F-actin dynamics couples sphingolipid metabolism to epithelial barrier integrity in chronic colitis
BackgroundIntestinal 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. ObjectiveWe aimed to examine actin cytoskeleton integrity during chronic colitis across mouse models and human patients and identify potential regulators of cytoskeleton dynamics. DesignF-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. ResultsDisrupted 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. ConclusionsF-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 TOPICO_LIEpithelial damage and increased paracellular permeability are key characteristics of inflammatory bowel diseases. C_LIO_LIParacellular permeability is partially attributed to the downregulation of junction proteins but this mechanism does not explain all clinical observations. C_LIO_LIIn 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. C_LI WHAT THIS STUDY ADDSO_LIF-actin dynamics is impaired in the intestinal epithelium across three different mouse models of chronic colitis and IBD patients. C_LIO_LIDisruption 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. C_LIO_LIInhibition of ceramide biosynthesis rescues F-actin polymerization and intestinal barrier integrity in mouse chronic colitis models. C_LI HOW THIS STUDY MIGHT AFFECT RESEARCH, PRACTICE OR POLICYO_LITargeting F-actin dynamics is a promising approach to improve gut epithelial integrity. C_LIO_LI"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. C_LI