Vanin-1 licenses inflammatory mediator production by gut epithelial cells and controls colitis by antagonizing peroxisome proliferator-activated receptor γ activity

Colitis involves immune cell–mediated tissue injuries, but the contribution of epithelial cells remains largely unclear. Vanin-1 is an epithelial ectoenzyme with a pantetheinase activity that provides cysteamine/cystamine to tissue. Using the 2,4,6-trinitrobenzene sulfonic acid (TNBS)-colitis model we show here that Vanin-1 deficiency protects from colitis. This protection is reversible by administration of cystamine or bisphenol A diglycidyl ether, a peroxisome proliferator-activated receptor (PPAR)γ antagonist. We further demonstrate that Vanin-1, by antagonizing PPARγ, licenses the production of inflammatory mediators by intestinal epithelial cells. We propose that Vanin-1 is an epithelial sensor of stress that exerts a dominant control over innate immune responses in tissue. Thus, the Vanin-1/pantetheinase activity might be a new target for therapeutic intervention in inflammatory bowel disease

Saccharomyces boulardii Improves Intestinal Cell Restitution through Activation of the α2β1 Integrin Collagen Receptor

Intestinal epithelial cell damage is frequently seen in the mucosal lesions of inflammatory bowel diseases such as ulcerative colitis or Crohn's disease. Complete remission of these diseases requires both the cessation of inflammation and the migration of enterocytes to repair the damaged epithelium. Lyophilized Saccharomyces boulardii (Sb, Biocodex) is a nonpathogenic yeast widely used as a therapeutic agent for the treatment and prevention of diarrhea and other gastrointestinal disorders. In this study, we determined whether Sb could accelerate enterocyte migration. Cell migration was determined in Sb force-fed C57BL6J mice and in an in vitro wound model. The impact on α2β1 integrin activity was assessed using adhesion assays and the analysis of α2β1 mediated signaling pathways both in vitro and in vivo. We demonstrated that Sb secretes compounds that enhance the migration of enterocytes independently of cell proliferation. This enhanced migration was associated with the ability of Sb to favor cell-extracellular matrix interaction. Indeed, the yeast activates α2β1 integrin collagen receptors. This leads to an increase in tyrosine phosphorylation of cytoplasmic molecules, including focal adhesion kinase and paxillin, involved in the integrin signaling pathway. These changes are associated with the reorganization of focal adhesion structures. In conclusion Sb secretes motogenic factors that enhance cell restitution through the dynamic regulation of α2β1 integrin activity. This could be of major importance in the development of novel therapies targeting diseases characterized by severe mucosal injury, such as inflammatory and infectious bowel diseases

Helicobacter pylori Counteracts the Apoptotic Action of Its VacA Toxin by Injecting the CagA Protein into Gastric Epithelial Cells

Infection with Helicobacter pylori is responsible for gastritis and gastroduodenal ulcers but is also a high risk factor for the development of gastric adenocarcinoma and lymphoma. The most pathogenic H. pylori strains (i.e., the so-called type I strains) associate the CagA virulence protein with an active VacA cytotoxin but the rationale for this association is unknown. CagA, directly injected by the bacterium into colonized epithelium via a type IV secretion system, leads to cellular morphological, anti-apoptotic and proinflammatory effects responsible in the long-term (years or decades) for ulcer and cancer. VacA, via pinocytosis and intracellular trafficking, induces epithelial cell apoptosis and vacuolation. Using human gastric epithelial cells in culture transfected with cDNA encoding for either the wild-type 38 kDa C-terminal signaling domain of CagA or its non-tyrosine-phosphorylatable mutant form, we found that, depending on tyrosine-phosphorylation by host kinases, CagA inhibited VacA-induced apoptosis by two complementary mechanisms. Tyrosine-phosphorylated CagA prevented pinocytosed VacA to reach its target intracellular compartments. Unphosphorylated CagA triggered an anti-apoptotic activity blocking VacA-induced apoptosis at the mitochondrial level without affecting the intracellular trafficking of the toxin. Assaying the level of apoptosis of gastric epithelial cells infected with wild-type CagA+/VacA+ H. pylori or isogenic mutants lacking of either CagA or VacA, we confirmed the results obtained in cells transfected with the CagA C-ter constructions showing that CagA antagonizes VacA-induced apoptosis. VacA toxin plays a role during H. pylori stomach colonization. However, once bacteria have colonized the gastric niche, the apoptotic action of VacA might be detrimental for the survival of H. pylori adherent to the mucosa. CagA association with VacA is thus a novel, highly ingenious microbial strategy to locally protect its ecological niche against a bacterial virulence factor, with however detrimental consequences for the human host

<em>Saccharomyces boulardii</em> Improves Intestinal Epithelial Cell Restitution by Inhibiting αvβ5 Integrin Activation State

<div><p>Intestinal epithelial cell damage is frequently seen in the mucosal lesions of infectious or inflammatory bowel diseases such as ulcerative colitis or Crohn's disease. Complete remission of these diseases requires both the disappearance of inflammation and the repair of damaged epithelium. <em>Saccharomyces boulardii</em> (<em>Sb</em>, Biocodex) is a non-pathogenic yeast widely used as a preventive and therapeutic probiotic for the prevention and treatment of diarrhea and other gastrointestinal disorders. We recently showed that it enhances the repair of intestinal epithelium through activation of α2β1 integrin collagen receptors. In the present study, we demonstrated that α2β1 integrin is not the sole cell-extracellular matrix receptor involved during <em>Sb</em>-mediated intestinal restitution. Indeed, by using cell adhesion assays, we showed that <em>Sb</em> supernatant contains heat sensitive molecule(s), with a molecular weight higher than 9 kDa, which decreased αvβ5 integrin-mediated adhesion to vitronectin by competing with the integrin. Moreover, <em>Sb</em>-mediated changes in cell adhesion to vitronectin resulted in a reduction of the αvβ5signaling pathway. We used a monolayer wounding assay that mimics <em>in vivo</em> cell restitution to demonstrate that down-modulation of the αvβ5 integrin-vitronectin interaction is related to <em>Sb</em>-induced cell migration. We therefore postulated that <em>Sb</em> supernatant contains motogenic factors that enhance cell restitution through multiple pathways, including the dynamic fine regulation of αvβ5 integrin binding activity. This could be of major importance in diseases characterized by severe mucosal injury, such as inflammatory and infectious bowel diseases.</p> </div

<i>Sb</i>S down-modulates cell adhesion to vitronectin.

<p>(A) Isolated HCT-8/E11 cells were treated with or without <i>Sb</i>S (dilution 1/8) and plated on either fibronectin or vitronectin at the indicated concentrations. Cell-ECM adhesion was evaluated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045047#s2" target="_blank">Methods</a>. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045047#s3" target="_blank">Results</a> are expressed as the percentage of cell adhesion. Data represent the mean+SD of 3 separate experiments. (B) HCT-8/E11 cells were treated with or without dilutions of <i>Sb</i>S and plated on vitronectin (Vn). Cell-vitronectin adhesion was evaluated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045047#s2" target="_blank">Methods</a>. (C) HCT-8/E11 cells were treated with or without <i>Sb</i>S, seeded in vitronectin-coated or fibronectin-coated wells and allowed to adhere for 2 h at 37°C. Spreading cells were counted microscopically. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045047#s3" target="_blank">Results</a> are expressed as the percentage of spreading cells. Data represent the mean+SD of 3 separate experiments. *** P<0.001.</p

αvβ5 integrin is required for HCT-8/E11 cell interaction with vitronectin.

<p>(A) Isolated HCT-8/E11 cells were incubated without (Ctrl) or with <i>Sb</i>S (<i>Sb</i>S) in the absence (-) or presence of either anti-αv or -α5 integrin mAbs. Cells were then seeded on vitronectin (3,15 µg/ml). Cell adhesion was evaluated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045047#s2" target="_blank">Methods</a>. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045047#s3" target="_blank">Results</a> are expressed as the percentage of cell adhesion compared to untreated cells (Ctrl). Data represent the mean+SD of 3 separate experiments. (B) HCT-8/E11 cells were transfected with anti -αv-integrin siRNAs (SiRNAαv) or scramble oligos (SiRNACtrl) for 48 h. Cells were then plated on vitronectin for 2 h. Cell-ECM adhesion was evaluated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045047#s2" target="_blank">Methods</a>. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045047#s3" target="_blank">Results</a> are expressed as the percentage of cell adhesion. Data represent the mean+SD of 3 separate experiments. ***P<0.001.</p

αvβ5 integrin is relocalized in the intestine of <i>SbS</i> force-fed mice.

<p>Mice were daily force-fed for one week with unused culture medium for <i>Sb</i> (Ctrl) or <i>Sb</i>S (<i>Sb</i>S). Frozen sections of small intestinal tissues were co-stained with a rabbit anti-β5-β integrin subunit (β5) and a rat anti E-cadherin (E-cad). Sections were then incubated with a mixture of Alexa 594-conjugated and Alexa 488-conjugated secondary antibodies against rabbit and mouse IgG, respectively. Rabbit Ig and rat Ig correspond to isotype controls Abs. Colocalized pixels appear in yellow in merged images. Each image is a representative image taken from tissue sections of four mice. The arrow and the arrowhead point out a villus and a crypt, respectively.</p