MUC2 mucin is the major glycoprotein in colonic mucus that separates intestinal microbiota from underlying host cells and serves as a food source for some eubacteria. MUC2 deficiency results in impaired epithelial barrier function, imbalance in gut microbiota, and spontaneous colitis. Probiotics have been shown to have a protective effect against colitis. In this study we used Muc2 mucin-deficient (Muc2^-/-) and Muc2^+/+ littermates to test whether the probiotic mixture VSL#3 requires an intact mucin barrier to exert its beneficial effect. VSL#3 alone reduced basal colonic proinflammatory cytokine levels and improved epithelial barrier function in Muc2^-/- animals. Similarly, in dextran sulfate sodium-induced colitis, VSL#3 dampened the proinflammatory chemokines KC, monocyte chemoattractant protein-1, and macrophage inflammatory protein-2 and upregulated the tissue regeneration growth factors transforming growth factor-β, fibroblast growth factor-1, and vascular endothelial growth factor-A, which accelerated resolution of colitis symptoms in Muc2^-/- animals. Importantly, improved colonic health in VSL#3-treated animals was associated with attenuated reactive oxygen species production by peritoneal macrophages, restoration of antimicrobial peptide gene expression in the small intestine, and increased abundance of bacterial commensals in the gut. The beneficial effects of VSL#3 in Muc2^-/- animals were mediated by acetate, an important short-chain fatty acid produced by gut bacteria. These studies provide evidence for the first time that VSL#3 can enhance epithelial barrier function by dampening the proinflammatory cytokine and chemokine response, accelerating restitution, and altering commensal microbiota in the absence of a functional mucus barrier.

New & noteworthy: It is unclear whether probiotics require an intact mucin barrier to first colonize and/or exert their protective functions. In this study we used mucin-deficient (Muc2^-/-) mice to interrogate if the multispecies probiotic mixture VSL#3 could enhance epithelial barrier function. In the absence of a mucus bilayer, VSL#3 dampened proinflammatory and chemokine production, accelerated restitution, and markedly improved gut permeability mediated by the short-chain fatty acid acetate in the colon.