Augmented Responses to Ozone in Obese Mice Require IL-17A and Gastrin-Releasing Peptide

Abstract

Ozone and obesity both increase IL-17A in the lungs. In mice, obesity augments the airway hyperresponsiveness and neutrophil recruitment induced by acute ozone exposure. Therefore, we examined the role of IL-17A in obesity-related increases in the response to ozone observed in obese mice. Lean wild-type and obese db/db mice were pretreated with IL-17A-blocking or isotype antibodies, exposed to air or ozone (2 ppm for 3 h), and evaluated 24 hours later. Microarray analysis of lung tissue gene expression was used to examine the mechanistic basis for effects of anti-IL-17A. Compared with lean mice, ozone-exposed obese mice had greater concentrations of BAL IL-17A and greater numbers of pulmonary IL-17A⁺ cells. Ozone-induced increases in BAL IL-23 and CCL20, cytokines important for IL-17A⁺ cell recruitment and activation, were also greater in obese mice. Anti-IL-17A treatment reduced ozone-induced airway hyperresponsiveness toward levels observed in lean mice. Anti-IL-17A treatment also reduced BAL neutrophils in both lean and obese mice, possibly because of reductions in CXCL1. Microarray analysis identified gastrin-releasing peptide (GRP) receptor (Grpr) among those genes that were both elevated in the lungs of obese mice after ozone exposure and reduced after anti-IL-17A treatment. Furthermore, ozone exposure increased BAL GRP to a greater extent in obese than in lean mice, and GRP-neutralizing antibody treatment reduced obesity-related increases in ozone-induced airway hyperresponsiveness and neutrophil recruitment. Our data indicate that IL-17A contributes to augmented responses to ozone in db/db mice. Furthermore, IL-17A appears to act at least in part by inducing expression of Grpr.

Keywords: CXCL1; airway hyperresponsiveness; gastrin-releasing peptide receptor; microarray; neutrophil.

Figure 1.

Ozone (O₃) exposure increases BAL IL-17A. BAL concentrations of (A and G) IL-17A, (C and H) IL-23, and (E) CCL20, as well as (F and I) serum concentrations of IL-17A, were measured by ELISA. (B) Lung IL-17A⁺CD45⁺ cells were measured by flow cytometry. (D) Correlation between BAL IL-17A and BAL IL-23 is shown. Data are derived from (A–F) db/db and wild-type (WT) mice or from (G–I) C57BL/6 mice fed either a high-fat diet (HFD) or regular mouse chow for 24 weeks. Mice were exposed to air or ozone (2 ppm for 3 h) and examined 24 hours after exposure. Results are mean ± SE of five to nine mice per group. *P < 0.05 versus air; ^#P < 0.05 versus lean mice.

Figure 2.

Anti–IL-17A reduces BAL neutrophils and BAL CXCL1 in obese O₃-exposed mice. (A) BAL neutrophils and BAL concentration of (B) CXCL1, (C) IL-6, and (D) granulocyte-colony stimulating factor (G-CSF) in db/db and WT mice treated intraperitoneally with IL-17A–neutralizing or isotype antibodies (4 μg/g) 24 hours before air or O₃ exposure. Results are mean ± SE of five to eight mice per group. *P < 0.05 versus air-exposed mice with same genotype and treatment; ^#P < 0.05 versus WT mice with same exposure and treatment; ^%P < 0.05 versus isotype-treated mice with same genotype and exposure.

Figure 3.

Anti–IL-17A reduces ozone-induced airway hyperresponsiveness in obese but not lean mice. To assess airway responsiveness (G), the coefficient of lung tissue damping, a measure of responses of the lung periphery, was measured by the forced oscillation technique in (A) WT mice or (B) db/db mice. Results are mean ± SE of five to eight mice per group. *P < 0.05 versus air-exposed mice with same genotype and treatment; ^#P < 0.05 versus WT mice with same exposure and treatment; ^%P < 0.05 versus isotype-treated mice with same genotype and exposure.

Figure 4.

Effect of anti–IL-17A in obese Cpe^fat/TNFR2^−/− mice exposed to ozone. Cpe^fat/TNFR2^−/− mice were treated intraperitoneally with IL-17A–neutralizing or isotype antibodies 24 hours before ozone exposure. Airway responsiveness was evaluated using G, the coefficient of lung tissue damping, a measure of responses of the lung periphery. Results are mean ± SE of seven to nine mice per group. *P < 0.05 versus isotype-treated mice.

Figure 5.

Anti–IL-17A attenuates O₃-induced increases in Grpr in obese mice. (A) Pulmonary mRNA abundance of Grpr in db/db and WT mice treated with IL-17A–neutralizing or isotype antibodies 24 hours before air or O₃ exposure. Results are mean ± SE of five to nine mice per group. (B) Pulmonary mRNA abundance of Grpr in obese Cpe^fat/TNFR2^−/− mice treated with IL-17A–neutralizing or isotype antibodies 24 hours before O₃ exposure. Results are mean ± SE of seven to nine mice per group. *P < 0.05 versus air-exposed mice with same genotype; ^#P < 0.05 versus WT mice with same exposure and antibody treatment; ^%P < 0.05 versus isotype-treated mice with the same genotype and exposure. Grpr = gastrin-releasing peptide receptor.

Figure 6.

Anti–gastrin-releasing peptide (anti-GRP) reduces responses to O₃ in obese mice. (A) Airway responsiveness using G, the coefficient of lung tissue damping, in db/db and WT mice treated intraperitoneally with anti-GRP or isotype antibodies 1 hour before O₃ exposure. (B) In db/db mice, airway responsiveness was also assessed after room air exposure. We also assessed (C) BAL neutrophils and (D) BAL IL-33 in the same mice. (E) Pulmonary Grpr mRNA abundance in db/db mice treated with anti-GRP or isotype antibody before O₃ exposure. ND = experiments with WT mice exposed to air were not done; consequently, statistical analysis of the effect of O₃ in the WT mice was not assessed. Results are mean ± SE of seven or eight mice per group. *P < 0.05 versus air-exposed mice with same genotype and treatment; ^#P < 0.05 versus WT mice with same exposure and treatment; ^%P < 0.05 versus isotype-treated mice with same genotype and exposure.

Figure 7.

Exposure to O₃ causes greater increases in BAL GRP in obese than in lean mice. (A) BAL GRP in WT and db/db mice exposed to air or O₃. N.D. = experiments with WT air-exposed mice were not done. (B) BAL GRP in HFD-fed versus chow-fed mice exposed to air or O₃. (C) BAL GRP in db/db and WT mice exposed to air or O₃ that had been pretreated with isotype versus anti–IL-17A antibodies. Results are mean ± SE of 4–14 mice per group. *P < 0.05 versus air-exposed mice with same genotype; ^#P < 0.05 versus WT mice with same exposure.