Intrinsic Differences Of Epithelial Tight Junctions In Asthmatic Airway Epithelium And Barrier Compromisation Following Human Rhinoviral Insult

Introduction: Junctional protein complexes such as tight junctions (TJ) provide a physical barrier against external insults. Althoughvarious aspects of TJs have been extensively assessed, few studies have directly addressed whether these are intrinsically different inasthmatic epithelium and the consequence on barrier function of disassembly post- human rhinovirus (HRV) infection. This study aimed toassess basal TJ expression and barrier function prior to and post-HRV infection in both monolayer and air-liquid interface (ALI) cultures ofhealthy and asthmatic epithelium. Methods: Primary airway epithelial cells from healthy and asthmatic children obtained via bronchial brushings and cultured as previouslydescribed (Kicic et al. 2006) were also differentiated into ALI and subsequently infected with HRV-1B over 24h. Tight junction geneexpression was assessed via qPCR, barrier integrity measured by TJ protein expression of claudin-1, occludin and zonula occluden-1 (ZO-1)via immunocytochemistry (ICC), in-cell western (ICW) and confocal microscopy while barrier function was assessed via transepithelialelectrical resistance (R ) measurement and a permeability assay. Results: Ex vivo TJ gene expression was significantly increased in asthmatic epithelium for claudin-1 and occludin, while ZO-1 was notsignificantly different when compared to healthy epithelium. In contrast, protein expression for claudin-1, occludin and ZO-1 observed viaICC was markedly reduced in asthmatics compared to healthy controls. Semi-quantitative assessment of claudin-1, occludin and ZO-1protein expression via ICW corroborated ICC findings and demonstrated significantly reduced basal membrane TJ expression inasthmatics compared to healthy controls (p<0.05). A greater effect on TJ disassembly was observed within the asthmatic epithelium postinfection which was concurrent with a marked increase in transepithelial permeability. Semi-quantitative assessment of occludin and ZO-1protein expression via confocal microscopy corroborated ICW findings and demonstrated reduced basal membrane TJ expression inasthmatic compared to healthy controls. A greater effect on TJ disassembly was observed within the ALI asthmatic epitheliumpost-infection and this was concurrent with a significant decrease in R and a marked increase in transepithelial permeability. Conclusion: This study demonstrates significant differences in basal membrane TJ protein expression between healthy and asthmaticepithelial cells, suggesting intrinsic differences between healthy and mild-asthmatic epithelium. Furthermore, post HRV infection, anexaggerated disassembly in the asthmatic epithelial cells, concomitant with increased transepithelial permeability suggests elevatedtrafficking of small sized aeroallergens into the sub-epithelial space, contributing to asthma exacerbations.