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NOD2 Mediates Susceptibility to Yersinia Pseudotuberculosis
course of bacterial localization and the immune response of antigen-specific CD4+ T cell in C.rodentium infection remain to be seen. Method and Result: C. rodentium which express GFP constitutively (CR/GFP) has been established by the transformation with the GFPexpression vector. The expression of GFP was confirmed by flow cytometry and fluorescence microscopy. To examine the localization of C.rodentium, 3-week-old wild-type mice were orally inoculated with CR/GFP. CR/GFP were detected on the cell surface at the colon at day 3 and some were also detected in the CD11b positive cells such as macrophages or dendtiric cells of the colon. The bacterial number in the section of the infected colon and in the feces were increased until day 14. To examine the antigen-specific immune response of CD4 + T cell in C.rodentium infection, OVA-expressing C.rodentium which produce chicken ovalbumin constitutively (CR/OVA) have also been established by the transformation with OVA producing plasmid. The antigenic expression of OVA was confirmed by western blot and T cell-proliferation assay. IgM and IgG response against OVA were detected 2 weeks after oral infection with CR/OVA. Next, to examine the C.rodentium-specific T cell response, CFSE systems were utilized. Wild-type mice were infected with CR/OVA at day 1. CFSE labeled OVA-specific CD4+ T cells (OT-II cells) were adoptively transferred at day 12 and then, MLN were collected at day 15. Proliferation of OVA-specific CD4+ T cells were demonstrated by the detection of multiple cell CD4+ division of CFSE-loaded transgenic T cells. Moreover, enhanced cytokine production of IFN-γ and IL-4 were also detected after In Vitro stimulation with OVA. Conclusion: These studies show the natural course and host immune response in C. rodentium infection. These model systems are useful to examine the mucosal immunity against C.rodentium infection in detail.
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Intro: Enterotoxigenic Bacteroides fragilis (ETBF), a molecular subtype of the human commensal B. fragilis, induce colitis. Stat3 (signal transducer and activator of transcription) is activated in some patients with inflammatory bowel disease (IBD). Stat3 regulates inflammation and cellular proliferation, processes key to IBD. To further evaluate the link between Stat3 activation and inflammation, we evaluated Stat3 activation in murine ETBF colitis. Methods: Mice were inoculated with 108 CFU of ETBF, non-toxigenic B. fragilis (NTBF) or buffer after receiving 3-5 days of antibiotics to enhance colonization. Stat3 activation was assessed in C57BL/6 mice, sacrificed at different time points from 16 hours to 1 month post-inoculation. CD4 Stat3 C57BL/6 knock-out mice were used to assess the role of CD4 Stat3 activation in inflammation at 7 days post-inoculation. Gastrointestinal (GI) tissue was ‘flash frozen' in liquid nitrogen for subsequent extraction of nuclear proteins or preserved in 10% formalin for routine histopathology (scored for inflammation and proliferation on a scale of 0-5). Immunohistochemistry (IHC), western blot and/or electrophoretic mobility shift assay (EMSA) were used to detect phosphorylated (activated) Stat3 (pStat3). Results: pStat3 was detected by western blot, IHC, or EMSA throughout the GI tract of mice at 16 hours post-inoculation with ETBF and persisted up to 1 month post-inoculation. ETBF induced colitis at all time points. At 16 hours, ETBF stimulated pStat3 in the esophagus, stomach, small bowel (except duodenum) as well as proximal and distal colon. Similar activation was evident at 1 month with extension to the spleen but not liver. pStat3 activation was epithelial in the small bowel but within epithelial and select inflammatory cells in the colon. The ceca (other small bowel and colon analyses pending) of ETBF-inoculated CD4 Stat3 KO mice demonstrated mild increases in inflammatory cell pStat3 as well as colitis histopathology scores compared to wild type ETBF-inoculated mice. NTBF and bufferinoculated mice did not demonstrate colitis or pStat3 at any time point. Discussion: Intestinal Stat3 is rapidly and persistently activated by ETBF. Cecal Stat3 activation by ETBF appears largely independent of CD4 cells, though, Stat3 activation in CD4 cells may contribute to the regulation of inflammation and serve to dampen ETBF-induced inflammation. Future work will define the specific cell types in which Stat3 is activated and their contribution to inflammation in ETBF colitis.
W1185 CpG Oligonucleotide Stimulation of TLR9 Inhibits Il17 Production from Mesenteric Lymph Node Cells Ahmed Metwali, Sarah Winckler, M.Nedim Ince, David E. Elliott Bacteria express DNA sequences containing unmethylated CpG motifs that stimulate Tolllike receptor 9 (TLR9) and promote Th1 responses. Although they promote Th1 responses, exposure to immunostimulantory CpG oligonucleotide (CpG) affords protection in some mouse models of colitis. Interleukin 17 is a pro-inflammatory cytokine that drives many immune-mediated diseases. IL17 is made by Th17 cells, a distinct linage separate from classical Th1 and Th2 cells. IL17 expression is increased in active Crohn's disease and ulcerative colitis. Blockade of IL17 inhibits murine colitis. We hypothesized that immunostimulatory CpG may regulate IL17 production. We measured IL17 production by ELISA in supernatants of cultured murine mesenteric lymph node (MLN) cells. We found that LPS (0.1 µg/ml) but not CpG (0.6 µg/ml) stimulates IL17 production by MLN cells. Addition of CpG to LPS-stimulated MLN cells abrogates IL17 production (cells + LPS = 1.42 ± 0.34 ng/ml, cells + CpG = below 30 pg/ml, cells + LPS + CpG = below 30 pg/ml, p<0.001). Inhibition of LPS-stimulated IL17 production occurs throughout a range of CpG doses. CpG is not toxic to cultured MLN cells. Inhibition of IL17 production requires immunostimulatory motifs in the oligonucleotide sequence. CpG does not inhibit LPS-stimulated IL17 production by MLN cells of TLR9-deficient mice. However, CpG does abrogate IL17 production in cocultures of wild type and TLR9-deficient MLN cells (p<0.001), suggesting indirect action. Suppression continues in transwell cultures with wild type cells separated from TLR9deficient MLN cells by a semiperimeable membrane, indicating CpG-stimulated production of a soluble inhibitory factor. Blockade of IL10 signaling with anti-IL10 receptor mAb partially restores LPS-stimulated MLN cell IL17 production inhibited by CpG. However, CpG significantly inhibits LPS-stimulated IL17 production in MLN cells from IL10-deficient mice though not as completely as it does in cells from wild type mice. CpG and rIL10 synergize to inhibit LPS-stimulated IL17 production by MLN cells from IL10-deficient mice. Conclusion: Stimulation of TLR9 with CpG oligonucleotide inhibits MLN cell IL17 production through two mechanisms one of which is mediated by induction of IL10. Grant Support: AI49382, VAMC and CCFA.
W1188 Dominant Genotypes in Mucosa-Associated Escherichia coli Strains from Pediatric Patients with Inflammatory Bowel Disease Valerio Iebba, Maria Pia Conte, Serena Schippa, Marta Aleandri, Catia Longhi, John Osborn, Osvaldo Borrelli, Paola Falconieri, Salvatore Cucchiara Escherichia coli (E. coli), a predominant facultative anaerobe of the human colonic flora, has been thought to participate in the pathogenesis of inflammatory bowel disease (IBD). Increased numbers of mucosa-associated E. coli have been observed in adult IBD patients; furthermore, some E. coli strains from Crohn's disease (CD) patients have been shown to adhere to intestinal epithelial cells and invade them, as well as to replicate extensively into macrophages. It has also been suggested that some E. coli genotypes are more likely than others to be associated with CD in adults. There are no studies on the molecular characterization of mucosa-associated bacteria in children with pediatric IBD. Aims: children with IBD were investigated in order to characterize mucosa-associated E. coli strains and to assess if a particular subset of E. coli strains could be associated with IBD in these subjects. Methods and subjects: we analyzed genomic profile, phylogenetic grouping, virulence-gene carriage, different biochemical and enzymatic properties, and adhesive/invasive abilities of 60 E. coli strains isolated from mucosal biopsies of pediatric patients affected by CD (12 cases), ulcerative colitis (UC) (7 cases), and from those of 19 children with functional intestinal disorders who served as controls. Median (and ranges) age (years) was 14.0 (12-17), 10.0 (8-13), 12.7 84-16), respectively. Patients underwent ileo-colonoscopy as a step of their diagnostic approach after parental written consensus. The study was approved by the ethical committee of the Faculty. Results: in E. coli strains deriving from IBD patients and from controls no noteworthy differences were found in the distribution of phylogroups, in the adhesive properties as well as in the occurrence of virulence-related genes . However, the genetic profile examination revealed two large clusters of genetically linked E. coli strains from IBD patients: in the first cluster were grouped 92% of E. coli strains isolated from CD patients, distributed between two sub-clusters; in the second cluster 78% of strains isolated from UC and 77% from controls, distributed between two sub-clusters, respectively. Conclusions: in children with IBD, whereas phenotypic characterization failed to identify peculiar pathogenetic strains, genomic analysis was able to define a close genetic relatedness among E. coli strains associated with CD and UC gut mucosa.
W1186 CARD15/NOD2 Mediates Susceptibility to Yersinia Pseudotuberculosis Ulrich Meinzer, Sophie Esmiol-Welterlin, Frederick Barreau, Dominique Berrebi, Monique Dussaillant, Stephane Bonacorsi, Fabrice Chareyre, Michiko Niwa-Kawakita, Corinne Alberti, Ghislaine Sterkers, Thecla Lesuffleur, Michel Peuchmaur, Michael Karin, Lars Eckmann, Marco Giovannini, Vincent Ollendorff, Hans Wolf-Watz, Jean-Pierre Hugot BACKGROUND AND AIMS: Nucleotide oligomerisation domain 2 (NOD2) is a component of the innate immunity which plays a role in the susceptibility for Crohn's disease (CD). Little is known about the In Vivo role of Nod2 during host-pathogen interactions. Because enteropathogenic Yersinia DNA has been found in CD lesions and a personal history of Yersiniosis has been shown to be a risk factor for CD, we explored the In Vivo response towards Yersinia pseudotuberculosis in NOD2 deficient mice. MATERIAL AND METHODS: Nod2 deleted mice (KO) were orally infected with 1x107 CFU Y. pseudotuberculosis and wild type controls (C57B6). The survival rate, intestinal inflammatory response, immune cell composition of the gut mucosa, cell death and KC concentrations were assessed using histology and immunohistochemistry experiments, FACS analyses and ELISA-assays. Oral survival curves were also established in mice carrying a Nod2 frame shift mutation homologous to the mutation associated with CD in Human. RESULTS: Nod2-deficient mice were more resistant to oral infection by Yersinia pseudotuberculosis, while no difference was seen after infection by the intraperiteonal route. This phenotype is characterized in Peyer's Patches by a decreased apoptosis of epithelial cells, an increased KC secretion, a stronger infiltration by phagocytes and, finally by a lower systemic bacterial dissemination. Increased resistance to Y. pseudotuberculosis was also seen in Crohn's disease-associated Nod2-mutant mice. CONCLUSIONS: Nod2-deficient mice are less susceptible to Y. pseudotuberculosis infection as a result of an alteration of the immune-response driven by the gut associated lymphoid tissues.
W1189 Crohn's Disease-Associated Escherichia coli Lf82 Aggravates Colitis in Injured Mouse Colon Via Signalling By Flagellin Frédéric Carvalho, Nicolas Barnich, Pierre Sauvanet, Claude Darcha, Agathe Gelot, Arlette Darfeuille-Michaud Background: Ileal lesions in Crohn's disease patients are colonized by pathogenic adherentinvasive Escherichia coli (AIEC) that harbour various virulence factors involved in adhesion to and invasion of intestinal epithelial cultured cells. Aim: Our aim is to investigate in a mouse model of colonic inflammation the behaviour of virulent AIEC reference bacteria LF82 compared to that of nonflagellated LF82 mutants. Methods: BALBc/J mice with intact or dextran sulfate sodium (DSS)-injured colon were orally challenged daily with 10e8 bacteria. The severity of colitis was assessed by determining disease activity index, colonic histological score, and myeoloperoxidase activity. Flagellin receptor and cytokine expression was measured by RT-PCR in colonic tissue. Results: In contrast to nonpathogenic E. coli, virulent LF82 bacteria exacerbated colitis in DSS-treated mice, substantially reducing survival
A-651
AGA Abstracts
AGA Abstracts
STAT3 Is Activated Throughout the Gastrointestinal Tract in Enterotoxigenic Bacteroides Fragilis Induced Colitis Shervin Rabizadeh, Shaoguang Wu, Emilia Albesiano, XinQun Wu, Laura Mirviss, KiJong Rhee, Hong-Rong Yen, Drew M. Pardoll, Cynthia Sears
W1187
Intro: Enterotoxigenic Bacteroides fragilis (ETBF), a molecular subtype of the human commensal B. fragilis, induce colitis. Stat3 (signal transducer and activator of transcription) is activated in some patients with inflammatory bowel disease (IBD). Stat3 regulates inflammation and cellular proliferation, processes key to IBD. To further evaluate the link between Stat3 activation and inflammation, we evaluated Stat3 activation in murine ETBF colitis. Methods: Mice were inoculated with 108 CFU of ETBF, non-toxigenic B. fragilis (NTBF) or buffer after receiving 3-5 days of antibiotics to enhance colonization. Stat3 activation was assessed in C57BL/6 mice, sacrificed at different time points from 16 hours to 1 month post-inoculation. CD4 Stat3 C57BL/6 knock-out mice were used to assess the role of CD4 Stat3 activation in inflammation at 7 days post-inoculation. Gastrointestinal (GI) tissue was ‘flash frozen' in liquid nitrogen for subsequent extraction of nuclear proteins or preserved in 10% formalin for routine histopathology (scored for inflammation and proliferation on a scale of 0-5). Immunohistochemistry (IHC), western blot and/or electrophoretic mobility shift assay (EMSA) were used to detect phosphorylated (activated) Stat3 (pStat3). Results: pStat3 was detected by western blot, IHC, or EMSA throughout the GI tract of mice at 16 hours post-inoculation with ETBF and persisted up to 1 month post-inoculation. ETBF induced colitis at all time points. At 16 hours, ETBF stimulated pStat3 in the esophagus, stomach, small bowel (except duodenum) as well as proximal and distal colon. Similar activation was evident at 1 month with extension to the spleen but not liver. pStat3 activation was epithelial in the small bowel but within epithelial and select inflammatory cells in the colon. The ceca (other small bowel and colon analyses pending) of ETBF-inoculated CD4 Stat3 KO mice demonstrated mild increases in inflammatory cell pStat3 as well as colitis histopathology scores compared to wild type ETBF-inoculated mice. NTBF and bufferinoculated mice did not demonstrate colitis or pStat3 at any time point. Discussion: Intestinal Stat3 is rapidly and persistently activated by ETBF. Cecal Stat3 activation by ETBF appears largely independent of CD4 cells, though, Stat3 activation in CD4 cells may contribute to the regulation of inflammation and serve to dampen ETBF-induced inflammation. Future work will define the specific cell types in which Stat3 is activated and their contribution to inflammation in ETBF colitis.
W1185 CpG Oligonucleotide Stimulation of TLR9 Inhibits Il17 Production from Mesenteric Lymph Node Cells Ahmed Metwali, Sarah Winckler, M.Nedim Ince, David E. Elliott Bacteria express DNA sequences containing unmethylated CpG motifs that stimulate Tolllike receptor 9 (TLR9) and promote Th1 responses. Although they promote Th1 responses, exposure to immunostimulantory CpG oligonucleotide (CpG) affords protection in some mouse models of colitis. Interleukin 17 is a pro-inflammatory cytokine that drives many immune-mediated diseases. IL17 is made by Th17 cells, a distinct linage separate from classical Th1 and Th2 cells. IL17 expression is increased in active Crohn's disease and ulcerative colitis. Blockade of IL17 inhibits murine colitis. We hypothesized that immunostimulatory CpG may regulate IL17 production. We measured IL17 production by ELISA in supernatants of cultured murine mesenteric lymph node (MLN) cells. We found that LPS (0.1 µg/ml) but not CpG (0.6 µg/ml) stimulates IL17 production by MLN cells. Addition of CpG to LPS-stimulated MLN cells abrogates IL17 production (cells + LPS = 1.42 ± 0.34 ng/ml, cells + CpG = below 30 pg/ml, cells + LPS + CpG = below 30 pg/ml, p<0.001). Inhibition of LPS-stimulated IL17 production occurs throughout a range of CpG doses. CpG is not toxic to cultured MLN cells. Inhibition of IL17 production requires immunostimulatory motifs in the oligonucleotide sequence. CpG does not inhibit LPS-stimulated IL17 production by MLN cells of TLR9-deficient mice. However, CpG does abrogate IL17 production in cocultures of wild type and TLR9-deficient MLN cells (p<0.001), suggesting indirect action. Suppression continues in transwell cultures with wild type cells separated from TLR9deficient MLN cells by a semiperimeable membrane, indicating CpG-stimulated production of a soluble inhibitory factor. Blockade of IL10 signaling with anti-IL10 receptor mAb partially restores LPS-stimulated MLN cell IL17 production inhibited by CpG. However, CpG significantly inhibits LPS-stimulated IL17 production in MLN cells from IL10-deficient mice though not as completely as it does in cells from wild type mice. CpG and rIL10 synergize to inhibit LPS-stimulated IL17 production by MLN cells from IL10-deficient mice. Conclusion: Stimulation of TLR9 with CpG oligonucleotide inhibits MLN cell IL17 production through two mechanisms one of which is mediated by induction of IL10. Grant Support: AI49382, VAMC and CCFA.
W1188 Dominant Genotypes in Mucosa-Associated Escherichia coli Strains from Pediatric Patients with Inflammatory Bowel Disease Valerio Iebba, Maria Pia Conte, Serena Schippa, Marta Aleandri, Catia Longhi, John Osborn, Osvaldo Borrelli, Paola Falconieri, Salvatore Cucchiara Escherichia coli (E. coli), a predominant facultative anaerobe of the human colonic flora, has been thought to participate in the pathogenesis of inflammatory bowel disease (IBD). Increased numbers of mucosa-associated E. coli have been observed in adult IBD patients; furthermore, some E. coli strains from Crohn's disease (CD) patients have been shown to adhere to intestinal epithelial cells and invade them, as well as to replicate extensively into macrophages. It has also been suggested that some E. coli genotypes are more likely than others to be associated with CD in adults. There are no studies on the molecular characterization of mucosa-associated bacteria in children with pediatric IBD. Aims: children with IBD were investigated in order to characterize mucosa-associated E. coli strains and to assess if a particular subset of E. coli strains could be associated with IBD in these subjects. Methods and subjects: we analyzed genomic profile, phylogenetic grouping, virulence-gene carriage, different biochemical and enzymatic properties, and adhesive/invasive abilities of 60 E. coli strains isolated from mucosal biopsies of pediatric patients affected by CD (12 cases), ulcerative colitis (UC) (7 cases), and from those of 19 children with functional intestinal disorders who served as controls. Median (and ranges) age (years) was 14.0 (12-17), 10.0 (8-13), 12.7 84-16), respectively. Patients underwent ileo-colonoscopy as a step of their diagnostic approach after parental written consensus. The study was approved by the ethical committee of the Faculty. Results: in E. coli strains deriving from IBD patients and from controls no noteworthy differences were found in the distribution of phylogroups, in the adhesive properties as well as in the occurrence of virulence-related genes . However, the genetic profile examination revealed two large clusters of genetically linked E. coli strains from IBD patients: in the first cluster were grouped 92% of E. coli strains isolated from CD patients, distributed between two sub-clusters; in the second cluster 78% of strains isolated from UC and 77% from controls, distributed between two sub-clusters, respectively. Conclusions: in children with IBD, whereas phenotypic characterization failed to identify peculiar pathogenetic strains, genomic analysis was able to define a close genetic relatedness among E. coli strains associated with CD and UC gut mucosa.
W1186 CARD15/NOD2 Mediates Susceptibility to Yersinia Pseudotuberculosis Ulrich Meinzer, Sophie Esmiol-Welterlin, Frederick Barreau, Dominique Berrebi, Monique Dussaillant, Stephane Bonacorsi, Fabrice Chareyre, Michiko Niwa-Kawakita, Corinne Alberti, Ghislaine Sterkers, Thecla Lesuffleur, Michel Peuchmaur, Michael Karin, Lars Eckmann, Marco Giovannini, Vincent Ollendorff, Hans Wolf-Watz, Jean-Pierre Hugot BACKGROUND AND AIMS: Nucleotide oligomerisation domain 2 (NOD2) is a component of the innate immunity which plays a role in the susceptibility for Crohn's disease (CD). Little is known about the In Vivo role of Nod2 during host-pathogen interactions. Because enteropathogenic Yersinia DNA has been found in CD lesions and a personal history of Yersiniosis has been shown to be a risk factor for CD, we explored the In Vivo response towards Yersinia pseudotuberculosis in NOD2 deficient mice. MATERIAL AND METHODS: Nod2 deleted mice (KO) were orally infected with 1x107 CFU Y. pseudotuberculosis and wild type controls (C57B6). The survival rate, intestinal inflammatory response, immune cell composition of the gut mucosa, cell death and KC concentrations were assessed using histology and immunohistochemistry experiments, FACS analyses and ELISA-assays. Oral survival curves were also established in mice carrying a Nod2 frame shift mutation homologous to the mutation associated with CD in Human. RESULTS: Nod2-deficient mice were more resistant to oral infection by Yersinia pseudotuberculosis, while no difference was seen after infection by the intraperiteonal route. This phenotype is characterized in Peyer's Patches by a decreased apoptosis of epithelial cells, an increased KC secretion, a stronger infiltration by phagocytes and, finally by a lower systemic bacterial dissemination. Increased resistance to Y. pseudotuberculosis was also seen in Crohn's disease-associated Nod2-mutant mice. CONCLUSIONS: Nod2-deficient mice are less susceptible to Y. pseudotuberculosis infection as a result of an alteration of the immune-response driven by the gut associated lymphoid tissues.
W1189 Crohn's Disease-Associated Escherichia coli Lf82 Aggravates Colitis in Injured Mouse Colon Via Signalling By Flagellin Frédéric Carvalho, Nicolas Barnich, Pierre Sauvanet, Claude Darcha, Agathe Gelot, Arlette Darfeuille-Michaud Background: Ileal lesions in Crohn's disease patients are colonized by pathogenic adherentinvasive Escherichia coli (AIEC) that harbour various virulence factors involved in adhesion to and invasion of intestinal epithelial cultured cells. Aim: Our aim is to investigate in a mouse model of colonic inflammation the behaviour of virulent AIEC reference bacteria LF82 compared to that of nonflagellated LF82 mutants. Methods: BALBc/J mice with intact or dextran sulfate sodium (DSS)-injured colon were orally challenged daily with 10e8 bacteria. The severity of colitis was assessed by determining disease activity index, colonic histological score, and myeoloperoxidase activity. Flagellin receptor and cytokine expression was measured by RT-PCR in colonic tissue. Results: In contrast to nonpathogenic E. coli, virulent LF82 bacteria exacerbated colitis in DSS-treated mice, substantially reducing survival
A-651
AGA Abstracts
AGA Abstracts
STAT3 Is Activated Throughout the Gastrointestinal Tract in Enterotoxigenic Bacteroides Fragilis Induced Colitis Shervin Rabizadeh, Shaoguang Wu, Emilia Albesiano, XinQun Wu, Laura Mirviss, KiJong Rhee, Hong-Rong Yen, Drew M. Pardoll, Cynthia Sears