MAIT细胞诱导HSC凋亡参与抑制CLD肝纤维化的探索性研究

Innate immunity has an obvious regulation on activation of hepatic satellite cells (HSC) in progression of cholestatic liver fibrogenesis. Mucosal-associated invariant T cells (MAIT cells) which are abundant in liver play important roles in innate immunity. However, the exact effect and underlying mechanism of MAIT cells regulating HSC remained obscure. Our previous research has confirmed that circulating MAIT cell frequency was dramatically reduced with an altered cytokine profile in cholestatic liver diseases (CLD) group. What's more, MAIT cells both in the liver and spleen of cholestatic mouse models were significantly decreased in frequency but were more activated compared with those in wild type mice. In this study, we will explore characters and functions of intrahepatic MAIT cells during progression of cholestatic fibrosis using in cholestatic mouse model and MAIT-deficient cholestatic mouse model by flow cytometry and liver pathology. Then we will investigate the pro-apoptotic effect of MAIT cells on HSC using a co-culture system, validating and interpreting the underlying mechanism by confocal staining, transfusion and transwell system. Our current study will be helpful to further understand the molecular mechanism of innate immunity regulating the progression of cholestatic fibrosis, and will provide more potential guidance for immune treatment aiming at CLD fibrogenesis.

慢性胆汁淤积性肝病（CLD）纤维化过程中，肝星状细胞（HSC）的激活与天然免疫反应密切相关。黏膜相关恒定T细胞（MAIT cells）在肝脏内较为丰富且在天然免疫反应中具有重要作用。目前MAIT细胞对HSC的作用及机制不明。我们前期的研究业已证实MAIT细胞在CLD初治患者外周血中的比例较正常下降且功能发生改变；胆汁淤积模型小鼠肝脏及脾脏内MAIT细胞比例下降而活性明显增加。本项目拟利用MAIT细胞缺失小鼠建立胆汁淤积模型，通过多色流式技术及肝组织病理学分析研究MAIT细胞对CLD模型鼠纤维化表型的影响；构建MAIT细胞与HSC共培养体系，结合激光共聚焦显微镜、transwell试验及MAIT细胞转输试验阐明MAIT细胞诱导HSC凋亡及其分子机制。该研究有助于深入理解CLD纤维化进展过程中天然免疫调控的分子机制，为针对CLD纤维化进行免疫治疗提供更多的思路。

我们通过饲喂含0.1%DDC和胆总管结扎（BDL）两种方式建立了胆汁淤积的小鼠模型。通过流式细胞分析，我们发现与对照组相比，胆汁淤积组小鼠肝脏的MAIT细胞比例显著降低，同时残留的MAIT细胞处于过度激活状态。通过功能分析我们发现胆汁淤积小鼠肝脏MAIT细胞 上调多种细胞因子的表达，包括IL-17A、IFN-γ、TNF-α、颗粒酶B、穿孔素等。我们随后利用MAIT细胞缺陷小鼠（MR1-/-）构建胆汁淤积模型，组织学分析显示MR1-/-小鼠肝内纤维化程度明显加重，并伴有肝星状细胞的激活增多及凋亡减少。提示MAIT细胞具有抑制胆汁淤积引起的肝纤维化作用。机制方面，DDC及BDL小鼠肝内NK细胞数量显著增加，且活化性受体分子表达增多。体外试验我们发现MAIT细胞可诱导NK细胞高表达细胞毒性分子TRAIL，并加强NK细胞对活化肝星状细胞的杀伤效应。且这一效应是通过直接接触及TNF-α所介导。此外，基于炎症和纤维化的关系，我们对MR1-/--DDC小鼠肝内的各项炎症指标也进行了检测。结果提示MR1-/--DDC汇管区炎症细胞浸润明显增多，同时伴有IL-17A、IL-6、TNF-α等炎症因子表达水平的增加。流式细胞分析结果显示MR1-/--DDC小鼠肝内CD8+ T细胞比例升高而CD4+ T细胞比例则显著降低。表明MAIT细胞也可能通过抑制PSC肝内炎症反应以达到抑制纤维化进展的作用。