Azithromycin mitigates human rhinovirus impact on barrier integrity and function in non-diseased airway epithelium

Kevin Looi^1,2,3*Erika Sutanto^1,2,3Thomas Iosifidis^1,2,3,4Luke Berry¹Anthony Kicic^1,2,3,4Stephen Michael Stick⁵

• ¹Wal-yan Respiratory Research Centre, The Kids Research Institute Australia, Perth, Australia
• ²Curtin School of Population Health, Faculty of Health Sciences, Curtin University, Perth, Australia
• ³Centre for Child Health Research, Telethon Kids Institute, University of Western Australia, Crawley, Western Australia, Australia
• ⁴Centre for Cell Therapy and Regenerative Medicine, School of Medicine, University of Western Australia, Perth, Western Australia, Australia
• ⁵Department of Respiratory and Sleep Medicine, Perth Children's Hospital, Nedlands, Western Australia, Australia

Introduction: Azithromycin improves symptomology in various chronic airway diseases exacerbated by viral infections. However, the mechanisms underlying the apparent antiviral effects of azithromycin remain unclear.Methods: Airway epithelial cells (AECs) from healthy children were cultured, expanded and differentiated into air-liquid interface (ALI) cultures. Submerged and differentiated primary cultures were treated with 10 µM of AZM for 24h and subsequently infected with human rhinovirus (HRV)-1b for 24h. Virus receptor expression, replication, progeny release and inflammatory cytokines (IL-1β, - 6, -8 and IP-10) were then measured. Barrier integrity was determined via qPCR, in-cell western (ICW), immunofluorescence confocal microscopy, transepithelial electrical resistance (RT) measurement and an apparent permeability (Papp) assay.Results: Treatment with AZM for 24h at the concentrations of 0.1, 1 and 10 µM did not have any significant impact on either cellular viability or cytotoxicity in un-infected cells. No significant effect on viral receptor, cytokine expression was observed in non-infected cells treated with 10 µM AZM. Similarly, there was no significant change in both occludin and ZO-1 expression in non-infected cells. However, claudin-1 gene expression was significantly reduced but corresponding protein expression was significantly increased following 10 µM AZM. Although RT was significantly lower, this was not corroborated by any significant change in epithelial permeability after 10 µM AZM treatment. Subsequent to HRV-1b infection, 10 µM AZM treatment significantly reduced cytotoxicity induced by infection. Viral receptor expression were not affected with AZM pre-treatment but a significant decrease in viral replication was observed. Except for IP-10, expression of IL-1β, -6, and -8 was significantly reduced. Gene and protein expression of key epithelial junctions were significantly higher in treated, infected cells, which were concomitant with epithelial barrier function.Discussions: This study identified that AZM can protect against HRV-1b-induced epithelial damage. Our data, demonstrating the antiviral, anti-inflammatory, and barrier-protective effects in vitro are strongly indicative of pleiotropic mechanisms of AZM for mitigating viral infection and its consequences. These effects are likely to contribute to the benefits observed in clinical trials of AZM in a number of chronic respiratory diseases.