Inflammatory bowel disease is a chronic gastrointestinal inflammatory disorder associated with changes in neuropeptide expression and function, including vasoactive intestinal peptide (VIP). VIP regulates intestinal vasomotor and secretomotor function and motility; however, VIP’s role in development and maintenance of colonic epithelial barrier homeostasis is unclear. Using VIP deficient (VIPKO) mice, we investigated VIP’s role in epithelial barrier homeostasis, and susceptibility to colitis. Colonic crypt morphology and epithelial barrier homeostasis were assessed in wildtype (WT) and VIPKO mice, at baseline. Colitic responses were evaluated following dinitrobenzene sulfonic acid (DNBS) or dextran-sodium sulfate (DSS) exposure. Mice were also treated with exogenous VIP. At baseline, VIPKO mice exhibited distorted colonic crypts, defects in epithelial cell proliferation and migration, increased apoptosis, and altered permeability. VIPKO mice also displayed reduced goblet cell numbers, and reduced expression of secreted goblet cell factors mucin 2 and trefoil factor 3. These changes were associated with reduced expression of caudal type homeobox 2 (Cdx2), a master regulator of intestinal function and homeostasis. DNBS and DSS-induced colitis were more severe in VIPKO than WT mice. VIP treatment rescued the phenotype, protecting VIPKO mice against DSS colitis, with results comparable to WT mice. In conclusion, VIP plays a crucial role in the development and maintenance of colonic epithelial barrier integrity under physiological conditions and promotes epithelial repair and homeostasis during 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.
Intestinal fibrosis is a serious complication of Crohn's disease (CD) that can lead to stricture formation, which requires surgery. Mechanisms underlying intestinal fibrosis remain elusive because of a lack of suitable mouse models. Herein, we describe a spontaneous mouse model of intestinal inflammation with fibrosis and the profibrotic role of arginase I. The Src homology 2 domain-containing inositol polyphosphate 5'-phosphatase-deficient (SHIP(-/-)) mice developed spontaneous discontinuous intestinal inflammation restricted to the distal ileum starting at the age of 4 weeks. Mice developed several key features resembling CD, including inflammation and fibrosis. Inflammation was characterized by abundant infiltrating Gr-1-positive immune cells, granuloma-like immune cell aggregates that contained multinucleated giant cells, and a mixed type 2 and type 17 helper T-cell cytokine profile. Fibrosis was characterized by a thickened ileal muscle layer, collagen deposition, and increased fibroblasts at the sites of collagen deposition. SHIP(-/-) ilea had increased arginase activity and arginase I expression that was inversely proportional to nitrotyrosine staining. SHIP(-/-) mice were treated with the arginase inhibitor S-(2-boronoethyl)-l-cysteine, and changes in the disease phenotype were measured. Arginase inhibition did not affect the number of immune cell infiltrates in the SHIP(-/-) mouse ilea; rather, it reduced collagen deposition and muscle hyperplasia. These findings suggest that arginase activity is a potential target to limit intestinal fibrosis in patients with CD.
The intestinal epithelium represents a critical barrier protecting the host against diverse luminal noxious agents, as well as preventing the uncontrolled uptake of bacteria that could activate an immune response in a susceptible host. The epithelial monolayer that constitutes this barrier is regulated by a meshwork of proteins that orchestrate complex biological function such as permeability, transepithelial electrical resistance, and movement of various macromolecules. Because of its key role in maintaining host homeostasis, factors regulating barrier function have attracted sustained attention from the research community. This paper will address the role of bacteria, bacterial-derived metabolism, and the interplay of dietary factors in controlling intestinal barrier function.
The human gastric pathogen Helicobacter pylori attaches to antral epithelial cells in vivo. Cultured human antral epithelial cells, AGS and NCI-N87 cell lines, were grown in the absence or presence of H. pylori and compared with respect to gene transcript levels, protein expression, organization of the actin cytoskeleton, and the regulation of cell migration. The Clontech Neurobiology array detected differentially expressed transcripts, while Western blots were used to investigate related changes in protein levels. Infection with H. pylori consistently upregulated annexin II, S100 A7, Rho-GTP, and IQGAP-1, whereas SSTR-1 was downregulated upon H. pylori infection. In the adherens junction, E-cadherin and IQGAP-1 were translocated from the plasma membrane to intracellular vesicles. The primary and NCI-N87 cells were similar with respect to cell-cell and cell-matrix adhesion and cell migratory behavior; in contrast the AGS cells were significantly different from the primary gastric epithelial cell preparations, and thus caution must be used when using this cell line for studies of gastric disease. These studies demonstrate a correlation between H. pylori infection and alterations to epithelial cell adhesion molecules, including increased levels of Rho-GTP and cell migration. These data indicate that destabilizing epithelial cell adherence is one of the factors increasing the risk of H. pylori-infected individuals developing gastric cancer.Helicobacter pylori is a spiral, gram-negative rod that attaches specifically to the gastric epithelial cells lining the antrum of the stomach (2). H. pylori is able to withstand the hostile environment of the stomach by secretion of urease buffers, which neutralize the pH of its immediate surroundings. Flagella allow these highly motile bacteria to cross the mucous lining of the stomach and to attach to the apical surface of the mucosal epithelial cells. These antral epithelial cells are linked together at the apical surface by a system of interacting proteins that comprise the tight and adherens junctions (12, 31). These junctions effectively seal off the lumen of the stomach, preventing access of gastric acid and pathogens to the interstitial space and, hence, to the general circulation.Individuals infected by H. pylori have increased gastrin levels and decreased levels of somatostatin (SST) hormones that regulate gastric acid secretion. As a result, infected individuals develop mucosal gastritis, increasing their risk of ulceration and, in the longer term, gastric cancer (10, 11). Gastric adenocarcinomas show characteristic changes in the expression of E-cadherin, a transmembrane protein forming the core of the adherens junction. Loss of the E-cadherin complex at the apical pole is thought to induce loss of cell-cell adhesion (34). Direct evidence of E-cadherin mutations triggering tumorigenesis has come from recent studies linking inactivating germ line mutations of the E-cadherin gene (CDH1) in hereditary diffuse gastric cancer (33). Furthermore, it has been shown that the ...
Enteropathogenic Escherichia coli (EPEC)-induced diarrhea is often associated with disruption of intestinal epithelial tight junctions. Although studies have shown alterations in the expression and localization of bicellular tight junction proteins during EPEC infections, little is known about whether tricellular tight junction proteins (tTJs) are affected. Using Caco-2 cell monolayers, we investigated if EPEC is capable of targeting the tTJ protein tricellulin. Our results demonstrated that at 4 h postinfection, EPEC induced a significant reduction in tricellulin levels, accompanied by a significant loss of transepithelial resistance (TEER) and a corresponding increase in paracellular permeability. Conversely, cells overexpressing tricellulin were highly resistant to EPEC-induced barrier disruption. Confocal microscopy revealed the distribution of tricellulin into the plasma membrane of infected epithelial cells and confirmed the localization of EPEC aggregates in close proximity to tTJs. Moreover, infections with EPEC strains lacking genes encoding specific type III secreted effector proteins demonstrated a crucial role for the effector EspG1 in modulating tricellulin expression. Complementation studies suggest that the EspG-induced depletion of tricellulin is microtubule dependent. Overall, our results show that EPEC-induced epithelial barrier dysfunction is mediated in part by EspG1-induced microtubuledependent depletion of tricellulin.
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.
NK cells obtained by exposing mouse fetal thymocytes to appropriate combinations of IL-4, IL-2, and PMA are phenotypically indistinguishable from cultured adult splenic NK cells with the exception that they generally lack measurable expression of all of the inhibitory Ly49 molecules that can currently be detected with Abs (Ly49A, -C, -G, and -I) and of the activating molecule Ly49D. Despite this deficiency, fetal NK cells have a similar specificity to Ly49-expressing adult splenic NK cells. Individual fetal NK cell clones display an essentially invariant and broad specificity similar to that of polyclonal populations of fetal or adult NK cells, although significant differences in the fine specificity of clones can occasionally be detected. Most remarkably, cloned fetal NK cell lines display heterogeneous expression of a restricted set of surface molecules that includes 10A7, Ly6C, 3C2, CD8, certain isoforms of CD45, and also, occasionally, Ly49 molecules. This heterogeneity is not related to the cell cycle or activation status of the cells, and micromanipulation recloning demonstrates unambiguously that it is not due to a lack of a single cell origin. Diversity is generated rapidly and the capacity for diversification appears to persist indefinitely in vitro. The expression of individual variable Ags is independent and stochastic, resulting in fetal NK “clones” being potentially composed of hundreds of phenotypically distinct cells. We hypothesize that fetal NK cells behave as progenitor cells that are undergoing a process of rapid, extensive, and continuous diversification and that are individually capable of generating and regenerating a complex NK cell repertoire.
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