乙型肝炎病毒激活NLRP3炎性体致肝损伤的作用研究

Innate immunity plays an important role in immunological liver injury induced by HBV infection. HBV is recognized by nonparenchymal cells of the liver, mainly by liver macrophages (Kupffer cells). Inflammasome is a platform triggering the activation of inflammatory caspases and processing of proIL-beta in macrophages. However, the role of inflammasome in HBV infection remains unkown so far. In the previous study, the applicant found that IL-1β levels in plasma from patients with hepatitis B were significantly higher than those of normal controls,and serum IL-1β levels of HBV transgenic mice were also higher than those of normal mice. The supernatant containing HBV could stimulate THP1 cells to produce IL-1β,and caspase-1 and NLRP3 were required in this process. NLRP3 inflammasome reconstitution assays showed that HBV could promote NLRP3 inflammasome activation and induce IL-1β secretion. In addition, enhanced NLRP3 expression was found in liver tissues from HBV transgenic mice. HBV could also regulate the expression of many immune molecules in host. Based on these new findings, we put forward the hypothesis: HBV infection induces liver injury by regulating NLRP3 inflammasome activation. In the present study, clinical samples will be used to analyze the correlation of HBV and NLRP3 inflammasome firstly. Then, cell models are used to explore the effects of HBV on NLRP3 inflammsome activation and related signal pathway. Cell models are also used to investigate the regulation of HBV on NLRP3 inflammasome expression and corresponding mechanisms. Finally, animal models will be involved in further identifying the role of HBV in regulating NLRP3 inflammasome and its role in liver injury induced by HBV infection. This study would provide novel theory for the pathogenesis of hepatitis B and provide new targets for intervene of HBV related liver diseases.

固有免疫在乙型肝炎病毒(HBV)感染致免疫性肝损伤中发挥重要作用。HBV主要由肝内巨噬细胞识别，炎性体是巨噬细胞触发炎症反应的分子平台，但在HBV感染中的作用未见报道。申请者发现乙肝患者及HBV转基因小鼠血清IL-1β升高，含HBV上清依赖于caspase-1及NLRP3刺激THP1细胞产生IL-1β，炎性体重构实验表明HBV可促进NLRP3炎性体活化，另发现HBV转基因小鼠肝组织中NLRP3蛋白的表达显著高于正常小鼠，且HBV可调控宿主多种免疫分子表达，由此提出假说：HBV可调节NLRP3炎性体活化进而致肝损伤进程。为论证该假说，本项目利用临床病例分析HBV与NLRP3炎性体的关系，深入研究HBV对NLRP3炎性体的激活及信号通路、HBV对NLRP3炎性体的调控及机制，利用动物模型验证其在HBV感染致肝损伤中的作用。该研究将为乙型肝炎发病机理提供新学说，并为HBV相关肝病干预提供新靶点。

乙型肝炎病毒(HBV)严重危害人类健康，但其致免疫性肝损伤的作用尚不十分清楚，HBV主要由肝内巨噬细胞识别，炎性体是巨噬细胞触发炎症反应的分子平台，其中NLRP3炎性体研究最为清楚。为明确NLRP3炎性体在HBV感染致肝损伤中的作用及其机制，在本项目中，建立急性乙型肝炎模型，确定HBV可促进IL-1β的产生，利用NLRP3基因敲除小鼠，明确HBV通过激活NLRP3炎性体促进IL-1β的产生。利用细胞模型，研究HBV对小鼠及人巨噬细胞产生IL-1β及IL-18的影响，干扰NLRP3可抑制HBV的效应。利用NLRP3炎性体重构体系，明确HBV各基因片段对NLRP3炎性体具有激活作用，通过抗原清除实验证实HBsAg参与促NLRP3炎性体活化的效应，加热、紫外线照射处理可完全消除HBV激活NLRP3炎性体的作用，纯化的HBV病毒及HBV各片段编码蛋白均可刺激THP-1细胞产生IL-1β，发现HBV促进IL-1β的产生与钙离子通道有关，另外，发现HBV可上调NLRP3蛋白的表达，并可促进ASC的分泌，相关作用机制仍在研究之中。通过该研究，明确HBV感染通过诱导NLRP3炎性体活化导致肝损伤的发生，为乙型肝炎发病机理提供了新的理论学说，并为HBV相关肝病干预提供新的思路。.通过拓展研究，发现免疫调节分子Tim-4可抑制NLRP3炎性体的活化，并发现其与II型糖尿病患者IL-1β呈负相关，通过该研究拓展了免疫分子Tim-4的生物学作用，获得国家自然科学基金面上项目一项。. 在本项目的资助下，培养博士研究生1名、硕士研究生4名，项目负责人及课题组成员多次参加国际及全国性会议，先后4次就本项目相关研究结果进行大会口头报告，多次进行书面交流，得到相关领域专家的认可与高度评价。现已发表SCI收录论文2篇，另有2篇论文正在发表之中。