IRAK-1调控NLRP3炎性体通路在狼疮肾炎进展中的作用机制

Inappropriate innate immune response is closely associated with the pathogenesis of SLE. We have shown that NLRP3 inflammasome activation accelerates lupus nephritis (LN) via promoting inflammatory infiltration and Th17/Tregs imbalance. Recent studies suggest that IRAK-1 mediates the activation of NLRP3 inflammasome in innate immune response. However, it remains unclear whether IRAK-1 is involved in the pathogenesis of LN by modulating NLRP3 inflammasome. Based on our preliminary experiments showing that IRAK-1 regulates NLRP3 activation in murine lupus macrophages, the present project aims to explore the roles of IRAK-1 in regulating NLRP3 inflammasome pathway and Th17/Tregs balance as well as its impact on LN development. IRAK-1 inhibitor or IRAK-1 gene silencing in vivo will be used to treat two murine models of SLE (MRL/lpr and NZB/WF1 mice). The therapeutic efficacy and its underlying mechanisms will be investigated. In addition, pristance-induced lupus model will be created in IRAK-1 knockout mice for further verification. In vitro, the self dsDNA-activated macrophages and dendritic cells will be utilized to study the effects of IRAK-1 on NLRP3 inflammasome activation and the differentiation of Th17 and Tregs. This project will help to determine the role of IRAK-1 in LN and search for novel targets for the treatment of LN.

固有免疫异常与狼疮发病密切相关。我们前期发现NLRP3炎症体通路活化促进炎症细胞浸润及Th17/Tregs失衡，加速狼疮肾炎(LN)进展。最新研究提示IRAK-1介导固有免疫反应中NLRP3炎症体通路活化，但IRAK-1是否通过调控NLRP3参与LN发病尚不明确。本课题在预实验提示IRAK-1介导狼疮巨噬细胞NLRP3活化的基础上，应用IRAK-1抑制剂和siRNA基因沉默等方法干预两种自发狼疮小鼠模型(MRL/lpr和NZB/WF1小鼠)，探讨在狼疮中IRAK-1对NLRP3炎性体通路活化、Th17和Tregs分化及对LN的影响；并用IRAK-1敲除小鼠诱导狼疮模型进一步验证。同时，体外探讨IRAK-1对自身dsDNA诱导的巨噬细胞和树突状细胞NLRP3炎性体通路活化的调控机制，及其对Th17和Tregs分化的作用。本项目有助于明确IRAK-1在LN中的作用机制并探索LN治疗新靶点。

研究背景：固有免疫与狼疮肾炎发病密切相关。NLRP3炎性小体是固有免疫的重要一员，介导IL-1β及IL-18的成熟释放，参与炎症进展。最新研究提示IRAK-1介导了NLRP3炎性小体通路的活化，但IRAK-1是否通过调控NLRP3炎性小体参与LN发病尚不明确。.研究内容：采用IRAK-1抑制剂及NLRP3抑制剂MCC950等治疗狼疮小鼠，收集小鼠肾脏进行肾脏病理染色及免疫荧光染色；Western blot观察IRAK-1/NLRP3炎性小体通路在肾脏的表达情况；流式细胞术分析肾脏浸润炎症细胞以及肾脏固有细胞NLRP3炎性小体活化变化。流式细胞术检测脾脏Tfh、Tfr、B细胞及浆细胞比例及Tfh细胞功能状态。收集小鼠血清、24小时尿液检测自身抗体、肾功能、尿蛋白及细胞因子。体外分离抗dsDNA抗体或去除IgG血清，体外刺激THP-1细胞或足细胞，探讨IRAK-1抑制剂、NLRP3抑制剂对抗dsDNA等自身抗体诱导的NLRP3炎性小体活化的抑制作用。收集SLE患者外周血标本，流式细胞术分析Tfh及Tfr细胞比例变化；检测单核细胞内NLRP3炎性小体活化情况；收集狼疮肾炎患者肾脏活检标本及尿液标本，免疫荧光染色分析NLRP3炎性小体在肾脏足细胞内的活化情况。.结果：发病狼疮小鼠肾脏IRAK-1、 NLRP3炎性小体显著活化；应用IRAK抑制剂、NLRP3抑制剂治疗后狼疮小鼠肾脏IRAK-1以及NLRP3炎性小体活化显著降低，肾损害明显减轻、免疫复合物沉积减少，蛋白尿、血自身抗体和血尿素氮水平明显下降，足细胞病变减轻；足细胞内NLRP3炎性小体活化显著降低。脾脏生发中心B细胞、浆细胞及Tfh细胞数目明显下降，Tfh增殖及功能被抑制。SLE患者外周血单核细胞内NLRP3炎性小体显著活化，抗dsDNA抗体等自身抗体可通过诱导ROS活化巨噬细胞及足细胞内NLRP3炎性小体，应用IRAK抑制剂或NLRP3抑制剂显著抑制自身抗体诱导的NLRP3炎性小体活化。.结论：IRAK-1/NLRP3炎性小体通路参与自发狼疮小鼠肾脏损害的发病；靶向IRAK-1/NLRP3炎性小体通路能抑制适应性免疫功能，减少自身抗体形成，抑制巨噬细胞及足细胞内NLRP3炎性小体活化，减轻狼疮肾炎。