Sterile inflammation and pyroptotic cell death in liver fibrosis

NLRP3炎症体是一种多蛋白胞质复合体，具有一定的模式功能 识别受体，已成为无菌炎症和细胞死亡的关键调节因子。 NLRP3激活后可分泌成熟的IL-1β和IL-18。Caspase 1的激活也可以 启动一种不同形式的程序性细胞死亡(上睑下垂)，通过处理 Gasdermin D和质膜上形成离散的气孔。使用单细胞RNA 转录组学(scRNA-seq)我们证明了时间控制的、可诱导的全局NLRP3 炎性小体激活导致1)肝巨噬细胞的移位，其特征是 KCS和单核细胞来源的巨噬细胞(MDM)的浸润性；2)肝脏的浸润性 促炎症中性粒细胞；3)增加激活的肌成纤维细胞；4)增强作用 对于IL-18驱动的NLRP3炎症体；5)这些变化与慢性 炎症、胶原沉积增多，发展为肝纤维化。基于我们的 我们发表了新的初步数据，提出了NLRP3的中心假设 炎症体调节的肝脏天然免疫细胞巢的变化是一个中心 触发肌成纤维细胞HSC激活的机制，以及IL-18驱动的肝纤维化。至 调查这一假设我们的建议有以下具体目标。首先，我们将确定 NLRP3激活导致KC耗竭的机制和后果。我们将测试 KCs中NLRP3炎性小体激活导致嗜热性细胞死亡和 MDMS在肝脏中的募集。我们将使用一些独特的、基因工程的 老鼠。为了研究NASH期间KC特异性的NLRP3功能丧失的影响，我们将使用我们的 新的Nlrp3-/-小鼠以模拟人类纤维性NASH的脂肪-果糖-胆固醇饮食。在……里面 体内实验将与使用纯化的KCs、IPSCs和sc的体外分析相补充。 核糖核酸转录学。其次，我们将剖析细胞特异性IL-18信号通路的作用 在NLRP3介导的HSC活化和肝纤维化中。我们将检验NLRP3的假设 中性粒细胞和MDMS的炎性小体激活，随后是IL-18信号通路 活化是HSC活化和肝纤维化的中心驱动因素。我们将描述其角色 IL-18信号转导和识别导致肝纤维化的新的下游介质。我们会 建立IL-18或IL-18R1或IL-18BP小鼠系，以研究细胞中的IL-18信号- 具体方式采用中性粒细胞或巨噬细胞特异性的NLRP3驱动模型。我们还将 研究NLRP3/IL-18信号在NASH相关肝纤维化中的细胞特异性调节作用 活着。最后，我们将研究针对IL-18途径在纤维化中的翻译机制。 肝病。

The NLRP3 inflammasome is a multi-protein cytoplasmic complex functioning as a pattern recognition receptor that has emerged as a key regulator of sterile inflammation and cell death. NLRP3 activation leads to secretion of mature IL-1β and IL-18. Caspase 1 activation can also initiate a distinct form of programmed cell death (pyroptosis) mediated via processing of Gasdermin D and formation of discrete pores in the plasma membrane. Using single cell RNA transcriptomics (scRNA-seq) we demonstrated that time-controlled, inducible global NLRP3 inflammasome activation results in 1) shifts in liver macrophages characterized by depletion of KCs and infiltration of monocyte-derived macrophages (MdMs); 2) liver infiltration with proinflammatory neutrophils; 3) increased activated myofibroblastic HSCs; 4) an enhanced role for NLRP3 inflammasome driven by IL-18; 5) These changes are associated with chronic inflammation, increased collagen deposition and development of liver fibrosis. Based on our published and novel preliminary data we propose the CENTRAL HYPOTHESIS that NLRP3 inflammasome regulated changes in the innate immune cell niche in the liver is a central mechanism that triggers myofibroblastic HSC activation, and liver fibrosis driven by IL-18. To investigate this hypothesis our proposal has following SPECIFIC AIMS. FIRST, we will determine the mechanisms and consequences of KC depletion induced by NLRP3 activation. We will test the hypothesis that NLRP3 inflammasome activation in KCs leads to pyroptotic cell death and the recruitment of MdMs to the liver. We will use a number of unique, genetically engineered mice. To study the effects of KC-specific loss of function of NLRP3 during NASH we will use our new floxed Nlrp3-/- mice on a fat-fructose-cholesterol diet that mimics human fibrotic-NASH. In vivo experiments will be complemented with ex vivo assays using purified KCs, iPSCs and sc- RNA transcriptomics. SECOND, we will dissect the role of cell-specific IL-18 signaling pathways in NLRP3 mediated HSC activation and liver fibrosis. We will test the hypothesis that NLRP3 inflammasome activation in neutrophils and MdMs, followed by IL-18 signaling pathway activation is a central driver of HSC activation and liver fibrosis. We will characterize the role of IL-18 signaling and identify novel downstream mediators responsible for liver fibrosis. We will generate floxed IL-18, or IL-18R1, or IL-18BP murine lines to study IL-18 signaling in a cell- specific manner using neutrophil or macrophage-specific, NLRP3-driven models. We will also assess cell-specific modulation of NLRP3/IL-18 signaling in murine NASH-associated fibrosis in vivo. Finally, we will investigate translational mechanisms targeting the IL-18 pathway in fibrotic liver disease.