Tu1702 A Balanced IL-1β Activity Is Required for Host Response to Citrobacter Rodentium Infection
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*p,0.001 vs. NC101 **p,0.01 vs. NC101-monoassociated mice Tu1699 Prenatal Methyl-Donor Micronutrient Supplementation Induces an Acute Colitis Prone Microbiome in Young Adult Mice Sabina Mir, Dorottya Nagy-Szakal, Reka Szigeti, Scot E. Dowd, C. W. Smith, Richard Kellermayer Background: Inflammatory bowel diseases (IBD) include Crohn disease and ulcerative colitis, which have become highly prevalent in developed countries around the world. The peak incidence of the disorders is in young adulthood. While the etiology of IBD is not known, an environmentally triggered exaggerated immune response against the intestinal microbiome is thought to mediate the diseases. The rapid emergence of IBD in the past decades has directed attention towards the potential dietary origins of the disease group. We examined how prenatal nutritional programming may impact predilection towards IBD in a murine model. Methods: C57Bl/6J mice were used. Dams were on control or methyl donor micronu- trient (MD) supplemented diets. Offspring were cross-fostered between these dams and subsequently weaned to a control diet. At 90 days of life (young adulthood) chemical colitis was induced by dextran sulfate sodium (DSS). Colonic mucosal Ppar-alpha (Ppara) expression was examined with real time RT-PCR in DSS Naïve cross-fostered mice. Colonic mucosal and cecal content microbiomes were interrogated with 454 based pyrosequencing of the 16S rRNA gene. The microbiome changes were tested functionally by colonizing germ free Swiss-Webster mice and exposing those to DSS colitis. Results: Prenatal MD supplementation was sufficient to modulate mucosal Ppara expression and worsen acute colitis in young adulthood. The prenatal dietary intervention induced the postnatal nurturing of a colitogenic microbiome. Conclusions: This is the first study to conclusively show that prenatal nutritional programming can modulate the mammalian host to harbor a colitogenic microbiome. These findings may be relevant for the nutritional developmental origins of IBD Tu1700 Increased Abundance of Bacteria Producing Hydrogen Sulfide in the Colonic Mucosa of Patients With Ulcerative Colitis and Crohn's Disease Franck Carbonero, Ann C. Benefiel, Jona Kristo, Jenna K. Leinberger, Maeve M. Leurck, Eugene Greenberg, H. Rex Gaskins The gut microbiota is increasingly suspected as a key factor in the etiology of inflammatory bowel disease (IBD). However, it is most often characterized in stool samples, which are only partially representative of the mucosal micro-environment where the inflammation originates. Here, a molecular-based approach was used to examine mucosa-associated microbiota in ileal, ascending and descending colon and rectal biopsies from ulcerative colitis (UC) and Crohn's disease (CD) patients, and healthy control subjects. We focused on sulfidogenic bacteria producing hydrogen sulfide, which is proinflammatory and a potent genotoxic agent. Targets included sulfate-reducing bacteria (SRB: dissimilatory sulfite reductase (dsrAB) gene of SRB and 16S rRNA genes of Desulfovibrio, Desulfobulbus, Desulfo- bacter and Desulfotomaculum), Bilophila wadsworthia (taurine:pyruvate aminotransferase (tpA)) and Fusobacterium nucleatum (Fn1419 and Fn1055 encoding enzymes that convert cysteine to hydrogen sulfide). These targets were quantified through qPCR targeting 16S rRNA genes and functional genes. A total of 321 biopsies, in some instances taken from inflamed and adjacent non-inflamed tissue, from 28 CD and 19 UC patients as well as 40 healthy subjects were quantified for the abundance of sulfidogenic microbes. A 10-fold greater abundance of SRB, in particular the genus Desulfotomaculum, was detected in inflamed tissue of CD and UC. Strikingly, all functional genes for sulfidogenic bacteria and SRB genera except Desulfobulbus were significantly more abundant in biopsies from IBD patients compared to healthy controls (at least 10 times more abundant for the more abundant genes). In addition, Bilophila wadsworthia was ubiquitous in IBD patients (99.5%) but much less prevalent for healthy controls (48%). This increased prevalence of SRB and genes mediating sulfide production from organic sulfur sources in IBD patients is consistent with the proinflammatory properties of hydrogen sulfide, which may contribute to chronic inflammation. As many of the patients examined were in remission, it can be hypothesized that they may be deficient in sulfide detoxification or other host pathways that provide tolerance to bacterial-produced sulfide. Together the data indicate an urgent need for system- atic study of the various microbial and host metabolic pathways involved. S-825 AGA Abstracts Tu1701 Pseudomonas Fluorescens Increases the Paracellular Permeability of Peyer's Patches and Ileal Mucosa by Il1r Dependent Mechanisms Ziad Alnabhani, Amar Madi, Monique Dussaillant, Marc Feuilloley, Jean-Pierre Hugot, Nathalie Connil, Frédérick Barreau Background & aims: Pseudomonas fluorescens is present in the intestinal lumen and has been proposed to play a role in Crohn's disease (CD). P. fluorescens alters epithelial permeabil- ity and translocates across Caco2/TC7 intestinal cells. As NOD2 is the main susceptibility CD gene and Nod2 is involved in the IL-1 β pathway, the aim of the study was to determine whether P. fluorescens and P. aeruginosa alter the paracellular permeability of Peyer's patches and ileal mucosa in mice and the respective roles of Nod2 and the IL-1 β pathways in these alterations. Methods: The behavior of the clinical strain P. fluorescens MFN1032 was compared to that of the psychrotrophic strain P. fluorescens MF37 and the opportunistic pathogen P. aeruginosa PAO1. Wild-type, Nod2-/- and IL1R-/- C57BL/6 mice models were inoculated intragastrically with 108 CFU of P. fluorescens or P. aeruginosa strains, and mice were killed 7 days after infection. Peyer's patches and ileal mucosa were removed and paracellular permeability (PP) was assessed in Ussing chamber (UC). PP was also studied ex vivo. For that, Peyer's patches and ileum biopsies were mounted in UC and then incubated with bacteria (10 8 CFU/mL) for 2 hours. Results: Seven days after infection with P. fluorescens MFN1032, P. fluorescens MF37 or P. aeruginosa PAO1 the PP of ileal mucosa was increased in WT mice but not in Nod2-/- and IL1R-/- mice. The ex vivo infection of ileal mucosa biopsies confirmed these findings. In Peyer's patches, P. fluorescens MFN1032 and P. aeruginosa PAO1 but not P. fluorescens MF37 increased the PP of WT mice. In contrast, PP of Peyer's patches was higher in Nod2-/- and IL1R-/- mice infected with P. fluorescens MF37 (but not with P. fluorescens MFN1032 and P. aeruginosa PAO1). These results indicate that Nod2 and IL1R pathway may prevent the alteration of the Peyer's patches PP induced by P. fluorescens MF37. Conclusion: We report here that P. fluorescens and P. aeruginosa are able to modify the homeostasis of the epithelial barrier function of Peyer's patches and ileum by IL1R dependent mechanisms. Tu1702 A Balanced IL-1β Activity Is Required for Host Response to Citrobacter Rodentium Infection Misagh Alipour, Yuefei Lou, Daniel Zimmerman, Aisha Baig, Daniel Muruve, Julia J. Liu, Eytan Wine Background and Aim: The intracellular nucleotide-binding oligomerization domain (NOD)- like receptors (NLRs) have emerged as important regulators of intestinal homeostasis, and play essential roles in the innate immune response to pathogens. In particular, the NLRP3 forms a multiprotein inflammasome complex responsible for the maturation and activation of caspase-1 and interleukin (IL)-1β. Recently, the Gram-negative mouse pathogen Citrobacter rodentium was shown to induce the NLRP3 inflammasome in macrophages and NLRP3-/- mice showed delayed clearance of the infection. This study was conducted to delineate the roles of NLRP3 inflammasome and IL-1 β in the host defense against acute C. rodentium infection in mice. Methods: NLRP3-/- and background C57BL/6 mice were infected by orogastric gavage with C. rodentium. After infection, mice received either IL-1 β (0.5 μg/ mouse; ip) or equal volume saline (control) on post-infection days 0, 2, and 4. On days 6 and 10, the mice were sacrificed. Bacterial counts in the intestinal tract and visceral organs were determined. Histopathology and crypt lengths of stained colon tissue were scored blindly. The epithelial integrity was assessed by biotin permeability and staining for C. rodentium. The inflammatory response was measured by cytokine levels. Colonic tissues were stained for macrophages and neutrophils as a measure of inflammatory cell infiltration, and cellular proliferation as a measure of colonic hyperplasia. Experiments were approved by the University of Alberta Animal Care Committee. Results: In WT mice, IL-1 β treatments failed to inhibit bacterial propagation to the spleen, lymph node, liver, and colon but significantly reduced the bacterial burden in NLRP3-/- mice over 10 days, as shown by bacterial counting and qPCR. On day 6-post infection NLRP3-/- mice had increased crypt length and there was a trend for reduced crypt length with IL-1 β treatment. In NLRP3-/- mice, the IL-1β treatments reduced C. rodentium attachment to the epithelial surface, decreased epithelial permeability and increased secretion of pro-inflammatory cytokines in the colon tissue, compared to WT mice. These findings were confirmed by histopathology and immune cell staining. Conclusion: Treatment of NLRP3-/- mice with IL-1 β appears to confer protection against C. rodentium infection by reducing bacterial colonization and epithelial permeability. Tu1703 Characterization of Humoral Immune Responses to C.difficile Toxins a and B in Patients With C. difficile-Associated Diarrhoea, Inflammatory Bowel Disease and Cystic Fibrosis Tanya M. Monaghan, Adrian Robins, Alan Knox, Herbert F. Sewell, Yashwant R. Mahida Patients with IBD are at increased risk of C. difficile infection, but those with cystic fibrosis (who are frequently admitted to hospital for antibiotics) may be protected against the disease. Methods. Four groups of patients were studied (i) C. difficile-associated diarrhoea (CDAD) in non-IBD patients (CDAD-NI), (ii) CDAD in IBD patients (CDAD-IBD), (iii) cystic fibrosis (CF) patients, without history of CDAD and (iv) healthy controls (HC). Serum antibody (IgG) levels were studied by ELISA and expressed as ELISA units (EU). Circulating antigen- activated B cells were investigated using Alexa Fluor 488-labelled toxin A (toxin A488) and flow cytometry using anti-CD19 and anti-IgD antibodies. Following induction of differentia- tion of memory B cells by polyclonal stimulation of peripheral blood mononuclear cells, toxin A- and B-specific antibody secreting cells (ASCs) were quantified using ELISPOT assays (and expressed as percentage of total IgG ASCs). In an additional group of IBD patients, with no history of C. difficile infection, secretion of toxin-specific IgG antibody by lamina propria cells was studied during culture of IBD small and large intestinal mucosal samples denuded of epithelial cells. Data are expressed as median (range). Results. Compared to AGA Abstracts
Transcript of Tu1702 A Balanced IL-1β Activity Is Required for Host Response to Citrobacter Rodentium Infection
*p,0.001 vs. NC101 **p,0.01 vs. NC101-monoassociated mice
Tu1699
Prenatal Methyl-Donor Micronutrient Supplementation Induces an AcuteColitis Prone Microbiome in Young Adult MiceSabina Mir, Dorottya Nagy-Szakal, Reka Szigeti, Scot E. Dowd, C. W. Smith, RichardKellermayer
Background: Inflammatory bowel diseases (IBD) include Crohn disease and ulcerative colitis,which have become highly prevalent in developed countries around the world. The peakincidence of the disorders is in young adulthood. While the etiology of IBD is not known,an environmentally triggered exaggerated immune response against the intestinal microbiomeis thought to mediate the diseases. The rapid emergence of IBD in the past decades hasdirected attention towards the potential dietary origins of the disease group. We examinedhow prenatal nutritional programming may impact predilection towards IBD in a murinemodel. Methods: C57Bl/6J mice were used. Dams were on control or methyl donor micronu-trient (MD) supplemented diets. Offspring were cross-fostered between these dams andsubsequently weaned to a control diet. At 90 days of life (young adulthood) chemical colitiswas induced by dextran sulfate sodium (DSS). Colonic mucosal Ppar-alpha (Ppara) expressionwas examined with real time RT-PCR in DSS Naïve cross-fostered mice. Colonic mucosaland cecal content microbiomes were interrogated with 454 based pyrosequencing of the16S rRNA gene. The microbiome changes were tested functionally by colonizing germ freeSwiss-Webster mice and exposing those to DSS colitis. Results: Prenatal MD supplementationwas sufficient to modulate mucosal Ppara expression and worsen acute colitis in youngadulthood. The prenatal dietary intervention induced the postnatal nurturing of a colitogenicmicrobiome. Conclusions: This is the first study to conclusively show that prenatal nutritionalprogramming can modulate the mammalian host to harbor a colitogenic microbiome. Thesefindings may be relevant for the nutritional developmental origins of IBD
Tu1700
Increased Abundance of Bacteria Producing Hydrogen Sulfide in the ColonicMucosa of Patients With Ulcerative Colitis and Crohn's DiseaseFranck Carbonero, Ann C. Benefiel, Jona Kristo, Jenna K. Leinberger, Maeve M. Leurck,Eugene Greenberg, H. Rex Gaskins
The gut microbiota is increasingly suspected as a key factor in the etiology of inflammatorybowel disease (IBD). However, it is most often characterized in stool samples, which areonly partially representative of the mucosal micro-environment where the inflammationoriginates. Here, a molecular-based approach was used to examine mucosa-associatedmicrobiota in ileal, ascending and descending colon and rectal biopsies from ulcerativecolitis (UC) and Crohn's disease (CD) patients, and healthy control subjects. We focusedon sulfidogenic bacteria producing hydrogen sulfide, which is proinflammatory and a potentgenotoxic agent. Targets included sulfate-reducing bacteria (SRB: dissimilatory sulfitereductase (dsrAB) gene of SRB and 16S rRNA genes of Desulfovibrio, Desulfobulbus, Desulfo-bacter and Desulfotomaculum), Bilophila wadsworthia (taurine:pyruvate aminotransferase(tpA)) and Fusobacterium nucleatum (Fn1419 and Fn1055 encoding enzymes that convertcysteine to hydrogen sulfide). These targets were quantified through qPCR targeting 16SrRNA genes and functional genes. A total of 321 biopsies, in some instances taken frominflamed and adjacent non-inflamed tissue, from 28 CD and 19 UC patients as well as 40healthy subjects were quantified for the abundance of sulfidogenic microbes. A 10-foldgreater abundance of SRB, in particular the genus Desulfotomaculum, was detected ininflamed tissue of CD and UC. Strikingly, all functional genes for sulfidogenic bacteria andSRB genera except Desulfobulbus were significantly more abundant in biopsies from IBDpatients compared to healthy controls (at least 10 times more abundant for the more abundantgenes). In addition, Bilophila wadsworthia was ubiquitous in IBD patients (99.5%) but muchless prevalent for healthy controls (48%). This increased prevalence of SRB and genesmediating sulfide production from organic sulfur sources in IBD patients is consistentwith the proinflammatory properties of hydrogen sulfide, which may contribute to chronicinflammation. As many of the patients examined were in remission, it can be hypothesizedthat they may be deficient in sulfide detoxification or other host pathways that providetolerance to bacterial-produced sulfide. Together the data indicate an urgent need for system-atic study of the various microbial and host metabolic pathways involved.
S-825 AGA Abstracts
Tu1701
Pseudomonas Fluorescens Increases the Paracellular Permeability of Peyer'sPatches and Ileal Mucosa by Il1r Dependent MechanismsZiad Alnabhani, Amar Madi, Monique Dussaillant, Marc Feuilloley, Jean-Pierre Hugot,Nathalie Connil, Frédérick Barreau
Background & aims: Pseudomonas fluorescens is present in the intestinal lumen and hasbeen proposed to play a role in Crohn's disease (CD). P. fluorescens alters epithelial permeabil-ity and translocates across Caco2/TC7 intestinal cells. As NOD2 is the main susceptibilityCD gene and Nod2 is involved in the IL-1 β pathway, the aim of the study was to determinewhether P. fluorescens and P. aeruginosa alter the paracellular permeability of Peyer's patchesand ileal mucosa in mice and the respective roles of Nod2 and the IL-1 β pathways inthese alterations. Methods: The behavior of the clinical strain P. fluorescens MFN1032 wascompared to that of the psychrotrophic strain P. fluorescens MF37 and the opportunisticpathogen P. aeruginosa PAO1. Wild-type, Nod2-/- and IL1R-/- C57BL/6 mice models wereinoculated intragastrically with 108 CFU of P. fluorescens or P. aeruginosa strains, and micewere killed 7 days after infection. Peyer's patches and ileal mucosa were removed andparacellular permeability (PP) was assessed in Ussing chamber (UC). PP was also studiedex vivo. For that, Peyer's patches and ileum biopsies were mounted in UC and then incubatedwith bacteria (10 8 CFU/mL) for 2 hours. Results: Seven days after infection with P.fluorescens MFN1032, P. fluorescens MF37 or P. aeruginosa PAO1 the PP of ileal mucosawas increased in WT mice but not in Nod2-/- and IL1R-/- mice. The ex vivo infection ofileal mucosa biopsies confirmed these findings. In Peyer's patches, P. fluorescens MFN1032and P. aeruginosa PAO1 but not P. fluorescens MF37 increased the PP of WT mice. Incontrast, PP of Peyer's patches was higher in Nod2-/- and IL1R-/- mice infected with P.fluorescens MF37 (but not with P. fluorescens MFN1032 and P. aeruginosa PAO1). Theseresults indicate that Nod2 and IL1R pathway may prevent the alteration of the Peyer'spatches PP induced by P. fluorescens MF37. Conclusion: We report here that P. fluorescensand P. aeruginosa are able to modify the homeostasis of the epithelial barrier function ofPeyer's patches and ileum by IL1R dependent mechanisms.
Tu1702
A Balanced IL-1β Activity Is Required for Host Response to CitrobacterRodentium InfectionMisagh Alipour, Yuefei Lou, Daniel Zimmerman, Aisha Baig, Daniel Muruve, Julia J. Liu,Eytan Wine
Background and Aim: The intracellular nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs) have emerged as important regulators of intestinal homeostasis, andplay essential roles in the innate immune response to pathogens. In particular, the NLRP3forms a multiprotein inflammasome complex responsible for the maturation and activation ofcaspase-1 and interleukin (IL)-1β. Recently, the Gram-negative mouse pathogen Citrobacterrodentium was shown to induce the NLRP3 inflammasome in macrophages and NLRP3-/-mice showed delayed clearance of the infection. This study was conducted to delineate theroles of NLRP3 inflammasome and IL-1β in the host defense against acute C. rodentiuminfection in mice. Methods: NLRP3-/- and background C57BL/6 mice were infected byorogastric gavage with C. rodentium. After infection, mice received either IL-1 β (0.5 μg/mouse; ip) or equal volume saline (control) on post-infection days 0, 2, and 4. On days 6and 10, the mice were sacrificed. Bacterial counts in the intestinal tract and visceral organswere determined. Histopathology and crypt lengths of stained colon tissue were scoredblindly. The epithelial integrity was assessed by biotin permeability and staining for C.rodentium. The inflammatory response was measured by cytokine levels. Colonic tissueswere stained for macrophages and neutrophils as a measure of inflammatory cell infiltration,and cellular proliferation as a measure of colonic hyperplasia. Experiments were approvedby the University of Alberta Animal Care Committee. Results: In WT mice, IL-1 β treatmentsfailed to inhibit bacterial propagation to the spleen, lymph node, liver, and colon butsignificantly reduced the bacterial burden in NLRP3-/- mice over 10 days, as shown bybacterial counting and qPCR. On day 6-post infection NLRP3-/- mice had increased cryptlength and there was a trend for reduced crypt length with IL-1 β treatment. In NLRP3-/-mice, the IL-1β treatments reduced C. rodentium attachment to the epithelial surface,decreased epithelial permeability and increased secretion of pro-inflammatory cytokines inthe colon tissue, compared to WT mice. These findings were confirmed by histopathologyand immune cell staining. Conclusion: Treatment of NLRP3-/- mice with IL-1 β appears toconfer protection against C. rodentium infection by reducing bacterial colonization andepithelial permeability.
Tu1703
Characterization of Humoral Immune Responses to C.difficile Toxins a and Bin Patients With C. difficile-Associated Diarrhoea, Inflammatory Bowel Diseaseand Cystic FibrosisTanya M. Monaghan, Adrian Robins, Alan Knox, Herbert F. Sewell, Yashwant R. Mahida
Patients with IBD are at increased risk of C. difficile infection, but those with cystic fibrosis(who are frequently admitted to hospital for antibiotics) may be protected against the disease.Methods. Four groups of patients were studied (i) C. difficile-associated diarrhoea (CDAD)in non-IBD patients (CDAD-NI), (ii) CDAD in IBD patients (CDAD-IBD), (iii) cystic fibrosis(CF) patients, without history of CDAD and (iv) healthy controls (HC). Serum antibody(IgG) levels were studied by ELISA and expressed as ELISA units (EU). Circulating antigen-activated B cells were investigated using Alexa Fluor 488-labelled toxin A (toxin A488) andflow cytometry using anti-CD19 and anti-IgD antibodies. Following induction of differentia-tion of memory B cells by polyclonal stimulation of peripheral blood mononuclear cells,toxin A- and B-specific antibody secreting cells (ASCs) were quantified using ELISPOT assays(and expressed as percentage of total IgG ASCs). In an additional group of IBD patients,with no history of C. difficile infection, secretion of toxin-specific IgG antibody by laminapropria cells was studied during culture of IBD small and large intestinal mucosal samplesdenuded of epithelial cells. Data are expressed as median (range). Results. Compared to
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