NLRP3 Inflammasome Activation, T Follicular Helper Cells and Autoimmunity

摘要 系统性红斑狼疮(SLE)是一种多器官系统自身免疫性疾病，主要影响 处于生育年龄的雌性。增生性肾小球肾炎(GN)患者常进展至末期 肾脏疾病分期(ESRD)。FDA已经50多年没有批准过治疗LN的新疗法了。 需要更好地了解LN的发病机制，以制定更有效的治疗策略。我们的 实验室已经证明，免疫复合体(IC)本身不足以引起ESRD，并结束 器官(肾脏)对损害的抵抗力是决定LN临床结局的重要因素。三款GN机型 已经建立起来了。通过一项新技术，我们已经在慢性肾小球肾炎中发现了独特的 CD11b+IA-F4/80-肾小球内巨噬细胞是产生肿瘤坏死因子α、足细胞的主要细胞 系膜细胞是产生IL-1β/IL-18的主要细胞，系膜细胞产生IL-6。提出了一种新的假设 肿瘤坏死因子α、白介素1β、白介素18和白介素6是由病变肾小球中不同的细胞(室)产生的。 这些细胞因子与它们的受体之间的相互作用助长了肾小球内持续的炎症。 导致足细胞减少和肾功能衰竭。我们已发表的和初步的数据显示NLRP3激活 足细胞中的炎性小体。此外，NLRP3激活发生在TFH细胞亚群中，这些细胞是有能力的 激活B细胞。除了抑制足细胞中NLRP3的激活外，MCC950，一种特异性的激酶 NLRP3的抑制剂抑制自身抗体的产生和生发中心(GC)的形成。我们提议审问 足细胞和Tfh细胞中NLRP3激活在狼疮肾炎发病机制中的不同作用三 提出了特定的目标：特定的目标1：利用具有CD4基因特异性缺失的突变小鼠 NLRP3激活途径检测CD4T细胞中这些基因失活对TFH细胞的影响 在B6背景下小鼠的发育，GC的形成，抗体的产生。类似的实验将会进行。 在以NZM2328为背景的小鼠中，增加了检测自身抗体的产生和发展 LN；特定目标2：确定足细胞缺失NLRP3激活途径中基因的影响 关于在没有减少自身抗体的情况下出现严重蛋白尿和早期死亡的情况；具体目标3： 为了证明狼疮株在caspase-1激活的Tfh细胞中固有地升高，并具有高反应性 免疫和特异性目标4：探讨MCC950抑制NLRP3的机制 活化、TFH细胞活化、GC形成和自身抗体的产生与严重蛋白尿的减轻 在肾毒血清诱导的肾炎模型中，干扰素𝛼可加速LN和自发性LN。这些研究的结果 拟议的实验将提供令人信服的数据来支持将NLRP3作为辅助治疗的靶向 治疗狼疮性肾炎的方法。他们将提供关于NLRPs激活和IL-1β作用的明确数据 和/或IL-18在TFH细胞激活和自身免疫中终止器官损伤。因此，这项建议具有重要意义 与我们对免疫反应和翻译潜能的理解有关的基础科学。

Abstract Systemic lupus erythematosus (SLE) is a multi-organ systemic autoimmune disorder affecting mainly females in their reproductive ages. Patients with proliferative glomerulonephritis (GN) often progress to end stages of renal disease (ESRD). No new therapy for LN has been approved by the FDA for more than 50 years. A better understanding of the pathogenesis of LN is needed to devise more effective therapeutic strategies. Our laboratories have shown that immune complexes (IC) by themselves are not sufficient to cause ESRD, and end organ (kidney) resistance to damage is important in determining the clinical outcome of LN. Three GN models have been established. With a novel technique, we have identified in chronic glomerulonephritis that unique CD11b+IA-F4/80- infiltrating intraglomerular macrophages are the dominant cells that make TNFα, podocytes are the dominant cells that made IL-1β/IL-18 and mesangial cells make IL-6. A novel hypothesis is proposed that TNFα, IL-1β, IL-18 and IL-6 are made by different cells (compartments) in the diseased glomeruli and that the interactions among these cytokines with their receptors fuel the on-going inflammation within glomeruli leading to podocyte effacement and renal failure. Our published and preliminary data show activation of NLRP3 inflammasome in podocytes. In addition, NLRP3 activation occurs in a subpopulation of Tfh cells that are potent to activate B cells. In addition to the inhibition of NLRP3 activation in podocytes, MCC950, the specific kinase inhibitor of NLRP3 inhibits auto-Ab production and germinal center (GC) formation. We propose to interrogate the differential roles of NLRP3 activation in podocytes and Tfh cells in the pathogenesis of lupus GN. Three specific aims are proposed: Specific Aim 1: To utilize mutant mice with CD4 specific deletion of genes in the NLRP3 activation pathway to determine the effect of inactivation of these gene in CD4 T cells on Tfh cell development, GC formation, Ab-production in mice on B6 background. Similar experiments will be carried out in mice on NZM2328 background with the addition of assays for auto-Ab production and the development of LN; Specific Aim 2: To determine the effects of podocyte deletion of genes in the NLRP3 activation pathway on the development of severe proteinuria and early mortality in absence of auto-Ab reduction; Specific Aim 3: To demonstrate that lupus strains are intrinsically elevated in caspase-1 activated Tfh cells and hyper-responsive to immunization and Specific Aim 4: To interrogate the mechanism by which MCC950 inhibits NLRP3 activation, Tfh cell activation, GC formation and auto-Ab production and the attenuation of severe proteinuria in nephrotoxic serum (NTS) induced nephritis, IFN𝛼 accelerated LN and spontaneous LN. The results of these proposed experiments will provide convincing data to support targeting NLRP3 as an adjunct therapeutic approach in treating LN. They will provide definitive data regarding the roles of NLRPs activation and IL-1β and/or IL-18 in Tfh cell activation and end organ damage in autoimmunity. Thus the proposal has significance in basic science relating to our understanding of immunological responses and translational potentials.