The neutrophil-activating protein of Helicobacter pylori promotes Th1 immune responses

Abstract

The Helicobacter pylori neutrophil-activating protein (HP-NAP) is a virulence factor of H. pylori that stimulates in neutrophils high production of oxygen radicals and adhesion to endothelial cells. We report here that HP-NAP is a TLR2 agonist able to induce the expression of IL-12 and IL-23 by neutrophils and monocytes. Addition in culture of HP-NAP, as an immune modulator, to antigen-induced T cell lines resulted in a remarkable increase in the number of IFN-gamma-producing T cells and decrease of IL-4-secreting cells, thus shifting the cytokine profile of antigen-activated human T cells from Th2 to a Th1 cytotoxic phenotype. We also found that in vivo HP-NAP elicited an antigen-specific Th1-polarized T cell response in the gastric mucosa of H. pylori-infected patients. These data indicate HP-NAP as an important factor of H. pylori able to elicit cells of the innate immune system to produce IL-12 and IL-23, and they suggest it as a new tool for promoting Th1 immune responses.

Figure 1. Kinetics of cytokine mRNA levels and IL-12p70 production in neutrophils and monocytes stimulated with HP-NAP.

Cytokine mRNAs in neutrophils (A–C) and monocytes (E–G) were determined by quantitative real-time PCR. The experiment shown is 1 representative of 7 experiments conducted with different cell preparations. The dotted lines represent the level of cytokine mRNA produced by mock cells. IL-12p70 protein levels were measured in the culture supernatants of the same neutrophils (D) and monocytes (H) harvested for messenger evaluation. Levels were assessed by a specific ELISA method. IL-12p70 protein levels at time 0 were under the lower limit of sensitivity of the assay (7.8 pg/ml). The kinetics of production were comparable among different experiments, whereas the amounts varied among different donors.

Figure 2. Flow cytometric analysis of HP-NAP–stimulated monocytes and DCs.

Solid and dotted lines correspond to HP-NAP–treated monocytes and to isotype controls, respectively. Results of 2 representative of 4 consecutive experiments are reported.

Figure 3. Activation of NF-κB in HEK 293 cells transfected with plasmid encoding human TLR2.

Parallel culture samples of hTLR2-transfected HEK 293 cells were stimulated with graded concentrations of HP-NAP (from 0.03 to 1.0 μM) (squares), or with graded concentrations of the specific hTLR2-positive control ligand PAM2 (from 0.1 to 10 ng/ml) (diamonds). A recombinant HEK 293 cell line for the reporter gene only was used as a negative control (data not shown). The NF-κB activation in each sample was quantified as OD values after 24 hours of stimulation. Results of a representative experiment are reported.

Figure 4. Conditioning with HP-NAP promotes IFN-γ production.

Addition in culture of HP-NAP together with antigen increases IFN-γ–producing T cells and reduces IL-4–secreting cells. TT-induced T cell lines were generated from PBMCs of 5 healthy donors in the presence of medium or HP-NAP. T cell blasts of each line were then stimulated with TT in the presence of irradiated autologous APCs, and IFN-γ– or IL-4–producing T cells were assessed by specific ELISPOT assays. Results represent mean numbers (± SD) of SFCs per million cultured cells counted using an automated ELISPOT reader.

Figure 5. Conditioning with HP-NAP promotes the Th1 polarization of antigen-specific T cells.

TT- or allergen-induced T cell lines (left and right panels, respectively) were generated in the presence of medium alone, HP-NAP, or IL-12. T cell blasts of each line were then cloned, and antigen-specific T cell clones were stimulated for 48Πhours with medium or the appropriate antigen in the presence of irradiated autologous APCs. Culture supernatants were then collected and assayed for their IFN-γ and IL-4 content. Clones able to produce IFN-γ, but not IL-4, were categorized as Th1; clones producing IL-4, but not IFN-γ, were coded as Th2, whereas clones producing both IFN-γ and IL-4 were categorized as Th0. Results represent mean percentage proportions (± SD) of clones with the indicated cytokine profile, obtained from series of 3 T cell lines for each condition.

Figure 6. WT, but not HP-NAP–null mutant, H. pylori promotes the Th1 shift of allergen-specific T cells.

Allergen-induced T cell lines were generated in the presence of medium alone, WT H. pylori, or HP-NAP–null H. pylori mutant (5 × 10⁵ CFUs/ml). T cell blasts of each line were then cloned, and allergen-specific T cell clones were stimulated for 48Πhours with medium or allergen in the presence of irradiated autologous APCs. Culture supernatants were then collected and assayed for their IFN-γ and IL-4 content. Results represent mean percentage proportions (± SD) of clones with the indicated cytokine profile, obtained in T cell lines from 3 donors.

Figure 7. Cytotoxic activity of Th clones derived from allergen-induced T cell lines conditioned with medium alone, HP-NAP, or IL-12.

Results represent the percentage specific ⁵¹Cr release induced by single clones in PHA-treated murine ⁵¹Cr-labeled P815 mastocytoma cells at an effector-to-target ratio of 5:1. Horizontal bars and boxes indicate mean values ± SD, respectively.