IL-17A impairs host tolerance during airway chronic infection by Pseudomonas aeruginosa

Abstract

Resistance and tolerance mechanisms participate to the interplay between host and pathogens. IL-17-mediated response has been shown to be crucial for host resistance to respiratory infections, whereas its role in host tolerance during chronic airway colonization is still unclear. Here, we investigated whether IL-17-mediated response modulates mechanisms of host tolerance during airways chronic infection by P. aeruginosa. First, we found that IL-17A levels were sustained in mice at both early and advanced stages of P. aeruginosa chronic infection and confirmed these observations in human respiratory samples from cystic fibrosis patients infected by P. aeruginosa. Using IL-17a(-/-) or IL-17ra(-/-) mice, we found that the deficiency of IL-17A/IL-17RA axis was associated with: i) increased incidence of chronic infection and bacterial burden, indicating its role in the host resistance to P. aeruginosa; ii) reduced cytokine levels (KC), tissue innate immune cells and markers of tissue damage (pro-MMP-9, elastin degradation, TGF-β1), proving alteration of host tolerance. Blockade of IL-17A activity by a monoclonal antibody, started when chronic infection is established, did not alter host resistance but increased tolerance. In conclusion, this study identifies IL-17-mediated response as a negative regulator of host tolerance during P. aeruginosa chronic airway infection.

Figure 1. P. aeruginosa infection and markers of immune response in the murine model of chronic airways infection and CF patients.

Two groups of minimum five C57Bl/6 mice were infected with 1 to 2 × 10⁶ CFU/lung of the P. aeruginosa strain AA43 embedded in agar beads and analyzed after 2 and 28 days of infection. Control (Ctrl) mice were treated with sterile agar-beads. (A) CFU were evaluated in total lung. Dots represent CFU in individual mice, horizontal lines represent mean values and the error bars represent the standard error of the mean (SEM). The data are pooled from at least two independent experiments (n = 4–12). Cytokines and chemokines, including IL-1β (B), IFN-γ (C), and IL-17A (D), were measured by Bioplex in murine lung homogenates. The data are pooled from at least two independent experiments (n = 3–9). Values represent the mean ± SEM. Levels of IL-17A (E), IFN-γ (F) and IL-4 (G) in sputa from CF patients never colonized by P. aeruginosa (free), with intermittent or chronic colonization up to two years (early) and with chronic colonization for at least four years (late) are compared. Dots represent values in individual patients and horizontal lines represent median values. Statistical significance is indicated: ^∗p < 0.05, ^∗∗p < 0.01.

Figure 2. Immune response in the murine model of chronic airways infection with P. aeruginosa.

C57Bl/6 mice were infected with 1 to 2 × 10⁶ CFU/lung of the P. aeruginosa strain AA43 embedded in agar beads and analyzed after 2 and 28 days of infection. Ctrl mice were treated with sterile agar-beads. The absolute numbers of leukocytes (A), alveolar macrophages (B), interstitial macrophages (C), neutrophils (D), dendritic cells (E), T cells (F), CD4⁺ T cells (G), CD8⁺ T cells (H) and B cells (I) were measured by flow cytometric analysis in cell suspensions of murine lungs. The frequency of IL-17A-, IFN-γ- and IL-4-producing CD4⁺ T (J) and CD8⁺ T (K) cells were measured in lung cell suspensions after PMA/ionomycin stimulation, by flow cytometric analysis. The data are pooled from at least two independent experiments (n = 4–12). Dots represent cells in individual mice, horizontal lines represent mean values and the error bars represent the SEM. Statistical significance is indicated: *p < 0.05, **p < 0.01, ***p < 0.001. (L) Lung immunohistochemistry was performed on challenged mice by staining with anti-CD3 and anti-B220 antibodies. Scale bars: 400 μm. Some BALT-like structures are indicated by arrows.

Figure 3. Virulence of P. aeruginosa and host immune response to chronic airways infection in IL-17a^−/−, IL-17ra^−/− and congenic wt mice.

Two groups of minimum five IL-17a^−/−, IL-17ra^−/− and congenic wt C57Bl/6 mice were infected with 1 to 2 × 10⁶ CFU/lung of the P. aeruginosa strain AA43 embedded in agar beads and analyzed 28 days post-infection. Ctrl mice were treated with sterile agar-beads. (A) CFU were evaluated in total lung. Dots represent CFU in individual mice, horizontal lines represent mean values and the error bars represent the SEM. The data are pooled from at least two independent experiments (n = 10–14). (B) Bacterial clearance (white) and incidence of colonization (green) were determined. The data are pooled from at least two independent experiments (n = 17–29). Cytokines and chemokines, including IL-17F (C), IL-17A (D), IL-21 (E), IL-22 (F), IL-23 (G), IFN-γ (H), IL-4 (I), IL-1β (J) and KC (K), were measured by Bioplex in lung homogenates of mice after 28 days of chronic lung infection with P. aeruginosa. The data are pooled from at least two independent experiments (n = 3–14). Values represent the mean ± SEM. Sections of airways from mice infected with AA43 for 28 days were stained with H&E, according to the standard procedure (L). Scale bars: 200 μm. Innate immune cells infiltration (M) and inflammation severity (N) were scored in tissue sections. Values represent the mean ± SEM. The data are pooled from two independent experiments (n = 3–5). Statistical significance is indicated: *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 4. Tissue damage after P. aeruginosa chronic airways infection in IL-17a^−/−, IL-17ra^−/− and congenic wt mice.

Two groups of minimum five IL-17a^−/−, IL-17ra^−/− and congenic wt C57Bl/6 mice were infected with 1 to 2 × 10⁶ CFU/lung of the P. aeruginosa strain AA43 embedded in agar beads and analyzed 28 days post-infection. Ctrl mice were treated with sterile agar-beads. (A) Sections of airway from mice infected with AA43 were stained with VEG for elastic fibers, according to the standard procedure. Scale bars: 25 μm. Scoring of elastin preservation was performed on slices stained with VEG (B). Levels of pro-MMP-9 (C) and TGF-β₁ (D) were measured by ELISA and Bioplex respectively in lung homogenates after 28 days of chronic lung infection. Values represent the mean ± SEM. The data are pooled from at least two independent experiments (n = 3–10). Statistical significance is indicated: *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 5. Anti-IL-17A mAb treatment in the murine model of chronic airways infection by P. aeruginosa.

C57Bl/6 mice were infected with 1 to 2 × 10⁶ CFU/lung of the P. aeruginosa strain AA43 embedded in agar beads. Every three days starting from the tenth day from infection, a group of mice was treated by intraperitoneal injection (i.p.) with 100 μg/mouse of anti-IL-17A mAb, while the other group was treated with 100 μg/mouse of control isotypic IgG and analyzed after 28 days of infection. Schematic view of the treatment schedule is shown (A). Leukocytes (B), T cells (C), CD4⁺ T cells (D), the frequency of IFN-γ- (E) and IL-17A-producing CD4⁺ T cells (F) after PMA/ionomycin stimulation, and neutrophils (G) were measured by flow cytometric analysis in cell suspensions of murine lungs. Dots represent cells in individual mice, horizontal lines represent mean values and the error bars represent the SEM. KC levels (H) were measured by Bioplex in the supernatants of murine lung cellular suspensions. Levels of pro-MMP-9 (I) by ELISA and TGF-β₁ (J) by Bioplex were measured in the supernatants of murine lung cellular suspensions. Values represent the mean ± SEM. The data are pooled from at least two independent experiments (n = 8–10). Statistical significance is indicated: ^*p < 0.05, ^**p < 0.01.