Imprinting of bronchial epithelial cells upon in vivo rhinovirus infection in people with asthma

Abstract

Background: Defective translocation of the translational repressor TIAR (T-cell internal antigen receptor) in bronchial epithelial cells (BECs) from asthma patients underlies epithelial hyperresponsiveness, reflected by an exaggerated production of a select panel of inflammatory cytokines such as CXCL-8, interleukin (IL)-6, granulocyte colony-stimulating factor, CXCL-10, upon exposure to tumour necrosis factor (TNF) and IL-17A. With this study we aimed to clarify whether epithelial hyperresponsiveness is a consistent finding, is changed upon in vivo exposure to rhinovirus (RV)-A16 and applies to the bronchoconstrictor endothelin-1.

Methods: BECs were obtained from asthma patients (n=18) and healthy individuals (n=11), 1 day before and 6 days post-RV-A16 exposure. BECs were cultured and stimulated with TNF and IL-17A and inflammatory mediators were analysed. The bronchoalveolar lavage fluid (BALF) was obtained in parallel with BECs to correlate differential cell counts and inflammatory mediators with epithelial hyperresponsiveness.

Results: Epithelial hyperresponsiveness was confirmed in sequential samples and even increased in BECs from asthma patients after RV-A16 exposure, but not in BECs from healthy individuals. Endothelin-1 tended to increase in BECs from asthma patients collected after RV-A16 exposure, but not in BECs from healthy individuals. In vitro CXCL-8 and endothelin-1 production correlated. In vivo relevance for in vitro CXCL-8 and endothelin-1 production was shown by correlations with forced expiratory volume in 1 s % predicted and CXCL-8 BALF levels.

Conclusion: Epithelial hyperresponsiveness is an intrinsic defect in BECs from asthma patients, which increases upon viral exposure, but not in BECs from healthy individuals. This epithelial hyperresponsiveness also applies to the bronchoconstrictor endothelin-1, which could be involved in airway obstruction.

FIGURE 1

Overview of study design. Screening was performed 14–56 days before rhinovirus (RV)-A16 challenge. Lung function tests, bronchoscopy and bronchoalveolar lavage fluid (BALF) were collected at the indicated time points, before and after RV-A16 challenge, in mild asthma patients and healthy individuals.

FIGURE 2

Tumour necrosis factor (TNF) and interleukin (IL)-17A-induced mediator production by bronchial epithelial cells (BECs) from asthma patients and healthy individuals, before and after in vivo rhinovirus (RV)-A16 challenge. BECs collected after in vivo RV-A16 challenge from mild asthma patients (n=18), as compared to BECs collected before the viral challenge, produced upon 16 h of exposure to TNF and IL-17A significantly higher levels of a) CXCL-8, b) IL-6, c) granulocyte colony-stimulating factor (G-CSF), d) CXCL-10 and, trend-wise, e) endothelin (ET)-1. In contrast, BECs from healthy individuals (n=11) did not show an increase. For statistical analyses, two-tailed paired t-tests (before versus after RV16) and two-tailed unpaired t-tests (healthy controls versus asthma patients) were used. Bars and whiskers depict mean±sd. f) ET-1 production correlated with that of CXCL-8 after stimulation with TNF and IL-17A in BECs, both before (left panel) and after (right panel) RV-A16 challenge. Correlations were done with Pearson's correlation coefficient. ns: nonsignificant. *: p<0.05; **: p<0.01.

FIGURE 3

In vitro and in vivo CXCL-8 measurements, their correlation, and that with forced expiratory volume in 1 s (FEV₁) % predicted and for endothelin (ET)-1. a) CXCL-8 bronchoalveolar lavage fluid (BALF) levels increased significantly after rhinovirus (RV)-A16 exposure in asthma patients (n=18), but not in healthy individuals (n=11). b) BALF CXCL-8 correlates with CXCL-8 released from bronchial epithelial cells (BECs) obtained after RV-A16 exposure when data for healthy and asthma participants are combined. c) RV-A16 infection in asthma patients (n=18) induced a significant drop in FEV₁ % predicted, but not for healthy individuals at day 4 after RV-A16 (n=11). d) ET-1 levels correlated significantly with FEV₁ % predicted before and not after exposure to RV-A16. e) The change (Δ) in CXCL-8 levels released in vitro by BECs obtained after RV-A16 correlated with FEV₁ measured after RV-A16, but not before RV-A16 exposure. For statistical analyses, two-tailed paired t-tests (before versus after RV16) and two-tailed unpaired t-tests (healthy controls versus asthma patients) were used. Nonparametric Spearman correlation test was performed with two-tailed analysis. ns: nonsignificant. *: p<0.05; ***: p<0.001.

FIGURE 4

The change in eosinophils, neutrophils, myeloperoxidase (MPO) and eosinophil cationic protein (ECP) at day 6 post-rhinovirus (RV)-A16 exposure. Comparison of deltas (Δ; percentage of cell numbers at day 6 after RV-A16 minus the baseline at day −1) in a) percentage eosinophils shows no significant difference, while b) the Δ of neutrophils in bronchoalveolar lavage fluid (BALF) of healthy individuals (n=11) was negative (lower during infection than at baseline) and significantly lower than the marginal increase (positive Δ) observed in mild asthmatics (n=18). The Δ of c) ECP and d) MPO levels in BALF were not statistically significantly different in mild asthma patients (n=18) and not in healthy individuals (n=10). The bars and whiskers depict mean±sd. For statistical analyses, two-tailed nonparametric Mann–Whitney t-tests were used for figure 4a and c; unpaired t-tests were used for figure 4b and d. ns: nonsignificant. **: p<0.01.