Helicobacter pylori γ-glutamyl transpeptidase and vacuolating cytotoxin promote gastric persistence and immune tolerance

Abstract

Infection with the gastric bacterial pathogen Helicobacter pylori is typically contracted in early childhood and often persists for decades. The immunomodulatory properties of H. pylori that allow it to colonize humans persistently are believed to also account for H. pylori's protective effects against allergic and chronic inflammatory diseases. H. pylori infection efficiently reprograms dendritic cells (DCs) toward a tolerogenic phenotype and induces regulatory T cells (Tregs) with highly suppressive activity in models of allergen-induced asthma. We show here that two H. pylori virulence determinants, the γ-glutamyl transpeptidase GGT and the vacuolating cytotoxin VacA, contribute critically and nonredundantly to H. pylori's tolerizing effects on murine DCs in vitro and in vivo. The tolerance-promoting effects of both factors are independent of their described suppressive activity on T cells. Isogenic H. pylori mutants lacking either GGT or VacA are incapable of preventing LPS-induced DC maturation and fail to drive DC tolerization as assessed by induction of Treg properties in cocultured naive T cells. The Δggt and ΔvacA mutants colonize mice at significantly reduced levels, induce stronger T-helper 1 (Th1) and T-helper 17 (Th17) responses, and/or trigger more severe gastric pathology. Both factors promote the efficient induction of Tregs in vivo, and VacA is required to prevent allergen-induced asthma. The defects of the Δggt mutant in vitro and in vivo are phenocopied by pharmacological inhibition of the transpeptidase activity of GGT in all readouts. In conclusion, our results reveal the molecular players and mechanistic basis for H. pylori-induced immunomodulation, promoting persistent infection and conferring protection against allergic asthma.

Fig. 1.

VacA and the enzymatic activity of GGT are required for DC tolerization. Bone marrow DCs were infected with H. pylori strains PMSS1, PMSS1Δggt, or PMSS1ΔvacA at a multiplicity of infection (MOI) of 50 and/or treated with 0.5 μg/mL E. coli LPS for 16 h before the analysis of CD11c and CD80 expression (A) and of IL-12p40 secretion by ELISA (B). Data are pooled from four independent experiments and represented as means ± SEM. MFI, mean fluorescence intensity. (C–E) Bone marrow DCs were infected as described in A and B; acivicin was added to the infections at 5 μg/mL where indicated. After 16 h, bacteria were killed with antibiotics. DCs were cocultured with CD4⁺CD25⁻ T cells for 3 d in the presence of rTGF-β, rIL-2, and anti-CD3ε mAb before the flow cytometric analysis of CD4, CD25, and FoxP3 expression. Representative plots of the CD4⁺ gate are shown in C. Pooled results from four (PMSS1ΔvacA) to seven (PMSS1Δggt) independent experiments are shown in D and from three independent wild-type infections performed with or without acivicin in E; in D and E, each symbol represents one coculture and horizontal lines indicate the medians. Uninfected DCs (ctrl), acivicin-treated DCs (acivicin), and T cells cultured in the absence of DCs (−) served as controls.

Fig. 2.

VacA and GGT are required for gastric colonization and DC tolerization in vivo. (A–F) C57BL/6 mice were infected at 6 wk of age with H. pylori PMSS1, PMSS1Δggt, or PMSS1ΔvacA for 1 (A–C) or 2 mo (D–F). Colony forming units (CFU) per stomach are shown in A and D. CD11c⁺ MLN DCs were immunomagnetically isolated from all mice killed at 1 mo p.i., cocultured with T cells, and subjected to flow cytometric analysis of CD4, CD25, and FoxP3 expression. CD25⁺FoxP3⁺ cells in the CD4⁺ gate are shown in B for representative donors and in C for all mice. Data in A–C are pooled from three independent experiments. DCs from uninfected mice (ctrl) and T cells cultured in the absence of DCs (−) served as controls. (E) Pathology scores assigned for inflammation, atrophy, intestinal metaplasia, and epithelial hyperplasia. (F) Representative micrographs of H&E-stained sections at 100 and 200× magnification. Arrowheads point to areas with inflammation; arrows indicate intestinal metaplastic glands. Uninfected controls are shown for comparison. (Scale bars, 100μm.) In A and C–E, each symbol represents one mouse; note that the mice in D–F are from the same infected cohort as a subset of mice shown in A–C.

Fig. 3.

VacA and the enzymatic activity of GGT contribute to neonatally acquired immune tolerance independently of T cells. (A) C57BL/6 mice were infected with H. pylori PMSS1 as neonates (iN) for 6 wk. One group received acivicin continuously every other day starting from the day of infection (acivicin_cont). Another group received acivicin only during the last 2 wk of infection. CD11c⁺ MLN DCs were cocultured with CD4⁺CD25⁻ T cells, and cocultures were stained for CD4, CD25, and FoxP3. CD25 and FoxP3 staining of the CD4⁺ gate is shown for all donors. (B) TCR-β^−/− mice were infected with H. pylori PMSS1, PMSS1Δggt, or PMSS1ΔvacA at 7 d (iN) or 6 wk of age (iA, infected as adults) for 1 mo. CD25⁺FoxP3⁺ cells in the CD4⁺ gate are shown for DC/T-cell cocultures of all donors. Data in B are pooled from two studies. In A and B, DCs from uninfected mice (ctrl) and T cells cultured in the absence of DCs (−) served as controls.

Fig. 4.

The H. pylori-mediated protection against asthma and generation of Tregs with protective activity depends on VacA. C57BL/6 mice were sensitized and challenged with ovalbumin to induce airway inflammation. Two groups of mice were neonatally infected with either wild-type PMSS1 (wt_iN) or PMSS1ΔvacA (ΔvacA_iN) before allergen sensitization. Two additional groups received CD4⁺CD25⁺ MLN T cells from neonatally wild-type– or PMSS1ΔvacA-infected donors (Treg_wt or Treg_ΔvacA) just before challenge. Negative controls were challenged without prior sensitization. (A) Airway hyper-responsiveness as assessed with increasing doses of methacholine. (B and C) Total cells and eosinophils in 1 mL of bronchoalveolar lavage fluid (BALF). (D) Tissue inflammation as assessed on H&E-stained tissue sections. (E) Goblet cell metaplasia as assessed on periodic acid Schiff (PAS)-stained sections. Data in A–E are pooled from three independent studies.