Dominique Velin

Tollip regulates proinflammatory responses to interleukin-1 and lipopolysaccharide.

ABSTRACT Activation of interleukin-1 (IL-1) receptor (IL-1R), Toll-like receptor 2 (TLR2), and TLR4 triggers NF-κB and mitogen-activated protein kinase (MAPK)-dependent signaling, thereby initiating immune responses. Tollip has been implicated as a negative regulator of NF-κB signaling triggered by these receptors in in vitro studies. Here, deficient mice were used to determine the physiological contribution of Tollip to immunity. NF-κB, as well as MAPK, signaling appeared normal in Tollip-deficient cells stimulated with IL-1β or the TLR4 ligand lipopolysaccharide (LPS). Similarly, IL-1β- and TLR-driven activation of dendritic cells and lymphocytes was indistinguishable from wild-type cells. In contrast, the production of the proinflammatory cytokines, IL-6 and tumor necrosis factor alpha was significantly reduced after IL-1β and LPS treatment at low doses but not at lethal doses of LPS. Tollip therefore controls the magnitude of inflammatory cytokine production in response to IL-1β and LPS.

Interleukin-17 Is a Critical Mediator of Vaccine-Induced Reduction of Helicobacter Infection in the Mouse Model

BACKGROUND & AIMS Despite the proven ability of immunization to reduce Helicobacter infection in mouse models, the precise mechanism of protection has remained elusive. This study explores the possibility that interleukin (IL)-17 plays a role in the reduction of Helicobacter infection following vaccination of wild-type animals or in spontaneous reduction of bacterial infection in IL-10-deficient mice. METHODS In mice, reducing Helicobacter infection, the levels and source of IL-17 were determined and the role of IL-17 in reduction of Helicobacter infection was probed by neutralizing antibodies. RESULTS Gastric IL-17 levels were strongly increased in mice mucosally immunized with urease plus cholera toxin and challenged with Helicobacter felis as compared with controls (654 +/- 455 and 34 +/- 84 relative units for IL-17 messenger RNA expression [P < .01] and 6.9 +/- 8.4 and 0.02 +/- 0.04 pg for IL-17 protein concentration [P < .01], respectively). Flow cytometry analysis showed that a peak of CD4(+)IL-17(+) T cells infiltrating the gastric mucosa occurred in immunized mice in contrast to control mice (4.7% +/- 0.3% and 1.4% +/- 0.3% [P < .01], respectively). Gastric mucosa-infiltrating CD4(+)IL-17(+) T cells were also observed in IL-10-deficient mice that spontaneously reduced H felis infection (4.3% +/- 2.3% and 2% +/- 0.6% [P < .01], for infected and noninfected IL-10-deficient mice, respectively). In wild-type immunized mice, intraperitoneal injection of anti-IL-17 antibodies significantly inhibited inflammation and the reduction of Helicobacter infection in comparison with control antibodies (1 of 12 mice vs 9 of 12 mice reduced Helicobacter infection [P < .01], respectively). CONCLUSIONS IL-17 plays a critical role in the immunization-induced reduction of Helicobacter infection from the gastric mucosa.

Granulocyte‐macrophage colony‐stimulating factor elicits bone marrow‐derived cells that promote efficient colonic mucosal healing

Background: Granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) therapy is effective in treating some Crohns disease (CD) patients and protects mice from colitis induced by dextran sulfate sodium (DSS) administration. However, its mechanisms of action remain elusive. We hypothesized that GM‐CSF affects intestinal mucosal repair. Methods: DSS colitic mice were treated with daily pegylated GM‐CSF or saline and clinical, histological, and inflammatory parameters were kinetically evaluated. Further, the role of bone marrow‐derived cells in the impact of GM‐CSF therapy on DSS colitis was addressed using cell transfers. Results: GM‐CSF therapy reduced clinical signs of colitis and the release of inflammatory mediators. GM‐CSF therapy improved mucosal repair, with faster ulcer reepithelialization, accelerated hyperproliferative response of epithelial cells in ulcer‐adjacent crypts, and lower colonoscopic ulceration scores in GM‐CSF‐administered mice relative to untreated mice. We observed that GM‐CSF‐induced promotion of mucosal repair is timely associated with a reduction in neutrophil numbers and increased accumulation of CD11b+ monocytic cells in colon tissues. Importantly, transfer of splenic GM‐CSF‐induced CD11b+ myeloid cells into DSS‐exposed mice improved colitis, and lethally irradiated GM‐CSF receptor‐deficient mice reconstituted with wildtype bone marrow cells were protected from DSS‐induced colitis upon GM‐CSF therapy. Lastly, GM‐CSF‐induced CD11b+ myeloid cells were shown to promote in vitro wound repair. Conclusions: Our study shows that GM‐CSF‐dependent stimulation of bone marrow‐derived cells during DSS‐induced colitis accelerates colonic tissue repair. These data provide a putative mechanism for the observed beneficial effects of GM‐CSF therapy in Crohns disease. (Inflamm Bowel Dis 2010;)

Results of the 2nd scientific workshop of the ECCO (III): basic mechanisms of intestinal healing.

The second scientific workshop of the European Crohns and Colitis Organization (ECCO) focused on the relevance of intestinal healing for the disease course of inflammatory bowel disease (IBD). The objective was to better understand basic mechanisms, markers for disease prediction, detection and monitoring of intestinal healing, impact of intestinal healing on the disease course of IBD as well as therapeutic strategies. The results of this workshop are presented in four separate manuscripts. This section describes basic mechanisms of intestinal healing, identifies open questions in the field and provides a framework for future studies.

Advances in vaccination against Helicobacter pylori

A vaccination against Helicobacter pylori may represent both prophylactic and therapeutic approaches to the control of H. pylori infection. Different protective H. pylori-derived antigens, such as urease, vacuolating cytotoxin A, cytotoxin-associated antigen, neutrophil-activating protein and others can be produced at low cost in prokaryote expression systems and most of these antigens have already been administered to humans and shown to be safe. The recent development by Graham et al. of the model of H. pylori challenge in humans, the recent published clinical trials and the last insight generated in animal models of H. pylori infection regarding the immune mechanisms leading to vaccine-induced Helicobacter clearance will facilitate the evaluation of immunogenicity and efficacy of H. pylori vaccine candidates in Phase II and III clinical trials.

Immunology of Helicobacter pylori infection.

Helicobacter pylori is a Gram-negative, spiral bacterium that colonizes the gastric mucosa of at least 50% of the world’s population and plays a causative role in the development of chronic gastritis as well as in gastric and duodenal ulcers. H. pylori triggers vigorous humoral and cellular immune responses in both systemic and mucosal compartments. In spite of this response, the vast majority of infected hosts are unable to clear the infection, and it persists for decades. Although Helicobacter is tolerated by a naïve host organism, preclinical studies have demonstrated that prophylactic or therapeutic vaccinations efficiently clear Helicobacter from the stomach. The understanding of the mechanisms leading to the Helicobacter persistence or the vaccine-induced eradication of Helicobacter in animal models will help to define optimal immunization strategies for future anti-Helicobacter vaccination clinical trials.

Immune responses to Helicobacter pylori infection

Helicobacter pylori (H. pylori) infection is one of the most common infections in human beings worldwide. H. pylori express lipopolysaccharides and flagellin that do not activate efficiently Toll-like receptors and express dedicated effectors, such as γ-glutamyl transpeptidase, vacuolating cytotoxin (vacA), arginase, that actively induce tolerogenic signals. In this perspective, H. pylori can be considered as a commensal bacteria belonging to the stomach microbiota. However, when present in the stomach, H. pylori reduce the overall diversity of the gastric microbiota and promote gastric inflammation by inducing Nod1-dependent pro-inflammatory program and by activating neutrophils through the production of a neutrophil activating protein. The maintenance of a chronic inflammation in the gastric mucosa and the direct action of virulence factors (vacA and cytotoxin-associated gene A) confer pro-carcinogenic activities to H. pylori. Hence, H. pylori cannot be considered as symbiotic bacteria but rather as part of the pathobiont. The development of a H. pylori vaccine will bring health benefits for individuals infected with antibiotic resistant H. pylori strains and population of underdeveloped countries.

Expression of Helicobacter pylori Virulence Factors and Associated Expression Profiles of Inflammatory Genes in the Human Gastric Mucosa

ABSTRACT Helicobacter pylori virulence factors have been suggested to be important in determining the outcome of infection. The H. pylori adhesion protein BabA2 is thought to play a crucial role in bacterial colonization and in induction of severe gastric inflammation, particularly in combination with expression of CagA and VacA. However, the influence of these virulence factors on the pathogenesis of H. pylori infection is still poorly understood. To address this question, the inflammatory gene expression profiles for two groups of patients infected with triple-negative strains (lacking expression of cagA, babA2, and vacAs1 but expressing vacAs2) and triple-positive strains (expressing cagA, vacAs1, and babA2 but lacking expression of vacAs2) were investigated. The gene expression patterns in the antrum gastric mucosa from patients infected with different H. pylori strains were very similar, and no differentially expressed genes could be identified by pairwise comparisons. Our data thus suggest that there is a lack of correlation between the host inflammatory responses in the gastric mucosa and expression of the babA2, cagA, and vacAs1 genes.

Toll-interacting protein modulates colitis susceptibility in mice.

Background:The intestinal epithelium accommodates with a myriad of commensals to maintain immunological homeostasis, but the underlying mechanisms regulating epithelial responsiveness to flora-derived signals remain poorly understood. Herein, we sought to determine the role of the Toll/interleukin (IL)-1 receptor regulator Toll-interacting protein (Tollip) in intestinal homeostasis. Methods:Colitis susceptibility was determined after oral dextran sulfate sodium (DSS) administration or by breeding Tollip−/− on an IL-10−/− background. The intestinal flora was depleted with 4 antibiotics before DSS exposure to assess its contribution in colitis onset. Bone marrow chimeras were generated to identify the cellular compartment, whereby Tollip may negatively regulate intestinal inflammation in response to DSS. Tollip-dependent epithelial barrier functions were studied in vitro by using Tollip-knockdown in Caco-2 cells and in vivo by immunohistochemistry and fluorescein isothiocyanate-labeled dextran gavage. Results:Genetic ablation of Tollip did not lead to spontaneous intestinal inflammatory disorders. However, Tollip deficiency aggravated spontaneous disease onset in IL-10−/− mice and increased susceptibility to DSS colitis. Increased colitis severity in Tollip-deficient mice was not improved by bacterial flora depletion using broad-spectrum antibiotics. In addition, DSS exposure of bone marrow chimeric mice revealed a protective role for Tollip in nonhematopoietic cells. Knockdown of Tollip in epithelial cells led to exaggerated NF&kgr;-B activity and proinflammatory cytokine secretion. Finally, DSS-treated Tollip−/− mice showed enhanced intestinal permeability and increased epithelial apoptosis when compared with wild-type controls, a finding that coincided with tight junction alterations on injury. Conclusion:Overall, our data show an essential role for Tollip on colitis susceptibility in mice.

Macrophages promote epithelial repair through hepatocyte growth factor secretion

Macrophages play a critical role in intestinal wound repair. However, the mechanisms of macrophage‐assisted wound repair remain poorly understood. We aimed to characterize more clearly the repair activities of murine and human macrophages. Murine macrophages were differentiated from bone marrow cells and human macrophages from monocytes isolated from peripheral blood mononuclear cells of healthy donors (HD) or Crohns disease (CD) patients or isolated from the intestinal mucosa of HD. In‐vitro models were used to study the repair activities of macrophages. We found that murine and human macrophages were both able to promote epithelial repair in vitro. This function was mainly cell contact‐independent and relied upon the production of soluble factors such as the hepatocyte growth factor (HGF). Indeed, HGF‐silenced macrophages were less capable of promoting epithelial repair than control macrophages. Remarkably, macrophages from CD patients produced less HGF than their HD counterparts (HGF level: 84 ± 27 pg/mg of protein and 45 ± 34 pg/mg of protein, respectively, for HD and CD macrophages, P < 0·009) and were deficient in promoting epithelial repair (repairing activity: 90·1 ± 4·6 and 75·8 ± 8·3, respectively, for HD and CD macrophages, P < 0·0005). In conclusion, we provide evidence that macrophages act on wounded epithelial cells to promote epithelial repair through the secretion of HGF. The deficiency of CD macrophages to secrete HGF and to promote epithelial repair might contribute to the impaired intestinal mucosal healing in CD patients.