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Upper graph: relative expression of IL-10+ Foxp3+ T cells by Flow cytometry. Lower graph:gene expression of IFN-gamma by qPCR.

Upper graph: Foxp3 gene expression by qPCR. Lower graph: IL-10 gene expression by qPCR.

Tu1711

Gpx1, Gpx2 and Gdac1 Each Have a Distinct Impact on Spontaneous andSalmonella-Induced Colitis in MiceRobert S. Esworthy, Byung-Wook Kim, James H. Doroshow, Thomas L. Leto, Fong-FongChu

Selenium deficiency increases the susceptibility of human and animal to infectious diseasesinvolving both viruses and bacteria. It is unclear which selenoprotein(s) are responsible forthis selenium effect in most cases, although both glutathione peroxidase (Gpx)-1 and Gpx2have been implicated. Here we show that both Gpx1 and Gpx2 have a role in spontaneouscolitis observed in 129 strain mice using multiple markers of disease signs and dysbiosis.Salmonella enterica serotype Typhimurium (S. Typhimurium) is a leading cause of foodpoisoning in humans. Whether selenoproteins also affect S. Typhimurium infection is notknown. We have tested S. Typhimurium susceptibility in mice deficient in either Gpx1 orGpx2 and found out both are highly susceptible to S. Typhimurium-induced colitis using

S-828AGA Abstracts

a metronidazole pre-treatment/low dose (2E7 CFU) oral inoculation model. The importanceof Gpx1 and Gpx2 in defending against infectious bacteria may help explain the spontaneousileocolitis in mice deficient in either or both Gpx1 and Gpx2 isoenzymes. Additionally, weexamined whether the Gdac1 locus, observed to moderate spontaneous colitis in 129 strainGpx1/2-KO (DKO) mice, had any impact on S. Typhimurium-colitis. We found the B6allele of Gdac1 promotes inflammation by increased TNF-alpha and NOD2 mRNA levels.Other inflammatory markers, including IFN-gamma, Nlrp3, IL1-beta, CD14, etc., showedelevated mean levels but failed to achieve statistical significance. Thus, we conclude thatGpx1 plays a prominent role in inflammatory responses against bacterial infection, but asubtle role in spontaneous colitis. Gpx2 has a strong impact on spontaneous colitis buthas a low impact on inflammatory responses. The Gdac1 locus impacts significantly bothspontaneous and bacteria-associated colitis in Gpx1-KO or Gpx2-KO mice.

Tu1712

Helicobacter pylori Affects Gastric Epithelial Barrier Integrity Independent ofIts Main Virulence Factors and Induces a Polarized Host InflammatoryCytokine Secretion in an In Vitro Human Cell ModelMaria R. Fiorentino, Hua Ding, Tom G. Blanchard, Marcelo Sztein, Alessio Fasano

Background: H. pylori infection is related to the development of diverse gastric pathologies,possibly by affecting epithelial junctional complexes that play critical roles in maintainingbarrier function, cell polarity and intercellular adhesion. Using a gastric epithelial cell model,we have elucidated the effects of H. pylori infection on the epithelial barrier integrity andhost response. Aim: To study gastric epithelial biological effects triggered by wild-type H.pylori and mutant strains. Methods: The NCI-N87 gastric cell line was inoculated with wild-type H. pylori (strains 26695 or M5), isogenic VacA-, CagPAI- and UreB- mutant strains,heat-killed bacteria or bacterial culture supernatants to evaluate initial host-pathogen interac-tions and the effect of exposure and colonization of these strains on mucosal barrier function.Barrier function was evaluated by TEER analysis, immunohistochemistry, and diffusion offluorescent proteins. Supernatants were collected for cytokine measurements. Results: Infec-tion of NCI-N87 monolayers with all live H. pylori strains used in this study caused aprogressive decrease in TEER compared to uninfected control and filtered/heat killed bacteriatreated monolayers. Cell viability was not altered by infection, suggesting that loss of barrierfunction was likely due to modulation of tight junctions. Immunohistochemistry on H. pyloriwild-type infected monolayers showed rearrangment of the tight junction proteins ZO-1and Claudin-1. Paracellular permeability assays showed a significantly increased net flux ofFITC-labeled markers through the infected monolayer, indicating that this increase in epithe-lial permeability can be accounted for a breakdown of the tight junction integrity. Cytokineproduction (IL-8, IL-6, TNF-α, INF-γ, IL-12p70, IL-1β, IL-10) was uniformly higher in thebasolateral compartment, although significant secretion was measured in the apical side aswell. H. pylori infected cells exhibit an approximately 4-fold increase in the production ofboth IL-8 and IL-10 in the basal side than uninfected cells. HK bacteria are capable toinduce the highest release of cytokines in both compartments. Conclusion: We have heredetermined that H. pylori dramatic alterations of the gastric epithelial barrier function occurvia modulation of tight junction cellular assembly and scaffolding and are independent ofVacA, CagPAI, and Urease virulence effectors. Moreover, we showed that the host respondsto H. pylori infection via a polarized secretion of immune mediators.

Tu1713

Helicobacter pylori Activates Signal Transducer and Activator of Transcription3 to Blunt Dendritic Cell MaturationDavid Rizzuti, Ted Wu, Ramzi Fattouh, Colin Reardon, Michelle Ang, Jillian Haight,Christian Schindler, Nicola L. Jones

AIMS: Helicobacter pylori causes a chronic infection and is the primary risk factor associatedwith the development of gastric cancer. Dendritic cells (DCs) are key orchestrators of hostimmunity, and modulation of DC function provides an attractive mechanism of immuneevasion by H. pylori. DCs depend on multiple signalling pathways to respond to pathogens,including cytokines and secreted vesicles such as exosomes. We previously identified asubset of infiltrating immune cells with increased STAT3 activation in H. pylori infectedgastric tissues. We therefore hypothesize that H. pylori infection induces STAT3 activationand therefore alters DC maturation through signalling dependent processes. METHODS:Wild-type and STAT3 KO bone-marrow derived DCs were incubated with H. pylori, exosomesisolated from H. pylori infected murine gastric epithelial cells, or the controls E. coli LPS,and IL-6. Stat3 activation was determined by immunoblotting and DC maturation wascharacterized by flow cytometry for the expression of maturation markers MHC-II, CD80and CD86. Cytokine profiles were determined by ELISA assays. RESULTS: Both H. pyloriinfection and incubation with H. pylori-infected cell-derived exosomes induced STAT3 activa-tion in wild-type DCs in aassociation with increased expression of MHC-II, CD80, andCD86 comparable to LPS-treated cells. H. pylori-infected DCs showed enhanced IL-10:IL-12 expression relative to control or LPS-treated cells. Interestingly, in comparison withwildtype DCs, infected STAT3 KO DCs showed higher maturation levels. In addition,incubation of H. pylori-infected DCs with neutralizing anti-IL10 antibodies inhibited STAT3activation and promoted increased maturation. CONCLUSIONS: Taken together our resultsindicate that H. pylori infection activates STAT3 in DCs to inhibit DC maturation in anautocrine/paracrine IL-10 dependent pathway. Furthermore, exosomes from H. pylori-infected cells can also activate STAT3 to blunt DC maturation. Thus we have identified anovel mechanism by which H. pylori evades host immunity.