The neutrophil-activating protein of Helicobacter pylori promotes Th1 immune responses (original) (raw)
HP-NAP stimulates IL-12 and IL-23 production in neutrophil granulocytes. Total RNA was extracted from neutrophils incubated with HP-NAP, retrotranscribed, and amplified by real-time PCR in the presence of specific primers. The amounts of mRNA for IL-12p35 and for IL-12p40 were remarkably increased (Figure 1, A and B), though with different kinetics and degree: IL-12p40 mRNA increased more than IL-12p35 messenger, in agreement with the well-known lower abundance of p35 transcripts even in activated inflammatory cells (15). While the expression of IL-12p40 peaked at 6 hours, the increased expression of p35 already observed at 6 hours reached its maximum at 12 hours. The kinetics of cytokine mRNA levels determined by quantitative RT-PCR were consistent with the kinetic of protein accumulation in culture supernatant (Figure 1D). A significant TNF-α mRNA expression was also found after 6 hours of incubation, before decreasing after 8 hours (data not shown).
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.
The p40 subunit associates not only with IL-12p35, but also with another molecule, p19, to form another heterodimeric cytokine, IL-23 (16). In neutrophils, treatment with HP-NAP resulted in a prompt upregulation of IL-23p19 mRNA that decreased thereafter at minimal levels at 24 hours (Figure 1C).
HP-NAP stimulates IL-12 and IL-23 production in monocytes. To further investigate the effect of HP-NAP on cells of the innate immunity, monocytes isolated from PBMCs of healthy donors were used as targets. HP-NAP was extremely efficient in upregulating the monocyte expression of IL-12p35, IL-12p40, and IL-23p19 mRNAs. In particular, IL-12p35 mRNA showed its peak at 30 minutes (Figure 1E), whereas IL-12p40 mRNA peaked between 8 and 12 hours and was still detectable at 24 hours (Figure 1F). The kinetics of IL-12p40 and IL-12p35 mRNA levels were consistent with the kinetic of IL-12 protein accumulation in the supernatant (Figure 1H). Finally, following HP-NAP stimulation, the monocyte expression of IL-23p19 mRNA (Figure 1G) peaked at 2 hours, followed by a progressive decrease down to baseline levels at 24 hours. These findings indicate that HP-NAP acting on either neutrophils or monocytes contributes to the creation of a cytokine milieu enriched in IL-12 and IL-23, which have the potential to drive the differentiation of antigen-stimulated T cells toward a polarized Th1 phenotype.
HP-NAP promotes MHC class II upregulation and IL-12 production in monocytes and DCs. While control monocytes incubated with medium progressively died between 28 and 36 hours of culture, monocytes incubated with HP-NAP survived longer. In addition, HP-NAP–treated monocytes revealed a progressive shape modification and a tendency to cluster and to detach from the substrate. In order to better characterize these observations, the expression of maturation markers in HP-NAP–stimulated monocytes was evaluated.
Compared with the corresponding baseline values, the expression of HLA-DR increased markedly at day 5 of incubation with HP-NAP (mean fluorescence intensity [MFI] 563 ± 81 versus 62.7 ± 0.7) and even more at day 7 (1,161 ± 117) (Figure 2). CD80 expression as well increased significantly at day 5 in comparison with its baseline (MFI 57.2 ± 15.7 versus 2.5 ± 1.5) and even more at day 7 (92 ± 12). Likewise, the expression of CD86, already increased after 5 days (MFI 53.7 ± 3.7), was remarkably higher at day 7 (148 ± 23), as compared with the baseline (9.1 ± 1.5). In contrast, the expression of CD40, B7-RP1, and B7-H1 was only weakly increased after 5–7 days of stimulation with HP-NAP, whereas the expression of B7-DC and CD83 remained unaffected. No significant change of the expression of the markers was observed in untreated control cells (data not shown). These results suggest that HP-NAP induces the differentiation of monocytes into mature DCs. Furthermore, following 24 hours of stimulation with HP-NAP, DCs produced detectable amounts of IL-12p70 in culture supernatants (mean ± SD, 250 ± 38 pg/ml).
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.
HP-NAP is a TLR2 agonist. Most TLR ligands are conserved microbial products (pathogen-associated molecular patterns, or PAMPs) that signal the presence of infection, and each TLR is triggered by a distinct set of microbial compounds (17–23). In order to define whether a given TLR was involved in the interaction with HP-NAP, we used human embryonic kidney (HEK) 293 cells transfected with plasmids encoding distinct human TLRs (24, 25). HEK 293 cell lines lack expression of endogenous TLRs, although their TLR signaling machine is fully functional (17, 26). NF-κB activation by HP-NAP was determined using an NF-κB–dependent reporter construct. Activation was observed only in cells expressing TLR2, whereas no activation was detectable in HEK 293 cells expressing TLR3, TLR4, TLR5, TLR7, TLR8, or TLR9 (Table 1). Moreover, the activation of NF-κB by HP-NAP was detectable in TLR2-expressing HEK cells in a range from 0.03 to 1.0 μM (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.
HP-NAP-induced activation of NF-κB in HEK 293 cells transfected with plasmid encoding distinct human TLRs
An important point to be addressed was whether other TLR2 agonists, such as PAM2 or PAM3, shared with HP-NAP the ability to induce IL-12 production by monocytes. To this end, graded concentrations of PAM2, PAM3, and HP-NAP were added in cultures of adherent monocytes, and cytokine production was assessed in supernatants after 24 hours. Like HP-NAP, both PAM2 and PAM3 were similarly efficient in inducing TNF-α, IL-6, and IL-8 production by monocytes (Table 2), but only HP-NAP was able to induce IL-12 production in a dose-dependent fashion. In order to rule out that the effects attributed to HP-NAP were due to a contaminating TLR2 ligand, an HP-NAP immune-depleted preparation was tested. As shown in Table 2, immune depletion with an anti–HP-NAP antibody abrogated the induction of cytokine synthesis; likewise, addition in culture of an anti-TLR2 antibody also resulted in abrogation of cytokine production by monocytes (data not shown).
Cytokine production by adherent monocytes induced by LPS or different agonists of human TLR2
Addition in culture of HP-NAP–null H.pylori mutant results in negligible IL-12 production by adherent monocytes. To better define the role of HP-NAP in the induction of IL-12 secretion by monocytes, an HP-NAP–null H. pylori mutant was compared with WT H. pylori for their efficiency in the induction of monocyte cytokine synthesis. Both WT and HP-NAP–null mutant bacteria were able to induce the production of comparable amounts of TNF-α, IL-6, and IL-8 by monocytes (Table 3). In contrast, stimulation with the highest dose of HP-NAP–null mutant H. pylori (5 × 105 CFUs/ml) resulted in very poor secretion of IL-12, which was lower than that induced by a 25 times lower concentration of WT H. pylori (0.2 × 105 CFUs/ml). These data suggest that a number of H. pylori components can activate monocytes to cytokine synthesis, but HP-NAP represents the critical molecule for the induction of substantial IL-12 secretion.
Cytokine production by adherent monocytes induced by WT H. pylori or HP-NAP-null H. pylori mutant
Addition in culture of HP-NAP results in preferential development of IFN-γ–producing T cells and reduction of IL-4–secreting cells. In view of its ability to induce IL-12 and IL-23 secretion by cells of the innate immunity, HP-NAP was tested for its capacity to affect the development of the cytokine profile of tetanus toxoid–specific (TT-specific) T cell responses. PBMCs from 5 TT-reactive donors were stimulated with TT in the presence of medium alone or HP-NAP. T cell blasts of each line were stimulated with the antigen (TT) in the presence of autologous APCs for 24 or 48 hours in ELISPOT microplates coated with anti–IFN-γ or anti–IL-4 antibody, respectively. At the end of the culture period, IFN-γ or IL-4 spot-forming cells (SFCs) were counted. Conditioning with HP-NAP resulted in a remarkable increase of IFN-γ–producing T cells and decrease of IL-4–secreting cells (P = 0.029 and P = 0.05, respectively) (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.
T cell blasts of each TT-induced line were recovered and cloned by limiting dilution according to a high-efficiency protocol (6, 27). A total of 168 CD4+ T cell clones were obtained from the TT-induced T cell lines in the presence of medium, whereas 152 CD4+ clones were obtained from TT-induced T cell lines generated in the presence of HP-NAP. Of the 168 CD4+ clones generated from the TT-induced lines conditioned with medium, 74 (44%) were reactive to TT, whereas the other 94 CD4+ clones of this series failed to proliferate in response to the challenge with TT. In the series of 152 clones generated from TT-induced cell lines conditioned with HP-NAP, 72 (47%) proliferated to the specific antigen stimulation. TT-specific clones of the 2 series were stimulated for 48 hours with TT in the presence of autologous APCs, and IFN-γ and IL-4 levels were measured in culture supernatants. In the series of 74 clones from the TT cell lines conditioned with medium, 22 (30%) expressed a Th1 profile, and 31 (42%) were Th0 producing both IFN-γ and IL-4, whereas 21 (28%) were Th2 clones (Figure 5, left panel). In contrast, in the series of 72 TT-specific clones from the TT-induced lines conditioned with HP-NAP, 49 (68%) were Th1 and 21 (29%) were Th0, whereas only 2 were Th2 (3%).
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.
Conditioning with PAM2 or PAM3 does not affect the development of IFN-γ– or IL-4–producing T cells induced by allergen. In subsequent experiments, PAM2 and PAM3 were compared with HP-NAP for their capacity to affect the development of the cytokine profile of T cell responses specific to the mite allergen Dermatophagoides pteronyssinus. PBMCs from 5 mite allergen–sensitive donors were stimulated with mite allergen in the presence of medium alone, PAM2 (50 ng/ml), PAM3 (50 ng/ml), or HP-NAP (3.0 μM). On day 6, allergen-induced T cell lines were expanded with IL-2. At day 12, T cell blasts of each line were stimulated with the specific allergen in the presence of autologous APCs for 24 or 48 hours in ELISPOT microplates coated with anti–IFN-γ or anti–IL-4 antibody, respectively. At the end of the culture period, IFN-γ or IL-4 SFCs were counted. As shown in Table 4, only conditioning with HP-NAP resulted in a remarkable increase of IFN-γ–producing T cells and decrease of IL-4–secreting cells (P < 0.0005 and P < 0.001, respectively), whereas PAM2 and PAM3 were not effective.
Allergen-induced IFN-γ and IL-4 SFCs in allergen-specific T cell lines conditioned with PAM2, PAM3, or HP-NAP
HP-NAP favors the shift of allergen-specific T cell clones from Th2 to Th1 phenotype. In order to assess whether HP-NAP substantially influenced the in vitro development of Th cell responses to allergens usually oriented to the Th2 pattern, allergen-induced T cell lines were generated from PBMCs of house dust mite allergen–sensitive donors, and medium, HP-NAP, or IL-12 was added at the time of allergen exposure in vitro. Stimulation with mite allergen resulted in the expansion of T cell lines. T cell blasts of each line were cloned as described previously (6, 27). A total of 38 CD4+ clones were obtained from the allergen-induced T cell lines in the presence of medium, whereas 40 and 55 CD4+ clones were obtained from allergen-induced lines generated in the presence of HP-NAP or IL-12, respectively. Of the 38 CD4+ clones generated from the allergen-induced lines conditioned with medium, 18 (47%) were reactive to mite allergen. In the series of clones generated from T cell lines conditioned with HP-NAP, 21 (52.5%) of the 40 CD4+ clones proliferated upon allergen challenge, whereas in the series of clones generated from T cell lines conditioned with IL-12, 42% of the CD4+ clones were allergen specific.
Allergen-specific T cell clones of the 3 series were stimulated for 48 hours with allergen in the presence of autologous APCs, and IFN-γ and IL-4 levels were measured in supernatants. In the series of allergen-specific clones from the T cell lines conditioned with medium, no clone expressed a Th1 profile, and 11% were Th0 producing both IFN-γ and IL-4, whereas 89% were Th2 clones (Figure 5, right panel). In contrast, in the series of allergen-specific clones from the allergen-induced lines conditioned with HP-NAP, as many as 38% were Th1, and 33% were Th0, whereas only 29% were Th2. As expected, conditioning with IL-12 resulted in the development of a series of allergen-specific clones including 22% Th1, 52% Th0, and 26% Th2. In conclusion, similarly to IL-12, addition in culture of HP-NAP resulted in a shift from preferential type 2 to predominant type 1 T cell responses, with remarkable expansion of IFN-γ–producing T cell clones and strong reduction (χ2 14.347, P < 0.0001) of allergen-specific clones with Th2 profile.
HP-NAP–null H. pylori mutant fails to shift to Th1 the development of allergen-specific T cell clones. In subsequent experiments, WT H. pylori and HP-NAP–null H. pylori mutant were compared for their ability to affect the development of the cytokine profile of mite allergen–specific T cell clones. PBMCs were obtained from 3 allergic donors, and for each donor 3 parallel allergen-induced T cell lines were started: the first was added with medium alone, the second with WT H. pylori (5 × 105 CFUs/ml), and the third with HP-NAP–null H. pylori mutant (5 × 105 CFUs/ml). After expansion with IL-2 and cloning, the 3 series of clones from each donor were compared for their IFN-γ and/or IL-4 production upon allergen stimulation. In the series of 143 allergen-specific clones from lines conditioned with medium, 3 (2%) were Th1, 54 (38%) were Th0, and 86 (60%) were Th2. In contrast, conditioning with WT H. pylori at the time of allergen exposure resulted in a series of 136 allergen-specific clones including 30 (22%) Th1, 80 (59%) Th0, and only 26 (19%) Th2 (P < 0.0005), whereas conditioning with HP-NAP–null H. pylori mutant resulted in 119 allergen-specific clones, of which only 5 (4%) were Th1, 56 (47%) Th0, and 58 (49%) Th2 (Figure 6). These data indicate that WT, but not HP-NAP–null mutant, H. pylori is able to induce a significant (P < 0.0005) Th1 shift of allergen-specific T cells and confirm that HP-NAP is a powerful Th1-polarizing agent in vitro.
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.
Addition in culture of HP-NAP results in preferential development of cytotoxic T cells producing high amounts of TNF-α. Since cytolytic activity is a common property of activated Th1 and of most Th0 clones but is usually missing in Th2 clones (28), the cytolytic potential of Th clones was assessed in a lectin-dependent (phytohemagglutinin M form–dependent) 51Cr-release assay with P815 murine mastocytoma cells as targets. At an effector-to-target ratio of 5:1, all the 6 Th0 but none of the 32 Th2 clones generated from allergen-induced T cell lines conditioned with medium were cytolytic (Figure 7). The mean (± SD) percentage specific 51Cr release induced by these Th0 clones was quite low (16.5% ± 11%). In the series of Th clones derived from T cell lines conditioned with HP-NAP, the 9 Th2 clones were not cytolytic, whereas all the 14 Th0 and the 17 Th1 clones expressed cytolytic activity with a significantly higher specific 51Cr release in comparison with that of clones from lines conditioned with medium (52% ± 21%, P < 0.0001). Significantly higher 51Cr release was still detectable at an effector-to-target ratio of 1:1 (28% ± 15%, P < 0.0001), suggesting that the cytolytic potential of those clones was high. Conditioning with HP-NAP, like IL-12, resulted in the outgrowth of allergen-specific clones, the majority of which showed cytolytic activity.
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.
With regard to the ability of T cell clones to produce TNF-α upon allergen stimulation, in the series of Th clones derived from the lines conditioned with medium, only 3 (9%) of the 34 Th2 and 5 (83%) of the 6 Th0 clones produced small amounts of TNF-α (0.56 ± 0.39 ng/ml, range 0.12–0.89, and 0.66 ± 0.43 ng/ml, range 0.18–1.35, respectively). In contrast, in the series of Th clones derived from the lines conditioned with HP-NAP, all the 17 Th1 and 13 of the 14 Th0 clones were able to produce high amounts of TNF-α (3.84 ± 2.62 ng/ml, range 1.40–9.50, and 3.25 ± 3.15 ng/ml, range 0.24–9.20), whereas only 3 of the 9 Th2 clones produced low amounts of that cytokine (0.12 ± 0.04 ng/ml, range 0.09–0.17). Likewise, in the series of clones derived from the lines conditioned with IL-12, all the 14 Th1 and the 24 Th0 clones produced high amounts of TNF-α (5.95 ± 3.28 ng/ml, range 0.84–10.50, and 2.90 ± 2.65 ng/ml, range 0.19–9.50), whereas only 9 of the 17 Th2 clones produced low amounts of TNF-α (0.17 ± 0.10 ng/ml, range 0.07–0.32).
HP-NAP–specific T cells from the _H. pylori_–induced gastric infiltrates are polarized cytotoxic Th1 cells producing TNF-α. In order to assess whether in vitro data reported above had any in vivo correlate, biopsy specimens of antral mucosa from 5 _H. pylori_–infected patients were cultured for 7 days in IL-2–conditioned medium in order to preferentially expand the in vivo–activated T cells present in their antral inflammatory infiltrates. T cell blasts were recovered and cloned by limiting dilution, as reported previously (6). In a series of 144 CD4+ T cell clones obtained from the 5 gastric biopsies, 27 (19%) showed significant proliferation to HP-NAP (Table 5).
Cytokine profile and cytolytic activity of HP-NAP-specific CD4+ T cell clones derived from the gastric mucosa of _H. pylori_-infected patients
Upon 48 hours of stimulation with HP-NAP, 100% of HP-NAP–specific gastric clones showed a clear-cut Th1 profile. All the 27 HP-NAP–specific clones produced high amounts of TNF-α (4.2 ± 3.3 ng/ml, range 0.75–11.4), and all expressed powerful cytolytic activity in the lectin-dependent 51Cr-release assay using P815 cells as target cells (Table 5).
In the same series of 144 CD4+ T cell clones obtained from the gastric biopsies, 30 clones (21%) were specific for H. pylori antigens other than HP-NAP, such as CagA, VacA, and urease. Upon stimulation with the specific H. pylori antigen, 25 of 30 clones (83%) showed a Th1 profile, whereas only 5 were Th0. All clones produced levels of TNF-α comparable to those produced by HP-NAP–specific clones, and 27 of 30 (90%) expressed cytotoxic activity.