− Mice Have Altered Intestinal Microbiota and Epithelia Oligosaccharide Patterns

− Mice Have Altered Intestinal Microbiota and Epithelia Oligosaccharide Patterns

Three weeks later, host responses were determined using histology, real time PCR, fluorescence in situ hybridization (FISH) and tissue culture. Bacter...

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Three weeks later, host responses were determined using histology, real time PCR, fluorescence in situ hybridization (FISH) and tissue culture. Bactericidal assay and proinflammatory gene expression in immune cells were analyzed in vitro. The nitric oxide (NO) donor sodium nitroprusside (SNP) was injected i.p. (2 mg/kg daily) to supplement NO. RESULTS: Il10-/; Nod2-/- mice are born healthy and do not develop any sign of spontaneous intestinal inflammation. However, C. jejuni infection induced severe intestinal inflammation in Il10-/; Nod2-/- compared to Il10-/- mice (colitis score 2.7 vs. 1.0, P= 0.01). The increased colitis was associated with elevated neutrophil accumulation, crypt abscesses, expression of the endogenous inflammatory mediators Il-1β (83% increase, P = 0.02), Tnf α (66% increase, P = 0.04) and Cxcl1 (39% increase, P = 0.04). FISH or culture assay showed enhanced C. jejuni invasion into the colon (232% increase, P= 0.03) and mesenteric lymph nodes (300% increase, P= 0.04) in Il10-/-; Nod2-/- compared to Il10-/- mice. Bone marrow derived macrophage (BMDM) generated from Il10-/-; Nod2-/- mice showed inferior bactericidal capacity compared to BMDM from Il10-/- mice. Interestingly, NOD2 ligand muramyl dipeptide enhanced C. jejuni-induced iNos mRNA expression in Il10-/- splenocyte, an effect loss in Nod2-/- mice. Splenocyte isolated from Il10-/-; Nod2-/- mice displayed reduced LC3 II and p62 expression following C. jejuni infection compared to Il10-/- splenocyte. In addition, peritoneal macrophage from Il10-/-; Nod2-/- mice produced less C. jejuni-induced bactericidal NO compared to macrophage from Il10-/- mice. Importantly, the NO donor SNP attenuated C. jejuni-induced colitis in Il10-/-; Nod2-/- mice (2.86 vs. 1.15, P= 0.02). This effect was associated with SNP enhanced clearance of C. jejuni in peritoneal macrophage and reduced Tnfα, Cxcl1 and iNos mRNA expression in splenocyte. CONCLUSION: Our findings suggest that NOD2 signaling is critical to control campylobacteriosis in Il10-/- mice, likely through NOD2-induced bactericidal responses (NO production and autophagy). 891 NHE8 Participates Intestinal Mucosal Protection Against Bacterial Infection Chang Liu, Hua Xu, Jing Li, Fayez K. Ghishan Introduction: NHE8 is expressed on the apical membrane of intestinal epithelial cells, and its expression is particularly abundant in the colon. Our previous study showed that Mucin2 expression was significantly reduced in NHE8-/- mice, suggesting that NHE8 may participate in mucosal protection in the colon. This study addresses the hypothesis that NHE8 participate in mucosal protection. Method: Colon tissue was collected from male wild type mice and NHE8-/- mice for RNA and DNA isolation. RNA was used to determine inflammatory cytokine expression with TaqMan probes. DNA was used to quantitate the major groups of bacteria adhered to epithelium layer with group-specific primers derived from 16S rDNA. NHE8 siRNA was used to knockdown NHE8 expression in Caco-2 cells. Salmonella typhimurium and Lactobacillus plantarum were used to study bacterial adherence in the presence or absence of NHE8 expression in Caco-2 cells. Result: NHE8-/- mice has an increase in total bacteria load adhered to the distal colon. The numbers of Firmicutes, Bacteroidetes, Lactobacillus, and segmented filament bacteria were significantly increased. Thus loss of NHE8 function changed the abundance and distribution of colonic microbiota. Cytokine expression profile showed that IL-1β, IL-4, IL-6 and TNFα gene expression were markedly increased in the distal colon of NHE8-/- mice. The resulting increased bacteria burden from the loss of NHE8 may be largely responsible for up-regulation of inflammatory cytokines. Interestingly, NHE8 expression was increased in wild-type cells exposed to S. typhimurium, while NHE8-deficient cells had more adherent S. typhimurium. L. plantarum showed similar adherence regardless of NHE8 expression. Under co-incubation conditions, L. plantarum decreased the number of S. typhimurium adhered to both NHE8-deficient and normal cells. On the other hand, S. typhimurium has no effect on the adherence of L. plantarum. These observations suggested that NHE8 protein could specifically block S. typhimurium adherence to the epithelial cells. Conclusion: NHE8 plays an important role in maintaining homeostasis of the intestinal bacterial family and protecting the host from infection via its effect on blocking infectious bacterial adherence to the intestinal epithelial cells.

889 NHE2-/- Mice Have Altered Intestinal Microbiota and Epithelia Oligosaccharide Patterns Melinda A. Engevik, Gary Shull, Roger T. Worrell Background and Objectives: Although it has been widely accepted that intestinal microbiota are able to affect ion transport, little data exists on the how changes in ion transport affects the microbiota niche development and maintenance. The ability to link changes in the micro-environment, such as ion composition and pH to specific bacterial proliferation is clinically advantageous for diseases that involve microbial dysbiosis, such as IBD, obesity and diabetes. In the human and mouse intestine, the apical Na+/H+ exchangers NHE2 and NHE3 affect both luminal Na+ and water as well as pH. Methods: Intestinal ion composition was measured in NHE2-/- mice by flame photometry and chloridometry and pH measured electrochemically. gDNA extracted from intestinal flushes or from mucosal scrapings were analyzed by qRT-PCR to examine luminal and mucosa-associated bacterial populations. Epithelial mucus oligosaccharide patterns were examined by histology with FIT-C labeled lectins. Results: Loss of NHE2 was compensated by increased NHE3 expression and NHE2-/mice have an acidic pH along the intestine length. In the NHE2-/- intestine Na+ composition was unchanged, however K+ was increased in the terinal ileum and Cl- was decreased in cecum and distal colon. Luminal and mucosa-associated total bacteria levels were unchanged in the NHE2-/-. However regional-specific changes were observed in specific phyla representation. In the luminal NHE2-/- population Firmicutes was increased and α-Proteobacteria was decreased only in the terminal ileum. In the NHE2-/- mucosa-associated population Actinobacteria was increased and γ-Proteobacteria was decreased in the ileum, while γ -Proteobacteria was increased and β-Proteobacteria was decreased in the cecum and αProteobacteria decreased in the distal colon. Studies in neonatal mice have shown that entrance of certain bacteria alter the mucus oligosaccharide composition. Corresponding to the regional NHE2-/- bacterial changes, differences were observed in host mucus oligosaccharide composition. Increased mannose, N-acetygalactosamine and galactose were observed in the NHE2-/- cecum. In contrast decreased mannose and galactose residues were observed in NHE2-/- distal colon. No changes were observed in fucose levels in any segment. Conclusion: Together these data indicate that changes in ion transport can induce region-specific bacterial changes which can then alter the host mucus oligosaccharide patterns. These host-bacterial interactions provide a possible mechanism of niche-development and shed insight on how certain groups proliferate in changing environments and maintain their proliferation by altering the host. Supported by NIH DK079979 to RTW and DK050594 to GES.

892 EPEC NleH1 and NleH2 Protect Against Host Inflammation and Improve DSSInduced Colitis and Survival in Mice Mai T. Nguyen, Ki-Jong Rhee, Gail A. Hecht Enteropathogenic E. coli (EPEC) non-LEE (locus of enterocyte effacement) effector proteins NleH1 and NleH2 inhibit inflammation by blocking host cell signaling pathways in vitro. This study aims to determine if these proteins attenuate intestinal inflammation induced by a non-infectious stimulus in vivo. The impact of EPEC infection and NleH1 and NleH2 expression on DSS-induced colitis was assessed. Initial studies determined if established EPEC infection reduced DSS colitis. Seven-week old uninfected male C57Bl6 control mice or those infected with wild-type EPEC or EPEC ΔnleH1/2 were given 3% DSS in drinking water for 6d. Colon length was measured as an indicator of colitis. WT EPEC infection significantly protected colon length as compared to uninfected controls (7.4±0.7 vs 6.0±0.5 cm, n=4, p,0.05). Infection with ΔnleH1/2, however, failed to afford protection (6.4±7 cm, n=5; p.0.05), suggesting that translocation of NleH1 and 2 by EPEC reduces the development of DSS-induced colitis in mice. We next addressed the impact of simultaneous EPEC infection and DSS administration on colitis. Mice were infected with ΔnleH1/2, ΔnleH1/ 2+pNleH1 or ΔnleH1/2+pNleH2 and DSS was added to drinking water for 9d. Survival was monitored for up to 14d. Only 17% of mice infected with ΔnleH1/2 and uninfected controls survived. In contrast, 83% infected with ΔnleH1/2+pNleH1 and 33% infected with ΔnleH1/ 2+pNleH2 survived indicating that both NleH1 and 2 protect against DSS-induced colitis however NleH1 is significantly more effective. Next, the therapeutic properties of EPEC and NleH1 and 2 in reversing colitis were tested. Mice were treated with DSS for 6d then DSS was withdrawn and mice were infected with WT EPEC, ΔnleH1/2, WT+pNleH1, ΔnleH1/2 +pNleH1 or ΔnleH1/2 +pNleH2. Controls included untreated mice and those receiving only DSS. Mice were monitored daily for weight changes for 12d at which time mice were sacrificed and intestinal length was measured and H&E staining performed. All mice infected with ΔnleH1/2 died or were sacrificed due to severe weight loss by 4d PI. However, complementation with either NleH1 or 2 improved survival significantly with 82% and 56% of mice infected with ΔnleH1/2+pNleH1 and ΔnleH1/2+pNleH2 surviving, resp. Overexpression

890 Defective NOD2-Induced Bactericidal Activity Exacerbates Campylobacter Jejuni-Induced Colitis in Il10-/- Mice Xiaolun Sun, Christian Jobin BACKGROUND: Although nucleotide-binding oligomerization domain-containing protein 2 (NOD2) mutation was first discovered in Crohn's Disease patients two decades ago, the contribution of this protein in regulating in vivo bacterial response is not well defined. Patients with IBD are often subject to relapsing episode following infection with enteric pathogens such as Campylobacter or Salmonella species. AIM: Investigate the role of NOD2 in Campylobacter jejuni-induced intestinal inflammation. METHODS: Specific pathogen free (SPF) C57BL/6 Il10-/- were crossed to Nod2-/- mice to generate Il10-/-; Nod2-/- mice. Mice were infected with C. jejuni (10^9 CFU/mouse) 24 hrs after a 7-day antibiotic treatment.

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AGA Abstracts

AGA Abstracts

pyrosequencing of 16S rRNA genes analyzed by UniFrac. Results: IV injection of G4 led to signal detection in all tissues, but ingested G4 was only detected at intestinal segments containing fecal material. The partial pressure of oxygen (pO2) was remarkably lower in the cecal feces than in the adjacent tissue. Inspiration of 100% O2 led to a rapid increase in cecal tissue pO2 and a delayed, more gradual increase in luminal pO2 (Figure). Both effects were reversible once animals were returned to ambient air. Importantly, time-dependent alterations in the gut microbiota were observed in HBOT treated, but not control mice. Conclusions: Using phosphorescence quenching oximetry, an innovative non-invasive approach to quantify gut luminal pO2 levels, we show, for the first time, that a dynamic oxygen equilibrium is maintained by interactions between the host and the gut microbiota whereby oxygen released by colonic tissue is consumed by the gut microbiota. Indeed, we have observed in healthy human subjects that there is an enrichment of aerobic and facultative anaerobic bacteria adherent to the rectal mucosa compared to the feces where obligate anaerobic bacteria predominated. Phosphorescence-based oximetry and imaging may help to determine whether the dysbiotic microbiota associated with IBD may, in part, be due to an alteration in the redox potential of the colonic environment due to hyperemia, bleeding, and/or the oxidative nature of the inflammatory response.