Neuroimmune Regulation of Intestinal Permeability in Inflammatory Bowel Disease (IBD) and Brain-Derived Neurotrophic Factor (BDNF) and Zonulin

Abstract

We have previously reported that human intestinal epithelial cells released on the basolateral side of culturedmonolayers, arginin-cleavage proteolytic activity upon stimulation by the bacterial motif LPS. Concomitantly, LPS was able to induce a significant increase in permeability of intestinal epithelial monolayers. Further we observed that LPS-induced increased permeability of cultured epithelial cells could be prevented by treatment with a large spectrum serine protease inhibitor. Here, we investigated the effects of pathogenic (EPEC) or commensal (HB101) forms of E. coli on the expression of the proteolytic enzyme mesotrypsin by intestinal epithelial cell lines, and the role of proteolytic activity in E. coliinduced increased permeability. Methods: Human intestinal epithelial cell line Caco-2 was polarized on Transwell chambers for 15 days. Cells were apically stimulated for 5 hours with bacterial suspension of E. coli HB101 and EPEC E2348/69 at 2.108 CFU/well, in the presence or not of the serine protease inhibitor FUT-175 (50μg/ml). Paracellular epithelium permeability was evaluated by dextran-FITC apical to basal passage method. We have evaluated the expression of the mesotrypsin/Trypsin IV gene by qRT-PCR for the mRNA and by western-blot using a rabbit polyclonal antibody against human trypsinogen IV protein. Results:We have observed that EPEC, but not HB101 was able to break the barrier function of epithelial cells in monolayers, increasing the transepithelial passage of dextran-FITC. The large spectrum serine protease inhibitor FUT-175 was able to completely prevent this increased permeability in response to EPEC. In parallel, we observed that the trypsinIV/ Mesotrypsin gene was expressed in Caco-2 cells infected with EPEC, at the mRNA levels. In contrast mRNA expression of trypsinIV/Mesotrypsin was significantly down-regulated when Caco-2 cells were exposed to the commensal form of E. coli: HB101. The trypsinogen IV protein was also detected in supernatants of epithelial cells exposed to EPEC, but not detected in supernatants of Caco-2 cells exposed to HB101. Conclusion: The intestinal epithelium infected by a pathogenic form of E. coli (EPEC), but not by a commensal form (HB101) becomes leaky, allowing the passage of macromolecules. The release of serine protease activity seems to play a major role in the breakage of this epithelial barrier function by pathogenic E. coli. Epithelial expression of trypsin IV/mesotrypsin, which is differentially regulated by pathogenic or commensal forms of E. coli, could play an important role in permeability changes and overall in alerting the immune system to pathogen exposure.