Interferon response to respiratory syncytial virus by bronchial epithelium from children with asthma is inversely correlated with pulmonary function

Abstract

Background: Respiratory viral infection in early childhood, including that from respiratory syncytial virus (RSV), has been previously associated with the development of asthma.

Objective: We aimed to determine whether ex vivo RSV infection of bronchial epithelial cells (BECs) from children with asthma would induce specific gene expression patterns and whether such patterns were associated with lung function among BEC donors.

Methods: Primary BECs from carefully characterized children with asthma (n = 18) and matched healthy children without asthma (n = 8) were differentiated at an air-liquid interface for 21 days. Air-liquid interface cultures were infected with RSV for 96 hours and RNA was subsequently isolated from BECs. In each case, we analyzed gene expression using RNA sequencing and assessed differences between conditions by linear modeling of the data. BEC donors completed spirometry to measure lung function.

Results: RSV infection of BECs from subjects with asthma, compared with uninfected BECs from subjects with asthma, led to a significant increase in expression of 6199 genes. There was significantly greater expression of 195 genes in BECs from children with asthma and airway obstruction (FEV₁/forced vital capacity < 0.85 and FEV₁ < 100% predicted) than in BECs from children with asthma without obstruction, or in BECs from healthy children. These specific genes were found to be highly enriched for viral response genes induced in parallel with types I and III interferons.

Conclusions: BECs from children with asthma and with obstructive physiology exhibit greater expression of types I and III interferons and interferon-stimulated genes than do cells from children with normal lung function, and expression of interferon-associated genes correlates with the degree of airway obstruction. These findings suggest that an exaggerated interferon response to viral infection by airway epithelial cells may be a mechanism leading to lung function decline in a subset of children with asthma.

Keywords: Asthma; RNA; epithelial cells; respiratory syncytial virus; sequence analysis; type I interferon.

FIG 1

Differential gene expression between children with airway obstruction and children without airway obstruction. A, A volcano plot showing group fold-change differences comparing the RSV A2-infected BECs between the group of children with obstruction and the group without obstruction. Red indicates genes with significantly higher expression in the obstruction group and blue indicates genes with significantly lower expression in the obstruction group (FDR < 0.05). B, List of differentially expressed genes including pattern recognition receptors upstream of type I interferon signaling, ISGs, type III interferons, key transcription factors downstream of type I interferon signaling, and interferon-stimulated chemoattractants regulating T cells and inflammatory leukocytes. C, A heat map of the differentially expressed genes. Gene expression levels are shown as row normalized Z scores with red reflecting higher expression and blue representing lower expression.

FIG 2

Correlation between expression of the viral response and interferon/ISG gene set and FEV₁ percentage predicted. Among children with asthma, the geometric means of the expression values of the 173 viral response and interferon/ISG genes by BECs show significant inverse correlation with degree of airway obstruction as defined by FEV₁ percentage predicted. The solid red line represents least squares regression and dotted lines represent the 95% confidence interval.

FIG 3

Reproducibility of findings with RSV line 19 stimulation and specificity to airway obstruction. A, Comparison of gene expression differences between asthma obstruction and asthma no obstruction groups were assessed separately in the RSV line 19-infected BECs and RSV A2-infected BECs. The fold-change values for each comparison are plotted demonstrating a high degree of correlation between the 2 datasets. Identical differences between the 2 comparisons would be along the 45° diagonal. Blue dots indicate the 195 statistically significant genes. B, A box plot of the geometric means of the upregulated genes in the asthma no obstruction (blue), asthma obstruction (red), and healthy children (gray) groups after RSV infection shows very similar responses to RSV A2 and RSV line 19 strains, and increased expression of this gene set specific to the asthma obstruction group. Airway obstruction was defined as an absolute FEV₁/FVC ratio <0.85 and FEV₁ percentage predicted <100%.

FIG 4

Protein concentrations were measured in BEC supernatant samples harvested 96 hours after RSV infection. IFNβ1 (A), CXCL10 (B), CXCL11 (C), IFNλ1/IL-29 (E), and CD40 (F) protein concentrations were significantly different among supernatants of BECs from children with asthma and airway obstruction, children with asthma and normal lung function, and healthy children (ANOVA, P< .05), and concentrations of these proteins were significantly greater in supernatants from BECs of children with asthma and airway obstruction as compared to supernatants of BECs from children with asthma and normal lung function (Dunn test, P < .05). There was not a significant difference in IFNλ2/IL-28A concentrations in supernatant by BECs between the 3 subject groups.