Alcohol disrupts spatial coordination of autophagy–lysosome pathways to impair intestinal barrier integrity

BACKGROUND: Alcohol use disorder (AUD) drives intestinal barrier dysfunction, endotoxemia, and liver injury, yet the epithelial mechanisms remain undefined. Autophagy and lysosomes maintain intestinal epithelial homoeostasis and barrier integrity, but their response to chronic alcohol exposure is unclear. METHODS: Human intestinal tissues from individuals with and without AUD were analysed alongside human intestinal organoids. Wild-type, CAG-RFP-EGFP-LC3, GFP-LC3B, and Atg16l1 hypomorphic mice underwent chronic-binge ethanol feeding. Organoids and Caco-2 cells were exposed to ethanol (50 mM) with or without bafilomycin A1 or Torin-2. Autophagy-lysosome pathways were assessed using transcriptomics, immunofluorescence, biochemical fractionation, and flux reporters. FINDINGS: Alcohol induced heterogeneous autophagy-lysosome responses characterised by lysosomal stress/adaptive remodelling. These changes occurred with limited autophagosome formation and maturation, resulting in impaired autophagic throughput. These responses differed along the crypt-villus axis and between intestinal regions. Disrupted autophagy was associated with altered trafficking and accumulation of claudin-2. Atg16l1 hypomorphic mice exhibited increased baseline intestinal permeability vs wild-type controls (p = 0.03). Following ethanol exposure, Atg16l1 hypomorphic mice showed greater intestinal permeability (p = 0.003), circulating and hepatic endotoxin (p = 0.002), hepatic triglycerides (p = 0.01), and serum ALT (p = 0.02) vs ethanol-fed wild-type mice. INTERPRETATION: Alcohol disrupts intestinal autophagy-lysosome homoeostasis in a spatially heterogeneous manner, accompanied by defective epithelial proteostasis and tight junction dysregulation. Impaired autophagosome formation exacerbates gut permeability and liver injury, supporting intestinal autophagy as a potential therapeutic target in alcohol-associated disease. Broader applicability of the human findings requires validation in larger cohorts. FUNDING: This study was supported by grants from the National Institutes of Health.