Phenotypical changes in a differentiating immortalized bronchial epithelial cell line after exposure to mainstream cigarette smoke and e-cigarette vapor

Experimental and Toxicologic Pathology xxx (xxxx) xxx–xxx

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Phenotypical changes in a differentiating immortalized bronchial epithelial cell line after exposure to mainstream cigarette smoke and e-cigarette vapor ⁎

Michaela Aufderheide , Makito Emura Cultex Laboratories GmbH, Feodor-Lynen-Str. 21, 30625 Hannover, Germany

A R T I C L E I N F O

A B S T R A C T

Keywords: Air-liquid interface (ALI) exposure CULTEX®RFS Immortalized normal human bronchial epithelial cells Phenotypical alterations Metaplastic changes

3D constructs composed of differentiated immortalized primary normal human bronchial epithelial (NHBE) cells (CL-1548) were repeatedly exposed at the air-liquid interface to non-lethal concentrations of mainstream cigarette smoke (4 cigarettes a day, 5 days/week, 8 repetitions in total) and e-cigarette vapor (50 puffs a day, 5 days/week, 8 repetitions in total) to build up a permanent burden on the cells. Samples were taken after 4, 6 and 8 times of repeated smoke exposure and the cultures were investigated using histopathological methods Compared to the clean air-exposed cultures (process control) and incubator control, the aerosol-exposed cultures showed a reduction of ciliated, mucus-producing and club cells. At the end of the exposure phase, we even found metaplastic areas positive for CK13 antibody in the cultures exposed to mainstream cigarette smoke and e-liquid vapor, commonly seen in squamous cells as a marker for non-cornified squamous epithelium. The control cultures (incubator cells) showed no comparable phenotypical changes. In conclusion, our in vitro model presents a valuable tool to study the induction of phenotypical changes after exposure to hazardous airborne material.

1. Introduction When e-cigarettes were introduced to the market in 2004, they were considered to be useful in two ways. One was that they could present a safer alternative to conventional cigarettes. The other was they could also provide a gateway for smokers to quit tobacco smoking. However, both expectations have not yet been satisfactorily met (Grana et al., 2014; Kaisar et al., 2016). For more than 50 years, there has been continuous cautioning against cigarette smoking as the cause of various chronic lung diseases, such as asthma, chronic obstructive pulmonary disease (COPD), bronchiolitis obliterans (BO), and various types of lung cancers (US Department of Health and Human Services, 2014). It has been generally accepted that these respiratory diseases tend to originate in specific regions of the airway. For example, COPD and BO occur in the distal small airways devoid of surrounding cartilage, whilst asthma occurs in the proximal large airways surrounded by cartilage (Emura et al., 2015). As to the lung cancers, we also see a similar region-specific vulnerability; squamous cell carcinomas, for instance, preferentially in the proximal large airways and adenocarcinomas in the distal small airways and terminal and intrapulmonary bronchioles (Travis et al., 2015; Emura and Aufderheide 2016). For some time, we have been interested in developing in vitro 3D cell culture models which will enable us to investigate specific biomarkers

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of COPD, BO and emphysema (Aufderheide et al., 2015; Rach et al., 2014; Scheffler et al., 2015a,b; Emura et al., 2015), and those of preneoplastic lesions (Emura and Aufderheide, 2016). For this purpose, we have used human distal small airway (bronchial) epithelial cells either in primary cultures or after immortalization with human telomerase reverse transcriptase (hTERT) (Ramirez et al., 2004; Delgado et al., 2011). In a model using primary cultures, we have been able to induce lesions resembling squamous cell metaplasia expressing cytokeratin 13 (CK13), one of the biomarkers of COPD, and severe ciliary toxicity as the result of repeated air-liquid interface exposure to cigarette smoke (Aufderheide et al., 2017, in press). As mentioned above, the future fate of e-cigarettes is still inconclusive or rather obscure. We have thus decided to study whether e-cigarettes exert effects equivalent to those exerted by conventional cigarettes in a similar cell model. Our short-term exposure studies have shown certain toxic effects (Scheffler et al., 2015a). The report being presented here confirms the previous observations and adds a more extensive range of toxicity-related findings. 2. Material & methods 2.1. Cells Normal human bronchial epithelial (NHBE) cells were isolated from

Corresponding author. E-mail address: [email protected] (M. Aufderheide).

http://dx.doi.org/10.1016/j.etp.2017.03.004 Received 9 March 2017; Accepted 21 March 2017 0940-2993/ © 2017 Published by Elsevier GmbH.

Please cite this article as: Aufderheide, M., Experimental and Toxicologic Pathology (2017), http://dx.doi.org/10.1016/j.etp.2017.03.004

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2.3. Histology

bronchus samples of a 75-year-old male patient with non-small cell lung cancer (NSCLC) (Scheffler et al., 2015a). Cells in the second passage were transduced with the third generation state-of-the-art lentiviral constructs containing cyclin-dependent kinase 4 (CDK4) and human telomerase reverse transcriptase (hTERT). This immortalized cell line CL-1548 is characterized by an in vivo-like mucociliary differentiation. The differentiation capacity of the cells and the presence of basal as well as progenitor cells recommend this cell line for repeated exposure studies to study phenotypical changes in the cell population after exposure to cigarette aerosols. CL-1548 cells, cultivated in regular flasks for expansion (80–90% confluence) using AEGM medium incl. supplements, G418 (50 g/mL) and Puromycin (0.3 μg/mL), were seeded on collagen IV coated inserts (seeding density: 1–1.5 × 105/cm2). The cells were cultivated under submerged conditions (37 °C and 5% CO2) and supplied with AEGM medium until reaching 100% confluence (4 days). Then, the apical medium was removed and the basal medium was replaced by differentiation medium (PneumaCult™-Maintenance, STEMCELL Technologies SARL, Köln, Germany). After 21 days of cultivation at the air-liquid interface, the exposure period started and the cells were transferred to the CULTEX® RFS module (age of the culture: 24 days). In total, the cells were cultivated for 35 days at the air-liquid interface, including an exposure phase with 8 smoke repetitions for the exposure groups (clean air and test aerosol). PBS, penicillin/streptomycin, DMEM from Biochrom (Cambridge, UK) and AEGM medium from Promocell (Heidelberg, Germany). All other cell culture reagents were purchased from Sigma Aldrich (St. Louis, MO, USA).

For histopathological staining, the exposed cell culture inserts were removed from the CULTEX®RFS Compact module as well as 3 inserts of the control cultures (process and incubator control), post-incubated for 24 h in differentiation medium (air-lifted) and subsequently fixed with 10% formalin for 1 h. Following the fixation, the membranes were washed twice with water, cut out of the inserts with a CULTEX® Insertomat (Cultex Laboratories GmbH, Germany) and embedded in paraffin. Using a microtome, sections of 5 μm thickness were prepared, beginning at the middle of the membrane. After deparaffinization, sections were stained with hematoxylin and eosin. Cell composition of the 3D construct was analyzed by immunohistochemical characterization of special cell types within the culture. Antibodies used for immunohistochemical staining of the sections were anti-p63 monoclonal (Abcam, Cambridge, MA, USA), antiMUC5AC and −MUC5 B as well as anti-CC10 (Santa Cruz Biotechnology Inc., Dallas, TX, USA). In addition, the anti-CK13 antibody from Abcam (USA) was also used to characterize the cellular phenotype of the 3D constructs of the different experimental groups. Prior to immunohistochemical staining, Heat-Induced antigen (Epitope) Retrieval (HIER) was performed to break cross-links often caused by formaldehyde fixation, which mask the antigenic sites of the proteins (antibody binding sites). 2.4. Immunohistochemical staining The NovoLink™ Polymer Detection System provided by Leica Microsystems (Milton Keynes, UK), was used for immunohistochemistry following the manufacturer’s protocol. This kit was specifically designed for immunostaining analyses on formalin-fixed, paraffin-embedded histological sections. In order to increase the throughput, the Shandon Coverplate™ Technology (Thermo Fisher Scientific, Schwerte, Germany) was used for immunohistochemical analyses. Here, the microscope slides are placed into the plastic cover plates, leaving only a small gap between the slides and the cover plate housing. This gap is sequentially filled with the reagents and buffers used for the immunohistochemical procedure. The liquid stays in the gap by capillary attraction until it is replaced by the following reagent. The cover plates are placed into slide racks, each holding 10 slides. The Shandon Coverplate™ Technology (Thermo Fisher Scientific, Germany) saves reagents and protects the histological sections during the staining procedure. The histological samples were analyzed microscopically (Axiophot, Carl Zeiss, Oberkochen, Germany) for phenotypical alterations, which were documented via a microscopic camera (Axiocam, Carl Zeiss, Germany).

2.2. Exposure at the air-liquid interface After 21 days of cultivation at the air-liquid interface, the cells were exposed repeatedly (daily for five days and after a recovery phase of two days again on three subsequent days, maximum exposure cycle: 8 smoke exposure repetitions) to clean air, mainstream cigarette smoke (4x K3R4F cigarettes per run according to ISO 3308, University of Kentucky, Lexington, KY, USA) and e-liquid vapor without nicotine (Tennessee Cured, Johnsons Creek, Hartland, WI, USA) using the CULTEX® RFS – Compact System (Cultex Laboratories GmbH, Germany). The exposure module is characterized by a radial flow distribution of the test atmosphere from one sampling point resulting in a homogeneous, stable and reproducible distribution and deposition of the airborne material on the surface of the cultivated cells. Two systems were operated in parallel in a clean bench, whereby one acted as a process control (cell culture inserts were exposed to clean air) (DIN 12021) and the other housed the smoke-exposed cell culture inserts. The K3R4F cigarettes were smoked by a smoking robot and operated as follows: 24 puffs with a volume of 35 mL in 2 s, a blowout time of 7 s and an inter-puff interval of 10s. The electronic cigarette type InSmoke Reevo Mini (InSmoke Shop, Switzerland) was handled in a comparable manner: 50 puffs (volume 35 mL, puff duration 2 seconds, blow-out time of 7 seconds) with an inter-puff interval of 10 s. The freshly generated mainstream smoke or vapor was diluted with synthetic air (1 L/min) and sucked into the module at a rate of 5 mL/ min/insert via a vacuum pump. The flow rate through the system (1 L/ min) as well as the flow above the cells (5 mL/min) was controlled by mass flow controllers (IQ+FLOW® and EL-FLOW®Select, Bronkhorst, Ruurlo, Netherlands). The exhaust air was led back to the fume hood. During the exposure period, samples were taken after 0, 4, 6 and 8 smoke exposure repetitions and analyzed microscopically after histopathological preparation of the cultures. As a reference control, cells cultivated air-lifted in the incubator were also analyzed (incubator control).

3. Results Throughout the whole exposure experiment, the incubator control (IC) cultures of NHBE CL-1548 cells showed a pronounced mucociliary differentiation. Cilia-bearing as well as mucus-secreting cells were distributed homogeneously within the culture (Fig. 1IC). The exposure of the cultures to the different test aerosols (8 exposure repetitions) resulted in phenotypical changes within the cell population (Fig. 1). Cultures exposed to mainstream cigarette smoke (CS) showed a clear reduction in mucus production and cilia bearing. A comparable, but less pronounced effect was observed for the cells treated with the e-liquid aerosol (EC). Figs. 2 and 3 especially show changes in the population of mucusproducing cells (immunohistochemical staining of MUC5AC and MUC5B) after 8 exposure repetitions. Cultures exposed to mainstream cigarette smoke (CS) and e-cigarette vapor (EC) exhibited a clear reduction in mucus-secreting cells and their secretion activity, whereby the effect was less pronounced for the cells treated with the e-liquid 2

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Fig. 1. Cross-section of cell culture insert membranes with HE (Hematoxylin and Eosin) stained immortalized NHBE cells (CL-1548) after 8 exposures to cigarette smoke (CS) and ecigarette vapor (EC). Non-exposed cells were used as an incubator control (IC).

cell layers. The exposure of the cultures to mainstream cigarette smoke also damaged this type of luminal cells and after 8 smoke repetitions the population was reduced significantly. A less pronounced effect was observed for the cells exposed to e-cigarette vapor (Fig. 4). The immunohistochemical staining of the basal cells to anti-p63

aerosol. A comparable effect could be observed for cells reacting positively with the anti-CC-10 antibody (Fig. 4). The unexposed 3D cultures included a large number of cells secreting club cell secretory protein (CCSP). These cells could be found mainly in the luminal and middle

Fig. 2. Cross-section of cell culture insert membranes with immunohistochemically (MUC5AC) stained immortalized NHBE cells (CL-1548) after 8 exposures to cigarette smoke (CS) and e-cigarette vapor (EC). Non-exposed cells were used as an incubator control (IC).

3

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Fig. 3. Cross-section of cell culture insert membranes with immunohistochemically (MUC5B) stained immortalized NHBE cells (CL-1548) after 8 exposures to cigarette smoke (CS) and ecigarette vapor (EC). Non-exposed cells were used as an incubator control (IC).

important to note here that, in this particular 3D ALI cell culture model without any co-cultured cell types other than epithelial cells, the exposure to both test atmospheres reduced the length of cilia and the frequency of ciliated cells, suppressed the frequency of cells expressing CC10, Muc5AC and Muc5B and induced the expression of CK13. The presence of p63-expressing cells was scarcely affected. On these bases, the effects of e-cigarette vapor and CS on the different cell types were compared after treatment of the immortalized cell line using the described repeated exposure regimen. In order to classify the changes, a brief overview of the known effects in vivo and in vitro is given by considering the different cell types of the bronchial epithelium.

antibody exhibited no fundamental changes in this part of the cell population (Fig. 5). The control cultures are characterized by positively marked cells showing a bead-like arrangement in the basal cell layer. This picture did not change during the exposure period to mainstream cigarette smoke. Within the e-vapor-exposed cultures, p63-positive cells also concentrated in the basal cell layers, although the number of cells seemed to be increased. The results of the investigation of the cultures for the induction of CK13-positive cells are shown in Fig. 6. In the incubator control cultures, no CK13 marked cells could be observed, whereas the aerosol-exposed cell populations exhibited positive cells. With regard to mainstream smoke, these cells were found in focal clusters scattered within the 3D culture. In the case of e-vapor, the cells are arranged in strips in the middle layers of the cultures (Fig. 6EC). The histopathological analysis of the exposure groups showed that mainstream cigarette smoke as well as e-cigarette vapor induced significant changes in the epithelial phenotype of the 3D cultures after repeated exposure to non-lethal doses. The cilia-bearing cell populations, interacting with the test aerosol, are affected, as demonstrated by a loss or shortening of the cilia. The luminal mucus or club cell secretory protein-secreting cells were reduced, pointing to additive effects of the persistent aerosol exposure. A marker for the sustainability of the effects was the induction of metaplastic cells within the 3D culture.

4.1. Ciliated cells A recent study shows that, independent of the sex, age, race and method of sample preparation, the ciliary length measured in endobronchial biopsy specimens (large airway epithelium) is statistically significantly shorter in healthy smokers than in healthy non-smokers (Leopold et al., 2009). Also, certain cilia-related genes are downregulated in smokers. The epithelial cells obtained from the human lower trachea and upper bronchi cultured at the ALI were exposed to the whole CS (1 cigarette/3 times) and 48 h later samples were collected for the evaluation of CS exposure. The number of ciliated cells, as determined by immunocytochemical positive reaction to a transcription factor Foxj1, was found to be reduced to approximately 30% of the unexposed control and the presence of Gefitinib, an EGFR tyrosine kinase inhibitor in the culture medium, clearly protected this reduction (Valencia-Gattas et al., 2016). In a similar 3D ALI culture system using human proximal bronchial epithelial cells, exposure to the whole CS (Ishikawa and Ito, 2017) or cigarette smoke extract (CSE) (Brekman et al., 2014;

4. Discussion The immortalized cell line (NHBE-CL1548) used in the present experiments showed a reasonably comparable response to the test materials, cigarette smoke (CS) and E-cigarette vapor, in terms of the morphological markers examined, such as CK13, ciliary toxicity, CC10 (CCSP: club cell secretory protein), Muc5AC, Muc5B and p63. It is 4

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Fig. 4. Cross-section of cell culture insert membranes with immunohistochemically (CC-10) stained immortalized NHBE cells (CL-1548) after 8 exposures to cigarette smoke (CS) and ecigarette vapor (EC). Non-exposed cells were used as an incubator control (IC).

patients compared with normal control subjects (Van Vyve et al., 1995; Guerra et al., 2016). Immunofluorescence intensity expressed by the staining with CC16 antibody was significantly reduced in large airway tissue sections of smokers (p < 0.05) and COPD patients (p < 0.05) (Laucho-Contreras et al., 2015). The above data from the asthma and COPD patients, smokers and non-smokers are worth taking into consideration, because the diseases have been regarded as being primarily associated with tobacco consumption (US Department of Health and Human Services, 2014). This is also true for the following description of other data concerning MUC5AC, MUC5B and p63 derived from human subjects, including asthma and COPD patients. An in vitro study using 3D ALI cultures of human large airway epithelial cells reported significantly (p < 0.01) increased numbers of CC10-positive (club cell marker) cells compared to the untreated control after 28 days of continuous treatment with 5% cigarette smoke extract (CSE), although the expression level of CC10 mRNA in the treated cultures was not significantly increased (Schamberger et al., 2015). In contrast to this study, similarly designed 3D ALI cultures of human large airway epithelial cells showed significantly (p < 0.001) reduced expression levels of SCGB1A1 (an alias of CC10) after continuous treatment with 3 or 6% CSE between 5 and 28 days of culture (Brekman et al., 2014). Similar results, namely significant (p < 0.05) reduction of SCGB1A1, were obtained by yet another study in which human large airway epithelial cells were cultured at 3D ALI in the presence of fibroblasts and after exposure to the whole CS (4 cigarettes/exposure/day; see also Aufderheide et al., 2015) every other day cultured up to the total of 21 days (Ishikawa and Ito, 2017).

Schamberger et al., 2015) interfered with the growth of the cilia themselves or differentiation (regeneration) of ciliated cells. Interestingly, overexpression of Foxj1, a cilia-related transcription factor, prevented the growth inhibition of cilia caused by CSE (Brekman et al., 2014). Epithelial cells obtained from normal human distal small airways were repeatedly exposed to the whole CS (4 cigarettes/ exposure/day) and samples were collected after 4, 6 and 8 exposures to measure the ciliary length. After 4 exposures to CS, the cilia were less than 3 μm long, while the control showed about 5 μm long cilia and after 6 and 8 exposures the cilia were hardly measurable on the cultured cells (Aufderheide et al., 2015). However, when the cigarettes were equipped with a charcoal filter, the average ciliary length was approximately 3.5 μm after 4 times of exposure, and even more than 4 μm after 6 and 8 exposures. 4.2. CC10 (CC16, CCSP, SCGB1A1) In a study on surgically resected specimens, the frequency of club (Clara) cells was found to be significantly (p < 0.01) lower in the bronchioles and terminal as well as respiratory bronchioles of cigarette smokers than in those of non-smokers (Lumsden et al., 1984). In the small airways of patients with asthma, the average number of cells positive for club cell secretory protein (CC10, CC16 or CCSP) antibody, calculated as percent to total epithelial cells, was significantly reduced (p = 0.0012) in comparison to the control subjects (Shijubo et al., 1999). Confocal immunofluorescence microscopy of small airway epithelial sections stained with a CC16 antibody revealed significantly (p < 0.05) reduced numbers of club cells in COPD patients compared to normal subjects (Zhu et al., 2015). Statistically significantly lower levels of CC16 were found in bronchoalveolar lavage fluid of asthma 5

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Fig. 5. Cross-section of cell culture insert membranes with immunohistochemically (p63) stained immortalized NHBE cells (CL-1548) after 8 exposures to cigarette smoke (CS) and ecigarette vapor (EC). Non-exposed cells were used as an incubator control (IC).

(Schamberger et al., 2015). In a similar culture model, but in this case in the presence of co-cultured human fibroblasts, the expression of MUC5AC was significantly (p < 0.05) high compared to the unexposed control, after whole CS exposure (2 cigarettes/exposure/day) at 2-day intervals up to 21 days (Ishikawa and Ito, 2017).

4.3. MUC5AC The number of bronchioles containing goblet cells up to 5% of the total number of epithelial cells counted in each sample bronchiole was significantly (p < 0.001) higher in the bronchioles of smokers than those of non-smokers (Lumsden et al., 1984). Semi-quantitative scoring of MUC5AC-positive cells in immunohistochemical peripheral lung sections was significantly (p < 0.01) higher in the smokers with or without COPD than the non-smokers (Caramori et al., 2004). Comparison of the percentages of bronchial epithelial area positively stained with MUC5AC antibody revealed that smokers with or without COPD expressed significantly (p < 0.001) more MUC5AC protein than nonsmokers (O’Donnell et al., 2004). Investigation of bronchial brushings for the level of mRNA of MUC5AC revealed a significantly higher (p < 0.05) level of MUC5AC expression in current smokers with chronic bronchitis than in never-smokers without chronic bronchitis (Mebratu et al., 2011). The epithelial cells obtained through brushing and bronchoscopy of the small airways were studied for the expression of MUC5AC core genes in order to compare healthy non-smokers and healthy smokers. The results showed that the latter subjects had a 1.4fold higher (p < 0.01) level of the gene expression than the former (Wang et al., 2012). In human bronchial epithelial cells 3D-cultured at ALI, MUC5AC expression was significantly (p < 0.001) lower in the cultures continuously exposed to 3 or 6% CSE for up to 28 days than in the unexposed control (Brekman et al., 2014). However, continuous exposure of human bronchial epithelial cells to 2.5 or 5% of CSE at ALI for up to 28 days led to a significantly (p < 0.001) increased frequency of cells stained positive for MUC5AC and also in the same study for a significantly (p < 0.05) higher level of MUC5AC mRNA at 2.5% of CSE exposure in comparison to the unexposed control

4.4. MUC5B Frequencies of MUC5B-positive cells were semi-quantitatively scored in immunohistochemical sections obtained from the peripheral lung of smokers with or without COPD and non-smokers, and it was found that there were no significant differences in the frequency between the two subject groups (Caramori et al., 2004). Comparison of sizes of epithelial area positively stained with MUC5B antibody revealed no statistically significant differences between smokers with or without COPD and non-smokers and the staining intensity was in general at a low level (O’Donnell et al., 2004). In human bronchial epithelial cells, 3D-cultured at ALI, MUC5B expression was significantly (p < 0.001) lower in the cultures continuously exposed to 3 or 6% CSE for up to 28 days than in the unexposed control (Brekman et al., 2014). Continuous exposure of human bronchial epithelial cells to 2.5 or 5% of CSE at ALI for up to 28 days did not give rise to a statistically significant increase of the MUC5B mRNA level compared to the unexposed control (Schamberger et al., 2015). In a similar culture model but co-cultured with human fibroblasts, the exposure of human bronchial epithelial cells to the whole CS (2 or 4 cigarettes/exposure/day) every other day up to 21 days caused a significant (p < 0.05) reduction of the MUC5B mRNA level compared to the unexposed control (Ishikawa and Ito, 2017). 6

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Fig. 6. Cross-section of cell culture insert membranes with immunohistochemically (CK13) stained immortalized NHBE cells (CL-1548) after 8 exposures to cigarette smoke (CS) and ecigarette vapor (EC). Non-exposed cells were used as an incubator control (IC).

ating basal cells of the proximal airway epithelium, suggesting different driving mechanisms in the squamous alteration between rats and humans (Bolton et al., 2009). Squamous metaplasia, as detected by the immunocytochemical reaction to involucrin (IVL), has been reported not only in the proximal large but also distal small airway epithelium of cigarette smokers and COPD patients (Burgel et al., 2011). In human bronchial epithelial cells cultured at ALI, the expression of IVL mRNA, examined by quantitative reverse transcription polymerase chain reaction (qRT-PCR), was statistically significantly elevated in comparison to the control level up to 28 days after exposure to 2.5%–6% of cigarette smoke extract (CSE) (Brekman et al., 2014; Schamberger et al., 2015). Taking into consideration the results and literature data, our observations obtained in the current study may allow the following statements. As far as our data on the development of CK13 (marker for squamous metaplasia), ciliated cells and CC10 (club cell marker) in the immortalized cell line CL1548 after exposure to CS are concerned, they are roughly consistent with the human data available in the literature. Other in vitro studies using primary human bronchial epithelial cells also show a similar tendency. However, slight differences between CS and EC vapor can still be seen in their effects on this cell line. As expected, the ciliated cells are less damaged by EC vapor than CS and CK13 is induced by CS as well as EC vapor. It may be interesting to know whether humans show a similar response to EC vapor. Histopathological studies on biopsies from EC users would seem to be warranted. As to the effects on the marker p63, there are no human data from studies on the small airways, making it difficult for direct comparison. However, other in vitro data are highly consistent with our current

4.5. Tp63 In the tracheobronchial biopsy specimens obtained from patients with with mild bronchial epithelial hyperplasia, severe hyperplasia and squamous metaplasia, and non-smoking human subjects, semi-quantitative scoring was carried out on the immunohistological sections for the grade of staining with a p63 antibody (a marker for basal cells). In comparison to the non-smokers, the patients with squamous metaplasia showed the highest grade (p < 0.001) of staining, which decreased in patients with severe (p < 0.001) and mild (p < 0.01) hyperplasia (Herfs et al., 2012). In 3D-cultured human bronchial epithelial cells, the continuous exposure to CSE of 3 or 6% (Brekman et al., 2014) or 2.5 or 5% (Schamberger et al., 2015) for up to 28 days had no statistically significant influence on the expression level of p63. Similarly, longterm exposure to the whole CS (2 or 4 cigarettes/exposure/day) at 2day intervals up to 21 days showed no statistically significant effects on the expression level of p63 even in the presence of co-cultured fibroblasts (Ishikawa and Ito, 2017).

4.6. Squamous cell metaplasia In the airway epithelium of cigarette smokers (with or without COPD), morphologically altered areas are not uncommon, and squamous cell metaplasia can also be frequently encountered containing variously sized groups of cells expressing CK13, not necessarily involving only the middle layer but also the luminal layer and, to a much lesser extent, the basal layer (Rigden et al., 2016). When spontaneously hypertensive rats were chronically exposed to tobacco smoke, they developed CK13-positive squamous metaplasia in the areas of prolifer7

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findings on CS and EC vapor, indicating that there are few adverse effects on the expression of p63. Inconsistent to a certain extent are our findings derived from in vitro cultured primary or immortalized airway epithelial cells exposed to CS and the observations made by others with the specimens derived directly from cigarette smokers, wherein MUC5AC expression was suppressed by CS in vitro as long as the exposure continues and smokers had always elevated MUC5AC production. However, when the exposed cultures were maintained for an additional 14 days after the cessation of the exposure for recovery, an overshoot of MUC5AC production towards hyperplasia and hypertrophy was observed (Aufderheide, 2016), resembling the overproduction of MUC5AC including MUC5AC cell hyperplasia seen in the cigarette smokers. Our in vitro data from EC vapor exposure are largely in agreement with the results of our CS exposure. In comparison with in vivo specimens directly derived from smokers we have some discrepancies in our in vitro model. One reason for the occurrence of such discrepancies may be that interleukin-13 (IL13), a cytokine known as a MUC5AC stimulator in asthma, is absent in our actual in vitro model, which purely consists of airway epithelial cells. This cytokine is usually released from an activated lymphoid cell type (a subset of T cells) under the stress of toxic compounds, e.g. cigarette smoke. Binding of this cytokine to epidermal growth factor (EGF) receptor of undifferentiated mucus cell progenitors triggers a chain reaction leading to the activation of the MUC5AC gene (Chen et al., 2014; Erde and Sheppard 2014). Another reason, in particular in the case of in vitro recovery, may also be that the pathway towards the overproduction of MUC5AC may be triggered by a surrogate stimulating factor, which could have given birth to the affected (injured) epithelial cells through the abrupt termination of CS stress and act on the mucous progenitor cells in a paracrine manner after secretion from the injured cells. In order to provide more reliable toxicological human data, the in vitro models can be combined with further cell types, for example lymphoid cells capable of releasing T helper 2 cytokines, to mimic the in vivo situation as far as possible. Particularly when products like EC vapor need to be examined for risk assessment.

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5. Conclusion The immortalized cell line (NHBE-CL1548) used in the present experiments showed a similar response to cigarette smoke (CS) and Ecigarette vapor after repeated exposure – effects also comparable with morphological alterations observed in smokers. Although the phenotypical changes induced by EC vapor were not so pronounced, we found a reduction in cilia frequency and length, a reduced frequency of cells expressing CC10, Muc5AC and Muc5B and the induction of CK13 expression. The presence of p63-expressing cells was scarcely affected. In summary, the EC vapor induced qualitative and quantitative morphological alterations comparable with those observed with conventional cigarette smoke thus pointing to a serious risk by this aerosol. References Aufderheide, M., Scheffler, S., Ito, S., Ishikawa, S., Emura, M., 2015. Ciliatoxicity in human primary bronchiolar epithelial cells after repeated exposure at the air-liquid interface with native mainstream smoke of K3R4F cigarettes with and without charcoal filter. Exp. Toxicol. Pathol. 67, 407–411. Aufderheide, M., Ito, S., Ishikawa, S., Emura, M., 2017. Metaplastic phenotype in human primary bronchiolar epithelial cells after repeated exposure to native mainstream smoke at the air-liquid interface. Exp. Toxicol. Pathol in press. Aufderheide M. (2016). Metaplastic phenotype in human primary bronchiolar epithelial cells after repeated exposure to mainstream cigarette smoke at the air-liquid interphase. European Society for Alternatives to Animal Testing (EUSAAT), Lecture in Toxicology I—Inhalation Toxicity, EUSAAT 2016, Austria Bolton, S.J., Pinnion, K., Oreffo, V., Foster, M., Pinkerton, K.E., 2009. Characterization of the proximal airway squamous metaplasia induic ed by chronic tobacco smoke exposure in spontaneously hypertensive rats. Respir. Res. 10, 118.

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