Aberrant expression and association of TJs and epithelial cell markers in nasal biopsy specimens of CRSwNP patients.
First, we investigated the expression and localization of TJs proteins (ZO-1, occludin and Cldn3) and epithelial cell marker MUC5AC (goblet cell) and βIV-tubulin (ciliated cell) in control subjects and CRSwNP patients. The epithelium in CRSwNP patients was damage as shown by loss of cilia and abnormal epithelium remodeling (goblet cell hyperplasia) by IF staining (figure 1A-F, G-L, P and Q ). In control subjects, ZO-1 was consistently expressed and localized at superficial side of the nasal epithelium (figure 1A and G ) and occludin was localized at both superficially and suprabasally at the cell boundaries but a higher intensity at the superficial layer (figure 1C and I ). Cldn3 was found at the cell boundaries and at the superficial layer of nasal epithelium (figure 1E and K ). On the other hand, in CRSwNP patients, protein expression of ZO-1, occludin and Cldn3 was reduced (all P <.001; figure 1B, H, M; D, J, N andF, L, O ). Similarly, the mRNA expression levels of ZO-1 ,occludin and Cldn3 were reduced in CRSwNP patients as compared to the control subject (figure 1R-T ).
Despite higher MUC5AC protein expression in CRSwNP, its mRNA expression was lower in tissue of CRSwNP (P <.001; figure 1U ) while there was no significant change in mRNA expression ofFoxj1 (figure 1V ). Furthermore, we found that MUC5AC expression was negatively correlated with occludin (r =-0.374,P =.003; figure 2B ) and Cldn3 (r =-0.386,P =.002; figure 2C ) but not with ZO-1 (r =-0.128,P =.328; figure 2A ) in nasal biopsies. βIV-tubulin expression was positively correlated with ZO-1 (r =.529, P<.001; figure 2D ), occludin (r =.566, P<.001; figure 2E ) and Cldn3 (r =.622, P<.001; figure 2F ). Then, we examined the expression of TJs in ciliated and goblet cells by single cell staining of primary nasal cells. We found that both ZO-1 (figure 2G and J ) and occludin (figure 2H and K ) were co-stained with goblet and ciliated cell. Interestingly, Cldn3 staining was only observed in ciliated cell (figure 2L ) but not goblet cell (figure 2I ).