NLRP3/ASC/Caspase-1通路调控Th17/Treg平衡在狼疮肾炎发病中的作用机制

Renal dendritic cells (rDCs) and Th17/Treg imbalance are closely associated with the pathogenesis of lupus nephritis (LN), and as the key downstream molecule of NLRP3/ASC/Caspase-1 pathway(primarily expressed on DCs), IL-1β is an important regulator of Th17/Treg balance. However, the role and mechanism of NLRP3/ASC/ Caspase-1 pathway in LN remains unclear. On the basis of our preliminary findings that NLRP3 expression was upregulated and correlated with disease activity in LN, this project intends to (1)To investigate the expression of NLRP3/ASC/Caspase-1 pathway-related molecules and the Th17/Treg balance(quantity, proportion, phenotype and function) in MRL/lpr murine kidney as well as the effects of inhibitors in vivo on the Th17/Treg balance and LN; (2)Through positive and negative regulation, the influence of this pathway on the Th17/Treg balance will be explored in the co-culture system of renal dendritic cells (rDCs) and splenic naive CD4+T cells; (3)The induced lupus model based on NLRP3, ASC and Caspase-1 knockout mice will be applied to further clarify the mechanism; (4)To investigate the correlation between this pathway and clinical data in in renal biopsies from LN patients. This project will help to explore the pathogenesis and find new therapeutic targets in LN.

肾脏树突状细胞(DCs)和Th17/Treg失衡与狼疮肾炎(LN)发病密切相关，而NLRP3/ASC/Caspase-1通路(主要表达于DCs)下游的关键分子IL-1β是调控Th17/Treg平衡的重要因素，但该通路在LN中作用不明。在前期发现狼疮肾炎NLRP3表达上调且与疾病活动相关的基础上，本课题拟(1)探讨MRL/lpr鼠肾脏NLRP3/ASC/Caspase-1通路表达及Th17/Treg平衡(数量、比例、表型和功能)变化及体内应用该通路抑制剂对Th17/Treg平衡的影响和对LN的治疗作用；(2)分离肾脏DCs与脾初始CD4+T细胞共培养，探讨正负调控该通路对Th17/Treg平衡的影响；(3)利用NLRP3、ASC和Caspase-1基因敲除鼠诱导狼疮模型进一步阐明其机制；(4)探讨LN患者肾活检标本中该通路与临床资料的相关性。本课题有助于探索LN的发病机制和发现新治疗靶点。

Th17/Treg失衡与狼疮肾炎(LN)发病有关，而NLRP3/ASC/Caspase-1通路活化产物IL-1β是调控Th17/Treg平衡的重要因素，但该通路在LN中作用不明。本项目发现自发性狼疮鼠肾脏该通路分子表达增高并伴有NLRP3炎性小体活化以及血和肾脏IL-1β升高，提示NLRP3与LN发病有关。体内抑制NLRP3活化降低MRL/lpr小鼠抗ds-DNA产生及其在肾脏沉积，减轻蛋白尿和肾损害，表明NLRP3在LN中起致病作用。为进一步了解LN中NLRP3的激活机制，我们还研究了NLRP3上游受体P2X7，发现狼疮小鼠肾脏P2X7表达上调；化学抑制剂体内阻断P2X7或用siRNA抑制其表达后，狼疮小鼠肾脏NLRP3活化受抑制，血清IL-1β、IL-17水平以及脾脏Th17/Treg比例降低，抗dsDNA抗体产生和肾脏沉积减少，蛋白尿和肾损害减轻，生存时间延长，提示P2X7通过激活NLRP3促进IL-1β产生及Th17细胞反应，参与LN发病。进一步研究发现：体内抑制激酶GSK-3活化可抑制狼疮小鼠肾脏NLRP3炎性小体活化，减轻肾损害，表明GSK3β激活NLRP3炎性小体通路参与LN发病。在人系统性红斑狼疮中，患者外周血单核细胞NLRP3活化程度与血中IL-1β和抗dsDNA水平以及狼疮疾病活动指数呈正相关。抗dsDNA抗体通过与人巨噬细胞表面TLR4结合，诱导活性氧产生而激活NLRP3。将人抗dsDNA抗体注射给NZB/W F1小鼠后，小鼠单核细胞NLRP3活化增强，IL-1β和IL-17水平升高，脾脏Th17/Treg细胞比例显著升高，表明抗dsDNA通过活化NLRP3促进IL-1β分泌，诱导Th17反应。本研究初步明确NLRP3炎性小体活化在LN中的致病作用及其调控因素，为LN治疗提出新靶点。