1. Volume 141, Issue 4, Pages 1273-1282.e1 (October 2011)
PAR2 Promotes Vaccine-Induced Protection Against Helicobacter Infection in Mice 
Dominique Velin, Sharmal Narayan, Eric Bernasconi, Nathalie Busso, Giancarlo Ramelli, Michel H. Maillard, Daniel Bachmann, Catherine Pythoud, Hanifa Bouzourene, Pierre Michetti, Alexander So 
Gastroenterology 
Volume 141, Issue 4, Pages e1 (October 2011)
DOI: /j.gastro
Copyright © 2011 AGA Institute Terms and Conditions

2. Figure 1
Vaccinated PAR2−/− mice failed to reduce Helicobacter burden following infection. PAR2−/− and WT littermates were vaccinated with urease and cholera toxin (CT). Negative controls were only treated with CT. Two weeks after vaccination, mice were challenged with either (C) H felis or (D) H pylori. Mice were killed at day 14 after bacterial challenge, and serum, spleen, and stomach samples were obtained to assess (A) serum anti-urease immunoglobulin (Ig) G antibody titers, (B) urease-specific proliferation, and (C) Helicobacter burden (RUT), respectively. Despite similar immune responses, WT mice had a lower infectious status than PAR2−/− mice. (D) Finally, mice were killed on day 14 following H pylori infection and bacterial colony-forming units calculated from stomach specimens. Although vaccinated WT mice demonstrated a lower infectious status in comparison with nonvaccinated WT mice, vaccinated PAR2−/− mice did not show significant reduction of H pylori infection as compared with nonvaccinated PAR2 mice (P > .4). Plots are representative of 2 independent experiments (n = 8–10 mice per group, *P < .05, ***P < .001).
Gastroenterology  , e1DOI: ( /j.gastro )
Copyright © 2011 AGA Institute Terms and Conditions

3. Figure 2
Reduced vaccine-induced protection against Helicobacter infection in PAR2−/− mice is associated with low levels of inflammatory mediators, neutrophil infiltration, and tissue damage in the gastric mucosa. PAR2−/− (white symbols) and WT littermates (black symbols) were vaccinated with urease and cholera toxin (CT) (n = 5–6 per group) and challenged with H felis 2 weeks subsequently. (A) Mice were killed 14 days following bacterial challenge and stomach tissue was recovered to perform quantitative PCR to assess mRNA expression of IL-17, IL-6, TGF-β, IFN-inducible protein 10 (IP-10), inducible nitric oxide synthase (iNOS), IL-1β, and IL-23p19. The relative expressions for IL-17, IL-1β, iNOS, and IL-6 were less than 0.08, 0.13, 0.16, and 0.06, respectively, in control WT CT and PAR2 CT mice. (B) Mice were killed 4 weeks after bacterial challenge and stomach tissue was recovered to perform quantitative PCR to assess mRNA expression of IL-17 and IL-1β. (C) Histopathologic damage index. (D) Histologic analysis was performed on stomach paraffin sections to evaluate tissue damages. The index of histopathologic damage was less than 1.5 in WT CT and PAR2−/− CT. (E) Gr-1 immunostaining was performed on stomach cryosections to detect inflammatory cells (bars = 100 μm). Experiments were performed twice.
Gastroenterology  , e1DOI: ( /j.gastro )
Copyright © 2011 AGA Institute Terms and Conditions

4. Figure 3
Impaired T-cell responses in vaccinated PAR2−/− mice. PAR2−/− and WT littermates were vaccinated with urease and cholera toxin (CT) and infected with H felis. Mice were killed 14 days following bacterial challenge and spleens were harvested to assess the activation status of (A) CD3+, (B) CD8+, and (C) CD4+ T cells by flow cytometry. Stomach tissue was also extracted to determine the frequency of infiltrating (D) CD4+ IL-17+ and (F) CD4+ IFN-γ+ cells. Following bacterial infection, vaccinated WT mice exhibited higher splenic percentages of activated T cells (CD3+ CD44+ CD62L−) and conventional CD8+ (CD8+ CD44+ CD62L−) and CD4+ T cells (CD4+ CD44+ CD62L−) when compared with vaccinated PAR2−/− mice. Vaccinated WT mice also had higher percentages of CD4+ IL17+ cells but equivalent frequency of CD4+IFN-γ+ infiltrating into the gastric mucosa. (E) Isolated stomach mononuclear cells were stimulated with urease for 5 days in vitro. IL-17 protein concentration was measured in the cell supernatant by ELISA. We observed that urease-stimulated WT mononuclear cells produce higher IL-17 levels than PAR2−/− counterparts. Plots are representative of at least 2 independent experiments (n = 5–6 mice per group, *P < .05).
Gastroenterology  , e1DOI: ( /j.gastro )
Copyright © 2011 AGA Institute Terms and Conditions

5. Figure 4
PAR2 deficiency is associated with DC dysfunctions. PAR2−/− (white symbols) and WT littermates (black symbols) were vaccinated with urease and cholera toxin (n = 5–6 per group) and challenged with H felis. Mice were killed 14 days following bacterial challenge and spleens harvested to assess (A) the DC activation status by flow cytometry. (B) Splenic DCs were isolated, by magnetic-activated cell sorter procedure, 2 weeks following infection to determine the expression levels of IL-23, IL-6, and TGF-β. (C) Splenic DCs were stimulated with H felis and OVA peptide for 2 hours and incubated for 5 days with ovalbumin-specific OT-II transgenic CD4+ T cells. IL-17 levels of cell supernatant were determined by ELISA.
Gastroenterology  , e1DOI: ( /j.gastro )
Copyright © 2011 AGA Institute Terms and Conditions

6. Figure 5
Adoptive transfer of WT DCs improves vaccine-induced clearance of Helicobacter in PAR2−/− mice. PAR2−/− and WT littermates were vaccinated with urease and cholera toxin (CT) or CT alone (n = 5–14 mice per group). Two weeks after vaccination, mice received adoptive transfer of 1 × 106 WT or PAR2−/− DCs and were subsequently challenged with H felis. Mice were killed 2 (A, left panel) or 4 (A, right panel) weeks after bacterial infection, where stomach samples were recovered (A) to assess bacterial burden (RUT) or (B) to perform quantitative PCR to assess mRNA expression of IL-17. Adoptive transfer of WT DCs into vaccinated PAR2−/− (A) enhances vaccine-induced Helicobacter clearance and (B) increases IL-17 expression in these animals. (C) Bone marrow–derived DCs were stimulated with H felis and OVA peptide for 2 hours and incubated for 5 days with ovalbumin-specific OT-II transgenic CD4+ T cells. IL-17 levels of cell supernatant were determined by ELISA. Plots are representative of at least 2 independent experiments.
Gastroenterology  , e1DOI: ( /j.gastro )
Copyright © 2011 AGA Institute Terms and Conditions

7. Supplementary Figure 1
Gastroenterology  , e1DOI: ( /j.gastro )
Copyright © 2011 AGA Institute Terms and Conditions

8. Supplementary Figure 2
Gastroenterology  , e1DOI: ( /j.gastro )
Copyright © 2011 AGA Institute Terms and Conditions