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Remodeling Gut Microbiota by Lactobacillus Reuteri DSM 17938 Suppresses Autoimmunity Induced by Treg Deficiency
1136
PGE2 PRODUCED BY MAST CELLS IS CRITICAL TO INDUCE VISCERAL HYPERSENSITIVITY IN IBS Gintautas Grabauskas, Xiaoyin Wu, D. K. Turgeon, Chung Owyang
CROHN'S DISEASE EXCLUSION DIET AND PARTIAL ENTERAL NUTRITION (CDED+PEN) VS EXCLUSIVE ENTERAL NUTRITION (EEN) MICROBIOME CHANGES OF A RANDOMIZED CLINICAL TRIAL (RCT) IN PEDIATRIC CD: REMISSION IS ASSOCIATED WITH SIMILAR STRUCTURAL AND FUNCTIONAL PROFILES Katherine A. Dunn, Rotem S. Boneh, Joseph P. Bielawski, Dan Turner, Johan E. Van Limbergen, Arie Levine
Visceral hypersensitivity (VH) is commonly observed in irritable bowel syndrome (IBS) patients; but the responsible mechanism is unclear. Low grade colonic mucosa inflammation occurs in IBS patients. Animal studies indicate that colonic mast cells play a critical role in mediating VH in IBS rodent models. Recently, we showed that PGE2 is responsible for pain in IBS-D patients. To provide a unifying hypothesis, which may explain this diverse group of observations, we propose that inflammatory mediators such as protease and histamine activate COX-2 to increase the synthesis of PGE2 from arachnoid acid (AA) by the mast cells. This process is enhanced by Substance P (sub P) released from intrinsic sensory nerves which increases the synthesis of AA, the substract for COX-2. To test this hypothesis, we obtained colonic biopsies from 17 IBS-D patients and 18 healthy controls (HC). Elisa data showed colonic biopsies from IBD patients contained 3-fold levels of PGE2 compared to HC (P<0.01). This was accompanied by a 2-3 fold increase in COX-2 gene expression. Similarly, colonic biopsies from IBS-D patients also contained a 2-3 fold increase in tryptase and histamine which stimulated a 2-fold increase in PGE2 from IBS colonic biopsies. Colonic distention to 40 mmHg caused a 30-fold increase in PGE2 which was blocked by the mast cell stabilizer, sodium cromolyn (i.p 40 mg/kg), celecoxib, a COX-2 inhibitor (i.p 7mg/kg) or sub P antagonist (SQ-140333 4 mg/kg). Immunohistochemical studies demonstrated that COX-2 immunoreactivities exclusively colocalized with mast cell tryptase immunoreactivities in colonic biopsy samples. Pain behavior studies showed a 3-4 fold increase in VMR to CRD at 20, 40, and 60 mmHg occurred 3h following IC infusion of IBS-D biopsy supernatants compared with supernatants from HC (P <0.01). This pronociceptive effect was blocked by cyclooxygenase inhibitor EP2 receptor antagonist (PF-04418948, i.p 1mg/kg), or sodium cromolyn, and sub P antagonist. In separate studies, we showed that intracolonic administration of IBS supernatant did not induce VH or cause a rise in PGE2 in response to colonic distention in mast cell deficient mice (W/WR mice) compared to control. To show that PGE2 is the final mediator to induce VH, we performed intrathecal injection (L5-DRG) of siRNA to reduce EP2 expression. This abolished VH in rats receiving IBS-D supernatant. In conclusion, our data showed that IBS colonic biopsies contain a number of inflammatory mediators. All act on the mast cells to activate COX-2 resulting in enhanced synthesis of PGE2. Colonic distention activates intrinsic sensory nerves to release sub P which is known to promote synthesis of AA, a precursor for PGE2 synthesis. These two events act in concert to increase release of PGE2 from mast cells and activate EP2 receptors in the DRG to induce VH.
Background & Aims: EEN is able to induce remission in CD patients, but can be difficult to maintain. A novel dietary intervention that combines partial enteral nutrition with an exclusion diet, that excludes foods proposed to trigger dysbiosis and inflammation (CDED+PEN) has been shown effective. We aim to compare the gut microbiome of CD participants in a prospective RCT comparing these diets, and to assess whether microbiome profiles can identify subgroups that are able to sustain remission. Methods: 24 pediatric patients received either CDED + 50% Modulen for 6 weeks, then CDED+25% Modulen for 6 weeks (Group 1) or EEN with Modulen for 6 weeks followed by free diet plus 25% Modulen (Group 2). Remission was present in 11/14 (78.5%) in Group 1, and 8/10 (80%) in Group 2. One patient from Group 1 relapsed by week 12. DNA from fecal samples collected at three time points (baseline, w6 and w12) was sequenced for 16S and whole metagenome. Taxonomic composition was inferred from 16S (QIIME) and from metagenomes (Metaphlan) and inferred for function (Diamond/HUMAnN). Results: Both interventions induced an increase in alpha diversity by w12. EEN patients experienced a transient reduction in diversity at w6, whereas CDED+PEN did not. Taxonomic and functional profiles were similar by w12. Pooling the results for both diets, the taxonomic composition of the 18 patients who sustained remission differed significantly from the 6 patients who did not. Twenty-two 16S-derived operational taxonomic units had different relative abundance (p ≤ 0.05), with a subgroup of eleven identified according to a false discovery threshold (q-value) of 0.15. Similar taxonomic results were inferred from the metagenome. Analysis of the functional repertoire yielded a similar pattern; 1811 genes differed between patients who sustained remission and those who did not, with 711 identified according to q-value < 0.05. To confirm that these results (taxonomic and functional) were not being driven by the signal associated with a single diet, we separately compared the remission patients for each diet to the pooled set of patients who did not maintain remission. Results were consistent for both diets, but with slightly smaller subgroups of genes. Supervised modeling is currently underway to investigate if functional and taxonomic profiles can be exploited to predict patient outcomes. Conclusion: Microbiome changes induced by CDED+PEN 50% are comparable with EEN in a pediatric RCT with active CD. Remission achieved with either dietary intervention is associated with similar structural and functional profiles.
1135 1137 REMODELING GUT MICROBIOTA BY LACTOBACILLUS REUTERI DSM 17938 SUPPRESSES AUTOIMMUNITY INDUCED BY TREG DEFICIENCY Yuying Liu, Baokun He, Thomas K. Hoang, Ting Wang, Christopher M. Taylor, XiangJun Tian, Meng Luo, Dat Q. Tran, Jain Zhou, Nina Tatevian, Thomas H. Gomez, Stefan Roos, J. Marc Rhoads
DIFFERENCES IN THE STOOL AND SKIN MICROBIOME, VIRULENCE FACTOR AND ANTIMICROBIAL RESISTANCE GENES IN A PRIVATE ROOM VERSUS A SHARED SPACE NEONATAL INTENSIVE CARE UNIT Suchitra K. Hourigan, Ta Allison, Elisabeth Z. Klein, Rajiv Baveja, Nassim Chettout, Nicole C. Clemency, Colin Heberling, Poorani Subramanian, Nur A. Hasan, John E. Niederhuber, Rita R. Colwell
Background: Regulatory T-cell (Treg) deficiency causes lethal, CD4+T cell-driven autoimmune diseases, such as IPEX syndrome (immunodysregulation, polyendocrinopathy, enteropathy, with X-linked inheritance) in humans and the scurfy (SF) phenotype mice. The gut microbiota drives host immune homeostasis by regulating the development of TH cells. Lactobacillus reuteri DSM17938 (LR) is effective for preventing or treating diseases in infants and children, including necrotizing enterocolitis, diarrhea, and infantile colic, hypothetically by modulating abnormal microbial communities. Objectives: To characterize the dynamic changes of autoimmunity and gut microbial dysbiosis over the short lifespan of the Tregdeficient SF mice; to examine if the gut microbial community is shifted by orally feeding LR; and to determine if LR inhibits autoimmunity in SF mice. Methods: Mice were given MRS as control, or LR in MRS (10^7 CFU/day) by gavage, daily, starting at d8 or d15 to d22 for sample analysis or long-term to observe survival. Lymphocyte infiltration in the lung and liver was evaluated. IFNγ+CD4+T (TH1) and IL4+CD4+T (TH2) cells were assessed by flow cytometry. Plasma IFNγ and IL4 were detected by ELISA. The microbiota was analyzed using 16s rRNA sequencing and QIIME. Results: Autoimmunity and gut microbiota developed early in SF mice, at d8, d15, and d22 with inflammation in many tissues (liver, lung, intestine, ear and tail) and an increased frequency of IFNγ+ and IL4+CD4+T cells in the spleen and mesenteric lymph nodes. Plasma levels of IFNγ and IL4 were increased as early as d8, at a time when the SF clinical phenotype was not seen. Decreased gut microbial diversity and a distinctly shifted microbial composition were demonstrated in SF mice compared to WT littermates at d22. Longitudinal analysis of relative abundance of gut microbial communities in these mice indicated broad population changes from the phylum to the genus levels in SF compared to WT mice, suggesting that Foxp3+Treg-deficiency shapes gut microbiota community structure and is associated with dysbiosis. The relative abundance of Lactobacillus was significantly lower, while Bacteroides was significantly higher in SF compared to that in WT stool. The decreased diversity associated with Treg-deficiency was reversed by LR, which specifically increased the relative abundance of the phylum Firmicutes and the genera Lactobacillus and Oscillospira, while decreasing the relative abundance of the phylum Tenericutes and the genus Bacteroides. LR significantly prolonged survival, reduced inflammation in the intestine, lung and liver and reduced TH1/TH2 cells and their associated cytokines in SF mice. Conclusions: LR reprograms gut microbiota and suppresses Treg deficiency-induced autoimmunity. This study demonstrates that the beneficial effects of LR are not contingent upon the presence of Tregs.
.Background: Critical microbiome development occurs early in life impacting future health. It is unknown whether there is differential microbiome development in infants in the neonatal intensive care unit (NICU) cared for in private rooms (PRs) vs. a shared space (SS) with other infants. Aims: To identify differences in the skin, stool and environmental microbiome, virulence factor and antimicrobial resistance genes in neonates in a PR vs. a SS NICU. Methods: As part of a longitudinal NICU microbiome study, stool was collected at 2 weeks and discharge along with a skin swab from the groin. 14 neonates from a PR NICU and 14 from a SS NICU were compared. Infants from each NICU were matched for gestational age, delivery mode and length of stay. Environmental swabs e.g. from sinks and isolettes were also obtained. DNA was extracted from samples and underwent shotgun metagenomic sequencing. Libraries were sequenced using Illumina HiSeq v3 chemistry for 100bp single end reads with the aim to generate 40M reads per sample. Metagenomic sequencing reads were directly analyzed by CosmosID bioinformatics software package (CosmosID Inc., Rockville, MD) to achieve bacterial identification at the species, subspecies, and/or strain level and quantification of relative abundance; similarly community resistome, virulence factors, viruses, fungi and parasites were also identified. Results: The likelihood ratio test comparing the 2 NICU cohorts (chi-squared) showed that the overall microbiome at species level was significantly different for skin (p= 0.0001) and environmental swabs (p=0.00003), but not for the 2 week and discharge stool (Figure 1). Several potential pathogen and virulence factor genes were found in all sample types from both NICUs including respiratory syncytial virus, clostridium difficile, Methicillin-resistant Staphylococcus aureus and parasites (Acanthamoeba polyphaga being most prevalent) although none resulted in clinical disease. STL polyomavirus and MW polyomavirus were significantly higher in the SS NICU environmental samples (Kruskal-Wallis, p=0.017) and papillomavirus trended towards significance in these samples (p=0.05). Virulence factor genes associated with Clostridium perfringens were significantly higher in the PR discharge stool; however the prevalence of Clostridium difficile was higher in the SS stool. Several antibiotic resistance genes were present in both cohorts but the prevalence was not significantly different. Conclusions: Using shotgun metagenomic sequencing and CosmosID technology to give a unique view of the infant microbiome, this pilot study shows that some differences exist in the microbiome and possibly microorganisms with pathogenic potential between a SS and PR NICU. Whether these confer differences in long term microbiome development or health outcomes will be examined in the extension of this study.
S-213
AGA Abstracts
AGA Abstracts
1134
PGE2 PRODUCED BY MAST CELLS IS CRITICAL TO INDUCE VISCERAL HYPERSENSITIVITY IN IBS Gintautas Grabauskas, Xiaoyin Wu, D. K. Turgeon, Chung Owyang
CROHN'S DISEASE EXCLUSION DIET AND PARTIAL ENTERAL NUTRITION (CDED+PEN) VS EXCLUSIVE ENTERAL NUTRITION (EEN) MICROBIOME CHANGES OF A RANDOMIZED CLINICAL TRIAL (RCT) IN PEDIATRIC CD: REMISSION IS ASSOCIATED WITH SIMILAR STRUCTURAL AND FUNCTIONAL PROFILES Katherine A. Dunn, Rotem S. Boneh, Joseph P. Bielawski, Dan Turner, Johan E. Van Limbergen, Arie Levine
Visceral hypersensitivity (VH) is commonly observed in irritable bowel syndrome (IBS) patients; but the responsible mechanism is unclear. Low grade colonic mucosa inflammation occurs in IBS patients. Animal studies indicate that colonic mast cells play a critical role in mediating VH in IBS rodent models. Recently, we showed that PGE2 is responsible for pain in IBS-D patients. To provide a unifying hypothesis, which may explain this diverse group of observations, we propose that inflammatory mediators such as protease and histamine activate COX-2 to increase the synthesis of PGE2 from arachnoid acid (AA) by the mast cells. This process is enhanced by Substance P (sub P) released from intrinsic sensory nerves which increases the synthesis of AA, the substract for COX-2. To test this hypothesis, we obtained colonic biopsies from 17 IBS-D patients and 18 healthy controls (HC). Elisa data showed colonic biopsies from IBD patients contained 3-fold levels of PGE2 compared to HC (P<0.01). This was accompanied by a 2-3 fold increase in COX-2 gene expression. Similarly, colonic biopsies from IBS-D patients also contained a 2-3 fold increase in tryptase and histamine which stimulated a 2-fold increase in PGE2 from IBS colonic biopsies. Colonic distention to 40 mmHg caused a 30-fold increase in PGE2 which was blocked by the mast cell stabilizer, sodium cromolyn (i.p 40 mg/kg), celecoxib, a COX-2 inhibitor (i.p 7mg/kg) or sub P antagonist (SQ-140333 4 mg/kg). Immunohistochemical studies demonstrated that COX-2 immunoreactivities exclusively colocalized with mast cell tryptase immunoreactivities in colonic biopsy samples. Pain behavior studies showed a 3-4 fold increase in VMR to CRD at 20, 40, and 60 mmHg occurred 3h following IC infusion of IBS-D biopsy supernatants compared with supernatants from HC (P <0.01). This pronociceptive effect was blocked by cyclooxygenase inhibitor EP2 receptor antagonist (PF-04418948, i.p 1mg/kg), or sodium cromolyn, and sub P antagonist. In separate studies, we showed that intracolonic administration of IBS supernatant did not induce VH or cause a rise in PGE2 in response to colonic distention in mast cell deficient mice (W/WR mice) compared to control. To show that PGE2 is the final mediator to induce VH, we performed intrathecal injection (L5-DRG) of siRNA to reduce EP2 expression. This abolished VH in rats receiving IBS-D supernatant. In conclusion, our data showed that IBS colonic biopsies contain a number of inflammatory mediators. All act on the mast cells to activate COX-2 resulting in enhanced synthesis of PGE2. Colonic distention activates intrinsic sensory nerves to release sub P which is known to promote synthesis of AA, a precursor for PGE2 synthesis. These two events act in concert to increase release of PGE2 from mast cells and activate EP2 receptors in the DRG to induce VH.
Background & Aims: EEN is able to induce remission in CD patients, but can be difficult to maintain. A novel dietary intervention that combines partial enteral nutrition with an exclusion diet, that excludes foods proposed to trigger dysbiosis and inflammation (CDED+PEN) has been shown effective. We aim to compare the gut microbiome of CD participants in a prospective RCT comparing these diets, and to assess whether microbiome profiles can identify subgroups that are able to sustain remission. Methods: 24 pediatric patients received either CDED + 50% Modulen for 6 weeks, then CDED+25% Modulen for 6 weeks (Group 1) or EEN with Modulen for 6 weeks followed by free diet plus 25% Modulen (Group 2). Remission was present in 11/14 (78.5%) in Group 1, and 8/10 (80%) in Group 2. One patient from Group 1 relapsed by week 12. DNA from fecal samples collected at three time points (baseline, w6 and w12) was sequenced for 16S and whole metagenome. Taxonomic composition was inferred from 16S (QIIME) and from metagenomes (Metaphlan) and inferred for function (Diamond/HUMAnN). Results: Both interventions induced an increase in alpha diversity by w12. EEN patients experienced a transient reduction in diversity at w6, whereas CDED+PEN did not. Taxonomic and functional profiles were similar by w12. Pooling the results for both diets, the taxonomic composition of the 18 patients who sustained remission differed significantly from the 6 patients who did not. Twenty-two 16S-derived operational taxonomic units had different relative abundance (p ≤ 0.05), with a subgroup of eleven identified according to a false discovery threshold (q-value) of 0.15. Similar taxonomic results were inferred from the metagenome. Analysis of the functional repertoire yielded a similar pattern; 1811 genes differed between patients who sustained remission and those who did not, with 711 identified according to q-value < 0.05. To confirm that these results (taxonomic and functional) were not being driven by the signal associated with a single diet, we separately compared the remission patients for each diet to the pooled set of patients who did not maintain remission. Results were consistent for both diets, but with slightly smaller subgroups of genes. Supervised modeling is currently underway to investigate if functional and taxonomic profiles can be exploited to predict patient outcomes. Conclusion: Microbiome changes induced by CDED+PEN 50% are comparable with EEN in a pediatric RCT with active CD. Remission achieved with either dietary intervention is associated with similar structural and functional profiles.
1135 1137 REMODELING GUT MICROBIOTA BY LACTOBACILLUS REUTERI DSM 17938 SUPPRESSES AUTOIMMUNITY INDUCED BY TREG DEFICIENCY Yuying Liu, Baokun He, Thomas K. Hoang, Ting Wang, Christopher M. Taylor, XiangJun Tian, Meng Luo, Dat Q. Tran, Jain Zhou, Nina Tatevian, Thomas H. Gomez, Stefan Roos, J. Marc Rhoads
DIFFERENCES IN THE STOOL AND SKIN MICROBIOME, VIRULENCE FACTOR AND ANTIMICROBIAL RESISTANCE GENES IN A PRIVATE ROOM VERSUS A SHARED SPACE NEONATAL INTENSIVE CARE UNIT Suchitra K. Hourigan, Ta Allison, Elisabeth Z. Klein, Rajiv Baveja, Nassim Chettout, Nicole C. Clemency, Colin Heberling, Poorani Subramanian, Nur A. Hasan, John E. Niederhuber, Rita R. Colwell
Background: Regulatory T-cell (Treg) deficiency causes lethal, CD4+T cell-driven autoimmune diseases, such as IPEX syndrome (immunodysregulation, polyendocrinopathy, enteropathy, with X-linked inheritance) in humans and the scurfy (SF) phenotype mice. The gut microbiota drives host immune homeostasis by regulating the development of TH cells. Lactobacillus reuteri DSM17938 (LR) is effective for preventing or treating diseases in infants and children, including necrotizing enterocolitis, diarrhea, and infantile colic, hypothetically by modulating abnormal microbial communities. Objectives: To characterize the dynamic changes of autoimmunity and gut microbial dysbiosis over the short lifespan of the Tregdeficient SF mice; to examine if the gut microbial community is shifted by orally feeding LR; and to determine if LR inhibits autoimmunity in SF mice. Methods: Mice were given MRS as control, or LR in MRS (10^7 CFU/day) by gavage, daily, starting at d8 or d15 to d22 for sample analysis or long-term to observe survival. Lymphocyte infiltration in the lung and liver was evaluated. IFNγ+CD4+T (TH1) and IL4+CD4+T (TH2) cells were assessed by flow cytometry. Plasma IFNγ and IL4 were detected by ELISA. The microbiota was analyzed using 16s rRNA sequencing and QIIME. Results: Autoimmunity and gut microbiota developed early in SF mice, at d8, d15, and d22 with inflammation in many tissues (liver, lung, intestine, ear and tail) and an increased frequency of IFNγ+ and IL4+CD4+T cells in the spleen and mesenteric lymph nodes. Plasma levels of IFNγ and IL4 were increased as early as d8, at a time when the SF clinical phenotype was not seen. Decreased gut microbial diversity and a distinctly shifted microbial composition were demonstrated in SF mice compared to WT littermates at d22. Longitudinal analysis of relative abundance of gut microbial communities in these mice indicated broad population changes from the phylum to the genus levels in SF compared to WT mice, suggesting that Foxp3+Treg-deficiency shapes gut microbiota community structure and is associated with dysbiosis. The relative abundance of Lactobacillus was significantly lower, while Bacteroides was significantly higher in SF compared to that in WT stool. The decreased diversity associated with Treg-deficiency was reversed by LR, which specifically increased the relative abundance of the phylum Firmicutes and the genera Lactobacillus and Oscillospira, while decreasing the relative abundance of the phylum Tenericutes and the genus Bacteroides. LR significantly prolonged survival, reduced inflammation in the intestine, lung and liver and reduced TH1/TH2 cells and their associated cytokines in SF mice. Conclusions: LR reprograms gut microbiota and suppresses Treg deficiency-induced autoimmunity. This study demonstrates that the beneficial effects of LR are not contingent upon the presence of Tregs.
.Background: Critical microbiome development occurs early in life impacting future health. It is unknown whether there is differential microbiome development in infants in the neonatal intensive care unit (NICU) cared for in private rooms (PRs) vs. a shared space (SS) with other infants. Aims: To identify differences in the skin, stool and environmental microbiome, virulence factor and antimicrobial resistance genes in neonates in a PR vs. a SS NICU. Methods: As part of a longitudinal NICU microbiome study, stool was collected at 2 weeks and discharge along with a skin swab from the groin. 14 neonates from a PR NICU and 14 from a SS NICU were compared. Infants from each NICU were matched for gestational age, delivery mode and length of stay. Environmental swabs e.g. from sinks and isolettes were also obtained. DNA was extracted from samples and underwent shotgun metagenomic sequencing. Libraries were sequenced using Illumina HiSeq v3 chemistry for 100bp single end reads with the aim to generate 40M reads per sample. Metagenomic sequencing reads were directly analyzed by CosmosID bioinformatics software package (CosmosID Inc., Rockville, MD) to achieve bacterial identification at the species, subspecies, and/or strain level and quantification of relative abundance; similarly community resistome, virulence factors, viruses, fungi and parasites were also identified. Results: The likelihood ratio test comparing the 2 NICU cohorts (chi-squared) showed that the overall microbiome at species level was significantly different for skin (p= 0.0001) and environmental swabs (p=0.00003), but not for the 2 week and discharge stool (Figure 1). Several potential pathogen and virulence factor genes were found in all sample types from both NICUs including respiratory syncytial virus, clostridium difficile, Methicillin-resistant Staphylococcus aureus and parasites (Acanthamoeba polyphaga being most prevalent) although none resulted in clinical disease. STL polyomavirus and MW polyomavirus were significantly higher in the SS NICU environmental samples (Kruskal-Wallis, p=0.017) and papillomavirus trended towards significance in these samples (p=0.05). Virulence factor genes associated with Clostridium perfringens were significantly higher in the PR discharge stool; however the prevalence of Clostridium difficile was higher in the SS stool. Several antibiotic resistance genes were present in both cohorts but the prevalence was not significantly different. Conclusions: Using shotgun metagenomic sequencing and CosmosID technology to give a unique view of the infant microbiome, this pilot study shows that some differences exist in the microbiome and possibly microorganisms with pathogenic potential between a SS and PR NICU. Whether these confer differences in long term microbiome development or health outcomes will be examined in the extension of this study.
S-213
AGA Abstracts
AGA Abstracts
1134