Deep Proteomic Profiling of Human Stem Cell-Derived Microglia: Insights Into Inflammatory and Alcohol-Induced Reactivity

Alcohol use disorder (AUD) is associated with significant neuroimmune dysregulation, with microglia emerging as a potential pathological driver of neurotoxicity. However, the molecular landscape driving this alcohol-induced reactivity has yet to be uncovered, especially in the context of human microglia. Here, we comprehensively characterized global-scale molecular changes of human stem cell-derived microglia (iMGLs) after exposure to ethanol and inflammatory mediators lipopolysaccharide (LPS) and TNFα using mass spectrometry-based proteomics. KOLF2.1J hiPSCs were differentiated to iMGLs utilizing commercially available kits and treated with multiple ethanol exposure paradigms, LPS, and TNFα. Proteomic profiles were assessed by data-independent acquisition (DIA) mass spectrometry, followed by a Luminex cytokine assay and a functional phagocytosis assay. Ethanol produced distinct, temporally dependent proteomic changes that differed markedly from the robust proinflammatory responses induced by LPS and TNFα. Cytokine measurement confirmed an overall lack of proinflammatory responses of iMGLs treated with ethanol in contrast to the elevated levels in LPS- and TNFα-treated cells. However, Ingenuity Pathway Analysis and flow cytometry revealed an increase in phagocytosis of iMGLs treated with ethanol for 48 hours. Our findings demonstrate that ethanol exposure induces distinct and temporally dependent molecular phenotypes in human iMGLs, which differ markedly from the robust proinflammatory responses elicited by LPS and TNFα. The novel pathways identified in this study will provide new insights into the human-specific response to ethanol and emphasizes the importance of utilizing human-specific models for neuroimmune interactions in AUD.