PROJECT SUMMARY Alcohol-associated liver disease (ALD), caused by excessive alcohol consumption, is a growing public health problem with a significant impact on the global population. In the United States, ALD is now a cause of rising liver-related mortality, especially among younger patients. Despite this severity, there are currently no FDA-approved therapies. The absence of consensus on optimal drug targets or strategies underscores gaps in our mechanistic understanding of disease drivers. While animal models have greatly contributed to our understanding of ALD pathogenesis, their utility and clinical relevance remain a subject of debate. To enhance diagnostic capabilities and advance effective therapeutics, it is imperative to establish reliable human-relevant models that mimic the liver's response to disease-specific challenges. The increasing number of genetic variants associated with ALD susceptibility further underscores the necessity for human-relevant systems. To address these limitations, we devised a human induced pluripotent stem cell (hiPSC)-derived model for ALD research, capitalizing on a multicellular in vitro liver culture system recently pioneered in our lab. Within this multicellular setup, we co-cultivated hiPSC-derived hepatocytes, macrophages, and hepatic stellate cells (HSCs), mirroring the intricate dynamics of liver physiology in both health and disease. Our investigations unveiled that subjecting the liver culture to clinically relevant blood-alcohol concentrations successfully replicated many pivotal alcohol (EtOH)-induced and endotoxin (i.e., LPS)-mediated ALD features. Deeper scrutiny of hepatocytes over time revealed a macrophage-mediated upregulation of hepatic progenitor markers, a dedifferentiation phenotype frequently observed in ALD patients. Furthermore, the liver cultures exhibited a heightened reaction to EtOH/LPS than EtOH or LPS alone, especially in terms of HSC activation. Subsequent investigation unveiled that EtOH exposure sensitized HSCs to LPS stimulation and TGFβ- induced activation, revealing a previously unexplored mechanism underlying ALD pathogenesis. Across three aims, employing cell and molecular approaches, we capitalize on the unique features of this platform to address questions that cannot be sufficiently explored through existing ex vivo human-relevant systems. We focus on our working hypothesis, postulating that activated macrophages induce hepatocyte dedifferentiation through the secretion of soluble factors (Aim 1), and that EtOH exposure elicits changes in specific genes and pathways, contributing to the heightened sensitivity of HSCs to LPS and TGFβ (Aim 2). Aim 3 seeks to unravel the roles of a genetic risk factor (i.e., rs738409 C>G variant in PNPLA3), in ALD development. These efforts will pave the way for more precise therapeutic interventions and targeted treatments. A better comprehension of the genetic influencers of ALD risk has the potential to enhance the identification o...