TLR通路激活γδT细胞参与BA胆管炎性损伤的机制研究

Biliary atresia (BA) is a pediatric liver disease characterized by progressive inflammation and fibrosis of both the extrahepatic and intrahepatic bile ducts. A current view of the pathogenesis of BA is that autoimmune response triggered by viral infection may play a predominant role in the progression of bile duct injury. Nevertheless, the mechanism is not fully understood. Our team reported for the first time that IL-17+T cells infiltrating into the portal areas play a role in immune damage of bile ducts. Further murine experiments showed that blocking IL-17A has a better effect than eliminating Th17 cells in ameliorating disease progression. It reveals that cell types other than Th17 might participate in immunological injury by producing IL-17A. Recent literatures demonstrate TLR mediated activation of γδT cells secrete Il-17A functioning in autoimmune diseases. Our results also show that γδT cells infiltrate around portal areas in BA infants and murine models. Therefore we speculate that γδT cells possibly play an important role in immunological damage of bile ducts in BA. In this research we aim at the following vital issues: 1) how IL-17A+γδT cells initiate and promote bile duct damage in BA and the secretion of IL-17A via TLR activation of γδT. 2) blockage of γδT cells at different levels and its impact to bile duct damage. This research may contribute to γδT-associated pathogenesis of BA and provide clues to BA treatment.

病毒感染触发自身免疫应答所致胆管进行性炎症损伤是胆道闭锁（BA）的重要原因，但机制尚未完全明了。申报者前期研究首次报道，BA肝汇管区浸润分泌IL-17A的T细胞，并证明IL-17+Th17细胞参与BA胆管炎症损伤。但在后续干预实验中发现，阻断IL-17A比清除Th17细胞更能减缓BA胆管炎症，提示其它细胞也可能通过产生IL-17A参与BA胆管损伤。近期文献报道，γδT细胞可通过Toll样受体（TLR）通路激活并分泌IL-17A，从而启动并延续自身免疫疾病。课题组前期研究也显示，BA肝汇管区出现明显的γδT细胞浸润。据此推测：胆管微环境中γδT细胞是参与BA胆管炎症损伤的效应细胞。为验证该假说，申报者拟开展如下研究：①证实IL-17+γδT细胞参与BA胆管炎症损伤；②阻断TLR通路或清除γδT细胞，观察对γδT细胞活化和/或对BA结局的影响，可望阐明γδT细胞参与BA发病的作用及机制。

研究证实病毒触发自身免疫应答所致胆管进行性炎症损伤是胆道闭锁（BA）的重要原因，但机制尚未完全阐明。前期研究表明，BA肝汇管区浸润分泌IL-17A的T细胞，并证明IL-17+Th17细胞参与BA胆管炎症损伤。但在后续干预实验中发现，阻断IL-17A比清除Th17细胞更能减缓BA胆管炎症，提示其它细胞也可能通过产生IL-17A参与BA胆管损伤。近期文献报道，γδT细胞可通过Toll样受体（TLR）通路激活并分泌IL-17A，从而启动并延续自身免疫疾病。为了探索胆管微环境中γδT细胞是参与BA胆管炎损伤的效应细胞，本课题组主要从两个方面进行研究，①证实IL-17+γδT细胞参与BA胆管炎症损伤；②阻断TLR通路或清除γδT细胞，观察对γδT细胞活化和/或对BA结局的影响，可望阐明γδT细胞参与BA发病的作用及机制。在我们的研究中，通过流式细胞术、免疫组化、ELISA等方法表明在人体标本和证实了IL-17+γδT细胞、TLR2,3,4、HMGB1及炎性细胞因子IL-17A在BA组明显升高；在动物水平上，我们应用猕猴轮状病毒（RRV）复制Balb/c野生小鼠BA模型，不同时间点分别或联合腹腔注射TLR2、3、4中和抗体(封闭TLR2、3、4)、进行腹腔注射，BA鼠黄疸明显变轻，上述炎症因子明显减轻。我们的研究结果表明在封闭TLR2、3、4受体时，IL-17+γδT细胞明显减少，验证了BA胆管微环境中γδT细胞通过TLR活化分泌IL-17A。项目投入经费58万元，支出37.47万元，各项支出基本与预算相符。剩余经费20.53万元，剩余经费计划用于本项目研究后续支出。