Table 1.
Primer Sequences.
Fig 1.
Cultured human nasal slices show unimpaired epithelium containing mucus-filled goblet cells.
A,B: Overview images of the established nasal slice culture system showing sliced nasal tissue cultured in culture plate inserts within a 12-well-plate. C,D: Immunohistochemical staining revealed the presence of acetyl-α-tubulin-positive cilia in nasal slice cultures. E: Hematoxylin and eosin-staining displayed the integrity of the ex vivo cultured epithelium containing ciliated epithelial cells (arrowheads), goblet cells (arrows) and a basal membrane (BM). Scale Bar: 20 μm. F, G: Mucin-filled goblet cells (arrows) were detected in cultivated nasal slices by Alcian Blue-staining and Periodic acid-Schiff stain. Scale Bar: 20 μm.
Fig 2.
Increased number of mucus-filled cells in LPS-treated nasal slice cultures is significantly reduced by co-treatment with 1,8-cineol.
A: Representative Alcian Blue-staining of an untreated nasal slice culture revealed no increased amount of mucus-filled goblet cells (arrows). B: Representative Alcian Blue-staining of LPS-treated nasal slices showed highly increased numbers of mucus-filled goblet cells (Arrows). C: Representative Alcian Blue-staining of cultured nasal slices co-treated with LPS and 1,8-cineol displayed a highly decreased number of mucus-filled goblet cells (Arrows). Scale Bar: 20 μm. D: Quantification of total areas of Alcian Blue-stained slice cultures from four independent donors revealed a significantly increased number of mucin-filled goblet cells in LPS-treated nasal slice cultures, which was significantly decreased after co-treatment with 1,8-cineol. *p < 0.5, **p < 0.01 were considered significant (t-test); ns: not significant (t-test).
Fig 3.
1,8-cineol-treamtent leads to significantly decreased levels of MUC gene expression after their LPS-dependent stimulation.
A: Real time PCR analyses of nasal slice culture depicted increased levels of MUC2 after LPS-treatment, which were significantly reduced in LPS- and 1,8-cineol-treated approaches. B: No significant changes in gene expression level of MUC5AC in nasal slice cultures after LPS- as well as LPS- and 1,8-cineol-treatment shown by real time PCR. C: Real time PCR analyses revealed decreased levels of MUC19 in nasal slice cultures co-treated with LPS- and 1,8-cineol in comparison to LPS-treated approaches. D: Real time PCR analyses showed decreased expression levels of TNFα in nasal slice cultures co-treated with LPS- and 1,8-cineol compared to LPS-treated approaches. *p < 0.5, **p < 0.01 were considered significant (t-test); ns: not significant (t-test). GAPDH: Glyceraldehyde 3-phosphate dehydrogenase.
Fig 4.
Nasal slice cultures exposed to 1,8-cineol show reduced activity of NF-κB.
A Immunocytochemistry of LPS-treated nasal slice cultures revealed nucleus localization of NF-κB-p65 (upper panels, arrows). Co-treatment with LPS and 1,8-cineol resulted in reduced amounts of nuclear NF-κB-p65 (lower panels, arrows) and localization of NF-κB-p65 in the cytoplasm (lower panels, arrowheads). Scale bar: 20μm. B: Quantification of immunocytochemical analysis showed significantly increased numbers of epithelial cells with cytoplasmic NF-κB-p65 after LPS and 1,8-cineol co-treatment in comparison to the LPS-approach, indicating a significantly reduced NF-κB-activity. ***p < 0.001 was considered significant (t-test). C: Schematic view of NF-κB activating MUC2 gene expression via binding to a κB-binding site in 5’ region of the MUC2 gene (31).
Fig 5.
Schematic view on nasal epithelium containing mucus-filled goblet cells during LPS-induced rhinosinusitis and after co-treatment with 1,8-cineol.
Co-treatment with LPS and 1,8-cineol leads to reduced production of mucin and decreased expression levels of mucin genes closely associated with attenuated NF-κB-activity.