F-actin dynamics couples sphingolipid metabolism to epithelial barrier integrity in chronic colitis

ABSTRACT Background Intestinal barrier dysfunction is a hallmark of inflammatory bowel diseases (IBD). This condition causes intoxication and immune hyperactivation. Understanding the events underlying epithelial barrier disruption during chronic inflammation is key to developing barrier-restoring therapies. Filamentous actin (F-actin) is essential for maintaining polarity and junctional integrity. However, the contribution of F-actin dynamics to IBD-associated barrier dysfunction remains unclear. Objective We aimed to examine actin cytoskeleton integrity during chronic colitis across mouse models and human patients and identify potential regulators of cytoskeleton dynamics. Design F-actin and junctional proteins were analyzed in three models of chronic colitis ( Muc2 KO, DSS-induced colitis, adoptive transfer colitis) using confocal microscopy. Claudin-3 interactors were identified by immunoprecipitation and proteomics. Intestinal organoids were used to assess the effect of F-actin disruption on barrier integrity. Metabolomic and gene expression analyses identified candidate pathways, further validated by chemical inhibition. Biopsies from patients with ulcerative colitis (UC) were examined using transmission electron microscopy and confocal microscopy. Results Disrupted actin dynamics emerged as a critical driver of epithelial barrier dysfunction in chronic colitis. An imbalance between polymeric and monomeric actin impaired barrier integrity in vivo and in 3D organoids. Immunoprecipitation identified actin and associated factors as the primary interactors of claudin-3 with reduced interaction during inflammation. Ceramide metabolism was revealed as a potential regulator of F-actin and intestinal barrier. In UC patients, we confirmed concurrent disruption of junctions and F-actin. Conclusions F-actin dysregulation contributes to barrier dysfunction in IBD and targeting its modulators, including ceramide biosynthesis, represents a novel therapeutic strategy. WHAT IS ALREADY KNOWN ON THIS TOPIC Epithelial damage and increased paracellular permeability are key characteristics of inflammatory bowel diseases. Paracellular permeability is partially attributed to the downregulation of junction proteins but this mechanism does not explain all clinical observations. In the Muc2 KO mouse model of chronic colitis, F-actin organization and membrane localization of tight junction protein claudin-3 are disrupted, although protein expression levels remain unchanged. WHAT THIS STUDY ADDS F-actin dynamics is impaired in the intestinal epithelium across three different mouse models of chronic colitis and IBD patients. Disruption of F-actin dynamics leads to impaired membrane localization of tight and adherens junction proteins and increased intestinal epithelial permeability in vivo and in colonic organoids. Inhibition of ceramide biosynthesis rescues F-actin polymerization and intestinal barrier integrity in mouse chronic colitis models. HOW THIS STUDY MIGHT AFFECT RESEARCH, PRACTICE OR POLICY Targeting F-actin dynamics is a promising approach to improve gut epithelial integrity. "Ceramide–F-actin–junction" axis is proposed as one of the mechanisms behind epithelial barrier disruption in colitis. Therapeutic targeting of this axis represents a promising path for restoring gut integrity.