AbstractAsthma affects 300 million children worldwide. Early age viral infection and allergen sensitization are associated with subsequent asthma development. We aimed to understand the consequences of respiratory syncytial virus (RSV) infection and/or house-dust mite (HDM) exposure on cytopathogenesis, virus replication, and innate immune responses in airway epithelium from young children. Well-differentiated primary nasal epithelial cell (WD-PNEC) cultures were derived from newborn infants and pre-school children (aged 1-6) and identified as; 1. healthy, 2. mild wheezers, and 3. severe wheezers [n=5 each]). They were infected with RSV following HDM stimulation. Virus growth kinetics, WD-PNEC cytopathology, innate immune responses, and effects of HDM treatment on IGF1R and nucleolin expression were analysed.
RSV infected WD-PNECs derived from either newborn and/or pre-school children resulted in similar growth kinetics regardless of age and wheeze phenotype. Interestingly, HDM pre-treatment significantly delayed viral growth between 24-48 hpi, independent of wheeze phenotype, but virus titres at 96 hpi were similar. While RSV infection resulted in significant cell loss across all age groups, cilia loss in WD-PNECs from severe wheezers in the pre-school cohort was most profound. HDM pre-treatment did not alter innate immune responses to infection. Expression of IL-29 and interferon-stimulated genes (ISGs), including Ifi6, Isg15, Duoxa2, Duox2, were significantly increased following infection, as were CEACAM1, TRAIL, CX3CL1, GM-CSF, CXCL8, and CXCL16 protein secretions. Interestingly, IRF9, IFI6, and ISG15 expression were significantly lower in mild compared to severe wheezers or healthy individuals. Furthermore, CEACAM1 and IL-33 secretions were highest, and GM-CSF was lowest, in WD-PNECs from severe wheezers. Surprisingly, genes associated with asthma and airway remodelling, including TSLP, HMGB1, and KRT5, were reduced following RSV infection in all cohorts. Moreover, Western blot and IF analysis of total IGF1R, PKCζ, and Nucleolin (important for RSV attachment/entry) showed a partial reduction following HDM treatment compared to DMEM controls at 3 hpi. These data may explain, in part, the attenuated RSV growth kinetics evident in WD-PNEC cultures pre-treated with HDM. However, although differential gene/protein expression was observed in children with no, mild or severe wheeze, the functional consequences of these responses on the progression of wheeze pathogenesis remain to be determined.
|Date of Award
|Aurelie Mousnier (Supervisor) & Ultan Power (Supervisor)