Activation of epithelial and inflammatory pathways in adolescent elite athletes exposed to intense exercise and air pollution

Abstract

Rationale: Participation in high-intensity exercise in early life might act as stressor to the airway barrier.

Objectives: To investigate the effect of intense exercise and associated exposure to air pollution on the airway barrier in adolescent elite athletes compared with healthy controls and to study exercise-induced bronchoconstriction (EIB) in this population.

Methods: Early-career elite athletes attending 'Flemish-Elite-Sports-Schools' (12-18 years) of 4 different sport disciplines (n=90) and control subjects (n=25) were recruited. Presence of EIB was tested by the eucapnic voluntary hyperventilation (EVH) test. Markers at mRNA and protein level; RNA-sequencing; carbon load in airway macrophages were studied on induced sputum samples.

Results: 444 genes were differentially expressed in sputum from athletes compared with controls, which were related to inflammation and epithelial cell damage and sputum samples of athletes contained significantly more carbon loaded airway macrophages compared with controls (24%, 95% CI 20% to 36%, p<0.0004). Athletes had significantly higher substance P (13.3 pg/mL, 95% CI 2.0 to 19.2) and calprotectin (1237 ng/mL, 95% CI 531 to 2490) levels as well as IL-6, IL-8 and TNF-α mRNA levels compared with controls (p<0.05). The incidence of EIB in athletes was 9%. The maximal fall in forced expiratory volume in 1 s (%) after EVH test in athletes was significantly associated with prior PM₁₀ and PM_2.5 exposure.

Conclusion: Early-career elite athletes showed increased markers of air pollution exposure, epithelial damage and airway inflammation compared with controls. Acute exposure to increased air pollution PM₁₀ levels was linked to increased airway hyper-reactivity.

Trial registration number: NCT03587675.

Keywords: airway epithelium; exercise.

Figure 1

Recruited subjects. (A) Included athletes (n=90) in elite Sports’ Schools in Antwerp, Leuven and Vilvoorde. *None were excluded. (B) Included controls (n=25). Exclusion criteria are shown.

Figure 2

Study design. Demonstrating order of different interventions at the study visit. EVH, eucapnic voluntary hyperventilation.

Figure 3

Increased carbon load in airway macrophages in athletes compared with controls. (A) Illustration of images captured for analysis showing airway macrophages stained by diff-Quick with increasing BC load. (B) The average particle count per macrophage, percentage of loaded macrophages and the percentage area occupied by BC for each participant was calculated by a blinded researcher. For each participant, 25 macrophages were counted. (Normality confirmed, unpaired t-test with Welch’s correction), controls (controls: n=14, athletes n=44) (C) Comparison of indoor (n=22) and outdoor (n=22) average BC load (normality confirmed, unpaired t-test). (D) Correlation between one week and one month average BC (µg/m³) and the average particle count observed in athletes (n=44). (Spearman correlation). BC, black carbon; ns, not significant.

Figure 4

Profiling of serum and induced sputum samples in controls and athletes. (A) Volcano plot with the magnitude expressed as log2 fold change (x-axis) and significance expressed as −log10 of the adjusted p value (y-axis) of differential expression analysis. Genes of interest are labelled. (B) Selected significantly enriched or downregulated pathways based on Ingenuity Pathways Analysis (IPA) analysis listed according their p value. (C) Significant enrichment plots at False Discovery Rate (FDR) <25%. (D) Following cytokine mRNA levels (controls: n=6, athletes: n=43) were measured via qPCR: IL-6, IL-8 and TNF-α. (E) Serum CC16 levels in controls vs athletes were measured via ELISA (controls: n=23, athletes: n=83). Sputum supernatants (controls: n=18, athletes: n=73) calprotectin (F) and substance P levels (G) were measured via ELISA. (Mann-Whitney U test).

Figure 5

Air pollution exposure prior EVH test. (A–C) Daily mean levels of each pollutant considered during the study period averaged over the three included Elite Sport’s Schools. The flashing red line refers to the maximum concentration of each pollutant established by WHO. The dotted blue lines refer to the time frame the athletes participated in the study. (G) Effect of the most robust air pollutant PM₁₀ on tight junction mRNA expression of CLDN1, OCLN and ZO-1. (Mann-Whitney) (PM₁₀low: n=22, PM₁₀high: n=20, outliers were removed based on Grubbs). EVH, eucapnic voluntary hyperventilation. O₃, ozone; PM_2.5, particulate matter < 2.5 µm; PM₁₀, particulate matter < 10 µm.