<i>Bifidobacterium bifidum</i>up-regulation of intestinal epithelial tight junction barrier is mediated by TLR-2/TLR-6 signal transduction pathway activation of occludin gene

Background: Defective intestinal tight junctions (TJ) barrier is a key pathogenic factor of inflammatory bowel diseases (IBD). Recent studies showed Bifidobacterium bifidum (BB) produces a strain-specific, rapid enhancement of intestinal TJ barrier: BB strain referred to as BB1 produced a marked enhancement of the TJ barrier while strain BB4 had no effect. The intracellular mechanism involved, inducing membrane receptors and the signaling pathways, that mediate the BB1 up-regulation of intestinal TJ barrier remain unclear. Aims: The aim of this study was to delineate the membrane receptor and the intracellular processes that mediate the BB1 up-regulation of the TJ barrier. 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: BB1 treatment (1x10 8 CFU/ml) produced a marked enhancement of Caco-2 TJ barrier, as evidenced by an increase in trans-epithelial resistance and a decrease in trans-epithelial flux of paracellular marker, inulin, in Caco-2 monolayers. BB4 did not affect Caco-2 TJ barrier, confirming that the BB1 up-regulation of TJ barrier was strain-specific. BB1, but not BB4, caused an increase in Caco-2 TLR-2 and TLR-6 expression at the apical membrane surface, without affecting TLR-1 expression. The siRNA induced knockdown (KD) of TLR-2 or TLR-6, but not TLR-1, prevented the BB1 up-regulation of TJ barrier. BB1 produced an increase in Caco-2 occludin protein level without affecting other TJ proteins. BB1 also produced an increase in occludin promoter activity and mRNA transcription. The siRNA-induced occludin KD prevented the BB1 up-regulation Caco-2 TJ barrier. The TLR-2 or TLR-6, but not TLR-1, KD prevented the BB1 increase in occludin gene activity and up-regulation of TJ barrier. Interestingly, BB1 activation of TLR-2/TLR-6 signal transduction pathway did not induce activation of MyD88 or IRAK4 but resulted in recruitment and activation of TOLLIP. Moreover, the siRNA-induced KD of TOLLIP, but not MyD88, prevented the BB1 up-regulation of occludin gene activity and TJ barrier. BB1 oral-gastric gavage in live mice also produced an increase in occludin mRNA and protein expression in mouse intestinal epithelial cells and up-regulation of intestinal TJ barrier (decrease in mouse intestinal permeability to dextran 10 KDa). To examine the regulatory role of intestinal epithelial TLR-2 receptor complex in BB1 up-regulation of intestinal TJ barrier in live mice, intestinal epithelial cell-specific knockout mice TLR-2 Δ IEC, was generated. The BB1 induced up-regulation of occludin and intestinal TJ barrier was prevented in TLR-2 Δ IEC mice. Conclusion: These results show for the first time that the BB1 enhancement of intestinal TJ barrier in -vitro and in-vivo is mediated by TLR-2/TLR-6 receptor complex recruitment of TOLLIP and TOLLIP dependent activation of occludin gene in intestinal epithelial cells. Our studies provide an important mechanistic insight into the regulatory role of TLR-2/TLR-6 receptor complex and TOLLIP in probiotic bacterial up-regulation of occludin gene activity and intestinal TJ barrier function. National Institute of Diabetes and Digestive and Kidney Diseases R01-DK-121073-01 This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.