Eosinophil-derived IL-13 promotes emphysema

Abstract

The inflammatory responses in chronic airway diseases leading to emphysema are not fully defined. We hypothesised that lung eosinophilia contributes to airspace enlargement in a mouse model and to emphysema in patients with chronic obstructive pulmonary disease (COPD).A transgenic mouse model of chronic type 2 pulmonary inflammation (I5/hE2) was used to examine eosinophil-dependent mechanisms leading to airspace enlargement. Human sputum samples were collected for translational studies examining eosinophilia and matrix metalloprotease (MMP)-12 levels in patients with chronic airways disease.Airspace enlargement was identified in I5/hE2 mice and was dependent on eosinophils. Examination of I5/hE2 bronchoalveolar lavage identified elevated MMP-12, a mediator of emphysema. We showed, in vitro, that eosinophil-derived interleukin (IL)-13 promoted alveolar macrophage MMP-12 production. Airspace enlargement in I5/hE2 mice was dependent on MMP-12 and eosinophil-derived IL-4/13. Consistent with this, MMP-12 was elevated in patients with sputum eosinophilia and computed tomography evidence of emphysema, and also negatively correlated with forced expiratory volume in 1 s.A mouse model of chronic type 2 pulmonary inflammation exhibited airspace enlargement dependent on MMP-12 and eosinophil-derived IL-4/13. In chronic airways disease patients, lung eosinophilia was associated with elevated MMP-12 levels, which was a predictor of emphysema. These findings suggest an underappreciated mechanism by which eosinophils contribute to the pathologies associated with asthma and COPD.

Figure 1.. Eosinophil-dependent alveolar destruction and modulation of MMP-12.

(A) H&E stained lung sections demonstrate increased alveolar spaces in I5/hE2 lungs relative to wild type and, their eosinophil deficient counterparts, I5/hE2/PHIL. Lung parenchyma 200x. (B) Quantification of average alveolar space revealed a two-fold increase in mean intercept length in I5/hE2 lungs (panel B, n=5 mice). (C) BAL fluid was collected and assessed by ELISA for MMP-12, a lung remodeling mediator with dramatically increased expression in I5/hE2 lungs (panel C, n=6 mice). Scale bar = 50μm. *:p<0.05. ****:p<0.0001. ns: not significant.

Figure 2.. MMP-12 mediates alveolar destruction in I5/hE2 model.

(A) H&E stained lung sections demonstrate enlarged airspaces in I5/hE2 mice relative to wild type and MMP-12^−/− mice. (B) H&E stained lung sections assessed for mean intercept length (L_m) demonstrate increased alveolar spaces in I5/hE2 mice relative to their MMP-12 deficient counterparts I5/hE2/MMP-12^−/−. Lung parenchyma 200x. (C). n≥3 mice. Scale bar = 50μm. **: p<0.01, ***:p<0.001, ****:p<0.0001.

Figure 3.. Eosinophils directly modulate alveolar macrophage production of MMP-12 through IL-13 dependent mechanisms.

ELISA measurement of MMP-12 in cell culture supernatant from in vitro 48h cultures of eosinophils (Eos) and/or alveolar macrophages (Macs). (A) Eosinophils cultured alone, macrophages cultured alone, and co-culture (Eos+Mac). Wild type eosinophils were activated (IL-33 for 24 hours then washed) and cultured with WT macrophages or MMP-12^−/− macrophages showing the MMP-12 is from macrophages. (B) Assessment of co-culture supernatants from resting eosinophils (Eos) vs. activated eosinophils (Eos(A)) with macrophages (Macs) demonstrated that pre-activation of eosinophils (IL-33 for 24 hours then washed) enhances macrophage MMP-12 production. (C) Transwell co-culture of activated eosinophils with macrophages shows that contact is not required for the enhanced macrophage MMP-12 production. (D) ELISA measurements from co-culture supernatants of IL-13^−/− vs. Wild Type (WT) eosinophils with WT macrophages demonstrates that eosinophil-derived IL-13 is promoting macrophage MMP-12 production. ***:p<0.001, ****:p<0.0001, ns: not significant.

Figure 4.. Eosinophil-derived IL-4/13 induces MMP-12 production and alveolar destruction in I5/hE2 mice.

(A) A genetic cross of I5/hE2 with the eosinophil-specific Cre mouse, eoCRE, x the available floxed IL-4/13 mouse (I5/hE2/eoCRE/4/13^fl/fl) revealed that enlarged alveolar spaces in I5/hE2 mice are dependent on eosinophil-derived IL-4/13. (B) H&E stained lung sections demonstrate enlarged airspaces in I5/hE2 mice relative to eosinophil-derived IL-4/13 deficient mice. (C) ELISA measurement of MMP-12 in BAL showed MMP-12 in I5/hE2 mice is dependent on eosinophil-derived IL-4/13. Lung parenchyma 200x. n≥3 mice. Scale bar = 50μm. *:p<0.05, **: p<0.01, ***:p<0.001, ****:p<0.0001.

Figure 5.. MMP-12 expression in eosinophilic emphysematous airways.

(A) EPX (B) MMP-12 expression in airways of n=40 patients with chronic airways disease (diagnosis of either asthma or COPD) classified based on eosinophilia. Mann Whitney Test . (C) Correlation of sputum MMP-12 with IL-13 (n=40). Spearman rank. (D) MMP-12 expression in airways of both asthma (n=16) and COPD (n=24), classified based on eosinophilia and (E) COPD samples only, classified with respect to eosinophilia and emphysema. Kruskal Wallis with Dunn’s multiple correction. (F) Correlation of sputum MMP-12 with FEV₁ % predicted in n=24 COPD patients. Spearman rank. Each symbol represents individual patient data, and median is indicated for each subset. Figure 5D, grey open circle indicates asthmatic patient with evidence of airspace enlargement. P≤0.05 considered as significant.