志贺毒素2激活NLRP3炎性体的分子机制及其在溶血性尿毒综合症中的作用研究

Infection with Shiga toxin (ST)-producing Escherichia coli can induce hemolytic uremic syndrome (HUS), which draw more attention worldwide due to the outbreaks caused by O157:H7 E.coli and O104:H4 E.coli occurring in United State and German respectively. Although ST2 is the major virulence factor associated with the development of HUS, the host inflammatory response can also contribute to the pathogenesis of ST-induced vascular lesion. Our previous work has shown that NLRP3 inflammasome is involved in the production of IL-1b induced by O157:H7 E. coli. Given the fact that ST can induce macrophage to produce IL-1b and there is no information available on how ST2 activates NLRP3 inflammasome and whether NLRP3 plays a great role in the development of HUS, we propose this project to address these questions. The aim of the project is to study the molecular multi-signal mechanisms of NLRP3 activation by ST in vitro and evaluate the effect of NLRP3 activation on pathogenesis of HUS in vivo using NLRP3 deficient mouse model of ST-HUS. We will firstly express and purify the ST2 for whole project. To study the effect of ST on the protein synthesis of pro-IL-1b, we will focus on the pathway of TLR/MyD88/MAPK/NF-κB using antibodies or inhibitors to block signaling or measuring phosphorylation of key proteins in this pathway. In order to know how ST2 affects activation of NLRP3 pathway, we will explore at least three distinct NLRP3 activation pathways including efflux of intracellular potassium ions (K+), release of cathepsin B and generation of reactive oxygen species (ROS). To clarify the role of NLRP3 activation in pathogenesis of ST-HUS in vivo, we will establish ST-HUS model to compare the difference in inflammatory response and pathology sign of HUS development between NLRP3 deficient and wild type mouse. The results of this project would provide more information about complex molecular mechanism of NLRP3 inflammation activation by ST, which will shed light on the role of host factors in progressing HUS and eventually benefit the better control of HUS in the future.

志贺毒素(ST)虽是引起溶血性尿毒综合征（HUS)的主要因素，但宿主炎症反应可增强ST毒性效应及促进HUS发生。我们前期工作发现NLRP3炎性体参与O157:H7诱导的IL-1b释放。鉴于ST能诱导巨噬细胞分泌IL-1b已被研究证实，ST如何激活NLRP3炎性体及其在HUS中的作用尚无报道，本项目以探索ST激活NLRP3炎性体的分子机制及评价NLRP3炎性体激活对ST-HUS的影响为研究目标。表达纯化ST2，体外实验研究ST2如何影响pro-IL-1b合成和激活NLRP3炎性体的信号通路，影响pro-IL-1b合成涉及TLR/MyD88、MAPK和NF-κB通路，激活NLRP3炎性体的信号通路包括钾离子外流，有机氧产生和组织蛋白酶B释放。同时利用NLRP3缺失小鼠建立ST-HUS模型，验证NLRP3对诱导宿主炎症反应以及发生HUS的影响。研究结果将为更好地防治HUS提供理论依据。

背景：产志贺毒素大肠杆菌感染可引起出血性肠炎和溶血性尿毒综合征（HUS), HUS病死率高愈后差，尚无有效措施控制HUS发生。虽然志贺毒素(ST)是最主要毒力因子，但细菌诱导宿主的炎症反应促进HUS发生的机制尚不明确。炎性体(inflammasome)是目前炎症机制研究中的科学热点，本课题组在研究NLRP3炎性体参与O157:H7诱导炎症反应的工作基础上，提出ST可能激活NLRP3，激活的NLRP3可能影响ST-HUS的假说，该内容尚无报道，故立项研究。研究内容包括: A）ST2全毒素的克隆表达纯化及其蛋白功能鉴定。B）体外研究ST2诱导宿主细胞IL-1表达、合成分泌的特征。C）利用NLRP3、caspase-1、ASC基因缺失的骨髓分化细胞，确认ST诱导的IL-1分泌是否依赖NLRP3/caspase-1。D）体外研究ST2激活NLRP3炎性体通路的分子机制。E）建立ST-HUS小鼠模型，利用NLRP3缺失小鼠体内验证NLRP3基因缺失对宿主发生STEC-HUS的影响。F）体内评价NLRP3抑制剂对小鼠STEC-HUS的保护作用。G)研究河弧菌和EHEC O57：H7的溶血素激活NLRP3的作用与机制。重要结果：利用炎性体的缺失小鼠体外确证了ST2全毒素诱导小鼠巨噬细胞释放IL-1依赖于NLRP3/caspase-1炎性体的激活，但不依赖于NLRC4和AIM2炎性体。发现ATP受体P2X7R阻断剂，K+外流，有机氧的产生是参ST2激活NLRP3炎性体的主要机制，组织蛋白酶B释放对ST2诱导IL-1的影响相对少。建立了ST2-HUS的C57/B6小鼠模型，发现NLRP3-caspase-1通路阻断的缺失小鼠的肾损伤显著轻于野生小鼠。体内初步评价特异性NLRP3抑制剂对ST-HUS有一定保护效果。对河弧菌和EHEC O57：H7激活NLRP3研究中阐明了溶血素是主要激活成分。预期发表SCI论文3篇，累计影响因子10分。已发表2篇合计影响因子8分。最后一篇投稿中预期影响因子5分。科学意义：本项目确证了NLRP3炎性体参与ST-HUS，对NLRP3特异性抑制剂保护ST-HUS的效果进行了评价，结果有助于寻找降低HUS发病率和病死率的防治对策。对其他细菌激活NLRP3参与致病的研究，有助阐明NLRP3参与不同致病菌诱导宿主炎性反应的免疫机制。

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