非经典炎症小体介导钩端螺旋体感染性炎症反应及细胞焦亡分子机制研究

Leptospirosis is an important global spreading zoonotic disease. The host animals such as rats infected with Leptospira only present mild symptoms but can shed leptospires from their urine for a long period of time. The human individuals infected with Leptospira present high fever and mononuclear macrophage infiltrating inflammation characterized by inflammatory injury of lung, liver and kidney tissues, but its mechanism remains unknown. Our previous study found that Leptospira could activate classical NLRP3 inflammasome but only caused low or moderate inflammatory reaction. The recent literatures reported that Gram-negative bacteria could induce cell pyroptosis and inflammatory responses through non-canonical inflammasomes mediated by caspase-11 in mice and caspase-4/5 in human. In the present research project, we will use laser confocal microscopy, gene silencing and Western Blot assay to determine the Leptospira and its LPS activating the caspase-4/5 of human mononuclear macrophages and caspase-11 of mouse mononuclear macrophages and their non-canonical inflammasomes to cause the expression of inflammatory cytokines (IL-1α, IL-1β, IL-18 and HMGB1) and cell pyroptosis as well as the enlarging inflammatory responses caused by IL-1α, IL-1β and HMGB1 through IL-1R-, TLR2/4- and RAGE-mediating autocrine and paracrine pathways. The results of this study not only illuminate the molecular mechanism of Leptospira-induced inflammatory responses but also provide novel strategies for prevention and therapy of leptospirosis, which have high creativeness and medical significance.

钩端螺旋体（简称钩体）病是全球流行人兽共患病。鼠类等动物感染钩体后症状轻微但长期尿液排菌，人感染钩体后引起高热及肺、肝、肾组织炎性损伤为特征的单核-巨噬细胞浸润性炎症，其机制不明。我们前期研究发现钩体激活NLRP3经典炎症小体但仅引起中低度炎症反应。近年报道革兰阴性菌经小鼠caspase-11或人caspase-4/5非经典炎症小体引发炎症反应和细胞焦亡（pyroptosis）。本项目拟采用激光共聚焦显微镜、基因沉默、Western Blot等方法确定钩体及其LPS激活人单核-巨噬细胞caspase-4/5和小鼠单核-巨噬细胞caspase-11 经非经典炎症小体表达IL-1α/1β、IL-18和HMGB1等炎性细胞因子和诱导细胞焦亡以及IL-1α/1β和HMGB1经IL-1R、TLR2/4、RAGE自分泌和旁分泌途径放大炎症反应分子机制，为钩体病防治提供新思路，具有较高创新性和医学意义。

钩端螺旋体（简称钩体）病是全球流行人兽共患病。鼠类等动物感染钩体后症状轻微但长期尿液排菌，感染钩体后引起高热及肺、肝、肾组织炎性损伤为特征的单核-巨噬细胞浸润性炎症，其机制不明。我们前期研究发现钩体激活NLRP3经典炎症小体但仅引起中低度炎症反应。近年报道革兰阴性菌经小鼠caspase-11或人caspase-4/5非经典炎症小体引发炎症反应和细胞焦亡（pyroptosis）。在国家自然科学基金项目的资助下，我们采用 Western blotting、ELISA、激光共聚焦、基因沉默、Real time RT-PCR 等多种细胞和分子生物学技术研究了非经典炎症小体介导人和小鼠巨噬细胞钩体感染性炎症反应及细胞焦亡的分子机制，研究结果发现，人单核巨噬细胞THP-1经钩体感染或转染钩体LPS（L-LPS）后，caspase-4非经典炎症小体活化并参与介导分泌炎性细胞因子。小鼠单核巨噬细胞J774A.1和小鼠腹腔巨噬细胞感染钩体或转L-LPS后caspase-11非经典炎症小体活化并参与介导炎性细胞因子IL-1β, IL-1α, IL-18 and HMGB-1的分泌。此外，还研究了IL-1α、IL-1β 和 HMGB1 结合 IL-1R、TLR2/4、RAGE 受体经自分泌和旁分泌途径放大炎症反应的分子机制以及单核-巨噬细胞吞噬问号钩体过程中产生的高水平 ROS 协同自分泌或旁分泌途径激活信号通路上调放大炎症反应的机制。本项目研究结果已发表相关论文8篇，其中SCI论文2篇，中文核心期刊论文6篇。另外有1篇英文论文已投稿至SCI收录杂志，另一篇论文已被核心期刊论文录用。本人受邀参加中国微生物学会人兽共患病病原生物学专业委员会螺旋体组成立大会并做了学术报告。对照项目预定的研究目标和考核指标，本项目课题组开展的研究工作已达到了预期的研究目标和考核指标。

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