Project Summary/Abstract Since the introduction of effective combination antiretroviral therapy (cART), HIV infection has been converted from a fatal disease into a chronic condition. Liver disease has emerged as the second leading cause of non- AIDS related death in persons living with HIV (PLWH). Nonalcoholic fatty liver disease (NAFLD) is the leading form of chronic liver disease in the U.S., afflicting approximately 30% of adults. PLWH have a high prevalence of NAFLD with rates as high as 53%, associated with not only higher frequency of the inflammatory non-alcoholic steatohepatitis (NASH) but also accelerated liver fibrosis progression. Moreover, in NAFLD/HIV, there is a disproportionate burden of progressive NASH and fibrosis compared with HIV(-) NAFLD. While recent studies have replicated the accelerated fibrosis progression seen in PLWH/NAFLD, the precise mechanisms remain poorly defined. We and others have led the field in demonstrating that HIV itself induces a profibrogenic program in the liver via several pathways. The Hippo signaling pathway controls cell proliferation and fibrogenesis through YAP transcriptional co-activators that regulate more than 400 genes through binding to TEAD, SMAD1/2/3 and other sites. Our recent work has shown increases in YAP-related gene expression among NAFLD patients with fibrosis and in mouse NASH models. We also observed a significant reduction in fibrosis-related parameters in hepatocyte-specific YAP knockout mice fed a NASH diet. We have also shown that HIV induces YAP activation in hepatocytes and macrophages via the LPA/PI3K and AKT pathways. Therefore, HIV and NAFLD likely cooperate at the level of YAP activation to contribute to fibrogenesis and liver damage. Hepatic and monocyte- derived macrophages, dysregulated by HIV infection and polarized during liver injury, are likely to contribute to NAFLD and HIV-related liver disease. Studies have observed increased macrophage content in the livers of PLWH and others have linked YAP activation to macrophage infiltration. However, comprehensive studies of the in vitro and in vivo effects of YAP activation among the key cell types (hepatocytes, macrophages, and HSCs) that drive fibrogenesis in PLWH are lacking. We hypothesize that HIV and NAFLD exert independent and additive effects on YAP activation in both macrophage and hepatocyte populations that in turn accelerate hepatic fibrosis progression through their action on HSCs. In this proposal, using novel coculture assays, ex vivo liver tissue, and a humanized mouse model and combining the efforts of liver pathogenesis and HIV virology/pathogenesis experts, we will: (1) define the contribution of YAP signaling to macrophage polarization, hepatocyte and hepatic stellate cell (HSC) activation in HIV/NAFLD.; (2) assess the effect of cART regimens on macrophage, hepatocyte and HSC phenotypes and YAP activation in models of HIV/NAFLD.; and (3) determine the effects of YAP inhibition on fibrogenesis in HIV/...