Alcoholic liver disease (ALD) continues to be a devastating disease in the United States and many other countries with the global mortality estimated to exceed 0.7 million per year. Chronic ethanol consumption results in lipid accumulation in hepatocytes and their organelle stress leading to inflammation and fibrosis in the progression of ALD. The crosstalk between hepatocytes and non- parenchymal cells - including hepatic macrophages (HMs) and hepatic stellate cells (HSCs) - is crucial to this process. However, the molecular mechanisms and signaling pathways involved in the crosstalk between lipid overloaded hepatocytes and non-parenchymal cells, remain incompletely understood. In particular, the mechanisms by which this crosstalk facilitates or regulates a transition from chronic asymptomatic alcoholic steatohepatitis (ASH) to fatal alcoholic hepatitis (AH) is a central issue for designing efficacious novel treatments for this devastating disease. While focused on the investigation of cell-to-cell communication we recently revealed that damaged hepatocytes release extracellular vesicles (EVs) and these EVs circulate in the blood in mouse models of ALD. EVs are efficiently internalized into target cells and transfer their cargo including miRNAs. The later is a key mechanism by which encapsulated miRNAs in EVs (EV-miRNAs) serve as ?functional extracellular RNAs? to regulate protein translation in target cells. EVs also contain sterile danger signal known as damage-associated molecular patterns (DAMPs). We found that EVs derived from hepatocytes from AH mice are enriched in mitochondrial DNA (mtDNA) that contribute to activation of an inflammatory process. Further, our pathway analysis of RNA-seq data comparing AH vs. preceding chronic (c)ASH mouse livers, identifies EV as one of the most significantly upregulated pathways. Based on these results, we propose the central hypothesis that EVs with a quantitatively and qualitatively distinct cargo are released from damaged hepatocytes in AH vs. preceding cASH to induce unique cellular crosstalk in the genesis of the AH pathologic phenotype. We also hypothesize that plasma EV analysis serves as a liquid liver biopsy providing a barcode for diagnosis and staging of ALD severity. To investigate these hypotheses our proposal has the following SPECIFIC AIMS. 1) Determine the role of EVs and their cargo derived from hepatocytes as barcodes for a transition from asymptomatic cASH to AH in murine models and human ALD. 2) Dissect the mechanisms involved in EV-mediated cell-to-cell communication in the cASH to AH transition. To address these central issues, we have put together a MPI investigative team with expertise in EV biology, ALD pathology, development of unique animal models, human ALD, and RNA therapeutics.