Role of intestinal epithelial-specific c-jun N-terminal kinase 2 (JNK2) in alcohol-induced tissue injury at the gut-liver axis

Background: Alcoholic hepatitis is associated with endotoxemia and systemic inflammation. Two critical factors that contribute to endotoxemia are gut microbiota dysbiosis and mucosal barrier dysfunction. There is no targeted therapy available to treat alcoholic hepatitis. Therefore, it is essential to define the mechanisms associated with alcohol-induced microbiota dysbiosis and intestinal epithelial barrier dysfunction. Our recent studies indicated that JNK2 plays a crucial role in alcohol-induced gut microbiota and mucosal barrier dysfunction. In the present study, we investigated the role of cell-specific JNK2 in alcohol-associated tissue injury by in vitro and in vivo approaches. Methods: Enteroids were grown from the crypts isolated from wild-type (WT) and JNK2 knockout (KO) mice. At 6-7 days after seeding, enteroids were exposed to varying concentrations of ethanol (EtOH; 30-50 mM) and acetaldehyde (AA; the toxic EtOH metabolic product; 100-200 μM). At varying times after EtOH+AA treatment, enteroids were fixed and stained for JNKT183Y185 (active JNK1/2) for JNK activation or co-stained for occludin and ZO-1 for tight junction (TJ) integrity assessment. Enterocyte (JNK2ΔIEC) and Paneth cell-specific (JNK2ΔPC) JNK2 knockout mice were generated by crossing JNK2Flox mice with VillinCre or Defa6Cre mice. Female JNK2ΔIEC, JNK2ΔPC, and JNK2Flox mice were fed a Lieber-DiCarli liquid diet with or without EtOH (0% 2d, 1% 2d, 2% 2d, 4% 1wk, 5% 1wk, & 6% 1wk). Control groups were pair-fed an isocaloric EtOH-free diet. Body weights were measured twice a week. Intestinal permeability was measured by the vascular-to-luminal flux of FITC-inulin (6 kDa) in vivo. Results: Immunofluorescence staining for JNKT183Y185 and confocal microscopy showed that EtOH+AA activated JNK with a peak activity at one-hour post-treatment, which began to decline by two hours, thus indicating a transient activation of JNK by EtOH+AA. EtOH+AA induced a dose-dependent reduction in TJ integrity, as demonstrated by redistribution of occludin and ZO-1 from intercellular junctions and a decrease in their fluorescence intensity. In contrast, enteroids derived from JNK2-KO mice displayed a stark resistance to alcohol toxicity across all tested concentrations. The KO enteroids maintained their morphology and structural integrity at the end of EtOH+AA treatment in contrast to WT enteroids. Chronic EtOH feeding in WT mice resulted in a dose-dependent reduction in body weight gain, whereas JNK2ΔIEC mice were resistant to EtOH-induced decrease in weight gain up to 5% EtOH. Intestinal mucosal permeability was significantly lower in the colon and ileum of JNK2ΔIEC mice compared to JNK2Flox control mice. However, EtOH feeding at doses up to 6% increased mucosal permeability in both JNK2ΔIEC and JNK2Flox mice. EtOH feeding (6%) elevated liver weight in both JNK2ΔIEC and JNK2Flox mice. These results indicate that intestinal protection by JNK2 deletion depends on the EtOH dose. It may be protective until 5% EtOH, but the mechanism of tissue injury at 6% EtOH is independent of JNK2. A similar study in JNK2ΔPC mice showed a significant protection against EtOH at doses up to 4%. Conclusion: These findings indicate that alcohol induces transient activation of JNK2 in the intestinal epithelium and plays a role in alcohol-induced disruption of TJ. In vivo studies suggest that the protective effect of JNK2 suppression depends on EtOH concentration and the cell types in the intestinal epithelium. Supported by NIH/NIAAA - R01AA012307, AA029270, and 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.