S09 Pathogen-mediated aggravation of DSS-colitis and cross-reactive antibodies in mannan-binding lectin deficient mice

22 5th International Meeting on Inflammatory Bowel Diseases, Capri, April 8 10, 2010 Session III The gut flora: homeostasis or inflammation S08 Oral presentation Crohn’s disease-associated adherent-invasive Escherichia coli target Peyer’s patches via long polar fimbriae B. Chassaing1 , N. Rolhion1 , A. de Vallée1 , S.Y. Salim2 , M. Prorok-Hamon3 , C. Neut4 , B.J. Campbell3 , J.D. Söderholm2 , J.P. Hugot5 , J.F. Colombel4 , A. Darfeuille-Michaud1 . 1 Université Auvergne, Clermont-Ferrand; France, 2 Linköping University, Linköping, Sweden, 3 University of Liverpool, Liverpool, UK, 4 INSERM U 795 Université Lille II, Lille, France, 5 INSERM U 843, Université Paris Diderot, Paris, France S09 Oral presentation Pathogen-mediated aggravation of DSS-colitis and crossreactive antibodies in mannan-binding lectin deficient mice 1 1 1 S. Müller , T. Schaffer , B. Flogerzi , B. SeiboldSchmid1 , J. Schnider1 , K. Takahashi2 , A.M. Schoepfer1,3 , F. Seibold1,4 . 1 Department of Clinical Research, Division of Gastroenterology, University of Bern, Switzerland, 2 Programs of Developmental Immunology, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA, 3 Farncombe Family Institute of Digestive Health Research, McMaster University, Hamilton, ON, Canada, 4 Department of Visceral Surgery and Medicine, Division of Gastroenterology, University Hospital Bern, Switzerland Background: Deficiency for mannan-binding lectin (MBL) associates with increased prevalence of anti-Saccharomyces cerevisiae antibodies (ASCA) and with complicated phenotypes of Crohn’s disease (CD). However, the origin of ASCA and the role of MBL in intestinal inflammation are currently unclear. Aim: To analyze local expression of MBL in the human intestine and to investigate consequences of MBL-deficiency in experimental colitis with concurrent intestinal mucosal exposure to pathogenic yeast and bacteria. S10 E. faecalis Gelatinase contributes to the development of intestinal inflammation by impairing epithelial barrier function: Role for bacteria-derived proteases in the pathology of IBD N. Steck1 , M. Hoffmann1 , C.M. Ferstl Hew3 , S.C. Kim4 , S.L. Tonkonogy2 , R.F. Vogel3 , R.B. Sartor4 , D. Haller1 . 1 Technische Universität München, Biofunctionality, ZIEL Research Center for Nutrition and Food Science, CDD-Center for Diet and Disease, Freising-Weihenstephan, Germany, 2 North Carolina State University, College of Veterinary Medicine, Raleigh, NC, USA, 3 Technische Universität München, Technische Mikrobiologie, Freising-Weihenstephan, Germany, 4 University of North Carolina, Center for Gastrointestinal Biology and Disease, Chapel Hill, NC, USA Background: Endogenous proteases contribute to the pathogenesis of IBD. Thereby the balance between proteolytic activities in the intestinal mucosa seems to be a key regulator for immune responses and barrier function. The aim of our study was the investigation of bacteria-derived proteases in the development of IBD. For this purpose we focused on the zinc dependent metalloprotease Gelatinase (GelE) from Enterococcus faecalis. Methods and Results: Monoassociation experiments of wild type (WT) and IL-10 / mice (129SvEv) with E. faecalis strain OG1RF showed increased mRNA expression levels of the bacterial GelE under conditions of experimental colitis. To further characterize the role of this bacterial protease in the pathogenesis of chronic intestinal inflammation, we monoassociated WT and IL-10 / mice for 15 weeks with E. faecalis strain OG1RF and isogenic mutant strains that lack GelE expression including TX5264 (gelE deletion) and TX5266 (fsrB deletion). Histopathological analysis revealed a significant reduction of colonic inflammation in the absence of bacterial GelE. This effect seems not to be a cause of an antigenic response triggered by GelE. Transwell experiments Background and Aims: Ileal lesions of patients with Crohn’s disease (CD) are colonized by adherent-invasive Escherichia coli (AIEC). The earliest observable lesions of recurrent CD are microscopic erosions at the specialized follicle-associated epithelium, which lines Peyer’s patches (PP). We aimed to quantify the PP-associated E. coli in CD patients compared to controls and to search for the presence of a virulence factor in AIEC that could be involved in the targeting of PP by these invasive bacteria. Methods: PP from CD patients and controls were analyzed by q-PCR for the presence of E. coli 16S r-RNA encoding gene, and the genome of AIEC strain LF82 was screened to search for putative virulence factors allowing bacteria to target PP. Wild-type bacteria and mutants were tested for their abilities to interact with murine and human isolated PP, and M cells. Results: Increased numbers of E. coli are associated with PP from CD patients compared to that from controls. A functional operon encoding long polar fimbriae (LPF) is present in AIEC strain LF82. The LF82-DlpfA mutant was impaired in its ability to interact with murine and human isolated PP and to translocate across M cell monolayers. Increased interaction of AIEC LF82 bacteria to PP was observed in Nod2 / mice due to increased number of M cells. The prevalence of subjects positive for ileum-associated AIEC strains harboring lpf operon was 21.8% in CD patients as against only 3.5% in controls (P = 0.027). Conclusion: Our findings, in addition to the suspected major role of PP in CD, show that CD-associated AIEC bacteria by expressing long polar fimbriae can use PP as an open gate to induce early stages of the disease. Material and Methods: Serum MBL and ASCA were measured by ELISA. Intestinal MBL was quantified by rtPCR. DSS-induced colitis was assessed in wild type and MBL-deficient mice orally challenged with C. albicans or adherent/invasive E. coli (AIEC) reference strain LF82. Wild type and MBL-deficient mice were infected with C. albicans to assess generation of cross-reactive ASCA. Viable C. albicans was determined from the kidneys of infected mice as a measure of yeast dissemination. Results and conclusion: Local, intestinal MBL expression was negligible compared to systemic MBL levels in patients and controls. Besides mannan, AIEC LF82-derived antigens were also retained by immobilized MBL and were recognized by affinity purified ASCA from CD patients. MBL-deficient but not wild type mice showed enhanced DSS colitis upon oral challenge with C. albicans or LF82. Infection of MBL-deficient mice with C. albicans led to prolonged persistence of antibodies crossreacting with S. cerevisiae mannan, by definition representing ASCA. Furthermore, systemic C. albicans infection led to increased dissemination in MBL-deficient mice, possibly due to reduced retention and clearance in circulation in the absence of MBL. These results suggest that systemic MBL helps to prevent excessive inflammation upon access of normally mild pathogens across the damaged intestinal epithelium. Lack of this innate defence promotes generation of abnormal, cross-re (...truncated)