Figure legends
Figure 1.Aberrant expression of TJs and epithelial cell markers in nasal biopsy specimens of CRSwNP patients. Representative IF staining of ZO-1 (A-B, G-H ), occludin (C-D, I-J ), Cldn3 (E-F, K-L ) with MUC5AC (A-F ) and βIV-tubulin (G-L ) in nasal biopsies from control subjects and CRSwNP patients. TFI level of ZO-1, occludin, Cldn3 and βIV-tubulin were decreased in CRSwNP (M-Oand Q respectively) whereas MUC5AC TFI level was increased in CRSwNP patients (P ). Similarly, mRNA levels of occludinand Cldn3 were significantly decreased in CRSwNP as compared to control subjects(S-T ). There was no significant change in mRNA expression of ZO-1 and Foxj1 between two groups (R, V ). Despite higher protein expression of MUC5AC as shown by IF staining in CRSwNP, its mRNA level was lower in CRSwNP tissue (U ). Relative expression of the target gene was normalized to 2-ΔCT with GAPDH . Statistical analysis was calculated using the Mann-Whitney U test. Data were presented as median with an interquartile range. Scale bar=20 μm.
Figure 2. Correlation of TJs with goblet and ciliated cell in nasal biospecimen. TFI level of MUC5AC was not correlated with ZO-1 (A ) but was negatively correlated with occludin and Cldn3 (B-C ). βIV-tubulin TFI level was positively correlated with ZO-1, occludin and Cldn3 (D-F ) in CRSwNP and control subjects. IF staining of single primary nasal cells showed that both ZO-1 (G, J ) and occludin (H, K ) were co-stained with goblet and ciliated cell. Interestingly, Cldn3 positive staining was only observed in ciliated cell (L ), but not goblet cell (I ). Spearman r characteristic was performed for statistical analysis. Black dot refers to CRSwNP (n=40) and red dot refers to control subjects (n=20). Scale bar=10 μm.
Figure 3. IL-13 induces epithelial remodeling and disrupts epithelial barrier integrity in hNECs. Representative IF images showed that IL-13 treatment induced more MUC5AC-secreting goblet cells and loss of ciliated cells in hNECs (A-D ). Western blotting results indicated that the protein expression of occludin and Cldn3 but not ZO-1 were significantly decreased with IL-13 treatment (E-H ). Damaged TJs proteins were shown by irregular staining patterns of ZO-1, occludin and Cldn3 in IL-13-matured hNECs as compared to untreated controls (I-N ). Intercellular epithelium permeability of IL-13-matured hNECs increased as compared to untreated hNECs (O-P ). IL-13 treatment of hNECs during differentiation also significantly reduced TEER at Day21 of ALI culture (Q ). Two-way ANOVA was used to analyze differences between hNECs with and without IL-13 treatment. Data were presented as median with an interquartile range. Fold change was quantified with reference to untreated hNECs. Scale bar=20 μm. hNECs, n=7.
Figure 4. Regulation of TJs protein expression during hNECs differentiation. IL-13 decreased the mRNA expression ofZO-1 , occludin , Cldn3 and Foxj1 but increased MUC5AC mRNA level in IL-13-matured hNECs as compared to untreated group (A-E ). IF staining was performed for Cldn3, MUC5AC, and βIV-tubulin from Day3 to Day21 during hNECs differentiation (F-G ). Cldn3 was only detected when ciliated cells were first observed at Day11 and Cldn3 was co-stained with βIV-tubulin during hNECs differentiation. Cldn3 localization is weakly detectable at poorly ciliated areas of hNECs induced by IL-13 whereas Cldn3 localization was more widespread in regions with more fully ciliated cells in untreated hNECs. The relative target gene was normalized to 2-ΔCT with RPL13A as a housekeeping gene. Two-way ANOVA was used to analyzed differences between hNECs with and without IL-13 treatment. Data were presented as median with an interquartile range. Scale bar=20 μm. hNECs, n=9.
Figure 5. Effects of RV infection on TJs and innate immune response on IL-13-treated hNECs. The RV progeny production and viral RNA expression were significantly increased for both untreated and IL-13-treated hNECs. Extent of RV progeny production and viral RNA expression were significantly lower in IL-13-treated hNECs as compared to untreated hNECs (A-B ). RV infection induced trend of reduction of Cldn3 mRNA expression in both IL-13-treated and untreated hNECs (C ). RV infection only significantly increased the mRNA expression of MUC5AC in untreated but not in IL-13-treated hNECs. RV downregulated Foxj1 mRNA levels for both untreated and IL-13-treated hNECs. With IL-13 treatment, regulation of mRNA level of MUC5AC and Foxj1 were significantly higher and lower respectively than in untreated hNECs (D-E ). RV infection significantly increased the mRNA expression of RV receptorICAM-1 , pathogen recognition receptor TLR3 and antiviralIFN-λ1 and CXCL10 in both untreated and IL-13-treated hNECs. With IL-13 treatment, upregulation of mRNA level of CXCL10was significantly lower than in untreated hNECs (F-I ). RV infection significantly upregulated mRNA expression of IL-25 andIL-33 for untreated hNECs but not IL-13-treated hNECs (J-K ). RV significantly regulated the mRNA expression ofTSLP in both untreated and IL-13-treated hNECs (L ). IF staining showed that RV infection induced slight decrease in expression of Cldn3 in IL-13-treated hNECs while expression of MUC5AC and βIV-tubulin was respectively higher and lower in IL-13-treated hNECs with RV infection (M-O ).The relative target gene was normalized to 2-ΔCT with RPL13A as a housekeeping gene. Two-tailed unpaired t-test was used to analyzed differences between hNECs with and without IL-13 treatment. Data were presented as median with an interquartile range. Fold change was quantified with reference to untreated hNECs. Scale bar=20 μm. hNECs, n=6.
Figure E1. Expression of TJ proteins during hNECs differentiation. Positive staining of ZO-1 and occludin was observed at cell to cell contact sites even in early stage of differentiation. Localization pattern was not linear but fragmented at cell to cell boundaries after IL-13 treatment (A-D ). Scale bar=20 μm.
Figure E2. Effects of RV infection on TJs and innate immune response on IL-13-treated hNECs. ZO-1 and occludinmRNA levels were increased in both untreated and IL-13-treated hNECs. RV significantly regulated ZO-1 and occludin mRNA levels in both untreated and IL-13-treated hNECs (A-B ). There was no significant change in mRNA expression of IL-5 (C ) whileIL-4 mRNA expression was undetectable (data not shown). RV infection significantly increased the mRNA expression of IL-13 in untreated but not IL-13-treated hNECs. RV significantly upregulated mRNA expression of IL-13 for untreated hNECs but not IL-13-treated hNECs (D ). There was no significant change in mRNA expression of IL-17A (E ). Representative images of IF staining showed that RV infection in both untreated and IL-13-treated hNECs induced slight increase in expression of ZO-1 and occludin (F-G ). The relative target gene was normalized to 2-ΔCT with RPL13A as a housekeeping gene. Two-tailed unpaired t-test was used to analyzed differences between hNECs with and without IL-13 treatment. Data were presented as median with an interquartile range. Fold change was quantified with reference to untreated hNECs. Scale bar=20 μm. hNECs, n=6.