调节性B细胞在肝纤维化进程中的免疫调控作用及机制研究

The immunological modulation in liver fibrosis is increasingly focused on and has shed new light on related researches. Since the immunoregulations of T cells, NK cells and NKT cells have been gradually clarified, do B cells, which represent 10% of the whole lymphocytes, also play regulatory roles in the process? Recent studies indicate B cells are no longer bystanders and influence the process of liver fibrosis independent on antibodies. State of the art, B cells are reported to differentiate into a new subset-regulatory B cells (Bregs), which can regulate T cell-mediated responses and induce the production of regulatory T cells (Tregs) via cell-contact or secreting IL-10, hence suppressing overactive immune responses and modulating immune tolerance. Supported by our research of the NSFC (No. 81070341), the process of hepatitis B-induced fibrosis inclined to a state of immune tolerance and the Treg/Th17 imbalance exhibiting as Treg dominance affected the fibrogenic function of HSCs, indicating there's a bidirectional regulation between the immune state and the process of liver fibrosis. Apart from Tregs, do Bregs contribute to immune tolerance in liver fibrosis as well? Bregs are proposed to be indispensable for Tregs-induced immune tolerance and in our preliminary experiments Bregs actually represented different frequencies in both spleen of chronic HBV-infected mice models and peripheral blood of chronic hepatitis B patients. Interestingly, our performed research of NSFC (No.30300151) also verified that HSCs could migrate into portal area and interact with lymphocytes there in the process of liver fibrosis. Therefore, this research will firstly observe the relation between B cell subsets especially Bregs and pathological grades and stages of liver fibrosis in chronic HBV-infected mice models and chronic hepatitis B patients. Secondly, Bregs-conducted influence on functions of CD4+T cells and HSCs will be mainly focused on via in vitro cells co-culture and adoptive transfer of Bregs, in details: 1) how Bregs affect CD4+T cell proliferation, maintenance, cytokine secretion as well as the balance of T cell subsets especially between Tregs and Th17; 2) how Bregs regulate the profibrotic functions of HSCs and reversely how HSCs affect proliferation and cytokine secretion of B cells in the process of liver fibrosis. Based on the above-mentioned research, we look forward to a better understanding of the role of Bregs in the immunoregulation in hepatitis B-induced fibrosis, hence enriching our acquisition about immune microenvironment of liver fibrosis and also providing theoretical foundations and application basis for its clinical immunotherapy.

肝纤维化的免疫调控机制受到重视，新近发现B细胞可分化为调节性B细胞(Bregs)，通过分泌IL-10或与细胞直接作用，调节免疫耐受及免疫应答。我们在研的国家自然科学基金课题表明乙肝肝纤维化进程趋于免疫耐受状态，Treg/Th17稳态向Treg的偏移可影响HSCs促纤维化功能；而Bregs是Tregs依赖的免疫耐受所必须的，我们预实验在HBV慢性感染小鼠与患者体内检测到Bregs差异表达。本次申请以观察慢性乙肝患者与HBV慢性感染小鼠各B细胞亚型(特别是Bregs)与肝纤维化分级分期关系为基础，以Bregs对CD4+T细胞与HSCs功能影响为主线，通过细胞共培养、获得性Bregs转移等途径探讨肝纤维化时Bregs调控CD4+T细胞功能、诱导Tregs生成影响Treg/Th17平衡及调节HSCs促纤维化功能及机制，深入阐述Bregs对肝纤维化进程的免疫调控作用，为免疫治疗肝纤维化提供理论基础。

新近发现B细胞可分化为具有特异性调节功能亚群即调节性B细胞（regulatory B cells, Bregs），可强有力分泌细胞因子IL-10或与其他细胞直接作用，调节免疫耐受及免疫应答。而我们前期研究发现乙肝肝纤维化进程趋于免疫耐受状态，Treg/Th17稳态向Tregs偏移，并可影响肝星状细胞（hepatic stellate cells, HSCs）促纤维化功能，这构成了以HSCs为中心环节的肝纤维化免疫微环境。本项目在此基础上，以B细胞及Bregs细胞在慢性乙型肝炎肝纤维化进程中的表达及其对CD4+ T细胞功能的影响为主线，运用多色流式细胞术等先进手段对慢性乙型肝炎患者及免疫小鼠开展了一系列体内外研究，发现：慢性乙型肝炎患者肝纤维化进程中，Bregs显著增加，且其表型为CD19+CD24hiCD38hi，增加的Bregs通过CD80/CD86及IL-10的分泌抑制效应性T细胞但促进Tregs的功能；此外，小鼠调节性B细胞（B10细胞）亦参与小鼠HBV加重肝纤维化进程的免疫调控。另一方面，我们发现慢乙肝患者增加的Tregs自噬水平有助于维持Tregs功能的完整性，而在慢乙肝中显著升高的高迁移率族蛋白1 (high mobility group box-1 protein, HMGB1) 可以通过HMGB1-RAGE轴及其下游ERK和mTOR信号通路激活Tregs自噬。我们的研究结果有助于阐述肝纤维化发生时肝脏局部的免疫调节机制，为免疫治疗肝纤维化，打破慢乙肝免疫耐受状态提供新的视角。本项研究按计划按时完成了预期目标，目前已在Clinical Science, Liver International等期刊发表SCI论文5篇，另有一篇文章刚被clinical science接收（见附件），研究结果在国际权威会议大会发言3次、壁报交流4次，国内权威学术会议大会发言和壁报交流各1次，并已培养毕业硕士研究生2名。