Diesel exhaust particle induction of IL-17A contributes to severe asthma

Abstract

Background: IL-17A has been implicated in severe forms of asthma. However, the factors that promote IL-17A production during the pathogenesis of severe asthma remain undefined. Diesel exhaust particles (DEPs) are a major component of traffic-related air pollution and are implicated in asthma pathogenesis and exacerbation.

Objective: We sought to determine the mechanism by which DEP exposure affects asthma severity using human and mouse studies.

Methods: BALB/c mice were challenged with DEPs with or without house dust mite (HDM) extract. Airway inflammation and function, bronchoalveolar lavage fluid cytokine levels, and flow cytometry of lung T cells were assessed. The effect of DEP exposure on the frequency of asthma symptoms and serum cytokine levels was determined in children with allergic asthma.

Results: In mice exposure to DEPs alone did not induce asthma. DEP and HDM coexposure markedly enhanced airway hyperresponsiveness compared with HDM exposure alone and generated a mixed T(H)2 and T(H)17 response, including IL-13(+)IL-17A(+) double-producing T cells. IL-17A neutralization prevented DEP-induced exacerbation of airway hyperresponsiveness. Among 235 high DEP-exposed children with allergic asthma, 32.2% had more frequent asthma symptoms over a 12-month period compared with only 14.2% in the low DEP-exposed group (P = .002). Additionally, high DEP-exposed children with allergic asthma had nearly 6 times higher serum IL-17A levels compared with low DEP-exposed children.

Conclusions: Expansion of T(H)17 cells contributes to DEP-mediated exacerbation of allergic asthma. Neutralization of IL-17A might be a useful potential therapeutic strategy to counteract the asthma-promoting effects of traffic-related air pollution, especially in highly exposed patients with severe allergic asthma.

Keywords: AHR; Airway hyperresponsiveness; Allergic asthma; BALF; Bronchoalveolar lavage fluid; DEP; Diesel exhaust particle; Forkhead box protein 3; Foxp3; GCPCR; Greater Cincinnati Pediatric Clinic Repository; HDM; House dust mite; IL-13 receptor; IL-13R; IL-17A; OR; Odds ratio; PE; PEES; Pediatric Environmental Exposures Study; Phycoerythrin; Regulatory T; SPT; Skin prick test; Treg; diesel exhaust particle; house dust mite; regulatory T cell.

Figure 1. DEP exacerbates HDM-induced airway responses

(A) Protocol. (B) Total BALF cell counts. (C) Airway resistance. Data from 2 separate experiments (n=10–18 mice per group; 1way ANOVA, *** p < 0.001, ** p < 0.01, * p < 0.05). (D) Representative, PAS stained airways. (E) HDM-specific IgG1 and (F) IgE titers.

Figure 2. DEP induces neutrophilia and exacerbates HDM-induced eosinophilia

(A) Differential counts. (B) BALF cell counts. Total BALF eosinophils (C) and neutrophils (F) assessed 24h after the last intratracheal exposure. BALF levels of (B) CCL11 (eotaxin-1), (C) IL5, (E) CxCL5 (ENA78) and (F) CxCL1 (KC) were assessed by ELISA (representative experiment with 6–7 mice / group; 1way ANOVA, ***p < 0.001, **p< 0.01, *p<0.05).

Figure 3. DEP induces IL-17A and exacerbate HDM-induced Th2 responses

(A) IFNγ (B) IL-4, (C) IL-13 and (D) IL-17A BALF levels assessed 24h after the last exposure. (E) IL-23p19 in lung homogenates. (F) IL-4, IL-17A and IL-17F mRNA lung levels (representative experiment with 5–7 mice / group; 1way ANOVA, ***p < 0.001, **p< 0.01, *p<0.05).

Figure 4. HDM+DEP exposures exacerbate HDM-induced T cell activation

(A) Representative FACS dot plots of the proportion of CD4⁺ cells versus CD8⁺ cells among CD3⁺ lung T-cells. (B) Percentages of CD69⁺Foxp3− cells among CD4⁺ lung T-cells. (C) Percentages of Foxp3⁺CD25⁺ cells among CD4+ lung T-cells (n=10–14 mice/group, 2 separate experiments; 1way ANOVA, ***p < 0.001, **p< 0.01, *p<0.05).

Figure 5. Exposure to HDM+DEP generates IL-17A⁺IL-13⁺CD4⁺ effector T-cells

(A) Representative FACS dot plots of lung CD44^highCD62^low effector T-cells. (B) Intracellular staining for CD4⁺ T cells producing IL-13 and IL-17A following ex vivo restimulation with PMA+ionomycin (n = 5–7 mice/goup). (C) Representative FACS dot plots of cytokine expression by CD4⁺CD44⁺effector T-cells and frequency of IL-13⁺ IL-17A⁺ double producers (n = 5–7 mice/goup; 1way ANOVA *p< 0.05, **p< 0.01, ***p<0.001).

Figure 6. IL-17A neutralization alleviated DEP exacerbation of HDM-induced airway responses

(A) Mice were given rat anti-murine IL-17A (200ug; M210) or an IgG1 control antibody intratracheally during FIDM±DEP challenges. (B) AHR was assessed 24h after the last exposure to HDM±DEP. (C) AHR following the last methacholine dose (50mg/ml). (D) Differential counts (2way ANOVA for panel B and D, 1way ANOVA for panel C, ***p < 0.001, **p<0.01, *p<0.05)

Figure 7. DEP exposure exacerbates asthma severity and increases IL-17A in childhood allergic asthma

(A) Among 235 GCPCR participants with allergic asthma, those with high DEP exposure (N=44) had more frequent asthma symptoms compared with those with low exposure. The observed association remained significant even after adjusting for age, race, sex, annual family income, type of health insurance, maternal education, being prescribed asthma controllers, adherence to asthma medication over the prior two weeks, and second-hand smoke exposure in multivariate logistic models (p=0.01). (B) Among 46 PEES asthmatics, 53% of children with high DEP exposure also had high levels of IL-17A. The association remained significant after adjustment for the above covariates and asthma symptom frequency.