乙肝相关慢加急性肝衰竭中IL-6/STAT3调控Tc17细胞介导肝细胞焦亡的作用及机制

The pathogenesis of HBV related acute-on-chronic liver failure (HBV-ACLF) remains unclear. We are the first to report the role of Tc17 cells in HBV-ACLF: Tc17 cells were elevated and involved in the process of liver injury; furthermore the elevated Tc17 cells could be an independent risk factor for predicting prognosis. However, the differentiation of Tc17 cells and the effects of Tc17 cells on hepatocyte pyroptosis in HBV-ACLF are still unknown. Further studies show that IL-6/STAT3 signaling pathway is involved in the differentiation of Tc17 cells, and Tc17 cells are associated with hepatocyte pyroptosis. Therefore, we propose a new scientific hypothesis that IL-6/STAT3 is involved in the differentiation of Tc17 cells, which in turn mediate hepatocyte pyroptosis and promote the progression of HBV-ACLF. This study will be carried out at multiple levels, including molecular, cellular, tissue and animal levels. First, the differences in phenotype and related cytokine profiles of Tc17 cells between intrahepatic and peripheral are compared. Next, CD8+ T cells are sorted by MACS. Then the mechanisms of IL-6/STAT3 pathway (up-regulating and blocking) on the differentiation of Tc17 cells in HBV-ACLF are explored. Third, Tc17 cells are induced in vitro. A vitro cell co-culture model and an in vivo liver failure model are established in order to clarify the effects of Tc17 cells on hepatocyte pyroptosis and related mechanism. This study will explore the pathogenesis of HBV-ACLF from the new perspective of Tc17 cells, and hope that new data for immunotherapy will be provided.

乙肝相关慢加急性肝衰竭（HBV-ACLF）的发病机制仍未阐明。申请人率先报道Tc17细胞在HBV-ACLF的作用：Tc17细胞表达上调，参与肝细胞损伤，是评估预后的独立危险因素。但Tc17细胞的分化机制及介导肝细胞焦亡的作用尚不清楚。预实验中发现IL-6/STAT3通路参与Tc17细胞分化，且Tc17细胞与细胞焦亡有关。因此提出科学假说：IL-6/STAT3调控Tc17细胞分化，进而介导肝细胞焦亡促进HBV-ACLF发生发展。本课题拟从分子、细胞、组织及动物水平等多层次：首先明确肝内外Tc17细胞表型及分泌细胞因子的差异；接着上调及阻断IL-6/STAT3通路，阐明该通路调控Tc17细胞分化的机制；进而诱导Tc17细胞分化，建立体外细胞模型及体内肝衰竭模型，揭示Tc17细胞介导肝细胞焦亡的作用及机制。本研究将从Tc17细胞这个新视点探讨HBV-ACLF的发病机制，为免疫治疗提供新思路。

乙肝相关慢加急性肝衰竭（HBV-ACLF）严重危害国民身体健康，HBV-ACLF发病机制复杂且仍未阐明是导致患者高病死率的关键。本项目在课题组前期关于HBV-ACLF免疫细胞应答失衡的基础上，深入研究Tc17细胞在HBV-ACLF的作用及调控机制。采用流式细胞术、免疫组化、Luminex、RT-PCR及构建细胞模型等成熟技术检测Tc17细胞表达、细胞表型、细胞因子谱，通过干预IL-6/STAT3信号通路研究其对Tc17细胞分化的影响及Tc17细胞对细胞焦亡的作用及可能机制。取得了以下关键发现：①Tc17细胞是HBV-ACLF分泌IL-17a的重要细胞来源，与血清中IL-17a表达成正相关，且高表达CCR6趋化受体。②Tc17细胞在HBV-ACLF表达明显升高，与疾病严重程度成正相关，能准确预测HBV-ACLF的临床转归，且Tc17细胞与国内外指南推荐的疾病严重程度评估模型具有等同的预测价值。③Tc17细胞高表达CD45RO、低表达CD62L、高表达细胞毒效应分子（CD107a、Perforin及Granzyme B）及表达促炎症因子（IL-22、TNF-α、IL-2及IFN-γ）。④Tc17细胞有助于清除乙肝病毒，IL-4是影响Tc17细胞清除乙肝病毒的关键分子。⑤IL-6/STAT3信号通路参与调控Tc17 细胞分化介导细胞焦亡。HBV-ACLF高表达IL-6；IL-6处理组CD8+T细胞增殖明显活跃，IL-17a表达明显升高，RORC(RORγt) mRNA表达亦明显升高，同时核内pSTAT3表达上调；此外，HBV-ACLF中IL-1β及IL-18表达明显升高，且Tc17 细胞与IL-1β和IL-18正相关。以上发现为进一步阐明HBV-ACLF的发病机制提供重要的依据支撑，亦为免疫干预治疗提供Tc17细胞这一新靶点。同时，也初步探索Tc17细胞与巨噬细胞及肝纤维化的关系，为进一步探索Tc17细胞的上游调控及临床应用转化提供基础。在课题研究过程中，共资助发表3篇SCI收录论文，培养研究生2名，完成了结题要求的指标。