- Email: [email protected]
Su1882 Effect of Sleep Fragmentation on Composition of Gut Microbiome and Mesenteric Lymph Node Signaling
the specific role of TNFR2 in regulating CD8+ T cell and the gut microbiome homeostasis could lead to refined therapeutic approaches especially for IBD patients that have CD8+ T cell dysfunction.
Su1881 Dietary Serine Controls the Competition Between Pathogenic and Commensal E. coli During Intestinal Inflammation Sho Kitamoto, Hiroko Nagao-Kitamoto, Peter Kuffa, Nobuhiko Kamada
AGA Abstracts
Su1879 Backgrounds: The Pathogenic E. coli, namely Adherent Invasive E. coli (AIEC), is accumulated in inflamed intestinal mucosa of patients with Crohn's disease (CD) and contributes to its pathogenesis. Previously we demonstrated that metabolically related gut commensal E. coli are able to outcompete the pathogenic E. coli for nutrients such as carbohydrates in steady state (Kamada et al., Science, 2012). However, the mechanisms of how pathogenic E. coli can gain the growth advantage over the commensal E. coli during inflammation remains poorly understood. Aims: We aimed to elucidate the mechanisms of how intestinal inflammation selectively fuels the growth of pathogenic E. coli as compared to that of non-pathogenic commensal E. coli in the inflamed gut. Methods: Human- and mouse-derived AIEC strains, LF82 and CUMT-8, were used in this study. To assess the competition of AIEC and commensal E. coli in vivo, AIEC was infected into specific-pathogen-free mice and AIEC and commensal E. coli strains were co-infected in germ-free mice. Dextran Sodium Sulfate (DSS) was used to induce intestinal inflammation. Bacterial burden of each strain in feces was monitored by the titration using antibiotics resistance. Luminal metabolites were analyzed by CE-TOF/ MS. To determine the preference of amino acids on the growth of E. coli, each E. coli strain was cultured in vitro in a minimal media supplemented with each single amino acid, and then bacterial proliferation was determined by density measurement. To access the importance of dietary amino acids on bacterial fitness in the gut, amino acid defined control diet, protein free diet and serine-glycine deficient diet were applied to bacterial competition assay in vivo. Results: The results we obtained in this study are as follows: 1) Both human and mouse AIEC strains can obtain the growth advantage over commensal E. coli during DSS colitis. 2) Inflammation alters the luminal nutrient availability including amino acids. 3) The deprivation of dietary amino acids prevents outgrowth of AIEC in the inflamed but not in the steady-state gut. 4) An amino acid serine selectively promotes growth of pathogenic, but not commensal, E. coli strains both in vitro and in vivo. Conclusion: Dietary serine controls the competitive fitness of pathogenic E. coli in the inflamed gut. These results provide novel insights into understanding of the pathophysiology of E. coli-driven intestinal inflammation in patients with CD, and expected to contribute to the development of new therapeutic approaches that specifically target the nutrients utilized by pathobionts.
Spatial 3D-Stereomicroscopic, Microbial and Metabolic Characterization of Intestinal Villous Erosions and Ulcerations in Mice Alexander Rodriguez-Palacios, Ludovica F. Buttó, Ilya R. Bederman, Dirk Haller, Fabio Cominelli Background & Aims: Recently, a method based on 3D-stereomicroscopic (SM) assessment of the gut mucosal surface (3D-SMAP gut) revealed the 3D-spatial complexity of focal lesion distribution with particular focus on cobblestone-lesions in mice. The aim of this study was to characterize for the first time the spatial, microbial and metabolic attributes of two other spontaneous 3D-SM lesions: i) villous tip erosions and ii) ulcers in the small intestine of mice. Methods: Lesions were assessed by 3D-SM retrospectively (147 mice) and prospectively (>54 mice) in >6 models of ileitis/colitis, in combination with 16S rRNA microbiome analysis of SM-dissected areas affected by these two pathologies, and with the infection of SPF and germ-free mice with ulcer-associated microbes. Of novel interest, we developed a method to test the role of mechanical disruption as a contributor to villous erosions and ulcer formation, and we discovered a strategy to harvest the villous intestinal epithelial cells that cover the intestinal villi maintaining the 3D-‘villuos-cap' shape architecture, providing an ex vivo assay to study villous-microbe interaction using a proposed scoring system. Results: Intestinal ulcer occurrence was dependent on mouse strain and experimental set ranging from 0-27% of mice indicating the potential role of the gut microbiota and mouse genetics in predisposition to ulcer formation. The ulcer shape that predominates was linear (1-3 villi wide, along circumferential gut axis). With aging, the ulcer occurrence/size did not increase, suggesting ulcers can be self-contained. With respect to villous erosions, at least one lesion was seen in 0-19% of mice/set. SM-dissection of lesions showed microbial communities enriched in Escherichia coli, Lactobacillus gasseri, Staphylococcus saprophiticus/ xylosus, and Achromobacter sp. Germ-free mice infected with these microbes displayed E. coli and Lactobacillus in feces and increased E. coli counts in ulcers (pair t-test, p<0.05). With respect to gut epithelial cells, glucose consumption in our in vitro ‘villous cap' assay using normal villi with a background mucosal-associated microbiome resulted in similar glucose consumption for three O2 conditions. In contrast, the ‘villous caps' incubated with ulcer-associated microbial lysates increased glucose consumption hypoxically (ANOVA, p<0.0046), and produce 3D-villous abnormalities. Conclusion: Intestinal villous erosions and ulcerations are unrecognized lesions in mice that should be expected to occur and confound experimental research. Our spatial, microbiological and metabolic analysis indicate that ulcer formation may be a self-contained pathology that may occur as a result of the combination of mechanical disruption and underlying metabolic and microbiologicallyenhanced immunological interactions that may vary with oxygen and carbon dioxide availability.
Su1882 Effect of Sleep Fragmentation on Composition of Gut Microbiome and Mesenteric Lymph Node Signaling Edward C. Oldfield, Rouzbeh Shams, Jennifer L. Copare, Laurie L. Wellman, Richard P. Ciavarra, Patric S. Lundberg, Larry D. Sanford, J. C. Ware, David A. Johnson Background: Both the gut microbiome and sleep fragmentation have been shown to influence the immune system, which can lead to altered expression of cytokines involved in gut integrity. This study evaluated the effect of sleep fragmentation on gut microbiome composition and cytokine regulation/expression. Methods: Sleep fragmentation was induced using a custom-designed cage with an automated motorized sweeper, which provided intermittent tactile stimulation every 2 minutes during either the light period (normal sleeping time), dark period (normal awake time), or control group (no sleep fragmentation). Control mice were allowed to sleep undisturbed in their home cage. Stool specimens were collected from 14 mice (5 control, 5 light, 4 dark) at 6 hour intervals (0000, 0600, 1200, 1800) for each of the following: 10 days sleep fragmentation, 20 days recovery, and after 10 days of a second sleep fragmentation period. DNA was then isolated from the collected stool specimen using a Mo Bio PowerFecal® DNA isolation kit and the samples were processed using nextgeneration sequencing methods. At the end of the study period, the mice were sacrificed and mesenteric lymph nodes were pooled by group and sampled using qPCR inflammatory cytokine arrays to assess changes in mucosal immunity essential for maintaining intestinal integrity. Results: Over 150 bacterial genera showed alterations with sleep fragmentation compared to control. Bacterial genera Lactobacillus and Parabacteroides showed significant circadian variability (with a reversal from normal pattern) (Figure 1). Across all groups, Parabacteroides peaked between 1800-0000 with a nadir around 0600, whereas Lactobacillus peaked between 1200-1800 with a nadir at 0000. Furthermore, the peak concentrations of both Parabacteroides and Lactobacillus were greater in the sleep fragmentation mice (both light and dark) compared to controls. Multiple pronounced changes in cytokines were induced by sleep fragmentation compared to controls (figure 2); among the most notable alterations was a decreased expression of cytokines CCR5, CCl3, and CxCl13 in the light group compared to the dark group, whereas the dark group showed an increased expression of Aimp1 compared to the light group. Conclusions: Sleep fragmentation imparted a significant effect on both the normal circadian variation in the gut microbiome and also in the levels of cytokine expression within the mesenteric lymph nodes. Compared to controls, both the light and dark groups had a significant dysbiotic effect on the peak levels of Parabacteroides and Lactobacillus, yet still maintained circadian rhythmicity albeit reversed from normal. Sleep fragmentation also resulted in altered mucosal immunity, demonstrated by decreased levels of cytokine expression compared to controls. Sleep fragmentation may have profound effects on gut dysbiosis and immune defense.
Su1880 RORgt-Dependent CD4+ T Cells Garden the Mucosa-Associated Microbiome of the Small Intestine and Colon Jonathan Jacobs, Lin Lin, Venu Lagishetty, Paul Ruegger, James Borneman, Jonathan Braun Background: The intestinal microbiome and the mucosal immune system have reciprocal effects on composition and function. It has previously been shown that segmented filamentous bacteria (SFB) induce Th17 cells - which require the transcription factor ROR gt - and that SFB in the feces and colonic lumen are affected by ROR gt-dependent innate lymphoid cells (ILCs). We investigated whether ROR gt-dependent T cells garden the microbiome of the small intestine and colon. Methods: 16S rRNA sequencing was performed for luminal and mucosal samples from the duodenum, jejunum, ileum, cecum, proximal colon, and distal colon of mice deficient in Rag1, ROR gt, T cell receptor alpha (TCR), and IL-17A, or mice treated with anti-IL17A antibody. Heterozygote littermates were used as controls. ROR gt/ Rag1 double knockout mice were engrafted with pooled small intestinal and colonic lamina propria CD4+ T cells (wild-type, RORgt-/-, IL-17A-/-) via retroorbital injection and sacrificed at 6 weeks of age for microbiome analysis. The MLI cohort, consisting of cecum and sigmoid colon lavage samples from 103 healthy individuals and 76 Crohn's disease patients, was used to investigate the relationship of the rs4845604 polymorphism of RORC (the gene encoding RORgt) with human microbiome composition. Multivariate models incorporating genotype (or treatment), gender, mouse litter, and sample site were used to identify OTUs associated with host genotype, antibody treatment, and CD4+ T cell engraftment. Results: Mice deficient in ROR gt had an altered small intestinal and colonic mucosa-associated microbiome characterized by overgrowth of SFB. Similar shifts were observed in the distal small intestinal and colonic mucosa of mice lacking an adaptive immune system (Rag1) or T cells (TCR), implying a role for ROR gt-dependent T cells. IL-17A deficiency or antibody neutralization did not affect the small intestinal microbiome but increased colonic SFB, arguing that IL-17A specifically shapes the colonic microbiome. Engraftment of lamina propria CD4+ T cells, or fate-mapped lamina propria Th17 cells, into the small intestine of RORgt/Rag1 double knockout mice reduced small intestinal SFB in a manner dependent upon RORgt but not IL-17A. In humans, the rs4845604 polymorphism of RORC - previously found to be protective for inflammatory bowel disease (IBD) - was associated with altered microbial composition in mucosal wash samples from Crohn's disease patients and healthy individuals. Conclusion: RORgt-dependent T cells garden the colonic microbiome via IL17A and the small intestinal microbiome through non-IL-17A-dependent mechanisms. In animal models, these processes affect abundance of a Th17-inducing microbe. The association of microbial composition in human cohorts with RORC genotype suggests that genetic variation in gardening could influence susceptibility to diseases such as IBD.
AGA Abstracts
X : 10052$CH01 03-28-16 00:57:27 PDFd : 10052B : e
S-578
Page 578
Su1884 Influence of the Microbiome on Epigenetic Mechanisms in Inflammatory Bowel Disease (IBD) Tiago Medina, Alexander J. Murison, Sebastian Scheer, Michelle I. Smith, Mark S. Silverberg, Colby Zaph, Mathieu Lupien, Cheryl Arrowsmith, Daniel DeCarvalho Inflammatory Bowel Disease is thought to occur as a result of a complex interplay between genetics and environmental factors such as the intestinal microbiota, which leads to a dysregulated immune response. Here, we describe a model that allowed us to evaluate the importance of early exposure of the offspring to their mother`s microbiota and determined how microbiota may alter the epigenetic program of infiltrating CD4 T cells. In this model, the microbiota of Foxp3-EGFP reporter breading mice was depleted with an antibiotic cocktail from the time of their weaning through the weaning on their offspring. The use of antibiotics prevented the transfer of a conventional maternal microbiota to the offspring who are then separated from the mother at weaning and given normal drinking water. At 8 weeks of age the offspring were sacrificed, their splenic naïve CD4 T cells were isolated via FACS and transferred into RAG1-/- mice, which lack T and B cells. A control group of breading mice were not given antibiotics and the splenic naïve CD4 T cells of their offspring were also transfer into RAG1-/- mice. RAG1-/- mice that received a T cell adoptive transfer from the offspring of antibiotic treated parents experienced intense gut inflammation as measured by histology and weight loss, while those that received the control group cells did not. This suggests that the inflammation was due to a microbiome difference and not the transfer itself. As further evidence, cohousing the offspring of antibiotic-treated and nontreated parents prior to adoptive transfer restored the microbiota of the treated offspring, as determined by qPCR of dominant taxa, and prevented their RAG1-/- recipient mice from developing intestinal inflammation. CD4 T cells from the offspring of antibiotic treated mothers strongly proliferated after in vitro stimulation compared to the control group. With the transfer of CD4 T cells into RAG1-/- mice, we saw a drastic change in the methylome between treated naïve CD4 T cells prior to transfer and those recovered from the RAG1-/mice after transfer, a change not observed in the control group naïve CD4 T cells. Preventing exposure of the offspring to their mother's microbiota by maternal antibiotic treatment was also able to modulate epigenetic changes in the CD4 T cells of the offspring, promoting the hypermethylation of anti-inflammatory genes, such as IL-10RB, ICOSL, IL-4R, HLA-G SOCS1, GRP83 (Treg inductor) and NKIRAS2 (NF- kB inhibitor) and the hypomethylation of pro-inflammatory genes, such as IFNG, IFNAR2, IL-17RB, IL-15RA, IL1RL1, GZMB, CD27, CD70, ALCAM (adhesion molecule), CCR4 and CCR5. In conclusion, the exposure of the offspring to their mother's microbiota during the parturition induces an epigenetic program capable of repressing pro-inflammatory genes and induces tolerogenic genes in CD4 T cells, preventing the inflammatory outcome.
Fig. 1. Circadian variation in microbiome in mice under control (CTRL, C), light SF (Light, L) and dark SF (Dark, D). Total reads for all microbiota (A). Select genera across conditions (B).
Su1885 Fig. 2. Ten days of light SF markedly suppresses mesenteric lymph node (MLN) cytokine gene expression. MLNs were pooled (4-5 mice/group), RNA isolated and cytokine profiles determined by qPCR array. Data are mean fold change relative to control (non-SF) mice for CCR5 and its ligands (A), Cxcr5/Cxcl13 (B) and multifunctional cytokine Aimp1 (C).
Colitis Vaccine: Flagellin-Elicited Immunity Keeps Motile Bacteria In-Check and Protects Against Intestinal Inflammation Benoit Chassaing, Hao Tran, Andrew T. Gewirtz BACKGROUND: Inflammatory Bowel Disease (IBD) is driven by a breakdown in the normally mutually beneficial host-microbiota relationship. Manipulating the host portion of this relationship, particularly antagonizing immune-promoting cytokines, has improved IBD management. We recently reported that disturbance of the intestinal microbiota using three different approaches (infectious agent, genetic predisposition and environmental factor) leads to a microbiota with increased pro-inflammatory potential and development of intestinal inflammation. Approaches to impact the microbiota to ameliorate IBD are not well developed despite the appreciation that microbiota composition has a great impact on disease outcome. GOAL: We hypothesize that manipulating the microbiota so as to make it inherently less pro-inflammatory (i.e. reduce levels of innate immune activators) may ultimately provide a novel approach to prevent and/or treat chronic intestinal inflammation. METHODS: Mice were treated with either PBS or purified flagellin (10µg) weekly by intraperitoneal injection. Feces were collected weekly and used for downstream analysis: fecal flagellin and LPS via cell-based reporter assay, inflammatory marker lipocalin-2 via ELISA, fecal and serum antiflagellin IgA and IgG via ELISA, and microbiota composition via 16S RNA Illumina Miseq sequencing. 12 weeks following the initial treatment, mice were either euthanized and intestinal samples processed to measure the intestinal inflammatory state (histopathology via hematoxylin & eosin staining, myeloperoxidase enzymatic activity, bacterial localization via confocal microscopy), or subjected to anti-IL-10 receptor treatment before tissue collection. RESULTS: Weekly treatment of mice with flagellin led to a strong induction of serum anti-flagellin IgG and fecal anti-flagellin IgA. Moreover, such immunization against bacterial flagellin was associated with strong modification of fecal microbiota composition that associated with a decrease in pro-inflammatory potential, as revealed by a decrease in the level of fecal bioactive flagellin. Such flagellin immunization and microbiota composition alteration protected mice against intestinal inflammation induced by anti-IL-10 receptor antibody treatment, as revealed by decreased spleen weight as well as decrease colon weight/length ratio in immunized animals compared to PBS-treated group. CONCLUSIONS: Flagellin inoculation alters microbiota composition and decreases level of fecal flagellin, and associated with protection against intestinal inflammation development. The mucosal immune system holds the power to reshape the microbiota and protect against inflammatory bowel disease.
Su1883 Loss of Tumor Necrosis Factor Receptor 1 Induces Severe Early-Onset Colitis in IL10 Knockout Mice in a Microbiome-Dependent Manner Sharon S. Tam, Philip E. Dubé, Rabea Alhosh, Shivesh Punit, Nandini Girish, Kay Washington, D. Brent Polk Tumor necrosis factor (TNF) is a therapeutic target in inflammatory bowel disease (IBD) and TNF receptor 1 (TNFR1) polymorphisms have been described in IBD patients. In fact, synergistic effects between TNFR1 and interleukin 10 (IL10) variants in early-onset IBD have been described. Although TNF and TNFR1 have been implicated in IBD pathogenesis, TNF also provides protective physiological roles in host defense and inflammatory resolution, suggesting that TNFR1 may regulate inflammatory responses to the commensal microbiome in IBD. Il10-/- mice provide a spontaneous model of colitis driven by the lack of immune tolerance to normal gut microbiota. We hypothesized that TNFR1 protects against colitis in Il10-/- mice by restraining dysbiosis and inflammation. Il10-/-Tnfr1+/- mice were crossed to generate Il10-/-Tnfr1-/- mice and control Il10-/-Tnfr1+/+ littermates. Co-housed mice underwent colonoscopy and barrier function assay (FD4 absorption) prior to euthanasia at 8 wk of age. A separate group of 8 wk-old Il10-/-Tnfr1-/- mice were treated with either antibiotics (neomycin and metronidazole) or water for 2 wk. Mice underwent colonoscopy before and after antibiotics, and endoscopic appearance was scored. Colon tissues were analyzed for histologic scoring, and mucosal immune cell populations were characterized by flow cytometry. Il10-/-Tnfr1-/- mice developed severe colitis in a highly reproducible manner by 8 wk of age, evidenced by both endoscopic and histologic scoring (p<0.01). Il10-/-Tnfr1-/- mice presented with extreme mucosal thickening and granularity by colonoscopy, and florid inflammation, crypt abscesses, marked enterocyte loss, and epithelial hyperplasia by histology, whereas Il10-/- littermates appeared healthy. FD4 absorption was increased in Il10-/Tnfr1-/- mice (p<0.0001). There were increases in colonic macrophages (F4/80+) and dendritic cells (MHCII+CD11c+) in mice lacking TNFR1. Furthermore, survival was reduced in Il10-/Tnfr1-/- mice vs. Il10-/- littermates (p<0.001), with 50% mortality by 12 wk. Antibiotics significantly improved colitis in Il10-/-Tnfr1-/- mice, inducing remission with reduced endoscopic (p<0.01) and histologic (p<0.01) scores. Antibiotics decreased mononuclear cells in
S-579
X : 10052$CH01 03-28-16 00:57:27 PDFd : 10052B : o
Page 579
AGA Abstracts
AGA Abstracts
the lamina propria, crypt abscesses, and hyperplasia. Il10-/-Tnfr1-/- mice develop early-onset severe colitis and antibiotic depletion rapidly induces remission. TNFR1 may inhibit colitis by restraining dysbiosis and mucosal immune responses, potentially macrophages and/or dendritic cells. Studies will determine how TNFR1 regulates disparate cell types in colitis and how these impact host-microbial interactions and which microbial factors determine disease severity in this model. Understanding how TNFR1 contributes to protection from colitis will lead to nuanced therapeutic avenues for IBD, particularly for early-onset IBD.
Su1881 Dietary Serine Controls the Competition Between Pathogenic and Commensal E. coli During Intestinal Inflammation Sho Kitamoto, Hiroko Nagao-Kitamoto, Peter Kuffa, Nobuhiko Kamada
AGA Abstracts
Su1879 Backgrounds: The Pathogenic E. coli, namely Adherent Invasive E. coli (AIEC), is accumulated in inflamed intestinal mucosa of patients with Crohn's disease (CD) and contributes to its pathogenesis. Previously we demonstrated that metabolically related gut commensal E. coli are able to outcompete the pathogenic E. coli for nutrients such as carbohydrates in steady state (Kamada et al., Science, 2012). However, the mechanisms of how pathogenic E. coli can gain the growth advantage over the commensal E. coli during inflammation remains poorly understood. Aims: We aimed to elucidate the mechanisms of how intestinal inflammation selectively fuels the growth of pathogenic E. coli as compared to that of non-pathogenic commensal E. coli in the inflamed gut. Methods: Human- and mouse-derived AIEC strains, LF82 and CUMT-8, were used in this study. To assess the competition of AIEC and commensal E. coli in vivo, AIEC was infected into specific-pathogen-free mice and AIEC and commensal E. coli strains were co-infected in germ-free mice. Dextran Sodium Sulfate (DSS) was used to induce intestinal inflammation. Bacterial burden of each strain in feces was monitored by the titration using antibiotics resistance. Luminal metabolites were analyzed by CE-TOF/ MS. To determine the preference of amino acids on the growth of E. coli, each E. coli strain was cultured in vitro in a minimal media supplemented with each single amino acid, and then bacterial proliferation was determined by density measurement. To access the importance of dietary amino acids on bacterial fitness in the gut, amino acid defined control diet, protein free diet and serine-glycine deficient diet were applied to bacterial competition assay in vivo. Results: The results we obtained in this study are as follows: 1) Both human and mouse AIEC strains can obtain the growth advantage over commensal E. coli during DSS colitis. 2) Inflammation alters the luminal nutrient availability including amino acids. 3) The deprivation of dietary amino acids prevents outgrowth of AIEC in the inflamed but not in the steady-state gut. 4) An amino acid serine selectively promotes growth of pathogenic, but not commensal, E. coli strains both in vitro and in vivo. Conclusion: Dietary serine controls the competitive fitness of pathogenic E. coli in the inflamed gut. These results provide novel insights into understanding of the pathophysiology of E. coli-driven intestinal inflammation in patients with CD, and expected to contribute to the development of new therapeutic approaches that specifically target the nutrients utilized by pathobionts.
Spatial 3D-Stereomicroscopic, Microbial and Metabolic Characterization of Intestinal Villous Erosions and Ulcerations in Mice Alexander Rodriguez-Palacios, Ludovica F. Buttó, Ilya R. Bederman, Dirk Haller, Fabio Cominelli Background & Aims: Recently, a method based on 3D-stereomicroscopic (SM) assessment of the gut mucosal surface (3D-SMAP gut) revealed the 3D-spatial complexity of focal lesion distribution with particular focus on cobblestone-lesions in mice. The aim of this study was to characterize for the first time the spatial, microbial and metabolic attributes of two other spontaneous 3D-SM lesions: i) villous tip erosions and ii) ulcers in the small intestine of mice. Methods: Lesions were assessed by 3D-SM retrospectively (147 mice) and prospectively (>54 mice) in >6 models of ileitis/colitis, in combination with 16S rRNA microbiome analysis of SM-dissected areas affected by these two pathologies, and with the infection of SPF and germ-free mice with ulcer-associated microbes. Of novel interest, we developed a method to test the role of mechanical disruption as a contributor to villous erosions and ulcer formation, and we discovered a strategy to harvest the villous intestinal epithelial cells that cover the intestinal villi maintaining the 3D-‘villuos-cap' shape architecture, providing an ex vivo assay to study villous-microbe interaction using a proposed scoring system. Results: Intestinal ulcer occurrence was dependent on mouse strain and experimental set ranging from 0-27% of mice indicating the potential role of the gut microbiota and mouse genetics in predisposition to ulcer formation. The ulcer shape that predominates was linear (1-3 villi wide, along circumferential gut axis). With aging, the ulcer occurrence/size did not increase, suggesting ulcers can be self-contained. With respect to villous erosions, at least one lesion was seen in 0-19% of mice/set. SM-dissection of lesions showed microbial communities enriched in Escherichia coli, Lactobacillus gasseri, Staphylococcus saprophiticus/ xylosus, and Achromobacter sp. Germ-free mice infected with these microbes displayed E. coli and Lactobacillus in feces and increased E. coli counts in ulcers (pair t-test, p<0.05). With respect to gut epithelial cells, glucose consumption in our in vitro ‘villous cap' assay using normal villi with a background mucosal-associated microbiome resulted in similar glucose consumption for three O2 conditions. In contrast, the ‘villous caps' incubated with ulcer-associated microbial lysates increased glucose consumption hypoxically (ANOVA, p<0.0046), and produce 3D-villous abnormalities. Conclusion: Intestinal villous erosions and ulcerations are unrecognized lesions in mice that should be expected to occur and confound experimental research. Our spatial, microbiological and metabolic analysis indicate that ulcer formation may be a self-contained pathology that may occur as a result of the combination of mechanical disruption and underlying metabolic and microbiologicallyenhanced immunological interactions that may vary with oxygen and carbon dioxide availability.
Su1882 Effect of Sleep Fragmentation on Composition of Gut Microbiome and Mesenteric Lymph Node Signaling Edward C. Oldfield, Rouzbeh Shams, Jennifer L. Copare, Laurie L. Wellman, Richard P. Ciavarra, Patric S. Lundberg, Larry D. Sanford, J. C. Ware, David A. Johnson Background: Both the gut microbiome and sleep fragmentation have been shown to influence the immune system, which can lead to altered expression of cytokines involved in gut integrity. This study evaluated the effect of sleep fragmentation on gut microbiome composition and cytokine regulation/expression. Methods: Sleep fragmentation was induced using a custom-designed cage with an automated motorized sweeper, which provided intermittent tactile stimulation every 2 minutes during either the light period (normal sleeping time), dark period (normal awake time), or control group (no sleep fragmentation). Control mice were allowed to sleep undisturbed in their home cage. Stool specimens were collected from 14 mice (5 control, 5 light, 4 dark) at 6 hour intervals (0000, 0600, 1200, 1800) for each of the following: 10 days sleep fragmentation, 20 days recovery, and after 10 days of a second sleep fragmentation period. DNA was then isolated from the collected stool specimen using a Mo Bio PowerFecal® DNA isolation kit and the samples were processed using nextgeneration sequencing methods. At the end of the study period, the mice were sacrificed and mesenteric lymph nodes were pooled by group and sampled using qPCR inflammatory cytokine arrays to assess changes in mucosal immunity essential for maintaining intestinal integrity. Results: Over 150 bacterial genera showed alterations with sleep fragmentation compared to control. Bacterial genera Lactobacillus and Parabacteroides showed significant circadian variability (with a reversal from normal pattern) (Figure 1). Across all groups, Parabacteroides peaked between 1800-0000 with a nadir around 0600, whereas Lactobacillus peaked between 1200-1800 with a nadir at 0000. Furthermore, the peak concentrations of both Parabacteroides and Lactobacillus were greater in the sleep fragmentation mice (both light and dark) compared to controls. Multiple pronounced changes in cytokines were induced by sleep fragmentation compared to controls (figure 2); among the most notable alterations was a decreased expression of cytokines CCR5, CCl3, and CxCl13 in the light group compared to the dark group, whereas the dark group showed an increased expression of Aimp1 compared to the light group. Conclusions: Sleep fragmentation imparted a significant effect on both the normal circadian variation in the gut microbiome and also in the levels of cytokine expression within the mesenteric lymph nodes. Compared to controls, both the light and dark groups had a significant dysbiotic effect on the peak levels of Parabacteroides and Lactobacillus, yet still maintained circadian rhythmicity albeit reversed from normal. Sleep fragmentation also resulted in altered mucosal immunity, demonstrated by decreased levels of cytokine expression compared to controls. Sleep fragmentation may have profound effects on gut dysbiosis and immune defense.
Su1880 RORgt-Dependent CD4+ T Cells Garden the Mucosa-Associated Microbiome of the Small Intestine and Colon Jonathan Jacobs, Lin Lin, Venu Lagishetty, Paul Ruegger, James Borneman, Jonathan Braun Background: The intestinal microbiome and the mucosal immune system have reciprocal effects on composition and function. It has previously been shown that segmented filamentous bacteria (SFB) induce Th17 cells - which require the transcription factor ROR gt - and that SFB in the feces and colonic lumen are affected by ROR gt-dependent innate lymphoid cells (ILCs). We investigated whether ROR gt-dependent T cells garden the microbiome of the small intestine and colon. Methods: 16S rRNA sequencing was performed for luminal and mucosal samples from the duodenum, jejunum, ileum, cecum, proximal colon, and distal colon of mice deficient in Rag1, ROR gt, T cell receptor alpha (TCR), and IL-17A, or mice treated with anti-IL17A antibody. Heterozygote littermates were used as controls. ROR gt/ Rag1 double knockout mice were engrafted with pooled small intestinal and colonic lamina propria CD4+ T cells (wild-type, RORgt-/-, IL-17A-/-) via retroorbital injection and sacrificed at 6 weeks of age for microbiome analysis. The MLI cohort, consisting of cecum and sigmoid colon lavage samples from 103 healthy individuals and 76 Crohn's disease patients, was used to investigate the relationship of the rs4845604 polymorphism of RORC (the gene encoding RORgt) with human microbiome composition. Multivariate models incorporating genotype (or treatment), gender, mouse litter, and sample site were used to identify OTUs associated with host genotype, antibody treatment, and CD4+ T cell engraftment. Results: Mice deficient in ROR gt had an altered small intestinal and colonic mucosa-associated microbiome characterized by overgrowth of SFB. Similar shifts were observed in the distal small intestinal and colonic mucosa of mice lacking an adaptive immune system (Rag1) or T cells (TCR), implying a role for ROR gt-dependent T cells. IL-17A deficiency or antibody neutralization did not affect the small intestinal microbiome but increased colonic SFB, arguing that IL-17A specifically shapes the colonic microbiome. Engraftment of lamina propria CD4+ T cells, or fate-mapped lamina propria Th17 cells, into the small intestine of RORgt/Rag1 double knockout mice reduced small intestinal SFB in a manner dependent upon RORgt but not IL-17A. In humans, the rs4845604 polymorphism of RORC - previously found to be protective for inflammatory bowel disease (IBD) - was associated with altered microbial composition in mucosal wash samples from Crohn's disease patients and healthy individuals. Conclusion: RORgt-dependent T cells garden the colonic microbiome via IL17A and the small intestinal microbiome through non-IL-17A-dependent mechanisms. In animal models, these processes affect abundance of a Th17-inducing microbe. The association of microbial composition in human cohorts with RORC genotype suggests that genetic variation in gardening could influence susceptibility to diseases such as IBD.
AGA Abstracts
X : 10052$CH01 03-28-16 00:57:27 PDFd : 10052B : e
S-578
Page 578
Su1884 Influence of the Microbiome on Epigenetic Mechanisms in Inflammatory Bowel Disease (IBD) Tiago Medina, Alexander J. Murison, Sebastian Scheer, Michelle I. Smith, Mark S. Silverberg, Colby Zaph, Mathieu Lupien, Cheryl Arrowsmith, Daniel DeCarvalho Inflammatory Bowel Disease is thought to occur as a result of a complex interplay between genetics and environmental factors such as the intestinal microbiota, which leads to a dysregulated immune response. Here, we describe a model that allowed us to evaluate the importance of early exposure of the offspring to their mother`s microbiota and determined how microbiota may alter the epigenetic program of infiltrating CD4 T cells. In this model, the microbiota of Foxp3-EGFP reporter breading mice was depleted with an antibiotic cocktail from the time of their weaning through the weaning on their offspring. The use of antibiotics prevented the transfer of a conventional maternal microbiota to the offspring who are then separated from the mother at weaning and given normal drinking water. At 8 weeks of age the offspring were sacrificed, their splenic naïve CD4 T cells were isolated via FACS and transferred into RAG1-/- mice, which lack T and B cells. A control group of breading mice were not given antibiotics and the splenic naïve CD4 T cells of their offspring were also transfer into RAG1-/- mice. RAG1-/- mice that received a T cell adoptive transfer from the offspring of antibiotic treated parents experienced intense gut inflammation as measured by histology and weight loss, while those that received the control group cells did not. This suggests that the inflammation was due to a microbiome difference and not the transfer itself. As further evidence, cohousing the offspring of antibiotic-treated and nontreated parents prior to adoptive transfer restored the microbiota of the treated offspring, as determined by qPCR of dominant taxa, and prevented their RAG1-/- recipient mice from developing intestinal inflammation. CD4 T cells from the offspring of antibiotic treated mothers strongly proliferated after in vitro stimulation compared to the control group. With the transfer of CD4 T cells into RAG1-/- mice, we saw a drastic change in the methylome between treated naïve CD4 T cells prior to transfer and those recovered from the RAG1-/mice after transfer, a change not observed in the control group naïve CD4 T cells. Preventing exposure of the offspring to their mother's microbiota by maternal antibiotic treatment was also able to modulate epigenetic changes in the CD4 T cells of the offspring, promoting the hypermethylation of anti-inflammatory genes, such as IL-10RB, ICOSL, IL-4R, HLA-G SOCS1, GRP83 (Treg inductor) and NKIRAS2 (NF- kB inhibitor) and the hypomethylation of pro-inflammatory genes, such as IFNG, IFNAR2, IL-17RB, IL-15RA, IL1RL1, GZMB, CD27, CD70, ALCAM (adhesion molecule), CCR4 and CCR5. In conclusion, the exposure of the offspring to their mother's microbiota during the parturition induces an epigenetic program capable of repressing pro-inflammatory genes and induces tolerogenic genes in CD4 T cells, preventing the inflammatory outcome.
Fig. 1. Circadian variation in microbiome in mice under control (CTRL, C), light SF (Light, L) and dark SF (Dark, D). Total reads for all microbiota (A). Select genera across conditions (B).
Su1885 Fig. 2. Ten days of light SF markedly suppresses mesenteric lymph node (MLN) cytokine gene expression. MLNs were pooled (4-5 mice/group), RNA isolated and cytokine profiles determined by qPCR array. Data are mean fold change relative to control (non-SF) mice for CCR5 and its ligands (A), Cxcr5/Cxcl13 (B) and multifunctional cytokine Aimp1 (C).
Colitis Vaccine: Flagellin-Elicited Immunity Keeps Motile Bacteria In-Check and Protects Against Intestinal Inflammation Benoit Chassaing, Hao Tran, Andrew T. Gewirtz BACKGROUND: Inflammatory Bowel Disease (IBD) is driven by a breakdown in the normally mutually beneficial host-microbiota relationship. Manipulating the host portion of this relationship, particularly antagonizing immune-promoting cytokines, has improved IBD management. We recently reported that disturbance of the intestinal microbiota using three different approaches (infectious agent, genetic predisposition and environmental factor) leads to a microbiota with increased pro-inflammatory potential and development of intestinal inflammation. Approaches to impact the microbiota to ameliorate IBD are not well developed despite the appreciation that microbiota composition has a great impact on disease outcome. GOAL: We hypothesize that manipulating the microbiota so as to make it inherently less pro-inflammatory (i.e. reduce levels of innate immune activators) may ultimately provide a novel approach to prevent and/or treat chronic intestinal inflammation. METHODS: Mice were treated with either PBS or purified flagellin (10µg) weekly by intraperitoneal injection. Feces were collected weekly and used for downstream analysis: fecal flagellin and LPS via cell-based reporter assay, inflammatory marker lipocalin-2 via ELISA, fecal and serum antiflagellin IgA and IgG via ELISA, and microbiota composition via 16S RNA Illumina Miseq sequencing. 12 weeks following the initial treatment, mice were either euthanized and intestinal samples processed to measure the intestinal inflammatory state (histopathology via hematoxylin & eosin staining, myeloperoxidase enzymatic activity, bacterial localization via confocal microscopy), or subjected to anti-IL-10 receptor treatment before tissue collection. RESULTS: Weekly treatment of mice with flagellin led to a strong induction of serum anti-flagellin IgG and fecal anti-flagellin IgA. Moreover, such immunization against bacterial flagellin was associated with strong modification of fecal microbiota composition that associated with a decrease in pro-inflammatory potential, as revealed by a decrease in the level of fecal bioactive flagellin. Such flagellin immunization and microbiota composition alteration protected mice against intestinal inflammation induced by anti-IL-10 receptor antibody treatment, as revealed by decreased spleen weight as well as decrease colon weight/length ratio in immunized animals compared to PBS-treated group. CONCLUSIONS: Flagellin inoculation alters microbiota composition and decreases level of fecal flagellin, and associated with protection against intestinal inflammation development. The mucosal immune system holds the power to reshape the microbiota and protect against inflammatory bowel disease.
Su1883 Loss of Tumor Necrosis Factor Receptor 1 Induces Severe Early-Onset Colitis in IL10 Knockout Mice in a Microbiome-Dependent Manner Sharon S. Tam, Philip E. Dubé, Rabea Alhosh, Shivesh Punit, Nandini Girish, Kay Washington, D. Brent Polk Tumor necrosis factor (TNF) is a therapeutic target in inflammatory bowel disease (IBD) and TNF receptor 1 (TNFR1) polymorphisms have been described in IBD patients. In fact, synergistic effects between TNFR1 and interleukin 10 (IL10) variants in early-onset IBD have been described. Although TNF and TNFR1 have been implicated in IBD pathogenesis, TNF also provides protective physiological roles in host defense and inflammatory resolution, suggesting that TNFR1 may regulate inflammatory responses to the commensal microbiome in IBD. Il10-/- mice provide a spontaneous model of colitis driven by the lack of immune tolerance to normal gut microbiota. We hypothesized that TNFR1 protects against colitis in Il10-/- mice by restraining dysbiosis and inflammation. Il10-/-Tnfr1+/- mice were crossed to generate Il10-/-Tnfr1-/- mice and control Il10-/-Tnfr1+/+ littermates. Co-housed mice underwent colonoscopy and barrier function assay (FD4 absorption) prior to euthanasia at 8 wk of age. A separate group of 8 wk-old Il10-/-Tnfr1-/- mice were treated with either antibiotics (neomycin and metronidazole) or water for 2 wk. Mice underwent colonoscopy before and after antibiotics, and endoscopic appearance was scored. Colon tissues were analyzed for histologic scoring, and mucosal immune cell populations were characterized by flow cytometry. Il10-/-Tnfr1-/- mice developed severe colitis in a highly reproducible manner by 8 wk of age, evidenced by both endoscopic and histologic scoring (p<0.01). Il10-/-Tnfr1-/- mice presented with extreme mucosal thickening and granularity by colonoscopy, and florid inflammation, crypt abscesses, marked enterocyte loss, and epithelial hyperplasia by histology, whereas Il10-/- littermates appeared healthy. FD4 absorption was increased in Il10-/Tnfr1-/- mice (p<0.0001). There were increases in colonic macrophages (F4/80+) and dendritic cells (MHCII+CD11c+) in mice lacking TNFR1. Furthermore, survival was reduced in Il10-/Tnfr1-/- mice vs. Il10-/- littermates (p<0.001), with 50% mortality by 12 wk. Antibiotics significantly improved colitis in Il10-/-Tnfr1-/- mice, inducing remission with reduced endoscopic (p<0.01) and histologic (p<0.01) scores. Antibiotics decreased mononuclear cells in
S-579
X : 10052$CH01 03-28-16 00:57:27 PDFd : 10052B : o
Page 579
AGA Abstracts
AGA Abstracts
the lamina propria, crypt abscesses, and hyperplasia. Il10-/-Tnfr1-/- mice develop early-onset severe colitis and antibiotic depletion rapidly induces remission. TNFR1 may inhibit colitis by restraining dysbiosis and mucosal immune responses, potentially macrophages and/or dendritic cells. Studies will determine how TNFR1 regulates disparate cell types in colitis and how these impact host-microbial interactions and which microbial factors determine disease severity in this model. Understanding how TNFR1 contributes to protection from colitis will lead to nuanced therapeutic avenues for IBD, particularly for early-onset IBD.