急性炎症环境诱导的成肌细胞/肌纤维的免疫特性及其与TGF-B2和NO的相关性研究

In vivo, under physiological conditions, neither MHC I nor MHC II antigens are detectable on normal mature muscle fibers, although MHC class I and/or II was observed in muscle tissue of patients with idiopathic inflammatory myopathies. Recently, we prepared acute myoinjury mice model using different ways (Notexin/Cardiotoxin i.m., or Crush). Unexpectedly, we found MHC-Class I Expression in regenerated myofiber is rather occasional, corresponding with that, is the rarely detected CD8+ T cell in injuried muscle tissue. It implies that, inflammatory condition induced by acute-myoinjury is completely different to that in chronic-pathologic inflammation conditions, and MHC expression in acute damage induced-regenerated myofibers is blocked. This leads to the following questions: i) Whether normal muscle cells（myoblasts and myotubes）have ability to function as immune cells in the inflammatory environment? ii) Why muscle cells don’t express MHC molecules in acute muscle inflammatory condition? and which factors involved in the inhibition of immune features of muscle cells.. In this program, C2C12 cells, primary mice and human myoblasts will be cultured, differentiated, and stimulated with IFN-，LPS，Alum，or CFA. Immunologic features of myoblasts/myotubes, including the expression of MHC-I/MHC-II and co-stimulatory molecules, the production of immunorelevant cytokines, adhesion molecule and chemokines, the formation of NLRP3 inflammasomes, and the effects of muscle cells on T cell activation, will be detected and analyzed. For exploring the effects of TGF-2，NO and NLRP3 inflammasomes on immunologic features of myoblasts/myotubes, cultured cells will be imposed with chemical interference, or lentivirus transfer. In vivo study will be performed using SM-OVA Tg mice received TA muscle damage, MHC gene over-expression or not, and finally OT-I/II cell transfer. The intramuscular infiltration of OT-I/II cells, MHC expression and NLRP3 inflammasomes formation in new myofibers will be analyzed.

炎性肌病患者的再生肌纤维表达MHC-I/II分子并具备抗原呈递功能，能主动参与肌内炎症反应。我们的前期研究却发现，小鼠急性肌损伤时，罕有肌纤维表达MHC分子。这提示，不同于慢性病理损伤，急性损伤时再生肌纤维MHC表达被抑制，缺乏针对肌细胞的自身免疫。本项目将体外培养C2C12细胞、小鼠和人的原代成肌细胞并进行炎性刺激，分析成肌细胞/分化肌管的免疫特性和T细胞激活效应、以及TGF-B2，NO，NLRP3炎症小体对肌细胞免疫特性的干扰效应。制备SM-OVA Tg小鼠急性肌损伤模型并诱导肌内MHC-I/II过表达，或化学干扰TGF-B2和NO体内水平; OT细胞获得性转移。分析再生肌纤维的免疫特性、OT细胞肌内渗出、炎症小体活性改变。通过上述研究, 明确炎性刺激能否诱导肌细胞转化为免疫细胞并表达MHC和共刺激分子，分析急性损伤后抑制肌纤维免疫特性的可能原因，为骨骼肌的免疫耐受机制提供新线索。

本项目以探寻骨骼肌损伤及炎症反应的干预因素为出发点，主要研究内容涉及：i) 炎性环境中骨骼肌纤维的免疫行为；ii) 肌纤维TGF-β条件敲除鼠（SM TGF-βr2−/−）、肌纤维特异表达OVA抗原转基因小鼠（MCK3E-OVA Tg）的构建及鉴定；iii) 炎性环境中肌组织及肌纤维ER Stress和UPR反应；iv) CAMKIV信号对肌内炎症及肌纤维免疫功能的干预；v) 骨骼肌TGF-b信号对UPR反应、肌内炎症和修复的影响。课题组成功构建SM TGF-βr2−/−小鼠、MCK3E-OVA Tg小鼠。同时，我们的研究证实，体外炎性环境中（IFN-γ刺激），分化肌管表达多种炎性分子（如，MHC-I/II、TLR3/7， CD40, CD86, ICAM-I, ICOS-L等）。持续促炎刺激将激活肌纤维UPR反应，通过IRE1α通路下调 p38 MAPK信号而实现对肌纤维免疫功能的抑制。 急性损伤诱导骨骼肌CaM、CaMKIV表达上调。该信号的持续活化可能激活肌纤维免疫功能，加重肌内炎症。此外，损伤骨骼肌TGF-β表达持续上调，有助于下调肌内炎性渗出，促进损伤肌组织再生。内源性TGF-β信号能直接干预肌纤维免疫特性，影响肌纤维内促炎细胞因子/趋化因子分泌。本项目的研究结果为治疗肌损伤导致的严重炎性肿胀提供了线索。