IL-17 is a critical component of vaccine-induced protection against lung infection by lipopolysaccharide-heterologous strains of Pseudomonas aeruginosa

Abstract

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 PA14DeltaaroA, was constructed. PA14DeltaaroA-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 PA14DeltaaroA-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, PA14DeltaaroA-immunized mice had more rapid recruitment of neutrophils to the airways early after challenge. T cells isolated from P. aeruginosa DeltaaroA-immunized mice proliferated and produced IL-17 in high quantities after coculture with gentamicin-killed P. aeruginosa. Six hours following challenge, PA14DeltaaroA-immunized mice had significantly higher levels of IL-17 in bronchoalveolar lavage fluid compared with unimmunized, Escherichia coli-immunized, or PAO1DeltaaroA-immunized mice. Antibody-mediated depletion of IL-17 before challenge or absence of the IL-17 receptor abrogated the PA14DeltaaroA 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.

Fig. 1

Absence of PAO1-targeted (A) and ExoU-specific (B) opsonic killing activity of sera from PA14ΔaroA-immunized mice 3 weeks 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 PA14ΔaroA mouse antiserum (0.4 mL i.p., n=5 mice/group) given 1 day prior to lung challenge with P. aeruginosa strain ExoU⁺ PAO1 (1.4 × 10⁷ CFU per mouse, a dose that resulted in 100% lethality of mice passively immunized with E. coli antiserum [not shown]).

Fig. 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, PAO1ΔaroA, or PA14ΔaroA bacteria. NMS: normal mouse serum. Each point is the mean of duplicates using pooled sera from 3-5 mice.

Fig. 3

Nasal immunization with P. aeruginosa ΔaroA vaccines primes splenic T cells. A. Proliferation of splenic T cells from unimmunized mice compared with mice immunized with E. coli, PAO1ΔaroA, or PA14ΔaroA. B. Proliferation of PAO1ΔaroA-immune T cells was significantly decreased by anti-CD4 monoclonal antibody. C. Proliferation of T cells from PAO1ΔaroA (left panel) or PA14ΔaroA (right panel) immunized mice was similar regardless of LPS serogroup or cytotoxic genotype of the stimulating gentamicin-killed, whole bacterial cell antigen (MOI: 10 bacteria per T cell). Bars represent means and error bars SEM. Cells were pooled from 3-5 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 to E. coli-immune T cells. In C, proliferation of ΔaroA-immune T cells was significantly greater (p<0.01, t test) compared to E. coli-immune T cells in the presence of P. aeruginosa. SEA: staphylococcal enterotoxin A, positive control; n.d., not determined.

Fig. 4

Nasal immunization with P. aeruginosa ΔaroA vaccine strains elicits Th17 cells in the spleen and lung. A. IL-17 (upper panel) and IFN-γ (lower panel) production by splenic T cells isolated 3 weeks after nasal immunization and then stimulated for 7 days in the presence of gentamicin-killed whole bacterial cells (MOI: 10 bacteria per T cell) of P. aeruginosa strains PAO1 or PA14 along with irradiated splenocytes. Cells were pooled from 3-5 mice per group. B. IL-17 secretion decreases to baseline when splenic T cells are co-cultured as in (A.) with strain PAO1 along with anti-CD4 monoclonal antibody (clone GK1.5, 1 μg/well). IC, isotype control IgG2b monoclonal antibody (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 (3 mice per group)18 h after challenge with strain ExoU⁺ PAO1 and then co-cultured for 7 days with gentamicin-killed PAO1 whole bacterial cells (MOI: 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 3 mice per group.

Fig. 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 non-immune mice (n=5-10 mice per group) with P. aeruginosa strain ExoU⁺ PAO1 (5×10⁶ 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 non-immune mice (n=5-15 mice per group) with strain ExoU⁺ PAO1 (5×10⁶ 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.

Fig. 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 prior to challenge with ExoU⁺ PAO1 (n=6 mice/group, 5×10⁷ 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 per group; p<0.05 by logrank test for comparison of PA14ΔaroA WT vs. PA14ΔaroA IL-17R KO mice at either challenge dose). C. Neutrophil recruitment in PA14ΔaroA-immunized mice is deficient in the absence of IL-17R signaling. BALF total neutrophil numbers were measured 6 h after challenge with ExoU⁺ PAO1 (7×10⁶ 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 per group, 2×10⁶ CFU). All mice (6 per group) survived after a challenge dose of 7×10⁵ CFU (not shown).