0006 Circadian Regulation of Epithelial Viral Load in Health and in Asthma

Abstract Introduction Molecular circadian rhythms regulate cellular processes underlying health including innate immune pathways. Prior studies have implicated circadian regulation of viral responses as contributing to airway inflammation in asthma, but investigation in human cell models of airway epithelium has been limited. Using primary human airway epithelial cells differentiated at an air-liquid interface as an ex vivo organotypic model from healthy donors and donors with asthma, we investigated circadian-time dependent regulation of viral replication of rhinovirus-16 (RV16) in airway epithelial cells. Methods Primary bronchial epithelial cells (BECs) from donors with pediatric asthma (n=5) or healthy donors (n=4) were differentiated at an air-liquid interface to an organotypic epithelium in temperature cycled incubators. Intrinsic circadian rhythmicity following temperature cycling was confirmed using BMAL1:Luciferase recordings and western blotting for BMAL1. At circadian time 0 hours and circadian time 12 hours, RV16 was applied to the apical surface at a multiplicity of infection of 0.5. BEC were harvested for RNA isolation at 12, 24, 48 and 96 hours after infection for genome copy number assessment with qPCR. Results Temperature cycles of 12 hours at 37C and 12 hours at 34C for 6 days reliably synchronized intrinsic circadian rhythms measured in luciferase assays in both healthy AECs and AECs from donors with pediatric asthma. Circadian amplitude and period was similar between healthy BECs and BECs from donors with asthma by gene expression and BMAL:Luciferase recording. Infection at time zero during the circadian cycle as compared to infection occurring 12 hours later was associated with lower viral replication at 24, 48, and 96 hours after infection in healthy BECs. Conclusion The core circadian clock genes maintain rhythmicity in healthy and asthma airway epithelia. Circadian time regulates epithelial viral replication in healthy BECs. Future work will measure viral replication in BECs with genetic ablation of core circadian genes and in correlation with in vivo circadian rhythm measurements. Support (if any) SRS (WTP), Parker B Francis Fellowship (WTP), NIH K24AI150991(JSD)