肠道LPS通过Rac1激活Kupffer细胞增加自身免疫性肝炎易感性：肠肝对话的新视角

The pathogenesis of autoimmune hepatitis (AIH) remains unclear, one of which mechanisms relies on the intestinal antigen induced hepatic immune disorders. In normal conditions, Kupffer cells (KC) show a tolerance to gut-derived LPS,while in case of the intestinal barrier impaired, gut-derived LPS increases the susceptibility of steatohepatitis and the severity of viral hepatitis. In our previous study, we found that the intestinal barrier of AIH patients was indeed impaired, and that the expression levels of TLR4 in Kupffer cells were enhanced. We also observed that prolonged LPS stimulation induced apoptosis of monocytes via Rac1 activation. However, it is still unknown that the role of gut-derived LPS on the functions of Kupffer cells in AIH pathogenesis. In the present study, we will first demonstrate in mice with DSS induced intestinal barrier defect that intestinal LPS aggravates Con A induced liver injury and fibrosis in wild-type rather than in TLR4-/- mice. Furthermore, by blocking KC in vivo and analyzing KC in vitro we will clarify in mice whether Kupffer cells are the cellular target of LPS during liver damage, with respect to the function of apoptosis and antigen presentation. Moreover, we will analyze TLR4-/- and wild-type mouse KC in vitro to address whether Rac1 is the molecular target of LPS in Kupffer cells mediating the indicated functions. Finally, the correlation of intestinal barrier, Rac1 activities of Kupffer cells, and hepatic T cell activation will be evaluated in the liver and intestinal biopsies of AIH patients. Our study will provide important information for AIH prevention and therapy from a new insight of immune cross-talk between gut and liver.

自身免疫性肝炎(AIH)发病机制未明，但肠道抗原诱导肝脏免疫紊乱是其机制之一。正常情况下肝Kupffer细胞（KC）对肠源性LPS呈耐受性，当肠屏障缺陷时，肠道LPS增加脂肪性肝炎易感性和病毒性肝炎严重度。我们发现，AIH患者存在肠屏障异常，且KC的TLR4表达增多，并发现LPS持续激活Rac1诱导单核细胞凋亡。然而，肠道LPS在AIH发生中如何影响KC的功能及其机制仍未知。为此，我们首先在DSS肠屏障缺陷鼠中证明肠道LPS加重野生鼠ConA诱导的肝损伤发生发展，对TLR4-/-鼠作用不明显；通过封闭鼠KC的体内实验和鼠KC体外实验证明KC凋亡、抗原递呈等功能改变是LPS加重ConA肝损伤的关键；再在TLR4-/-和野生鼠离体 KC中证明Rac1是LPS影响KC功能的靶分子；并在AIH患者肝和肠组织中验证肠屏障、肝脏炎症和KC Rac1活性的关系；旨在探索基于肠肝免疫对话的AIH防治策略。

在本项目资助下，天津医科大学总医院自身免疫性肝病团队有了显著进步。根据本项目设计内容，我们首先证明了AIH患者肝组织巨噬细胞数量增多、TLR4表达量增加，巨噬细胞向M1型极化，活化的巨噬细胞程序性坏死相关通路RIP3信号蛋白表达水平增高，且与IL-6的调控紧密相关。进一步我们发现，肝组织巨噬细胞中RIP3信号通路上下游蛋白VAV1、PAK1的表达水平显著增加，并与疾病活动度正相关，同时RIP3是自身免疫性肝病一线免疫抑制剂硫唑嘌呤的作用靶点，这提示巨噬细胞RIP3介导的炎性信号通路参与了AIH的发生、发展并成为潜在治疗靶点（硕士论文，已发表IF 3.7分）。为了阐释巨噬细胞活化的机制，我们研究了AIH和对照者肠屏障功能，发现AIH患者无肝硬化和有肝硬化者外周血 D-Lac、DAO 水平均升高，肠道黏膜形态存在异常，肠道黏膜组织免疫组化染色显示 ZO-1、Occludin表达量下降，提示AIH患者肠道的机械屏障破坏。动物实验发现预先给予小鼠极低浓度DSS（1%）破坏肠粘膜，可显著加重ConA小鼠的肝脏炎症（硕士论文、部分结果已发表IF 3.9分）。进一步细胞实验发现细菌成分PGN显著增加巨噬细胞Rac1的活性，脂多糖可显著激活巨噬细胞RIP3信号通路，而硫唑嘌呤有效分子6-硫基嘌呤可以显著抑制Rac1和RIP3信号的活性且其促进和抑制的作用均呈剂量依赖性（博士论文，部分结果已发表IF 1.7分）。综上所述，我们的研究提示AIH患者的肠屏障异常可能出现于肝功能异常之前，且与肝脏巨噬细胞数量增多、活性增强密切相关，受巨噬细胞RIP3信号通路调控。未来我们将深入探索基于肠道的AIH干预策略及其进一步机制研究。