共刺激分子OX40通过调控CD4+T 细胞活化加重对乙酰氨基酚急性肝损伤的机制研究

Drug induced liver injury (DILI) is an important threaten of public health. Acetaminophen(APAP) –induced liver injury is a classic model of DILI. The role of adaptive immune response in the pathogenesis of APAP-induced liver injury has not been thoroughly studied. In the previous study, we have found that CD4+ T cells and CD8+ T cells in the liver were activated significantly. The plasmatic OX40 concentration and the expression of OX40 in CD4+ T cells both increased obviously. Meanwhile, the APAP-induced liver injury of OX40-/- mice was milder than WT mice. Thus, we presume that OX40 promotes the activation of CD4+T cells. At the same time, the activated CD4+ T cells and plasmatic OX40 further promotes the activation of innate immune reaction, which promotes the injury of liver simultaneously. To verify the hypothesis, we will build APAP-induced liver injury models with WT and OX40-/- mice. First, we will compare the proliferation and activation of innate immune cells and adaptive immune cells of the two groups systemically. Secondly, we will testify whether OX40 can aggravate APAP-induced hepatotoxicity by promoting the activation of CD4+T cells. Thirdly, we will testify whether plasmatic OX40 protein can directly increase the injury of liver induced by APAP. Last but not least, we plan to test the concentration of plasmatic OX40 and the expression of OX40 in CD4+T cells of patients suffered from APAP-induced liver injury. In this study, we aim to verify that OX40, a co-stimulating factor, plays an important role in the pathogenesis of APAP-induced liver injury.

药物性肝损伤(DILI)是威胁公众健康安全的重要疾病。对乙酰氨基酚（APAP）模型是DILI的经典模型，适应性免疫在其发病中的作用研究较少。我们的前期工作提示APAP模型中，CD4+T、CD8+T细胞活化增强，血浆OX40浓度及CD4+T细胞OX40表达增加，OX40-/-小鼠APAP肝损伤减轻。据此我们提出假设：APAP肝损伤中，OX40促进了CD4+T细胞增殖活化，活化的CD4+T细胞及可溶性OX40激活固有免疫，共同参与APAP肝损伤。为此，本课题拟首先对比WT小鼠与OX40-/-小鼠APAP模型肝固有免疫及适应性免疫细胞增殖活化异同；其次，探索OX40是否通过影响CD4+T细胞加重肝损伤；再次，验证OX40分子是否可直接加重APAP肝损伤。最后，检测APAP肝损伤患者血浆OX40及外周血CD4+T细胞OX40表达水平。本研究证明OX40在APAP诱导的DILI模型中的作用。

药物性肝损伤(DILI)是威胁公众健康安全的重要疾病。对乙酰氨基酚（APAP）模型是DILI 的经典模型，适应性免疫在其发病中的作用研究较少。我们的前期工作提示APAP模型中，CD4+ T、CD8+T细胞活化增强，血浆OX40浓度及CD4+T细胞OX40表达增加，OX40-/-小鼠APAP肝损伤减轻。据此我们提出假设：APAP肝损伤中，OX40促进了CD4+T细胞增殖活化，活化的CD4+T细胞及 可溶性OX40激活固有免疫，共同参与APAP肝损伤。首先我们利用 WT 小鼠、OX40-/-小鼠建立APAP 肝损伤模型，发现APAP肝损伤小鼠肝组织CD4+T活化增加（表达CD69分子增多）、分泌IL-17增多，而OX40敲除后CD4+T细胞表达CD69分子、分泌IL-17均明显减少，提示OX40可能是通过调控CD4+T细胞活化、促进其向Th17分化而加重APAP肝损伤。其次，建立过继转移模型，发现与过继转移B6-CD4+T细胞相比，过继转移OX40-/- CD4+T细胞的小鼠，APAP肝损伤更轻、转氨酶更低，提示OX40通过促进CD4+T细胞分化、增殖、凋亡及炎症因子分泌而加重肝损伤。第三，我们发现APAP肝损伤单核细胞来源的巨噬细胞（MC细胞）浸润明显增多、MC细胞分泌TNF-α明显增多，且在OX40敲除后MC浸润显著减少、分泌TNF-α明显减少，提示OX40通过促进肝内MC细胞浸润并促进其向M1型巨噬细胞极化加重肝损伤。第四，将OX40/Fc以及对照IgG分别与巨噬细胞系RAW264.7、分选的肝内单核细胞共培养，发现OX40/Fc直接刺激巨噬细胞系RAW264.7，增强其抗原提呈能力（上调OX40L、MHCII、TLR4、CD40）、迁移趋化能力（上调CCR2、CCR5、CCR7、CCR9、CXCR3、CXCR5等趋化因子受体）、促进其分泌促炎因子（TNFα、TGFβ、IL-1β、IFNγ等）。第五，我们发现患者血清OX40水平明显高于健康对照组，提示OX40参与了人DILI的发病过程，更进一步验证了小鼠实验的发现。