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Mo1791 Ulcerative Colitis-Associated Escherichia coli Colonize the Ileum and Cecum of Infected Mice by Adhering to the Intestinal Epithelium
Mo1790
inflammatory scores were separated into different groups based on their microbiota composition as detected by CCA separation based on TRFLP analysis. In general, control and L colitis grouped closer to each other while the M and H colitis groups were more clearly separated. The main differences between WT and Gai2-/- mice in the semi-quantification of bacteria were observed as decreased amounts of Bacteroides (colon/H group and faeces/L group), C. leptum group (faeces/L group), Lactobacillus (caecum and colon/H group, and faeces/L group) and SFB (caecum/L and M group, and faeces/H group). The mRNA levels of IL-17, IFN-g, IL-27 were significantly increased in both caecum and colon of Gai2-/compared with WT mice. IL-10 showed significant decrease only in caecum of mice with high colitis score. TGF- β1 was unaffected. Significant negative correlations between Bacteroides-IL-27 and Lactobacillus-IFN-y, IL-27 were detected in caecum. Conclusion: Different inflammation levels in IBD are linked with clear changes in the GI microbiota specifically with members of Bacteroides and Lactobacillus and with inflammatory cytokines such as IL-27 and IFN-g. These changes are not limited to the colon but were also detected in the caecum. Our results show that also the caecum is affected by the disease in a mouse model resembling ulcerative colitis. The reported changes here might have an important role in the pathogenesis of IBD.
AGA Abstracts
Butyrate Induces Intestinal Epithelial Cell to Express Thrombospondin 1 and Suppress Intestinal Mucosal Inflammation Leilei Fang, Zhanju Liu Background: Monocytes (Mo) play an important role in the pathogenesis of intestinal mucosal inflammation. This study aims to investigate into the mechanism whereby the intestinal epithelial cell (IEC)-derived thrombospondin 1 (TSP1) modulates Mo properties and regulates intestinal inflammatory responses. Methods: The production of TSP1 by IEC was evaluated by quantitative real-time PCR and Western blotting. The properties of Mos were analyzed by flow cytometry. A mouse model of colitis was established to assess the role of epithelium-derived TSP1 induced by C. butyricum in the suppression of intestinal inflammation. Results: The results demonstrated that mouse IECs expressed TSP1, which was markedly upregulated by butyrate or feeding with C. butyricum. Coculture the butyrateprimed IECs and Mos or exposure of Mos to TSP1 in the culture induced expression of transforming growth factor (TGF)-β in Mos. These TGF-β+ Mos had tolerogenic properties that could promote generation of inducible regulatory T cells. Importatnly, adoptive transfer with TSP1-primed Mos, or feeding C. butyricum could prevent experimental colitis in mice. Conclusions: C. butyricum induces IECs to produce TSP1 and induces generation of TGFβ+ Mos, which further suppress experimental colitis in mice. The results implicate that administration of C. butyricum or butyrate may have the potential to ameliorate chronic intestinal inflammation through inducing immune suppressive Mos.
Mo1793 Gut Microbiome Composition Change in Crohn's Patients Following Ileal Resection Surgery Zoya Grigoryan, Mitchell Bernstein, Antonio Galvao Neto, Arielle Radin, Lea Ann Chen Introduction: Inflammatory Bowel Disease (IBD), and Crohn's Disease in particular, is known to be associated with dysbiosis in the gut microbiome. However, the characterization and implications of changes in the gut microbiome of patients undergoing treatment such as ileal resection surgery are not well understood. We present preliminary data from an ongoing study of Crohn's disease patients before and after undergoing ileal resection for stricturing disease. Methods: Crohn's patients undergoing ileal resection for the treatment of complicated IBD were recruited by physician referral. Stool samples were collected prior to surgery, at the first bowel movement after surgery, four weeks after surgery, and every four weeks afterward. Quality of life surveys, medication information, and clinical data were collected with each stool sample. 16S rRNA sequencing of the v4 region was performed on extracted stool DNA using 2x150 bp amplicon paired-end sequencing on the Illumina Miseq platform. Data processing and analysis was performed using QIIME software v1.8 referencing the Greengenes database (version gg_13_8). Results: Data for three patients (2 male) are presented. Taxa plots showing relative abundance of gut microbiota on the family level show instability in microbiome composition in individual patients over available timepoints [Figure 1]. Principal coordinate analysis (PCoA) of samples from before surgery (baseline) through 12 weeks following surgery reveals that samples cluster primarily by individual by both weighted and unweighted Unifrac distance analysis ((p=0.001 for both, R2 =0.79 and 0.33, respectively) [Figure 2]. A trend of recovery to baseline can be observed, with samples at 4 weeks after surgery more closely clustering to the baseline than immediately after surgery for all patients [Figure 2]. Individual changes in taxa composition and shifts in PCoA correspond with changes in clinical symptoms and medications [Figures 1,2]. Conclusions: These preliminary data suggest that ileal resection in Crohn's patients may have implications for gut microbiome changes, which may be transient following surgery, and that individual enterotype plays a very strong role in microbiome composition. Ongoing analysis will seek to elucidate the relationship between the gut microbiome, surgery, and clinical outcomes and symptoms.
Mo1791 Ulcerative Colitis-Associated Escherichia coli Colonize the Ileum and Cecum of Infected Mice by Adhering to the Intestinal Epithelium Hengameh Chloé Lauridsen, Hyungjun Yang, Else Bosman, Hongbing Yu, Xiujuan Wu, Caixia Ma, Carsten Struve, Andreas M. Petersen, Karen A. Krogfelt, Kevan Jacobson, Bruce Vallance Introduction Specific subtypes of Escherichia coli, of the phylogenetic groups B2 and D have been found in increased numbers in the intestine of patients with Inflammatory Bowel Disease (IBD). Previously, we have shown that the phylogroup B2 Ulcerative Colitis (UC) associated E. coli p19A induce cell death in dendritic cells and disrupt tight junctions in Caco-2 cells in vitro. In this study, the goal was to investigate whether orally delivered p19A colonizes the intestine of wildtype C57BL/6 mice, and mice deficient (-/-) in SIGIRR, a negative regulator of TLR/IL-1R signalling. Materials and Methods Sigirr -/- mice were chosen because they exhibit a heightened intestinal inflammatory tone, and show increased susceptibility to enteric bacterial pathogens. 10-12 week old C57BL/6 and Sigirr -/- mice were orally gavaged with 5 mg vancomycin /mouse. Six hours later, the mice were orally gavaged with 3 x108 colony forming units (CFU) of an overnight culture of luciferase expressing UC-associated E. coli p19A (p19A-lux) grown in LB broth. Control mice received LB broth only. Tissue and fecal samples were homogenized and plated onto selective agar plates to enumerate CFU, An In Vivo Imaging System (IVIS) was used to visualize the location of the p19A bacteria in intact intestinal tissues while immunohistochemistry and histology were used to localize the bacteria. Results The first day after infection with the UC-associated E. coli p19A, mice of both genotypes showed signs of illness, with modest levels of weight loss and displaying a hunched appearance. Over a time course of 7 days, both C57BL/6 and Sigirr -/- mice shed significant levels of bacteria (108 - 109 CFU/gram stool). Following euthanization of the infected mice, luciferase expressing p19A bacteria were found adherent to the mucosal surface of the ileum and cecum of C57BL/6 mice. Interestingly, p19A-lux expressed luminescent signals were substantially (10-50x) greater in the Sigirr -/- mice, but in the same locations, indicating that this microbe adheres more readily to the Sigirr -/- tissues. Using the immunostaining method, we confirmed that P19A were adherent to the intestinal epithelium of infected mice. Moreover, p19A was found at systemic sites (spleen and liver) in large numbers (103 - 106 CFU/gram tissue) suggesting that p19A disrupts epithelial barrier function in vivo, similar to the observations in vitro. Conclusion This study shows that the UC-associated E. coli p19A colonizes the intestines of Sigirr -/- and C57BL/6 mice, adhering to the intestinal epithelium and causing signs of illness. The higher inflammatory tone of the Sigirr -/- mice may underlie the greater adherence of P19A to their tissues. The resulting infection model will be used to help define the potential role of this pathobiont in the pathogenesis of UC. Mo1792 Alterations in Gut Microbiota Composition in Tissues and Faeces in Parallel With Enhanced Cytokine Gene Expression in the Gai2-/- Mouse Model of Spontaneous Colitis Ignacio Rangel, John-Peter Ganda Mall, Fei Sjöberg, Roger Willén, Elisabeth Hultgren Hörnquist Background: A key factor in the pathogenesis of Inflammatory Bowel Diseases (IBD) is an altered gut microbiota composition. The aim of the present study was to investigate the variations in the GI microbiota and mucosal cytokine gene expression in relation to different degrees of colitis in a spontaneous mouse model of IBD, the Gai2-/- mice. We applied non-culture techniques to analyze the microbiota of the Gai2-deficient mouse model of spontaneous colitis and their wild type (WT) littermates in colonic and caecal tissues and faeces. In parallel, we analysed the cytokine gene expression from the same tissues. Method: Analysis of the microbiota in small intestine, caecum, colon and faeces from Gai2-/- mice (n=18) and their wild type (WT) littermates (n=22) was done by Terminal Restriction Fragment Length Polymorphism (TRFLP) and qPCR targeting Bacteroides, C. leptum group, Lactobacillus and segmented filamentous bacteria (SFB). Colitis was scored histologically and the degree of colitis were referred to as 1-2=low (L), 3= medium (M) and 4-5=high (H). Overall analysis of TRFLP results and histological scores was made with a constrained correspondence analysis (CCA). Differences in cytokine gene expression and bacterial qPCR data were determined by Mann-Whitney U test. Spearmans correlation coefficient was used for analysing the correlation between cytokines and bacteria. Results: Mice with different
AGA Abstracts
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inflammatory scores were separated into different groups based on their microbiota composition as detected by CCA separation based on TRFLP analysis. In general, control and L colitis grouped closer to each other while the M and H colitis groups were more clearly separated. The main differences between WT and Gai2-/- mice in the semi-quantification of bacteria were observed as decreased amounts of Bacteroides (colon/H group and faeces/L group), C. leptum group (faeces/L group), Lactobacillus (caecum and colon/H group, and faeces/L group) and SFB (caecum/L and M group, and faeces/H group). The mRNA levels of IL-17, IFN-g, IL-27 were significantly increased in both caecum and colon of Gai2-/compared with WT mice. IL-10 showed significant decrease only in caecum of mice with high colitis score. TGF- β1 was unaffected. Significant negative correlations between Bacteroides-IL-27 and Lactobacillus-IFN-y, IL-27 were detected in caecum. Conclusion: Different inflammation levels in IBD are linked with clear changes in the GI microbiota specifically with members of Bacteroides and Lactobacillus and with inflammatory cytokines such as IL-27 and IFN-g. These changes are not limited to the colon but were also detected in the caecum. Our results show that also the caecum is affected by the disease in a mouse model resembling ulcerative colitis. The reported changes here might have an important role in the pathogenesis of IBD.
AGA Abstracts
Butyrate Induces Intestinal Epithelial Cell to Express Thrombospondin 1 and Suppress Intestinal Mucosal Inflammation Leilei Fang, Zhanju Liu Background: Monocytes (Mo) play an important role in the pathogenesis of intestinal mucosal inflammation. This study aims to investigate into the mechanism whereby the intestinal epithelial cell (IEC)-derived thrombospondin 1 (TSP1) modulates Mo properties and regulates intestinal inflammatory responses. Methods: The production of TSP1 by IEC was evaluated by quantitative real-time PCR and Western blotting. The properties of Mos were analyzed by flow cytometry. A mouse model of colitis was established to assess the role of epithelium-derived TSP1 induced by C. butyricum in the suppression of intestinal inflammation. Results: The results demonstrated that mouse IECs expressed TSP1, which was markedly upregulated by butyrate or feeding with C. butyricum. Coculture the butyrateprimed IECs and Mos or exposure of Mos to TSP1 in the culture induced expression of transforming growth factor (TGF)-β in Mos. These TGF-β+ Mos had tolerogenic properties that could promote generation of inducible regulatory T cells. Importatnly, adoptive transfer with TSP1-primed Mos, or feeding C. butyricum could prevent experimental colitis in mice. Conclusions: C. butyricum induces IECs to produce TSP1 and induces generation of TGFβ+ Mos, which further suppress experimental colitis in mice. The results implicate that administration of C. butyricum or butyrate may have the potential to ameliorate chronic intestinal inflammation through inducing immune suppressive Mos.
Mo1793 Gut Microbiome Composition Change in Crohn's Patients Following Ileal Resection Surgery Zoya Grigoryan, Mitchell Bernstein, Antonio Galvao Neto, Arielle Radin, Lea Ann Chen Introduction: Inflammatory Bowel Disease (IBD), and Crohn's Disease in particular, is known to be associated with dysbiosis in the gut microbiome. However, the characterization and implications of changes in the gut microbiome of patients undergoing treatment such as ileal resection surgery are not well understood. We present preliminary data from an ongoing study of Crohn's disease patients before and after undergoing ileal resection for stricturing disease. Methods: Crohn's patients undergoing ileal resection for the treatment of complicated IBD were recruited by physician referral. Stool samples were collected prior to surgery, at the first bowel movement after surgery, four weeks after surgery, and every four weeks afterward. Quality of life surveys, medication information, and clinical data were collected with each stool sample. 16S rRNA sequencing of the v4 region was performed on extracted stool DNA using 2x150 bp amplicon paired-end sequencing on the Illumina Miseq platform. Data processing and analysis was performed using QIIME software v1.8 referencing the Greengenes database (version gg_13_8). Results: Data for three patients (2 male) are presented. Taxa plots showing relative abundance of gut microbiota on the family level show instability in microbiome composition in individual patients over available timepoints [Figure 1]. Principal coordinate analysis (PCoA) of samples from before surgery (baseline) through 12 weeks following surgery reveals that samples cluster primarily by individual by both weighted and unweighted Unifrac distance analysis ((p=0.001 for both, R2 =0.79 and 0.33, respectively) [Figure 2]. A trend of recovery to baseline can be observed, with samples at 4 weeks after surgery more closely clustering to the baseline than immediately after surgery for all patients [Figure 2]. Individual changes in taxa composition and shifts in PCoA correspond with changes in clinical symptoms and medications [Figures 1,2]. Conclusions: These preliminary data suggest that ileal resection in Crohn's patients may have implications for gut microbiome changes, which may be transient following surgery, and that individual enterotype plays a very strong role in microbiome composition. Ongoing analysis will seek to elucidate the relationship between the gut microbiome, surgery, and clinical outcomes and symptoms.
Mo1791 Ulcerative Colitis-Associated Escherichia coli Colonize the Ileum and Cecum of Infected Mice by Adhering to the Intestinal Epithelium Hengameh Chloé Lauridsen, Hyungjun Yang, Else Bosman, Hongbing Yu, Xiujuan Wu, Caixia Ma, Carsten Struve, Andreas M. Petersen, Karen A. Krogfelt, Kevan Jacobson, Bruce Vallance Introduction Specific subtypes of Escherichia coli, of the phylogenetic groups B2 and D have been found in increased numbers in the intestine of patients with Inflammatory Bowel Disease (IBD). Previously, we have shown that the phylogroup B2 Ulcerative Colitis (UC) associated E. coli p19A induce cell death in dendritic cells and disrupt tight junctions in Caco-2 cells in vitro. In this study, the goal was to investigate whether orally delivered p19A colonizes the intestine of wildtype C57BL/6 mice, and mice deficient (-/-) in SIGIRR, a negative regulator of TLR/IL-1R signalling. Materials and Methods Sigirr -/- mice were chosen because they exhibit a heightened intestinal inflammatory tone, and show increased susceptibility to enteric bacterial pathogens. 10-12 week old C57BL/6 and Sigirr -/- mice were orally gavaged with 5 mg vancomycin /mouse. Six hours later, the mice were orally gavaged with 3 x108 colony forming units (CFU) of an overnight culture of luciferase expressing UC-associated E. coli p19A (p19A-lux) grown in LB broth. Control mice received LB broth only. Tissue and fecal samples were homogenized and plated onto selective agar plates to enumerate CFU, An In Vivo Imaging System (IVIS) was used to visualize the location of the p19A bacteria in intact intestinal tissues while immunohistochemistry and histology were used to localize the bacteria. Results The first day after infection with the UC-associated E. coli p19A, mice of both genotypes showed signs of illness, with modest levels of weight loss and displaying a hunched appearance. Over a time course of 7 days, both C57BL/6 and Sigirr -/- mice shed significant levels of bacteria (108 - 109 CFU/gram stool). Following euthanization of the infected mice, luciferase expressing p19A bacteria were found adherent to the mucosal surface of the ileum and cecum of C57BL/6 mice. Interestingly, p19A-lux expressed luminescent signals were substantially (10-50x) greater in the Sigirr -/- mice, but in the same locations, indicating that this microbe adheres more readily to the Sigirr -/- tissues. Using the immunostaining method, we confirmed that P19A were adherent to the intestinal epithelium of infected mice. Moreover, p19A was found at systemic sites (spleen and liver) in large numbers (103 - 106 CFU/gram tissue) suggesting that p19A disrupts epithelial barrier function in vivo, similar to the observations in vitro. Conclusion This study shows that the UC-associated E. coli p19A colonizes the intestines of Sigirr -/- and C57BL/6 mice, adhering to the intestinal epithelium and causing signs of illness. The higher inflammatory tone of the Sigirr -/- mice may underlie the greater adherence of P19A to their tissues. The resulting infection model will be used to help define the potential role of this pathobiont in the pathogenesis of UC. Mo1792 Alterations in Gut Microbiota Composition in Tissues and Faeces in Parallel With Enhanced Cytokine Gene Expression in the Gai2-/- Mouse Model of Spontaneous Colitis Ignacio Rangel, John-Peter Ganda Mall, Fei Sjöberg, Roger Willén, Elisabeth Hultgren Hörnquist Background: A key factor in the pathogenesis of Inflammatory Bowel Diseases (IBD) is an altered gut microbiota composition. The aim of the present study was to investigate the variations in the GI microbiota and mucosal cytokine gene expression in relation to different degrees of colitis in a spontaneous mouse model of IBD, the Gai2-/- mice. We applied non-culture techniques to analyze the microbiota of the Gai2-deficient mouse model of spontaneous colitis and their wild type (WT) littermates in colonic and caecal tissues and faeces. In parallel, we analysed the cytokine gene expression from the same tissues. Method: Analysis of the microbiota in small intestine, caecum, colon and faeces from Gai2-/- mice (n=18) and their wild type (WT) littermates (n=22) was done by Terminal Restriction Fragment Length Polymorphism (TRFLP) and qPCR targeting Bacteroides, C. leptum group, Lactobacillus and segmented filamentous bacteria (SFB). Colitis was scored histologically and the degree of colitis were referred to as 1-2=low (L), 3= medium (M) and 4-5=high (H). Overall analysis of TRFLP results and histological scores was made with a constrained correspondence analysis (CCA). Differences in cytokine gene expression and bacterial qPCR data were determined by Mann-Whitney U test. Spearmans correlation coefficient was used for analysing the correlation between cytokines and bacteria. Results: Mice with different
AGA Abstracts
S-712