Muscle cells could serve as antigen-presenting cells, and participate in the activation of immune response. Immunological characteristics of muscle cells, and their capacities to equip themselves with immunorelevant molecules, remain to be elucidated. In this study, we investigated the immunological properties of myoblasts and differentiated myotubes in vitro and in vivo, under the IFN-γ induced inflammatory condition. We found that the fused C C myotubes are more sensitive to inflammatory stimulation, and significantly upregulated the expression levels of MHC-I/II and TLR3/7 molecules, than that of proliferated myoblasts. As well, some co-stimulatory/-inhibitory molecules, including CD40, CD86, ICAM-I, ICOS-L, and PD-L1, were prominently upregulated in IFN-γ induced myotubes. Notably, we detected the protein levels of ASC, NLRP3, and Caspase-1 increased in stimulated myotubes, and IL-1β in cell culture supernatant, implying the activation of NLRP3 inflammasomes in IFN-γ treated myotubes. The pro-inflammatory cytokines and chemokines mRNA levels in IFN-γ induced C C myotubes and myoblasts, involving IL-1, IL-6, and MCP-1, increased markedly. T cell activation test further verified IFN-γ induced C C myotubes prompt to the proliferation of the splenic CD4 and CD8 T cells. In Cardiotoxin-damaged tibialis anterior (TA) muscle, some regenerated myofibers expressed both MHC class I and class II molecules under IFN-γ enhanced inflammatory condition. Thus, our work demonstrates that muscle cells are active participants of local immune reactions. Anat Rec, 301:1551-1563, 2018. © 2018 Wiley Periodicals, Inc.

The objective of this study is to investigate the role of Calmodulin-dependent protein kinase IV (CaMKIV) in Cardiotoxin (CTX)-induced mice muscle inflammation. CTX injection i.m. was performed to induce B6 mice acute tibialis anterior (TA) muscle injury. The mice were then injected i.p. with the recombinant CaMKIV protein or its antagonist KN-93. Immunoblotting was used to assess Calmodulin (CaM) and CaMKIV levels. Immunofluorescence was used to detect intramuscular infiltration or major histocompatibility complex (MHC)-I expression in damaged muscle. The extent of infiltration was evaluated by fluorescent intensity analysis. Cytokines/chemokines levels were determined by qPCR. CaMKIV gene knockdown in C2C12 cells was performed in order to evaluate the effects of CaMKIV on immuno-behavior of muscle cells. CTX administration induced a strong up-regulation of CaM and p-CaMKIV levels in infiltrated mononuclear cells and regenerated myofibers. In vivo adding of the recombinant CaMKIV protein enhanced intramuscular infiltration of monocytes/macrophages in damaged muscle and increased the number of proinflammatory Ly-6CF4/80 macrophage cells. CaMKIV protein treatment induced a striking up-regulation of mRNA levels of IL-1, IL-6, MCP-1, and MCP-3 in CD45 cells sorted from damaged muscle; increased the infiltration of CD8 T cells; and induced the up-regulation of MHC-I in partial regenerated myofibers, which was rarely observed in muscle damage alone. Additionally, CaMKIV protein treatment diminished the regulatory T cells (Tregs) number and led to the damaged TA muscle repair delay. In vitro CaMKIV gene knockdown reversed IFN-γ-induced up-regulation of MHC-I/II and TLR3 in the differentiated C2C12 myotubes. CaMKIV can act as an immunostimulation molecule and enhances the acute muscle inflammatory responses.

Key points There is a close relationship between skeletal muscle physiology and Ca2+/calmodulin (CaM) signalling. Despite the effects of Ca2+/CaM signalling on immune and inflammatory responses having been extensively explored, few studies have investigated the role of CaM pathway activation on the post‐injury muscle inflammatory response. In this study, we investigated the role of CaM‐dependent signalling in muscle inflammation in cardiotoxin induced myoinjuries in mice. The Ca2+/calmodulin‐dependent protein kinase II (CaMII), Ca2+/calmodulin‐dependent protein kinase IV (CaMKIV), and nuclear factor of activated T cells (NFAT) pathways are likely to be simultaneously activated in muscle cells and in infiltrating lymphocytes and to regulate the immune behaviours of myofibres in an inflammatory environment, and these pathways ultimately affect the outcome of muscle inflammation. Abstract Calcium/calmodulin (Ca2+/CaM) signalling is essential for immune and inflammatory responses in tissues. However, it is unclear if Ca2+/CaM signalling interferes with muscle inflammation. Here we investigated the roles of CaM‐dependent signalling in muscle inflammation in mice that had acute myoinjuries in the tibialis anterior muscle induced by intramuscular cardiotoxin (CTX) injections and received intraperitoneal injections of either the CaM inhibitor calmidazolium chloride (CCL) or CaM agonist calcium‐like peptide 1 (CALP1). Multiple inflammatory parameters, including muscle autoantigens and toll‐like receptors, mononuclear cell infiltration, cytokines and chemokines associated with peripheral muscle inflammation, were examined after the injury and treatment. CALP1 treatment enhanced intramuscular infiltration of monocytes/macrophages into the damaged tibialis anterior muscle and up‐regulated mRNA and protein levels of muscle autoantigens (Mi‐2, HARS and Ku70) and Toll‐like receptor 3 (TLR3), and mRNA levels of tumor necrosis factor α (TNF‐α), interleukin‐6 (IL‐6), Monocyte chemoattractant protein‐1 (MCP1), Monocyte chemoattractant protein‐3 (MCP3) and Macrophage inflammatory protein‐1(MIP‐1α) in damaged muscle. In contrast, CCL treatment decreased the intramuscular cell infiltration and mRNA levels of the inflammatory mediators. After CALP1 treatment, a substantial up‐regulation in Ca2+/calmodulin‐dependent protein kinase II (CaMKII), Ca2+/calmodulin‐dependent protein kinase IV (CaMKIV) and nuclear factor of activated T cells (NFAT) activity was detected in CD45+ cells isolated from the damaged muscle. More pro‐inflammatory F4/80+Ly‐6C+ cells were detected in CD45‐gated cells after CALP1 treatment than in those after CCL treatment or no treatment. Consistently, in interferon‐γ‐stimulated cultured myoblasts and myotubes, CALP1 treatment up‐regulated the activities of CaMKII, CaMKIV and NFAT, and levels of class I/II major histocompatibility complexes (MHC‐I/II) and TLR3. Our findings demonstrated that CaM‐dependent signalling pathways mediate the injury‐induced acute muscle inflammatory response.

Background/Aims: Whether calcium/calmodulin-dependent protein kinase IV (CaMKIV) plays a role in regulating immunologic features of muscle cells in inflammatory environment, as it does for immune cells, remains mostly unknown. In this study, we investigated the influence of endogenous CaMKIV on the immunological characteristics of myoblasts and myotubes received IFN-γ stimulation. Methods: C₂C₁₂ and murine myogenic precursor cells (MPCs) were cultured and differentiated in vitro, in the presence of pro-inflammatory IFN-γ. CaMKIV shRNA lentivirus transfection was performed to knockdown CaMKIV gene in C₂C₁₂ cells. pEGFP-N1-CaMKIV plasmid was delivered into knockout cells for recovering intracellular CaMKIV gene level. CREB1 antagonist KG-501 was used to block CREB signal. qPCR, immunoblot analysis, or immunofluorescence was used to detect mRNA and protein levels of CaMKIV, immuno-molecules, or pro-inflammatory cytokines and chemokines. Co-stimulatory molecules expression was assessed by FACS analysis. Results: IFN-γ induces the expression or up-regulation of MHC-I/II and TLR3, and the up-regulation of CaMKIV level in muscle cells. In contrast, CaMKIV knockdown in myoblasts and myotubes leads to expression inhibition of the above immuno-molecules. As well, CaMKIV knockdown selectively inhibits pro-inflammatory cytokines/chemokines, and co-stimulatory molecules expression in IFN-γ treated myoblasts and myotubes. Finally, CaMKIV knockdown abolishes IFN-γ induced CREB pathway molecules accumulation in differentiated myotubes. Conclusions: CaMKIV can be induced to up-regulate in muscle cells under inflammatory condition, and positively mediates intrinsic immune behaviors of muscle cells triggered by IFN-γ.

Inorganic/organic hybrid silica-chitosan (CS) scaffolds have promising potential for bone defect repair, due to the controllable mechanical properties, degradation behavior, and scaffold morphology. However, the precise in vivo immuno-reactivity of silica-CS hybrids with various compositions is still poorly defined. In this study, we fabricated the three-dimensional (3D) interconnected porous chitosan-silica (CS/SiO ) and chitosan-silica-hydroxyapatite (CS/SiO /HA) hybrids, through sol-gel process and 3D plotting skill, followed by the naturally or freeze drying separately. Scanning electron microscopy demonstrated the hybrids possessed the uniform geometric structure, while, transmission electron microscopy displayed nanoscale silica, or HA nanoparticles dispersed homogeneously in the CS matrix, or CS/silica hybrids. After intramuscular implantation, CS/SiO and CS/SiO /HA hybrids triggered a local and limited monocyte/macrophage infiltration and myofiber degeneration. Naturally dried CS/SiO hybrid provoked a more severe inflammation than the freeze-dried ones. Dendritic cells were attracted to invade into the implants embedded-muscle, but not be activated to prime the adaptive immunity, because the absence of cytotoxic T cells and B cells in muscle received the implants. Fluorescence-activated cell sorting (FACS) analysis indicated the implanted hybrids were incapable to initiate splenocytes activation. Plasma complement C3 enzyme linked immunosorbent assay (ELISA) assay showed the hybrids induced C3 levels increase in early implanting phase, and the subsequent striking decrease. Thus, the present results suggest that, in vivo, 3D plotted porous CS/SiO and CS/SiO /HA hybrids are relatively biocompatible in vivo, which initiate a localized inflammatory procedure, instead of a systematic immune response. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 1223-1235, 2018.

Digital skin defects resulting from trauma are often associated with dysfunction of the digital nerve and the extensor and flexor tendons in the affected fingers. The repair of these complex tissue defects requires a graft containing multiple tissues that can be used to reconstruct the tendons and nerves and restore the skin. Such procedures can cause multiple injuries and significant damage to the donor site. The current study used a novel technique to repair complex dorsal and palmar digital soft‐tissue defects. First, multiple tissues were cut and collected from the donor site. Then, part of the flexor carpi ulnaris tendon was transplanted to repair the tendon defect, and a medial antebrachial cutaneous nerve graft was used to repair the digital nerve defect. Finally, a skin flap was used to cover the skin defect. This paper reports on 31 cases of complex soft‐tissue digital defects, with defect areas of 2–18 cm2. One patient presented with a postoperative arterial crisis in the flap. All other patients recovered without experiencing a vascular crisis, flap necrosis, or wound infection. The postoperative flaps were similar in texture to the original digital skin. The sensation and the extension/flexion functions in the affected fingers recovered well. The effect on grip strength, wrist flexion, and forearm sensation was minor and the postoperative total active motion scores of the affected digits were good or excellent in 96.77% of the cases. The flap sensation recovery rate was also excellent in 83.87% of the cases. The present technique facilitates the repair of multiple dorsal and palmar digital soft‐tissue, tendon and nerve defects, reduces the damage to the donor site, and significantly improves the success of surgical repair.

Background Proinflammatory microglia rely predominantly on glycolysis to maintain cytokine production during an inflammatory response. However, during ischemia, where glucose supply is low, inducible nitric oxide synthase (iNOS) production remains high accompanied by an increase in monocarboxylate transporter 1 (MCT1) expression. In this study, we explored whether there is a link between iNOS and MCT1 expressions, and whether pyruvate can act as an energy source to sustain the M1 phenotype. Methods Using a mouse model with laser-induced brain ischemia and cell culture with low-glucose treatment, we examined responses from microglia. Results The expressions of iNOS and MCT1, as well as arginase-1 (ARG1), were increased in the brain of the ischemic mouse model. In the BV2 microglial cell line and primary microglia treated under low glucose condition, iNOS and MCT1 also increased, while ARG1 decreased. The addition of pyruvate under low-glucose or lipopolysaccharide (LPS) treatment enhanced iNOS and MCT1 expressions compared with groups without pyruvate. MCT1 knockdown resulted in decreased iNOS while MCT1 overexpression increased iNOS. Furthermore, inhibitor of nuclear factor-kappaB (NF-κB) reduced both iNOS and MCT1. Conclusion Our data suggested that after proinflammatory microglial polarization, MCT1 is upregulated through the NF-κB signaling pathway, which leads to iNOS production. We speculate that microglia may continuously pick up monocarboxylates such as pyruvate through MCT1 to sustain the M1 phenotype.

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