmin, a major intermediate filament protein, expressed abundantly in muscle tissue, was identified as the target of one IBM muscle-derived rIgG. Collectively, these data support the view that IBM includes a humoral immune response in both the periphery and at the site of tissue damage that is directed towards self-antigens.     

FOXP3+ T Cells Recruited to Sites of Sterile Skeletal Muscle Injury Regulate the Fate of Satellite Cells and Guide Effective Tissue Regeneration

Muscle injury induces a classical inflammatory response in which cells of the innate immune system rapidly invade the tissue. Macrophages are prominently involved in this response and required for proper healing, as they are known to be important for clearing cellular debris and supporting satellite cell differentiation. Here, we sought to assess the role of the adaptive immune system in muscle regeneration after acute damage. We show that T lymphocytes are transiently recruited into the muscle after damage and appear to exert a pro-myogenic effect on muscle repair. We observed a decrease in the cross-sectional area of regenerating myofibers after injury in Rag2^-/- γ-chain^-/- mice, as compared to WT controls, suggesting that T cell recruitment promotes muscle regeneration. Skeletal muscle infiltrating T lymphocytes were enriched in CD4⁺CD25⁺FOXP3⁺ cells. Direct exposure of muscle satellite cells to in vitro induced Treg cells effectively enhanced their expansion, and concurrently inhibited their myogenic differentiation. In vivo, the recruitment of Tregs to acutely injured muscle was limited to the time period of satellite expansion, with possibly important implications for situations in which inflammatory conditions persist, such as muscular dystrophies and inflammatory myopathies. We conclude that the adaptive immune system, in particular T regulatory cells, is critically involved in effective skeletal muscle regeneration. Thus, in addition to their well-established role as regulators of the immune/inflammatory response, T regulatory cells also regulate the activity of skeletal muscle precursor cells, and are instrumental for the proper regeneration of this tissue.     

Mitochondrial dynamic remodeling in strenuous exercise-induced muscle and mitochondrial dysfunction: regulatory effects of hydroxytyrosol

Physical exercise is considered to exert a positive effect on health, whereas strenuous or excessive exercise (Exe) causes fatigue and damage to muscle and immune functions. The underlying molecular mechanisms are still unclear. We designed a protocol to mimic Exe and explore the ensuing cellular damage and involvement of mitochondrial dynamics. We found that Exe was prone to decrease endurance capacity and induce damage to renal function and the immune system. Muscle atrophy markers atrogin-1 and MuRF1 mRNA were increased by Exe, accompanied by increased autophagy and mitochondrial fission in skeletal muscle. Exe caused a decrease in PGC-1α and complex I expression; it also activated JNK and Erk1/2 pathways and consequently induced p53, p21, and MnSOD expression in skeletal muscle. The involvement of oxidant-induced autophagy and mitochondrial dysfunction was confirmed in C2C12 myoblasts. Hydroxytyrosol (HT), a natural olive polyphenol, efficiently enhanced endurance capacity and prevented Exe-induced renal and immune system damage. Also, HT treatment inhibited both the Exe-induced increase in autophagy and mitochondrial fission and the decrease in PGC-1α expression. In addition, HT enhanced mitochondrial fusion and mitochondrial complex I and II activities in muscle of Exe rats. These results demonstrate that Exe-induced fatigue and damage to muscle and immune functions may be mediated via the regulation of mitochondrial dynamic remodeling, including the downregulation of mitochondrial biogenesis and upregulation of autophagy. HT supplementation may regulate mitochondrial dynamic remodeling and enhance antioxidant defenses and thus improve exercise capacity under Exe conditions.     

Induction of innate immune gene expression following methyl methanesulfonate-induced DNA damage in sea urchins

Sea urchins are noted for the absence of neoplastic disease and represent a novel model to investigate cellular and systemic cancer protection mechanisms. Following intracoelomic injection of the DNA alkylating agent methyl methanesulfonate, DNA damage was detected in sea urchin cells and tissues (coelomocytes, muscle, oesophagus, ampullae and gonad) by the alkaline unwinding, fast micromethod. Gene expression analyses of the coelomocytes indicated upregulation of innate immune markers, including genes involved in NF-κB signalling. Results suggest that activation of the innate immune system following DNA damage may contribute to the naturally occurring resistance to neoplastic disease observed in sea urchins.     

In vitro models to study compressive strain-induced muscle cell damage

Skeletal muscle tissue is highly susceptible to sustained compressive straining, eventually leading to tissue breakdown in the form of pressure sores. This breakdown begins at the cellular level and is believed to be triggered by sustained cell deformation. To study the relationship between compressive strain-induced muscle cell deformation and damage, and to investigate the role of cell-cell interactions, cell-matrix interactions and tissue geometry in this process, in vitro models of single cells, monolayers and 3D tissue analogs under compression are being developed. Compression is induced using specially designed loading devices, while cell deformation is visualised with confocal microscopy. Cell damage is assessed from viability tests, vital microscopy and histological or biochemical analyses. Preliminary results from a 3D cell seeded agarose model indicate that cell deformation is indeed an important trigger for cell damage; sustained compression of the model at 20% strain results in a significant increase in cell damage with time of compression, whereas damage in unstrained controls remains constant over time.     

Covalent labeling of immune cells

Inflammation is a common, fast, and innate response of the immune system to sterile or infectious tissue damage or autoimmune triggers. It aims at minimizing tissue destruction and maintaining organ function, hence is vital to life. Therefore, the immune system comprises the concerted action of a variety of different immune cells with specific tasks in the initiation, maintenance, and termination of inflammation. Visualizing their localization, trafficking, and interaction is of utmost importance to unravel the dynamics of inflammation in the living organism and requires tools for cell-specific labeling and imaging. Many concepts for covalent cell-type or protein-specific labeling have been developed, but only few have been implemented for labeling immune cells. Here, we review approaches that were already successful for fluorescent reporters and radioactive nuclides. We also provide a glimpse on emerging technologies that bear potential for immune cell labeling and imaging in vivo.     

Effect of T3 on metabolic response and oxidative stress in skeletal muscle from sedentary and trained rats

We investigated whether swim training modifies the effect of T₃-induced hyperthyroidism on metabolism and oxidative damage in rat muscle. Respiratory capacities, oxidative damage, levels of antioxidants, and susceptibility to oxidative challenge of homogenates were determined. Mitochondrial respiratory capacities, H₂O₂ release rates, and oxidative damage were also evaluated. T₃-treated rats exhibited increases in muscle respiratory capacity, which were associated with enhancements in mitochondrial respiratory capacity and tissue mitochondrial protein content in sedentary and trained animals, respectively. Hormonal treatment induced muscle oxidative damage and GSH depletion. Both effects were reduced by training, which also attenuated tissue susceptibility to oxidative challenge. The changes in single antioxidant levels were slightly related to oxidative damage extent, but the examination of parameters affecting the susceptibility to oxidants indicated that training was associated with greater effectiveness of the muscle antioxidant system. Training also attenuated T₃-induced increases in H₂O₂ production and, therefore, oxidative damage of mitochondria by lowering their content of autoxidizable electron carriers. The above results suggest that moderate training is able to reduce hyperthyroid state-linked tissue oxidative damage, increasing antioxidant protection and decreasing the ROS flow from the mitochondria to the cytoplasmic compartment.     

Skeletal muscle damage and impaired regeneration due to LPL-mediated lipotoxicity

According to the concept of lipotoxicity, ectopic accumulation of lipids in non-adipose tissue induces pathological changes. The most prominent effects are seen in fatty liver disease, lipid cardiomyopathy, non-insulin-dependent diabetes mellitus, insulin resistance and skeletal muscle myopathy. We used the MCK(m)-hLPL mouse distinguished by skeletal and cardiac muscle-specific human lipoprotein lipase (hLPL) overexpression to investigate effects of lipid overload in skeletal muscle. We were intrigued to find that ectopic lipid accumulation induced proteasomal activity, apoptosis and skeletal muscle damage. In line with these findings we observed reduced Musculus gastrocnemius and Musculus quadriceps mass in transgenic animals, accompanied by severely impaired physical endurance. We suggest that muscle loss was aggravated by impaired muscle regeneration as evidenced by reduced cross-sectional area of regenerating myofibers after cardiotoxin-induced injury in MCK(m)-hLPL mice. Similarly, an almost complete loss of myogenic potential was observed in C2C12 murine myoblasts upon overexpression of LPL. Our findings directly link lipid overload to muscle damage, impaired regeneration and loss of performance. These findings support the concept of lipotoxicity and are a further step to explain pathological effects seen in muscle of obese patients, patients with the metabolic syndrome and patients with cancer-associated cachexia.     

Biological approaches to improve skeletal muscle healing after injury and disease

Skeletal muscle injury and repair are complex processes, including well‐coordinated steps of degeneration, inflammation, regeneration, and fibrosis. We have reviewed the recent literature including studies by our group that describe how to modulate the processes of skeletal muscle repair and regeneration. Antiinflammatory drugs that target cyclooxygenase‐2 were found to hamper the skeletal muscle repair process. Muscle regeneration phase can be aided by growth factors, including insulin‐like growth factor‐1 and nerve growth factor, but these factors are typically short‐lived, and thus more effective methods of delivery are needed. Skeletal muscle damage caused by traumatic injury or genetic diseases can benefit from cell therapy; however, the majority of transplanted muscle cells (myoblasts) are unable to survive the immune response and hypoxic conditions. Our group has isolated neonatal skeletal muscle derived stem cells (MDSCs) that appear to repair muscle tissue in a more effective manner than myoblasts, most likely due to their better resistance to oxidative stress. Enhancing antioxidant levels of MDSCs led to improved regenerative potential. It is becoming increasingly clear that stem cells tissue repair by direct differentiation and paracrine effects leading to neovascularization of injured site and chemoattraction of host cells. The factors invoked in paracrine action are still under investigation. Our group has found that angiotensin II receptor blocker (losartan) significantly reduces fibrotic tissue formation and improves repair of murine injured muscle. Based on these data, we have conducted a case study on two hamstring injury patients and found that losartan treatment was well tolerated and possibly improved recovery time. We believe this medication holds great promise to optimize muscle repair in humans. (Part C) 96:82–94, 2012. © 2012 Wiley Periodicals, Inc.     

Gene expression and immune response kinetics using electroporation-mediated DNA delivery to muscle

BACKGROUND: Injection of DNA encoding exogenic proteins into muscle tissue combined with electroporation often results in a transient increase of the encoded protein concentration in the muscle and the blood. The reduction is normally due to an immune response against the exogenic protein but other factors may also be involved. How various electroporation parameters affect the concentration kinetics of syngenic and exogenic proteins is studied in relation to immune response and muscle damage after electroporation-mediated DNA transfer to muscle. METHODS: Electroporation was applied to mouse quadriceps and rat tibialis anterior muscles after injection of DNA encoding either secreted alkaline phosphatase (SEAP), beta-galactosidase (beta-gal), luciferase or a mouse IgG molecule. Protein concentrations in blood or muscle and antibody responses were measured for a period up to 3 months. Tissue inflammation and muscle cell damage were studied on muscle cross-sections and assessed by measuring the concentrations of creatine phosphokinase (CPK) in blood. RESULTS: Mice with the highest SEAP concentration in blood at day 7 also had the highest rate of decrease afterwards, the strongest antibody responses against SEAP and the highest acute levels of CPK in blood. DNA-transfected muscle fibers were significantly reduced in number from days 7 to 14. Mononuclear cells surrounded the reporter gene expressing muscle fibers, thus indicating a cellular immune response. When using DNA encoding a syngenic protein the protein concentration in blood was relatively stabile over a 3-month period, but showed different kinetics for various electroporation parameters. CONCLUSIONS: Our findings suggest that the optimal electroporation parameters for DNA vaccination may be different from the optimal parameters for long-term expression of genes encoding syngenic proteins.     

Trace Element Concentrations in Beef Cattle Related to the Breed Aptitude

Environmental and occupational mercury exposure is considered a major public health issue. Despite being well known that MeHg exposure causes adverse effects in several physiologic functions, MeHg effects on salivary glands still not completely elucidated. Here, we investigated the cellular MeHg-induced damage in the three major salivary glands (parotid, submandibular, and sublingual) of adult rats after chronic, systemic and low doses of MeHg exposure. Rats were exposed by 0.04 mg/kg/day over 60 days. After that, animals were euthanized and all three glands were collected. We evaluated total Hg accumulation, metallothionein I/II (MT I/II), α-smooth muscle actin (α-SMA), and cytokeratin 18 (CK18) immune expression. Our results have showed that MeHg is able to disrupt gland tissue and to induce a protective mechanism by MT I/II expression. We also showed that cell MT production is not enough to protect gland tissue against cellular structural damage seen by reducing marking of cytoskeletal proteins as CK18 and α-SMA. Our data suggest that chronic MeHg exposure in low-daily doses is able to induce cellular damage in rat salivary glands.     

The free diffusion of macromolecules in tissue-engineered skeletal muscle subjected to large compression strains

Pressure-related deep tissue injury (DTI) represents a severe pressure ulcer, which initiates in compressed muscle tissue overlying a bony prominence and progresses to more superficial tissues until penetrating the skin. Individual subjects with impaired motor and/or sensory capacities are at high risk of developing DTI. Impaired diffusion of critical metabolites in compressed muscle tissue may contribute to DTI, and impaired diffusion of tissue damage biomarkers may further impose a problem in developing early detection blood tests. We hypothesize that compression of muscle tissue between a bony prominence and a supporting surface locally influences the diffusion capacity of muscle. The objective of this study was therefore, to determine the effects of large compression strains on free diffusion in a tissue-engineered skeletal muscle model. Diffusion was measured with a range of fluorescently labeled dextran molecules (10, 20, 150kDa) whose sizes were representative of both hormones and damage biomarkers. We used fluorescence recovery after photobleaching (FRAP) to compare diffusion coefficients (D) of the different dextrans between the uncompressed and compressed (48-60% strain) states. In a separate experiment, we simulated the effects of local partial muscle ischemia in vivo, by reducing the temperature of compressed specimens from 37 to 34 degrees C. Compared to the D in the uncompressed model system, values in the compressed state were significantly reduced by 47+/-22% (p<0.02). A 3 degrees C temperature decrease further reduced D in the compressed specimens by 10+/-6% (p<0.05). In vivo, the effects of large strains and ischemia are likely to be summative, and hence, the present findings suggest an important role of impaired diffusion in the etiology of DTI, and should also be considered when developing biochemical screening methods for early detection of DTI.     

Adipose tissue,the immune system and exercise fatigue: how activated lymphocytes compete for lipids

Adipose depots that contain lymph nodes, and probably intermuscular fat in skeletal and cardiac muscle, are specialized to provision adjacent tissue in a paracrine mode. Perinodal adipocytes respond selectively to various cytokines and incorporate proportionately more polyunsaturated fatty acids. Lipolysis in the adipocytes of node-containing depots can be stimulated via inflammation of the enclosed lymph nodes. Repeated immune stimulation elicits properties characteristic of perinodal adipocytes in those elsewhere in the same depot, and hours later in other node-containing depots, but not in nodeless depots. Such site-specific properties of adipose tissue enable partitioning of dietary and metabolic supplies of fatty acids between competing tissues. Local interactions emancipate the peripheral immune system from competing with other tissues for lipids during immune responses, and may be especially important during periods of high demand, such as strenuous exercise. Biopsies of subcutaneous adipose tissue from sites remote from lymph nodes do not adequately represent the composition of fatty acids available to the immune system in situ, and perhaps that supplied to other tissues. Intermuscular fat in skeletal and cardiac muscle may also indicate paracrine relationships between adipocytes and "end-user" tissues. The concept of paracrine interactions between certain adipocytes and "user" tissue may account for the widespread contiguity between these tissues in vivo.     

免疫的代价

免疫系统发育的代价主要是为B淋巴细胞和T淋巴细胞发育抗原识别多样性时的低效率过程提供能量 ,为开始形成白血球提供基质 (如氨基酸和脂肪 ) ,并为胚胎和雏鸟提供白细胞。维持免疫系统的代价与持续产生白细胞、免疫球蛋白以及其它血浆蛋白时的营养分配有关 ,这些血浆蛋白用于取代在正常的细胞代谢中丢失的蛋白质以及细胞间的蛋白质。利用免疫系统阻碍可能的病源体入侵的代价有两种主要形式。第一 ,由于白细胞参与效应子机制时发生的损害、组织整合性以及寄主细胞的存活力的损伤 ,组织功能有一些损失。第二 ,在活化相应类型的细胞并产生其效应子作用时存在着营养耗费。病源体挑战所带来的主要代价是系统的急性期反应(特别是肝脏的复原 )通过产生保护性的蛋白质来帮助免疫系统     

Regulatory T cells in skeletal muscle repair and regeneration: recent insights

Skeletal muscle repair and regeneration after injury is a multi-stage process, involving a dynamic inflammatory microenvironment consisting of a complex network formed by the interaction of immune cells and their secreted cytokines. The homeostasis of the inflammatory microenvironment determines whether skeletal muscle repair tissues will ultimately form scar tissue or regenerative tissue. Regulatory T cells (Tregs) regulate homeostasis within the immune system and self-immune tolerance, and play a crucial role in skeletal muscle repair and regeneration. Dysregulated Tregs function leads to abnormal repair. In this review, we discuss the role and mechanisms of Tregs in skeletal muscle repair and regeneration after injury and provide new strategies for Treg immunotherapy in skeletal muscle diseases.Subject terms: Trauma, Immunotherapy     

Innate immune cells are dispensable for regenerative growth of imaginal discs

Following tissue damage the immune response, including inflammation, has been considered an inevitable condition to build the host defense against invading pathogens. The recruitment of innate immune leukocytes to injured tissue is observed in both vertebrates and invertebrates. However, it is still not conclusive whether the inflammatory response is also indispensable for the wound healing process by itself, in addition to its role in microbial clearance. In this study we determine the requirement of innate immune cells, both hemocytes and fat body cells, in Drosophila imaginal disc regeneration. We investigate wound healing and regenerative cell proliferation of damaged imaginal discs under immunodeficient conditions. To delay development of Drosophila at matured third instar larval stage we used a sterol-mutant erg2 knock-out yeast strain in the medium. This dietary-controlled developmental arrest allowed us to generate larvae free of immune cells without interfering with their larval development. In addition, this approach allowed uncoupling regenerative cell proliferation of damaged discs from their normal developmental growth. We furthermore examined the regenerative cell proliferation of fragmented imaginal discs by transplantation into host flies deficient of immune cells. We demonstrate that the damaged/fragmented discs in immune cells deficient conditions still exhibit regenerative cell proliferation comparable to those of control samples. These results suggest that recruitment of immune cells is not a prerequisite for the regenerative growth of damaged imaginal discs.     

Emerging role of immune cell network in autoimmune skin disorders: An update on pemphigus,vitiligo and psoriasis

Autoimmune skin diseases are a group of disorders that arise due to a deregulated immune system resulting in skin tissue destruction. In the majority of these conditions, either autoreactive immune cells or the autoantibodies are generated against self-antigens of the skin. Although the etiology of these diseases remains elusive, biochemical, genetic, and environmental factors such as infectious agents, toxins damage the skin tissue leading to self-antigen generation, autoantibody attack and finally results in autoimmunity of skin. Immune dysregulation, which involves predominantly T helper 1/17 (Th1/Th17) polarization and the inability of regulatory T cells to regress immune response, is implicated in autoimmune skin diseases.The emerging roles of immune cells, cytokines, and chemokines in the pathogenesis of common autoimmune skin diseases like pemphigus, vitiligo, and psoriasis are discussed in this review. The main focus is on the interplay between immune cell network including the innate and adaptive immune system, regulatory cells, immune checkpoints and recently identified tissue-resident memory cells (TRMs) in disease pathogenesis and relapse. We also attempt to highlight on the immune mechanisms common to these diseases which can be targeted for designing novel therapeutics.     

Envenomations by Bothrops and Crotalus snakes induce the release of mitochondrial alarmins

Skeletal muscle necrosis is a common manifestation of viperid snakebite envenomations. Venoms from snakes of the genus Bothrops, such as that of B. asper, induce muscle tissue damage at the site of venom injection, provoking severe local pathology which often results in permanent sequelae. In contrast, the venom of the South American rattlesnake Crotalus durissus terrificus, induces a clinical picture of systemic myotoxicity, i.e., rhabdomyolysis, together with neurotoxicity. It is known that molecules released from damaged muscle might act as 'danger' signals. These are known as 'alarmins', and contribute to the inflammatory reaction by activating the innate immune system. Here we show that the venoms of B. asper and C. d. terrificus release the mitochondrial markers mtDNA (from the matrix) and cytochrome c (Cyt c) from the intermembrane space, from ex vivo mouse tibialis anterior muscles. Cyt c was released to a similar extent by the two venoms whereas B. asper venom induced the release of higher amounts of mtDNA, thus reflecting hitherto some differences in their pathological action on muscle mitochondria. At variance, injection of these venoms in mice resulted in a different time-course of mtDNA release, with B. asper venom inducing an early onset increment in plasma levels and C. d. terrificus venom provoking a delayed release. We suggest that the release of mitochondrial 'alarmins' might contribute to the local and systemic inflammatory events characteristic of snakebite envenomations.     

An essential role for TH2-type responses in limiting acute tissue damage during experimental helminth infection

Helminths induce potent T helper 2 (TH2)-type immune responses that can mediate worm expulsion, but the role of this response in controlling the acute tissue damage caused by migrating multicellular parasites through vital tissues remains uncertain. We used a helminth infection model in which parasitic nematode larvae migrate transiently through the lung, resulting in hemorrhage and inflammation. We found that IL-17 initially contributed to inflammation and lung damage, whereas subsequent IL-4 receptor (IL-4R) signaling reduced elevations in IL-17 mRNA levels, enhanced the expression of insulin-like growth factor 1 (IGF-1) and IL-10 and stimulated the development of M2 macrophages, all of which contributed to the rapid resolution of tissue damage. These studies indicate an essential role for TH2-type immune responses in mediating acute wound healing during helminth infection.     

Epigenetic Control of Immunity

Immunity relies on the heterogeneity of immune cells and their ability to respond to pathogen challenges. In the adaptive immune system, lymphocytes display a highly diverse antigen receptor repertoire that matches the vast diversity of pathogens. In the innate immune system, the cell''s heterogeneity and phenotypic plasticity enable flexible responses to changes in tissue homeostasis caused by infection or damage. The immune responses are calibrated by the graded activity of immune cells that can vary from yeast-like proliferation to lifetime dormancy. This article describes key epigenetic processes that contribute to the function of immune cells during health and disease.