Pathogenic Escherichia coli in inflammatory bowel diseases : Proceedings of the 1st International Meeting on E. coli and IBD, June 2007, Lille, France

National audienceSeveral different groups have recently reported the presence of pathogenic E. call associated with ileal. and/or colonic mucosa of patients with inflammatory bowel diseases. Given the important role of the gut microflora and enteropathogens in the initiation and perpetuation of intestinal inflammation, important issues now arise. Are these IBD associated E. coli pathogenic? Have they evolved from commensal bacteria? What is their reservoir? Are these bacteria sufficient to drive IBD pathogenesis? Which immunological defects may predispose to colonization by such bacteria? In June 2007, clinicians and basic scientists met in Lille, France with the goal of exchanging ideas and materials on this emerging topic. State-of-the-art lectures given by widely recognized international experts were associated with debates, case discussions and expert opinions. This paper summarizes most data that were exchanged during this day and represents an update on the potential role of pathogenic E. coli in inflammatory bowel diseases

Replication of Crohn’s disease-associated adherent-invasive E. coli within macrophages is dependent on TNF-α and impaired by TNF-α antagonist

International audienc

Replication of Crohn's disease-associated AIEC within macrophages is dependent on TNF-alpha secretion

International audienceAdherent and invasive Escherichia coli (AIEC) associated with Crohn's disease are able to survive and to replicate extensively in active phagolysosomes within macrophages. AIEC-infected macrophages release large amounts of tumour necrosis factor-alpha (TNF-alpha) and do not undergo cell death. The aim of the present study was to determine what benefit AIEC bacteria could gain from inducing the release of large amounts of TNF-alpha by infected macrophages and to what extent the neutralization of TNF-alpha could affect AIEC intramacrophagic replication. Our results showed that the amount of TNF-alpha released by infected macrophages is correlated with the load of intramacrophagic AIEC bacteria and their intracellular replication. TNF-alpha secretion was not related to the number of bacteria entering host cells because when the number of bacteria internalized in macrophage was decreased by blocking lipid raft-dependent and clathrin-coated pits-dependent endocytosis, the amount of TNF-alpha secreted by infected macrophages was not modified. Interestingly, dose-dependent increases in the number of intracellular AIEC LF82 bacteria were observed when infected macrophages were stimulated with exogenous TNF-alpha, and neutralization of TNF-alpha secreted by AIEC-infected macrophages using anti-TNF-alpha antibodies induced a significant decrease in the number of intramacrophagic bacteria. These results indicate that AIEC bacteria use TNF-alpha as a Trojan horse to ensure their intracellular replication because replication of AIEC bacteria within macrophages induces the release of TNF-alpha, which in turn increases the intramacrophagic replication of AIEC. Neutralizing TNF-alpha secreted by infected macrophages may represent an effective strategy to control AIEC intracellular replication. Laboratory Investigation (2012) 92, 411-419; doi:10.1038/labinvest.2011.156; published online 31 October 201

Enterocolitis due to immune checkpoint inhibitors: a systematic review

International audienceImmune checkpoint inhibitors targeting cytotoxic T-lymphocyte-associated protein-4 (CTLA-4) and programmed death-1 (PD-1)/ ligand are increasingly used to treat several types of cancer. These drugs enhance antitumour T-cell activity and therefore induce immunerelated adverse effects (irAE), of which gastrointestinal (GI) irAE are among the most frequent and severe. This systematic literature review summarises the clinical manifestations, management and pathophysiology of GI irAE due to immune checkpoint inhibitors. GI irAE induced by anti-CTLA-4 are frequent, potentially severe and resemble IBD, whereas those induced by PD-1 blockade seem to be less frequent and clinically more diverse. Baseline symbiotic gut microbiota is associated with an enhanced antitumour response to immune checkpoint inhibitors and an increased susceptibility to developing enterocolitis, in patients treated with anti-CTLA-4. These findings open new perspectives for possible manipulation of the gut microbiota in order to better identify responders to immune checkpoint inhibitors and to increase their efficacy and safety

Presence of adherent Escherichia coli strains in ileal mucosa of patients with Crohn's disease

International audienceBackground & Aims: Infectious agents are suspected of being involved in the pathogenesis of Crohn's disease. This study was designed to look for the presence of virulent Escherichia coli strains associated with the ileal mucosa of patients with Crohn's disease. Methods: E. coli strains were recovered from resected chronic ileal lesions (n = 20), neoterminal ileum after surgery from patients with (n = 19) and without (n = 11) endoscopic recurrence, and controls (n = 13). Bacterial adhesion was determined in vitro using intestinal cell lines; other associated virulence factors were assessed by DNA hybridization and polymerase chain reaction experiments. Results: None of the strains harbored any of the virulence factor–encoding genes of E. coli involved in acute enteric diseases. However, mannose-resistant adhesion to differentiated Caco-2 cells was found for 84.6% and 78.9% of the E. coli strains isolated from chronic and early recurrent lesions, respectively, compared with 33% of controls (P < 0.02). In addition, 21.8% of the strains induced a cytolytic effect by synthesis of an α-hemolysin. Conclusions: E. coli strains isolated from the ileal mucosa of patients with Crohn's disease adhere to differentiated intestinal cells and may disrupt the intestinal barrier by synthesizing an α-hemolysin

High prevalence of adherent-invasive Escherichia coli associated with ileal mucosa in Crohn's disease

International audienceBACKGROUND & AIMS: Adherent-invasive Escherichia coli (AIEC) pathovar has been identified in the intestinal mucosa of patients with Crohn's disease (CD). AIEC reference strain LF82 is able to adhere to intestinal epithelial cells, to invade epithelial cells via a mechanism involving actin polymerization and microtubules, and to survive and replicate within macrophages. This study was performed to assess the prevalence of AIEC associated with intestinal mucosa of patients with CD, ulcerative colitis (UC), and of controls. METHODS: A search for E. coli strains was performed with ileal specimens of 63 patients with CD and 16 controls without inflammatory bowel disease (IBD), and with colonic specimens of 27 patients with CD, 8 patients with UC, and 102 controls. The abilities of E. coli strains to invade epithelial cells and to survive and replicate within macrophages were assessed using the gentamicin protection assay. Bacterial uptake by epithelial cells was analyzed using cytoskeletal inhibitors. Bacterial adhesion was quantified with Caco-2 and Intestine-407 cells. The presence of known E. coli virulence genes was assessed by polymerase chain reaction and DNA hybridization. RESULTS: In ileal specimens, AIEC strains were found in 21.7% of CD chronic lesions vs. in 6.2% of controls. In neoterminal ileal specimens, AIEC strains were found in 36.4% of CD early lesions (P = 0.034 vs. controls) and 22.2% of healthy mucosa of CD patients. In colonic specimens, AIEC strains were found in 3.7% of CD patients, 0% of UC patients, and 1.9% of controls. CONCLUSIONS: AIEC strains are associated specifically with ileal mucosa in CD

Humoral immunity links Candida albicans infection and celiac disease.

The protein Hwp1, expressed on the pathogenic phase of Candida albicans, presents sequence analogy with the gluten protein gliadin and is also a substrate for transglutaminase. This had led to the suggestion that C. albicans infection (CI) may be a triggering factor for Celiac disease (CeD) onset. We investigated cross-immune reactivity between CeD and CI.Serum IgG levels against recombinant Hwp1 and serological markers of CeD were measured in 87 CeD patients, 41 CI patients, and 98 healthy controls (HC). IgA and IgG were also measured in 20 individuals from each of these groups using microchips sensitized with 38 peptides designed from the N-terminal of Hwp1.CI and CeD patients had higher levels of anti-Hwp1 (p=0.0005 and p=0.004) and anti-gliadin (p=0.002 and p=0.0009) antibodies than HC but there was no significant difference between CeD and CI patients. CeD and CI patients had higher levels of anti-transglutaminase IgA than HC (p=0.0001 and p=0.0039). During CI, the increase in anti-Hwp1 paralleled the increase in anti-gliadin antibodies. Microchip analysis showed that CeD patients were more reactive against some Hwp1 peptides than CI patients, and that some deamidated peptides were more reactive than their native analogs. Binding of IgG from CeD patients to Hwp1 peptides was inhibited by γIII gliadin peptides.Humoral cross-reactivity between Hwp1 and gliadin was observed during CeD and CI. Increased reactivity to Hwp1 deamidated peptide suggests that transglutaminase is involved in this interplay. These results support the hypothesis that CI may trigger CeD onset in genetically-susceptible individuals

Distribution of antibodies in patients with Celiac disease (CeD), invasive <i>Candida</i> infection (CI), and healthy controls (HC).

<p><b>(A)</b> Anti-Hwp1 IgG; <b>(B)</b> anti-gliadin IgA and IgG; <b>(C)</b> anti-transglutaminase IgG; <b>(D)</b> anti-transglutaminase IgA; <b>(E)</b> anti-deamidated gliadin IgA and IgG. Anti-Hwp1 IgG, anti-gliadin IgA and IgG results are expressed as optical density (450 nm), and anti-transglutaminase IgG and IgA <b>as log of arbitrary units (log AU)</b> and anti-deamidated IgA and IgG results as arbitrary units (AU). The significance of discrimination between the different groups is represented below each figure as the <i>p</i> value.</p

Antibody reactivity of sera from Celiac disease (CeD) patients according to adherence to a gluten-free diet (GFD).

<p><b>(A)</b> Anti-Hwp1 IgG; <b>(B)</b> anti-gliadin IgA and IgG; <b>(C)</b> anti-transglutaminase IgG; <b>(D)</b> anti-transglutaminase IgA; <b>(E)</b> anti-deamidated gliadin IgA and IgG. Anti-Hwp1 IgG, anti-gliadin IgA and IgG results are expressed as optical density (450 nm) anti-tTG IgG as log of arbitrary units (log AU) anti-IgA tTG and anti-deamidated gliadin as arbitrary units (AU). YES: strict adherence to a GFD in the previous 2 months. NO: no strict adherence in the previous 2 months.</p

(A) Analysis of rNtermHwp1 purification by SDS-PAGE and immunoblotting.

<p>After migration in polyacrylamide gels, only a single band was stained with Coomassie blue in the sample containing purified rNtermHwp1 (lane 1). This band was detected with anti-HisTag (lane 2) and mAb 16B1 (lane 3) antibodies, showing purity of the sample. As deduced from its amino acid sequence, rNtermHwp1 has a theoretical molecular mass of 19.5 kDa but migrates in SDS PAGE as a 45 kDa protein. This molecular mass shift is a result of the rigid coiled structure of Hwp1. <b>(B) Photomicroscopy. (1)</b> Bright field microscopy of <i>C</i>. <i>albicans</i> yeast forms producing germ tubes. <b>(2)</b> Same microscopic field as <b>(1)</b> using immunofluorescence staining with mAb 16B1, which selectively binds to the surface of germ tubes. <b>(C) Reactivity of mAb 16B1 with rNterm Hwp1 by ELISA, expressed as optical density</b>.</p