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Tu1695 Saccharomyces Boulardii Prevents the Antibiotic Induced Changes in Colonic Microbiota
rather than a primary cause of colitis and that IBD is dependent on interacting microbial and genetic factors.
Tu1697 Viral Pathogens Pull the Necrotic Trigger in a TNF-Receptor Independent Manner in Caspase-8ΔIEC Mice Claudia Günther, Helmut Neumann, Guiwei He, Eva Martini, Markus F. Neurath, Christoph Becker
AGA Abstracts
Tu1695 Saccharomyces Boulardii Prevents the Antibiotic Induced Changes in Colonic Microbiota Alexander Swidsinski, Vera Loening-Baucke, Sonja Swidsinski
Background: Intestinal epithelial necroptosis has recently been described as a potential pathogenic mechanism driving Crohn's disease. It was shown that lack of caspase-8 in intestinal epithelial cells leads to enhanced necroptosis and loss of immune homeostasis in the gut, resulting in spontaneous development of terminal ileitis. However, the mechanism triggering small intestinal epithelial necroptosis is still unknown. Aims: In this study we investigated the impact of death receptor ligands (DRL) and PAMPs (Pathogen-associated molecular pattern) on Rip3 mediated intestinal necroptosis. Methods: Caspase-8 ΔIEC, Rip3-/Caspase-8ΔIEC, Tnf-R1-/-Caspase-8ΔIEC mice and intestinal organoids were treated with PIC (PolyIC) and LPS (Lipopolysaccharide). Mice were examined by measuring body temperature, weight loss and monitoring development of diarrhoea. Tissue damage was investigated by immunohistochemistry of gut specimen, western blot and qPCR. Results: We could demonstrate that programmed necrosis of intestinal epithelial cells, induced in the absence of caspase-8, is mediated via the Rip3 since Rip3-/-Caspase-8 ΔIEC double knock out mice were completely rescued regarding the loss of Paneth cells and intestinal inflammation as well as cell death. We could further demonstrate that additional deletion of Tnf-R1 did not rescue the phenotype of Caspase-8 ΔIEC mice, suggesting that caspase-8 in vivo is either activated by a Tnf-R1 independent pathway or that redundant receptor signals are present in the steady state gut. Indeed we could show that targeting of Toll like receptors (TLR) by injection of PIC and LPS into mice induced a fast and dramatic villous atrophy and severe destruction of the small bowel of Caspase-8 ΔIEC mice as compared to control littermates, leading to the death of the former mice within 6 hours. Immunohistochemistry analysis revealed an excessive number of TUNEL positive but caspase-3 negative dying epithelial cells after TLR-stimulation in Caspase-8ΔIECmice, but not in Rip3-/-Caspase-8ΔIEC, indicating that this form of cell death is due to Rip3-mediated necroptosis. Moreover we discovered that PIC triggered necroptosis was directly mediated via TLR3, since Tnf-R1-/-Caspase8ΔIEC mice were not protected against PAMP induced cell death. At least we could show in vitro that PIC induced necroptosis was independent of immune cells since Caspase-8 ΔIEC derived organoids, but not control organoids, were sensitive to PAMP induced Rip3-mediated cell death. Conclusion: Our data demonstrate that Rip3-mediated cell death in the small intestine of Caspase-8ΔIEC mice is triggered by Pattern-Recognition Receptors. Moreover we could show that dsRNA activation of the TLR3 induce epithelial cell death in a Tnfindependent manner. This indicates that viral pathogens are the main triggering effectors for small intestinal inflammation in Caspase-8 ΔIEC mice.
Background: The impact of antibiotics on colonic microbiota is poorly characterized. Methods: Three groups of women (N=10 each) treated for bacterial vaginosis were investigated . Metronidazole 3x400mg/day + ciprofloxacin 2x500mg/day were given for 2 weeks. Group I received antibiotics only, Gr.II received Saccharomyces boulardii(Sb) concomitant to, and Gr.III received Sb subsequent to antibiotic therapy. A 250 mg capsule Sb Perenterol® Biocodex was given 3xdaily for two weeks. Microbiota were investigated using structure functional FISH analysis of Carnoy fixated stool cylinders. Stools were collected at days -90,-60,-30,7,14,28,42,56, and 70 as related to the start of antibiotic therapy. 250 bacterial FISH probes were tested and 82 were selected for longitudinal analysis. Results: Bacteroides, F. prausnitzii and Roseburia (habitual groups) were present in all patients (10-30% of the fecal mass each). The occurrence of other bacteria was occasional. The composition of microbiota prior to antibiotics was stable in repeated investigations of the same patient, despite high interindividual variability. Antibiotic therapy suppressed all bacterial groups, and some of the microbiota previously not detected, emerged in marginal concentrations (C.viridae, Streptococcus, Staphylococcus, Bif. longum ). Bifidobacteriaceae, Enterobacteriacae or C.difficile groups increased in the first two to four weeks after the end of the therapy (p ,0.01) and returned to initial levels with recovery of the habitual bacterial groups. Sb concomitant with antibiotics reduced the antibiotic associated suppression of bacteria. After one week of antibiotic therapy, concentrations of F.prausnitzii dropped in Gr.I, II, and III, respectively, from a median of 13.6±3.7 to 3.1±2.0, 8±4.1, and 3±2.1 x 109bacteria/ml. The decrease in microbial concentrations in Gr. II receiving Sb concomitant to antibiotics was moderate and differed significantly from the decrease in Gr.I and III (p ,0.001). The microbiota recovered quicker in groups receiving Sb. The habitual bacteria reached initial values 4 weeks after the end of antibiotics in all groups treated with Sb In Group I, F. prausnitzi was still reduced to 9.8±2.8 x 109bacteria/ml and was significantly lower than prior to antibiotics (p=0.04). The composition of microbiota returned quicker to the individual initial profiles in the groups receiving Sb In regard to the 82 investigated bacterial groups, the median number of mismatches in composition of microbiota prior to and after antibiotic therapy was 8.7; 5.2 (p=0.05) and 2.4 (p ,0.001) in Gr.I, II and III, respectively. The difference between pre- and postantibiotic period was significantly higher in Gr.I, receiving no Sb. Conclusions: S. boulardii prevents/reduces effectively the antibiotic-associated changes in colonic microbiota, when given concomitant/subsequent to antibiotic therapy.
Tu1698 Tu1696
Inflammation-Induced Regulatory Pathways of Oxidative Stress-Responses in Commensal Escherichia coli Impair Bacterial Motility, Biofilm Formation and Attenuate Experimental Colitis Sandrine Y. Tchaptchet, Ting-Jia Fan, Laura E. Goeser, Ryan B. Sartor, Jonathan J. Hansen
Folate Prevents CEACAM6 Abnormal Expression and Subsequent AdherentInvasive E. Coli- Induced Inflammation in Ceabac10 Mice Jérémy Denizot, Alexis Desrichard, Allison Agus, Arlette Darfeuille-Michaud, Nicolas Barnich
BACKGROUND: Dysregulated immune responses to commensal intestinal bacteria contribute to the development of human inflammatory bowel diseases (IBDs) and experimental colitis, conditions that are characterized by increased reactive oxygen intermediates in inflamed tissues. Experimental colitis is associated with increased expression of the small regulatory RNA oxyS in luminal Escherichia coli that protects bacteria from oxidative stress and inhibits expression of rpoS, a subunit of RNA polymerase that impairs growth of E. coli in mouse intestine. In addition to potentially enhancing growth of E. coli in the intestine, oxyS also reduces bacterial motility, which is linked to decreased biofilm formation. HYPOTHESIS: Expression of oxyS in commensal luminal E. coli during colitis inhibits rpoS expression, promotes bacterial survival, but decreases bacterial motility/biofilm formation and therefore host inflammatory responses. METHODS: OxyS and rpoS expression in cecal bacteria from wild-type (WT) and Il-10-/- (KO) germ-free mice monoassociated with the commensal murine E. coli isolate, NC101, was measured by real-time PCR. Biofilm formation and motility of NC101 and NC101 lacking oxyS (NC101 ΔoxyS) or rpoS (NC101ΔrpoS) were determined by crystal violet staining of biofilms on a polystyrene surface and by measuring colony diameter on soft agar, respectively. Histological inflammation in colon sections, concentrations of NC101 in luminal contents, and adaptive immune responses to NC101 antigens in ex vivo-stimulated mesenteric lymph node cells (MLN) from KO and WT mice monoassociated with NC101, NC101ΔoxyS or NC101ΔrpoS were determined using blinded scoring, quantitative culture, and ELISA for IFN- γ, respectively. RESULTS: Cecal E. coli oxyS expression was proportional to the severity of colitis in monoassociated mice, but differing from our hypothesis did not correspond to decreased rpoS expression. Biofilm formation and motility rates of NC101 were significantly reduced compared to NC101 ΔoxyS and NC101ΔrpoS (Table). Contrary to our hypothesis, luminal bacterial densities were not different in mice monoassociated with NC101, NC101 ΔoxyS or NC101ΔrpoS. However, histological inflammation scores were lower in NC101- vs. NC101 ΔoxyS- and NC101ΔrpoSmonoassociated KO mice and IFN-γ secretion by NC101 lysate-stimulated MLN cells from NC101- vs. NC101 ΔoxyS- and NC101 ΔrpoS-monoassociated KO mice was decreased (Table). CONCLUSIONS: Intestinal inflammation causes commensal E. coli to increase oxyS expression, which does not affect luminal bacterial survival, but similar to rpoS, impairs bacterial motility and biofilm formation in vitro and decreases host immune responses in vivo. Further investigation of luminal microbial adaptation to immune-mediated inflammation may provide novel insights into the pathogenesis and treatment of IBDs. Table
Background: Abnormal expression of CEACAM6 in Crohn's disease (CD) patients allows Adherent-Invasive Escherichia coli (AIEC) to colonize gut mucosa, leading to the development of inflammation. Folate are key vitamins involved in maintenance of DNA methylation patterns in mammalians and folate deficiency is often observed in CD patients, especially those with ileal involvement of the disease. In vitro, CEACAM6 expression is regulated by Hypoxia Inductible Factor-1 (HIF-1) in a DNA methylation dependent manner. We thus investigated the importance of folate in the control of CEACAM6 expression in vivo and in AIEC-induced inflammation Methods: Global methylation status of CEACAM6 promoter was analyzed using bisulfite/SnapShot sequencing in ileal or colonic enterocytes. Transgenic CEABAC10 mice expressing human CEACAM6 fed normal or methyl-donor deficient diet MDD (folate and B12 vitamin deficient) were supplemented with folic acid (FA). Colonic enterocytes were isolated and CEACAM6 and hif-1 α expression were measured by RT-qPCR and Western-blot. AIEC colonization and gut inflammation were evaluated in mice orally challenged with 10e9 bacteria. Results: Methyl-donor deficient diet (MDD) led to a significant decrease in methylation levels of CpG-containing HIF-1 Responsive Elements (HRE) in CEACAM6 promoter, leading to significant increase in CEACAM6 mRNA and protein in colonic enterocytes of CEABAC10 mice compared to a normal diet. After AIEC LF82 infection, important weight loss was observed in mice fed a MDD but not in mice fed a conventional diet. Three days post-infection, higher numbers of AIEC LF82 were associated with colonic mucosa of MDD-fed mice compared to normal diet-fed mice, due to a higher CEACAM6 protein expression. This was associated with significant higher release of pro-inflammatory cytokines IL-6, KC and IL-1β from colonic mucosa in MDD-fed mice compared to normal diet-fed mice. Folate supplementation increased methylation level of CpG-containing HRE in CEACAM6 promoter, leading to decrease CEACAM6 expression. Conclusion: Folate deficiency induces hypomethylation of HRE-containing CpG in CEACAM6 promoter, which correlates with higher CEACAM6 expression. These findings indicate that abnormal CEACAM6 expression in ileal mucosa of CD patients could be related to folate deficiency. Folate supplementation could be effective to achieve and maintain inflammation remission in patients with ileal involvement of the disease by preventing CEACAM6 abnormal expression and subsequent AIEC-mediated inflammation.
AGA Abstracts
S-824
Tu1697 Viral Pathogens Pull the Necrotic Trigger in a TNF-Receptor Independent Manner in Caspase-8ΔIEC Mice Claudia Günther, Helmut Neumann, Guiwei He, Eva Martini, Markus F. Neurath, Christoph Becker
AGA Abstracts
Tu1695 Saccharomyces Boulardii Prevents the Antibiotic Induced Changes in Colonic Microbiota Alexander Swidsinski, Vera Loening-Baucke, Sonja Swidsinski
Background: Intestinal epithelial necroptosis has recently been described as a potential pathogenic mechanism driving Crohn's disease. It was shown that lack of caspase-8 in intestinal epithelial cells leads to enhanced necroptosis and loss of immune homeostasis in the gut, resulting in spontaneous development of terminal ileitis. However, the mechanism triggering small intestinal epithelial necroptosis is still unknown. Aims: In this study we investigated the impact of death receptor ligands (DRL) and PAMPs (Pathogen-associated molecular pattern) on Rip3 mediated intestinal necroptosis. Methods: Caspase-8 ΔIEC, Rip3-/Caspase-8ΔIEC, Tnf-R1-/-Caspase-8ΔIEC mice and intestinal organoids were treated with PIC (PolyIC) and LPS (Lipopolysaccharide). Mice were examined by measuring body temperature, weight loss and monitoring development of diarrhoea. Tissue damage was investigated by immunohistochemistry of gut specimen, western blot and qPCR. Results: We could demonstrate that programmed necrosis of intestinal epithelial cells, induced in the absence of caspase-8, is mediated via the Rip3 since Rip3-/-Caspase-8 ΔIEC double knock out mice were completely rescued regarding the loss of Paneth cells and intestinal inflammation as well as cell death. We could further demonstrate that additional deletion of Tnf-R1 did not rescue the phenotype of Caspase-8 ΔIEC mice, suggesting that caspase-8 in vivo is either activated by a Tnf-R1 independent pathway or that redundant receptor signals are present in the steady state gut. Indeed we could show that targeting of Toll like receptors (TLR) by injection of PIC and LPS into mice induced a fast and dramatic villous atrophy and severe destruction of the small bowel of Caspase-8 ΔIEC mice as compared to control littermates, leading to the death of the former mice within 6 hours. Immunohistochemistry analysis revealed an excessive number of TUNEL positive but caspase-3 negative dying epithelial cells after TLR-stimulation in Caspase-8ΔIECmice, but not in Rip3-/-Caspase-8ΔIEC, indicating that this form of cell death is due to Rip3-mediated necroptosis. Moreover we discovered that PIC triggered necroptosis was directly mediated via TLR3, since Tnf-R1-/-Caspase8ΔIEC mice were not protected against PAMP induced cell death. At least we could show in vitro that PIC induced necroptosis was independent of immune cells since Caspase-8 ΔIEC derived organoids, but not control organoids, were sensitive to PAMP induced Rip3-mediated cell death. Conclusion: Our data demonstrate that Rip3-mediated cell death in the small intestine of Caspase-8ΔIEC mice is triggered by Pattern-Recognition Receptors. Moreover we could show that dsRNA activation of the TLR3 induce epithelial cell death in a Tnfindependent manner. This indicates that viral pathogens are the main triggering effectors for small intestinal inflammation in Caspase-8 ΔIEC mice.
Background: The impact of antibiotics on colonic microbiota is poorly characterized. Methods: Three groups of women (N=10 each) treated for bacterial vaginosis were investigated . Metronidazole 3x400mg/day + ciprofloxacin 2x500mg/day were given for 2 weeks. Group I received antibiotics only, Gr.II received Saccharomyces boulardii(Sb) concomitant to, and Gr.III received Sb subsequent to antibiotic therapy. A 250 mg capsule Sb Perenterol® Biocodex was given 3xdaily for two weeks. Microbiota were investigated using structure functional FISH analysis of Carnoy fixated stool cylinders. Stools were collected at days -90,-60,-30,7,14,28,42,56, and 70 as related to the start of antibiotic therapy. 250 bacterial FISH probes were tested and 82 were selected for longitudinal analysis. Results: Bacteroides, F. prausnitzii and Roseburia (habitual groups) were present in all patients (10-30% of the fecal mass each). The occurrence of other bacteria was occasional. The composition of microbiota prior to antibiotics was stable in repeated investigations of the same patient, despite high interindividual variability. Antibiotic therapy suppressed all bacterial groups, and some of the microbiota previously not detected, emerged in marginal concentrations (C.viridae, Streptococcus, Staphylococcus, Bif. longum ). Bifidobacteriaceae, Enterobacteriacae or C.difficile groups increased in the first two to four weeks after the end of the therapy (p ,0.01) and returned to initial levels with recovery of the habitual bacterial groups. Sb concomitant with antibiotics reduced the antibiotic associated suppression of bacteria. After one week of antibiotic therapy, concentrations of F.prausnitzii dropped in Gr.I, II, and III, respectively, from a median of 13.6±3.7 to 3.1±2.0, 8±4.1, and 3±2.1 x 109bacteria/ml. The decrease in microbial concentrations in Gr. II receiving Sb concomitant to antibiotics was moderate and differed significantly from the decrease in Gr.I and III (p ,0.001). The microbiota recovered quicker in groups receiving Sb. The habitual bacteria reached initial values 4 weeks after the end of antibiotics in all groups treated with Sb In Group I, F. prausnitzi was still reduced to 9.8±2.8 x 109bacteria/ml and was significantly lower than prior to antibiotics (p=0.04). The composition of microbiota returned quicker to the individual initial profiles in the groups receiving Sb In regard to the 82 investigated bacterial groups, the median number of mismatches in composition of microbiota prior to and after antibiotic therapy was 8.7; 5.2 (p=0.05) and 2.4 (p ,0.001) in Gr.I, II and III, respectively. The difference between pre- and postantibiotic period was significantly higher in Gr.I, receiving no Sb. Conclusions: S. boulardii prevents/reduces effectively the antibiotic-associated changes in colonic microbiota, when given concomitant/subsequent to antibiotic therapy.
Tu1698 Tu1696
Inflammation-Induced Regulatory Pathways of Oxidative Stress-Responses in Commensal Escherichia coli Impair Bacterial Motility, Biofilm Formation and Attenuate Experimental Colitis Sandrine Y. Tchaptchet, Ting-Jia Fan, Laura E. Goeser, Ryan B. Sartor, Jonathan J. Hansen
Folate Prevents CEACAM6 Abnormal Expression and Subsequent AdherentInvasive E. Coli- Induced Inflammation in Ceabac10 Mice Jérémy Denizot, Alexis Desrichard, Allison Agus, Arlette Darfeuille-Michaud, Nicolas Barnich
BACKGROUND: Dysregulated immune responses to commensal intestinal bacteria contribute to the development of human inflammatory bowel diseases (IBDs) and experimental colitis, conditions that are characterized by increased reactive oxygen intermediates in inflamed tissues. Experimental colitis is associated with increased expression of the small regulatory RNA oxyS in luminal Escherichia coli that protects bacteria from oxidative stress and inhibits expression of rpoS, a subunit of RNA polymerase that impairs growth of E. coli in mouse intestine. In addition to potentially enhancing growth of E. coli in the intestine, oxyS also reduces bacterial motility, which is linked to decreased biofilm formation. HYPOTHESIS: Expression of oxyS in commensal luminal E. coli during colitis inhibits rpoS expression, promotes bacterial survival, but decreases bacterial motility/biofilm formation and therefore host inflammatory responses. METHODS: OxyS and rpoS expression in cecal bacteria from wild-type (WT) and Il-10-/- (KO) germ-free mice monoassociated with the commensal murine E. coli isolate, NC101, was measured by real-time PCR. Biofilm formation and motility of NC101 and NC101 lacking oxyS (NC101 ΔoxyS) or rpoS (NC101ΔrpoS) were determined by crystal violet staining of biofilms on a polystyrene surface and by measuring colony diameter on soft agar, respectively. Histological inflammation in colon sections, concentrations of NC101 in luminal contents, and adaptive immune responses to NC101 antigens in ex vivo-stimulated mesenteric lymph node cells (MLN) from KO and WT mice monoassociated with NC101, NC101ΔoxyS or NC101ΔrpoS were determined using blinded scoring, quantitative culture, and ELISA for IFN- γ, respectively. RESULTS: Cecal E. coli oxyS expression was proportional to the severity of colitis in monoassociated mice, but differing from our hypothesis did not correspond to decreased rpoS expression. Biofilm formation and motility rates of NC101 were significantly reduced compared to NC101 ΔoxyS and NC101ΔrpoS (Table). Contrary to our hypothesis, luminal bacterial densities were not different in mice monoassociated with NC101, NC101 ΔoxyS or NC101ΔrpoS. However, histological inflammation scores were lower in NC101- vs. NC101 ΔoxyS- and NC101ΔrpoSmonoassociated KO mice and IFN-γ secretion by NC101 lysate-stimulated MLN cells from NC101- vs. NC101 ΔoxyS- and NC101 ΔrpoS-monoassociated KO mice was decreased (Table). CONCLUSIONS: Intestinal inflammation causes commensal E. coli to increase oxyS expression, which does not affect luminal bacterial survival, but similar to rpoS, impairs bacterial motility and biofilm formation in vitro and decreases host immune responses in vivo. Further investigation of luminal microbial adaptation to immune-mediated inflammation may provide novel insights into the pathogenesis and treatment of IBDs. Table
Background: Abnormal expression of CEACAM6 in Crohn's disease (CD) patients allows Adherent-Invasive Escherichia coli (AIEC) to colonize gut mucosa, leading to the development of inflammation. Folate are key vitamins involved in maintenance of DNA methylation patterns in mammalians and folate deficiency is often observed in CD patients, especially those with ileal involvement of the disease. In vitro, CEACAM6 expression is regulated by Hypoxia Inductible Factor-1 (HIF-1) in a DNA methylation dependent manner. We thus investigated the importance of folate in the control of CEACAM6 expression in vivo and in AIEC-induced inflammation Methods: Global methylation status of CEACAM6 promoter was analyzed using bisulfite/SnapShot sequencing in ileal or colonic enterocytes. Transgenic CEABAC10 mice expressing human CEACAM6 fed normal or methyl-donor deficient diet MDD (folate and B12 vitamin deficient) were supplemented with folic acid (FA). Colonic enterocytes were isolated and CEACAM6 and hif-1 α expression were measured by RT-qPCR and Western-blot. AIEC colonization and gut inflammation were evaluated in mice orally challenged with 10e9 bacteria. Results: Methyl-donor deficient diet (MDD) led to a significant decrease in methylation levels of CpG-containing HIF-1 Responsive Elements (HRE) in CEACAM6 promoter, leading to significant increase in CEACAM6 mRNA and protein in colonic enterocytes of CEABAC10 mice compared to a normal diet. After AIEC LF82 infection, important weight loss was observed in mice fed a MDD but not in mice fed a conventional diet. Three days post-infection, higher numbers of AIEC LF82 were associated with colonic mucosa of MDD-fed mice compared to normal diet-fed mice, due to a higher CEACAM6 protein expression. This was associated with significant higher release of pro-inflammatory cytokines IL-6, KC and IL-1β from colonic mucosa in MDD-fed mice compared to normal diet-fed mice. Folate supplementation increased methylation level of CpG-containing HRE in CEACAM6 promoter, leading to decrease CEACAM6 expression. Conclusion: Folate deficiency induces hypomethylation of HRE-containing CpG in CEACAM6 promoter, which correlates with higher CEACAM6 expression. These findings indicate that abnormal CEACAM6 expression in ileal mucosa of CD patients could be related to folate deficiency. Folate supplementation could be effective to achieve and maintain inflammation remission in patients with ileal involvement of the disease by preventing CEACAM6 abnormal expression and subsequent AIEC-mediated inflammation.
AGA Abstracts
S-824