IL-23 mediates inflammatory responses to mucoid Pseudomonas aeruginosa lung infection in mice

Abstract

Patients with cystic fibrosis (CF) develop chronic Pseudomonas aeruginosa lung infection with mucoid strains of P. aeruginosa; these infections cause significant morbidity. The immunological response in these infections is characterized by an influx of neutrophils to the lung and subsequent lung damage over time; however, the underlying mediators to this response are not well understood. We recently reported that IL-23 and IL-17 were elevated in the sputum of patients with CF who were actively infected with P. aeruginosa; however, the importance of IL-23 and IL-17 in mediating this inflammation was unclear. To understand the role that IL-23 plays in initiating airway inflammation in response to P. aeruginosa, IL-23p19(-/-) (IL-23 deficient) and wild-type (WT) mice were challenged with agarose beads containing a clinical, mucoid isolate of P. aeruginosa. Levels of proinflammatory cytokines, chemokines, bacterial dissemination, and inflammatory infiltrates were measured. IL-23-deficient mice had significantly lower induction of IL-17, keratinocyte-derived chemokine (KC), and IL-6, decreased bronchoalveolar lavage (BAL) neutrophils, metalloproteinase-9 (MMP-9), and reduced airway inflammation than WT mice. Despite the reduced level of inflammation in IL-23p19(-/-) mice, there were no differences in the induction of TNF and interferon-gamma or in bacterial dissemination between the two groups. This study demonstrates that IL-23 plays a critical role in generating airway inflammation observed in mucoid P. aeruginosa infection and suggests that IL-23 could be a potential target for immunotherapy to treat airway inflammation in CF.

Fig. 1

A: expression of IL-23p19 mRNA measured by TaqMan real-time PCR. Mice were killed after being infected with Pseudomonas aeruginosa strain PA M57–15-laden agarose beads for 0, 3, 5, and 7 days or uninfected control beads (Cntrl). IL-23p19^−/− exhibited no detectable IL-23p19, whereas the wild-type (WT) infected mice had significantly increased IL-23p19 expression at days 3 and 5. There was no detectable IL-23p19 production in control mice. B: IL-12p40 was measured in the bronchoalveolar lavage (BAL) fluid at the protein level. There were no significant differences between infected WT and IL-23p19^−/− mice. There is detectable production at baseline and for control mice of both groups and noted increase in IL-12p40 production in both WT and IL-23p19^−/− mice at day 3. N = 3–5 per group for A and B.*P < 0.05.

Fig. 2

IL-17 protein measured by ELISA in BAL. IL-17 was measured at 0, 3, 5, and 7 days post-infection (n = 5 per group); *P < 0.05. IL-23p19^−/− mice exhibited no detectable IL-17, whereas the WT infected mice had significantly increased IL-17 expression at day 3. IL-17 remained elevated at day 5 in WT infected mice but was not significantly greater than the other groups. There was no detectable production of IL-17 in the WT or IL-23p19^−/− control mice.

Fig. 3

Chemokine and metalloproteinase (MMP) levels in lung homogenate and BAL fluid during chronic P. aeruginosa infection. Keratinocyte-derived chemokine (KC) (A) and MMP-9 (E) from lung homogenate were measured by LINCOplex and reported for day 5 after infection (n = 3–5 per group); *P < 0.05. LPS-induced CXC chemokine (LIX) (D) from BAL was measured by ELISA and reported for day 5 after infection (n = 5); *P < 0.05. Macrophage inflammatory protein-1α (MIP-1α) (B) and IFN-γ-inducible protein 10 (IP-10) (C) from BAL were measured by LINCOplex and reported for day 5 after infection. (n = 5); *P < 0.05. Levels in the mice treated with control beads were either not detectable (ND) or not significantly different from baseline. In addition, WT controls were not significantly different from IL-23p19^−/− controls.

Fig. 4

Cytokine levels in BAL fluid during chronic P. aeruginosa infection. IL-6 (A), IFN-γ (D), and TNF-α (C), measured by Luminex, are reported for day 5 after infection. IL-6 was significantly increased in infected WT mice compared with IL-23p19^−/− infected mice. Infected WT mice had significantly elevated levels over control WT mice. There was no significant difference between WT and IL-23p19^−/− control mice (n = 5 per group); *P < 0.05.

Fig. 5

Cellular recruitment during chronic P. aeruginosa infection. A: absolute (abs.) numbers of recovered macrophages in BAL fluid from WT and IL-23p19^−/− mice on days 0, 3, 5, and 7. B: percent macrophages in BAL fluid from WT and IL-23p19^−/− on days 0, 3, 5, and 7 after infection. C: absolute numbers of recovered neutrophils (PMNs) in BAL fluid from WT and IL-23p19^−/− mice on days 0, 3, 5, and 7. D: percent neutrophils in BAL fluid from WT and IL-23p19^−/− mice on days 0, 3, 5, and 7 after infection. Where significant differences are noted, the infected WT mice demonstrate higher levels than the IL-23p19^−/− infected mice and the WT control mice; there is no significant difference between WT and IL-23p19^−/− control mice (n = 10 per group); *P < 0.05.

Fig. 6

Representative photomicrographs of lung from WT and IL-23p19^−/− mice infected with PA M57–15-laden beads at days 3, 5, and 7. Top panels for each time point are at ×50 magnification, and bottom panels are at ×1,000 magnification. Day 3 (a–d): black arrows indicate airways with marked inflammation, and blue arrows indicate beads. Day 5 (e–h): B marks agarose bead in the airway lumen. i–l: day 7.

Fig. 7

Quantitative scoring of intraluminal (A) and peribronchial (B) infiltrate: comparing WT and IL-23p19^−/− infected and control demonstrates significant differences at days 3, 5, and 7 for the infected WT mice compared with the IL-23p19^−/− infected and WT control groups (n = 5 per group); *P < 0.05.

Fig. 8

Quantitative comparisons of bacterial burden in the lung by comparison of colony-forming units (CFU) (A) and percent of mice originally infected from which bacteria are recovered (B) (n = per group).