Epithelial MLCK deficiency alleviates alcohol-associated liver disease via dendritic–Th17 cell axis

Background & Aims Intestinal dendritic cells (DCs) play a key role in immune regulation, especially during injury. The role of intestinal epithelial paracellular permeability in DCs antigen presentation and its regulation is unclear. Myosin light-chain kinase (MLCK) regulates barrier permeability, and this study examines its role in DCs function and immune responses in alcohol-associated liver disease (ALD). Methods We used systemic and intestinal epithelial-specific Mylk knockout models ( n = 6 mice per group) to investigate the MLCK-mediated leak pathway in ALD. The effects of constitutively active MLCK in intestinal epithelial cells were also assessed ( n = 6). Mechanistic investigations focused on LPA-LPAR2-Ca 2+ -dependent MLCK activation, while also evaluating tight junction integrity, DC antigen uptake and presentation by flow cytometry, and pathogenic Th17 cell responses ( n = 3). Results The MLCK-mediated leak pathway is upregulated in ALD ( n = 6, p < 0.05). Both systemic and intestinal epithelial-specific Mylk knockout attenuated ALD progression, as evidenced by reduced liver injury markers (ALT decreased by 50%, n = 6, p < 0.05), while constitutively active MLCK accelerated disease ( n = 6, p < 0.05). LPA-LPAR2 signaling activated MLCK via Ca 2+ /CaM-dependent pathways, disrupted tight junctions, and enhanced DC antigen uptake and antigen-presenting capacity (by 1.4-fold, n = 3, p < 0.05). This promoted pathogenic Th17 cell responses and GM-CSF production (by 2-fold, n = 6, p < 0.05) associated with liver inflammation. Conclusions This study highlights the role of MLCK-regulated intestinal permeability in DCs function and Th17 cells differentiation in ALD. Targeting this pathway may provide new therapeutic strategies for ALD. Impact and implications This study reveals the crucial role of MLCK-regulated intestinal paracellular permeability in alcohol-associated liver disease (ALD), expanding our understanding of the gut-liver interaction. The findings provide new scientific insights into immune regulation and barrier function, offering potential therapeutic implications for the treatment of ALD. By highlighting the role of MLCK-mediate barrier function in dendritic cells (DCs) function and Th17 cells differentiation, this research opens up new avenues for clinical application, particularly in developing targeted therapies for ALD. For researchers, these findings offer a fresh perspective on the interplay between the gut microbiota and immune responses, advancing the field of immunology and gastrointestinal diseases.