Project Summary In the era of highly active antiretroviral therapy (ART), liver disease has become a common reason for hospitalization and one of the leading causes of mortality among HIV positive individuals. The etiologies of hepatotoxicity in HIV are multifaceted. HIV mono-infection, HIV co-infection with hepatotropic viruses (HBV and HCV), HIV and alcohol-induced hepatotoxicity and ART-induced hepatotoxicity are known causes of liver disease among HIV-infected individuals. Among all causes of hepatotoxicity, the mechanisms of alcohol- induced hepatotoxicity in HIV-infected cells are unknown. Given that all HIV-infected individuals are on ART due to high ART availability and accessibility, it becomes difficult to evaluate the single effects of alcohol on the liver of HIV-infected individuals. That is why I am adopting an invitro system with the help of my mentors (Drs. Osna and Poluektova) to explore alcohol-induced hepatotoxicity in HIV-infected hepatocytes. My preliminary data under the supervision of my mentors has revealed the following findings: Alcohol metabolites enhanced HIV accumulation in hepatocytes which triggers reactive oxygen species (ROS) that lead to hepatocyte death via apoptotic pathway. When Hepatic Stellate Cells (HSC) were exposed to HIV-infected apoptotic hepatocytes, an upregulation of profibrotic genes such as Col 1A1, TIMP 1 and TGFβ was established after 2 hours of exposure. This implies that there might be a cross talk between hepatocytes and HSC in the premises of HIV and alcohol via apoptotic hepatocytes which results in liver fibrosis. These observations were confirmed in the liver of humanized mice exposed to HIV and alcohol. For therapeutic purposes it has become necessary to understand the mechanisms of alcohol-induced hepatotoxicity in HIV- infected cells. To understand this mechanism, we are confronted with research questions which seek to understand the receptors for HIV entry into hepatocytes and the influence of alcohol metabolites to increase HIV entry. Moreover, the impact of alcohol on HIV degradation in hepatocytes needs to be explored. It is not known if hepatocyte death is due to apoptosis or other cell death mechanisms (such as necrosis or necroptosis) are involved. Hence understanding the type of cell death mechanism will help devise strategies to block/prevent the hepatocyte death, which mediates liver fibrosis. Also, we will explore the pathways involved in the activation of profibrotic pathways to understand if blocking these pathways will prevent activation of fibrotic genes. All the research questions will be tested in both in vitro and in vivo models. Skills that will be gained during the 3-year period dedicated to completing the 3 aims of the research proposal are laboratory techniques such as proteomics, flow cytometry, Real Time Polymerase Chain Reaction (RT PCR) for HIV RNA, mRNA, HIV DNA, digital droplet PCR (ddPCR), flow cytometry, cell culture, and animal handling. During this period,...