IL-13 induces epithelial remodeling and disrupts epithelial barrier integrity in hNECs.
To analyze the direct effect of IL-13 on nasal epithelial barrier functions, IL-13 was added throughout hNECs differentiation in ALI culture. Cilia loss and mucus over-secretion were observed in IL-13-matured hNECs (all P =.016; figure 3A-D ). Similar to the trend observed in nasal biospecimen, the protein expression of occludin and Cldn3 but not ZO-1 were significantly decreased in hNECs with IL-13 treatment (all P =.016; figure 3E-H ). IL-13 also disrupted ZO-1, occludin and Cldn3 as shown by an irregular staining patterns as compared to untreated hNECs (figure 3I-N ). In addition, epithelium permeability assay using Sulfo-NHS-Biotin as a tracer revealed that IL-13 increased the paracellular permeability of hNECs (figure 3O-P ). Long-term exposure of hNECs to IL-13 resulted in a reduction of TEER (figure 3Q ).
IL-13 induced regulation of TJ genes during hNECs differentiation.
Next, we analyzed the role of IL-13 on regulation of the TJs formation during hNECs differentiation. Cldn3 mRNA expression level was significantly reduced from Day11 onwards while the ZO-1 andoccludin mRNA expression were significantly reduced only when hNECs were fully differentiated at Day21(figure 4A-C ). IL-13 upregulated and downregulated mRNA expression of MUC5AC andFoxj1 during hNECs differentiation respectively (figure 4D-E ).
Positive IF staining of Cldn3 was detected when ciliated cells were first observed at Day11 and was colocalized with βIV-tubulin-positively stained cells during differentiation of untreated hNECs, while Cldn3 staining was only detected from Day15 onwards in IL-13-matured hNECs (figure 4F-G ). In addition, there was lower expression of Cldn3 observed in IL-13-matured hNECs with less ciliated cells as compared to more widespread expression pattern of Cldn3 in untreated controls with more ciliated cells. On the other hand, positive stained ZO-1 and occludin were observed to localize at cell-to-cell contact sites as early as Day7 and Day3 at early stage of differentiation of hNECs while their localizations were non-linear and fragmented at cell-to-cell boundaries in IL-13-matured hNECs (figure E1A-D ).
IL-13 modulates the effects of RV infection in hNECs
To better understand the effect of RV on TJs of inflammatory airway model, we next examine the nasal epithelial barrier integrity, remodeling (cilia and goblet cells) and immune responses of IL-13-treated hNECs against high dose of acute RV infection. Firstly, we found that RV progeny production and viral RNA expression were significantly increased with or without IL-13 treatment. Interestingly, RV progeny production and viral RNA expression were significantly lower in IL-13-treated hNECs as compared to RV-infected hNECs without IL-13 (all P =.031; figure 5A-B ).
RV infection upregulated mRNA levels of ZO-1 and occludinbut IF staining showed only slight decrease with or without IL-13 treatment (figure E2A-B and F-G ). However, RV infection showed reducedCldn3 mRNA expression in both IL-13-treated and untreated hNECs, albeit not statistically significant (P =.219; figure 5C ). With IL-13 stimulation, RV infection further reduced Cldn3mRNA expression as compared to untreated group (P =.063;figure 5C ). Similar to ZO-1 and occludin, IF staining showed that RV infection induced slight decrease in expression of Cldn3 in both IL-13-treated and untreated hNECs (figure 5M ).
We also investigated the effect of RV infection on the cell type of hNECs. RV infection upregulated the mRNA expression of MUC5AC in untreated hNECs but not in IL-13-treated hNECs as compared to their respective mock-infected controls (all P =.031; figure 5D ). Interestingly, RV infection downregulated Foxj1 mRNA levels in both untreated and IL-13-treated hNECs (all P =.031;figure 5E ). Representative images of IF staining showed that RV infection increased expression of MUC5AC and reduced βIV-tubulin expression in IL-13-treated hNECs as compared to uninfected IL-13-treated hNECs while RV infection induced slight changes of expression in untreated group (figure 5N-O ).
We also examine the innate immune responses of IL-13-treated hNECs against acute RV infection. The mRNA expression of RV receptor ICAM-1 was reduced in both IL-13-treated and untreated hNECs as compared to the respective mock-infected controls (all P =.031; figure 5F ). With IL-13 stimulation, RV infection further reduced ICAM-1 mRNA expression as compared to infected hNECs without IL-13 (P =.094;figure 5F ). Similarly, RV pathogen recognition receptorTLR3 was reduced in both IL-13-treated and untreated hNECs as compared to the respective uninfected controls (all P =.031;figure 5G ). Additionally, RV infection increased the mRNA expression of antiviral type III IFN (IFN-λ1 ) and chemokineCXCL10 in both IL-13-treated and untreated hNECs (all P =.031;figure 5H-I ). However, IL-13-treated hNECs has reduced capacity for upregulation of antiviral responses against RV infection as compared to RV infection in untreated hNECs as showed by lower extent of mRNA upregulation for both IFN-λ1 and CXCL10 (P =.063 and .031; figure 5H-I ). Moreover, RV infection upregulated mRNA expression of IL-25 and IL-33 only in untreated hNECs but not in IL-13-treated group (P =.031; figure 5J-K ), whereas TSLP mRNA was upregulated in both IL-13-treated and untreated groups. (all P =.031; figure 5L ). With IL-13 stimulation, RV infection further increased TSLP mRNA expression as compared to untreated group (P =.063; figure 5L ). Similar to IL-25 and IL-33 , RV infection increased the mRNA expression of type-2 cytokine IL-13 in untreated but not IL-13-treated hNECs (P =.031; figure E2D ). There was no significant change in mRNA expression of IL-5 and IL-17A(figure E2C and E ) while IL-4 mRNA expression was undetectable (data not shown).