The effect of human rhinovirus-16 infection on microRA, tissue barrier and innate immunity in human bronchial epithelium from healthy and asthmatic airways

Abstract

Background: Rhinovirus (RV) is a common human respiratory pathogen. In healthy people RV infection causes the development of upper respiratory tract symptoms of limited duration, whilst in asthmatic patients the symptoms may be severe, prolonged and cause exacerbation of disease. Asthmatics were reported to have dysfunctional bronchial epithelial barrier with decreased junctional protein expression and impaired innate immune responses to infection, which may contribute to symptom exacerbation. MicroRNA (miR) are small RNAs that specifically bind to the 3’ untranslated regions (3’UTRs) of target messenger RNAS (mRNA) and alter protein expression by influencing mRNA stability and translation. It was hypothesised that RV infection of human bronchial epithelial cells (HBEC) alters the expression of miRNA-23a, -200b, -200c and -429—miRs which are predicted to target mRNAs encoding proteins relevant to epithelial barrier integrity and innate immunity.

Hypotheses: HRV-16 infection of culture differentiated airway epithelia alters levels of miR-23a and the miR-200 family (miR-200b, -200c, -429). MicroRNA responses will be different in healthy and asthmatic patients, potentially contributing to decreased epithelial integrity and impaired innate immune responses observed in asthmatics.

Methods: Primary human bronchial epithelial cells from healthy and asthmatic donors were differentiated for 21 days at an air-liquid interface (ALI) before being infected with HRV-16. Supernatants and cells were harvested at 24, 48 and 72 hours. Cells were lysed for RNA extraction and levels of miRNAs and in silico predicted targets (Zinc finger E-box-binding homeobox 1 (ZEB1), TNF-α Inhibitory Protein 3 (TNFAIP3), E-cadherin and occludin) were assessed. Supernatants were tested for interferon (IFN)-λ protein release. Differentiated HBECs were treated for 72 hours with IFN-λ to assess the effect of HRV-16 induced antiviral mediators on expression of microRNAs and target mRNA/protein in intact epithelial barrier. Differentiating HBECs were treated for 21 days with IFN-λ to assess the effect of HRV-16 induced antiviral mediators on expression of miRNAs and target mRNA/protein in differentiating, ‘repairing’ barrier. Additional epithelial barrier characteristics were assessed in response to IFN-λ treatment through imunoflourescent staining, cell counts, fluorescein isothocynate (FITC) 4kD dextran assays and transepithelial resistance (TER). Dual luciferase reporter assays were utilised to confirm miR-23a binding to predicted sequence target in 3’UTR of TNFAIP3.

Results: MiR-200b, -429 and -23a were significantly decreased in response to HRV-16—with a trend for greater reductions in asthmatic donors. Predicted miR-23a target TNFAIP3, predicted miR-429 target occludin and E-cadherin had significantly altered mRNA in response to HRV-16. There were no alterations to ZEB1. HRV-16 lead to significant inductions of IFN-λ mRNA and protein release. IFN-λ treatment of differentiated ALI cultures showed significant reductions of miR-23a and miR-429 in cultures from healthy donors with elevated TNFAIP3 and occludin mRNA and elevated ZEB1 protein expression. In cultures from asthmatic donors, miR-23a was significantly decreased in response to IFN-λ but there were no significant reductions in miR-429 with elevated TNFAIP3 expression and elevated ZEB1 protein expression. IFN-λ treatment decreased TER and changed cell morphology with less pronounced effects in asthmatic individuals. In differentiating ALI cultures, miR-23a and miR-429 were significantly decreased in healthy individuals, but no significant alterations were observed in asthmatics. ZEB1, E-cadherin, TNFAIP3 and occludin mRNA were significantly changed in response to IFN-λ in healthy donors with significant changes to E-cadherin and TNFAIP3 observed in asthmatic donors. Western blotting results were inconclusive. IFN-λ treatment decreased TER, increased FITC passage, decreased cell numbers and changed cell morphology with less pronounced effects in asthmatic individuals. Dual reporter luciferase assays confirmed miR-23a binding to predicted target sequence in 3’UTR of TNFAIP3/A20, which may affect NF-κB activation.

Conclusions: HRV-16 significantly reduces miR-200b, -429 and -23a which may account for alterations in epithelial barrier integrity and innate immune responses via microRNA targets ZEB1, E-cadherin, occludin and TNFAIP3/A20. IFN-λ protein, released in response to HRV-16, effects epithelial barrier properties in both intact and repairing barrier which may be mediated by significant reductions in miRNAs of interest and aforementioned targets. Asthmatic donors appear to be less responsive to IFN-λ which may underpin the impaired epithelial barrier and impaired innate immune responses to HRV-16 infection observed in these patients.

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ORCID for Tilman Sanchez-Elsner: orcid.org/0000-0003-1915-2410

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