sregulon is upregulated in luminal E. coli from long-term monoassociated IL-10-/- mice (KO)with colitis compared to bacteria from healthy wild-type (WT) controls. Since colonicinflammation is associated with lower luminal pH, we chose to focus on two members ofthe stress response regulon that encode enzymes important for bacterial fitness in acidicenvironments, gadA and B. We hypothesized that gadAB expression varies with the phaseof colitis and may also be enhanced by biologically relevant pro-inflammatory factors.METHODS: WT and KO mice were monoassociated with a commensal E. coli strain (NC101).Cecal bacterial gadAB expression was measured using real-time PCR, histological inflamma-tion was quantified using a validated scoring system, and spontaneous secretion of IL-12/23p40 from intestinal explant cultures was assessed using ELISA. GadAB expression incultured NC101 treated with conditioned media from stimulated splenocytes or recombinantcytokines was measured using real-time PCR. The effect of phagocytosis of NC101 bymacrophages on gadAB transcription was also assessed. RESULTS: Interestingly, during thefirst 2 weeks of monoassociation, prior to onset of histological inflammation, gadAB expressionis higher in NC101 from WT than KO mice. However, at later time points, when aggressivehistological inflammation is present, expression of gadAB in NC101 from KO mice is greatercompared to WT. NC101 gadAB mRNA was also significantly increased in bacteria grownIn Vitro and stimulated with recombinant TNF and IFNγ compared to media control.Furthermore, NC101 ingested by macrophages increase gadAB transcription 1 hour post-infection, coinciding with the predicted time of phagolysosomal acidification. CONCLU-SIONS: The E. coli stress response genes, gadAB, are differentially regulated in a bimodalfashion during the early phases of experimental colitis and are increased in response to pro-inflammatory cytokines and macrophage phagocytosis In Vitro. Further investigation of theimpact of E. coli gadAB expression on the development of colitis is warranted and willprovide novel insights into the pathogenesis of IBD.GadAB expression relative to bacterial 16S (fold difference)
Dysbiosis in Inflammatory Bowel Disease: A Link With Antimicrobial PeptidesSecretion?Sylvie Rajca, Henri Duboc, Harry Sokol, Lydie Humbert, Virginie Grondin, GinetteThomas, Chantal Bridonneau, Philippe Langella, Camille Mayeur, Laurent Beaugerie,Jacques Cosnes, Joelle Masliah, Dominique Rainteau, Philippe Seksik
Aim: Alterations of intestinal microbiota (dysbiosis) are involved in inflammatory boweldiseases (IBD) pathogenesis. Antimicrobial peptides (AMP) such as defensins play a pivotalrole in the regulation of the intestinal microbiota. It has been also shown that microbiotaparticipates in AMP secretion giving a picture of a regulation loop. Besides, defensinsdeficiency (mRNA, immunohistochemistry), has been incriminated in IBD pathogenesis.More precisely, the deficiency implied human beta-defensin 1 (hBD1) in IBD and alpha-defensin 5 (HD5) in ileal Crohn's disease. The aim of our study was to look for a linkbetween IBD associated dysbiosis and gut intraluminal expression of hBD1 and HD5 humandefensins. Materials and Methods: Faecal samples were collected from 31 healthy subjectsand 41 patients with colonic IBD including 12 patients with Crohn's disease, 29 withulcerative colitis and among them 18 were in remission and 23 with active disease. Exclusioncriteria were the use of antibiotics within 3 months before sampling and ileal disease orresection. Bacterial composition of the microbiota in term of main phylogenetic groups andbacterial species, was performed using quantitative real-time polymerase chain reaction. Indry stools, hBD1 and HD5 were measured by liquid chromatography coupled to tandemmass spectrometry. Using Pearson test, correlation between the bacterial composition of themicrobiota and intraluminal rate of human defensins has been investigated. Results: Dysbiosishas been observed in IBD characterized by a low counts of Firmicutes (Clostridium leptump<0.0001, Clostridium coccoides p<0.0001 groups) and Faecalibacterium prausnitzii p=0.0006 at the species level. Enterobacteria group (p=0.0003) and Escherichia coli (p=0.0002)were more abundant in patients with an active IBD. In parallel, hBD1 was decreased infaecal samples of IBD patients (168.2+/-158.7 ng/g feces) compared to healthy subjects(296.8 +/- 180.9ng/g feces, p=0.005). It was the same for HD5 (16.8+/-15.3 ng/g feces inIBD vs 66.2+/-35.8 ng/g feces in healthy subjects, p<0.0001). We observed that hBD1expression was positively correlated to F. prausnitzii counts and negatively correlated to theBifidobacteria counts. HD5 expression was positively correlated to F. prausnitzii and C.leptum counts and negatively correlated to E. coli, Enterobacteria and C. coccoïdes counts.Conclusion Our results confirmed a dysbiosis in IBD centered by low counts in Firmicutesand especially F. prausnitzii. This dysbiosis was associated with an intraluminal decrease ofhBD1 and HD5 rates in colonic IBD. Correlations between some bacterial groups and thedrop of hBD1 and HD5 suggest a direct or indirect regulation of intraluminal AMP byintestinal bacteria or its metabolites. IBD dysbiosis could therefore play a role in a chronicintestinal inflammation disturbing AMP secretion.
High-Fat Diets Cause a Shift From Anti-Inflammatory IgA to PRO-Inflammatory IgG Responses Against Commensal Gut Bacteria: A NovelMechanism for Diet-Induced Metabolic InflammationNadeem Mohammed, Lihua Tang, Willem de Villiers, Erik Eckhardt
BACKGROUND/AIM: High-fat diets cause obesity, metabolic syndrome and inflammation(“metabolic inflammation”). However, germfree mice are resistant to high-fat diet inducedmetabolic inflammation, which suggests that intestinal bacteria mediate pro-inflammatoryeffects of dietary fat. We aimed to test the hypothesis that high-fat dieting may causeinflammation by breaking down immunological tolerance to commensal micro-organisms.METHODS: C57Bl/6 mice were put on low- or high- fat diets (LFD or HFD; 10 or 60% ofKcal from fat) for up to 10 weeks. Plasma anti- Escherichia coli and anti-cecal content IgGlevels were determined with immunoblotting and ELISA. Total and anti- E. coli IgA weremeasured in the stool by ELISA. Intestinal lamina propria CD11C+ (dendritic) cells wereanalyzed for activation by flow cytometry. Lastly, plasma samples from lean (BMI 24.8 ±3.0), obese (BMI 41.1 ± 10.3), and obese / diabetic (BMI 40.6 ± 7.0) patients were screenedfor anti- E. coli antibodies by ELISA. RESULTS: High-fat dieting significantly increasedplasma levels of anti- E. coli IgG and anti-cecal extract IgG. Conversely, stool IgA levelsdecreased almost 4-fold, both for total IgA and for anti- E. coli IgA. Two weeks into high-fat dieting, there was an almost threefold increase in the percentage of CD80+CD11C+ andCD86+CD11C+ cells in the lamina propria of the small intestine. However, high-fat dietsdid not affect the percentage CD80/CD86+ CD11C+ cells in the colon. Lastly, plasma fromobese individuals contained more anti- E. coli IgG than plasma from lean controls. Strikingly,obese-diabetic patients had significantly higher anti- E.coli IgG than obese non-diabetics.CONCLUSION: High-fat diets cause a switch from anti-inflammatory mucosal IgA responsesagainst gut bacteria to pro-inflammatory systemic IgG responses. This has the potential toset the stage for systemic inflammation, which can disturb metabolism. The switch to IgGwas associated with activation of dendritic cells in the lamina propria of fat exposed smallintestine. We speculate that increased intestinal absorption of dietary fat impairs the defaultresponse of the intestinal mucosal immune system towards commensal bacteria. Lastly,blood tests for anti-E. coli IgG could be used to identify obese individuals at risk for diabetes.
Immune Profile of Gut Mucosa T Cells Associated With E.Coli LadenMacrophages in Crohn's DiseaseVanessa Avgousti, Neil B. Rayment, Tim Elliott, Barry N. Hudspith, Christos Karaiskos,Jonathan Brostoff, Jeremy D. Sanderson
Although the etiology of Crohn's disease (CD) remains unknown, there is increasing evidencethat a genetically determined dysregulated immune response to the resident intestinal micro-flora plays a significant role in the pathogenesis of CD. We have shown that in these patients,a population of intestinal macrophages harbors viable E.coli and it is therefore possible thatthis failure to kill and remove bacteria would contribute to the observed inflammatoryresponse. In previous studies we have shown that these E.coli laden macrophages produceless TNFα compared to those not infected with bacteria and increased T cells are seenclustering around these infected macrophages. It is therefore possible that this could leadto T cell activation and a defective cross-talk between the innate and adaptive immunesystems. In this study we have used co-localisation immunofluorescent techniques to investig-ate the immunological profile of T cells associated with E.coli laden macrophages, T cellsassociated with macrophages free of bacteria and those within control mucosa. Snap frozenmucosal biopsies were taken at routine colonoscopy from patients with CD and controlswith normal colorectal mucosa and cryostat sections made. Laden/unladen macrophageswere identified using CD-68 staining and a polyclonal anti E.coli and the T cells wereidentified using an anti CD3. In serial section the T cells were labeled with anti IL10, IL17,TNFα, TGFβ, FoxP3 or IL23receptor and the results analysed using confocal microscopy.We found that the profile of T cells surrounding laden macrophages compared to un-ladendemonstrated an elevated number of cells with a regulatory phenotype. More of these cellsexpressed IL-10 (67% versus 11%; p=0.001) and FoxP3 (14% versus 2%; p= 0.001). Incontrast, increased numbers of pro-inflammatory T cells lineage were seen surrounding un-laden macrophages as measured by cells staining positive for TNFα (22%), IL-17 (26%)and IL23 (30%) but were undetected in association with laden macrophages. We haveobserved both an increased number and distribution of T cells in CD and an altered profileof regulatory verses inflammatory lymphocytes within the lamina propria. This would suggestan underlying defect in the role of macrophage-to-T cell signaling, however more work isrequired to identify the mechanisms of this defective cross-talk and how an increase inregulatory cells leads to the obsereved inflammatory state seen in Crohn's Diesease.
Direct Methane Detection (DMD) in Breath Correlates With Quantitative StoolReal Time Polymerase Chain Reaction (RT-PCR) for Methane ProducingArchaea (MPA) in HumansKunal Gupta, Rachel P. Smith, Gianna Vannelli, Jonathan E. Nucum, André-Denis G.Wright, Peter L. Moses
Background: MPA have been implicated in a spectrum of pathogenic disorders in humansincluding inflammatory bowel disease, gastrointestinal (GI) cancer, functional GI disorders,anorexia and obesity. In previous studies, breath methane has been measured using avariety of protocols which often include pre-measurement diet restriction and/or ingestionof carbohydrate (CHO). Breath methane has been assumed to be a surrogate for gut MPAwithout validation or correlation with quantification of gut microorganisms in previousstudies. Using DMD and quantitative RT-PCR with mcrA primers, we compared exhaledmethane in parts per million (ppm) and the quantity of MPA in extracted DNA derivedfrom stool. Methods: Informed consent was obtained from subjects who agreed to providestool and breath samples. The protocol and consent form was approved by the institutionalreview board at the University of Vermont College of Medicine. Genomic DNA was extracted
from stool samples. RT- PCR was used to detect and quantify MPA by targeting the ArchaealQmcrA-F and QmcrA-R primers targeting the methyl-coenzyme M reductase subunit foundonly in MPAmcrA gene present in methanogenic Archea species. Direct measurement ofbreath methane was performed using the Quintron Breath Tracker SC (Quintron InstrumentCompany, Milwaukee, WI, USA) with subjects maintaining their usual diet. Results: 16subjects are reported: 9 M, 7F. Age: mean 34 years (18-53), BMI: mean 24.4 (20-31).MPAwere present at some level in 16/16 (100%) subjects by RT-PCR. 12/16 (75%) subject'sdemonstrated PCR detection with a threshold cycle value (Ct) greater than 30.22. Breathmethane was detected in 4/16 (25%) of the subjects and concentrations of exhaled methanecorrelated with RT-PCR Ct values ≤ 30.22. Conclusions: Breath methane has been assumedto be an accurate surrogate for the presence and quantity of MPA. However, only a singlereport by Stewart et al has compared methane production and stool RT-PCR in individuals.This study utilizes an improved strategy for identification of MPA by RT-PCR. Breath methanewas detected using DMD with subjects with a Ct value ≤ 30.22 by PCR. Exhaled methaneconcentration correlated with PCR detection of MPA (1.33-30.67 ppm). These data suggestthat DMD done in an ambulatory setting over a short time period on un-prepped subjectsmay correlate with MPA concentration. Identification of individuals with high concentrationsof MPA may impact on a spectrum of clinical conditions. Expansion of this data base hasthe potential to validate the correlative relationship between MPA concentration and breathmethane measurement by DMD.
Intestinal Alkaline Phosphatase Detoxifies Bacterial ComponentsAngela K. Moss, Madhu S. Malo, Halim Yammine, Sayeda Nasrin Alam, SundaramRamasamy, Rakesh Biswas, Nur Muhammad, Kanakaraju Kaliannan, Obaida H. Batal, H.Shaw Warren, Elizabeth Hohmann, Richard A. Hodin
Background Intestinal alkaline phosphatase (IAP) is an enzyme known to protect hostsagainst a variety of conditions including Gram-negative bacteremia, peritoneal sepsis, andinflammatory bowel disease. Recent studies using individual, purified molecules have identi-fied three possible physiological targets for IAP: lipopolysaccharide (LPS), flagellin, and CpGDNA. Little is known, however, regarding how IAP interacts with actual bacterial components.Methods Increasing doses of LPS were incubated +/- IAP or polymixin B (a potent neutralizerof LPS) and applied to THP-1 monocytic cells. Tissue culture media were assayed at severaltime points for IL-8 concentration by ELISA. A variety of bacteria were then studied: Gram-negative (E. coli and Salmonella) and Gram-positive (Enterococcus, S. aureus, and Listeria).Overnight bacterial cultures were centrifuged, and the resuspended bacteria were thensonicated. The resulting bacterial lysates were sterilized by passage through a 0.2um filter,incubated +/- IAP or polymixin B overnight, then applied to THP-1 cells for 24 hours, andthe media were assayed for IL-8 concentration. Results Detoxification of LPS by both IAPand polymixin B was found to be dose- and time-dependent. Polymixin B was significantlymore effective, with IAP resulting in a 32% decrease in IL-8 level and polymixin B providinga 99% decrease in IL-8 production at 24 hours. As expected, IAP and polymixin B had nodetoxifying effect on PBS, LB broth, or TNF-alpha. IL-8 production induced by E. coli lysatewas decreased 16% by IAP and 84% by polymixin B (p=0.04 and 0.00004 respectivelycompared with control). Enterococcus lysates were not significantly detoxified by either IAPor polymixin B. IAP was effective at detoxifying S. aureus, Listeria, and Salmonella lysateswith 24%, 25%, and 36% decreases in IL-8 production, respectively (p<0.0001 for all),whereas polymixin only caused a 7% decrease for S. aureus and had no inhibitory effectson Listeria and Salmonella. Conclusion Whereas polymixin B only inhibits LPS, the IAPenzyme is effective at detoxifying pro-inflammatory products from both Gram-positive andGram-negative bacteria. These results suggest a broader physiologic role than previouslyrecognized for IAP in protection against inflammation caused by pathogenic bacteria.
Enhanced Susceptibility to an Inflammatory Insult Following Escherichia coliColonization is Associated With Changes in the Density and SpatialDistribution of the MicrobiotaAmanda E. Ramer-Tait, Anne-Marie C. Overstreet, Todd A. Atherly, Jesse M. Hostetter,Cherie J. Ziemer, Albert Jergens, Michael J. Wannemuehler
While it is clear that aberrant mucosal immune responses to microbial antigens significantlycontribute to inflammatory bowel disease, as do genetic and environmental factors, themechanisms by which resident enteric bacteria elicit pathognomonic responses are not wellunderstood. Our laboratory has previously demonstrated that prior colonization with theotherwise non-pathogenic bacterial provocateur, Escherichia coli SWW33, perturbs mucosalhomeostasis and predisposes to disease depending on the severity of a subsequent inflammat-ory trigger. Specifically, immunocompetent C3H/HeN mice that harbor a defined microbiota(DM; the altered Schaedler flora [ASF]) and are devoid of Escherichia species are subsequentlycolonized with E. coli SWW33 are not sensitive to a modest inflammatory insult deliveredin the form of low-dose (1.5%) dextran sulfate sodium (DSS) in drinking water. However,E. coli SWW33-colonized DM mice develop severe typhlitis when given a higher dose (2.5%)DSS while control DM mice do not. In addition, ASF antigen-specific IL-17 production wassignificantly increased in cultures of mesenteric lymph node CD4+ T cells from E. coli-colonized DM mice treated with high-dose DSS as compared to cultures of CD4+ T cellsfrom E. coli-colonized mice treated with low-dose DSS. The aim of this study was to furtherdefine the mechanisms by which the resident microbiota elicit pathognomonic responsesand how alterations in the gut microbiota selectively impact disease risk and severity in asusceptible host. We hypothesized that changes in the density and spatial distribution ofthe ASF would correlate with the enhanced antigen-specific adaptive immune responseobserved in E. coli-colonized DM mice treated with high-dose DSS. E. coli-colonized micetreated with high-dose DSS had significantly more E. coli present in the lumen of the cecumand proximal colon than sham and low-dose DSS treated DM mice colonized with E. coli.Colonization with E. coli and treatment with high-dose DSS also altered the density of theresident ASF, causing increases in the numbers of luminal Lactobacilli and Clostridium spp.as compared to treatments that caused no or mild disease. Clostridium spp. were often foundmucosally-associated within the adherent mucus layer and occasionally within the mucosa.
S-329 AGA Abstracts
Together, our data show that the more severe disease observed in DM mice treated withhigh-dose DSS is associated with an increase in the number of E. coli colonizing the host.Moreover, alterations to the microbial community following colonization with E. coli andexposure to an inflammatory insult may trigger enhanced immune responses directed againstthe microbiota.
Faecalibacterium Prausnitzii in Crohn's Disease: Hope or Hype? A SystematicReviewManeesh Dave, Emmanuel C. Gorospe, Jay Luther
Background: Growing evidence supports the critical role for intestinal microbiota in chronicinflammation observed in inflammatory bowel disease (IBD). It has been suggested thatalterations in the composition of intestinal microbiota modifies disease course. Faecalibacter-ium prausnitzii, a member of the firmicute family, is a commensal gut bacterium found inhumans. Recent data have illustrated lower levels of F prausnitzii in both stool and intestinalmucosal samples of patients with Crohn's disease, suggesting the bacteria offers a protectiveeffect against chronic inflammation. Aim: To perform a systematic review of studies comparingthe levels of F prausnitzii in patients with and without Crohn's diseases. Methods: A searchof MEDLINE, EMBASE, SCOPUS, DARE, and abstracts from key GI meetings (DDW, UEGWetc) was performed by two independent reviewers. Inclusion criteria required moleculardetection of F prausnitzii using polymerase chain reaction or terminal restriction fragmentlength polymorphism in mucosal biopsy or stool specimens of patients with and withoutCrohn's disease. Results: Of the 102 initial abstracts reviewed, 21 promising articles werereviewed in detail. 6 papers (N=293 subjects) met the inclusion criteria. One paper wasexcluded as it only reported lower recurrence of F prausnitzii in Crohn's patient with relapseafter surgery and had no control population. The total number of Crohn's and controlpatient's in the included studies was 183 and 110, respectively. All 6 papers in the analysisnoted a highly statistically significant reduction in F prausnitzii in Crohn's patients ascompared to controls. Data on the precise number of patients in each group was availablein only 4 papers (shown in table). Four studies used stool samples and two used biopsysamples for analysis. Variation in study design, differences in patient and control subjectsbaseline characteristics, variation in molecular method used to quantify F prausnitzii, andvariation in sample collection (mucosal versus stool) contributed to significant heterogeneity,precluding meta-analysis. Conclusion: The available data suggest lower levels of intestinalF prausnitzii in patients with Crohns disease, however significant inter study variabilitylimits the certainty of this finding. Our systematic review reveals the need for larger prospect-ive studies that better define patient populations, both in the Crohn's group (duration ofdisease, previous treatments) as well in the control group (age and sex-matched). Futurestudies should also standardize molecular techniques and obtain biopsy samples from intest-inal mucosa, and not just feces.Faecalibacterium prausnitzii in Crohn's disease
Blastocystis and Dientamoeba Fragilis in Active and Inactive InflammatoryBowel DiseaseAndreas M. Petersen, Henrik V. Nielsen, Christen Rune Stensvold, Jørgen H. Engberg,Alice Friis-Møller, Inge Nordgaard-Lassen, Signe Wildt, Karen A. Krogfelt
Background: The role of Blastocystis and of Dientamoeba fragilis in intestinal disease iscontroversial; studies have suggested a possible role of these parasitic infections in irritablebowel syndrome. Their possible role in inflammatory bowel disease (IBD) is, however,unknown. Aim: To identify possible differences in prevalence of infection with Blastocystisand D. fragilis in patients with active and inactive ulcerative colitis (UC) and Crohn's disease(CD), in patients with an ileal pouch anal anastomosis after UC unresponsive to medicaltherapy (IPAA) with or without pouchitis, and in controls (healthy controls and patientswith chronic diarrhoea due to other causes than IBD). Methods: All fecal samples weresubjected to microscopy for parasites, culture for Blastocystis and PCR for D. fragilis. Diseaseactivity was evaluated by disease ac-tivity indexes (Harvey Bradshaw Index, Clinical ActivityIndex and Modified Pouchitis Disease Activity Index); data were analyzed by chi-squaretest, two-sided. Results: Fecal samples were collected from a total of 117 patients andcontrols, consisting of 41 patients with UC (20 with active disease and 21 in remission),42 patients with CD (22 with active disease, 20 in remission), 17 patients with IPAA (9with pouchitis and 8 without), and 17 controls (8 healthy controls and 9 with chronicdiarrhoea from other causes than IBD). Blastocystis was significantly more prevalent inpatients with inactive UC (19 %) compared to patients with active UC (0 %), (p<0.05). Thedetection of Blastocystis in inactive UC did not differ significantly from the detection ratein controls (12 %). Blastocystis was not detected in patients with CD or IPAA, neither withactive nor with inactive dis-ease. D. fragilis was found significantly more often in patientswith inactive UC (30 %) compared to patients with active UC (5 %), (p<0.05), and the