TLR4在抗磷脂综合征病理机制中的双重作用研究

The relationship between TLR4 and antiphospholipid syndrome (APS) has been attracting much attention recently. However, the definite roles of TLR4 in the pathological mechanisms of APS have not thoroughly understood. Our previous study has demonstrated that TLR4 and its signal transduction pathway could mediate β2GPI and anti-β2GPI complex inducing TF expression in monocyte, which is contributed to the thrombosis in APS. Reviewing the references and our previous results, we hypothesize that TLR4 may have dual roles in the activation of autoimmunity as well as in antibody-induced thrombosis in APS. In the current study, the mice with TLR4 intact or TLR4 defect will be used in different assays including cellular and molecular biology, immunology, as well as intravital image in order to explore the roles of TLR4 in β2GPI-stimulated differentiation and maturation of the immunocytes and the production of anti-β2GPI antibody in mice, and in APL-induced arterial and venous thrombosis in vivo. The specific inhibitors of TLR4 or TLR2 will be used to further demonstrate the roles of TLR4 and analyze whether TLR2 is involved. This study will become a new step for exploring the pathological mechanisms of APS. Therefore, the new target molecule for immune intervention and thrombosis therapy in APS will be discovered in the study.

TLR4与抗磷脂综合征（APS）的关系正受到愈来愈多的关注。然而TLR4在APS病理机制中的确切作用并未阐明。本课题组前期研究证明TLR4及其信号通路能够介导β2GPI/抗β2GPI复合物刺激单核细胞表达TF，有助于APS血栓形成。基于文献及前期工作，本项目提出"TLR4在APS的自身免疫激活及其抗体诱发的血栓形成中发挥双重作用"假说。本研究拟利用纯系TLR4正常及TLR4缺陷型小鼠，结合细胞生物学、分子免疫学、分子生物学以及分子影像学、活体成像等研究技术，深入分析TLR4在β2GPI刺激小鼠免疫系统激活及其抗体产生、以及在抗磷脂抗体诱发小鼠动静脉血栓形成中的作用，并利用TLR4及TLR2拮抗剂进一步求证TLR4的作用，分析是否涉及TLR2。研究内容是APS病理机制探讨的新进程，将为APS的免疫干预及血栓形成的防治提供新靶点依据。

TLR4与抗磷脂综合征（APS）的关系正受到愈来愈多的关注, 然而TLR4在APS病理机制中的确切作用并未阐明。本研究利用纯系TLR4正常（C3H/HeN）及TLR4缺陷型小鼠（C3H/HeJ），采用β2GPI蛋白免疫接种二型小鼠，分析小鼠免疫系统激活及其抗体产生情况。结果显示TLR4正常小鼠的树突状细胞（DC）成熟、T细胞活化、B细胞活化乃至自身抗体的产生显著强于TLR4缺陷型小鼠。另一方面，采用抗β2GPI抗体刺激TLR4正常及TLR4缺陷型小鼠，利用分子生物学以及分子影像学、活体成像等研究技术，深入分析了TLR4在抗磷脂抗体诱发小鼠动静脉血栓形成中的作用。结果表明抗β2GPI抗体能够诱导TLR4正常小鼠EAPS模型形成，促进小鼠动脉及静脉血栓形成，小鼠血管内皮表达TF、vWF、及黏附分子VCAM-1、ICAM-1和E-selectin增强，腹腔巨噬细胞表达炎性分子TNF-α、IL-1β和IL-6显著上升。而TLR4缺陷小鼠的血栓形成明显减弱，血管内皮及腹腔巨噬细胞表达活性分子的能力也大大降低。本研究还发现TLR4/ NF-κB信号通路有助于oxLDL/β2GPI/anti-β2GPI复合物诱导TLR4正常小鼠腹腔巨噬细胞泡沫化及表达动脉粥样硬化相关分子，从而促进动脉粥样硬化的进程。茶多酚（EGCG）及他汀类药物、TLR4拮抗剂TAK-242、NF-κB抑制剂PDTC及c-Jun/AP-1抑制剂Curcumin能够明显降低抗β2GPI抗体对小鼠以上的刺激效应。TLR2拮抗剂anti-mTLR2-IgG能够部分抑制抗β2GPI抗体对小鼠巨噬细胞的刺激效应，但弱于TAK-242的抑制效应。本研究结论：TLR4作为关键受体介导机体免疫系统识别并结合β2GPI蛋白，激活免疫细胞直至抗β2GPI抗体产生；TLR4及其信号通路有助于抗β2GPI抗体诱发体内动静脉血栓形成。本研究结果提示, TLR4及其信号通路的关键分子可作为APS的免疫干预及血栓防治的新靶点。