Project Summary Dysfunctional inflammatory immune responses to modified lipids/cholesterol and bacterial components such as LPS leads to unresolved and chronic inflammation, promoting various metabolic and genetic diseases. One prime example of chronic inflammatory disease is atherosclerosis, a major cause of cardiovascular disease (CVD) related mortalities. Despite progress in treatments, CVD still accounts for ~1 out of every 3 deaths in the USA. Another disease related to dysregulated cholesterol metabolism and unresolved extrahepatic inflammation is progressive familial intrahepatic cholestasis 1 (PFIC1), where mutations in ATP8b1 (a member of the type 4 subfamily of P-type ATPases) perturbs the detergent-resistant state of the hepatic canalicular membrane, leading to hepatic injury. While much is known about the pathophysiology of PFIC1, the underlying mechanism for how the loss of ATP8b1 leads to compromised integrity of the canalicular membrane is not clear. A high throughput proteomic analysis suggests that ATP8b1 flippase activity and phosphoinositide metabolism may be interconnected, but the mechanistic link between ATP8b1 and phosphatidylinositol metabolism is not clear. Our data provide novel insights into the mechanism that leads to plasma membrane remodeling in PFIC1 macrophages and hepatocytes by establishing role of ATP8b1 in PIP2 trafficking. We are proposing cutting-edge methods to unequivocally establish PIP2 flippase activity of ATP8b1, making it the first known PIP2 flippase from any system. PFIC1 patients often present with extrahepatic inflammatory manifestations, suggesting role of ATP8b1 in regulating inflammation. Recent studies have highlighted the major role of pyroptosis executor Gasdermin D (GsdmD), a pore-forming protein, in promoting a variety of inflammatory diseases. Our data identified ATP8b1 as the first known negative regulator of GsdmD cleavage and we propose to decipher the mechanism of this effect in human induced pluripotent stem cells (iPSCs) and mouse models of inflammation. In addition, we will determine the therapeutic efficacy of targeting GsdmD in reducing atherosclerosis and determine the tissue-specific role of GsdmD in CVD. Successful completion of these aims will reveal new biology underlying PFIC1 extrahepatic inflammatory manifestations and GsdmD-induced progression of atherosclerosis. Our data showing GsdmD cleavage to be the primary mechanism behind increased IL-1β release in ATP8b1-/- monocytes and macrophages may allow the design of anti-GsdmD therapeutics for treating extrahepatic inflammation in PFIC1. Our data showing the efficacy of Disulfiram as an anti-atherosclerotic reagent in hyperlipidemic mice provide pre-clinical data for targeting GsdmD for treating atherosclerosis. Elucidation of tissue-specific role of GsdmD will open up new fields of investigation and therapeutic interventions for treating atherosclerosis.