IL-17 and TNF-α Are Key Mediators of Moraxella catarrhalis Triggered Exacerbation of Allergic Airway Inflammation

Abstract

Alterations of the airway microbiome are often associated with pulmonary diseases. For example, detection of the bacterial pathogen Moraxella catarrhalis in the upper airways is linked with an increased risk to develop or exacerbate asthma. However, the mechanisms by which M. catarrhalis augments allergic airway inflammation (AAI) remain unclear. We here characterized the cellular and soluble mediators of M. catarrhalis triggered excacerbation of AAI in wt and IL-17 deficient as well as in animals treated with TNF-α and IL-6 neutralizing antibodies. We compared the type of inflammatory response in M. catarrhalis infected, house dust mite (HDM)-allergic and animals infected with M. catarrhalis at different time points of HDM sensitization. We found that airway infection of mice with M. catarrhalis triggers a strong inflammatory response with massive neutrophilic infiltrates, high amounts of IL-6 and TNF-α and moderate levels of CD4⁺ T-cell-derived IFN-γ and IL-17. If bacterial infection occurred during HDM allergen sensitization, the allergic airway response was exacerbated, particularly by the expansion of Th17 cells and increased TNF-α levels. Neutralization of IL-17 or TNF-α but not IL-6 resulted in accelerated clearance of M. catarrhalis and effectively prevented infection-induced exacerbation of AAI. Taken together, our data demonstrate an essential role for TNF-α and IL-17 in infection-triggered exacerbation of AAI.

Keywords: IL-17; Moraxellaceae infections; TNF-α; exacerbation of allergic reactions; exacerbation of pulmonary inflammation; infection and allergy; microbial exacerbation of pulmonary inflammation; pulmonary inflammation.

Figure 1

Pulmonary immune response after intranasal M. catarrhalis infection. C57Bl/6 animals were infected (i.n.) with 2 × 10⁸ CFU M. catarrhalis and BAL and lungs were harvested at indicated time points. (A) Bacterial CFU were determined in lung homogenates. (B) Total cell counts in BALF. (C) Differential cell counts in BALF. (D) CXCL1 and CXCL10 chemokines in BALF. (E) Cytokines in BALF. (F) Quantitative expression of IL-17 mRNA (qPCR) in lungs. (G) Frequency of IL-17A⁺CD4⁺ T cells in lungs. (H) Analysis of γδ T cells and CD4⁺ T cells secreting IL-17A at day 7 after PBS treatment or infection. n = 6 mice per group and two independent experiments were performed. ***P = 0.001, **P = 0.01, and *P = 0.05 (one-way ANOVA). BAL, bronchoaleveolar lavage; BALF, BAL fluid; qPCR, quantitative polymerase chain reaction; PBS, phosphate-buffered saline; ANOVA, analysis of variance.

Figure 2

Reduced inflammatory response to M. catarrhalis infection in IL-17 KO mice. IL-17 KO (black bars) and wt mice (white bars) were infected (i.n.) with 2 × 10⁸ CFU M. catarrhalis. BAL, lungs, and serum were harvested at indicated time points. (A) Bacterial CFU in lung homogenates were counted at different time points after infection. (B) Airway inflammation represented by BAL total cell counts. (C) Differential cell counts in BAL. (D) Representative periodic acid Schiff-stained airways of wt and IL-17 KO mice (E) Amounts of IL-6, TNF-α, and IL-1β in BALF. (F) IL-6 and TNF-α in serum. (G) Survival of IL-17 KO (squares) and wt animals (circles) after infection. Data were from two independent experiments (n = 10 mice per group). ***P = 0.001, **P = 0.01, and *P = 0.05 (one-way ANOVA). BAL, bronchoaleveolar lavage; BALF, BAL fluid; ANOVA, analysis of variance.

Figure 3

Neutralization of TNF-α protects against respiratory M. catarrhalis infection. C57BL/6 animals received a single i.p. injection of 100 µg of anti-TNF-α (black bars) or IgG1 control mAbs (gray bars) and were 4 h later intranasally infected with 2 × 10⁸ CFU M. catarrhalis. (A) Amounts of IL-6, TNF-α, IL-1β, and IL-17 in BALF of untreated or anti-TNF-α-treated mice at indicated time points. (B) As in (A) but in serum (C) Survival of anti-TNF-α-treated animals (circle) or control IgG-treated (square) mice after infection with M. catarrhalis. Data were from two independent experiments, n = 6 mice per group. ***P = 0.001, **P = 0.01, and *P = 0.05 (one-way ANOVA). BALF, BAL fluid; ANOVA, analysis of variance.

Figure 4

Neutralization of IL-6 enhances M. catarrhalis triggered inflammation. C57BL/6 animals received a single i.p. injection of 100 µg of anti-IL-6 or IgG1control mAbs and 4 h later were infected (i.n.) with M. catarrhalis (2 × 10⁸ CFU) (A) Amounts of TNF-α, IL-6, IL-1β, and IL-17 in BALF. (B) Amount of TNF-α in serum. (C) Survival of PBS-treated control mice (PBS), control-IgG1-treated animals infected with M.catarrahlis (Wt/M.cat) and anti-IL-6-mAb-treated animals infected with M. catarrhalis (anti IL-6). Data were from two independent experiments, n = 3 mice per group. ***P = 0.001, **P = 0.01, and *P = 0.05 (one-way ANOVA). BALF, BAL fluid; PBS, phosphate-buffered saline; ANOVA, analysis of variance.

Figure 5

IL-17 is a key cytokine of M. catarrhalis-induced exacerbation of AAI. C57BL/6 (white bars) and IL-17 KO animals (black bars) were infected (i.n.) with 2 × 10⁸ CFU M. catarrhalis after the second HDM exposure. BAL and lungs were analyzed at day 23. (A) Protocol. (B) Total cell counts in BAL. (C) Differential cell counts. (D) Representative periodic acid Schiff-stained airways. (E) Goblet cell counts in lung tissues. (F) Inflammation score. (G) Intracellular staining of CD4⁺ lung T cells for IL-17A, IFN-γ, IL-5, and IL-13. Data were from two independent experiments, n = 8 mice per group. ***P = 0.001, **P = 0.01, and *P = 0.05 (one-way ANOVA). AAI, allergic airway inflammation; HDM, house dust mite; BAL, bronchoaleveolar lavage; ANOVA, analysis of variance.

Figure 6

Anti TNF-α treatment alleviates M. catarrhalis-induced exacerbation of HDM AAI. C57BL/animals were infected (i.n.) with 2 × 10⁸ CFU M. catarrhalis after the second HDM exposure. Mice were administered a single dose of 100 µg anti TNF-α or IgG1 control mAb 4 h prior to M. catarrhalis infection. BAL and lungs were analyzed at day 23. (A) Protocol (B) Total cell counts in BAL. (C) Differential cell counts. (D) Representative periodic acid Schiff-stained airways. (E) Goblet cell counts in lung tissues. (F) Inflammation score. (G) Intracellular staining of lung CD4⁺ T cells for IL-17A, IFN-γ, and IL-5/IL-13 positive cells. Data were from two independent experiments, n = 8 mice per group. ***P = 0.001, **P = 0.01, and *P = 0.05 (one-way ANOVA). HDM, house dust mite; AAI, allergic airway inflammation; BAL, bronchoaleveolar lavage; ANOVA, analysis of variance.