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365 The Lymphatic System Controls Intestinal Inflammation and Inflammation-Associated Colon Cancer Through the Chemokine Decoy Receptor D6
the intestine was examined. Confocal microscopy co-localized IFN-lambda and mouse mast cell protease-1 (a marker of mouse mast cells) in the ileum and colon. Profound infiltration of mononuclear cells, mastocytosis, ulcers and crypt necrosis in the intestinal mucosa were observed. Significantly increases in the numbers and cytokines of Th1 cells were noted. It is noteworthy that the flagellin-specific Th1 cell proliferation was increased drastically in the presence of flagellin in culture. The IBD-like Th1 pattern inflammation in mouse intestine could be abolished by pretreatment with antibodies against IFN-lambda receptors or using mast cell deficient mice. Conclusions: (i) IFN-lambda was identified and localized in mast cells of IBD biopsy. (ii) Microbial antigen specific immune responses induce IFN-lambda expression in mast cells of mouse intestinal mucosa. (iii) IFN-lambda is involved in the pathogenesis of Th1 pattern inflammation in the intestine. 365 The Lymphatic System Controls Intestinal Inflammation and InflammationAssociated Colon Cancer Through the Chemokine Decoy Receptor D6 Stefania Vetrano, Elena M. Borroni, Raffaella Bonecchi, Carmen Correale, Adelaida Sarukhan, Vincenzo Arena, Massimo C. Fantini, Massimo Roncalli, Alberto Malesci, Annunciata Vecchi, Alberto Mantovani, Massimo Locati, Silvio Danese D6 is a chemokine receptor expressed on leucocytes and lymphatic endothelial cells. It plays an important role in the control of inflammation by acting as a decoy and scavenger receptor for inflammatory chemokines. The chronic mucosal inflammation that occurs in IBD which is also associated with an increased risk of colorectal cancer, could be the result of an inappropriate resolution of the inflammatory response and removal of proinflammatory chemokines. The aims of this study were to investigate the D6 expression in human IBD mucosa and the In Vivo functional role of D6 receptor in experimental colitis and inflammation-associated colon cancer. D6 was analyzed by confocal microscopy in human IBD, colon cancer and control tissues. D6-/- and WT C57BL/6 mice were treated with AOM and 3 oral cycles of DSS. Mice were monitored daily for weight loss, fecal blood and diarrhea and a DAI was calculated. The damage of murin colonic mucosa was evaluated by endoscopical and histological scores . The chemokines scavanged by D6 receptor were measured in colonic organ culture by ELISA. Bone-marrow chimeric mice were produced by transplanting bone marrow cells isolated from C57BL/6-CD45.1 mice into lethally irradiated D6-/- and WT hosts. D6 was expressed mainly by lymphatic vessels and leukocytes, in IBD, colonic cancer and control tissues. D6 -/- mice were significantly more susceptible to experimental colitis as assessed by body weight loss (p<0.001), DAI (p<0.001), endoscopical and histological inflammation (p<0.05) compared to WT mice. D6 -/- inflamed mucosa displayed a significant increase in CD3, CD11C and CD68 positive cells, but not MPO positive cells compared to inflamed WT mucosa. The lack of D6 resulted in significantly higher levels of CCL-2, -5, MIP2, KC and JE (all p<0.05) than WT mice. Furthermore, D6 -/- mice showed a higher (p<0.01) number of tumor incidence in the distal segment of the colon characterized by a high grade (HG) of differentiation in comparison to WT mice. C57BL/6-CD45.1 →D6 -/chimeras displayed the same phenotype as D6 deficient mice after DSS treatment, on the contrary no differences were observed between D6 -/- → C57BL/6-CD45.1 and WT→ C57BL/6-CD45.1 chimeras. Our results unveil the lymphatic system as a novel and active player in IBD and colitis-associated colon cancer pathogenesis, acting as a scavanger of inflammatory chemokines through the D6 decoy receptor. The ineffective scavenging of proinflammatory chemokines in the inflamed colon significantly increases tumor incidence, suggesting D6 as a novel and potential tool in modulating intestinal inflammation and susceptibility to tumor formation in the colon.
363 Post-Infective Dyspepsia Eight Years After the Walkerton Outbreak of Waterborne Gastroenteritis (GE) Alexander C. Ford, Marroon Thabane, Stephen M. Collins, Paul Moayyedi, Amit X. Garg, William F. Clark, John K. Marshall Background and Aim: In May 2000, municipal water contamination by E. coli 0157:H7 and Campylobacter species caused a large outbreak of acute GE in Walkerton, Ontario. We assessed the prevalence of post-infective dyspepsia. Methods: All Walkerton Health Study participants were invited for follow-up in 2008. During standardized interviews, adult participants were asked to complete a validated short -form Leeds dyspepsia questionnaire (S-FLDQ), with dyspepsia defined by a summed frequency score ≥4 (APT 2007;25:477486). Subjects with symptoms pre-dating the outbreak by self-report, were defined as having premorbid dyspepsia and were excluded from the study cohort. Subjects enrolled in 2003 or earlier who were age > 18 at the time of interview and permanent residents of Walkerton in May 2000 were included in the analysis. Sensitivity analysis was performed using the Rome II definition of dyspepsia, which excluded individuals with predominant heartburn or regurgitation. The prevalence of post-infective dyspepsia in 2008 was compared among participants with vs. without GE in May 2000. Risk factors for post-infective dyspepsia were identified by univariate and multivariate logistic regression. Results: Of 1997 subjects who completed the S-FLDQ, 908 were excluded (546 were non-residents of Walkerton, 181 had participated by proxy [telephone interview], 131 had joined the study after 2003 and 244 had premorbid dyspepsia). Of 1089 eligible subjects (60.1% female; mean age 55.0±17.6), 706 were exposed to GE in May 2000. The prevalence of dyspepsia was higher in exposed vs. non-exposed subjects (49.3% vs. 30.0%, odds ratio (OR) 2.27, 95% CI 1.74-2.95, p<0.0001). A similar increase was seen using the Rome II definition of dyspepsia (29.9% vs. 14.6%, OR 2.45, 95% CI 1.80-3.45, p<0.0001). In univariate analysis, independent risk factors for dyspepsia included a history of anxiety/depression, younger age, and specific features of the acute GE (abdominal cramps, fever, peak stool frequency, and longer duration of diarrhea). In multivariate analysis history of anxiety/depression, abdominal cramps, and longer duration of diarrhea during acute GE remained significant risk factors for dyspepsia. The multivariate model remained similar when a composite outcome of dyspepsia and/or current use of PPI/H2RA was used. Conclusion: The prevalence of dyspepsia is increased in exposed individuals 8 years after a waterborne outbreak of acute GE. These data suggest that, similar to irritable bowel syndrome, there is a post-infective etiology to dyspepsia in some individuals, and that features of acute GE can predict risk.
366 TL1A Enhances the Differentiation of Human TH17 Cells Kathrin S. Michelsen, Qi T. Yu, Brian Ko, Lisa S. Thomas, Hidetoshi Takedatsu, Carol J. Landers, Stephan R. Targan Background and significance: TL1A is a member of the TNF superfamily that mediates a strong co-stimulation of TH1 cells by enhancing IFN-γ production by peripheral and mucosal CD4+ T cells. The expression of TL1A and its receptor DR3 is increased in inflamed mucosa of Crohn's Disease (CD) patients and in murine models of ileitis. We have previously shown that TL1A is induced in human antigen-presenting cells (APCs) by Fc γ Receptor but not Toll-like receptor signaling. Furthermore, we have shown that in a chronic model of colitis neutralizing TL1A antibodies attenuate clinical signs of colitis by attenuating TH1 and TH17 responses. However, it remains to be elucidated if TL1A enhances de novo differentiation of TH17 cells. Aim: To determine if TL1A enhances the differentiation of TH17 cells in humans. Methods: To determine if TL1A enhances the differentiation of human TH17 cells we isolated naive CD4+ T cells and stimulated under TH17-driving conditions in the absence or presence of TL1A. IL-17, RORC, RORA, TL1A, IL-6, TGF-β, and IL-23 mRNA expression was measured by real-time PCR. IL-17 secretion by TH17 cells was measured by ELISA. Intracellular cytokine production of IFN-γ and IL-17 in TH17 cells was measured by flow cytometry. Results: FcγR signaling in monocytes or dendritic cells leads to the concomitant induction of TL1A, IL-6, TGF-β, and IL-23, a cytokine milieu that has been demonstrated to foster the development of TH17 cells. In contrast, TLR2 and TLR4 signaling did neither induce TL1A nor TGF-β suggesting that FcγR but not TLR signaling in APCs plays an important role in supporting TH17 differentiation. TL1A, in combination with TGF-β and IL-6, promotes the differentiation of human TH17 cells from naive CD4+ T cells. Stimulation of CD4+ T cells with TL1A and TGF-β/IL-6 leads to a unique cell population of IL-17/IFNγ producing TH17 cells. Furthermore, TL1A enhances the IL-17 production by committed CD45RO+CCR6+ TH17 cells. When we analyzed the induction of the transcription factors RORA and RORC, both have been shown to be important in the differentiation of TH17 cells, we observed that TL1A enhances the induction of RORA but has no enhancing effect on RORC mRNA. However, the enhanced RORA mRNA levels corresponded to an increase in IL-17 mRNA under the same stimulatory conditions suggesting that the enhancing effects TL1A has on RORA gene expression is sufficient to enhance the production of IL-17. Conclusion: Our findings establish an important role of TL1A in promoting human TH17 cell differentiation and function and may provide a potential target for therapeutic intervention in chronic inflammatory TH17 driven diseases, such as CD.
364 Role of Mast Cell-Derived Interferon Lambda in Intestinal Inflammation Induced By Microbial Antigens Xiao Chen, Shao-Heng He, Bai-Sui Feng, Ping-Chang Yang Background: IFN-lambda is a newly identified molecule that is involved in the promotion of Th1 responses and plays an important role in the regulation of immune responses. This study aimed to identify and localize the IFN-lambda in the intestinal mucosa of patients with IBD (inflammatory bowel disease) and its pathogenic role in the induction of IBD-like inflammation in the intestine of mice. Methods and Results: Human study: Thirty-two intestinal biopsies and 6 samples from surgical removed intestinal tissue were collected from patients with IBD; “normal” intestinal tissue was sampled from surgical removed intestinal segments (the non-disease part) from 6 patients with intestinal cancer (using as controls). Based on the information supplied by their physicians, the diagnosis of IBD was verified according to standard criteria of IBD. Intestinal sections were stained with anti-tryptase (mast cell marker) antibody or/and antibodies against IFN-lambda (IL-28, or IL-29). Double positive stained cells were 81% (64~94%) in IL-28/tryptase staining and 92% (88~96%) in IL-29/tryptase staining. In normal intestinal mucosa, significantly less number of mast cells and double positive stained cells (4.4% mast cells expressed IL-28 and 5.6% mast cells expressed IL-29) were observed. The expression of IL-28 and IL-29 in the intestine was confirmed by Western blotting. Animal model study: To further elucidate the pathogenic role of IFN-lambda in IBD-like inflammation in the intestine, Balb/c and C57B/6 mice were sensitized to bacterial antigen flagellin (with complete Freund adjuvant) and challenged with flagellin daily for 7 days. The immune inflammation and IFN-lambda expression in
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upper endoscopy for multiple indications as part of a prospective eosinophilic esophagitis prevalence study. As part of our initial protocol, multiple biopsies were obtained from esophagus, stomach and duodenum on each patient. Non-ulcer dyspepsia patients were identified via a detailed chart review strictly using Rome III criteria for non-ulcer dyspepsia. A blinded pathologist counted eosinophils in 5 high-power fields in both the stomach and duodenum. The presence of eosinophil clusters, eosinophil degranulation and Helicobacter pylori were also collected. Results: 93.5% (374/400) patients completed endoscopy with biopsies. The group had a mean age (SD) of 52(15) years, 51% male and 56% Caucasian. 28.3% (106/374) of patients met criteria for non-ulcer dyspepsia. Mean (SD) eosinophil count in the proximal and distal esophagus, stomach and duodenum were 0.96 (5.3), 0.99 (6.7), 5.3 (8.6) and 20.5 (10.3), respectively. When comparing patients with non-ulcer dyspepsia versus all other patients, there was no difference in mean gastric (5.4 vs. 5.2, p = 0.81) or duodenal eosinophil count (19.8 vs. 20.8, p=0.41). There was no increased frequency of eosinophil degranulation, eosinophil clusters or Helicobacter pylori in patients with nonulcer dyspepsia. Patients with Helicobacter pylori (N=46) had a higher mean gastric (10.6 vs. 4.5, p<0.001) and duodenal eosinophil count (24.3 vs. 20.0, p=0.01) compared to patients who were Helicobacter pylori negative. Conclusion: Amongst patients undergoing outpatient upper endoscopy, there was no difference in mean stomach or duodenum eosinophil count between patients with and without non-ulcer dyspepsia. Only Helicobacter pylori was associated with elevated eosinophil counts in stomach and duodenum. Further studies are need to determine the role of gastric and duodenal eosinophilia as biomarkers for nonulcer dyspepsia. References 1. Talley NJ, Walker MM, Aro P, Ronkainen J, Storskrubb T, Hindley LA, Harmsen WS, Zinsmeister AR, Agreus L. Non-ulcer dyspepsia and duodenal eosinophilia: an adult endoscopic population-based case-control study. Clin Gastroenterol Hepatol 2007;5:1175-83.
363 Post-Infective Dyspepsia Eight Years After the Walkerton Outbreak of Waterborne Gastroenteritis (GE) Alexander C. Ford, Marroon Thabane, Stephen M. Collins, Paul Moayyedi, Amit X. Garg, William F. Clark, John K. Marshall Background and Aim: In May 2000, municipal water contamination by E. coli 0157:H7 and Campylobacter species caused a large outbreak of acute GE in Walkerton, Ontario. We assessed the prevalence of post-infective dyspepsia. Methods: All Walkerton Health Study participants were invited for follow-up in 2008. During standardized interviews, adult participants were asked to complete a validated short -form Leeds dyspepsia questionnaire (S-FLDQ), with dyspepsia defined by a summed frequency score ≥4 (APT 2007;25:477486). Subjects with symptoms pre-dating the outbreak by self-report, were defined as having premorbid dyspepsia and were excluded from the study cohort. Subjects enrolled in 2003 or earlier who were age > 18 at the time of interview and permanent residents of Walkerton in May 2000 were included in the analysis. Sensitivity analysis was performed using the Rome II definition of dyspepsia, which excluded individuals with predominant heartburn or regurgitation. The prevalence of post-infective dyspepsia in 2008 was compared among participants with vs. without GE in May 2000. Risk factors for post-infective dyspepsia were identified by univariate and multivariate logistic regression. Results: Of 1997 subjects who completed the S-FLDQ, 908 were excluded (546 were non-residents of Walkerton, 181 had participated by proxy [telephone interview], 131 had joined the study after 2003 and 244 had premorbid dyspepsia). Of 1089 eligible subjects (60.1% female; mean age 55.0±17.6), 706 were exposed to GE in May 2000. The prevalence of dyspepsia was higher in exposed vs. non-exposed subjects (49.3% vs. 30.0%, odds ratio (OR) 2.27, 95% CI 1.74-2.95, p<0.0001). A similar increase was seen using the Rome II definition of dyspepsia (29.9% vs. 14.6%, OR 2.45, 95% CI 1.80-3.45, p<0.0001). In univariate analysis, independent risk factors for dyspepsia included a history of anxiety/depression, younger age, and specific features of the acute GE (abdominal cramps, fever, peak stool frequency, and longer duration of diarrhea). In multivariate analysis history of anxiety/depression, abdominal cramps, and longer duration of diarrhea during acute GE remained significant risk factors for dyspepsia. The multivariate model remained similar when a composite outcome of dyspepsia and/or current use of PPI/H2RA was used. Conclusion: The prevalence of dyspepsia is increased in exposed individuals 8 years after a waterborne outbreak of acute GE. These data suggest that, similar to irritable bowel syndrome, there is a post-infective etiology to dyspepsia in some individuals, and that features of acute GE can predict risk.
366 TL1A Enhances the Differentiation of Human TH17 Cells Kathrin S. Michelsen, Qi T. Yu, Brian Ko, Lisa S. Thomas, Hidetoshi Takedatsu, Carol J. Landers, Stephan R. Targan Background and significance: TL1A is a member of the TNF superfamily that mediates a strong co-stimulation of TH1 cells by enhancing IFN-γ production by peripheral and mucosal CD4+ T cells. The expression of TL1A and its receptor DR3 is increased in inflamed mucosa of Crohn's Disease (CD) patients and in murine models of ileitis. We have previously shown that TL1A is induced in human antigen-presenting cells (APCs) by Fc γ Receptor but not Toll-like receptor signaling. Furthermore, we have shown that in a chronic model of colitis neutralizing TL1A antibodies attenuate clinical signs of colitis by attenuating TH1 and TH17 responses. However, it remains to be elucidated if TL1A enhances de novo differentiation of TH17 cells. Aim: To determine if TL1A enhances the differentiation of TH17 cells in humans. Methods: To determine if TL1A enhances the differentiation of human TH17 cells we isolated naive CD4+ T cells and stimulated under TH17-driving conditions in the absence or presence of TL1A. IL-17, RORC, RORA, TL1A, IL-6, TGF-β, and IL-23 mRNA expression was measured by real-time PCR. IL-17 secretion by TH17 cells was measured by ELISA. Intracellular cytokine production of IFN-γ and IL-17 in TH17 cells was measured by flow cytometry. Results: FcγR signaling in monocytes or dendritic cells leads to the concomitant induction of TL1A, IL-6, TGF-β, and IL-23, a cytokine milieu that has been demonstrated to foster the development of TH17 cells. In contrast, TLR2 and TLR4 signaling did neither induce TL1A nor TGF-β suggesting that FcγR but not TLR signaling in APCs plays an important role in supporting TH17 differentiation. TL1A, in combination with TGF-β and IL-6, promotes the differentiation of human TH17 cells from naive CD4+ T cells. Stimulation of CD4+ T cells with TL1A and TGF-β/IL-6 leads to a unique cell population of IL-17/IFNγ producing TH17 cells. Furthermore, TL1A enhances the IL-17 production by committed CD45RO+CCR6+ TH17 cells. When we analyzed the induction of the transcription factors RORA and RORC, both have been shown to be important in the differentiation of TH17 cells, we observed that TL1A enhances the induction of RORA but has no enhancing effect on RORC mRNA. However, the enhanced RORA mRNA levels corresponded to an increase in IL-17 mRNA under the same stimulatory conditions suggesting that the enhancing effects TL1A has on RORA gene expression is sufficient to enhance the production of IL-17. Conclusion: Our findings establish an important role of TL1A in promoting human TH17 cell differentiation and function and may provide a potential target for therapeutic intervention in chronic inflammatory TH17 driven diseases, such as CD.
364 Role of Mast Cell-Derived Interferon Lambda in Intestinal Inflammation Induced By Microbial Antigens Xiao Chen, Shao-Heng He, Bai-Sui Feng, Ping-Chang Yang Background: IFN-lambda is a newly identified molecule that is involved in the promotion of Th1 responses and plays an important role in the regulation of immune responses. This study aimed to identify and localize the IFN-lambda in the intestinal mucosa of patients with IBD (inflammatory bowel disease) and its pathogenic role in the induction of IBD-like inflammation in the intestine of mice. Methods and Results: Human study: Thirty-two intestinal biopsies and 6 samples from surgical removed intestinal tissue were collected from patients with IBD; “normal” intestinal tissue was sampled from surgical removed intestinal segments (the non-disease part) from 6 patients with intestinal cancer (using as controls). Based on the information supplied by their physicians, the diagnosis of IBD was verified according to standard criteria of IBD. Intestinal sections were stained with anti-tryptase (mast cell marker) antibody or/and antibodies against IFN-lambda (IL-28, or IL-29). Double positive stained cells were 81% (64~94%) in IL-28/tryptase staining and 92% (88~96%) in IL-29/tryptase staining. In normal intestinal mucosa, significantly less number of mast cells and double positive stained cells (4.4% mast cells expressed IL-28 and 5.6% mast cells expressed IL-29) were observed. The expression of IL-28 and IL-29 in the intestine was confirmed by Western blotting. Animal model study: To further elucidate the pathogenic role of IFN-lambda in IBD-like inflammation in the intestine, Balb/c and C57B/6 mice were sensitized to bacterial antigen flagellin (with complete Freund adjuvant) and challenged with flagellin daily for 7 days. The immune inflammation and IFN-lambda expression in
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upper endoscopy for multiple indications as part of a prospective eosinophilic esophagitis prevalence study. As part of our initial protocol, multiple biopsies were obtained from esophagus, stomach and duodenum on each patient. Non-ulcer dyspepsia patients were identified via a detailed chart review strictly using Rome III criteria for non-ulcer dyspepsia. A blinded pathologist counted eosinophils in 5 high-power fields in both the stomach and duodenum. The presence of eosinophil clusters, eosinophil degranulation and Helicobacter pylori were also collected. Results: 93.5% (374/400) patients completed endoscopy with biopsies. The group had a mean age (SD) of 52(15) years, 51% male and 56% Caucasian. 28.3% (106/374) of patients met criteria for non-ulcer dyspepsia. Mean (SD) eosinophil count in the proximal and distal esophagus, stomach and duodenum were 0.96 (5.3), 0.99 (6.7), 5.3 (8.6) and 20.5 (10.3), respectively. When comparing patients with non-ulcer dyspepsia versus all other patients, there was no difference in mean gastric (5.4 vs. 5.2, p = 0.81) or duodenal eosinophil count (19.8 vs. 20.8, p=0.41). There was no increased frequency of eosinophil degranulation, eosinophil clusters or Helicobacter pylori in patients with nonulcer dyspepsia. Patients with Helicobacter pylori (N=46) had a higher mean gastric (10.6 vs. 4.5, p<0.001) and duodenal eosinophil count (24.3 vs. 20.0, p=0.01) compared to patients who were Helicobacter pylori negative. Conclusion: Amongst patients undergoing outpatient upper endoscopy, there was no difference in mean stomach or duodenum eosinophil count between patients with and without non-ulcer dyspepsia. Only Helicobacter pylori was associated with elevated eosinophil counts in stomach and duodenum. Further studies are need to determine the role of gastric and duodenal eosinophilia as biomarkers for nonulcer dyspepsia. References 1. Talley NJ, Walker MM, Aro P, Ronkainen J, Storskrubb T, Hindley LA, Harmsen WS, Zinsmeister AR, Agreus L. Non-ulcer dyspepsia and duodenal eosinophilia: an adult endoscopic population-based case-control study. Clin Gastroenterol Hepatol 2007;5:1175-83.