Sterile inflammation and pyroptotic cell death in liver fibrosis

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.