Ting-Jia Fan

Inflammation-induced acid tolerance genes gadAB in luminal commensal Escherichia coli attenuate experimental colitis.

ABSTRACT Dysregulated immune responses to commensal intestinal bacteria, including Escherichia coli, contribute to the development of inflammatory bowel diseases (IBDs) and experimental colitis. Reciprocally, E. coli responds to chronic intestinal inflammation by upregulating expression of stress response genes, including gadA and gadB. GadAB encode glutamate decarboxylase and protect E. coli from the toxic effects of low pH and fermentation acids, factors present in the intestinal lumen in patients with active IBDs. We hypothesized that E. coli upregulates gadAB during inflammation to enhance its survival and virulence. Using real-time PCR, we determined gadAB expression in luminal E. coli from ex-germfree wild-type (WT) and interleukin-10 (IL-10) knockout (KO) (IL-10−/−) mice selectively colonized with a commensal E. coli isolate (NC101) that causes colitis in KO mice in isolation or in combination with 7 other commensal intestinal bacterial strains. E. coli survival and host inflammatory responses were measured in WT and KO mice colonized with NC101 or a mutant lacking the gadAB genes (NC101ΔgadAB). The susceptibility of NC101 and NC101ΔgadAB to killing by host antimicrobial peptides and their translocation across intestinal epithelial cells were evaluated using bacterial killing assays and transwell experiments, respectively. We show that expression of gadAB in luminal E. coli increases proportionately with intestinal inflammation in KO mice and enhances the susceptibility of NC101 to killing by the host antimicrobial peptide cryptdin-4 but decreases bacterial transmigration across intestinal epithelial cells, colonic inflammation, and mucosal immune responses. Chronic intestinal inflammation upregulates acid tolerance pathways in commensal E. coli isolates, which, contrary to our original hypothesis, limits their survival and colitogenic potential. Further investigation of microbial adaptation to immune-mediated inflammation may provide novel insights into the pathogenesis and treatment of IBDs.

Environmental Factors Modify the Severity of Acute DSS Colitis in Caspase-11-Deficient Mice

Background Human and mouse studies implicate the inflammasome in the pathogenesis of inflammatory bowel diseases, though the effects in mice are variable. The noncanonical inflammasome activator caspase-11 (Casp11) reportedly attenuates acute dextran sodium sulfate (DSS) colitis in mice. However, the effects of Casp11 on chronic experimental colitis and factors that influence the impact of Casp11 on acute DSS colitis are unknown. Methods We studied the role of Casp11 in Il10-/- mice and acute and chronic DSS colitis mouse models. We quantified colonic Casp11 mRNA using quantative polymerase chain reaction and colitis using weight loss, blinded histological scoring, IL-12/23p40 secretion by colonic explants, and fecal lipocalin-2. We determined fecal microbial composition using 16S amplicon sequencing. Results We detected increased colonic Casp11 mRNA in Il10-/- mice with chronic colitis, but not in mice with DSS colitis. The presence of Casp11 did not alter the severity of chronic colitis in DSS-treated or Il10-/- mice. Contrary to prior reports, we initially observed that Casp11 exacerbates acute DSS colitis. Subsequent experiments in the same animal facility revealed no effect of Casp11 on acute DSS colitis. There were pronounced stochastic changes in the fecal microbiome over this time. The majority of bacterial taxa that changed over time in wild-type vs Casp11-/- mice belong to the Clostridiales. Conclusions Casp11 does not impact chronic experimental colitis, and its effects on acute DSS colitis vary with environmental factors including the microbiota, particularly Clostridiales. Stochastic drifts in intestinal microbiota composition, even in mice in the same housing facility, should be considered when interpreting studies of acute DSS colitis models.

Sa1773 Caspase-11 Enhances Killing of Colitogenic Escherichia coli by Macrophages, but Does Not Affect Development of Experimental Colitis

WT Balb/C mice (p<0.001 for each cytokine btw uninfected control and helminth-infected; N=at least 3 independent experiments), while no regulation of IFNγ and modest induction of IL4 or IL10 was seen in STAT6-/mice. Helminths did not induce TGFβ production in STAT6-/animals. In an acute GVHD and colitis model, helminths regulated WT donor T cell Th1 cytokine generation, serum Th1 cytokines, colitis and survival in WT and not STAT6-/recipients (p<0.05 for each parameter btw. helminth infected WT and helminthinfected STAT6-/recipients, multiple experiments). Helminth infection was associated with the induction of donor and recipient FoxP3+ regulatory T cells (Tregs) in WT and not STAT6-/bone marrow transplanted mice (p<0.05 btw. helminth infected WT and helminthinfected STAT6-/recipients, multiple experiments). Addition of TGFβ to STAT6-/T cell cultures restored T cell IL10 production and Treg generation that regulate Th1 inflammation. Conclusions: STAT6 is a critical transcription factor directing mucosal TGFβ producing Th3 cell generation. Helminths utilize STAT6 pathway to induce mucosal Tregs, trigger mucosal T cell IL10 secretion and regulate alloreactive colitis in a TGFβ dependent manner.

316 Acid-Tolerance Genes Gadab in Commensal Escherichia coli Impair Luminal Bacterial Growth, Alter Bacteria-Epithelial Cell Interactions and Attenuate Experimental Colitis

G A A b st ra ct s the waaWVL operon is not present in non pathogenic E. coli strains but is present in most AIEC from our collection and in two other sequenced AIEC strains NRG 857C and UM146. CONCLUSION: The presence of waaWVL operon is essential for AIEC bacteria to form biofilm at the surface of the intestinal mucosa. The search for the presence of the waaWVL operon could represent a useful molecular tool to identify pathogenic AIEC and targeting waaWVL operon expression could be a very potent therapeutic strategy to interfere with the ability of AIEC to form biofilm on the gut mucosa of Crohns disease patients.

Mo1770 Expression of the Oxys Small Regulatory RNA in Luminal Commensal Escherichia coli Attenuates Experimental Colitis

BACKGROUND: Although the etiology of human inflammatory bowel diseases (IBD) remains elusive, they are due in part to dysregulated innate and adaptive immune responses to luminal commensal bacteria in genetically predisposed individuals. Host inflammation reciprocally affects luminal bacterial functions. Genes that protect Escherichia coli (E. coli) from oxidative stress, including the small regulatory RNA oxyS, are upregulated in luminal E. coli from monoassociated IL-10-deficient mice (IL10-/-) with colitis compared to healthy wildtype (WT) controls. AIMS: We aimed to investigate how intestinal inflammation alters expression of E. coli oxyS In Vivo, characterize innate immune pathways that are affected by oxyS expression In Vitro, and examine the ability of E. coli lacking oxyS to induce experimental colitis. METHODS: Gene expression was measured by real-time PCR of luminal bacteria from WT and IL10-/mice monoassociated with the commensal murine isolate E. coli NC101. Bacterial survival and pro-inflammatory cytokine secretion in WT and IL10-/bone-marrow derived macrophages infected with NC101, NC101 lacking oxyS (NC101 ΔoxyS), or NC101 overexpressing oxyS (NC101 oxyS+) was determined using gentamicinprotection assays and ELISA, respectively. Histological inflammation in colon sections, concentrations of E. coli NC101 in luminal contents, and adaptive immune responses to NC101 antigens in mesenteric lymph node cells (MLN) from IL10-/and WT mice monoassociated with NC101 or NC101 ΔoxyS were determined using blinded scoring, quantitative plating on BHI agar, and ELISA for IFN-γ, respectively. RESULTS: Cecal E. coli NC101 increase expression of oxyS proportional to the degree of colitis in monoassociated IL10-/mice. Infection of primary IL-10-/and WT macrophages In Vitro by NC101, NC101 ΔoxyS and NC101 oxyS+ bacteria results in similar intracellular bacterial survival and secretion of TNF. Bacterial densities in cecal contents from IL-10-/mice monoassociated for 10 wks with NC101 and NC101 ΔoxyS were not significantly different (1.28x10^10 ± 0.12 and 1.65x10^10 ± 0.65 colony forming units/g content, respectively; p=0.54). However, composite histological colon inflammation scores were lower inNC101vs. NC101ΔoxyS-monoassociated IL-10-/mice (10.33 ± 0.31 and 12.33 ± 0.79, respectively; p<0.05); and IFN-γ secretion by NC101 lysate-stimulatedMLN cells fromNC101vs. NC101ΔoxyS-monoassociated IL-10-/mice was reduced (12.2x10^5 ± 0.8 and 21.9x10^5 ± 1.7 pg/ml, respectively; p<0.001). CONCLUSIONS: Commensal E. coli responds to intestinal inflammation by upregulating oxyS, which when expressed, decreases host immune responses independently of luminal bacterial concentrations. Elucidating mechanisms of this host-microbial dialogue will provide novel insights into the pathogenesis of IBD and potentially reveal novel therapeutic targets.

Inflammation Impacts the Development of Colorectal Cancer Through Changes in Microbial Composition and Associated Genotoxic Capacity

Background: Selenoproteins, many of which protect against oxidative injury, have been postulated to affect the development of GI inflammation and cancer. Glutathione peroxidase3 (Gpx3) is an extracellular selenoprotein that is elevated in the plasma of patients with inflammatory bowel disease. Also, mice treated with Dextran Sodium Sulfate (DSS), a model of murine colitis, have increased plasma Gpx3. Based on these observations we hypothesized that Gpx3 could modify inflammatory carcinogenesis (CAC) in the colon. Methods: We selected the azoxymethane (AOM)/cyclic DSS rodent model for our studies as this is a robust model for interrogating genetic modifiers of CAC. 11 Gpx3-/and 12 WT mice were injected with 12 mg/kg AOM followed by four cycles of 3% DSS ad lib. An additional cohort of 18 Gpx3-/and 13 WT mice were subjected to cyclic DSS therapy without AOM initiation. At necropsy, colons were isolated and tumor burden and distribution were scored. TUNEL staining to evaluate apoptosis and IHC for BrdU and β-catenin was performed on colon sections. Results: When treated with AOM and cyclic DSS ad lib, both Gpx3-/and WT mice developed polyps and had mucosal injury localized to the distal colon. However, Gpx3-/mice had increased polyposis (21.1 ± 1.6 vs 10.8 ± 1.5 polyps per colon, P<0.0001), without a difference in polyp size (7.9 ± 1.1 vs 6.4 ± 0.3, P=0.20). There appeared to be an increase in polyp burden in DSS only treated Gpx3-/mice, although this did not reach statistical significance (1.0 ± 0.3 vs 0.4 ± 0.2, P=0.15). Polyp sizes were similar between both groups of mice. Histologic injury was more severe in the Gpx3-/mice (16.2 ± 2.5 vs 7.8 ±0.64, P<0.01, combined histologic injury score). Overall, there was no difference in grade of lesions, with high-grade dysplasia present in both groups of mice. While there was no difference in intra-tumoral apoptosis observed, there was significantly higher proliferation in Gpx3-/tumors (164.0 ± 46.7 vs 73.9 ±54.4 Ki67+ cells/HPF, P<0.01). Consistent with this we observed increased cytoplasmic and nuclear β-catenin staining in Gpx3-/tumors (P<0.001). Conclusions: These studies demonstrate that Gpx3 modifies tumor initiation in inflammatory carcinogenesis. This suggests that Gpx3may serve a protective role in inflammation associated colitis perhaps via decreasing levels of H2O2 and hydroperoxides in the colonic mucosa, thus reducing oxidative DNA damage.