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Mo1783 Faecalibacterium prausnitzii Inhibiting the TNBS-Induced Colitis by Regulating NLRP3 Inflammasome
AGA Abstracts
unknown. Hence, we investigated here whether the gut microbiota can affect colonic miRNAs secretion and profile. Results: High throughput technology screening the expression levels of 752 known mouse miRNAs revealed that miRNA profiles were different in the feces of GF mice compared to conventionalized mice, with 13 miRNAs deregulated in the absence of a microbiota. In order to investigate if the microbiota composition play a role in fecal miRNA profiles, germ-free C57/Bl6 mice were "conventionalized" with microbiota of WT mice, non colitic IL10-/- mice or colitic IL10-/- mice, and fecal miRNAs profiles were analyzed. Microbiota composition analysis confirmed that we successfully transferred the difference in composition, with clear clustering according to the microbiota transferred. Mice receiving microbiota from colitic IL10-/- mice harbor a proinflammatory microbiota 49 days post conventionalization, as assayed by fecal LPS and flagellin loads, and proinflammatory potential of mice receiving microbiota from non-colitic IL10-/- mice were found to be moderately elevated compare to mice receiving microbiota from WT mice. Intestinal inflammatory status of mice from the three groups was unchanged, as assessed by MPO. However, mice colonized with colitic IL10-/- microbiota displayed a slight but significant increase in blood glucose level, as well as other parameters of low-grade intestinal inflammation, such as fecal Lcn-2. miRNA profile was analyzed and Principal component analysis showed a clear clustering between the 3 groups at 49 days post conventionalization, that was not observed at day 0 (before conventionalization). Interestingly, the level of some miRNAs correlate with the level of some members of the gut microbiota found to drive the difference in the microbiota composition previously observed. Conclusion: Altogether, these data showed that the microbiota composition induces drastic changes in the dynamic of fecal miRNA. Fecal miRNA signature could be used as tools for assessing the gut microbiota composition and its inflammatory potential.
and the stool of the transplanted mice consisted in 30% C4 genotype and had a high level of Bacteroidetes cluster in comparison with the mice transplanted with bacteria from HC. Conclusions: The fecal microbiota of CD patients is different from those of HC in that they present a wide variety of Bacteroidetes cluster genotypes. The C4 genotype by itself, or together with the C1 genotype, seems to be intimately related to the activity of the disease. These results were also confirmed in transplanted mice. Mo1783 Faecalibacterium prausnitzii Inhibiting the TNBS-Induced Colitis by Regulating NLRP3 Inflammasome Airong Tang, Ping Cao, Tao Zhang, Chenggong Yu Background and aims:NLRP3 inflammasome plays an important role in the development of experimental colitis. Faecalibacterium prausnitzii (F. prausnitzii) and its cultured supernatant have anti-inflammatory effect. The study was to explore the effects and mechanism of F. prausnitzii and its supernatant on colitis rats. Method: Fifty Sprague-Dawley rats were divided into normal control group (N=10) and experimental group (N=40). The experimental groups were treated with 5% TNBS (100mg/kg) to induce UC model. After 24 hours, experimental group were randomly divided into four groups (N=10 in each group), which treated with PBS, culture medium, F. prausnitzii and F. prausnitzii supernatant by gavage once a day for seven days, respectively; and control group treated with PBS in the same way. Levels of weight, fecal score, length and pathological score of colon were evaluated. Protein levels of NLRP3, ASC, caspase-1 in colonic mucosa were detected by Western blot. mRNA levels of NLRP3,ASC,caspase-1,IL-1β and IL-18 in colonic tissue was detected by real time PCR. The plasma levels of IL-1β and IL-18 were detected by ELISA.Results: The weight of experimental group decreased significantly compared to control group (P<0.05). The value of fecal and histological score in experimental groups was higher than that of control group, but was lower in F. prausnitzii and supernatant group than that of PBS group and medium group (P<0.05). Protein levels of NLRP3, ASC, caspase-1, mRNA levels of NLRP3, ASC, caspase1, IL-1β in colon tissue and level of IL-1β in plasma were significantly higher in experimental groups than that of control group (P<0.05). The levels of above parameter in F. prausnitzii group and supernatant group had significantly lower than that of PBS group and medium group (P<0.05), but the level of IL-18 in tissue and plasma were significantly lower in the experimental groups than that of control group (P<0.05). Conclusions: F. prausnitzii and its cultured supernatant could decreased the degree of TNBS colitis by inhibiting the expression of NLRP3, and the effect of cultured supernatant of F. prausnitzii is more obvious than that of F. prausnitzii.
Mo1781 Crohn's Disease-Associated Adherent-Invasive Escherichia coli Induce Secretion of Exosomes With Pro-Inflammatory Activity by Intestinal Epithelial Cells Jessica Carriere, Alexis Bretin, Nicolas Barnich, Hang T. Nguyen Background & Aims: Crohn's disease (CD) is a chronic inflammatory bowel disease of which the etiology involves environmental, genetic and microbial factors. Our group and others have shown a high prevalence of the invasive Escherichia coli strains, designated adherent-invasive E. coli (AIEC), in the intestinal mucosa of CD patients. Exosomes are small endosomal-derived vesicles involved in cell to cell communication and have been implicated in various diseases including cancer and infectious disorders. It has been reported that mammalian cells infected with pathogens can release exosomes containing microbial compounds. Here, we investigated the capacity of CD-associated AIEC bacteria to induce secretion of exosomes by intestinal epithelial cells and to determine the inflammatory characteristics of the released exosomes. Methods: Human intestinal epithelial T84 cells were infected with the AIEC reference strain LF82. Exosomes were purified using the ExoQuick exosome precipitation reagent. Exosomes released by LF82-infected T84 cells were tested for their ability to promote pro-inflammatory responses in naïve macrophagic cells. Identification of exosomal proteins was performed by mass spectrometry. Results: Electron microscopy and immunogold-labeling analyses for CD63, an exosomal marker, showed that differentiated T84 cells infected with AIEC LF82 secreted an increased amount of exosomes compared to uninfected cells. This was confirmed by increased levels of CD63 as assessed by Western blot. Stimulation of human macrophages with exosomes secreted by LF82-infected T84 cells, but not by uninfected cells, significantly induced production of the pro-inflammatory cytokines TNF-alpha and IL-6, and this was not due to the presence of lipopolysaccharide, known to induce a pro-inflammatory response. Mass spectrometry analysis revealed that exosomes released by T84 cells upon LF82 infection carried microbial antigens such as the outer membrane protein C, known to be involved in AIEC adhesion and invasion. Conclusion: Our study shows that in response to CD-associated AIEC infection, intestinal epithelial cells release exosomes that can trigger pro-inflammatory responses in naïve macrophagic recipient cells.
Mo1784 Inflammatory Macrophages Response to Stimulation by Beta-1,3-Glucan and May Contributes to the Pathogenesis of Inflammatory Bowel Disease Kiyoto Mori, Tadakazu Hisamatsu, Hiroaki Suzuki, Mina Kitazume, Katsuyoshi Shimamura, Nobuhiro Nakamoto, Hirotoshi Ebinuma, Katsuyoshi Matsuoka, Makoto Naganuma, Takanori Kanai Background and Aim : Fungi represent a diverse microbial community in the normal human intestine. Recent research has highlighted the importance of fungi residing in the gut and the interaction between commensal fungi and intestinal inflammation. In feces, any species of fungi were significantly more abundant in Crohn's disease (CD) patients in compared with the healthy controls. We previously reported that, macrophages derived from CD patients produced a large amount of inflammatory cytokine in response to intestinal commensal bacteria. Here, we investigated the response of macrophages to Curdlan (beta-1,3-glucan), which is one of the cell wall components of commensal fungus . The aim of this study is to reveal the interaction between commensal fungi and host immune cells in inflammatory bowel disease (IBD). Method: CD14+ monocytes were isolated from peripheral blood cells of healthy human and were differentiated in the presence of M-CSF (named as M-macrophages, M-Mϕ) or M-CSF and IFN-γ (named as M-gamma macrophages, Mγ-Mϕ) . Cytokine production of macrophages derived from peripheral blood cells of healthy human in response to beta-(1,3)-glucan was analysed using a flow cytometry. The expression of Dectin-1, which was the receptor of beta-(1,3)-glucan, was examined on flow cytometry. The expression of Dectin-1 was also investigated using western blotting. The gene expression of Dectin-1 was examined using quantitative RT-PCR. Cytokine production of PBMC derived macrophages in response to beta-(1,3)-glucan was measured in the presence of anti-Dectin-1 receptor antagonist or isotype controls using a flow cytometry. Cytokine production of lamina propria mononuclear cells (LPMC) derived from CD and ulcerative colitis (UC) patients in response to beta-(1,3)-glucan was analyzed using a flow cytometry. Results: Mγ-Mϕ produced a large amount of inflammatory cytokine in response to Curdlan(beta-(1,3)-glucan). Dectin-1 was expressed in both M-Mϕ and Mγ-Mϕ, but the expression was significantly higher in MγMϕ than in M-Mϕ. Dectin-1 mRNA expression was higher in Mγ-Mϕ than in M-Mϕ. Recognition of beta-(1,3)-glucan was mediated by Dectin-1 receptor in macrophages. LPMC derived from CD patients stimulated with Curdlan produced a large amount of TNF-α compared with those derived from UC patients. Conclusion: Beta-(1,3)-glucan induced inflammatory cytokines in Mγ-Mϕ, suggesting the potential involvement of commensal fungal microbiota in the pathogenesis of IBD.
Mo1782 Presence of Genotypes of Bacteroidetes Associated With Intestinal Microbiota in Patients With Crohn's Disease and Evaluation of Its Role in the Induction of Intestinal Inflammation in Mice Manuel Barreiro-de Acosta, Rosana Sueiro, Ana Paula De Felipe, Laura Uribarri-González, Rocio Ferreiro, José Manuel Leiro, Enrique Dominguez-Munoz Background: The pathogenesis of inflammatory bowel disease (IBD) involves an imbalance of the gut microbiota generating an inappropiate activation of the mucosal immune system in genetically predisposed individuals. The human commensal microbiota contains a large number of Bacteroidetes species that may cause inflammation in animal models. The aim of this study was to detect and evaluate the influence of different Bacteroidetes genotypes on the activity of Crohn disease (CD) patients. In addition, the effect of these bacteria isolated from CD patients on gut inflammation was evaluated in mice. Methods: We performed a case control study on the intestinal bacteria of the phylum Bacteroidetes from faeces of CD and healthy controls (HC) using a polymerase chain reaction (PCR) designed to detect human-specific genetic markers targeting Bacteroidetes-like 16S rRNA genes in fecal DNA samples. The PCR products from the 16S rRNA genes were digested with HinfI, PciI, DpnII and AciI enzymes and restriction fragment length polymorphism (RFLP) were determined. RFLP and sequencing analysis indicated that a total of 6 bacterial genotypes do exist: N1, C1, C2, C3, C4 and C5 (of which N1 genotype is probably a strain of Bacteroides dorei and C1, and maybe C2, strains of B. vulgatus). The relationship between CD activity (CDAI>150) and microbiota was evaluated. Microbiota from CD patients were transplanted into mice gut to evaluate their ability to induce inflammation. Results are shown in percentages. Results: 11 CD patients (8 with active CD -aCD- (CDAI>150), and 3 with inactive CD -iCD-), and 11 HC were included. The predominant Bacteroidetes genotype in feces from HC and iCD was N1 (present in 100% of samples), whereas this genotype was found in only 28% of patients with aCD. 18% aCD patients showed the C1 genotype, 9% the C1 and C3 genotypes together, 18% the C4 genotype, and 27% the C1 and C4 genotypes together. The transplant of bacteria from CD patients to mice led to large bowel inflammation,
AGA Abstracts
Mo1785 Intestinal Epithelial Cells Contribute to Citrobacter Rodentium Induced Infectious Colitis Through an NF-κB-Dependent Mechanism Amy Mackos, Prosper N. Boyaka, Michael T. Bailey Gastrointestinal disorders, including the inflammatory bowel diseases and enteric infectious diseases, are exacerbated by inflammation contributed by inflammatory monocytes. These monocytes are recruited to the site of infection to help clear pathogens. However an increased accumulation of inflammatory monocytes can lead to excessive inflammation, tissue damage, and loss of function. As a result, the recruitment of inflammatory monocytes to the colon
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unknown. Hence, we investigated here whether the gut microbiota can affect colonic miRNAs secretion and profile. Results: High throughput technology screening the expression levels of 752 known mouse miRNAs revealed that miRNA profiles were different in the feces of GF mice compared to conventionalized mice, with 13 miRNAs deregulated in the absence of a microbiota. In order to investigate if the microbiota composition play a role in fecal miRNA profiles, germ-free C57/Bl6 mice were "conventionalized" with microbiota of WT mice, non colitic IL10-/- mice or colitic IL10-/- mice, and fecal miRNAs profiles were analyzed. Microbiota composition analysis confirmed that we successfully transferred the difference in composition, with clear clustering according to the microbiota transferred. Mice receiving microbiota from colitic IL10-/- mice harbor a proinflammatory microbiota 49 days post conventionalization, as assayed by fecal LPS and flagellin loads, and proinflammatory potential of mice receiving microbiota from non-colitic IL10-/- mice were found to be moderately elevated compare to mice receiving microbiota from WT mice. Intestinal inflammatory status of mice from the three groups was unchanged, as assessed by MPO. However, mice colonized with colitic IL10-/- microbiota displayed a slight but significant increase in blood glucose level, as well as other parameters of low-grade intestinal inflammation, such as fecal Lcn-2. miRNA profile was analyzed and Principal component analysis showed a clear clustering between the 3 groups at 49 days post conventionalization, that was not observed at day 0 (before conventionalization). Interestingly, the level of some miRNAs correlate with the level of some members of the gut microbiota found to drive the difference in the microbiota composition previously observed. Conclusion: Altogether, these data showed that the microbiota composition induces drastic changes in the dynamic of fecal miRNA. Fecal miRNA signature could be used as tools for assessing the gut microbiota composition and its inflammatory potential.
and the stool of the transplanted mice consisted in 30% C4 genotype and had a high level of Bacteroidetes cluster in comparison with the mice transplanted with bacteria from HC. Conclusions: The fecal microbiota of CD patients is different from those of HC in that they present a wide variety of Bacteroidetes cluster genotypes. The C4 genotype by itself, or together with the C1 genotype, seems to be intimately related to the activity of the disease. These results were also confirmed in transplanted mice. Mo1783 Faecalibacterium prausnitzii Inhibiting the TNBS-Induced Colitis by Regulating NLRP3 Inflammasome Airong Tang, Ping Cao, Tao Zhang, Chenggong Yu Background and aims:NLRP3 inflammasome plays an important role in the development of experimental colitis. Faecalibacterium prausnitzii (F. prausnitzii) and its cultured supernatant have anti-inflammatory effect. The study was to explore the effects and mechanism of F. prausnitzii and its supernatant on colitis rats. Method: Fifty Sprague-Dawley rats were divided into normal control group (N=10) and experimental group (N=40). The experimental groups were treated with 5% TNBS (100mg/kg) to induce UC model. After 24 hours, experimental group were randomly divided into four groups (N=10 in each group), which treated with PBS, culture medium, F. prausnitzii and F. prausnitzii supernatant by gavage once a day for seven days, respectively; and control group treated with PBS in the same way. Levels of weight, fecal score, length and pathological score of colon were evaluated. Protein levels of NLRP3, ASC, caspase-1 in colonic mucosa were detected by Western blot. mRNA levels of NLRP3,ASC,caspase-1,IL-1β and IL-18 in colonic tissue was detected by real time PCR. The plasma levels of IL-1β and IL-18 were detected by ELISA.Results: The weight of experimental group decreased significantly compared to control group (P<0.05). The value of fecal and histological score in experimental groups was higher than that of control group, but was lower in F. prausnitzii and supernatant group than that of PBS group and medium group (P<0.05). Protein levels of NLRP3, ASC, caspase-1, mRNA levels of NLRP3, ASC, caspase1, IL-1β in colon tissue and level of IL-1β in plasma were significantly higher in experimental groups than that of control group (P<0.05). The levels of above parameter in F. prausnitzii group and supernatant group had significantly lower than that of PBS group and medium group (P<0.05), but the level of IL-18 in tissue and plasma were significantly lower in the experimental groups than that of control group (P<0.05). Conclusions: F. prausnitzii and its cultured supernatant could decreased the degree of TNBS colitis by inhibiting the expression of NLRP3, and the effect of cultured supernatant of F. prausnitzii is more obvious than that of F. prausnitzii.
Mo1781 Crohn's Disease-Associated Adherent-Invasive Escherichia coli Induce Secretion of Exosomes With Pro-Inflammatory Activity by Intestinal Epithelial Cells Jessica Carriere, Alexis Bretin, Nicolas Barnich, Hang T. Nguyen Background & Aims: Crohn's disease (CD) is a chronic inflammatory bowel disease of which the etiology involves environmental, genetic and microbial factors. Our group and others have shown a high prevalence of the invasive Escherichia coli strains, designated adherent-invasive E. coli (AIEC), in the intestinal mucosa of CD patients. Exosomes are small endosomal-derived vesicles involved in cell to cell communication and have been implicated in various diseases including cancer and infectious disorders. It has been reported that mammalian cells infected with pathogens can release exosomes containing microbial compounds. Here, we investigated the capacity of CD-associated AIEC bacteria to induce secretion of exosomes by intestinal epithelial cells and to determine the inflammatory characteristics of the released exosomes. Methods: Human intestinal epithelial T84 cells were infected with the AIEC reference strain LF82. Exosomes were purified using the ExoQuick exosome precipitation reagent. Exosomes released by LF82-infected T84 cells were tested for their ability to promote pro-inflammatory responses in naïve macrophagic cells. Identification of exosomal proteins was performed by mass spectrometry. Results: Electron microscopy and immunogold-labeling analyses for CD63, an exosomal marker, showed that differentiated T84 cells infected with AIEC LF82 secreted an increased amount of exosomes compared to uninfected cells. This was confirmed by increased levels of CD63 as assessed by Western blot. Stimulation of human macrophages with exosomes secreted by LF82-infected T84 cells, but not by uninfected cells, significantly induced production of the pro-inflammatory cytokines TNF-alpha and IL-6, and this was not due to the presence of lipopolysaccharide, known to induce a pro-inflammatory response. Mass spectrometry analysis revealed that exosomes released by T84 cells upon LF82 infection carried microbial antigens such as the outer membrane protein C, known to be involved in AIEC adhesion and invasion. Conclusion: Our study shows that in response to CD-associated AIEC infection, intestinal epithelial cells release exosomes that can trigger pro-inflammatory responses in naïve macrophagic recipient cells.
Mo1784 Inflammatory Macrophages Response to Stimulation by Beta-1,3-Glucan and May Contributes to the Pathogenesis of Inflammatory Bowel Disease Kiyoto Mori, Tadakazu Hisamatsu, Hiroaki Suzuki, Mina Kitazume, Katsuyoshi Shimamura, Nobuhiro Nakamoto, Hirotoshi Ebinuma, Katsuyoshi Matsuoka, Makoto Naganuma, Takanori Kanai Background and Aim : Fungi represent a diverse microbial community in the normal human intestine. Recent research has highlighted the importance of fungi residing in the gut and the interaction between commensal fungi and intestinal inflammation. In feces, any species of fungi were significantly more abundant in Crohn's disease (CD) patients in compared with the healthy controls. We previously reported that, macrophages derived from CD patients produced a large amount of inflammatory cytokine in response to intestinal commensal bacteria. Here, we investigated the response of macrophages to Curdlan (beta-1,3-glucan), which is one of the cell wall components of commensal fungus . The aim of this study is to reveal the interaction between commensal fungi and host immune cells in inflammatory bowel disease (IBD). Method: CD14+ monocytes were isolated from peripheral blood cells of healthy human and were differentiated in the presence of M-CSF (named as M-macrophages, M-Mϕ) or M-CSF and IFN-γ (named as M-gamma macrophages, Mγ-Mϕ) . Cytokine production of macrophages derived from peripheral blood cells of healthy human in response to beta-(1,3)-glucan was analysed using a flow cytometry. The expression of Dectin-1, which was the receptor of beta-(1,3)-glucan, was examined on flow cytometry. The expression of Dectin-1 was also investigated using western blotting. The gene expression of Dectin-1 was examined using quantitative RT-PCR. Cytokine production of PBMC derived macrophages in response to beta-(1,3)-glucan was measured in the presence of anti-Dectin-1 receptor antagonist or isotype controls using a flow cytometry. Cytokine production of lamina propria mononuclear cells (LPMC) derived from CD and ulcerative colitis (UC) patients in response to beta-(1,3)-glucan was analyzed using a flow cytometry. Results: Mγ-Mϕ produced a large amount of inflammatory cytokine in response to Curdlan(beta-(1,3)-glucan). Dectin-1 was expressed in both M-Mϕ and Mγ-Mϕ, but the expression was significantly higher in MγMϕ than in M-Mϕ. Dectin-1 mRNA expression was higher in Mγ-Mϕ than in M-Mϕ. Recognition of beta-(1,3)-glucan was mediated by Dectin-1 receptor in macrophages. LPMC derived from CD patients stimulated with Curdlan produced a large amount of TNF-α compared with those derived from UC patients. Conclusion: Beta-(1,3)-glucan induced inflammatory cytokines in Mγ-Mϕ, suggesting the potential involvement of commensal fungal microbiota in the pathogenesis of IBD.
Mo1782 Presence of Genotypes of Bacteroidetes Associated With Intestinal Microbiota in Patients With Crohn's Disease and Evaluation of Its Role in the Induction of Intestinal Inflammation in Mice Manuel Barreiro-de Acosta, Rosana Sueiro, Ana Paula De Felipe, Laura Uribarri-González, Rocio Ferreiro, José Manuel Leiro, Enrique Dominguez-Munoz Background: The pathogenesis of inflammatory bowel disease (IBD) involves an imbalance of the gut microbiota generating an inappropiate activation of the mucosal immune system in genetically predisposed individuals. The human commensal microbiota contains a large number of Bacteroidetes species that may cause inflammation in animal models. The aim of this study was to detect and evaluate the influence of different Bacteroidetes genotypes on the activity of Crohn disease (CD) patients. In addition, the effect of these bacteria isolated from CD patients on gut inflammation was evaluated in mice. Methods: We performed a case control study on the intestinal bacteria of the phylum Bacteroidetes from faeces of CD and healthy controls (HC) using a polymerase chain reaction (PCR) designed to detect human-specific genetic markers targeting Bacteroidetes-like 16S rRNA genes in fecal DNA samples. The PCR products from the 16S rRNA genes were digested with HinfI, PciI, DpnII and AciI enzymes and restriction fragment length polymorphism (RFLP) were determined. RFLP and sequencing analysis indicated that a total of 6 bacterial genotypes do exist: N1, C1, C2, C3, C4 and C5 (of which N1 genotype is probably a strain of Bacteroides dorei and C1, and maybe C2, strains of B. vulgatus). The relationship between CD activity (CDAI>150) and microbiota was evaluated. Microbiota from CD patients were transplanted into mice gut to evaluate their ability to induce inflammation. Results are shown in percentages. Results: 11 CD patients (8 with active CD -aCD- (CDAI>150), and 3 with inactive CD -iCD-), and 11 HC were included. The predominant Bacteroidetes genotype in feces from HC and iCD was N1 (present in 100% of samples), whereas this genotype was found in only 28% of patients with aCD. 18% aCD patients showed the C1 genotype, 9% the C1 and C3 genotypes together, 18% the C4 genotype, and 27% the C1 and C4 genotypes together. The transplant of bacteria from CD patients to mice led to large bowel inflammation,
AGA Abstracts
Mo1785 Intestinal Epithelial Cells Contribute to Citrobacter Rodentium Induced Infectious Colitis Through an NF-κB-Dependent Mechanism Amy Mackos, Prosper N. Boyaka, Michael T. Bailey Gastrointestinal disorders, including the inflammatory bowel diseases and enteric infectious diseases, are exacerbated by inflammation contributed by inflammatory monocytes. These monocytes are recruited to the site of infection to help clear pathogens. However an increased accumulation of inflammatory monocytes can lead to excessive inflammation, tissue damage, and loss of function. As a result, the recruitment of inflammatory monocytes to the colon
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