Human α-defensin-5 (HD5) supplementation attenuates alcohol-induced tissue injury at the gut-liver axis

Background: Alcohol misuse results in hepatitis, pancreatitis, and neurodegenerative disorders. Alcohol consumption alters gut microbiota and increases gut permeability, leading to endotoxemia and multiple organ damage. However, the cellular and molecular mechanisms of alcohol-induced microbiota dysbiosis and barrier dysfunction remain poorly defined. Antimicrobial peptides such as α-defensins, secreted by Paneth cells, regulate gut microbiota homeostasis. We previously reported that ethanol (EtOH) feeding reduces α-defensin expression in mice. α-defensin expression is regulated by Wnt signaling and T-cell factor 4 (TCF4). Paneth cell TCF4 deficiency exacerbates alcohol-induced tissue injury at the Gut-Liver axis. Therefore, we investigated whether the effect of Paneth cell TCF4 deficiency can be prevented by HD5 supplementation and whether HD5 supplementation affects alcohol-induced tissue injury. Methods: Male TCF4(fl/wt)-Defa6Cre (TCF4-deficient; heterozygous Paneth cell-specific knockout of TCF4) mice were fed a Lieber-DiCarli liquid diet with or without EtOH (0% 2d, 1% 2d, 2% 2d, 4% 1wk, 5% 1wk, & 6% 1wk) (EF). Control groups were pair-fed (PF) an isocaloric EtOH-free diet. Starting at 1% EF, mice were given a solution of HD5 (5 mg/kg mouse body weight) in either the PF or the EF diet. Intestinal permeability was measured by the vascular-to-luminal flux of FITC-inulin (6 kDa) in vivo. Tight junction (TJ) and adherens junction (AJ) integrity were assessed by confocal microscopy for occludin, ZO-1, E-cadherin, and β-catenin. Ileum TCF4 mRNA was assessed by qPCR. Plasma lipopolysaccharide (LPS) was measured to evaluate endotoxemia. Liver damage was assessed by analyzing liver weight, histopathology, plasma ALT, plasma AST, Oil Red stain, and cytokine mRNA (TNF-α, IL-6, IL-10, IL-1β, and MCP1). The levels of luminal defensins were assessed by mass spectrometry and proteomics analyses. Results: HD5 supplementation significantly reduced EtOH-induced changes in body weight. EtOH-induced mucosal permeability was associated with reduced staining for TJ and AJ proteins, indicating mucosal barrier dysfunction. HD5 supplementation attenuated EtOH-induced disruption of epithelial TJs and AJs and mucosal barrier dysfunction in TCF4-deficient mice compared with EF alone. Ileum TCF4 mRNA is reduced by ethanol. Interestingly, HD5 supplementation increased TCF4 mRNA levels, suggesting upregulation of Wnt signaling in EtOH-fed TCF4-deficient mice. EtOH-induced plasma LPS elevation was attenuated through HD5 supplementation. Elevations in liver weight, plasma ALT, plasma AST, and liver cytokine mRNA indicated EtOH-induced liver damage, which was attenuated by HD5 supplementation. Furthermore, liver histopathology and Oil Red staining show that HD5 supplementation reduced fat droplet deposits and steatosis in the TCF4-deficient mouse liver compared to EF. EtOH feeding reduced the levels of α-defensins (DEFA1, DEFA5, DEFA20, DEFA21, and DFAR1) in the colonic lumen. Once again, HD5 supplementation elevated these α-defensins, indicating that HD5 directly or indirectly regulated the Paneth cell function. Conclusion: These data indicate that HD5 supplementation alleviates alcohol-induced mucosal barrier dysfunction, endotoxemia, and liver damage, suggesting that Paneth cell dysfunction and suppression of α-defensin expression play a crucial role in alcohol-induced tissue injury. Therefore, Paneth cell α-defensin and TCF4 have therapeutic potential in treating alcohol-induced tissue injury at the Gut-Liver axis. Support or Funding Information: NIH/NIAA - R01AA012307, AA029270; Veterans Administration - IO1BX003014. This abstract was presented at the American Physiology Summit 2026 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.