Inflammatory epithelial cytokines after in vitro respiratory syncytial viral infection are associated with reduced lung function

Morphology of epithelial cells post respiratory syncytial virus (RSV) infection in cystic fibrosis and control cells. Homozygous p.Phe508del cystic fibrosis transmembrane conductance regulator (CFTR) and control primary human nasal epithelial cells (PNECs) in mock and RSV infection were stained at 72 h post infection. Representative immunofluorescence images demonstrate β-tubulin staining in epithelial cilia, E-cadherin and β-catenin staining in cell–cell adherens junctions and ZO-1 staining in tight junctions. Scale bar=10 µm.

Protein expression and transepithelial resistance (TER) post respiratory syncytial virus (RSV) infection in cystic fibrosis and control cells. Protein was harvested from homozygous p.Phe508del cystic fibrosis transmembrane conductance regulator (CFTR) and control primary human nasal epithelial cells in mock and RSV infection at 72 h post infection. Western blots were performed. a) Epithelial cilia marker β-tubulin, cell–cell adherens markers E-cadherin and β-catenin and tight junctions marker ZO-1. b) Quantification of Western blots demonstrates that expressions of the proteins are not significant between p.Phe508del CFTR and control cells (n=5–6), by Mann–Whitney test. c) The TER between homozygous p.Phe508del CFTR and control cells is not significant at 24, 48 and 72 hpi. The data were analysed with two-way ANOVA analysis followed by post hoc Bonferroni multiple comparisons. *p<0.05; ns: not significant; M: Mock; R: RSV; CNX: calnexin.

Pattern recognition receptors gene expression is increased in cystic fibrosis (CF) and control cells post respiratory syncytial virus (RSV). RSV increases RIG-I, MDA-5, TLR2, TLR3, TLR4 and TLR7 mRNA expression. TLR9 mRNA is unchanged and TLR8 mRNA is undetectable using Qiagen SBH0265788-200 TLR8 primers. The relative mRNA levels of RIG-I and MDA-5 are significantly higher in homozygous p.Phe508del cystic fibrosis transmembrane conductance regulator (CFTR) cells compared to control cells post RSV infection. No significant differences are seen in TLR genes post RSV between CF and control cells. Interferon-stimulated gene ISG56 shows more mRNA expression in homozygous p.Phe508del CFTR cells. Non-paired data were analysed by Mann–Whitney test, and paired data were analysed by matched Wilcoxon test (n=11–12). **p<0.01; ***p<0.001; ns=not significant.

Cystic fibrosis cells produce higher amounts of cytokines post respiratory syncytial virus (RSV) infection as compared to control cells. The production of cytokines was measured by Luminex in basal media at 72 hpi in primary human nasal epithelial cells. Among the 36 cytokines measured, the levels of interleukin (IL)-8, IL-9, IL-10, IL-12p70, IL-15, IL-17A, tumour necrosis factor-α (TNF-α), vascular endothelial growth factor (VEGF) and granulocyte colony-stimulating factor (G-CSF) are significantly higher in homozygous p.Phe508del cystic fibrosis transmembrane conductance regulator (CFTR) cells than in control cells. Non-paired data were analysed by Mann–Whitney test, and paired data were analysed by matched Wilcoxon test (n=10). *p<0.05; **p<0.01.

The production of Type III interferons (IFNs) is not reduced in cystic fibrosis cells. The production of Type III IFNs interleukin (IL)-28A and IL-29 were measured. IL-28A and IL-29 are not different between respiratory syncytial virus (RSV)-infected p.Phe508del cystic fibrosis transmembrane conductance regulator cells and control cells. The production of IL-28A and IL-29 in mock cells were below the limit of detection. Non-paired data were analysed by Mann–Whitney test, and paired data were analysed by matched Wilcoxon test (n=9–10). **p<0.01.