TLR9 activation suppresses inflammation in response to Helicobacter pylori infection

Abstract

Helicobacter pylori (H. pylori) induces chronic gastritis in humans, and infection can persist for decades. One H. pylori strain-specific constituent that augments disease risk is the cag pathogenicity island. The cag island encodes a type IV secretion system (T4SS) that translocates DNA into host cells. Toll-like receptor 9 (TLR9) is an innate immune receptor that detects hypo-methylated CpG DNA motifs. In this study, we sought to define the role of the H. pylori cag T4SS on TLR9-mediated responses in vivo. H. pylori strain PMSS1 or its cagE^- mutant, which fails to assemble a T4SS, were used to infect wild-type or Tlr9^-/- C57BL/6 mice. PMSS1-infected Tlr9^-/- mice developed significantly higher levels of inflammation, despite similar levels of colonization density, compared with PMSS1-infected wild-type mice. These changes were cag dependent, as both mouse genotypes infected with the cagE^- mutant only developed minimal inflammation. Tlr9^-/- genotypes did not alter the microbial phenotypes of in vivo-adapted H. pylori strains; therefore, we examined host immunological responses. There were no differences in levels of T_H1 or T_H2 cytokines in infected mice when stratified by host genotype. However, gastric mucosal levels of IL-17 were significantly increased in infected Tlr9^-/- mice compared with infected wild-type mice, and H. pylori infection of IL-17A^-/- mice concordantly led to significantly decreased levels of gastritis. Thus loss of Tlr9 selectively augments the intensity of IL-17-driven immune responses to H. pylori in a cag T4SS-dependent manner. These results suggest that H. pylori utilizes the cag T4SS to manipulate the intensity of the host immune response.

Keywords: Helicobacter pylori; Toll-like receptor 9; gastritis.

Fig. 1.

Loss of Tlr9 exacerbates Helicobacter pylori (H. pylori)-induced inflammation in vivo. Wild-type or Tlr9^−/− C57BL/6 mice were challenged with either vehicle (Brucella broth), H. pylori strain PMSS1, or the PMSS1 isogenic cagE⁻ mutant for 6 wk. A: inflammation scores of wild-type or Tlr9^−/− mice infected with or without H. pylori. Each data point represents the inflammation score from an individual animal. Means ± SE are shown. **P < 0.01, ****P < 0.0001. B: representative hematoxylin and eosin images of H. pylori-infected or uninfected wild-type (WT) and Tlr9^−/− mice at ×20 magnification. Scale bar = 100 μm.

Fig. 2.

Tlr9 deficiency does not alter H. pylori colonization density or virulence phenotypes. A: stomach sections were homogenized and serially diluted on blood-agar plates to quantify H. pylori colonization. Each data point represents the colonization density from an individual animal. ****P < 0.0001. Toll-like receptor 9 (TLR9) activation (B) and CagA translocation (C) were quantified for each output strain. A–C: means ± SE are shown. D: Spearman correlation plot of T4SS function (as quantified by CagA translocation) vs. TLR9 activation of in vivo-adapted H. pylori isolates. Each data point represents 1 output strain (n = 15 isolates per group).

Fig. 3.

H. pylori-induced immune responses in wild-type and Tlr9^−/− mice. A: ELISA of H. pylori-specific serum IgGs in H. pylori-infected mice. B–G: stomach sections from uninfected or H. pylori-infected wild-type or Tlr9^−/− mice were subjected to a multiplex cytokine and chemokine array. Expression levels were normalized to total protein (mg) and square root transformed. Expression levels of representative T_H1 cytokines are shown in IFN-γ (B) and IL-2 (C), representative T_H1 associated chemokines are shown in keratinocyte chemoattractant (KC) (D) and IP-10 (E), and representative T_H2 cytokines are shown in IL-4 (F) and IL-10 (G). Each data point represents an individual animal. Means ± SE are shown. *P < 0.05, **P < 0.01.

Fig. 4.

H. pylori-mediated TLR9 responses and IL-17. A: IL-17 expression in gastric mucosa was determined by cytokine and chemokine array, and results are represented as picograms per milligram of protein (square root transformed). B: mRNA expression of IRF-4 in gastric mucosa was quantified and normalized relative to levels of GAPDH. C: representative images of FoxP3 staining in wild-type and Tlr9^−/− mice. White arrows indicate FoxP3⁺ cells; ×400 magnification, scale bar = 50 μm. D: FoxP3⁺ cells were enumerated in 5 high-powered fields spanning the antrum and corpus from each animal. E: inflammation scores from wild-type or IL1-7A^−/− mice infected with H. pylori strain PMSS1 for 12 wk. A–E: each data point represents 1 animal. Means ± SE are shown. *P < 0.05, **P < 0.01.