1,8-Cineol Reduces Mucus-Production in a Novel Human Ex Vivo Model of Late Rhinosinusitis

Abstract

Inflammatory diseases of the respiratory system such as rhinosinusitis, chronic obstructive pulmonary disease, or bronchial asthma are strongly associated with overproduction and hypersecretion of mucus lining the epithelial airway surface. 1,8-cineol, the active ingredient of the pharmaceutical drug Soledum, is commonly applied for treating such inflammatory airway diseases. However, its potential effects on mucus overproduction still remain unclear.In the present study, we successfully established ex vivo cultures of human nasal turbinate slices to investigate the effects of 1,8-cineol on mucus hypersecretion in experimentally induced rhinosinusitis. The presence of acetyl-α-tubulin-positive cilia confirmed the integrity of the ex vivo cultured epithelium. Mucin-filled goblet cells were also detectable in nasal slice cultures, as revealed by Alcian Blue and Periodic acid-Schiff stainings. Treatment of nasal slice cultures with lipopolysaccharides mimicking bacterial infection as observed during late rhinosinusitis led to a significantly increased number of mucin-filled goblet cells. Notably, the number of mucin-filled goblet cells was found to be significantly decreased after co-treatment with 1,8-cineol. On a molecular level, real time PCR-analysis further showed 1,8-cineol to significantly reduce the expression levels of the mucin genes MUC2 and MUC19 in close association with significantly attenuated NF-κB-activity. In conclusion, we demonstrate for the first time a 1,8-cineol-dependent reduction of mucin-filled goblet cells and MUC2-gene expression associated with an attenuated NF-κB-activity in human nasal slice cultures. Our findings suggest that these effects partially account for the clinical benefits of 1,8-cineol-based therapy during rhinosinusitis. Therefore, topical application of 1,8-cineol may offer a novel therapeutic approach to reduce bacteria-induced mucus hypersecretion.

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.