Tolerance rather than immunity protects from Helicobacter pylori-induced gastric preneoplasia

Abstract

Background & aims: Chronic infection with the bacterial pathogen Helicobacter pylori causes gastric disorders, ranging from chronic gastritis to gastric adenocarcinoma. Only a subset of infected persons will develop overt disease; most remains asymptomatic despite lifelong colonization. This study aims to elucidate the differential susceptibility to H pylori that is found both across and within populations.

Methods: We have established a C57BL/6 mouse model of H pylori infection with a strain that is capable of delivering the virulence factor cytotoxin-associated gene A (CagA) into host cells through the activity of a Cag-pathogenicity island-encoded type IV secretion system.

Results: Mice infected at 5-6 weeks of age with CagA(+)H pylori rapidly develop gastritis, gastric atrophy, epithelial hyperplasia, and metaplasia in a type IV secretion system-dependent manner. In contrast, mice infected during the neonatal period with the same strain are protected from preneoplastic lesions. Their protection results from the development of H pylori-specific peripheral immunologic tolerance, which requires transforming growth factor-β signaling and is mediated by long-lived, inducible regulatory T cells, and which controls the local CD4(+) T-cell responses that trigger premalignant transformation. Tolerance to H pylori develops in the neonatal period because of a biased ratio of T-regulatory to T-effector cells and is favored by prolonged low-dose exposure to antigen.

Conclusions: Using a novel CagA(+)H pylori infection model, we report here that the development of tolerance to H pylori protects from gastric cancer precursor lesions. The age at initial infection may thus account for the differential susceptibility of infected persons to H pylori-associated disease manifestations.

Figure 1

H. pylori PMSS1 injects CagA into gastric epithelial cells, and rapidly induces gastric cancer precursor lesions in a type IV secretion system-dependent manner. (A) AGS cells were co-cultured with PMSS1 or PMSS1ΔCagE for 16 hours. 3D-confocal immunofluorescence images show elongation and scattering of cells co-cultured with the wild-type strain, but not the ΔCagE mutant. Extracts of the same cells were subjected to immunoblotting with anti-CagA and anti-phospho-CagA antibodies. (B) The hummingbird phenotype induced by clones re-isolated from 1, 3, 6 and 9 month infected mice was quantified on a scale of 0–4 and compared to the input strain. Between 6 and 11 independent re-isolated clones per animal were analyzed for 2 to 4 mice per time point. Individual mice are plotted on the x-axis; each square represents one re-isolated clone. Medians for all clones from one individual and for all mice of a time point are indicated by black and dotted bars. (C) CagA delivery and phosphorylation was confirmed by immunoblotting for selected re-isolates. (D–F) C57/BL6 mice were infected at 6 weeks of age with PMSS1 or PMSS1ΔCagE and sacrificed at 1 month and 3 months post infection (p.i.). (D) Colony-forming units (CFU) per stomach; bars indicate the medians. (E) Gastric IFN-γ expression as determined by real time RT-PCR compared to an age-matched uninfected control group. (F) Low and high magnification micrographs of one representative mouse per group; pathology scores were assigned for the four parameters chronic inflammation, atrophy, hyperplasia and metaplasia; every mouse is represented by four data points. Results representative of 2–3 independent experiments are shown for all panels except B and C.

Figure 2. Neonatally infected mice fail to mount local and systemic immune responses to H. pylori infection

(A–F) C57BL/6 mice were infected with H. pylori PMSS1 at either 7 days (infected as neonates, iN) or 5 weeks of age (infected as adults, iA) and sacrificed at 1, 2 and 4 months p.i. A shared, uninfected control group consisted of mice that were 1, 2, 4 and 6 months old at the time of sacrifice. (A) CFU per stomach; bars indicate medians. (B) Gastric IFN-γ expression as determined by real time RT-PCR. (C) Serum titers to H. pylori as determined by ELISA. (D, E) Gastric mucosal infiltration of CD4⁺IFN-γ⁺ T-cells and all CD4⁺ T-cells as determined by intracellular cytokine and surface staining for IFN-γ and CD4 at 1 month p.i. (F) Production of IFN-γ by MLN cells after 4 days in culture as determined by ELISA. Data are representative of 5 experiments.

Figure 3. Neonatally infected mice are protected from gastric preneoplastic pathology

(A–D) Gastric corpus histopathology of the neonatally and adult-infected mice described in Figure 2 was assessed on Giemsa-, Alcian blue- and Periodic Acid-Schiff-stained paraffin sections. Representative micrographs are shown in A, C and D and histopathology scores are shown in B. Low and high magnification micrographs are shown in C and D. Data are representative of 5 experiments.

Figure 4. The ability of mouse-colonizing H. pylori to inject CagA in vitro is lost in mice infected as adults, but not in neonatally infected mice

(A) H. pylori PMSS1 clones were isolated from neonatally or adult-infected mice 4 and 6 months p.i. (iN, 39 clones; iA, 31 clones). Re-isolates derived from 3 independent experiments were assessed quantitatively for hummingbird phenotype induction in AGS cells. Individual mice are plotted on the x-axis; each square represents one clone. Medians for individual mice and time points are indicated by black and dotted bars. (B) The delivery and phosphorylation of CagA by selected 6 month isolates was confirmed by phospho-Cag-specific immunoblotting of infected AGS cell extracts. (C) Representative micrographs of the analysis shown in A.

Figure 5. The priming of T-effector cell responses is not impaired in neonatally infected compared to adult-infected mice

(A–C) CD4⁺CD25⁻ effector T-cells from the spleens of neonatally infected and adult-infected animals (Teff (iN) and Teff (iA)) were adoptively transferred into 6 week old TCR-β^−/− hosts. All recipients and controls were infected 1 day post-transfer. Colonization and gastric histopathology was determined 4 weeks p.i. Colony counts (A), representative micrographs (B) and histopathology scores (C) are shown. (D) Neonatal and adult mice were immunized 4 times in weekly intervals with an H. pylori vaccine (VacN and VacA) prior to challenge infection. All animals were sacrificed 2 weeks p.i. (see schematic, left panel) and colonization levels were compared to non-immunized control mice (iN, iA). Data are representative of 2 independent experiments.

Figure 6. Tolerance induction and maintenance requires peripheral regulatory mechanisms

(A–C) Neonatal FoxP3-eGFP:DTR mice ( Dereg, triangles) and their non-transgenic littermates (B6 WT, circles) were infected with PMSS1. Treg were depleted by i.p. administration of diphtheria toxin (DT) starting on the day of infection, either for the entire 4 week time course of the experiment (DT_d7–d35, black triangles) or only during the first 2 weeks of infection (DT_d7–d21, white triangles). Treg depletion was on average 98% effective in the mesenteric lymph nodes draining the GI tract. All animals were assessed with respect to H. pylori colonization (A) and gastric histopathology (B, C) 4 weeks p.i. and compared to uninfected transgenic mice depleted of Treg for 4 weeks (inverted triangles). (D–F) TCR-β^−/− mice were infected as neonates and, at 5 weeks of age, received immunomagnetically purified effector T-cells from the spleen of an adult infected animal (TCR-β^−/−(iN) +Teff (iA), white circles). H. pylori colonization (D) and gastric histopathology (E, F) was assessed four weeks post adoptive transfer in comparison to a group that had not received cells (TCR-β^−/−(iN), grey circles; compare also to Fig. 5A-C). All results are representative of 2–3 experiments.

Figure 7. Tolerance induction requires the activity of TGF-β-responsive, long-lived CD4⁺ T-cells

(A–C) Neonatal CD4-dnTβRII mice (black circles) and their WT littermates (grey circles) were infected for 1 month. All mice were analyzed with respect to H. pylori colonization and gastric histopathology and compared to respective adults (grey and black squares). Colony counts (A), representative micrographs (B) and histopathology scores (C) are shown. (D) Neonatally infected mice were subjected to antibiotic eradication therapy followed by re-infection five weeks later (schematic, left panel). Eradication was verified in a control group (1). The remaining mice were killed 4 weeks after re-infection (2) along with groups that had been infected as neonates or adults in week 9 (3, 4). Colony counts are shown. Pooled data from two studies are shown in AC; data in D are representative of 2 independent experiments.