Probiotics prevent zonulin-mediated intestinal barrier dysfunction secondary to bacterial colonization

Abstract

Background: In the mammalian host, the intestinal epithelium along with indigenous microflora serves as a powerful protective barrier. During pathologic conditions, this ecosystem gets disrupted leading to adverse consequences. We have previously shown that Gram (-) normal gut flora can adhere, invade, and trigger necrotizing enterocolitis-like changes in weanling rabbits. Recently, we described a novel eukaryotic protein, zonulin, and its regulatory role in gut permeability via tight junction disassembly. In this study, we evaluated the effect of a candidate probiotic strain, Lactobacillus plantarum strain PP-217 (Lp) on zonulin-mediated changes in the gut mucosa. Methods: Explant cultures from rabbit intestine were used to investigate the ability of a normal flora E. coli to induce zonulin release. Polarity of zonulin release, changes in transepithelial electrical resistance (TEER), transport of inulin, and transcytosis of E. coli were studied in the snapwell system using rabbit and rhesus monkey gut. Zonulin concentration was measured by sandwich ELISA. Results: E. coli induced significant release of zonulin from rabbit organ cultures (1.68 2.2 ng/mg protein.mrrr') compared to uninfected controls. Co-infection with Lp, resulted in a 3-8-fold reduction in zonulin release. Cultures of E. coli alone did not produce zonulin. In the snapwell systems, addition of 10 E. colilml to the apical side of the intestinal segments resulted in zonulin release from the luminal side, but not from the basolateral (BL) side, a drop in TEER (ll 20-57 n crrr'), an increased passage of inulin (11-34 ~mol.h/ crrr'), and transcytosis of E. coli to the BL side (3.3 XlO 1.8XlO in 1-6 hr). Lp reduced the release of zonulin (10-100 fold), prevented the drop in TEER (>50%), reduced inulin transcytosis (>75%), and caused a 4-100fold blockage of E. coli transcytosis. Addition of FIIO, a synthetic octapeptide analogue of zonulin provided similar protection in gut permeability. Conclusions: These results demonstrate that normal flora bacteria can induce zonulin release by the small intestine resulting in a reduction in barrier function and increased bacterial transcytosis. Candidate probiotic strains have the ability to prevent such changes. While the mechanism of action of probiotics in this system remains unidentified, the implications and its potential use can range from enteric bacterial infections to a myriad of conditions involving reduced intestinal permeability. Supported by NIH grants HD 29735 & 34974.