Introduction
The normal sinonasal epithelium comprises of ciliated cell, goblet cell, club cell and basal cell1. Tight junctions (TJs), which is located at the most apical part of intercellular junction between these epithelial cells, serves as a physical barrier of nasal airway epithelium to protect it from the external environment2. The main components of TJs are occludin, claudins, junctional adhesion molecules and scaffold protein zonula occludens (ZO). These TJ proteins are the key structural proteins which maintain epithelial polarity, regulate pericellular permeability and participate in epithelial cell proliferation, differentiation and migration3,4. These functions play an important role in distinct tissue compartmentalization and homeostasis of nasal epithelium. Disruption of TJs may cause reduction in epithelial cohesion and integrity which may lead to a variety of pathological conditions. It has been demonstrated that TJ proteins are involved in the pathophysiology of chronic airway inflammatory disorders including chronic rhinosinusitis with nasal polyps (CRSwNP), allergic rhinitis and asthma5-7.
IL-13 is the key regulator in type-2 mediated inflammation in CRSwNP. IL-13 induces goblet cell hyperplasia, loss of cilia, inducible nitric oxide syntheses production and fibrosis to accelerate inflammation and promote remodeling8,9. The elevated levels of pro-inflammatory cytokines contributing to diseases pathophysiology and barrier dysfunctions have been implicated in a variety of tissues9-11. Studies have shown that IL-13 impaired barrier function by reducing expression of occludin, ZO-1 and β-catenin in primary bronchial epithelial cells of asthmatic patients12 and also disrupted intestinal barrier by upregulation of claudin-213. Additionally, it was found that with IL-4/IL-13 and IL-5 stimulation, E-cadherin and ZO-1 were downregulated in cultured epithelial cells of patients with allergic rhinitis14. Hence, these studies further support the hypothesis that change in expression and localization of TJs accounts for epithelial barrier dysfunctions in chronic inflammatory diseases.
In CRSwNP, persistent and prolonged mucosal inflammation is well characterized and is closely related to elevated type-2 cytokines. However, the role of these cytokines on impairing the nasal epithelial barrier is still unknown. On the other hand, human nasal epithelial cells (hNECs), being the primary entry point of most inhaled pathogens, are the key players in regulating inflammatory responses and are an important source of pro-inflammatory cytokines15. Among respiratory viruses, rhinovirus (RV) is most commonly associated with exacerbation of chronic airway disease16,17. It was found that RV infection damaged TJs integrity in airway epithelial cells of asthmatic patients by reducing ZO-1, occludin and claudin-1 protein expression7. In addition, while it is reported that RV caused transient barrier disruption in a model of normal air-liquid interface (ALI) differentiated airway epithelium, RV infection at initial stage of differentiation in an injury model prolonged barrier dysfunction by decreasing transepithelial electrical resistance (TEER) and occludin level18. Furthermore, recent study has shown that RV infection increased production of pro-inflammatory mediators and contributed to the exaggerated inflammatory response in in vitro cancer cell line19. While RV is reported highly prevalent in chronic rhinosinusitis patients20, the underlying mechanism of the association of airway disease with chronic inflammation and virus comorbidity is still poorly understood.
Our previously established in vitro IL-13-matured hNECs model using IL-13 stimulation closely mimicked the physiological condition and epithelium responses of in vivo nasal mucosa in CRSwNP21. Using this model, we investigate the direct effect of IL-13 on hNECs and TJ proteins expression in pseudostratified layers to analyze the nasal epithelial barrier functions. Moreover, to better define the effects of respiratory viruses on TJs of inflammatory airway model, we have also analyzed the nasal epithelial barrier integrity, remodeling and immune responses of IL-13-treated hNECs against RV acute infection.