Discussion
Our study demonstrates an impaired epithelial barrier function in CRSwNP patients along with decreased protein and mRNA expression of ZO-1, occludin and Cldn3. Long-term exposure of IL-13 altered hNECs composition in conjunction with nasal epithelial barrier dysfunction and enhanced mucosal inflammation, as well as reduced capacity for antiviral response against acute RV infection. These observations extend the knowledge of dysregulation of TJs in chronic airway diseases such as CRSwNP. Most importantly, ALI-cultured hNECs are shown to form functional barriers similar to that found in nasal biopsies. Using the IL-13-matured hNECs model, we have demonstrated that pro-inflammatory mediator IL-13 regulates the formation TJs and thereby disrupts the nasal epithelial barrier functions.
Nasal epithelial cells form a functional barrier which is mainly regulated by TJs. Recent studies have highlighted multiple defective TJs in patients with chronic airway diseases26 and reported that ZO-1, claudin-1, claudin-4 and occludin contributed to the leaky barrier of airway epithelium14,27,28. In our study, we found that the expression of Cldn3, similar to ZO-1 and occludin, is significantly lower in sinonasal tissues of CRSwNP as compared to healthy controls. Moreover, we noticed that occludin and Cldn3 protein expression level negatively correlates with MUC5AC expression and positively correlates with βIV-tubulin expression in nasal biopsies specimens. It appears that alteration of nasal epithelial homeostasis during inflammation may impair TJs integrity. For this purpose, we used ALI-cultured hNECs with IL-13 stimulation which mimics the remodeling of nasal epithelium during chronic inflammation to explore the formation and disruption of TJs and thereby the IL-13-induced barrier damage.
We demonstrated that long-term exposure of hNECs to IL-13 results in overproduction of mucus and cilia loss along with impaired epithelial barrier function which is evident by increased epithelium permeability and decreased TEER. ZO-1, occludin and Cldn3 structures were also disrupted by IL-13 treatment during hNECs differentiation. In addition to enhanced inflammation, defective barrier function allows foreign substance to infiltrate the sinonasal submucosa and causes aggravation of airway inflammation and remodeling. Hence, TJs play an important in the pathophysiology of chronic airway disease. Taken together, these data indicate that impaired formation of TJs protein ZO-1, occludin and Cldn3 is largely associated with barrier dysfunction.
Although previous studies have examined TJs expression following various cytokine insults, differentiated epithelial cell lines or primary airway epithelial cells were commonly used14. However, these cell models were not able to elucidate the processes of TJs formation during epithelium differentiation. Therefore, we investigate the effect of IL-13 on TJs protein formation during the time course of ALI cultivation. We found that positively stained ZO-1 and occludin were observed as early as Day7 and Day3 respectively at early stage of differentiation of hNECs with or without IL-13 treatment. Interestingly,Cldn3 gene expression only increased when ciliated cells were first observed at Day11 and is expressed only on ciliated cells of hNECs. Hence, the reduced levels of Cldn3 may be attributed to loss of cilia in airway epithelium of chronic inflammatory airway disease. Additionally, as key barrier function proteins, claudins serve as paracellular barrier29 and are shown to have differential tissue-specific expression patterns which account for the differences in paracellular tightness and ion selectivity11,30,31. Although Cldn3 is expressed in the airways, its role in airway barrier function has not been fully defined. Studies in lower airways have demonstrated that the expression level and function of Cldn3 are not comparable between type-I and type-II alveolar cells32-34. Therefore, consistent with study which reported varying TJ composition in different airway epithelial cell type, the differential expression of Cldn3 in our study implies that Cldn3 may be involved in regulation of epithelial barrier as well as in epithelial differentiation. However, involvement Cldn3 in ciliogenesis during differentiation is currently unknown. Thus, further studies will be needed to investigate how TJ composition and epithelial cell differentiation interrelate for regulation of barrier permeability within stratified epithelia.
As the nasal airway is the primary target site for most respiratory viral infections, impaired epithelial barrier could lead to greater susceptibility against viral infection and dysregulation of host innate immune responses35. RV is the most prevalent respiratory virus in CRSwNP patients and is the most commonly associated with exacerbation of chronic airway disease16,17,20. As RV infection may have significant implications in regulating the epithelial barrier function and mucosal inflammation of CRSwNP, we further investigate the effect of RV infection on TJs of IL-13-matured hNECs and the effects of IL-13 on the host responses of hNECs against RV infection. We found that the altered hNECs composition (cilia loss and mucus overproduction) in the presence of IL-13 is associated with the reduced RV replication (viral RNA level) and viral particle formation as compared to RV infection without IL-13 at the same initial infectious dose. As our previous study found that RV almost exclusively infected ciliated cells but not goblet and basal cells in in vitro hNECs24, cilia loss due to IL-13 may impede RV infection by reducing target cells for viral replication. However, despite lower viral replication and production, RV infection of IL-13-treated hNECs worsened the mucociliary function by further inducing loss of cilia as shown by reduction of Foxj1mRNA level and IF staining. While studies have reported that RV infection disrupted TJs in primary airway epithelial cells16,36, our data showed that RV infection induced minimal alteration to TJs proteins in hNECs with and without IL-13 treatment. In addition, we investigated the effects of IL-13 on innate immune responses of hNECs against RV infection. We found that viral entry receptor ICAM-1 , RV-induced host pathogen sensor (TLR3) and antiviral immune responses (IFN-λ1 and CXCL10) were upregulated in both untreated and IL-13-treated hNECs, suggesting that RV infection induced immune surveillance and antiviral responses even in inflammatory hNECs model. However, the capacity for interferon activation and chemokine signaling were impaired when hNECs is predisposed with IL-13 environment as shown by lower IFN-λ1 and CXCL10 expression as compared untreated hNECs. In contrast, RV-induced greater upregulation ofTSLP expression in IL-13-treated hNECs suggesting that RV infection in nasal epithelium predisposed with type-2 cytokine environment could lead to enhanced allergic inflammation which may further drive inflammation during RV-induced exacerbation of disease. Our current study investigated the effects of mild respiratory virus RV-16, which commonly associated to chronic respiratory disease exacerbations, on epithelium barrier function and immune capacity of hNECs in type-2 cytokine environment. While RV infection induced minimal change to TJs dysfunction, the impairment of efficient antiviral response in nasal epithelium may be attributed to IL-13-induced change in hNECs composition. More pathogenic respiratory viruses could be studied using this model to assess the epithelia barrier function and antiviral responses of chronic inflammatory airway disease.
In conclusion, IL-13, a typical type-2 cytokine, contributes to diminished barrier function and airway inflammation that are seen in CRSwNP patients. Knowledge about the dysregulation of TJs will help to better understand the pathophysiology of CRSwNP and define the specific mechanisms that link allergic inflammation and antiviral responses, which could lead to new strategies for the prevention and treatment of the disease.