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The Journal of Immunology, 2008, 181, 4965 -4975
Copyright © 2008 by The American Association of Immunologists, Inc.
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IL-17 Is a Critical Component of Vaccine-Induced Protection against Lung Infection by Lipopolysaccharide-Heterologous Strains of Pseudomonas aeruginosa1

Gregory P. Priebe2,*,{dagger},{ddagger}, Rebecca L. Walsh*, Terra A. Cederroth*, Akinobu Kamei*, Yamara S. Coutinho-Sledge*, Joanna B. Goldberg§ and Gerald B. Pier*,{ddagger}

* Channing Laboratory, Department of Medicine, Brigham and Women’s Hospital, {dagger} Departments of Anesthesia (Critical Care) and Medicine (Infectious Diseases), Children’s Hospital Boston, and {ddagger} Harvard Medical School, Boston, MA 02115; and § Department of Microbiology, University of Virginia, Charlottesville, VA 22908


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
In a murine model of acute fatal pneumonia, we previously showed that nasal immunization with a live-attenuated aroA deletant of Pseudomonas aeruginosa strain PAO1 elicited LPS serogroup-specific protection, indicating that opsonic Ab to the LPS O Ag was the most important immune effector. Because P. aeruginosa strain PA14 possesses additional virulence factors, we hypothesized that a live-attenuated vaccine based on PA14 might elicit a broader array of immune effectors. Thus, an aroA deletant of PA14, denoted PA14{Delta}aroA, was constructed. PA14{Delta}aroA-immunized mice were protected against lethal pneumonia caused not only by the parental strain but also by cytotoxic variants of the O Ag-heterologous P. aeruginosa strains PAO1 and PAO6a,d. Remarkably, serum from PA14{Delta}aroA-immunized mice had very low levels of opsonic activity against strain PAO1 and could not passively transfer protection, suggesting that an antibody-independent mechanism was needed for the observed cross-serogroup protection. Compared with control mice, PA14{Delta}aroA-immunized mice had more rapid recruitment of neutrophils to the airways early after challenge. T cells isolated from P. aeruginosa {Delta}aroA-immunized mice proliferated and produced IL-17 in high quantities after coculture with gentamicin-killed P. aeruginosa. Six hours following challenge, PA14{Delta}aroA-immunized mice had significantly higher levels of IL-17 in bronchoalveolar lavage fluid compared with unimmunized, Escherichia coli-immunized, or PAO1{Delta}aroA-immunized mice. Antibody-mediated depletion of IL-17 before challenge or absence of the IL-17 receptor abrogated the PA14{Delta}aroA vaccine’s protection against lethal pneumonia. These data show that IL-17 plays a critical role in antibody-independent vaccine-induced protection against LPS-heterologous strains of P. aeruginosa in the lung.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
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 PAO1{Delta}aroA, 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 PAO1{Delta}aroA 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 PAO1{Delta}aroA 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 PA14{Delta}aroA. Remarkably, we found that active intranasal immunization with PA14{Delta}aroA 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.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Bacterial strains

The bacterial strains and plasmids used in these experiments, along with their relevant characteristics and sources, are listed in Table I. PA14{Delta}aroA was constructed as previously described (6).


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  		Table I. Bacterial strains and plasmids used in this study

 
Preparation of bacterial inocula for in vivo challenge studies

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.

Immunization of mice

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 {Delta}aroA vaccine strain or live Escherichia coli HB101 as a control on each nostril. Escalating doses of 1 x 108, 5 x 108, and 1 x 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.

Murine pneumonia model

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.

Opsonophagocytic assays

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.

Anti-P. aeruginosa IgG titers

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

T cell proliferation, stimulation, and cytokine measurements

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 x 105 T cells, 1 x 105 irradiated (1500 rads) splenocytes isolated from nonimmunized C3H/HeN mice and purified using density centrifugation over Lympholyte-M (Accurate Chemical), and 1 x 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 x 105 T cells and 1 x 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-{gamma} 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 CD3{epsilon} (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.

Statistical analyses

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.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Protective efficacy of PA14{Delta}aroA vaccination

We initially evaluated the protective efficacy of the PA14{Delta}aroA vaccine against LPS-homologous and LPS-heterologous strains. In our prior work, we showed that the serogroup O2/O5 vaccine PAO1{Delta}aroA 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 PA14{Delta}aroA 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 PAO1{Delta}aroA-immunized mice with a lower dose of strain PA14. Notably, PA14{Delta}aroA-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.


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  		Table II. Protective efficacy against acute fatal P. aeruginosa pneumonia following active nasal immunization of C3H/HeN mice with E. coli HB101, PAO1{Delta}aroA, or PA14{Delta}aroA

 
Opsonic killing activity in antisera raised to P. aeruginosa PA14{Delta}aroA

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 PA14{Delta}aroA 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. 1A, there was minimal opsonic killing of strain PAO1 in serum from mice immunized with the PA14{Delta}aroA strain, whereas good killing was achieved using sera from mice immunized with the LPS-homologous PAO1{Delta}aroA strain. Opsonic killing activity in antisera raised to PA14{Delta}aroA 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 PAO1{Delta}aroA against its parental strain PAO1 (7). Because PA14{Delta}aroA possesses the genes for ExoU while PAO1{Delta}aroA 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. 1B, there was no significant opsonic killing of an ExoU-producing strain compared with the strain containing the empty vector.


Figure 1
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  		FIGURE 1. Absence of PAO1-targeted (A) and ExoU-specific (B) opsonic killing activity of sera from PA14{Delta}aroA-immunized mice 3 wk after the final immunization. Bars are means and error bars the SEM. p-values calculated by ANOVA with Tukey’s multiple comparison test. C, Survival after passive immunization with PA14{Delta}aroA mouse antiserum (0.4 ml i.p., n = 5 mice/group) given 1 day before lung challenge with P. aeruginosa strain ExoU+ PAO1 (1.4 x 107 CFU per mouse, a dose that resulted in 100% lethality of mice passively immunized with E. coli antiserum (not shown)).

 
Passive immunization and whole-cell ELISA experiments

Next, we determined whether passive immunization with pooled antisera from PA14{Delta}aroA-immunized mice mediated protection against lethal pneumonia due to the LPS-heterologous strain ExoU+ PAO1. Mice given the PA14{Delta}aroA 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 PAO1{Delta}aroA strain (Fig. 1C). These results suggested, surprisingly, that an antiserum-independent mechanism is a key component of the heterologous protection engendered by active vaccination with the PA14{Delta}aroA vaccine. We also tested for O Ag-specific and ExoU-specific Abs by comparing IgG titers of the PAO1{Delta}aroA- and PA14{Delta}aroA-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 PA14{Delta}aroA antisera had the expected lower reactivity than did the antisera raised to PAO1{Delta}aroA 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 PA14{Delta}aroA vaccine elicited a comparable level of Ab to non-O side-chain Ags as did the PAO1{Delta}aroA vaccine.


Figure 2
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  		FIGURE 2. Comparison of IgG binding curves to P. aeruginosa strains PAO1 (with empty vector pUCP19), ExoU+ PAO1, and their LPS-rough isogenic galU mutant cells by ELISA using antisera from C3H/HeN mice immunized with live E. coli, PAO1{Delta}aroA, or PA14{Delta}aroA bacteria. NMS, normal mouse serum. Each point is the mean of duplicates using pooled sera from three to five mice.

 
T cell responses elicited by P. aeruginosa PA14{Delta}aroA vaccine

To determine whether immune T cells are induced by nasal immunization with the P. aeruginosa {Delta}aroA 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. 3A, there was a high level of proliferation of both PAO1{Delta}aroA- and PA14{Delta}aroA-immune T cells to either PAO1 or PA14, although the PA14{Delta}aroA-immune T cells proliferated less well when stimulated with PAO1 compared with the PAO1{Delta}aroA-immune T cells. Proliferation of PAO1{Delta}aroA-immune T cells was inhibited by an Ab to CD4 but not to CD8 (Fig. 3B). Proliferation of PAO1{Delta}aroA- and PA14{Delta}aroA-immune T cells was high when stimulated with multiple P. aeruginosa strains regardless of LPS serogroup or ability to produce ExoU (Fig. 3C). Interestingly, the P. aeruginosa {Delta}aroA-immune T cells proliferated when cultured with E. coli, but the E. coli-immune T cells were not significantly stimulated by P. aeruginosa strains.


Figure 3
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  		FIGURE 3. Nasal immunization with P. aeruginosa {Delta}aroA vaccines primes splenic T cells. A, Proliferation of splenic T cells from nonimmunized mice compared with mice immunized with E. coli, PAO1{Delta}aroA, or PA14{Delta}aroA. B, Proliferation of PAO1{Delta}aroA-immune T cells was significantly decreased by anti-CD4 mAb. C, Proliferation of T cells from PAO1{Delta}aroA (left panel) or PA14{Delta}aroA (right panel) immunized mice was similar regardless of LPS serogroup or cytotoxic genotype of the stimulating gentamicin-killed, whole bacterial cell Ag (multiplicity of infection of 10 bacteria per T cell). Bars represent means and error bars SEM. Cells were pooled from three to five mice per group. In A, *, p < 0.05 and #, p < 0.01 in comparison to E. coli control group by ANOVA with Dunnett’s multiple comparison test. In B, *, p < 0.05 compared with IgG control of corresponding vaccine group and #, p < 0.01 by t test compared with E. coli-immune T cells. In C, proliferation of {Delta}aroA-immune T cells was significantly greater (p < 0.01, t test) compared with E. coli-immune T cells in the presence of P. aeruginosa. SEA, staphylococcal enterotoxin A, positive control; n.d., not determined.

 
Cytokine secretion by P. aeruginosa {Delta}aroA vaccine-elicited T cells

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-{gamma}, 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 {Delta}aroA-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. 4B), 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. 4C and were remarkably higher in the {Delta}aroA-immune groups, with the PAO1{Delta}aroA-immune and PA14{Delta}aroA-immune T cells having ~10-fold and 40-fold higher IL-17, respectively, than did the control T cell groups.


Figure 4
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  		FIGURE 4. Nasal immunization with P. aeruginosa {Delta}aroA vaccine strains elicits Th17 cells in the spleen and lung. A, IL-17 (upper panel) and IFN-{gamma} (lower panel) production by splenic T cells isolated 3 wk after nasal immunization and then stimulated for 7 days in the presence of gentamicin-killed whole bacterial cells (multiplicity of infection of 10 bacteria per T cell) of P. aeruginosa strains PAO1 or PA14 along with irradiated splenocytes. Cells were pooled from three to five mice per group. B, IL-17 secretion decreases to baseline when splenic T cells are cocultured as in A with strain PAO1 along with anti-CD4 mAb (clone GK1.5, 1 µg/well). IC, isotype control IgG2b mAb (1 µg/well). Bars indicate means of triplicate wells, error bars the SEM. #, p < 0.05 compared with E. coli control group by ANOVA with Dunnett’s multiple comparison test. *, p < 0.01 compared with corresponding vaccine group treated with IC by ANOVA with Tukey’s multiple comparison test. C, IL-17 production by lung leukocytes isolated from vaccinated and unvaccinated mice (three mice per group)18 h after challenge with strain ExoU+ PAO1 and then cocultured for 7 days with gentamicin-killed PAO1 whole bacterial cells (multiplicity of infection of 10 bacteria per T cell). Bars indicate means of triplicate wells, error bars the SEM. #, p < 0.01 and *, p < 0.05 compared with E. coli control group by ANOVA with Dunnett’s multiple comparison test. D, Intracellular staining for IL-17 in lymphocytes isolated from collagenase-digested lungs of vaccinated and unvaccinated mice 2 h after challenge with P. aeruginosa strain ExoU+ PAO1. Cells were pooled from three mice per group.

 
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. 4D, there was a small population of IL-17-producing TCRβ-positive cells in the lungs of {Delta}aroA-immune mice, with 3-fold more in the PA14{Delta}aroA-immunized group. Similar proportions of IL-17-positive cells were seen in CD3{epsilon}-positive cells, and no IL-17-positive cells were seen outside the lymphocyte gate of {Delta}aroA-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).

Neutrophil recruitment to the lungs of P. aeruginosa {Delta} aroA-immunized mice

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 {Delta}aroA-immunized mice and whether the different P. aeruginosa {Delta}aroA vaccines elicited different neutrophil recruitment. As shown in Fig. 5A, there were significantly more neutrophils in BALF 6 h after challenge of P. aeruginosa {Delta}aroA-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. 5B) and homogenized lung tissue (not shown) in the P. aeruginosa {Delta}aroA-immunized mice compared with the control groups. IL-17 levels in BALF (Fig. 5C) obtained 6 h after challenge with a lethal dose of ExoU+ PAO1 were significantly higher in the PA14{Delta}aroA-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.


Figure 5
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  		FIGURE 5. Increased neutrophil recruitment to airways early after bacterial challenge is coincident with lower bacterial CFU and higher IL-17 levels in BALF. Total BALF neutrophils (A) and bacterial CFU (B) isolated after infection of immune and nonimmune mice (n = 5–10 mice/group) with P. aeruginosa strain ExoU+ PAO1 (5 x 106 CFU). Bars represent medians and error bars the interquartile range. p-values are by Kruskal-Wallis with Dunn’s multiple comparison test. C, IL-17 concentration in BALF following infection of immune and nonimmune mice (n = 5–15 mice/group) with strain ExoU+ PAO1 (5 x 106 CFU). Bars depict means and error bars the SEM. *, p < 0.05 compared with E. coli control group by ANOVA with Dunnett’s multiple comparison test.

 
Effect of loss of IL-17 on protective efficacy of P. aeruginosa {Delta}aroA vaccines against LPS-heterologous strain challenge

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 PA14{Delta}aroA 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. 6A, left panel). However, there was significantly higher mortality (p < 0.02, logrank test) in the PA14{Delta}aroA-immunized mice that received anti-IL-17 IgG compared with the mice given control IgG (Fig. 6A, right panel), indicating that depletion of IL-17 abrogates vaccine-induced protection. In contrast, mice immunized with the PAO1{Delta}aroA 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 PA14{Delta}aroA (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. 6B, right panel). We observed lower percentage survival in the PA14{Delta}aroA-immunized wild-type C57BL/6 mice challenged with 5 x 107 CFU/mouse (40% survival, Fig. 6B) compared with PA14{Delta}aroA-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).


Figure 6
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  		FIGURE 6. Neutralization of IL-17 or absence of its receptor diminishes vaccine-induced protection from P. aeruginosa pneumonia. A, Survival of immunized wild-type C3H/HeN mice after administration of IL-17-specific rabbit IgG (solid line) or control IgG (dashed line) 1 day before challenge with ExoU+ PAO1 (n = 6 mice/group, 5 x 107 CFU). B, Survival of immunized wild-type C57BL/6 (WT, solid line) or IL-17R KO (dotted line) mice after challenge with ExoU+ PAO1 (n = 5–7 mice/group; p < 0.05 by logrank test for comparison of PA14{Delta}aroA WT vs PA14{Delta}aroA IL-17R KO mice at either challenge dose). C, Neutrophil recruitment in PA14{Delta}aroA-immunized mice is deficient in the absence of IL-17R signaling. BALF total neutrophil numbers were measured 6 h after challenge with ExoU+ PAO1 (7 x 106 CFU) and normalized to bacterial CFU. Each point represents one mouse. p-value by Mann-Whitney U test. D, Innate immunity to P. aeruginosa pneumonia is intact in IL-17R KO mice. Survival of unimmunized wild-type C57BL/6 (solid line) and IL-17R KO (dashed line) mice after challenge of with ExoU+ PAO1 (n = 6 mice/group, 2 x 106 CFU). All mice (six per group) survived after a challenge dose of 7 x 105 CFU (not shown).

 
Consistent with the IL-17 neutralization experiments in PAO1{Delta}aroA-immunized C3H/HeN mice, IL-17R KO mice immunized with the PAO1{Delta}aroA vaccine and then challenged with the LPS-homologous strain ExoU+ PAO1 were fully protected (not shown). These PAO1{Delta}aroA-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 PA14{Delta}aroA-immunized wild-type C57BL/6 mice compared with PA14{Delta}aroA-immunized IL-17R KO mice. We again used P. aeruginosa strain ExoU+ PAO1 (7 x 106 CFU/mouse) as the challenge strain. As shown in Fig. 6C, 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 x 106 CFU of strain ExoU+ PAO1 (Fig. 6D) or 7 x 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.


   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
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 PAO1{Delta}aroA vaccine, active immunization with the PA14{Delta}aroA 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-{alpha}) is also required for Th17 polarization (29).

The mechanisms whereby the PA14{Delta}aroA vaccine induces more robust IL-17 responses than the PAO1{Delta}aroA 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-{alpha}, 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-{alpha}, 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-{gamma} 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 PA14{Delta}aroA. 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.


   Disclosures
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflicts of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by National Institutes of Health Grants K08 AI50036 and R21 HL079423 (to G.P.P.), R01AI068112 (to J.B.G.), and AI22535 (to G.B.P.). Back

2 Address correspondence and reprint requests to Dr. Gregory P. Priebe, Brigham and Women’s Hospital, Channing Laboratory, 181 Longwood Avenue, Boston, MA 02115. E-mail address: gpriebe{at}rics.bwh.harvard.edu Back

3 Abbreviations used in this paper: TSA, tryptic soy agar; KO, knockout; BALF, bronchoalveolar lavage fluid. Back

Received for publication March 25, 2008. Accepted for publication August 1, 2008.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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Characterization of Protective Mucosal and Systemic Immune Responses Elicited by Pneumococcal Surface Protein PspA and PspC Nasal Vaccines against a Respiratory Pneumococcal Challenge in Mice
Clin. Vaccine Immunol., May 1, 2009; 16(5): 636 - 645.
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