BIFIDOBACTERIUM BIFIDUM ENHANCEMENT OF INTESTINAL EPITHELIAL TIGHT JUNCTION BARRIER IS MEDIATED BY TLR-2/TLR-6 SIGNAL TRANSDUCTION PATHWAY-DEPENDENT INCREASE IN TOLL-INTERACTING PROTEIN (TOLLIP) AND OCCLUDIN GENE ACTIVITY

Background: Dysfunctional intestinal tight junctions (TJ) barrier is a key pathogenetic factor of inflammatory bowel diseases. We recently showed that a particular strain of Bifidobacterium bifidum, strain BB1, produces a strain-specific marked enhancement in intestinal TJ barrier in a TLR-2 dependent manner. TOLLIP is a potent inhibitory, cytosolic adaptor protein that represses MyD88. However, the intracellular mechanism and the involvement of membrane receptors and adaptor proteins remain unclear. Aims: The major aim of this study was to delineate the intracellular mechanisms that mediate the BB1 enhancement of the intestinal epithelial TJ barrier by examining the interaction of BB1 with the enterocyte apical membrane receptor TLR-2 and the adaptor proteins TOLLIP/MyD88.
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: 1) Treatment of BB1 (1x10⁸ CFU/ml) produced an increase in Caco-2 transepithelial resistance (TER) (80% increase) and a decrease in trans-epithelial flux of paracellular marker, inulin (50% decrease). BB4 did not affect Caco-2 TER or inulin flux, confirming that the BB1 enhancement of TJ barrier was strain-specific. 2) BB1, but not BB4, caused an increase in Caco-2 TOLLIP expression, activity and siRNA induced knockdown (KD) of TOLLIP prevented the BB1 induced increase in Caco-2 TJ barrier. 3) BB1 did not affect expression or activity of MyD88, and siRNA MyD88 KD transfection did not inhibit the BB1 enhancement of the TJ barrier. 4) BB1 caused an increase in TLR-2 expression and TLR-2 KD prevented the BB1 increase in TOLLIP expression, activity, and TJ barrier enhancement. 5) BB1 produced an increase in occludin gene activity (promoter activity) and protein expression. Occludin KD prevented the BB1 increase in TJ barrier. 6) BB1 (but not BB4) oral-gastric gavage in live mice also produced an enhancement of intestinal TJ barrier as evidenced by the decrease (50%) in mouse intestinal permeability to dextran 10 kDa. 7) The BB1 enhancement of mouse intestinal TJ barrier was prevented in TLR-2 intestinal epithelial cell-specific KO mice (TLR-2 ^fl/fl Villin-cre mice). 8) BB1 produced an increase in occludin mRNA and protein expression in mouse enterocytes and the BB1 increase in occludin expression was abolished in TLR-2 ^fl/fl Villin-cre mice. Conclusion: These studies provide a novel insight into the role of adaptor proteins and intracellular mechanisms that mediate the BB1 enhancement of intestinal epithelial TJ barrier in in vitro and in vivo. These data show that TLR-2 signal transduction pathway mediates the BB1 enhancement of intestinal TJ barrier via a TOLLIP-dependent up-regulation of occludin gene activity, highlighting TOLLIP as a potential therapeutic target for enhancing the intestinal TJ barrier function.