Human nasal turbinate specimens (middle and inferior turbinates) were obtained from patients during minimal-invasive surgery after informed written consent and according to the principles of the Declaration of Helsinki (1964) and local and international guidelines (Bezirksregierung Detmold/Münster). Fifty percent of the patients were pre-treated with decongestant spray containing Xylometazolin (Nasic, pharmacological half-life of 12h, Klosterfrau Healthcare Group) prior to obtaining nasal inferior turbinate tissue. Isolation and further experimental procedures were ethically approved by the ethics commission of the Ärztekammer Westfalen-Lippe and the medical faculty of the Westfälische Wilhems-Universität (Münster, Germany) (approval reference number 2012-15-fS).

Specimens obtained during surgery were directly placed on ice in Dulbecco modified Eagle medium (PAA, Pasching, Austria) supplemented with penicillin and streptomycin. For tissue culture, specimens were sliced (200μm thickness) using McIlwain tissue chopper (Ted Pella, Inc., Redding, CA) before being transferred to culture plate inserts comprising a 0.4 μm nitrocellulose membrane (Millipore/Greiner, Billerica, MA, USA). Slice-containing membranes were placed at the interface of air and B-ALI differentiation medium (Lonza, Basel, Switzerland) within respective cell culture well-plates (TPP Techno Plastic Products, Trasadingen, Austria) and DMEM High Glucose (Biochrom, Berlin, Germany) followed by culture in a humidified incubator (Binder, Tuttlingen, Germany) at 37°C and 5% CO₂. Nasal slices were cultured for up to 4 weeks and fed every two days by replacing the medium with fresh B-ALI differentiation medium.

Human nasal slices cultivated for 7 days were treated with LPS (100 ng/ml, rough strains from Salmonella enterica Re 595, cat. no. L9764, Sigma-Adrich St. Louis, MO, USA) or LPS and 1,8-Cineol (10⁻⁵ M, Klosterfrau Healthcare Group, Cassella-med GmbH & Co. KG, Cologne, Germany as described in [24] for 60 minutes followed by respective stainings or real time PCR.

Nasal slice cultures were fixed using 4% formalin, treated with water for 60 minutes and dehydrated and embedded by applying 50% EtOH, 75% EtOH, 95% EtOH, 100% EtOH, Xylol and Xylol:Paraplast (1:1, Leica Biosystems Nussloch GmbH, Nussloch, Germany) for 60 minutes each followed by application of Paraplast over night. Afterwards, 2 μm thick sections were prepared. Prior to immunohistochemistry, sections were deparaffinized in Xylene and afterwards rehydrated in 100% EtOH for 3 minutes, 95% EtOH for 3 minutes and 70% EtOH for 3 minutes followed by application of distilled water.

Alternatively, 10 μm thick cryostat sections were prepared from nasal slices cultures and inferior turbinate tissue followed by fixation using 4% paraformaldehyde for 20 min at RT prior to immunohistochemistry.

Blocking was performed using 5% goat serum for 30 minutes followed by incubation with primary rabbit anti-acetyl-α-tubulin antibody (1:800, Cell Signaling Technology, Danvers, MA, USA), anti-Caspase 3 antibody (1:500, Cell Signaling) or anti-p65 antibody (Santa Cruz Biotechnology, Heidelberg, Germany) for 2 hours at RT. Secondary fluorochrome-conjugated antibody (goat anti rabbit conjugated with Alexa 555, 1:300; Molecular Probes, Göttingen, Germany) applied for 1 hour at room temperature (RT). Nuclear counterstaining was performed using SYTOX green (1:20000, Invitrogen, Carlsbad, CA, USA) for 30 minutes at RT. Fluorescence imaging was performed using confocal laser scanning microscopy (LSM 510, Carl Zeiss, and DM IRB, Leica, Wetzlar, Germany).

Cultured nasal slices were embedded in paraffin and sectioned as described above. For Hematoxylin and Eosin (HE) staining sections were deparaffinized in Xylene and afterwards rehydrated in 100% EtOH for 3 minutes, 95% EtOH for 3 minutes and 70% EtOH for 3 minutes. After rinsing the sections in distilled water, Mayer’s alum haematoxylin was applied for 5 minutes and rinsed in running tap water. Afterwards, sections were stained for 2 minutes using 2% Eosin solution and immersed once in water. Sections were dehydrated by rinsing in 70% Ethanol for 3 minutes, 90% Ethanol for 3 minutes and two times 100% Ethanol for 3 minutes each. Finally, the slides were cleared using Xylene and mounted in Entellan.

For Alcian Blue staining sections were deparaffinized and rehydrated as described above. Rehydrated sections were stained for 8 minutes in Alcian blue solution (1g Alcian blue, 100 ml 3% acetic acid, pH 2.5) and rinsed in running tap water for 2 minutes. Afterwards, 0.1% Nuclear fast red solution (0.1g nuclear fast red, 5g Aluminum sulfate, 100ml distilled water) was applied for 10 minutes followed by rinsing in running water for 3 minutes. Sections were dehydrated, cleared and finally mounted in Entellan as described above.

Sections were deparaffinized and hydrated to water. 0.5% periodic acid solution was applied for 5 minutes. After rinsed in distilled water Schiff reagent was placed for 15 minutes. Washing in lukewarm tap water for 5 minutes was followed by a counterstaining in Mayer's hematoxylin for 1 minute. Again the sections were washed in tap water for 5 minutes, followed by dehydration and embedding using a synthetic mounting medium.

Total RNA was isolated from tissue cultures of three donors after respective LPS- or LPS and 1,8-cineol treatment using the MasterPure RNA Purification Kit (Biozym Scientific GmbH, Hessisch Oldendorf, Germany) followed by cDNA synthesis by First Strand cDNA Synthesis Kit (Thermofisher, St. Leon-Rot, Germany) according to the manufacturer’s guidelines. qPCR reactions were performed in technical triplicates using myBudget 5x EvaGreen QPCR-Mix II (ROX) (BioBudget Technologies GmbH, Krefeld, Germany) according to the manufacturer’s guidelines and assayed with a ABI PRISM 7000 Sequence detection system (Thermo Fisher Scientific, Bonn, Germany). For primer sequences see Table 1.

To investigate potential effects of 1,8-cineol on mucus hypersecretion, we applied ex vivo cultures of sliced human nasal turbinate tissue (Fig 1A and 1B). Determining the integrity of the cultured nasal tissue, immunohistochemical stainings revealed the presence of acetyl-α-tubulin-positive cilia in nasal slices cultured for 32 days (Fig 1C and 1D). Hematoxylin and eosin-stained ciliated epithelial cells forming an intact ciliated surface (Fig 1E, arrowheads), goblet cells (Fig 1E, arrows) as well as the presence of a basal membrane (Fig 1E, BM) further confirmed the integrity of the ex vivo cultured epithelium [25, 26]. In addition, cultured nasal tissue showed no changes in Caspase 3-expression compared to inferior turbinate tissue, suggesting unaffected viability of the cultured nasal tissue (S1 Fig). The presence of goblet cells was further investigated using Alcian Blue-staining and Periodic acid-Schiff stain detecting mucins [27]. As depicted in Fig 1E–1G, mucin-filled goblet cells were visible in cultivated nasal slices (arrowheads), suggesting the applicability of the here established culture system to investigate potential effects of 1,8-cineol on mucus overproduction.

Simulating the presence of a bacterial cell wall, we treated cultivated nasal slices with LPS to determine potential effects of 1,8-cineol on mucin overproduction during experimentally induced late rhinosinusitis [28]. Here, Alcian Blue-staining revealed a highly increased number of mucin-filled goblet cells in cultured tissue after LPS-treatment (Fig 2B, arrows) in contrast to the untreated control approach (Fig 2A, arrows). Notably, the number of mucin-filled goblet cells was decreased in cultured nasal slices treated with LPS and 1,8-cineol (Fig 2C, arrows). Determining these effects in more detail, quantification of mucus-filled goblet cells revealed a significantly increased number of mucin-filled cells after LPS-treatment, which was significantly decreased after co-treatment with 1,8-cineol (Fig 2D). In addition, cultured nasal slices co-treated with LPS and 1,8-cineol showed unchanged low numbers of Caspase 3-expressing cells compared to untreated and LPS-treated cultures as well as to inferior turbinate tissue, suggesting unaffected viability of the cultured nasal tissue (S1 Fig).

Investigating the effects of 1,8-cineol on mucus overproduction on a molecular level, we examined LPS as well as LPS- and 1,8-cineol-treated nasal slice cultures with real time PCR analysis to determine levels of mucin gene expression. Here, co-treatment of nasal tissue with LPS and 1,8-cineol was significantly reduce the expression level of the mucin gene MUC2 in comparison to LPS-stimulated samples (Fig 3A). However, the expression levels of the mucin genes MUC5AC and MUC5B were not significantly reduced in LPS-treated nasal slice cultures after co-treatment with 1,8-cineol (Fig 3B, data not shown). Interestingly, the expression level of MUC19 was also shown to be significantly reduced in slice cultures treated with LPS and 1,8-cineol in comparison to the LPS-treated approach (Fig 3C).

The transcription factor NF-κB is an essential regulatory factor of MUC gene-expression [29, 30]. Therefore, its activity was determined in ex vivo cultured nasal slices exposed to LPS. Nasal slice cultures co-treated with LPS and 1,8-cineol exhibited significantly reduced expression levels of the NF-κB-target gene TNFα in comparison to its LPS-dependent stimulation (Fig 3D). Immunocytochemistry of LPS-treated nasal slice cultures revealed localization of NF-κB-p65 within the nucleus, further indicating NF-κB-activity (Fig 4A upper panels, arrows). Co-treatment with LPS and 1,8-cineol resulted in reduced amounts of nuclear NF-κB-p65 (Fig 4A, lower panels, arrows) and localization of NF-κB-p65 in the cytoplasm (Fig 4A, lower panels, arrowheads). Quantification of immunocytochemical analysis showed significantly increased numbers of epithelial cells with cytoplasmic NF-κB-p65 in response to 1,8-cineol co-treatment in comparison to the LPS-approach (Fig 4B). The significantly reduced NF-κB-activity is suggested to result in the observed decrease in MUC2-gene expression levels (Fig 4C)[31].