从CD4+T细胞的分化探讨由α7-nAChR介导的胆碱能抗炎通路抑制VMC炎症反应的机制

CD4+ helper T cell types 1, 2, 17 and Treg have been implicated in the pathogenesis of myocarditis after viral infection. Recently, we have found that excessive sympathetic activation in viral myocarditis aggravates inflammatory lesions, and vagus nerve stimulation and activation of cholinergic antiinflammatory pathway attenuate inflammation in viral myocarditis through α7 nicotinic acetylcholine receptor (α7-nAChR). Therefore, the results provide us a novel insight about the mechanisms involved in the pathogenesis of viral myocarditis and suggest that dysfunctional autonomic neural-immune circuit (sympathetic activation and vagal withdrawal) is associated with the development of viral myocarditis. However, the effects of cholinergic antiinflammatory pathway on CD4+ T cell responses in viral myocarditis are unknown. Our previous studies have demonstrated that activation of cholinergic antiinflammatory pathway via α7-nAChR downregulates the mRNA expression of Th1 and Th17 cytokines, and α7-nAChR is expressed by CD4+ T cells. Hence, we postulate that activation of cholinergic antiinflammatory pathway could ameliorate the severity of myocarditis by regulating the differentiation of Th cell subsets through CD4+ T cell α7-nAChR. In the present study, we will investigate the effects of cholinergic antiinflammatory pathway on CD4+ T cell responses in a coxsackievirus B3 murine myocarditis model. In addition, CD4+ T cells were isolated from murine splenocytes by immunomagnetic beads and cultured in vitro. The agonist and antagonist of α7-nAChR will be administered, and the right cervical vagus nerve will be cut off in the infected mice. The proportion of the Th cell subsets, levels of CD4+ T cell α7-nAChR, inflammatory cytokines, NF-κB, pJAK and pSTAT protein expression within the myocardium will be determined. The most novel insight of the study is that the nervous system may modulate the inflammatory response in viral myocaditis. The study will help elucidate the novel mechanism involved in the pathogenesis of viral myocarditis, and open new possibilities in the therapeutic management of viral myocarditis.

Th细胞亚群比例及分化异常与病毒性心肌炎（VMC）的发生发展密切相关。我们前期研究发现，交感神经系统和迷走神经相关的胆碱能抗炎通路（CAP）在VMC中分别介导促炎和抗炎作用，因此提出了自主神经-免疫调节系统失衡参与VMC发生发展的新观点。然而，CAP能否通过影响Th细胞亚群分化来抑制VMC炎症尚未见报道。申请者发现CAP兴奋后可抑制VMC小鼠Th1和Th17细胞相关炎症因子的表达，而且CD4+T细胞表面表达α7nAChR，这些启示我们CAP很可能通过影响Th分化来抑制VMC的炎症反应。本项目以VMC小鼠及其脾脏CD4+T细胞为研究对象，从兴奋/阻滞α7nAChR及切断颈部迷走神经着手，通过在体和离体两方面观察激活及阻断CAP对Th细胞亚群分化的影响，解析CAP调控VMC小鼠Th细胞亚群分化的机制。本项目最大的特色是将VMC的机制研究深入神经免疫学领域，从而为VMC的治疗提供一个崭新的思路。

CD4+T细胞(help T cell，Th细胞)亚群比例和分化异常与病毒性心肌炎(VMC)的发生发展密切相关。我们前期的研究表明，迷走神经相关的胆碱能抗炎通路(CAP)能够减弱VMC的炎症反应，下调Th1和Th17细胞相关炎症因子的表达，而且CD4+T细胞表面表达α7烟碱型乙酰胆碱受体（α7nAChR）。这提示迷走神经相关的CAP可能通过改变Th细胞的分化来减弱VMC的炎症反应。本研究的目的是探讨迷走神经相关的CAP对VMC小鼠CD4+T细胞分化的影响。我们首先获得VMC小鼠脾脏CD4+T细胞，并在有α7nAChR激动剂或抑制剂存在的情况下培养。收集细胞，流式细胞术分析各Th细胞亚群百分比，Western blot分析转录因子释放情况。然后，我们在体内检测了迷走神经相关的CAP对Th细胞分化的影响。在急性病毒性心肌炎中，分别用α7nAChR激动剂、抑制剂或切断颈部迷走神经来激活和阻断CAP。激活CAP可增加Th2和Treg细胞比例，降低Th1和Th17细胞在脾脏中的比例，下调T-bet, Ror-γ、IL-1和IL-17在心脏中的表达，而 Gata3、IL–4和Foxp3的表达上调。同时，我们还观察到激活CAP能使促炎细胞因子水平下降，心肌病变严重程度和细胞浸润程度减轻，VMC小鼠心功能缓解。此外,激活CAP后能增强CD4+T细胞中JAK2-STAT3信号通路的激活，抑制NF-κB的激活。而阻断CAP则起到了相反的效果。我们的研究结果表明，迷走神经相关的CAP能有效保护心肌免受病毒感染，这可能与调节Th细胞分化有关。本研究将VMC的机制研究深入神经免疫学领域，有助于阐明VMC发病机制，为VMC的治疗提供新的可能。