Balenine, Imidazole Dipeptide Promotes Skeletal Muscle Regeneration by Regulating Phagocytosis Properties of Immune Cells.

Balenine is one of the endogenous imidazole dipeptides derived from marine products. It is composed of beta-alanine and 3-methyl-L-histidine, which exist mainly in the muscles of marine organisms. The physiological functions of dietary balenine are not well-known. In this study, we investigated whether the supplementation of dietary balenine was associated with muscle function in a cardiotoxin-indued muscle degeneration/regeneration model. Through morphological observation, we found that the supplementation of balenine-enriched extract promoted the regeneration stage. In addition, the expression of regeneration-related myogenic marker genes, such as paired box protein 7, MyoD1, myogenin, and Myh3, in a group of mice fed a balenine-enriched extract diet was higher than that in a group fed a normal diet. Moreover, the supplementation of balenine-enriched extract promoted the expression of anti-inflammatory cytokines as well as pro-inflammatory cytokines at the degeneration stage. Interestingly, phagocytic activity in the balenine group was significantly higher than that in the control group in vitro. These results suggest that balenine may promote the progress of muscle regeneration by increasing the phagocytic activity of macrophages.

Mushroom supplements triggering a flare of HMGCR immune mediated necrotising myopathy.

Hydroxyl-methyl-glutaryl-Co-A reductase (HMGCR) immune mediated necrotising myopathy (IMNM) is a rare autoimmune myositis that is thought to be triggered by statins and responds to immunomodulation. We report a case of a woman in her 30s with HMGCR IMNM without a history of statin exposure who had a clear flare of her myositis after beginning mushroom supplements. Mushrooms are natural HMGCR inhibitors, and this is the first case to demonstrate a flare triggered by mushrooms in a patient with known HMGCR IMNM. This case highlights the importance of reviewing diet and supplements in patients with IMNM. It also emphasises the importance of strict statin avoidance for patients with IMNM even when the myositis is under good control.

Integrative analyses of hub genes and their association with immune infiltration in adipose tissue, liver tissue and skeletal muscle of obese patients after bariatric surgery.

Bariatric surgery (BS) is an effective treatment for obesity. Adipose tissue, liver tissue and skeletal muscle are important metabolic tissues. This study investigated hub genes and their association with immune infiltration in these metabolic tissues of obese patients after BS by bioinformatic analysis with Gene Expression Omnibus datasets. Differentially expressed genes (DEGs) were identified, and a protein-protein interaction network was constructed to identify hub genes. As a result, 121 common DEGs were identified and mainly enriched in cytokine-cytokine receptor interactions, chemokine signaling pathway, neutrophil activation and immune responses. Immune cell infiltration analysis showed that the abundance of M1 macrophages was significantly lower in adipose and liver tissue after BS (p<0.05). Ten hub genes (TYROBP, TLR8, FGR, NCF2, HCK, CCL2, LAPTM5, MNDA and S100A9) that were all downregulated after BS were also associated with immune cells. Consistently, results in the validated dataset showed that the expression levels of these hub genes were increased in obese patients and mice, and decreased after BS. In conclusion, this study analysed the potential immune and inflammatory mechanisms of BS in three key metabolic tissues of obese patients, and revealed hub genes associated with immune cell infiltration, thus providing potential targets for obesity treatment.

Neutrophil and natural killer cell imbalances prevent muscle stem cell-mediated regeneration following murine volumetric muscle loss.

Volumetric muscle loss (VML) overwhelms the innate regenerative capacity of mammalian skeletal muscle (SkM), leading to numerous disabilities and reduced quality of life. Immune cells are critical responders to muscle injury and guide tissue resident stem cell­ and progenitor-mediated myogenic repair. However, how immune cell infiltration and intercellular communication networks with muscle stem cells are altered following VML and drive pathological outcomes remains underexplored. Herein, we contrast the cellular and molecular mechanisms of VML injuries that result in the fibrotic degeneration or regeneration of SkM. Following degenerative VML injuries, we observed the heightened infiltration of natural killer (NK) cells as well as the persistence of neutrophils beyond 2 wk postinjury. Functional validation of NK cells revealed an antagonistic role in neutrophil accumulation in part via inducing apoptosis and CCR1-mediated chemotaxis. The persistent infiltration of neutrophils in degenerative VML injuries was found to contribute to impairments in muscle stem cell regenerative function, which was also attenuated by transforming growth factor beta 1 (TGFß1). Blocking TGFß signaling reduced neutrophil accumulation and fibrosis and improved muscle-specific force. Collectively, these results enhance our understanding of immune cell­stem cell cross talk that drives regenerative dysfunction and provide further insight into possible avenues for fibrotic therapy exploration.

TLR4 is a regulator of trained immunity in a murine model of Duchenne muscular dystrophy.

Dysregulation of the balance between pro-inflammatory and anti-inflammatory macrophages has a key function in the pathogenesis of Duchenne muscular dystrophy (DMD), a fatal genetic disease. We postulate that an evolutionarily ancient protective mechanism against infection, known as trained immunity, drives pathological inflammation in DMD. Here we show that bone marrow-derived macrophages from a murine model of DMD (mdx) exhibit cardinal features of trained immunity, consisting of transcriptional hyperresponsiveness associated with metabolic and epigenetic remodeling. The hyperresponsive phenotype is transmissible by bone marrow transplantation to previously healthy mice and persists for up to 11 weeks post-transplant. Mechanistically, training is induced by muscle extract in vitro. The functional and epigenetic changes in bone marrow-derived macrophages from dystrophic mice are TLR4-dependent. Adoptive transfer experiments further support the TLR4-dependence of trained macrophages homing to damaged muscles from the bone marrow. Collectively, this suggests that a TLR4-regulated, memory-like capacity of innate immunity induced at the level of the bone marrow promotes dysregulated inflammation in DMD.

TLR4-Mediated Inflammatory Responses Regulate Exercise-Induced Molecular Adaptations in Mouse Skeletal Muscle.

Endurance exercise induces various adaptations that yield health benefits; however, the underlying molecular mechanism has not been fully elucidated. Given that it has recently been accepted that inflammatory responses are required for a specific muscle adaptation after exercise, this study investigated whether toll-like receptor (TLR) 4, a pattern recognition receptor that induces proinflammatory cytokines, is responsible for exercise-induced adaptations in mouse skeletal muscle. The TLR4 mutant (TLR4m) and intact TLR4 control mice were each divided into 2 groups (sedentary and voluntary wheel running) and were housed for six weeks. Next, we removed the plantaris muscle and evaluated the expression of cytokines and muscle regulators. Exercise increased cytokine expression in the controls, whereas a smaller increase was observed in the TLR4m mice. Mitochondrial markers and mitochondrial biogenesis inducers, including peroxisome proliferator-activated receptor beta and heat shock protein 72, were increased in the exercised controls, whereas this upregulation was attenuated in the TLR4m mice. In contrast, exercise increased the expression of molecules such as peroxisome proliferator-activated receptor-gamma coactivator 1-alpha and glucose transporter 4 in both the controls and TLR4m mice. Our findings indicate that exercise adaptations such as mitochondrial biogenesis are mediated via TLR4, and that TLR4-mediated inflammatory responses could be involved in the mechanism of adaptation.

Targeting necroptosis in muscle fibers ameliorates inflammatory myopathies.

Muscle cell death in polymyositis is induced by CD8+ cytotoxic T lymphocytes. We hypothesized that the injured muscle fibers release pro-inflammatory molecules, which would further accelerate CD8+ cytotoxic T lymphocytes-induced muscle injury, and inhibition of the cell death of muscle fibers could be a novel therapeutic strategy to suppress both muscle injury and inflammation in polymyositis. Here, we show that the pattern of cell death of muscle fibers in polymyositis is FAS ligand-dependent necroptosis, while that of satellite cells and myoblasts is perforin 1/granzyme B-dependent apoptosis, using human muscle biopsy specimens of polymyositis patients and models of polymyositis in vitro and in vivo. Inhibition of necroptosis suppresses not only CD8+ cytotoxic T lymphocytes-induced cell death of myotubes but also the release of inflammatory molecules including HMGB1. Treatment with a necroptosis inhibitor or anti-HMGB1 antibodies ameliorates myositis-induced muscle weakness as well as muscle cell death and inflammation in the muscles. Thus, targeting necroptosis in muscle cells is a promising strategy for treating polymyositis providing an alternative to current therapies directed at leukocytes.

From skeletal muscle damage and regeneration to the hypertrophy induced by exercise: what is the role of different macrophage subsets?

Macrophages are one of the top players when considering immune cells involved with tissue homeostasis. Recently, increasing evidence has demonstrated that macrophages could also present two major subsets during tissue healing: proliferative macrophages (M1-like), which are responsible for increasing myogenic cell proliferation, and restorative macrophages (M2-like), which are involved in the end of the mature muscle myogenesis. The participation and characterization of these macrophage subsets are critical during myogenesis to understand the inflammatory role of macrophages during muscle recovery and to create supportive strategies that can improve mass muscle maintenance. Indeed, most of our knowledge about macrophage subsets comes from skeletal muscle damage protocols, and we still do not know how these subsets can contribute to skeletal muscle adaptation. Thus, this narrative review aims to collect and discuss studies demonstrating the involvement of different macrophage subsets during the skeletal muscle damage/regeneration process, showcasing an essential role of these macrophage subsets during muscle adaptation induced by acute and chronic exercise programs.

Fermented rice bran supplementation attenuates chronic colitis-associated extraintestinal manifestations in female C57BL/6N mice.

Patients with inflammatory bowel disease (IBD) have higher incidence of extraintestinal manifestations (EIM), including liver disorders, sarcopenia, and neuroinflammation. Fermented rice bran (FRB), generated from rice bran (RB), is rich in bioactive compounds, and exhibits anti-colitis activity. However, its role in EIM prevention is still unclear. Here, for the first time, we investigated whether EIM in female C57Bl/6N mice is attenuated by FRB supplementation. EIM was induced by repeated administration of 1.5% dextran sulfate sodium (DSS) in drinking water (4 d) followed by drinking water (12 d). Mice were divided into 3 groups-control (AIN93M), 10% RB, and 10% FRB. FRB ameliorated relapsing colitis and inflammation in muscle by significantly lowering proinflammatory cytokines Tnf-α and Il-6 in serum and advanced glycation end product-specific receptor (Ager) in serum and muscle when compared with the RB and control groups. As FRB reduced aspartate aminotransferase levels and oxidative stress, it might prevent liver disorders. FRB downregulated proinflammatory cytokine and chemokine transcripts responsible for neuroinflammation in the hippocampus and upregulated mRNA expression of G protein coupled receptors (GPRs), Gpr41 and Gpr43, in small and large intestines, which may explain the FRB-mediated protective mechanism. Hence, FRB can be used as a supplement to prevent IBD-associated EIM.

Belimumab for Immune-Mediated Necrotizing Myopathy Associated With Anti-SRP Antibodies: A Case Report and Retrospective Review of Patients Treated With Anti-B-Cell Therapy in a Single Center and Literature.

Background: Immune-mediated necrotizing myopathy (IMNM) is characterized by markedly elevated creatinine kinase and histologically scattered necrotic muscle fibers and generally associated with autoantibodies against signal recognition particle (SRP) or 3-hydroxy-3-methylglutaryl-coA-reductase (HMGCR). Poor clinical response to conventional therapies and relapses commonly occur in severe cases. Anti-B-cell therapies have been used in refractory/relapsing cases. Methods: The characteristics of a patient with IMNM associated with anti-SRP antibodies including physical examination, laboratory tests, and disease activity assessment were evaluated. Conventional therapy, belimumab treatment schedule, and follow-up data were recorded. Medical records of IMNM patients treated in our department from September 2014 to June 2021 were reviewed to evaluate the efficacy and safety of anti-B-cell therapy for anti-SRP IMNM. A literature review of patients with anti-SRP IMNM treated with anti-B-cell therapies was performed. Results: We describe a case of a 47-year-old woman with IMNM associated with anti-SRP antibodies who relapsed twice after conventional therapy but showed good response and tolerance to belimumab at 28 weeks follow-up. In this review, three patients from our department were treated with rituximab. Two of the three patients rapidly improved after treatment. Twenty patients and five retrospective studies were included in the literature review. All patients were administered rituximab as an anti-B-cell drug. Conclusion: Despite a lack of rigorous clinical trials, considerable experience demonstrated that anti-B-cell therapy might be effective for patients with IMNM associated with anti-SRP antibodies. Belimumab in association with steroids might be an encouraging option for refractory/relapsing cases.

Programmed Cell Death Pathways in the Pathogenesis of Idiopathic Inflammatory Myopathies.

Idiopathic inflammatory myopathy (IIM) is a heterogeneous group of acquired, autoimmune muscle diseases characterized by muscle inflammation and extramuscular involvements. Present literatures have revealed that dysregulated cell death in combination with impaired elimination of dead cells contribute to the release of autoantigens, damage-associated molecular patterns (DAMPs) and inflammatory cytokines, and result in immune responses and tissue damages in autoimmune diseases, including IIMs. This review summarizes the roles of various forms of programmed cell death pathways in the pathogenesis of IIMs and provides evidence for potential therapeutic targets.

Research progress in immune microenvironment regulation of muscle atrophy induced by peripheral nerve injury.

Denervated skeletal muscular atrophy is primarily characterized by loss of muscle strength and mass and an unideal functional recovery of the muscle after extended denervation. This review emphasizes the interaction between the immune system and the denervated skeletal muscle. Immune cells such as neutrophils, macrophages and T-cells are activated and migrate to denervated muscle, where they release a high concentration of cytokines and chemokines. The migration of these immune cells, the transformation of different functional immune cell subtypes, and the cytokine network in the immune microenvironment may be involved in the regulatory process of muscle atrophy or repair. However, the exact mechanisms of the interaction between these immune cells and immune molecules in skeletal muscles are unclear. In this paper, the immune microenvironment regulation of muscle atrophy induced by peripheral nerve injury is reviewed.

Cas9-specific immune responses compromise local and systemic AAV CRISPR therapy in multiple dystrophic canine models.

Adeno-associated virus (AAV)-mediated CRISPR-Cas9 editing holds promise to treat many diseases. The immune response to bacterial-derived Cas9 has been speculated as a hurdle for AAV-CRISPR therapy. However, immunological consequences of AAV-mediated Cas9 expression have thus far not been thoroughly investigated in large mammals. We evaluate Cas9-specific immune responses in canine models of Duchenne muscular dystrophy (DMD) following intramuscular and intravenous AAV-CRISPR therapy. Treatment results initially in robust dystrophin restoration in affected dogs but also induces muscle inflammation, and Cas9-specific humoral and cytotoxic T-lymphocyte (CTL) responses that are not prevented by the muscle-specific promoter and transient prednisolone immune suppression. In normal dogs, AAV-mediated Cas9 expression induces similar, though milder, immune responses. In contrast, other therapeutic (micro-dystrophin and SERCA2a) and reporter (alkaline phosphatase, AP) vectors result in persistent expression without inducing muscle inflammation. Our results suggest Cas9 immunity may represent a critical barrier for AAV-CRISPR therapy in large mammals.

Dynamics of Polarized Macrophages and Activated CD8+ Cells in Heart Tissue of Atlantic Salmon Infected With Piscine Orthoreovirus-1.

Piscine orthoreovirus (PRV-1) infection causes heart and skeletal muscle inflammation (HSMI) in farmed Atlantic salmon (Salmo salar). The virus is also associated with focal melanized changes in white skeletal muscle where PRV-1 infection of macrophages appears to be important. In this study, we studied the macrophage polarization into M1 (pro-inflammatory) and M2 (anti-inflammatory) phenotypes during experimentally induced HSMI. The immune response in heart with HSMI lesions was characterized by CD8+ and MHC-I expressing cells and not by polarized macrophages. Fluorescent in situ hybridization (FISH) assays revealed localization of PRV-1 in a few M1 macrophages in both heart and skeletal muscle. M2 type macrophages were widely scattered in the heart and were more abundant in heart compared to the skeletal muscle. However, the M2 macrophages did not co-stain for PRV-1. There was a strong cellular immune response to the infection in the heart compared to that of the skeletal muscle, seen as increased MHC-I expression, partly in cells also containing PRV-1 RNA, and a high number of cytotoxic CD8+ granzyme producing cells that targeted PRV-1. In skeletal muscle, MHC-I expressing cells and CD8+ cells were dispersed between myocytes, but these cells did not stain for PRV-1. Gene expression analysis by RT-qPCR complied with the FISH results and confirmed a drop in level of PRV-1 following the cell mediated immune response. Overall, the results indicated that M1 macrophages do not contribute to the initial development of HSMI. However, large numbers of M2 macrophages reside in the heart and may contribute to the subsequent fast recovery following clearance of PRV-1 infection.

Roles of Skeletal Muscle-Derived Exosomes in Organ Metabolic and Immunological Communication.

Skeletal muscles secrete various factors, such as proteins/peptides, nucleotides, and metabolites, which are referred to as myokines. Many of these factors are transported into extracellular bodily fluids in a free or protein-bound form. Furthermore, several secretory factors have been shown to be wrapped up by small vesicles, particularly exosomes, secreted into circulation, and subsequently regulate recipient cells. Thus, exosome contents can be recognized as myokines. In recipient cells, proteins, microRNAs, and metabolites in exosomes can regulate the expression and activity of target proteins associated with nutrient metabolism and immune function. The levels of circulating exosomes and their contents are altered in muscle disorders and metabolic-related states, such as metabolic dysfunction, sarcopenia, and physical fitness. Therefore, such circulating factors could mediate various interactions between skeletal muscle and other organs and may be useful as biomarkers reflecting physiological and pathological states associated with muscular function. Here, this review summarizes secretory regulation of muscle-derived exosomes. Their metabolic and immunological roles and the significance of their circulating levels are also discussed.

Transcriptome Analysis of Immune Receptor Activation and Energy Metabolism Reduction as the Underlying Mechanisms in Interleukin-6-Induced Skeletal Muscle Atrophy.

Background: Inflammation may trigger skeletal muscle atrophy induced by cancer cachexia. As a pro-inflammatory factor, interleukin-6 may cause skeletal muscle atrophy, but the underlying molecular mechanisms have not been explored. Methods: In this experimental study, we used adult male ICR mice, weighing 25 ± 2 g, and the continuous infusion of interleukin-6 into the tibialis anterior muscle to construct a skeletal muscle atrophy model (experimental group). A control group received a saline infusion. RNA-sequencing was used to analyze the differentially expressed genes in tissue samples after one and three days. Gene Ontology and the Kyoto Encyclopedia of Genes and Genomes analysis were applied to define the function of these genes, and protein-protein interaction analysis was performed to identify potential transcription factors. Fluorescence microscopy was used to determine the muscle fiber cross-sectional area after 14 days. Results: Continuous infusion of interleukin-6 for 14 days caused significant muscle atrophy. RNA-sequencing found 359 differentially expressed genes in the 1- and 3-day tissue samples and 1748 differentially expressed genes only in the 3-day samples. Functional analysis showed that the differentially expressed genes found in both the 1- and 3-day samples were associated with immune receptor activation, whereas the differentially expressed genes found only in the 3-day sample were associated with reduced energy metabolism. The expression of multiple genes in the oxidative phosphorylation and tricarboxylic acid cycle pathways was down-regulated. Furthermore, differentially expressed transcription factors were identified, and their interaction with interleukin-6 and the differentially expressed genes was predicted, which indicated that STAT3, NF-κB, TP53 and MyoG may play an important role in the process of interleukin-6-induced muscle atrophy. Conclusions: This study found that interleukin-6 caused skeletal muscle atrophy through immune receptor activation and a reduction of the energy metabolism. Several transcription factors downstream of IL-6 have the potential to become new regulators of skeletal muscle atrophy. This study not only enriches the molecular regulation mechanism of muscle atrophy, but also provides a potential target for targeted therapy of muscle atrophy.

Distinct mechanisms involving diacylglycerol, ceramides, and inflammation underlie insulin resistance in oxidative and glycolytic muscles from high fat-fed rats.

This study investigated whether oxidative and glycolytic rat skeletal muscles respond differently to a high-fat (HF) sucrose-enriched diet with respect to diacylglycerol (DAG) and ceramides accumulation, protein kinase C (PKC) activation, glucose metabolism, and the expression of inflammatory genes. HF diet (8 weeks) suppressed insulin-stimulated glycogen synthesis and glucose oxidation in soleus (Sol), extensor digitorum longus (EDL) and epitrochlearis (Epit) muscles. However, DAG and ceramides levels increased in Sol and EDL, but not in Epit muscles of HF-fed rats. Additionally, membrane-bound PKC-delta and PKC-theta increased in Sol and EDL, whereas in Epit muscles both PKC isoforms were reduced by HF diet. In Epit muscles, HF diet also increased the expression of tumor necrosis factor-α (TNF-α) receptors (CD40 and FAS), toll-like receptor 4 (TLR4), and nuclear factor kappa light polypeptide gene enhancer in B cells (NF-kB), whereas in Sol and EDL muscles the expression of these inflammatory genes remained unchanged upon HF feeding. In conclusion, HF diet caused DAG and ceramides accumulation, PKC activation, and the induction of inflammatory pathways in a fiber type-specific manner. These findings help explain why oxidative and glycolytic muscles similarly develop insulin resistance, despite major differences in their metabolic characteristics and responsiveness to dietary lipid abundance.

Dulaglutide improves muscle function by attenuating inflammation through OPA-1-TLR-9 signaling in aged mice.

Dulaglutide, a glucagon-like peptide-1 receptor (GLP-1R) agonist, is widely used to treat diabetes. However, its effects on muscle wasting due to aging are poorly understood. In the current study, we investigated the therapeutic potential and underlying mechanism of dulaglutide in muscle wasting in aged mice. Dulaglutide improved muscle mass and strength in aged mice. Histological analysis revealed that the cross-sectional area of the tibialis anterior (TA) in the dulaglutide-treated group was thicker than that in the vehicle group. Moreover, dulaglutide increased the shift toward middle and large-sized fibers in both young and aged mice compared to the vehicle. Dulaglutide increased myofiber type I and type IIa in young (18.5% and 8.2%) and aged (1.8% and 19.7%) mice, respectively, compared to the vehicle group. Peroxisome proliferator-activated receptor-gamma coactivator-1α (PGC-1α), a master regulator of mitochondrial biogenesis, decreased but increased by dulaglutide in aged mice. The expression of atrophic factors such as myostatin, atrogin-1, and muscle RING-finger protein-1 was decreased in aged mice, whereas that of the myogenic factor, MyoD, was increased in both young and aged mice following dulaglutide treatment. In aged mice, optic atrophy-1 (OPA-1) protein was decreased, whereas Toll-like receptor-9 (TLR-9) and its targeting inflammatory cytokines (interleukin-6 [IL-6] and tumor necrosis factor-α [TNF-α]) were elevated in the TA and quadriceps (QD) muscles. In contrast, dulaglutide administration reversed this expression pattern, thereby significantly attenuating the expression of inflammatory cytokines in aged mice. These data suggest that dulaglutide may exert beneficial effects in the treatment of muscle wasting due to aging.

Immunomodulation and Biomaterials: Key Players to Repair Volumetric Muscle Loss.

Volumetric muscle loss (VML) is defined as a condition in which a large volume of skeletal muscle is lost due to physical insult. VML often results in a heightened immune response, resulting in significant long-term functional impairment. Estimates indicate that ~250,000 fractures occur in the US alone that involve VML. Currently, there is no active treatment to fully recover or repair muscle loss in VML patients. The health economics burden due to VML is rapidly increasing around the world. Immunologists, developmental biologists, and muscle pathophysiologists are exploring both immune responses and biomaterials to meet this challenging situation. The inflammatory response in muscle injury involves a non-specific inflammatory response at the injured site that is coordination between the immune system, especially macrophages and muscle. The potential role of biomaterials in the regenerative process of skeletal muscle injury is currently an important topic. To this end, cell therapy holds great promise for the regeneration of damaged muscle following VML. However, the delivery of cells into the injured muscle site poses a major challenge as it might cause an adverse immune response or inflammation. To overcome this obstacle, in recent years various biomaterials with diverse physical and chemical nature have been developed and verified for the treatment of various muscle injuries. These biomaterials, with desired tunable physicochemical properties, can be used in combination with stem cells and growth factors to repair VML. In the current review, we focus on how various immune cells, in conjunction with biomaterials, can be used to promote muscle regeneration and, most importantly, suppress VML pathology.