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A4704 - A Physiological Relevant Rhinovirus Infection Model in Differentiated Human Primary Bronchial Epithelial Cells from Healthy, Asthmatic and COPD Donors
Author Block: P. Veerati1, A. Reid2, K. Nichol1, L. Ngan1, W. Teresa1, P. A. B. Wark3, D. A. Knight4, C. L. Grainge3, N. W. Bartlett5; 1Respiratoty Division, Hunter Medical Research Institute, New Lambton Heights, Australia, 2Biomedical Sciences and Pharmacy, University of Newcastle, New Lambton Heights, Australia, 3Respiratory and Sleep Medicine, John Hunter Hosp, New Lambton NSW 2322, Australia, 4Biomedical Sciences and Pharmacy, University of Newcastle, Callaghan, Australia, 5University of Newcastle - Aus, Newcastle, Australia.
Rationale:
The majority of asthma and COPD exacerbations are caused by viral infections, rhinoviruses (RV) being the most common. Differentiated air-liquid interface (ALI) bronchial epithelial cell cultures are commonly used for viral infection studies however there has been concern that ALI cultures are difficult to infect with RV at low multiplicities of infection (MOI) that better reflect naturally occurring infections. We hypothesized that 1: infection with RV of both major and minor groups at ultra-low MOI (0.001) is possible in ALI cultured bronchial epithelium from donors without respiratory disease (healthy) and donors with asthma and COPD 2: ultra-low MOI infection would confirm previously demonstrated innate anti-viral immune deficiencies and increased viral replication in asthma and COPD.
Methods:
pBECs from healthy, asthmatic and COPD donors were obtained at bronchoscopy, cultured and differentiated at ALI. Cells were infected with both major (RV16) and minor group (RV1B) viruses at an ultra-low MOI of 0.001. Samples were collected at 0, 24, 48, 72 and 96 hours following infection and were analysed for viral RNA, interferon-beta (IFN-β), -lambda (IFN-λ), IL-25 and Muc5AC gene expression. IFN-β, IFN-λ, Muc5AC, IL-8, CXCL10, IL-6, and CCL5 proteins in apical supernatants were also measured. Paraffin-embedded sections were stained for mucin-containing goblet cells and viral capsid protein.
Results:
ALI cultures infected with RV1B showed 3-4 log increase in viral RNA peaking at 24 hours post infection (p.i.) which was also the time of maximal detection of viral capsid protein. After 24 hours viral RNA levels plateaued and capsid protein was almost undetectable. Infection induced the release of IFN-β and IFN-λ proteins from epithelial cells, maximal at 48 hours p.i. and this was reduced in asthma and COPD. RV infection also induced CXCL10 protein release from 48 hours p.i. Similar replication kinetics and induction of anti-viral and inflammatory cytokines were observed with RV16 infection.
Conclusion:
We are able to reliably infect fully differentiated ALI epithelial cultures with RV using an MOI 0.001. Replication peaked at 24 h before shutting down and this directly preceded peak induction of type I and III interferons. Whilst the peak levels of IFN were lower in asthma and COPD, IFN expression kinetics were not different (increased at 48 h in all groups) to that of healthy donor cells which might explain why viral replication was not increased in asthma and COPD despite significantly lower levels of IFN.