PROJECT SUMMARY/ABSTRACT Liver disease is the second-leading cause of mortality in HIV-infected patients, but its significance as a component of this infection requires further study. HIV infection damages liver cells, including resident macrophages (i.e., Kupffer cells) and hepatocytes. HIV-induced hepatotoxicity is potentiated by second hits, including alcohol. Alcohol consumption enhances the pathological features of HIV infection by increasing viremia, suppressing immune responses, promoting non-adherence to treatment, and causing poor HIV treatment outcomes. In addition, antiretroviral therapy (ART) is less effective in individuals who consume large quantities of alcohol, and certain drugs also provide hepatotoxic effects, leading to treatment cessation. Meanwhile, it is now clear that the liver plays an important role in the pathogenesis of HIV infection. It is possible that in HIV- infected alcohol abusers, the dissemination of infection and liver pathology results from cell-to-cell communication via extracellular vesicles (EVs), including apoptotic bodies (ABs) and exosomes containing viral nucleic acids, miRNAs as well as HIV- and alcohol-modified host cell cargos, which promote liver injury. Moreover, EVs from liver cells can spread inflammation to other organs. In this study, we hypothesize that ethanol metabolism amplifies HIV-triggered communication between hepatocytes and macrophages via EVs, thereby promoting liver inflammation and fibrosis. Furthermore, extrahepatic systemic distribution of these EVs may contribute to the systemic spread of HIV and dissemination of inflammation to other organs. This hypothesis will be tested in the following three Specific Aims: 1. Examine potentiating effects of alcohol on HIV-induced hepatocyte death and the role of large extracellular vesicles (i.e., apoptotic bodies) in promoting liver inflammation by macrophages. 2. Define how alcohol accelerates HIV-induced exosome release from hepatocytes and the role of exosomes in cross-talk between hepatocytes and macrophages in the development of liver inflammation 3. Elucidate the contribution of EVs to liver injury caused by both HIV-infection and ethanol consumption in vivo This pre-clinical investigation will provide the foundation for future clinical trials and translation. Furthermore, these findings could be readily translated to clinical practice by discovering potential biomarkers of liver injury progression, which accompany HIV-infection in those who abuse alcohol. By blocking EV release, we will seek additional treatment modalities that prevent the development of liver disease and systemic inflammation.