Vasoactive intestinal peptide ameliorates intestinal barrier disruption associated with Citrobacter rodentium-induced colitis.

Attaching and effacing bacterial pathogens attach to the apical surface of epithelial cells and disrupt epithelial barrier function, increasing permeability and allowing luminal contents access to the underlying milieu. Previous in vitro studies demonstrated that the neuropeptide vasoactive intestinal peptide (VIP) regulates epithelial paracellular permeability, and the high concentrations and close proximity of VIP-containing nerve fibers to intestinal epithelial cells would support such a function in vivo. The aim of this study was to examine whether VIP treatment modulated Citrobacter rodentium-induced disruption of intestinal barrier integrity and to identify potential mechanisms of action. Administration of VIP had no effect on bacterial attachment although histopathological scoring demonstrated a VIP-induced amelioration of colitis-induced epithelial damage compared with controls. VIP treatment prevented the infection-induced increase in mannitol flux a measure of paracellular permeability, resulting in levels similar to control mice, and immunohistochemical studies demonstrated that VIP prevented the translocation of tight junction proteins: zonula occludens-1, occludin, and claudin-3. Enteropathogenic Escherichia coli (EPEC) infection of Caco-2 monolayers confirmed a protective role for VIP on epithelial barrier function. VIP prevented EPEC-induced increase in long myosin light chain kinase (MLCK) expression and myosin light chain phosphorylation (p-MLC). Furthermore, MLCK inhibition significantly attenuated bacterial-induced epithelial damage both in vivo and in vitro. In conclusion, our results indicate that VIP protects the colonic epithelial barrier by minimizing bacterial-induced redistribution of tight junction proteins in part through actions on MLCK and MLC phosphorylation.

Conlin VS ，Wu X ，Nguyen C ，Dai C ，Vallance BA ，Buchan AM ，Boyer L ，Jacobson K ... -  《-》

Saccharomyces boulardii ameliorates Citrobacter rodentium-induced colitis through actions on bacterial virulence factors.

Wu X ，Vallance BA ，Boyer L ，Bergstrom KS ，Walker J ，Madsen K ，O'Kusky JR ，Buchan AM ，Jacobson K ... -  《AMERICAN JOURNAL OF PHYSIOLOGY-GASTROINTESTINAL AND LIVER PHYSIOLOGY》

Mechanism of extracellular calcium regulation of intestinal epithelial tight junction permeability: role of cytoskeletal involvement.

Recent studies suggest that an abnormal increase in intestinal tight junction (TJ) permeability may be an important etiologic factor in number of diseases including Crohn's disease, NSAID-associated enteritis, and various infectious diarrheal syndromes. The intracellular processes involved in regulation of intestinal epithelial TJ permeability, however, remain poorly understood. In this study, we used cultured Caco-2 intestinal epithelial cells to examine the intracellular processes involved in extracellular Ca(++) modulation of intestinal epithelial monolayer TJ barrier. Incubation of the filter-grown Caco-2 intestinal monolayers in Ca(++)-free solution (CFS), consisting of modified Krebs-buffer solution containing 0 mM Ca(++) and 1 mM EGTA, resulted in a rapid drop in Caco-2 epithelial resistance and increase in epithelial permeability to paracellular markers mannitol and inulin, indicating an increase in TJ permeability. The increase in Caco-2 TJ permeability was rapidly reversed by the re-introduction of Ca(++) (1.8 mM) into the incubation medium. The CFS-induced increase in Caco-2 TJ permeability was associated with separation of the cytoplasmic and transmembrane TJ proteins, ZO-1 and occludin, and formation of large intercellular openings between the adjoining cells. The CFS-induced modulation of TJ barrier was associated with activation of myosin light chain kinase (MLCK) activity and centripetal retraction of peri-junctional actin and myosin filaments. The inhibition of CFS-induced activation of Caco-2 MLCK with MLCK inhibitor (ML-7) prevented the CFS-induced retraction of actin and myosin filaments and the subsequent alteration of TJ barrier function and structure. Our results suggested that the CFS-induced alteration of TJ proteins and functional increase in TJ permeability was mediated by Caco-2 MLCK activation and the resultant contraction of the peri-junctionally located actin-myosin filaments. Consistent with the role of MLCK in this process, selected inhibitors of Mg(++)-myosin ATPase and metabolic energy, but not protein synthesis inhibitors, also prevented the CFS-induced retraction of actin and myosin filaments and the subsequent increase in TJ permeability. In conclusion, our results indicate that extracellular Ca(++) is crucial for the maintenance of intestinal epithelial TJ barrier function. The removal of extracellular Ca(++) from the incubation medium causes activation of Caco-2 MLCK, which in turn leads to an increase in intestinal monolayer TJ permeability.

Ma TY ，Tran D ，Hoa N ，Nguyen D ，Merryfield M ，Tarnawski A ... -  《MICROSCOPY RESEARCH AND TECHNIQUE》

Vasoactive intestinal peptide prevents PKCε-induced intestinal epithelial barrier disruption during EPEC infection.

We previously showed that vasoactive intestinal peptide (VIP) protects against bacterial pathogen-induced epithelial barrier disruption and colitis, although the mechanisms remain poorly defined. The aim of the current study was to identify cellular pathways of VIP-mediated protection with use of pharmacological inhibitors during enteropathogenic Escherichia coli (EPEC) infection of Caco-2 cell monolayers and during Citrobacter rodentium-induced colitis. EPEC-induced epithelial barrier disruption involved the PKC pathway but was independent of functional cAMP, Rho, and NF-κB pathways. VIP mediated its protective effects by inhibiting EPEC-induced PKC activity and increasing expression of the junctional protein claudin-4. Short-term treatment with TPA, which is known to activate PKC, was inhibited by VIP pretreatment, while PKC degradation via long-term treatment with TPA mimicked the protective actions of VIP. Immunostaining for specific PKC isotypes showed upregulated expression of PKCθ and PKCε during EPEC infection. Treatment with specific inhibitors revealed a critical role for PKCε in EPEC-induced barrier disruption. Furthermore, activation of PKCε and loss of barrier integrity correlated with claudin-4 degradation. In contrast, inhibition of PKCε by VIP pretreatment or the PKCε inhibitor maintained membrane-bound claudin-4 levels, along with barrier function. Finally, in vivo treatment with the PKCε inhibitor protected mice from C. rodentium-induced colitis. In conclusion, EPEC infection increases intracellular PKCε levels, leading to decreased claudin-4 levels and compromising epithelial barrier integrity. VIP inhibits PKCε activation, thereby attenuating EPEC-induced barrier disruption.

Morampudi V ，Conlin VS ，Dalwadi U ，Wu X ，Marshall KC ，Nguyen C ，Vallance BA ，Jacobson K ... -  《-》

Citrobacter rodentium infection causes iNOS-independent intestinal epithelial dysfunction in mice.

Skinn AC ，Vergnolle N ，Zamuner SR ，Wallace JL ，Cellars L ，MacNaughton WK ，Sherman PM ... -  《CANADIAN JOURNAL OF PHYSIOLOGY AND PHARMACOLOGY》