Interferon-gamma and alcohol produce cellular injury in a hepatocyte spheroid model

Background: Alcohol misuse is twice as common in People with HIV (PWH) than in the general population. PWH on antiretroviral therapy (ART) have chronic immune activation that contributes to comorbidities including liver disease, which is a leading cause of non-AIDS mortality. However, the effects of alcohol misuse on liver pathogenesis in PWH are not clear. Cross talk with other cell types may significantly contribute to hepatocyte injury, which contributes to the development of alcohol-related liver disease (ARLD). Alcohol misuse and HIV infection both increase proinflammatory CD4 Th1 cells and cytokine secretion, which can contribute to hepatocyte injury. Specifically, plasma interferon gamma (IFN-g) levels correlate with serum bilirubin levels and liver disease severity. IFN-g can affect mitochondrial function, reactive oxygen species (ROS) generation, and induce apoptosis. Therefore, in this study, we hypothesized that IFN-g and ethanol increase hepatocyte injury and the development of ARLD in PWH. Methods: Hepatocyte spheroids were formed by culturing the HepaRG human hepatocyte cell line in ultra-low attachment plates for 4 days. After spheroid formation, a 10-day treatment with 50 mM ethanol was initiated. On day 7 of ethanol exposure, IFN-g was added at concentrations of 2.5 ng/ml or 5 ng/ml IFN-g. Hepatocyte injury was assessed after 72 hours of treatment by quantifying apoptosis, ATP content, and ROS production. The study was conducted using six technical replicates and two biological replicates. Statistical analyses were performed using two-way ANOVA in GraphPad Prism. Results: Ethanol treatment of hepatocyte spheroids cultured with IFN-g resulted in a significant dose-dependent increase of apoptosis (p<0.0001), a significant increase in ROS production (p<0.0001), and a significant decrease in ATP content with an interaction of ethanol and IFN-g (p=0.038). Additionally, there was a significant negative correlation between ATP content and apoptosis (r=-0.79, p<0.0001). Conclusions: Proinflammatory cytokines produced by CD4 T cells may contribute to hepatocyte injury and ARLD in PWH as reflected by IFN-g induced apoptosis and oxidative stress. Decreased ATP production by surviving hepatocytes may indicate inability to energetically compensate for inflammatory stress. Supported by CARC & T32 (P60-AA009803, T32-AA007577). This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.