Musculin对Th17分化的作用及其调控机制

CD4+ T cells play important roles in fighting against infection, cancer and maintaining tolerance to self as well as transplanted organs. They are divided into Th1, Th2, Th17 and regulatory T (Treg) subsets, whose differentiation is orchestrated by disctinct transcription factors. Identifying those factors preferentially induced in a lineage-specific manner will shed light on the regulation of phenotype and function of each subset. Our prior work has identified critical extracellular cytokine signals that reciprocally regulate the differentiation of regulatory T cells and pathogenic Th17 cells. Following this line of innovative research, we now find that Musculin (MSC), a basic helix-loop-helix transcription factor that regulates muscle development and whose expression and function in T cells has never been reported, is highly expressed in Th17 cells. MSC correlates with stronger expression of RORγt, IL-17A/F and transcription factor Id2 in Th17 cells, and is also induced in Tregs when they are deprogrammed into Th17 cells. Because Th17 cells are the main effector cells in immune disorders caused by trauma, inflammation, infection and autoimmunity, we propose to further study the intracellular determining factors for its differentiation and function. We hypothesize that MSC plays an crucial role in Th17 cell differentiation by interacting with Id2 and regulating the RORγt promotor activity. In this application, we will use IL-17A GFPKI-MusculinKI-DsRed, Foxp3-RFPKI-IL-17A-GFPKI, IL-17RKO, MusculinKO-DsRed mice imported to our lab to perform a series of in vivo experiments. We will treat the animals with yeast cell wall component Zymosan to mimic the induction of Th17 cells during yeast infection, which is a serious life-threatening condition in burn patients. We will also explore the influence of proinflammatory cytokines, abundantly expressed in trauma conditions, on MSC-induced Th17 differentiation and Treg deprogramming. The use of immunological techniques including FACS, qPCR and lentivirus infection, and the study of MSC function at molecular, cellular and animal levels will enable us to gain insights on the transcriptional regulation of Th17 and Tregs, two important CD4+ T helper subsets that are involved in maintaining homeostasis. The knowledge will also help design novel treatment to rebuild immunological balance after inflammatory reactions.

作为介导机体免疫功能变化的效应细胞，Th17在创伤、感染、疾病等因素引起的炎症反应中发挥了重要作用。目前调控Th17分化的转录因子研究多集中于RORγt方面，尚不清楚是否还有其它转录因子参与了该过程。申请人前期工作首次报道，在肌肉发育过程中发挥关键作用的转录因子Musculin在Th17细胞中高表达，且诱发调节性T细胞的失稳，其机制可能与Musculin和Id2之间以及对RORγt启动子的作用有关。本课题拟采用以Foxp3-RFPKI-IL-17A-GFPKI和MusculinKO-DsRed为代表的一批业已引进和自制的转基因小鼠为主要实验动物，在复制酵母多糖刺激小鼠模型诱导Th17发生的基础上，探讨模拟炎症反应条件下Musculin对Th17分化的影响和作用规律，揭示Musculin和Id2共同调控Th17功能的分子机制，为炎症反应后重建机体内环境稳定和维持免疫平衡提供理论基础和新思路。

作为介导机体免疫功能变化的效应细胞，Th17 在创伤、感染、疾病等因素引起的炎症反应中发挥了重要作用，目前调控Th17 分化的转录因子研究多集中于RORγt 方面，尚不清楚是否还有其它转录因子参与了该过程。本项目从美国成功引进了IL-17A 绿色荧光蛋白敲入（green fluorescent protein knockin, GFPKI）小鼠；建立了拥有自主知识产权的基因分型方法；完成了内核糖体（Internal ribosome entry site, IRES）进入位点突变序列的构建。在此基础上，我们委托北京百奥赛图公司基因生物技术有限公司完成了Musculin基因敲除/基因嵌入模式小鼠的研发工作，目前该品系小鼠及CMV-Cre和CD4-Cre小鼠已到位。同时，我们开展了从初始T细胞分化以及调节性T细胞转分化为Th17的角度完成了正常和炎症情况下Musculin和Th17的关系研究，结果发现：正常和炎症条件下初始T细胞向Th17分化过程中伴随着Musculin的高表达，并与Th17的数量和百分比呈正相关，但在Th1、Th2和调节性T细胞（Treg）的分化方向则无此现象；而且，在Treg转分化为Th17的过程中，Musculin的表达规律与前述条件相似，并诱发调节性T细胞的失稳。提示在肌肉发育过程中发挥关键作用的转录因子Musculin可能参与了Th17在炎症反应中的分化过程。另外，我们还探讨了正常和炎症反应条件下Musculin对骨髓来源树突状细胞（bone marrow derived dendritic cell, BMDC）功能的体外调节以及在诱导初始T细胞向Th17分化过程中的作用规律，目前该研究正在进行中。通过上述研究工作的开展，进一步验证和探讨炎症反应条件下Musculin 对Th17 分化的影响和作用规律，为炎症反应后重建机体内环境稳定和维持免疫平衡提供理论基础和新思路。本项目发表国际SCI论文1篇，国内CSCD论文2篇（含英文论文1篇）；参加全国性学术会议1次并做大会发言；获国家发明专利授权1项；培养技术员2名。