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665 The Role of the Intestinal Microbiota in Colonic Barrier Dysfunction Induced By Neonatal Stress
AGA Abstracts
n=12). In contrast, no differences in splenic cytokine secretion was observed in the EPStreated animals. Conclusions: A polysaccharide enriched fraction from Bifidobacterium infantis 35624 enhances the expression of Foxp3+ T regulatory cells In Vivo and is not pro-inflammatory In Vitro. The results provide further support for the distinct nature and beneficial aspects of this strain of Bifidobacterium and underscore the importance of strain-specificity in the context of probiotic function. 664 Sub-Therapeutic Antibiotic Treatment Alters the Murine Gut Microbiome and Affects Early-Life Metabolism and Body Composition Ilseung Cho, Barbara Methe, Sean Raj, Yael R. Nobel, Martin J. Blaser Background: The gut microbiome has an essential role in host metabolic homeostasis. Subtherapeutic antibiotics have been used for decades for growth promotion in the agricultural industry, but the relevant mechanisms are not known. We hypothesized that manipulating the gut microbiome in sub-therapeutic antibiotic treated (STAT) mice will alter their metabolic homeostasis. Methods: 21 day-old C57B6 (Jackson Laboratories) with ad libitum access to water and standard chow were given no antibiotics or sub-therapeutic doses of penicillin, vancomycin, penicillin+vancomycin, or chlortetracycline at a dose of 1μg antibiotic/g body weight in their drinking water (n=10/group). All mice were sacrificed at 9 weeks, scanned by densitometry and serum collected for gut hormone analysis by Luminex assays. DNA extracted from cecal and fecal specimens was subjected to PCR using barcoded universal primers interrogating the V3 region of the 16S rRNA gene followed by 454 sequencing and taxonomic analysis. Results: All 4 groups of STAT mice showed significantly increased fat mass and % body fat when compared to controls (See table). STAT mice had 14.1±2.5% more fat mass than controls [range: 8.1±3.4% (chlortetracycline) to 22.9±8.1% (penicillin)]. Lean mass was not significantly different between groups. Leptin, insulin, and IGF-1 were not significantly different between STAT mice and controls but gastric inhibitory polypeptide (GIP) was significantly elevated in the STAT mice vs controls (36.2±16.5pg/mL vs 24.4±13.3pg/ml, p<0.01). 454 sequencing yielded 555,000 readable sequences (5784±676 seq/sample with mean length 188±2.5bp) that were analyzed at the Order level. The Bacteroidales/Clostridiales (B/C) ratios in the fecal specimens from the control and STAT mice (medians: 19.5 vs 6.7) were significantly different (p<0.01). Unsupervised hierarchical clustering analysis at multiple taxonomic levels showed antibiotic-specific effects. Conclusions: STAT mice have altered early-life body composition with significantly increased fat mass and % body fat. GIP, a small intestinal incretin, may be one mediator of these metabolic changes. 454 results show that STAT mice have specific microbiota changes, with the direction of the B/C ratio changes consistent with prior findings in murine and human obesity.
*Values are mm Hg unless stated otherwise. #Values only provided for parameters in model 662 High Fat Diet Determines the Composition of the Gut Microbiome Independent of Host Genotype and Phenotype Marie A. Hildebrandt, Christian Hoffmann, Micah Hamady, Ying-Yu Chen, Rob Knight, Frederic D. Bushman, Rexford S. Ahima, Gary D. Wu Background: The co-evolution of the gut microbiome with its host has resulted in both commensal and symbiotic relationships. Nevertheless, increasing evidence suggests that gut microbial flora may play a role in the pathogenesis of several disease processes including inflammatory bowel disease, diabetes, and diet-induced obesity. Factors that determine the composition of the gut microbiome are just beginning to be elucidated. In this study, we examined the role of RELMβ, an intestinal-specific protein that is constitutively expressed in the colon due to the presence of commensal gut bacteria, on the composition of the gut microbiome. Methods and Results: Secreted apically into the lumen of the bowel by goblet cells, RELMβ protein expression is not only found at high levels in the stool but can also be detected in the serum. Interestingly, a high fat diet induces the expression and secretion of RELMβ into the stool. Wild-type and RELMβ KO mice were phenotypically similar on a normal chow diet, but metabolic characterization revealed that RELMβ KO mice remained lean on a high fat diet compared to wild-type controls due to an increase in energy expenditure. There were no differences in food intake, fat absorption, core body temperature, or physical activity. Using deep sequencing of 16S and metagenomic samples, we determined the composition of the gut microbiome in wild-type and RELMβ KO mice on both diets. Bacterial phylotyping revealed that there were large alterations in the gut microbiome between the two diets, irrespective of the differences in host phenotype. Effects of a high fat diet included an increase in the Firmicutes, a corresponding decrease in Bacteroidetes, and characteristic changes in bacterial gene content. More subtle, but reproducible effects of RELMβ genotype on the gut microbiome could also be observed on both diets. Conclusion: These results demonstrate that, although both host genotype and diet can independently influence gut microbiome composition, dietary factors may be the principal determinant.
665 The Role of the Intestinal Microbiota in Colonic Barrier Dysfunction Induced By Neonatal Stress Adam J. Moeser, Anthony T. Blikslager, Susan L. Tonkonogy, Temitope O. Keku
663
Alterations in the commensal gut microbiota have been reported in stress-related gastrointestinal disorders however the exact role of the microbiota is poorly understood. The objective of this study was to investigate the role of the intestinal microbiota in neonatal stress-induced colonic barrier dysfunction. Conventional (CONV) and germ-free (GF)-reared C57BL/6 pups were subjected to neonatal maternal separation (NMS) stress by removing pups from their dams for three hours daily during postnatal days 1-18. Control litters for each CONV and GF mice groups were left undisturbed with their dams. At postnatal days 10, 18, 30, and 60, In Vivo colonic permeability assays were performed in control and NMS mice within CONV and GF groups (n=6 mice/treatment group at each postnatal time point). Tissues were harvested for histological and Western blot analyses. In CONV mice, NMS induced elevations in colonic permeability that were evident at postnatal day 10 and persisted throughout the 60 day postnatal measurement period (p<0.05 vs. controls at each time point). In GF mice, NMS failed to cause significant alterations in colonic mucosal barrier function at any time point measured. Villous morphology and mucosal architecture was similar between the control and NMS in both CONV and GF mice. Mucosal mast cell numbers were similar in CONV and GF mice and were not influenced by NMS. However, colonic levels of mucosal mast cell tryptase were increased in CONV but not GF mice subjected to NMS. Analysis of the microbiota in CONV mice by TRFLP revealed no significant differences between control and NMS groups (n=3 samples/treatment/ postnatal time point). Overall these data demonstrate a critical role of the intestinal microbiota in neonatal stressinduced mucosal barrier dysfunction. The exact mechanisms by which the microbiota contributes to development of barrier dysfunction remain largely unknown but may be related to mast cell tryptase activity.
A Secreted Polysaccharide from Bifidobacterium Infantis 35624 Promotes Development of FOXP3+ T Regulatory Cells in Gnotobiotic Mice In Vivo. Liam O'Mahony, David O'Mahony, Anne Lyons, David Groeger, Ray Grant, Linda Drummond, Frances O'Brien, Barry Kiely, Fergus Shanahan Background & Aim: The enteric microbiota condition and prime the host immune response. We and others have previously demonstrated that selected microbial strains, such as Bifidobacterium infantis 35624, drive the development of immune-regulatory networks involving the induction of regulatory T cells. While the exact mechanisms underpinning this immunological effect are unclear, recent reports suggest that commensal polysaccharides modulate host immune maturation. Thus, the aim of our study was to determine if polysaccharides from Bifidobacterium infantis 35624 could stimulate T regulatory cells In Vivo. Methods: Cell-free supernatants from Bifidobacterium infantis 35624 were generated and a high molecular weight extra-cellular polysaccharide (EPS)-rich fraction was isolated using standard biochemical techniques. THP-1 dendritic cells were exposed to EPS and NF-κB activation measured using the secreted embryonic alkaline phosphatase (SEAP) reporter system. Cell viability was determined using the alamar blue assay while cytokine production was measured using MSD kits. EPS was co-incubated with LPS to assess if EPS could inhibit LPS-induced cellular activation. In addition, EPS was administered orally (20mg/kg) to Swiss Webster germ-free animals and induction of Foxp3+ T regulatory cells monitored using RT-PCR and flow cytometry. Cytokine production was measured using RT-PCR and MSD kits. Results: The EPS preparation was confirmed to be DNA-free and LPS-free with minimal protein contamination. EPS did not induce NF-κB activation or cytokine secretion by THP-1 cells and was non-toxic over a wide range of concentrations tested. A modest attenuation of LPS-induced NF-κB activation (approx. 40% reduction) was observed with ≥250pg/ml EPS. Administration of the EPS fraction to germ-free mice resulted in the increased expression of Foxp3 gene expression within the ileum. Flow cytometric assessment of splenic CD4+/CD25+/ Foxp3+ T cell subsets revealed a significant increase in regulatory T cell numbers in EPSfed germ-free animals (3.4+/-0.3%, n=15) compared to saline-treated controls (1.6+/-0.2%,
AGA Abstracts
A-102
n=12). In contrast, no differences in splenic cytokine secretion was observed in the EPStreated animals. Conclusions: A polysaccharide enriched fraction from Bifidobacterium infantis 35624 enhances the expression of Foxp3+ T regulatory cells In Vivo and is not pro-inflammatory In Vitro. The results provide further support for the distinct nature and beneficial aspects of this strain of Bifidobacterium and underscore the importance of strain-specificity in the context of probiotic function. 664 Sub-Therapeutic Antibiotic Treatment Alters the Murine Gut Microbiome and Affects Early-Life Metabolism and Body Composition Ilseung Cho, Barbara Methe, Sean Raj, Yael R. Nobel, Martin J. Blaser Background: The gut microbiome has an essential role in host metabolic homeostasis. Subtherapeutic antibiotics have been used for decades for growth promotion in the agricultural industry, but the relevant mechanisms are not known. We hypothesized that manipulating the gut microbiome in sub-therapeutic antibiotic treated (STAT) mice will alter their metabolic homeostasis. Methods: 21 day-old C57B6 (Jackson Laboratories) with ad libitum access to water and standard chow were given no antibiotics or sub-therapeutic doses of penicillin, vancomycin, penicillin+vancomycin, or chlortetracycline at a dose of 1μg antibiotic/g body weight in their drinking water (n=10/group). All mice were sacrificed at 9 weeks, scanned by densitometry and serum collected for gut hormone analysis by Luminex assays. DNA extracted from cecal and fecal specimens was subjected to PCR using barcoded universal primers interrogating the V3 region of the 16S rRNA gene followed by 454 sequencing and taxonomic analysis. Results: All 4 groups of STAT mice showed significantly increased fat mass and % body fat when compared to controls (See table). STAT mice had 14.1±2.5% more fat mass than controls [range: 8.1±3.4% (chlortetracycline) to 22.9±8.1% (penicillin)]. Lean mass was not significantly different between groups. Leptin, insulin, and IGF-1 were not significantly different between STAT mice and controls but gastric inhibitory polypeptide (GIP) was significantly elevated in the STAT mice vs controls (36.2±16.5pg/mL vs 24.4±13.3pg/ml, p<0.01). 454 sequencing yielded 555,000 readable sequences (5784±676 seq/sample with mean length 188±2.5bp) that were analyzed at the Order level. The Bacteroidales/Clostridiales (B/C) ratios in the fecal specimens from the control and STAT mice (medians: 19.5 vs 6.7) were significantly different (p<0.01). Unsupervised hierarchical clustering analysis at multiple taxonomic levels showed antibiotic-specific effects. Conclusions: STAT mice have altered early-life body composition with significantly increased fat mass and % body fat. GIP, a small intestinal incretin, may be one mediator of these metabolic changes. 454 results show that STAT mice have specific microbiota changes, with the direction of the B/C ratio changes consistent with prior findings in murine and human obesity.
*Values are mm Hg unless stated otherwise. #Values only provided for parameters in model 662 High Fat Diet Determines the Composition of the Gut Microbiome Independent of Host Genotype and Phenotype Marie A. Hildebrandt, Christian Hoffmann, Micah Hamady, Ying-Yu Chen, Rob Knight, Frederic D. Bushman, Rexford S. Ahima, Gary D. Wu Background: The co-evolution of the gut microbiome with its host has resulted in both commensal and symbiotic relationships. Nevertheless, increasing evidence suggests that gut microbial flora may play a role in the pathogenesis of several disease processes including inflammatory bowel disease, diabetes, and diet-induced obesity. Factors that determine the composition of the gut microbiome are just beginning to be elucidated. In this study, we examined the role of RELMβ, an intestinal-specific protein that is constitutively expressed in the colon due to the presence of commensal gut bacteria, on the composition of the gut microbiome. Methods and Results: Secreted apically into the lumen of the bowel by goblet cells, RELMβ protein expression is not only found at high levels in the stool but can also be detected in the serum. Interestingly, a high fat diet induces the expression and secretion of RELMβ into the stool. Wild-type and RELMβ KO mice were phenotypically similar on a normal chow diet, but metabolic characterization revealed that RELMβ KO mice remained lean on a high fat diet compared to wild-type controls due to an increase in energy expenditure. There were no differences in food intake, fat absorption, core body temperature, or physical activity. Using deep sequencing of 16S and metagenomic samples, we determined the composition of the gut microbiome in wild-type and RELMβ KO mice on both diets. Bacterial phylotyping revealed that there were large alterations in the gut microbiome between the two diets, irrespective of the differences in host phenotype. Effects of a high fat diet included an increase in the Firmicutes, a corresponding decrease in Bacteroidetes, and characteristic changes in bacterial gene content. More subtle, but reproducible effects of RELMβ genotype on the gut microbiome could also be observed on both diets. Conclusion: These results demonstrate that, although both host genotype and diet can independently influence gut microbiome composition, dietary factors may be the principal determinant.
665 The Role of the Intestinal Microbiota in Colonic Barrier Dysfunction Induced By Neonatal Stress Adam J. Moeser, Anthony T. Blikslager, Susan L. Tonkonogy, Temitope O. Keku
663
Alterations in the commensal gut microbiota have been reported in stress-related gastrointestinal disorders however the exact role of the microbiota is poorly understood. The objective of this study was to investigate the role of the intestinal microbiota in neonatal stress-induced colonic barrier dysfunction. Conventional (CONV) and germ-free (GF)-reared C57BL/6 pups were subjected to neonatal maternal separation (NMS) stress by removing pups from their dams for three hours daily during postnatal days 1-18. Control litters for each CONV and GF mice groups were left undisturbed with their dams. At postnatal days 10, 18, 30, and 60, In Vivo colonic permeability assays were performed in control and NMS mice within CONV and GF groups (n=6 mice/treatment group at each postnatal time point). Tissues were harvested for histological and Western blot analyses. In CONV mice, NMS induced elevations in colonic permeability that were evident at postnatal day 10 and persisted throughout the 60 day postnatal measurement period (p<0.05 vs. controls at each time point). In GF mice, NMS failed to cause significant alterations in colonic mucosal barrier function at any time point measured. Villous morphology and mucosal architecture was similar between the control and NMS in both CONV and GF mice. Mucosal mast cell numbers were similar in CONV and GF mice and were not influenced by NMS. However, colonic levels of mucosal mast cell tryptase were increased in CONV but not GF mice subjected to NMS. Analysis of the microbiota in CONV mice by TRFLP revealed no significant differences between control and NMS groups (n=3 samples/treatment/ postnatal time point). Overall these data demonstrate a critical role of the intestinal microbiota in neonatal stressinduced mucosal barrier dysfunction. The exact mechanisms by which the microbiota contributes to development of barrier dysfunction remain largely unknown but may be related to mast cell tryptase activity.
A Secreted Polysaccharide from Bifidobacterium Infantis 35624 Promotes Development of FOXP3+ T Regulatory Cells in Gnotobiotic Mice In Vivo. Liam O'Mahony, David O'Mahony, Anne Lyons, David Groeger, Ray Grant, Linda Drummond, Frances O'Brien, Barry Kiely, Fergus Shanahan Background & Aim: The enteric microbiota condition and prime the host immune response. We and others have previously demonstrated that selected microbial strains, such as Bifidobacterium infantis 35624, drive the development of immune-regulatory networks involving the induction of regulatory T cells. While the exact mechanisms underpinning this immunological effect are unclear, recent reports suggest that commensal polysaccharides modulate host immune maturation. Thus, the aim of our study was to determine if polysaccharides from Bifidobacterium infantis 35624 could stimulate T regulatory cells In Vivo. Methods: Cell-free supernatants from Bifidobacterium infantis 35624 were generated and a high molecular weight extra-cellular polysaccharide (EPS)-rich fraction was isolated using standard biochemical techniques. THP-1 dendritic cells were exposed to EPS and NF-κB activation measured using the secreted embryonic alkaline phosphatase (SEAP) reporter system. Cell viability was determined using the alamar blue assay while cytokine production was measured using MSD kits. EPS was co-incubated with LPS to assess if EPS could inhibit LPS-induced cellular activation. In addition, EPS was administered orally (20mg/kg) to Swiss Webster germ-free animals and induction of Foxp3+ T regulatory cells monitored using RT-PCR and flow cytometry. Cytokine production was measured using RT-PCR and MSD kits. Results: The EPS preparation was confirmed to be DNA-free and LPS-free with minimal protein contamination. EPS did not induce NF-κB activation or cytokine secretion by THP-1 cells and was non-toxic over a wide range of concentrations tested. A modest attenuation of LPS-induced NF-κB activation (approx. 40% reduction) was observed with ≥250pg/ml EPS. Administration of the EPS fraction to germ-free mice resulted in the increased expression of Foxp3 gene expression within the ileum. Flow cytometric assessment of splenic CD4+/CD25+/ Foxp3+ T cell subsets revealed a significant increase in regulatory T cell numbers in EPSfed germ-free animals (3.4+/-0.3%, n=15) compared to saline-treated controls (1.6+/-0.2%,
AGA Abstracts
A-102