IL-17 Is a Critical Component of Vaccine-Induced Protection against Lung Infection by Lipopolysaccharide-Heterologous Strains of Pseudomonas aeruginosa¹ Free

Pseudomonas aeruginosa is a significant pathogen for hospitalized and immunocompromised patients. It is the most commonly isolated Gram-negative rod in the setting of hospital-acquired pneumonia (1) and, in the setting of ventilator-associated pneumonia, has a strikingly high attributable mortality (2). Vaccine development for LPS-smooth strains of P. aeruginosa, which are the type of strains that cause nosocomial infections and are the initial colonizing strains in patients with cystic fibrosis (3), has been hampered by the inability to achieve broad protection across different LPS O Ag types (called serotypes or serogroups). In both mice (4) and humans (5), O Ag-based vaccine candidates have largely been abandoned due to lack of LPS-heterologous protection.

We are developing live-attenuated P. aeruginosa strains as intranasal vaccines and have shown previously that an unmarked aroA deletion mutant of the P. aeruginosa laboratory strain PAO1, denoted PAO1ΔaroA, is highly attenuated and, after intranasal immunization of mice, engenders high levels of serum opsonic Ab directed against LPS-homologous strains (6). Protection studies showed that after active immunization the PAO1ΔaroA vaccine prevented lethal pneumonia after inoculation with the parental strain PAO1, its cytotoxic variant ExoU⁺ PAO1, or some LPS subtype-related strains; however, there was no protection after inoculation with LPS-heterologous strains (7). Passive immunization using antisera generated in nasally immunized rabbits protected only against lethal pneumonia due to ExoU⁺ PAO1 and failed to protect against LPS-heterologous strains or even against the parental strain PAO1 despite serum opsonic Ab titers that were identical to or higher than those observed after active immunization where protection was achieved (6). These results suggested both that the immunodominant nature of the LPS O Ag of PAO1ΔaroA may be hindering a broad Ab response and also that immune effectors elicited by active vaccination in addition to Ab are likely to be important for full protection against pneumonia.

Because of the broad host range of P. aeruginosa strain PA14 (8), we hypothesized that a live-attenuated vaccine based on this strain might elicit a broader array of immune effectors and we thus constructed and tested an aroA deletion mutant in PA14 called PA14ΔaroA. Remarkably, we found that active intranasal immunization with PA14ΔaroA could protect against lethal pneumonia caused by LPS-heterologous strains in the absence of opsonic Ab. Additional experiments showed that the neutrophil-attracting, T cell-secreted cytokine IL-17 is critical for the protective efficacy of this vaccine. Our results suggest that when opsonic Ab levels are low, rapid recruitment of neutrophils by IL-17-secreting vaccine-induced Th17 cells can achieve broad-based protections against P. aeruginosa pneumonia.

The bacterial strains and plasmids used in these experiments, along with their relevant characteristics and sources, are listed in Table I. PA14ΔaroA was constructed as previously described (6).

Frozen bacterial stocks were plated and grown overnight at 37°C on tryptic soy agar (TSA)³ or TSA containing the appropriate antibiotics (carbenicillin at 400 μg/ml, gentamicin at 150 μg/ml). For intranasal inoculation experiments, bacteria were suspended in PBS. For some survival experiments, bacteria were suspended in PBS containing 1% heat-inactivated FCS (HyClone). Concentrations were adjusted spectrophotometrically and confirmed after serially diluting in PBS containing 1% FCS and enumerating growth on TSA after overnight incubation at 37°C.

Six- to 8-wk-old female C3H/HeN mice were purchased from Harlan Sprague Dawley, while C57BL/6 mice were purchased from Taconic Farms. The C57BL/6 mice were age- and gender-matched with IL-17R knockout (KO) mice (also in C57BL/6 background) that were originally obtained from Amgen and bred in-house. Mice were housed under virus-free conditions, and all animal experiments complied with institutional and federal guidelines regarding the use of animals in research. Immunizations were performed as described previously (6). Briefly, mice anesthetized with ketamine and xylazine were inoculated by placing 10 μl of the live P. aeruginosa ΔaroA vaccine strain or live Escherichia coli HB101 as a control on each nostril. Escalating doses of 1 × 10⁸, 5 × 10⁸, and 1 × 10⁹ CFU were administered at weekly intervals. For passive immunization experiments, mice were given pooled mouse antiserum (0.4 ml i.p.) 1 day before challenge. IL-17 depletion studies were done using IgG (1 mg i.p.) purified with protein G-linked agarose (Invitrogen) from an anti-mouse IL-17 rabbit antiserum kindly provided by A. Tzianabos (9). Control rabbit IgG was purchased from Sigma-Aldrich.

All animal experiments complied with institutional and federal guidelines regarding the use of animals in research. We used our previously described model of acute fatal pneumonia following intranasal inoculation (10). In this model, 67–100% of the bacterial inoculum is consistently and rapidly translocated to the lungs from the nose of anesthetized 6- to 8-wk-old mice (10). For quantitation of CFU, neutrophils, and cytokines in bronchoalveolar lavage fluid (BALF), mice were sacrificed using CO₂ at the indicated time points and then BALF was obtained by cannulation of the trachea followed by two instillations of 1 ml of cold PBS containing 0.5 mM EDTA. For CFU measurements, BALF was diluted in 1% proteose peptone (BD Biosciences) and then plated on TSA for enumeration of CFU after overnight growth at 37°C. The limit of detection was 1 CFU in 100 μl of the undiluted BALF, which corresponded to 10 CFU/ml. BALF was centrifuged and the supernatant frozen at −80°C before measurement of IL-17 by ELISA (R&D Systems). Erythrocytes in the cell pellets were lysed using a mouse erythrocyte lyse kit (R&D Systems) according to the manufacturer’s instructions. Total cell counts were determined with a hemacytometer and trypan blue counterstaining. Percentage neutrophils was measured by flow cytometry using PE-labeled anti-Ly6G (1A8) after treatment with anti-CD16/CD32 (2.4G2, FcBlock, BD Biosciences). For survival analyses, mice were followed daily for at least 7 days to assess mortality. Moribund animals were sacrificed and considered nonsurvivors.

Standard methods were employed, as described (6), with infant rabbit serum (Accurate Chemical) as the complement source and polymorphonuclear leukocytes isolated from human volunteers. By convention, the serum concentrations listed were the input rather than final concentration (the initial serum dilution is further diluted 4-fold in the assay tube). Under routine conditions, killing of ≥50% is considered biologically significant and therefore serves to classify a serum as positive for opsonic killing activity. Although killing <50% is sometimes statistically significant, this level of killing is not considered biologically significant.

P. aeruginosa-specific IgG binding curves were measured by ELISAs using plates coated with whole live bacteria as previously described (6).

Splenic T cells were isolated using CD3 cell selection columns from R&D Systems. Cell purity was routinely >90% CD3⁺ cells as assessed by flow cytometry (not shown). Lung leukocytes were isolated after perfusion of the lungs in situ with PBS containing heparin (10 U/ml) followed by incubation with EDTA, then digestion with collagenase and DNase, and then sedimentation over a discontinuous Percoll gradient, all as previously reported (11). Bacteria used as Ags were killed by incubation of a 10⁹ CFU/ml suspension with gentamicin (1 mg/ml in PBS) for 1 h at 37°C, with killing verified by absence of growth of 100 μl samples plated on TSA. Staphylococcal enterotoxin A was purchased from Sigma-Aldrich and used at final concentration of 10 ng/ml. For proliferation experiments, each well of a 96-well round-bottom plate was seeded with 1 × 10⁵ T cells, 1 × 10⁵ irradiated (1500 rads) splenocytes isolated from nonimmunized C3H/HeN mice and purified using density centrifugation over Lympholyte-M (Accurate Chemical), and 1 × 10⁶ gentamicin-killed bacteria, all suspended in RPMI 1640 containing 10% heat-inactivated FCS as well as standard concentrations of glutamine, sodium pyruvate, nonessential amino acids, penicillin, streptomycin, and 1 mM 2-ME (all from Invitrogen). On day 5 of stimulation, wells were pulsed with [³H]thymidine (1 μCi/well) for 8 h before scintillation counting.

For cytokine secretion experiments, 96-well plates were used containing 1 × 10⁵ T cells and 1 × 10⁵ irradiated splenocytes as feeder cells. For both proliferation and cytokine secretion experiments, anti-CD4 (GK1.5), anti-CD8 (53-6.7), and rat IgG isotype controls were purchased from BD Biosciences and used at 1 μg per well. For stimulation of bulk lung leukocytes isolated 18 h after challenge with live bacteria, no feeder cells were added. IL-17 and IFN-γ were measured in supernatants after 7 days of stimulation using ELISA (R&D Systems). Intracellular cytokine staining was performed using a kit from BD Biosciences according to the manufacturer’s instructions. Lung leukocytes isolated as described above 2 h after challenge with live bacteria were stimulated for 15 h ex vivo with PMA and ionomycin (Leukocyte Activation Cocktail, BD Biosciences) in the presence of brefeldin A (GolgiPlug, BD Biosciences). After treatment with anti-CD16/CD32, cells were stained with FITC-labeled Abs to mouse TCRβ (H57–597) or CD3ε (145-2C11), followed by fixation and permeabilization and then staining with PE-labeled anti-mouse IL-17 (TC11–18H10.1) and analysis on a MoFlo (DakoCytomation) flow cytometer. Abs and appropriate isotype controls were obtained from BD Biosciences.

Nonparametric data were evaluated by Mann-Whitney U test (for two-group comparisons) or Kruskal Wallis (for three or more groups) with Dunn’s multiple comparison test for pairwise comparisons. Parametric data were analyzed by t test (for two-group comparisons) or ANOVA with Dunnett’s multiple comparison test using E. coli-immunized mice or cells as the control comparator group. All analyses were performed using Prism software (GraphPad Software). Survival data were analyzed by Fisher’s exact test or by survival analysis with the Kaplan-Meier method, also using Prism.

We initially evaluated the protective efficacy of the PA14ΔaroA vaccine against LPS-homologous and LPS-heterologous strains. In our prior work, we showed that the serogroup O2/O5 vaccine PAO1ΔaroA elicited high levels of opsonic Ab as well as protection from lethality after lung challenge with LPS-homologous strains but not with LPS-heterologous strains. As shown in Table II, nasal immunization with PA14ΔaroA resulted in significantly better survival compared with E. coli-immunized mice after challenge with a high dose of the parental strain PA14. There was no protection after challenge of PAO1ΔaroA-immunized mice with a lower dose of strain PA14. Notably, PA14ΔaroA-immunized mice were protected from lethality after challenge with ExoU-producing strains of the LPS-heterologous strains PAO1 (serogroup O₂/O5) and PAO6a,d (serogroup O6). There was no protection, however, after challenge with the noncytotoxic strains Fisher IT-7 (serogroup O2/O5) or 6294 (serogroup O6). This is likely related to the higher LD₅₀s of the noncytotoxic strains in this model and thus the very high challenge doses required to achieve 100% mortality in the E. coli-immunized control mice.

For most vaccines effective against P. aeruginosa infections, serum opsonic Ab directed against the LPS O Ag has been the most important immune effector (12). To assess the role of serum Abs and Abs to specific Ags such as the ExoU cytotoxin in the protection afforded by the PA14ΔaroA vaccine, we first tested the opsonic killing activity of pooled sera obtained from immunized mice 3 wk after the third (final) nasal immunization. As depicted in Fig. 1,A, there was minimal opsonic killing of strain PAO1 in serum from mice immunized with the PA14ΔaroA strain, whereas good killing was achieved using sera from mice immunized with the LPS-homologous PAO1ΔaroA strain. Opsonic killing activity in antisera raised to PA14ΔaroA was present against the parental PA14 strain, although the opsonic titer (the calculated serum dilution at which the killing activity dropped to 50% as determined by linear regression) was ∼50 (not shown), which is much lower than the titer of ∼1000 seen with antiserum to PAO1ΔaroA against its parental strain PAO1 (7). Because PA14ΔaroA possesses the genes for ExoU while PAO1ΔaroA does not, and thus the PA14-based vaccine could induce Ab to this Ag, we next evaluated whether Ab to ExoU could be mediating opsonic killing if for unexpected reasons this Ag was retained on the bacterial cell surface. As shown in Fig. 1 B, there was no significant opsonic killing of an ExoU-producing strain compared with the strain containing the empty vector.

Next, we determined whether passive immunization with pooled antisera from PA14ΔaroA-immunized mice mediated protection against lethal pneumonia due to the LPS-heterologous strain ExoU⁺ PAO1. Mice given the PA14ΔaroA antiserum i.p. 1 day before challenge had significantly higher mortality compared with the 0% mortality of mice given identical amounts of antisera raised to the LPS-homologous PAO1ΔaroA strain (Fig. 1,C). These results suggested, surprisingly, that an antiserum-independent mechanism is a key component of the heterologous protection engendered by active vaccination with the PA14ΔaroA vaccine. We also tested for O Ag-specific and ExoU-specific Abs by comparing IgG titers of the PAO1ΔaroA- and PA14ΔaroA-immunized mouse antisera to whole cells of the wild-type parental strain PAO1 (containing the empty vector) compared with strain ExoU⁺ PAO1 as well as ExoU-producing and nonproducing LPS-rough galU mutants of strain PAO1 (galU mutants are O Ag deficient with a truncated outer LPS core) (13). As shown in Fig. 2, the PA14ΔaroA antisera had the expected lower reactivity than did the antisera raised to PAO1ΔaroA against P. aeruginosa strains PAO1 (pUCP19) and ExoU⁺ PAO1 based on its different LPS serogroup. The titers to the O Ag-deficient galU mutants were identical, suggesting that the PA14ΔaroA vaccine elicited a comparable level of Ab to non-O side-chain Ags as did the PAO1ΔaroA vaccine.

To determine whether immune T cells are induced by nasal immunization with the P. aeruginosa ΔaroA vaccines, we isolated splenic T cells 3 wk after immunization (before challenge) and cocultured them with irradiated splenocytes as APCs and gentamicin-killed P. aeruginosa as Ag and measured T cell proliferation by [³H]thymidine incorporation after 5 days. As shown in Fig. 3,A, there was a high level of proliferation of both PAO1ΔaroA- and PA14ΔaroA-immune T cells to either PAO1 or PA14, although the PA14ΔaroA-immune T cells proliferated less well when stimulated with PAO1 compared with the PAO1ΔaroA-immune T cells. Proliferation of PAO1ΔaroA-immune T cells was inhibited by an Ab to CD4 but not to CD8 (Fig. 3,B). Proliferation of PAO1ΔaroA- and PA14ΔaroA-immune T cells was high when stimulated with multiple P. aeruginosa strains regardless of LPS serogroup or ability to produce ExoU (Fig. 3 C). Interestingly, the P. aeruginosa ΔaroA-immune T cells proliferated when cultured with E. coli, but the E. coli-immune T cells were not significantly stimulated by P. aeruginosa strains.

As neutrophils are known to be essential mediators of protective immunity in the lung against P. aeruginosa infection (14, 15), we hypothesized that the recently described Th17 subset of CD4 helper T cells, which secrete the neutrophil-attracting cytokine IL-17, might play a role in rapid recruitment of neutrophils to the airways. We thus evaluated IL-17 secretion after 7-day coculture of splenic T cells from naive and immunized mice cultured with irradiated splenocytes and gentamicin-killed P. aeruginosa strains PAO1 and PA14 as Ags (Fig. 4, A and B). For comparison, we also measured levels of IFN-γ, the predominant Th1 effector cytokine, and found them not to be significantly different among the vaccine groups. In contrast, the levels of IL-17 in the supernatants of the P. aeruginosa ΔaroA-immune T cells stimulated with P. aeruginosa were significantly higher than those of control T cells. Addition of the anti-CD4 mAb (clone GK1.5, which blocks class II MHC Ag-specific binding to CD4 and thereby blocks CD4 T cell function) (16) during coculture returned the IL-17 levels to those of the control T cells (Fig. 4,B), indicating that CD4 T cells are the predominant source of IL-17 in this system. We also isolated intraparenchymal and airway lung leukocytes 18 h after challenge of immune and nonimmune mice by digesting the lung tissue with collagenase and DNase followed by a discontinuous Percoll gradient. Numbers of T cells in the lungs before challenge were too low to allow accurate assessment of cytokine secretion from cells before challenge, so we therefore only evaluated cytokine secretion from lung T cells after challenge. At the 18 h time point after challenge, the cellular infiltrate is ∼35% lymphocytes (with the remainder neutrophils) as assessed by forward- and side-scatter FACS analysis, and the composition was not significantly different between the experimental groups (not shown). The isolated lung leukocytes were cocultured with gentamicin-killed PAO1 for 7 days. The IL-17 levels in the supernatants of these in vivo-immunized, ex vivo-stimulated lung leukocytes are shown in Fig. 4 C and were remarkably higher in the ΔaroA-immune groups, with the PAO1ΔaroA-immune and PA14ΔaroA-immune T cells having ∼10-fold and 40-fold higher IL-17, respectively, than did the control T cell groups.

To determine the cellular source of the IL-17 in vivo, we measured intracellular staining for IL-17 in lung parenchymal and airway lymphocytes isolated as described above but from mice sacrificed just 2 h after challenge with live bacteria. Again, numbers of lymphocytes in the lungs of immunized mice before challenge were too low to allow intracellular cytokine analysis. Cells were stimulated for 15 h with PMA and ionomycin in the presence of brefeldin A. As shown in Fig. 4 D, there was a small population of IL-17-producing TCRβ-positive cells in the lungs of ΔaroA-immune mice, with 3-fold more in the PA14ΔaroA-immunized group. Similar proportions of IL-17-positive cells were seen in CD3ε-positive cells, and no IL-17-positive cells were seen outside the lymphocyte gate of ΔaroA-immune mice (not shown). We were unable to assess CD4 staining in these experiments due to apparent decreased expression of CD4 during stimulation (not shown). Unstimulated cells showed no detectable IL-17 (not shown). Lung leukocytes (lymphocytes and nonlymphocytes) isolated 18 h after challenge with live bacteria showed no intracellular staining for IL-17 with or without PMA/ionomycin stimulation (not shown).

Because IL-17 is known to stimulate neutrophil recruitment to the lung, we tested whether the numbers of neutrophils recruited to the airways are greater in the P. aeruginosa ΔaroA-immunized mice and whether the different P. aeruginosa ΔaroA vaccines elicited different neutrophil recruitment. As shown in Fig. 5,A, there were significantly more neutrophils in BALF 6 h after challenge of P. aeruginosa ΔaroA-immunized mice compared with the E. coli-immunized and unimmunized controls. Notably, even at this early time point after challenge, there were significantly fewer CFU of P. aeruginosa in the BALF (Fig. 5,B) and homogenized lung tissue (not shown) in the P. aeruginosa ΔaroA-immunized mice compared with the control groups. IL-17 levels in BALF (Fig. 5 C) obtained 6 h after challenge with a lethal dose of ExoU⁺ PAO1 were significantly higher in the PA14ΔaroA-immunized compared with the E. coli-immunized mice (p < 0.01, ANOVA with Dunnett’s multiple comparison test). At the later time point (18 h), neutrophil numbers and IL-17 levels in BALF were highest in the E. coli-immunized mice. Bacterial CFU were very high in the control groups at this time after challenge, indicating that these neutrophils were not effective at clearing the challenge strain.

We next determined the effects of neutralization of IL-17 before lung challenge with the LPS-heterologous strain PAO1 ExoU⁺ in mice immunized intranasally with the P. aeruginosa PA14ΔaroA vaccine or with E. coli as control. As expected, there was no effect of neutralization of IL-17 on survival following challenge of the E. coli-immunized mice (Fig. 6,A, left panel). However, there was significantly higher mortality (p < 0.02, logrank test) in the PA14ΔaroA-immunized mice that received anti-IL-17 IgG compared with the mice given control IgG (Fig. 6,A, right panel), indicating that depletion of IL-17 abrogates vaccine-induced protection. In contrast, mice immunized with the PAO1ΔaroA vaccine and then similarly depleted of IL-17 had no change in median survival and were fully protected against LPS-homologous strain PAO1 ExoU⁺ because these mice had high levels of serum opsonic Ab specific for the LPS of strain PAO1 (not shown). We also found that when we immunized mice genetically deficient in production of the IL-17 receptor (IL-17R KO) with PA14ΔaroA (or with E. coli as control) and then challenged them with ExoU⁺ PAO1, the IL-17R KO mice had significantly decreased survival compared with wild-type mice (Fig. 6,B, right panel). We observed lower percentage survival in the PA14ΔaroA-immunized wild-type C57BL/6 mice challenged with 5 × 10⁷ CFU/mouse (40% survival, Fig. 6,B) compared with PA14ΔaroA-immunized wild-type C3H/HeN mice given the same challenge dose (88%, Table II). This was likely due to the high susceptibility of mice in the C57BL/6 background to P. aeruginosa lung infection (17) and the relative resistance of C3H/HeN mice (18).

Consistent with the IL-17 neutralization experiments in PAO1ΔaroA-immunized C3H/HeN mice, IL-17R KO mice immunized with the PAO1ΔaroA vaccine and then challenged with the LPS-homologous strain ExoU⁺ PAO1 were fully protected (not shown). These PAO1ΔaroA-immunized IL-17R KO mice had normal levels of opsonic Ab to strain PAO1 (not shown), which very likely accounted for the lack of an impact of deficient IL-17 signaling. We next evaluated the neutrophil recruitment to the airways 6 h after challenge of PA14ΔaroA-immunized wild-type C57BL/6 mice compared with PA14ΔaroA-immunized IL-17R KO mice. We again used P. aeruginosa strain ExoU⁺ PAO1 (7 × 10⁶ CFU/mouse) as the challenge strain. As shown in Fig. 6,C, there were significantly fewer BALF neutrophils normalized to bacterial load in the IL-17R KO mice. Of note, nonimmunized IL-17R KO and wild-type C57BL/6 mice had similar survival curves when challenged with 2 × 10⁶ CFU of strain ExoU⁺ PAO1 (Fig. 6 D) or 7 × 10⁵ CFU (100% survival in both groups, n = 6 mice/group, not shown), indicating that IL-17R signaling is not important for the innate host defense against acute lethal pneumonia caused by P. aeruginosa.

In the present study, we tested the hypothesis that a live-attenuated P. aeruginosa vaccine based on the broad-host range P. aeruginosa strain PA14 would elicit a broader array of immune effectors than a vaccine derived from strain PAO1. We found that unlike the PAO1ΔaroA vaccine, active immunization with the PA14ΔaroA vaccine could protect against acute lethal pneumonia caused by LPS-heterologous P. aeruginosa strains. Surprisingly, we observed that this protection was not due to serum opsonic activity but rather was dependent on the T cell-secreted cytokine IL-17. The protective efficacy was coincident with a rapid reduction in bacterial CFU in BALF as well as higher neutrophil numbers and higher IL-17 in the BALF 6 h after lung challenge. These findings indicate that active immunization with P. aeruginosa vaccines that induce T cells secreting IL-17 could be a key component of full immunity to lung infection by this pathogen and, furthermore, might be an explanation for why a large clinical trial of passively administered hyperimmune IgG obtained from volunteers given a multivalent P. aeruginosa O Ag conjugate vaccine did not reduce the incidence of infection in recipients of the Ab (5).

Recent evidence suggests that IL-17-secreting CD4⁺ Th cells comprise a distinct lineage called Th17 (19, 20) and that they play an important role in innate antibacterial host defense in the lung (21, 22, 23) as well as in autoimmunity (24). IL-17 is a proinflammatory cytokine expressed primarily by activated memory CD4⁺ T cells and is the prototypic member of a family of cytokines named IL-17A–F (where IL-17A is what is typically denoted IL-17, and IL-17E is IL-25) (25). IL-17-secreting NKT cells (specifically, CD1d-restricted, NK1.1-negative NKT cells with invariant TCR) have also recently been shown to recognize glycolipid ligands derived from Sphingomonas wittichii and Borrelia burgdorferi and to be involved in neutrophil recruitment to the airways after exposure to LPS (26). Th17 cell development has been linked to IL-23, an IL-12 cytokine family member that shares a common subunit with IL-12 (IL-12p40) but has a distinct subunit, p19 (27). TGF-β appears to be critical for commitment to Th17 development by increasing IL-23R expression, thereby conferring responsiveness to IL-23 (28). An inflammatory milieu (in particular the proinflammatory cytokine IL-6, with amplification by IL-1β and TNF-α) is also required for Th17 polarization (29).

The mechanisms whereby the PA14ΔaroA vaccine induces more robust IL-17 responses than the PAO1ΔaroA vaccine after subsequent challenge with live bacteria are not clear. Presumably, the PA14-based vaccine induces higher levels of IL-23, IL-6, IL-1β, TNF-α, and/or TGF-β during the priming of the immune response in the lung or local lymph nodes. This could be related to differential inflammatory activity of the different LPS O Ags of the two vaccine strains and/or to one or several of the additional genes and gene clusters possessed by strain PA14 that are absent from strain PAO1, including the pathogenicity islands PAPI-1 (108 kb) and PAPI-2 (11 kb) (30). Notably, a subtractive hybridization technique called representational difference analysis found not only that several genes, including pilA and pilC, in strain PA14 differed substantially from their counterparts in strain PAO1 but also that only strain PA14 possessed a homolog of the gene for the siderophore-associated ABC transporter protein YbtQ of Yersinia pestis that, when mutated, led to significant attenuation in G. mellonella and in a burned mouse model of sepsis (31). While some siderophores are known to suppress T cell proliferation and function (32), others such as the siderophore lactoferrin have been shown to induce Th responses to the bacillus Calmette-Guérin vaccine (33), so it is conceivable that differences in siderophore production might skew a vaccine-induced T cell response.

The action of IL-17 in the lung centers on neutrophil recruitment via induction of CXC chemokine secretion (KC and MIP-2 in rodents, growth-related oncogene and IL-8 in humans) along with granulopoietic factors (such as G-CSF) that lead to increased bone marrow production and/or prolonged survival of neutrophils (21). In studies of innate immunity to Klebsiella pneumoniae, Kolls and coworkers found that IL-17R-deficient mice were highly susceptible in an acute pneumonia model, with increased mortality and bacterial dissemination associated with delayed neutrophil recruitment to the lung as well as lower levels of G-CSF and MIP-2 in the knockout mice (23, 34). In acute P. aeruginosa pneumonia, on the other hand, we found that nonimmune IL-17R-deficient mice had similar susceptibility compared with wild-type mice, although the IL-17R-deficient mice were much more susceptible in the setting of vaccination. In a different lung infection model, the Kolls group has shown that IL-23-deficient mice had less lung inflammation after challenge with a mucoid P. aeruginosa strain encased in agar beads (35).

Overexpression of IL-17 in the lung via a recombinant adenoviral vector led to local induction of TNF-α, MIP-2, and G-CSF and augmented neutrophil recruitment and bacterial clearance after challenge with K. pneumoniae (22). In this model, signaling through TLR4 was shown to be critical for IL-23 generation (36). Interestingly, cystic fibrosis patients were found to have high levels of IL-17 in BALF, particularly during pulmonary exacerbations (37). Whether IL-17 plays a role in the excessive neutrophilic infiltration and IL-8 secretion characteristic of cystic fibrosis lung disease (38) remains to be determined.

While the time course of IL-17 production in innate immunity models is relatively slow (12–16 h) (23), the production of IL-17 in settings wherein the adaptive response has been activated is far more rapid (as early as 3 h) (39). There is a growing body of evidence that there is a critical role of IL-17 in adaptive immunity to extracellular bacterial respiratory pathogens, particularly Streptococcus pneumoniae and Bordetella pertussis. Malley and coworkers have shown that IL-17 mediates antibody-independent, vaccine-induced protection against nasopharyngeal pneumococcal colonization in mice immunized with the species-common cell wall polysaccharide called the C polysaccharide (40). Higgins et al. have demonstrated that T cell secretion of IL-17 is critical for protective efficacy of the whole-cell B. pertussis vaccine and is also dependent on intact TLR4 signaling (41). Antibody-mediated neutralization of IL-17 in the pertussis studies resulted in an ∼10-fold increase in CFU 10 days after infection, but the impact on survival was not assessed. Those workers also demonstrated that exposure of peritoneal macrophages or macrophage cell lines to IL-17 enhanced bactericidal activity (41). Evaluation of IL-17R-deficient mice has not been reported in either of these models.

A number of investigators have demonstrated T cell-mediated control of P. aeruginosa infections in rodent models. A high-molecular weight polysaccharide Ag prepared from the LPS O Ag elicited P. aeruginosa-specific T cells capable of killing the organism in vitro and protecting neutropenic mice against infection in a mechanism whereby anti-LPS IgG on the T cell surface mediated antibody-dependent cellular cytotoxicity (42). Other workers have observed CD4⁺ T cell-mediated control of P. aeruginosa pneumonia after vaccination with paraformaldehyde-killed P. aeruginosa given to rats orally or injected into Peyer’s patches (43, 44). Intravenous immunization of mice with P. aeruginosa-pulsed dendritic cells has also been shown to protect against subsequent P. aeruginosa pneumonia (45). The P. aeruginosa-protective T cell epitopes have not been defined, although studies in humans in the early 1980s reported that the alkaline phosphatase of P. aeruginosa, a secreted protein, was the principal Ag recognized by clones of T cells derived by stimulation of PBMC from healthy volunteers using crude bacterial culture fluids (46). More recently, immunization with a replication-deficient adenoviral vector expressing a 14-aa epitope of P. aeruginosa outer membrane protein OprF showed that CD4⁺ and CD8⁺ T cell responses to the OprF peptide could be elicited as well as protection against lethal pneumonia in mice (47). Investigations into whether Th1 (IFN-γ dominated) vs Th2 (IL-4 and IL-10 dominated) CD4⁺ T cell responses are more important for protective immunity against P. aeruginosa lung infections have yielded conflicting results, with some reports suggesting that Th1-prone strains of mice are more susceptible and other reports concluding just the opposite (14, 17, 18, 48). None of those studies evaluated Th17 cells or the cytokine IL-17 as they preceded identification of the Th17 lineage.

The importance of a clear understanding of the role of IL-17 in vaccine-induced protection against bacterial pneumonia relates not only to the design of more effective vaccines but also to the fact that inhibitors of the IL-17 pathway are under active development as antiinflammatory agents for treatment of a host of autoimmune disorders ranging from rheumatoid arthritis to multiple sclerosis (49, 50). Our results suggest that disabling the IL-17 response could nullify the protective effects of some vaccines, a situation that will need to be studied extensively and weighed carefully in the risk-benefit analysis of IL-17-directed treatment of autoimmune diseases.

The present study suggests that IL-17 is a critical component of vaccine-induced immunity to LPS-heterologous strains of P. aeruginosa and thus paves the way toward a broadly protective P. aeruginosa vaccine using either mixtures of multiple live-attenuated aroA deletants or potentially by expressing heterologous LPS O Ags from a single live-attenuated strain such as PA14ΔaroA. The importance of the neutrophil in control of P. aeruginosa infections is also underscored by our results, as is the critical interplay between components of innate and adaptive immunity (neutrophils with T cells and antibody) during vaccine-induced protection from infection.

The authors have no financial conflicts of interest.