IMMUNE-MEDIATED HEPATOCYTE INJURY: INTERPLAY OF ALCOHOL AND T CELLS - ABSTRACT THE PRIMARY PURPOSE OF THIS RUTH L. KIRSCHSTEIN NRSA F30 APPLICATION IS TO PROVIDE THE GROUNDWORK THAT WILL PREPARE THE APPLICANT FOR AN ACADEMIC MEDICAL CAREER. MORE THAN 10% OF THE POPULATION IN THE UNITED STATES MEET THE CRITERIA FOR ALCOHOL USE DISORDER, AND EXCESSIVE ALCOHOL USE IS ONE OF THE LEADING CAUSES OF PREVENTABLE DEATHS. SPECIFICALLY, ALCOHOL-ASSOCIATED LIVER DISEASE (ALD) IS A LEADING CAUSE OF MORBIDITY AND MORTALITY IN PEOPLE WITH ALCOHOL MISUSE, AND ALD-RELATED DEATHS ARE PROJECTED TO CONTINUE TO RISE 4.7% ANNUALLY. ALCOHOL MISUSE CAUSES SIGNIFICANT ALTERATIONS TO THE IMMUNE SYSTEM THAT CONTRIBUTE TO THE DEVELOPMENT AND PROGRESSION OF ALD. WHILE THE EFFECTS OF ALCOHOL ON INNATE IMMUNE CELLS LIKE KUPFFER CELLS ARE WELL DESCRIBED, THE ROLE OF ADAPTIVE IMMUNE CELLS ON LIVER PATHOGENESIS IS NOT CLEAR. PREVIOUS STUDIES IN THE LABORATORY INDICATE THAT CHRONIC ALCOHOL TREATMENT DOES NOT INCREASE LIPID CONTENT IN HEPATOCYTE SPHEROIDS. THESE RESULTS AND PUBLISHED DATA SUGGEST THAT CROSS TALK WITH OTHER CELL TYPES SIGNIFICANTLY CONTRIBUTE TO LIPID ACCUMULATION IN HEPATOCYTES, WHICH IS THE FIRST STAGE OF ALD. ADDITIONALLY, PUBLISHED WORK FROM THE GROUP DEMONSTRATE THAT ALCOHOL INCREASES DIFFERENTIATION OF CD4 T CELLS TO PROINFLAMMATORY TH1 CELLS. ALCOHOL MISUSE INCREASES INFLAMMATORY SIGNALING FROM IMMUNE CELLS, INCLUDING EXTRACELLULAR VESICLES (EVS), MICRORNA-155 IN EVS, AND PROINFLAMMATORY CYTOKINES, WHICH CAN CONTRIBUTE TO HEPATOCYTE INJURY. MIR-155 TARGETS LIPID AND INFLAMMATORY GENES AND HAS BEEN IMPLICATED IN ALD. LIKEWISE, PROINFLAMMATORY CYTOKINE INTERFERON GAMMA (IFN-G) IS CYTOTOXIC AND CONTRIBUTES TO BIOENERGETIC DYSFUNCTION, REACTIVE OXYGEN SPECIES PRODUCTION, AND CELL DEATH OF HEPATOCYTES. PRELIMINARY DATA GENERATED FOR THIS PROPOSAL INDICATE PLASMA IFN-G SIGNIFICANTLY PREDICTS AST/ALT RATIOS IN PEOPLE WITH RECENT ALCOHOL MISUSE. ADDITIONAL RESULTS SUGGEST THAT COCULTURING HEPARG HEPATOCYTE SPHEROIDS WITH NAIVE STIMULATED CD4 T CELLS OR IFN-G SIGNIFICANTLY INCREASES HEPATOCYTE CELL DEATH, WHICH IS FURTHER INCREASED IN ETHANOL-TREATED SPHEROIDS. TAKEN TOGETHER, PRELIMINARY DATA AND PUBLISHED LITERATURE PROVIDE EVIDENCE FOR ALCOHOL AND T CELL-MEDIATED HEPATOCYTE DAMAGE LEADING TO END-ORGAN INJURY. THUS, THE OVERALL HYPOTHESIS FOR THIS PROJECT IS THAT ALCOHOL MISUSE INCREASES CD4 T CELL SECRETION OF BOTH MIR-155 IN EXTRACELLULAR VESICLES (EVS) AND IFN-G THAT CONTRIBUTE TO HEPATOCYTE INJURY. THE PROPOSED STUDY WILL EMPLOY A VARIETY OF STATE-OF-THE-ART TECHNIQUES TO TEST THE HYPOTHESIS THAT ALCOHOL MISUSE (1) INCREASES MIR-155 IN CD4 T CELL-DERIVED EVS, AND (2) INCREASES IFN-G SECRETION FROM CD4 T CELLS, WHICH INCREASES MARKERS OF CELLULAR INJURY IN HEPATOCYTE SPHEROIDS. WITH A STRONG MENTORING TEAM DEDICATED TO DEVELOPING A WELL-ROUNDED PHYSICIAN SCIENTIST, COMPLETION OF THE PROPOSED TRAINING PLAN IN ALCOHOL MISUSE-RELATED RESEARCH WILL ENSURE THAT THE APPLICANT IS EQUIPPED FOR A SUCCESSFUL CAREER IN ACADEMIC MEDICINE.