Immunological Behaviors Of Myoblastes/Differentiated Myotubes In Vitro Under Inflammatory Environment

Skeletal muscle is the largest cell bank in human body, as well as a common place for immune responses (such as autoimmune and infectious muscle diseases, intramuscular vaccines and gene therapy, etc.). Muscle satellite cells and mature muscle fibers do not express detectable levels of MHC molecules in physiological conditions. But research has shown that, human myoblasts cultured in vitro can express MHC-Ⅰ (HLA I, HLA class Ⅰ) and pro-inflammatory cytokines (such as IFN-γ, TNF-α, IL-1α, IL-1β,or MIP-1α) will further up-regulate the expression of HLA-Ⅰ. Myoblasts and differentiated myotubes will express MHC-Ⅱ molecules (HLA class-Ⅱ) under the IFN-y induced inflammatory condition.Therefore, myoblasts and myocytes have been regarded to own the intrinsic capacity acting as facultative antigen presenting cells (APCs).Myoblasts cultured in vitro can express immune proteasome subunit (eg. LMP2, LMP7, MECL1), hand the endogenous antigen (via MHC-Ⅰ pathway) and recognize CD8+T cell. Myoblasts also express key enzymes relevant to MHC-Ⅱ antigen processing and presentation (eg Cathepsins) under inflammatory condition, participate exogenous or endogenous antigens presenting to CD4+T cells. Human myoblasts after inflammatory stimulation continued express adhesion molecules (such as LFA-1, ICAM-1) and costimulatory molecules (such as ICOS-L, PD-L1 and CD40). All these researches supported that skeletal muscle cells have capacity acting as APCs in an inflammatory environment and participate in driving the process of adaptive immune responses.Researches of the immunological behaviors of skeletal muscle cells have mostly used human myoblasts and differentiated myotubes. Rarely reported to use myoblast cell lines (such as C2C12, L6 cells). Skeletal muscle cells themselves are the targets of autoimmune attack in inflammatory muscle diseases and in myasthenia gravis. The functional properties of muscle cells relevant for driving the immune response within the muscular tissue, and the capacities of muscle cells to equip themselves with immunorelevant molecules, remains to be elucidated.In this study, we explored systematically the immunological characteristics of C2C12 myoblasts and differentiated myotubes in vitro, under the IFN-y induced inflammatory condition. Our data indicate that, the fused myotubes are more sensitive to inflammatory stimulation, and significantly high up-regulated MHC-Ⅰ/Ⅱ molecules and TLR3/7 under the IFN-y inducing, than that of proliferated myoblasts. As well, some co-stimulatory/-inhibitory molecules, including CD40, CD86, ICAM-I, ICOS-L, and PD-L1, were prominently up-regulated in IFN-y induced myotubes. Notably, we detected the protein level increase of ASC, NLRP3 and Caspase-1 in stimulated myotubes, and of IL-1β in cell supernatant, which imply the activation of NLRP3 inflammasomes in IFN-y treated myotubes. The pro-inflammatory cytokines and chemokines mRNA expression in IFN-y induced myotubes and myoblasts, involving IL-1, IL-6, and MCP-1, increased markedly. Using T cell activation test, we further proved APC function of myotubes in vitro, since we verified IFN-y induced C2C12 cells prompt to the proliferation of the isolated B6 mice splenic CD4+and CD8+T cells. Our work provides supportive evidences for the immunological capabilities of skeletal muscle cells (especially the differentiated myotubes), in this context, skeletal muscle cells, especially the fused myotubes, should be considered as active participants of immune reactions.Four parts of our research:Part one:C2C12 cell’s culture and differentiation in vitroObjective:Get cultured C2C12 myoblasts and in differentiated myotubes.Methods:After thawed by general procedure, C2C12 cells were implanted in the culture bottle and cultivated in DMEM/F12 complete medium supplemented with 100 U/mL penicillin,100 ug/mL streptomycinand 10% fetal bovine serum. They were routine cultured in the incubator at 37℃and with 5% CO2. C2C12 Cells were grown to approximately 70-80% confluence (myoblasts) and then differentiated by 2% horse serum (myotubes) about 48h. Cells were viewed with a microscope everyday.Results:The undifferentiated C2C12 cells grow as fusiform, grow rapidly. Changing the volume fraction of 2% horse serum in DMEM/F12 medium 3 to 4 days, cell fusion occurs and myotube formation can be seen under the microscope, as long as cell density increases, more than two nuclear myotubes appears.Conclusions:, C2C12 cell growth well under conventional in vitro culture conditions, C2C12 cells are well-differentiated by the induce of horse serum, can be used for further analysis.Part two:The express of MHC molecules and TLRS in C2C12 myoblasts and myotubes in vitro under inflammatory environment.Objective:Investigated the expression of MHC-Ⅰ/Ⅱ molecules and Toll-like receptors (TLRs) in cultured myoblasts and in differentiated myotubes (myofibers) under inflammatory environment, and analyzed whether there skeletal muscle cells have the potential to become antigen presenting cells.Methods:For proinflammatory stimulation, myoblasts or myotubes were treated with IFN-y in starved medium with or without horse serum, respectively, and analyzed after 24h and 48h. C2C12 cells were cultured in IFN-y-constructed inflammatory environment. We investigated the expression of MHC-I/II molecules and Toll-like receptors (TLRs) in cultured myoblasts and in differentiated myotubes (myofibers) by using Western blot, FACS analysis and immunofluorecence staining.Results:MHC-I molecule H-2Kb, Toll-like receptors 3 and 7 (TLR3/TLR7) continuously low-expressed in C2C12 cells, and their protein levels significantly up-regulated under IFN-y treatment. We noticed under the inflammatory condition, house serum differentiated myotubes expressed H-2Kb and TLR3/7 more highly, than cultured myoblasts, and reached the peak at 48h after IFN-y stimulation. Instead, MHC-Ⅱ molecule H2-Ea expressed in myotubes, and the level is significantly increased by IFN-γ treatment. Using q-PCR、FACS analysis and immunofluorecence staining, we further confirmed the IFN-γ induced expression increase of TLR3/7 and of MHC-Ⅰ/Ⅱ in C2C12 myotubes.Conclusions:These data indicate that, in vitro, muscle cells are inducible to express immune-related molecules. Of note, myotubes express immune molecules more markedly than that of myoblasts under pro-inflammatory condition, suggesting differentiated myotubes are more sensitive to inflammatory stimulation.Part three:Other immunological behaviors of skeletal muscle cells in vitro under inflammatory environmentObjective:To investigate the immunological characteristics of C2C12 myoblasts and differentiated myotubes in vitro, under the IFN-γ induced inflammatory condition, including NLRP3 inflammasome, some pro-inflammatory cytokines, chemokines and co-stimulatory/-inhibitory molecules.Methods:C2C12 cells were cultured in IFN-γ-constructed inflammatory environment, added with or without 2% horse serum. We investigated the expression of mRNA expression of some pro-inflammatory cytokines by qRT-PCR, including IL-1α、IL-1β、IL-6、IL-8、IL-10、IL-15、IL-18、TGF-β、MCP-1、MIP-1a and TNF-α. And investigated the expression of some co-stimulatory/-inhibitory molecules, including CD40, CD86, ICAM-I, ICOS-L and PD-L1 by FACS analysis. We detected the expression of ASC, NLRP3 and Caspase-1 by immunofluorecence staining and Western blot, and IL-1β level in the cell culture medium was testified by ELISA assay.Results:Immunofluorecence staining、 Western blot and q-PCR analysis showed that, NLRP3, ASC and mature Caspase-1 was induced in IFN-y treated-myotubes (especially in 48h-cultured cells). Our qRT-PCR analysis demonstrated that, un-stimulated myoblasts and myotubes expressed low constitutive levels of pro-inflammatory IL-1, IL-6, IL-18, and MCP-1, but not for IL-8 and IL-15. In IFN-γ constructed inflammatory milieu, house serum-differentiated myotubes, but not myoblasts, up-regulated mRNA levels of IL-1, IL-6, and MCP-1 dramatically. Besides, we confirmed IFN-γ induced-myoblasts and -myotubes were cellular source of anti-inflammatory IL-10, because IL-10 mRNA level was detected to be up-regulated markedly in these cells, especially in IFN-γ induced-myotubes. Very little change was detected for TGF-β levels in C2C12 cells after IFN-γ stimulation. ELISA analysis showed that, by the presence of IFN-γ, IL-1β concentration was dramatically higher in differentiated myotubes, than that of myoblasts.Our FACS analysis showed that, cultured C2C12 myoblasts and myotubes express CD80 constitutively, but IFN-γ treatment had no effects on the cell number with CD80+ phenotype. In contrast with CD80 expression, a higher proportion of CD86+ cells were found, involving both myoblasts and myotubes, under IFN-γ stimulation. Un-stimulated C2C12 cells express CD40, ICOS-L and ICAM-1, and positive cell numbers dramatically increased by the proinflammatory stimuli. Interestingly, we found PD-L1 expressed in muscle cells with an IFN-γ-dependent way completely, demonstrating dramatically increase of the positive proportion following IFN-γ stimulation.Conclusions:Our data indicated the immunobiology of muscle cells in inflammatory condition in vitro, through the activation of NLRP3 inflammasome pathway.Muscle cells, especially myotubes, are capable of selectively expressing a variety of cytokines/chemokines in response to inflammation microenvironment in vitro. And the presence of these co-stimulatory molecules further emphasizes the important immunological capacities of muscle cells and their role in muscle immune interactions.Part four:Inflammatory-induced differentiated myotubes promote T cell activation and proliferationObjective:Evaluating whether muscle cells could be responsible for the activation and proliferation of T cells. To verify its potential as antigen-presenting cells.Methods:CFSE-labeled CD8+/CD4+ T cells obtained from the spleen of wild B6 mice and purified by magnetic sorting using a negative isolation kit, were co-cultured with the 48h-IFN-γ-pulsed myotubes, in the presence of Con A. Proliferation of gated CD8+, or CD4+cells was evaluated by flow cytometry at 48h after co-culturing.Results:Con A induced CD8+and CD4+T cells to prime and proliferate in vitro. Not surprisingly, CD8+and CD4+T cells co-cultured with IFN-γ-treated myotubes appeared to proliferate more intensely, than that with untreated cells.Conclusions:These data suggested that, muscle cells, especially the differentiated myotubes, can be induced to obtain immunological properties in vitro, be recognized by T cells in a MHC-restricted fashion, and therefore can promote T cell proliferation.