Background: Estrogen signaling is indispensable for muscle regeneration, yet the role of estrogen in the development of muscle inflammation, especially in the intramuscular T cell response, and the influence on the intrinsic immuno-behaviors of myofibers remain largely unknown. We investigated this issue using the mice model of cardiotoxin (CTX)-induced myoinjury, with or without estrogen level adjustment. Methods: CTX injection i.m. (tibialis anterior, TA) was performed for preparing mice myoinjury model. Injection s.c. of 17β-estradiol (E₂) or estrogen receptor antagonist 4-OHT, or ovariectomy (OVX), was used to change estrogen level of animal models in vivo. Serum E₂ level was evaluated by ELISA. Gene levels of estrogen receptor (ERs) and cytokines/chemokines in inflamed muscle were monitored by qPCR. Inflammatory infiltration was observed by immunofluorescence. Macrophage and T cell phenotypes were analyzed by FACS. Immunoblotting was used to assess protein levels of ERs and immunomolecules in C₂C₁₂ myotubes treated with E₂ or 4-OHT, in the presence of IFN-γ. Results: We monitored the increased serum E₂ level and the upregulated ERβ in regenerated myofibres after myotrauma. The absence of estrogen in vivo resulted in the more severe muscle inflammatory infiltration, involving the recruitment of monocyte/macrophage and CD4⁺ T cells, and the heightened proinflammatory (M1) macrophage. Moreover, estrogen signaling loss led to Treg cells infiltration decrease, Th1 response elevation in inflamed muscle, and the markedly expression upregulation of immunomolecules in IFN-γ-stimulated C₂C₁₂ myotubes in vitro. Conclusion: Our data suggest that estrogen is a positive intervention factor for muscle inflammatory response, through its effects on controlling intramuscular infiltration and phenotypes of monocytes/macrophages, on affecting accumulation and function of Treg cells, and on suppressing Th1 response in inflamed muscle. Our findings also imply an inhibition effect of estrogen on the intrinsic immune behaviors of muscle cells.

Hydroxyapatite (HAp) particles with different morphology but similar physicochemical properties were prepared by a novel and facile route. The interaction between the particles with BMSCs was investigated, and results revealed that particles’ shape has a strong influence on cellular behavior.

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 C₂C₁₂ 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 C₂C₁₂ myotubes and myoblasts, involving IL‐1, IL‐6, and MCP‐1, increased markedly. T cell activation test further verified IFN‐γ induced C₂C₁₂ 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.

Excessive inflammatory response may delay the regeneration and damage the normal muscle fibers upon myoinjury. It would be important to be able to attenuate the inflammatory response and decrease inflammatory cells infiltration in order to improve muscle regeneration formation, resulting in better muscle functional recovery after myoinjury. This study was undertaken to explore the role of Nitric oxide (NO) during skeletal muscle inflammatory process, using a mouse model of Notexin induced myoinjury. Intramuscular injection (tibialis anterior, TA) of Notexin was performed for preparing mice myoinjury. NO synthase inhibitor (L-NAME) or NO donor (SNP) was intraperitoneally injected into model mice. On day 4 and 7 post-injury, expression of muscle-autoantigens and toll-like receptors (TLRs) was evaluated from muscle tissue by qRT-PCR and Western Blot; the intramuscular infiltration of monocytes/macrophage (CD11b⁺ or F4/80⁺ cells), CD8⁺ T cell (CD3ε⁺CD8α⁺), apoptotic cell (CD11b⁺caspase3⁺), and MHC-I molecule H-2K^b-expressing myofibers in damaged muscle were assessed by imunoflourecence analysis; the mRNAs expression of cytokines and chemokines associated with the preferential biological role during the muscle damage-induced inflammation response, were assessed by qRT-PCR. We detected the reduced monocytes/macrophages infiltration, and increased apoptotic cells in the damaged muscle treated with SNP comparing to untreatment. As well, SNP treatment down-regulated mRNA and protein levels of muscle autoantigens, TLR3, and mRNA levels of TNF-α, IL-6, MCP-1, MCP-3, and MIP-1α in damaged muscle. On the contrary, L-NAME induced more severe intramuscular infiltration of inflammatory cells, and mRNA level elevation of the above inflammatory mediators. Notably, we observed an increased number of MHC-I (H2-K^b) positive new myofibers, and of the infiltrated CD8⁺ T cells in damaged muscle at the day 7 after L-NAME treatment. The result herein shows that, NO can act as an endogenous anti-inflammatory molecule during the ongoing muscle inflammation. Our finding may provide new insight to optimize NO-based therapies for improving muscle regeneration after myoinjury.