基于肠-肝轴研究肠道菌群驱动TLRs信号转导通路调控自身免疫性肝炎滤泡TFR/TFH失衡的研究

Autoimmune hepatitis (AIH) is a complex autoimmune disease characterized by disturbances in T lymphocytes, B lymphocytes, and exocrine. Notably, our previous studies have shown that the loss of the balance between follicular regulatory T (TFR) cells and follicular helper T (TFH) cell in blood and liver tissue was found to be predictive of systemic and sustained B cell activation as well as autoantibody abnormal secretion. Collectively, these data indicate that loss of the TFR to TFH balance is suggested to contribute to the pathogenesis of AIH. Therefore, development of therapeutic strategies to increase the numbers and function of TFR may be highly promising for the intervention of AIH. Interestingly, increasing evidence indicates that intestinal microbial play a critical role in the regulation of immune function and liver diseases. In the pre-experiment, we have confirmed the intestinal microflora imbalance in AIH patients by intestinal microflora RT-PCR, which was the maintenance and severity of AIH. However, the specific mechanism is unclear. Based on the presence of the “intestine-liver” axis in the body, we hypothesized that the “dysregulated” intestinal flora-driven TLRs signal transduction pathway resulted in TFR/TFH balance shift, induced AIH onset, and “reconstituted” intestinal flora balance can "block" the pathogenesis. To verify the hypothesis, we use modern techniques and methods, such as microbiology and immunology, to study the role of intestinal flora imbalance in the pathogenesis of AIH by animal experiments, to study the role of intestinal flora and TLRs-mediated MyD88-dependent and MyD88-independent signaling pathways in follicular TFR/ TFH cells, and to explain the key targets and key signaling molecules for the treatment of AIH in the balance of intestinal microflora, and to provide experimental data and theoretical basis for the clinical treatment of AIH.

免疫紊乱是导致自身免疫性肝炎（AIH）发病的重要机制之一。研究显示滤泡调节性T细胞和辅助性T细胞（TFR/TFH）制约平衡，TFR缺失导致TFH细胞过度增殖，并诱导B细胞功能异常产生大量自身抗体，共同引发AIH，提示TFR/TFH平衡调控是治疗AIH的关键切入点。肠道微生物参与调节免疫功能和肝脏疾病，我们预实验证实AIH患者存在肠道菌群失调，是AIH发病的维持和重症化因素，但机制不明。基于机体内“肠-肝”轴的存在，我们推测“失调”的肠道菌群驱动TLRs信号转导通路导致TFR/TFH平衡偏移，诱发AIH的发病，而“重建”肠道菌群平衡可阻断发病过程。为验证假说，本课题采用微生物学等现代技术和方法，通过动物实验，深入研究肠道菌群和TLRs介导的MyD88依赖型和MyD88非依赖型信号通路对滤泡TFR/TFH细胞的调节机制，阐释重建肠道菌群平衡治疗AIH的关键靶点，为临床治疗AIH提供理论基础。

研究背景：自身免疫性肝炎（AIH）是一种复杂的免疫性疾病，发病率高，目前缺乏有效的治疗，是医学界一大顽症。研究显示滤泡调节性T细胞和辅助性T细胞（TFR/TFH）制约平衡，TFR缺失导致TFH细胞过度增殖，并诱导B细胞功能异常产生大量自身抗体，共同引发AIH，提示TFR/TFH平衡调控是治疗AIH的关键切入点。肠道微生物参与调节免疫功能和肝脏疾病，我们预实验证实AIH患者存在肠道菌群失调，是AIH发病的维持和重症化因素。基于机体内“肠-肝”轴的存在，我们推测“失调”的肠道菌群驱动TLRs信号转导通路导致TFR/TFH平衡偏移，诱发AIH的发病，而“重塑”肠道菌群平衡可阻断发病过程。.主要内容：①阐明“重塑”肠道菌群平衡对自身免疫性肝损伤的影响；②研究肠道菌群对滤泡 TFR/TFH细胞免疫平衡的影响；③探索肠道菌群调控TFR/TFH细胞平衡的信号转导机制。.重要结果：①AIH患者和EAH模型小鼠血清中肝功（ALT/AST/TBIL）和肠源性毒素（DAO/ET/LPS）明显升高；粪便组织内双歧杆菌、乳酸杆菌、弱芽孢杆菌、柔嫩梭菌含量减少，而大肠杆菌含量增加，存在菌群紊乱；体内TFR细胞比例下降，TFH细胞比例升高，存在TFR/TFH细胞失衡；②口服乳酸杆菌或粪菌移植可减轻肝脏炎症反应；减少肠源性毒素的吸收；修复肠道菌群紊乱；调控滤泡TFR/TFH细胞失衡；③机制研究中发现乳酸杆菌可能通过TLR4/MyD88/NF-κB信号转导通路调控EAH小鼠滤泡TFR/TFH失衡，存在DCs依赖性；④肠道菌群紊乱加剧生发中心中滤泡TFR/TFH细胞的失衡，而粪菌移植可“逆转”EAH小鼠生发中心的滤泡TFR/TFH失衡，进而减轻肝脏炎症反应。.关键数据及其科学意义：本研究从肠道微生态角度揭示了“重塑”肠道菌群平衡通过TLR4/MyD88/NF-κB信号转导路调控EAH小鼠滤泡TFR/TFH失衡，进而减轻肝脏炎症反应，为揭示AIH发病的部分分子机制及发现新的防治措施提供科学依据。