Tricellular Tight Junction Protein Tricellulin Is Targeted by the Enteropathogenic Escherichia coli Effector EspG1, Leading to Epithelial Barrier Disruption.

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 microtubule-dependent depletion of tricellulin.

Vasoactive intestinal polypeptide promotes intestinal barrier homeostasis and protection against colitis in mice.

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.

SHIP-deficient mice develop spontaneous intestinal inflammation and arginase-dependent fibrosis.

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.

Helicobacter pylori infection targets adherens junction regulatory proteins and results in increased rates of migration in human gastric epithelial cells.

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.