载脂蛋白A4调控T细胞免疫应答抑制炎症的作用机制研究

Apolipoprotein A4 (APOA4) has the functions in regulating glycolipid metabolism and suppressing inflammatory diseases. T cells play the pivotal roles in immune and inflammatory response in host which associated with cellular energy metabolism. We found that APOA4 gene knockout (A4KO) mice, with inflammation induced by carbon tetrachloride (CCl4), had much less CD4+ T cells in liver and spleen, as well as the copy numbers of mitochondria in liver, but the liver inflammation aggravated, compared with the WT control. Currently, with a proteomic analysis, it was reported that mitochondrial glycolysis, as well as the APOA4 amount in the cells impacted the activation, proliferation and the tuned balance between human Treg and Tconv cells. It was suggested that APOA4 may be involved in regulating the proliferation and differentiation of T cells through energy metabolic pathway, but the specific mechanism is still unknown. In this study, firstly, to confirm the effect of APOA4 on T cells, we will analyze the cellular energy metabolism, activation and differentiation of T cell subsets in liver, spleen and blood from wild type and A4KO mice with LPS-induced inflammation, as well as human peripheral blood with inflammation induced by LPS with or without recombinant APOA4 protein. Secondly, to find APOA4’s target proteins, pathway and target CD4+ T cell subsets, Proteomics technology will be used to explore its mechanism of APOA4 suppressing inflammation via regulating the immune response of T lymphocytes. Finally, chronic liver injury induced by CCl4 in APOA4 target knockout (A4tKO) and A4KO mice will be established to verify the mechanism of the effect of APOA4 on CD4+T cells. This study is expected to link APOA4, immunity and energy metabolism, explore the molecular mechanism of APOA4’s role on anti-inflammation via regulating T cell subsets, and provide evidence for the diagnosis and treatment of inflammatory diseases.

载脂蛋白A4（APOA4）具有调节糖脂代谢及抗炎功能。T细胞在能量代谢的影响下参与机体免疫应答和炎症反应。我们发现，CCl4诱导的肝损伤中，APOA4基因敲除（A4KO）小鼠CD4+T细胞和肝线粒体显著减少，肝脏炎症加重。新近报道，静息和增殖状态的调节性T细胞和常规T细胞线粒体功能、糖酵解、脂肪酸氧化各有不同，而各细胞亚群中APOA4的含量也随之变化。提示APOA4可能通过能量代谢途径参与调控T细胞亚群的增殖和分化而发挥抑制炎症作用，但具体机制不明。本研究拟①用LPS诱导A4KO小鼠及人外周血炎症模型，明确APOA4对T细胞的调控作用；②用蛋白质组学技术寻找APOA4作用靶点和靶T细胞亚群，并揭示其作用机制；③用A4KO及靶点敲除小鼠建立CCl4诱导的慢性肝损伤模型，验证APOA4调控CD4+T细胞抑制炎症的作用机制。本研究将揭示APOA4与免疫和代谢的关系，为炎症性疾病的诊治提供依据。

肥胖相关的代谢性炎症和肝炎，都是威胁人类健康的重要问题。载脂蛋白A4 (Apoa4)，具有抗炎和抗氧化作用，调节脂质代谢和葡萄糖稳态。我们推测Apoa4在炎症中发挥积极作用。.本研究对Apoa4敲除（KO）小鼠和WT小鼠，进行高脂饮食诱导肥胖（DIO）小鼠模型。发现，Apoa4缺乏导致小鼠体重增加，胰岛素抵抗(IR)和血浆游离脂肪酸(FFA)增加，但Apoa4重组蛋白稳定表达可部分逆转上述指征。与DIO-WT相比，内脏白色脂肪组织(WAT)和血液中促炎巨噬细胞增加。WAT的组织RNA测序分析发现，细胞因子-细胞因子受体相互作用，T细胞受体和B细胞受体，以及 IL-17和TNF-α在DIO-KO小鼠中上调。体外培养发现, 补充Apoa4蛋白抑制巨噬细胞中LPS诱导的IKK和JNK磷酸化，增加3T3-L1脂肪细胞的胰岛素敏感性。提示，在肥胖性炎症中, Apoa4通过减少WAT中M1巨噬细胞堆积、抑制IL17-IKK和IL17-JNK通路激活保护小鼠。.CCl4诱导小鼠慢性肝损伤发现，与WT-CCl4相比，KO-CCl4小鼠肝脏SOD活性降低，血清MDA活性增强，肝脏纤维化相关基因α-SMA、TIMP-1和COL1A1表达升高，病理检测肝损伤和纤维化加重；而且巨噬细胞堆积，炎症因子TNF-α、IL-6和CCL5表达升高，但IL-10降低。另外，KO-CCl4小鼠肝脏、血细胞和脾脏中CD3+、CD4+和CD8+T细胞百分比降低，而单核细胞增多，且ApoA4蛋白和T细胞注射小鼠可部分逆转以上指标。提示，ApoA4可能通过抑制纤维化介质和炎症因子、减少促炎巨噬细胞堆积和调节CD8+ T和CD4+ T细胞作用参与肝脏保护。.总之，Apoa4在肥胖性炎症和肝脏炎症中表现有抗炎和免疫调节作用，是炎症防治的新靶点。.