0006 Circadian Cyclic Gene Expression in Human Airway Epithelial Cells in Asthma and Viral Infections

Abstract Introduction Cellular circadian rhythms regulate gene expression and innate immune pathways related to airway diseases in animal models of disease. However, circadian regulation of gene expression remains uninvestigated in human airway epithelial cells. Primary human airway epithelial cells can be grown at an air-liquid interface as an ex vivo organotypic model to characterize molecular circadian rhythms in the human airway. Using cells from healthy and donors with disease, we hypothesized that circadian cyclic gene expression would be altered in asthma and would display altered viral responses. Methods Cells were synchronized with temperature cycled incubators and RNA isolated every 4 hours over a 48 hour period for RNA-sequencing from primary human airway epithelial cells from healthy and asthmatic children. CompareRhythms in R was used to identify differential rhythmicity using the cosinor method. EnrichR pathway analysis for Reactome, Panther, GO molecular functions, and GO biological processes was used to identify relevant biological pathways with altered circadian rhythmic expression. Human rhinovirus 16 was applied to the apical surface at a multiplicity of infection of 0.5 and RNA isolated 96 hours later for genome copy number assessment with PCR. Results Circadain clock genes were rhythmic in airway epithelial cells from health and donors with asthma with preserved phase relationships indicating an intact core circadian clock. Analysis of circadian cyclic gene expression identified 4% of genes with circadian cyclic gene expression following temperature synchronization. Approximately 100 genes demonstrated altered circadian rhythmicity in airway epithelial cells from donors with asthma. Circadian rhythm and nuclear receptors had common rhythmicity in healthy and asthma. IL-17 signalling, cytokine receptor, and neutrophil chemotaxis pathways had altered circadian rhythmicity in asthma. Infection at time zero (end of temperature cycling) was associated with a two-fold lower viral replication than infection 12 hours later in healthy airway epithelial cells. Conclusion The core circadian clock genes maintain rhythmicity in healthy and asthma airway epithelia. Circadian regulation in immune and cytokine signaling pathways is altered in asthma. Support (if any) Sleep Research Society Foundation Career Development Award (WTP), ATS ASPIRE (WTP), Parker B Francis Fellowship (WTP), NIH R01AI163160 (JSD); NIH K24AI150991 (JSD)