Differential effects of intense exercise and pollution on the airways in a murine model

Abstract

Background: Exercise-induced bronchoconstriction (EIB) is a transient airway narrowing, occurring during or shortly after intensive exercise. It is highly prevalent in non-asthmatic outdoor endurance athletes suggesting an important contribution of air pollution in the development of EIB. Therefore, more research is necessary to investigate the combination of exercise and pollutants on the airways.

Methods: Balbc/ByJ mice were intranasally challenged 5 days a week for 3 weeks with saline or 0.2 mg/ml diesel exhaust particles (DEP), prior to a daily incremental running session or non-exercise session. Once a week, the early ventilatory response was measured and lung function was determined at day 24. Airway inflammation and cytokine levels were evaluated in bronchoalveolar lavage fluid. Furthermore, innate lymphoid cells, dendritic cells and tight junction mRNA expression were determined in lung tissue.

Results: Submaximal exercise resulted in acute alterations of the breathing pattern and significantly improved FEV_0.1 at day 24. DEP exposure induced neutrophilic airway inflammation, accompanied with increased percentages of CD11b⁺ DC in lung tissue and pro-inflammatory cytokines, such as IL-13, MCP-1, GM-CSF and KC. Occludin and claudin-1(Cldn-1) expression were respectively increased and decreased by DEP exposure. Whereas, exercise increased Cldn-3 and Cldn-18 expression. Combining exercise and DEP exposure resulted in significantly increased SP-D levels in the airways.

Conclusion: DEP exposure induced typical airway neutrophilia, DC recruitment and pro-inflammatory cytokine production. Whereas, intensive exercise induced changes of the breathing pattern. The combination of both triggers resulted in a dysregulation of tight junction expression, suggesting that intensive exercise in polluted environments can induce important changes in the airway physiology and integrity.

Keywords: Dendritic cells; Diesel exhaust particles; Exercise-induced bronchoconstriction; Non-allergic; Tight junctions.

Fig. 1

Study design used in experiment 1 and 2

Fig. 2

Longitudinal changes of pre-exposure breathing pattern at day 1, 8, 15 and 22. Breathing parameters were measured on days 1, 8, 15 and 22 prior to the exposure and running or rest session using a double chamber plethysmograph. a The average inspiratory time (Ti) pre-exposure. b The average peak inspiratory flow (PIF) pre-exposure. c The average minute volume (MV) pre-exposure. d The average breathing frequency (f) pre-exposure. Data is shown as mean (n = 7–9 mice per group). *p < 0.05 for saline exposed exercised mice compared with DEP exposed exercised mice, **p < 0.01 for DEP exposed non-exercised mice compared with DEP exposed exercised mice (Two-Way ANOVA)

Fig. 3

Exposure-induced changes of the breathing pattern. Breathing parameters were measured on days 1, 8, 15 and 22 prior to and immediately after the exposure and running or rest session using a double chamber plethysmograph. The exposure-induced changes were determined by calculating the difference between the measured values pre- and post-exposure. If the difference > 0, an exercise-induced increase is identified. If the difference < 0, an exercise-induced decrease was identified, as shown in (a) for the inspiratory time at day 22. b The average exposure-induced changes of the inspiratory time (ΔTi). c The average exposure-induced changes of the expiratory time (ΔTe). d The average exposure-induced changes of the peak inspiratory flow (ΔPIF). e The average exposure-induced changes of the peak expiratory flow (ΔPEF). f The average exposure-induced changes of the minute volume (ΔMV). g The average exposure-induced changes of the breathing frequency (Δf). Data are shown as mean with SD (n = 7–8 mice/group). *p < 0.05, **p < 0.01, ***p < 0.001 (Two-Way ANOVA)

Fig. 4

Baseline lung function and airway hyperreactivity on day 24. Lung function parameters and airway hyperreactivity were assessed at day 24, 24 h after the last exposure and running or rest session, using the FlexiVent system. a Baseline Tiffeneau-index_0.1 was calculated as FEV_0.1/FVC. (p = 0.0756, Two-Way ANOVA). b Correlation between baseline Tiffeneau-index_0.1 and running distance of saline exposed exercised mice (r = 0.5449, p = 0.1293, Pearson correlation). c Correlation between baseline Tiffeneau-index_0.1 and running distance of DEP exposed exercised mice (r = − 0.7313, p = 0.0252, Pearson correlation). d The group average dose-response of the airway resistance (Rn) to methacholine (0–20 mg/ml) was measured using a forced oscillation technique (QP3). **p < 0.01 for Sal/NE compared with Sal/E, ***p < 0.001 for Sal/E compared with DEP/E (Two-Way ANOVA). e The group average dose-response of FEV_0.1 (%) to methacholine (0–20 mg/ml) was measured using the negative pressure forced expiration (NPFE) manoeuvre. *p < 0.05 DEP/NE compared with DEP/E (Two-Way ANOVA). f The provocative methacholine concentration inducing a 20% decrease in FEV_0.1, relative to the baseline FEV_0.1, was calculated based on the dose-response curve of FEV_0.1 (%), shown in (e). (exercise-effect with ^##p = 0.0080, Two-Way ANOVA). Individual data point in figure a and f are shown with mean (n = 7–9 mice per group)

Fig. 5

Broncho-alveolar lavage (BAL) and DEP-uptake. a Total number of macrophages, neutrophils and eosinophils was counted in bronchoalveolar lavage fluid of experiment 1 and 2. **p < 0.01 and ***p < 0.001 (One-Way ANOVA). b The percentage of macrophages loaded with DEP was counted. For each mice of experiment 1 and 2, 100 macrophages were counted in duplicate and an average of the duplicates was calculated per mice. Data is shown as mean with SD in (a and b) (n = 20–21 mice per group). c The number of particles taken up by the macrophages was calculated for each mice of experiment 1. Hundred macrophages per mice were counted and an average of the 100 macrophages per mice was calculated, shown in (c). *p < 0.05 (Unpaired t-test) (n = 8–9 mice per group). d Macrophages in bronchoalveolar lavage fluid loaded with DEP, representing DEP/E mice (40 X amplification). e Macrophages in bronchoalveolar lavage fluid, representing Sal/E mice. (40 X amplification)

Fig. 6

Dendritic cells in lung tissue. Antigen presenting cells in lung tissue were measured using flow cytometry. Cells were analyzed as a auto fluorescent macrophages, b CD45⁺ low auto fluorescent MHCII⁺CD11c⁺ DC (Total DC), c CD45⁺ low auto fluorescent MHCII⁺CD11c⁺CD11b⁺CD103⁻ conventional DC (CD11b⁺ cDC or cDC2), d CD45⁺ low auto fluorescent MHCII⁺CD11c⁺CD11b⁻CD103⁺ cDC (CD103⁺ cDC or cDC1), e CD45⁺ low auto fluorescent MHCII⁺CD11c⁺CD11b⁺CD64⁺ monocyte derived DC (moDC) and f CD45⁺ low auto fluorescent MHCII⁺CD11c⁺SiglecH⁺ plasmacytoid DC (pDC). Data is shown as mean with SD (n = 12 mice per group. *p < 0.05 (Two-Way ANOVA). Detailed gating strategy available in supplementary Figure S3

Fig. 7

Airway permeability and integrity. a Surfactant protein D (SP-D) concentrations in serum were measured using ELISA (R&D Systems). b Uric acid concentrations in serum were measured using ELISA (ThermoFisher). c Protein levels in bronchoalveolar lavage fluid were measured using a Bradford assay (Biorad). d OCLN mRNA expression, e ZO-1 mRNA expression, f Cldn-1 mRNA expression, g Cldn-3 mRNA expression, h Cldn-4 mRNA expression and i Cldn-18 mRNA expression in lung tissue were measured using RT-qPCR. Data shown as mean with SD (n = 9 mice per group). *p < 0.05, **p < 0.01, ***p < 0.001 comparing individual groups. & = DEP-effect, # = Exercise-effect, + = Interaction-effect (Two-Way ANOVA)

Fig. 8

Breathing pattern pre- and post-exposure. Based on the breathing parameters measured pre and post exposure and running, a breathing pattern was reconstructed. Dark green indicates the breathing pattern before exercise and light green after exercise. Ti: inspiratory time, Te: expiratory time, PIF: peak inspiratory flow, PEF: peak expiratory flow, TV: tidal volume, EV: expiratory volume