<i>BIFIDOBACTERIUM BIFIDUM</i> PREVENTS CYTOKINE INDUCED INCREASE IN INTESTINAL TIGHT JUNCTION PERMEABILITY BY A NOVEL MECHANISM: CROSSTALK BETWEEN PPAR-γ, NF-κB AND MLCK

Background A defective gastrointestinal epithelial tight junction (TJ) barrier has been implicated as a key pathogenic factor in inflammatory bowel diseases (IBD) and other gut inflammatory conditions. An important therapeutic target to treat IBD is to enhance or restore the intestinal TJ barrier function. We recently showed that Bifidobacterium bifidum (BB) causes a marked enhancement of the intestinal epithelial TJ barrier in a strain-specific manner. However, the role of BB (and the possible mechanism involved) in protecting against cytokine-induced increase in intestinal permeability remains unclear. Aims: The major purpose of this study was to delineate the protective effect of BB against the proinflammatory cytokines (TNF-α and IL-1β) induced increase in intestinal TJ permeability and the mechanism involved. Methods: Filter-grown Caco-2 monolayers (in vitro) and recycling intestinal perfusion of live mice (in vivo) were used to assess intestinal TJ permeability. Results: TNF-α and IL-1β caused rapid activation of NF-kB and NF-kB-dependent activation of myosin light chain kinase (MLCK) gene expression and activity in Caco-2 monolayers and mouse enterocytes in live mice. BB inhibited the TNF-α and IL-1β increase in intestinal TJ permeability in a strain-specific manner. BB preservation of the TJ barrier was preceded by increased peroxisome proliferator-activated receptor-gamma (PPAR-γ) activity, phosphorylation, and inhibition of NF-kB activation. BB also inhibited the TNF-α and IL-1β increase in MLCK expression and activity in Caco-2 monolayers and in mouse enterocytes. BB inhibition of TNF-α and IL-1β induced an increase in NF-kB activation, MLCK expression and activity, and TJ permeability was abolished by siRNA-induced knockdown of PPAR-γ in Caco-2 monolayers. The Villin-cre intestinal epithelial-specific PPAR-γ knock-out mice were generated to study the role of mouse enterocytes PPAR-γ in BB protective effect. BB did not inhibit the TNF-α and IL-1β induced NF-kB activation, increase MLCK expression and activity, or increased mouse intestinal permeability in PPAR-γ intestinal epithelial cell-deficient mice (Villin Cre). Conclusion: These studies provide a novel insight into the mechanism of BB preservation of the intestinal TJ barrier and protection against cytokine-induced increase in intestinal TJ permeability. These data show for the first time, BB (or probiotic bacteria) preserves and protects the intestinal barrier function by PPAR-γ-dependent inhibition of NF-kB activation and the subsequent MLCK gene activation and phosphorylation.