Limosilactobacillus reuteri metabolites modulate immune pathways and intestinal barrier repair after 5 fluorouracil exposure

Antimetabolites such as 5 fluorouracil are known to induce inflammation in the gut and oral cavity, underscoring the need for strategies that mitigate chemotherapy-associated toxicity. The aim of this study was to determine whether secreted components from the probiotic bacterium Limosilactobacillus reuteri DSM 17938, specifically cell-free supernatant, exopolysaccharides, and extracellular membrane vesicles, can support epithelial barrier recovery following 5 fluorouracil-induced injury. Exposure to 5 fluorouracil impaired viability, metabolic activity, and barrier integrity, and shifted the functional responses of Caco-2 cells toward increased inflammation. Stimulation with exopolysaccharides after removal of 5 fluorouracil significantly improved barrier integrity in both enterocyte-like Caco-2 cells and primary human intestinal epithelial cells, while paradoxically inducing an inflammatory protein profile in the enterocyte-like cells. Transcriptomic analysis revealed that exopolysaccharides modulate gene programs associated with extracellular matrix organization and structural remodelling. Furthermore, cell-free supernatant, membrane vesicles, and exopolysaccharides differentially influenced monocyte polarization pathways when monocytes were cultured with supernatant from 5 fluorouracil-exposed Caco-2 cells. Together, these findings demonstrate that bacterial metabolites such as exopolysaccharides influence intestinal barrier recovery upon inflammation and activate immune cell recruitment that could have consequences for the intestinal epithelial integrity during inflammation.