Background: An increasing number of studies using primary human bronchial epithelial cells (BECs) have reported intrinsic differences in the expression of several genes between cells from asthmatic and non-asthmatic donors. The stability of gene expression by primary BECs with increasing cell passage number has not been well characterized. Methods: To determine if expression by primary BECs from asthmatic and non-asthmatic children of selected genes associated with airway remodeling, innate immune response, immunomodulatory factors, and markers of differentiated airway epithelium, are stable over increasing cell passage number, we studied gene expression patterns in passages 1, 2, 3, 4, and 5 BECs from asthmatic (n = 6) and healthy (n = 6) subjects that were differentiated at an air-liquid interface. RNA was harvested from BECs and RT-PCR was performed for TGFβ1, TGFβ2, activin A, FSTL3, MUC5AC, TSLP, IL-33, CXCL10, IFIH1, p63, KT5, TUBB4A, TJP1, OCLN, and FOXJ1. Results: Expression of TGFβ1, TGFβ2, activin A, FSTL3, MUC5AC, CXCL10, IFIH1, p63, KT5, TUBB4A, TJP1, OCLN, and FOXJ1 by primary BECs from asthmatic and healthy children was stable with no significant differences between passages 1, 2 and 3; however, gene expression at cell passages 4 and 5 was significantly greater and more variable compared to passage 1 BECs for many of these genes. IL-33 and FOXJ1 expression was also stable between passages 1 through 3, however, expression at passages 4 and 5 was significantly lower than by passage 1 BECs. TSLP, p63, and KRT5 expression wasstableacrossBEC passages1through5for bothasthmatic andhealthy BECs. Conclusions: These observations illustrate the importance of using BECs from passage ≤3whenstudyinggene expression by asthmatic and non-asthmatic primary BECs and characterizing the expression pattern across increasing cell passage number for each new gene studied, as beyond passage 3 genes expressed by primary BECs appear to less accurately model in vivo airway epithelial gene expression. Keywords: Asthma, Children, Airway remodeling, Epithelial cells Background asthmatic airway disease . Additionally, understanding Asthma continues to be one of the most prevalent and of the importance of BECs in immune surveillance and costly diseases of childhood throughout the world . In coordination of the immune response to infections and recent years, our understanding of asthma pathogenesis environmental antigens has grown to include an import- has grown to include a central role for bronchial epithe- ant role for BEC-derived cytokines and direct cell-to-cell lial cells (BECs) in the establishment and maintenance of communication beyond their role in barrier function and innate immunity . As a result, BECs have become the focus of many recent studies aimed to elucidate mech- * Correspondence: firstname.lastname@example.org Stephen R. Reeves and Kaitlyn A. Barrow contributed equally to this work. anisms underlying asthma pathogenesis in children . Center for Immunity and Immunotherapies, Seattle Children’s Research Studying BECs in children with and without asthma Institute, Seattle, WA, USA presents a unique set of challenges. Unlike studies in Pulmonary and Sleep Medicine Division, Department of Pediatrics, University of Washington, Seattle, WA, USA adults, obtaining cells via bronchoscopic airway biopsy © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Reeves et al. BMC Pulmonary Medicine (2018) 18:91 Page 2 of 11 from children is problematic as most institutional review expression differences that occur in BECs over subse- boards cannot approve such studies in children as the quent passages. In order to better characterize the stabil- risk of general anesthesia and the lack of direct benefit ity of gene expression by primary BECs over multiple to the subject precludes such investigations. While passages, we tested the hypothesis that the expression of transformed and immortalized cell lines exist and are a panel of genes involved in airway remodeling, immune commercially available for in vitro studies, none of the regulation, innate immune response, airway epithelial currently available cell lines are derived from pediatric basal cells, ciliogenesis, and epithelial tight junctions donors, and very little clinical data is provided about the would be stable through increasing cell passages in an subjects from whom such lines were obtained. Further- ex vivo organotypic culture model system using primary more, immortalized BEC cell lines are variable in their BECs obtained from well-phenotyped children with or ability to recapitulate the anatomical and physiologic without asthma that were differentiated at an air-liquid features observed in the in vivo condition . Direct interface (ALI). comparisons of airway epithelial behavior in the asth- matic vs. non-asthmatic conditions is critical to further- Methods ing our understanding of the pathophysiology of asthma, Subjects which therefore requires use of primary airway epithelial For this study, we recruited asthmatic and healthy children cells. In addition, given the recognition that asthma is a ages 6-18 years undergoing an elective surgical procedure complex and heterogeneous disease with multiple endo- that required endotracheal intubation and general anesthesia types with likely unique aspects driving pathophysiology for a clinically indicated procedure. Children with asthma , it is increasingly important that donors of primary had at least a 1-year history of physician-diagnosed asthma, epithelial cells used in basic and translational studies be used a short-acting beta-agonist ≥ twice a month or were carefully clinically characterized so as to allow investiga- taking a daily maintenance medication (inhaled corticoster- tions of the role of the airway epithelium within unique oid or montelukast), and were born ≥36 weeks gestation. asthma endotypes. Additionally,childrenwithasthmahad oneormoreof the Given the limitations of in vitro models utilizing cell following atopic features: history of a positive skin prick test lines and the difficulty obtaining biopsy specimens in the or positive radioallergosorbent testing (RAST) for a pediatric population, ex vivo cultures of primary BECs common aeroallergen, elevated serum IgE (> 100 IU/mL), obtained via bronchial brushings have become a useful history of physician-treated allergic rhinitis, history of model in which to perform studies related to the airway physician-treated atopic dermatitis. Healthy subjects were epithelium in children . Collection of bronchial epi- born ≥36 weeks gestation, and lacked a history of asthma, thelial cells using brushings performed through an endo- reactive airway disease, chronic cough, chronic lung disease, tracheal tube while a patient is under general anesthesia or past treatment with bronchodilators, systemic or inhaled (taking advantage of a planned anesthesia for a separate, steroids, or oxygen. A detailed medical history was obtained clinically indicated procedure) have been described and to ensure the subjects met these inclusion criteria. importantly pose minimal additional risk to the subject At the time of anesthesia, a blood sample was drawn [7, 8]. While this methodology has been utilized success- from each subject and used to measure total serum IgE fully in children and adults for more than a decade and and RAST allergen-specific IgE to dust mites (D. farina provides a reliable, safe, and relatively non-invasive way and D. pteronyssinus), cat epithelium, dog epithelium, to obtain primary BECs [7–10] that can be utilized to Alternaria tenuis, Aspergillus fumigatus, and timothy support ex vivo studies, the major trade-off is that the grass. At a subsequent follow-up visit the fraction of number of cells obtained using this methodology is rela- exhaled nitric oxide (FE ) was measured according to NO tively low. In most cases expansion of the cell number in American Thoracic Society (ATS) guidelines using a culture is an essential step required to produce sufficient NIOX MINO nitric oxide analyzer (Aerocrine®, Sweden) material to conduct experiments. . Spirometry was performed using a VMAX® series While the passage of cells in culture poses a theoret- 2130 spirometer (VIASYS Healthcare, Hong Kong) to ical loss of cellular phenotype the further cells are prop- quantify forced vital capacity (FVC), forced expiratory vol- agated beyond the donor, several studies including work ume in 1 s (FEV ), and forced expiratory flow between 25 from our lab have demonstrated phenotypic differences and 75% of FVC (FEF ) according to ATS guidelines. 25-75 in primary cells obtained from children with and without asthma (reviewed by McLellan et al.) suggesting that cell Ethics, consent and permissions phenotype is preserved at least in the initial 2-3 passages Written consent was obtained from parents of subjects . While there have been anecdotal reports of cells per- and assent was obtained for children ≥ age 10 years. The forming poorly in cell cultures at later passages, we are work presented in this study was approved by the Seattle not aware of studies that have rigorously examined gene Children’s Hospital Institutional Review Board. Reeves et al. BMC Pulmonary Medicine (2018) 18:91 Page 3 of 11 Establishment of bronchial epithelial cell cultures RNA extraction and real-time PCR Following the induction of anesthesia and securing of the TotalRNA wasisolatedfromBECsdifferentiatedatanALI. endotracheal tube, three samples of BECs were obtained Three wells from each experimental condition were from each subject using 4 mm Harrell® unsheathed bron- harvested and pooled to isolate RNA using the RNAqueous choscope cytology brushes (CONMED® Corporation). The kit for total RNA purification from Ambion®-Applied brush was inserted through an endotracheal tube, ad- Biosystems (Austin, TX). RNA concentration and quality vanced until resistance was felt, and rubbed against the was determined using a NanoDrop ND-1000 spectropho- airway surface for 2-3 s as described previously [7, 8]. Cells tometer Thermo Fisher Scientific). RNA samples (1 μg) were seeded onto type I collagen coated T-25 cell culture were reverse-transcribed using the SuperScript® VILO flasks and proliferated under submerged culture condi- cDNA Synthesis Kit (Life Technologies, Grand Island, NY). tions. Cultures were proliferated in a humidified incubator Samples were diluted up to a final volume of 100 μl (10 ng/ at 37 °C in an atmosphere of 5% CO in PneumaCult™-Ex μl). Quantitative real-time PCR was performed using bronchial epithelial growth medium (BEGM) (StemCell™ validated TaqMan® probes (Life Technologies) for trans- Technologies) containing gentamicin and amphotericin B, forming growth factor beta (TGFβ)1 (Hs00998133_m1), and supplemented with penicillin-streptomycin (100 μg/ml; TGFβ2 (Hs00234244_m1), activin A (Hs01081598_m1), Invitrogen®). Fluconazole (25 μg/mL) was added to P0 follistatin-like-3 (FSTL3, Hs00610505_m1), mucin 5 AC medium for the first 96 h, after which medium was aspi- (MUC5AC, Hs01365616_m1), thymic stromal rated and replaced with medium without fluconazole. lymphopoietin (TSLP, Hs00263639_m1), interleukin-33 Medium was thereafter changed every 48 h until the cul- (IL-33, Hs01125942_m1), C-X-C motif chemokine 10 ture reached ~ 70-90% confluence. All primary BEC lines (CXCL10, Hs00171042_m1), interferon induced with heli- were screened for mycoplasma using MycoAlert™ PLUS case C domain (1IFIH1, Hs00223420_m1), p63 (TP63, Mycoplasma Detection Kit (Lonza, Inc) and found to Hs00978340_m1), cytokeratin 5 (KRT5, Hs00361185_m1), be negative. beta-tubulin (TUBB4A, Hs00760066_s1), forkhead box J1 (FOXJ1, Hs00230964_m1), zona occluden-1 (TJP1, Air-liquid Interface (ALI) epithelial cell cultures Hs01551861_m1), occludin (OCLN, Hs05465837_g1), and BECs were used for these studies at each passage corre- glyceraldehyde 3-phosphate dehydrogenase (GAPDH, sponding number. Once cells were ~ 70-90% confluence in Hs02758991_g1). Assays were performed using the TaqMan® flasks, they were trypsinized with 1 mL of 0.025% Fast Advanced Master Mix reagents and accompanying Trypsin-EDTA and then seeded onto collagen I pre-coated protocol and the Applied Biosystems StepOnePlus™ (Collagen Solution, STEMCELL™ Technologies) Corning Real-Time PCR System with StepOne Software v2.2.2 Costar 12 mm 0.4 μm Transwells® (Corning® Life Sciences) (Life Technologies). The primary quantitative PCR data- at a concentration of 100,000 cells per transwell. Cells were sets used and/or analyzed for this study are provided as a then kept in submerged culture using BEGM in both the (Additional file 1:Appendix I). apical and basolateral well chambers for 7 days or until confluent. Once confluent, cells were then changed to Statistical analysis Pneumacult™ ALI Medium (StemCell™ Technologies) in For clinical parameters, the paired t-test was used for the lower basolateral chamber only and the remaining ap- comparisons that were normally distributed within each ical media was aspirated. ALI media in the basolateral com- subject group. For non-normally distributed data, the partment was changed every other day and cells were Wilcoxon signed-rank test was used. For RT-PCR studies, differentiated at an ALI for 21 days. the relative expression of genes were normalized using glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as a Study design non-regulated reference gene. Gene expression at P2-P5 is BECs seeded into the initial T25 flasks were designated reported as fold change compared to the gene expression passage 0 (P0) and allowed to proliferate until cells were at P1. Analyses of RT-PCR results were performed using ~ 70-90% confluent at which point cells were passaged. GenEx version 5.0.1 (MultiD Analyses AB, Göteborg, One of the three flasks was then passaged into a trans- Sweden) based on methods described by Pfaffl . Statis- well plate in order to establish P1 ALI cultures for ex- tical significance was set at P < 0.05. One-way ANOVA periments. Another P0 T25 flask was split into 3 (ordinary one-way ANOVA with Sidak’smutlitple com- additional T25 flasks (also P1) to carry forward into sub- parisons test for normally distributed data; Kruskal-Wallis sequent experiments. The remaining P0 T25 was either ANOVA with Dunn’s multiple comparisons test for preserved in liquid nitrogen or also carried forward for non-normally distributed data) was used to compare separate studies. This paradigm was carried forward for expression of genes at P2-P5 to expression at P1 using each passage (P + 1) until reaching ALI cultures up Prism® 6.0 software (GraphPad Software Inc., San Diego, through passage 5 (P5) to conduct experiments (Fig. 1). CA). Two-way ANOVA was used to determine if gene Reeves et al. BMC Pulmonary Medicine (2018) 18:91 Page 4 of 11 Fig. 1 Schematic depicting experimental design for passaging primary bronchial epithelial cells over 5 passages in ex vivo cell culture for gene expression studies expression patterns over increasing cell passage were dif- similar in age (11.16 ± 3.7 years vs. 11.98 ± 5.1 years, re- ferent between BECs from asthmatic and healthy subjects. spectively, p= NS). There was a 2/3 male predominance Statistical analyses of clinical and lung function data were with both groups (no difference between asthma vs. also performed using Prism®. healthy). All asthmatic subjects displayed atopic features including eczema (50%), allergic rhinitis (83%), or positiv- Results ity to aeroallergen by RAST IgE testing (83%). No history Bronchial brushings were obtained from both asthmatic of eczema or allergic rhinitis was reported in the (n = 6) and healthy donors (n = 6) to generate ALI cultures non-asthmatic group. One subject in the healthy group used in this study. Clinical characteristics for each group did display a positive RAST result. The majority of asth- are shown in Table 1. Asthmatic and healthy donors were matic subjects were using inhaled corticosteroids (83%) at Table 1 Subject Characteristics Asthmatic Subjects Healthy Subjects p Value n= 6 n= 6 Age yrs. (mean ± SD) 11.16 (3.7) 11.98 (5.1) 0.76 Sex (female/male) 4/6 4/6 Currently using daily asthma controller (%) 5 (83%) N/A History of atopy, n; (%) 6 (100%) 1 (17%) < 0.01 Positive RAST, n; (%) 5 (83%) 1 (17%) 0.02 IgE IU/mL (median ± SD) 306.5 (387.32) 185.83 (403.47) 0.6 FVC % predicted (mean ± SD) 106.4 (13.1) 95.0 (13.9) 0.22 FEV /FVC Ratio (mean ± SD) 0.78 (0.03) 0.89 (0.07) 0.02 FEV % predicted (mean ± SD) 94.4 (14.1) 97.25 (13.9) 0.77 FEF % predicted (mean ± SD) 78.4 (4.9) 103.9 (5.5) 0.03 25-75 FE ppb (mean ± SD) 31.2 (24.2) 12.0 (5.1) 0.26 NO RAST Radioallergosorbent testing, FVC Forced vital capacity, FEV Forced expiratory volume in one second, FEF Forced expiratory flow between 25 and 75% 1 25-75 of expiration Reeves et al. BMC Pulmonary Medicine (2018) 18:91 Page 5 of 11 the time of enrollment. There was a non-significant trend expression with increasing passage between BECs from toward higher FENO levels in asthmatic subjects com- asthmatic and healthy subjects (p = 0.9). TGFβ2 expres- pared to healthy subjects (31.2 ± 24.2 ppb vs. 12.0 ± sion was also not significantly different from P1 through 5.1 ppb, p = 0.26). Total serum IgE levels were not signifi- P3, but similar to TGFβ1, expression was significantly cantly different between asthmatic and healthy subjects increased in both P4 and P5 compared to P1 among (306.5 ± 387.32 IU/mL vs. 185.83 ± 403.47 IU/mL, p =0.6). both asthmatic and healthy subjects (p < 0.05, Fig. 2b), Measures of lung function by spirometry demonstrated without significant differences in the pattern of gene ex- no differences in FVC or FEV between the groups; how- pression by BECs from the two subject groups (p = 0.08). ever, FEF (78.4% ± 4.9% vs 103.9% ± 5.5%, p =0.03) Expression of MUC5AC displayed marked variability be- 25-75 and FEV /FVC (0.78% ± 0.03% vs. 103.9% ± 5.5%, p =0.02) ginning with P3, with the greatest variability observed at were significantly lower in the asthmatic group and con- P4 and P5. However, given the high degree of variability, sistent with airway obstruction. MUC5AC expression was not significantly different at Expression of genes associated with airway remodeling P2-P5 compared to P1 (p = 0.3, Fig. 2c) by BECs, nor by differentiated primary BECs over successive passages was there a difference in the pattern of gene expression (P1-P5) is depicted in Fig. 2. TGFβ1 expression was not by BECs from asthmatic and healthy donors (p = 0.4). significantly different from P1 through P3; however, Gene expression of both activin A and FSTL3 were expression was increased in P4 and P5 compared to P1 orders of magnitude greater at P4 and P5 compared to BECs from both asthmatic and healthy donors (p < 0.05, expression at P1 (p < 0.01 and p < 0.001, respectively) for Fig. 2a), and there was not a difference in the pattern of BECs from both asthmatic and healthy donors, without a b TGFβ1 TGFβ2 MUC5AC 5 30 * * 80 * 40 0 0 P1 P2 P3 P4 P5 P1 P2 P3 P4 P5 P1 P2 P3 P4 P5 P1 P2 P3 P4 P5 P1 P2 P3 P4 P5 P1 P2 P3 P4 P5 Asthma Healthy Asthma Healthy Asthma Healthy * Expression significantly different than P1 (p<0.05) * Expression significantly different than P1 (p<0.05) d e activin A FSTL3 * * 0.1 0.1 P1 P2 P3 P4 P5 P1 P2 P3 P4 P5 P1 P2 P3 P4 P5 P1 P2 P3 P4 P5 Asthma Healthy Asthma Healthy * Expression significantly different than P1 (p<0.001) * Expression significantly different than P1 (p<0.01) Fig. 2 Expression of genes related to airway remodeling by primary BECs. Expression of TGFβ1(a), TGFβ2(b), MUC5AC (c), activin A (d), and FSTL3 (e) by BECs at P1 (n = 6 asthma donors, n = 6 healthy donors), P2 (n = 6 asthma donors, n = 6 healthy donors), P3 (n = 4 asthma donors, n = 6 healthy donors), P4 (n = 6 asthma donors, n = 6 healthy donors), and P5 (n = 6 asthma donors, n = 6 healthy donors) are presented as box- and-whisker plots which depict the interquartile range and median (the ends of each box represent the upper and lower quartiles, error bars represent the maximum and minimum, and the horizontal line within the box represents the median). To compare expression of genes at P2-P5 to expression at P1, and to compare patterns of gene expression between asthmatic and healthy donors, ordinary two-way ANOVA with Dunnett’s multiple comparisons test was used for normally distributed data, and Kruskal-Wallis ANOVA with Dunn’s multiple comparisons test was used for non-normally distributed data Relative expression compared to Passage 1 Relative expression compared to Passage 1 (normalizedtoGAPDH) (normalized to GAPDH) Relative expression compared to Passage 1 Relative expression compared to Passage 1 (normalized to GAPDH) (normalized to GAPDH) Relative expression compared to Passage 1 (normalized to GAPDH) Reeves et al. BMC Pulmonary Medicine (2018) 18:91 Page 6 of 11 pattern differences between the two subject groups (acti- significantly greater at P4 and P5 as compared to P1 (p < vin A: p = 0.08; FSTL3: p = 0.3); however, expression for 0.01; Fig. 4d), expression of TUBB4A and OCLN were sig- both were not significantly different at P2 or P3 compared nificantly greater at P5 as compared to P1 (p = 0.05; Fig. 4c, to P1 (Fig. 2d and e). Although the study was not designed e), and there were not significant differences in the overall or powered to assess differences in the expression of patterns of expression of these genes with increasing cell specific genes between asthmatic and healthy BECs, at P1 passage between the asthmatic and healthy subject groups expression of TGFβ2 and MUC5AC, normalized to (TJP1: p =0.5; TUBB4A: p =0.6; OCLN: p =0.8). In con- GAPDH, were significantly greater by asthmatic as com- trast to the other studied genes associated with epithelial pared to healthy BECs (Additional file 2:FigureS2). differentiation, expression of FOXJ1 decreased in later pas- In addition to genes associated with airway remodeling, sage ALI cultures, and expression was significantly lower at expression of several genes involved in innate immune re- P4 and P5 compared to expression at P1 by BECs from sponse (IFIH1, CXCL10) and immunomodulation (TSLP, both asthmatic and healthy BECs (p < 0.01; Fig. 4f), without IL-33) were also analyzed over increasing passages by pattern differences between the subject groups (p =0.2). Al- BECs. There was significant variability in CXCL10 expres- though the study was not powered to assess differences in sion by BECs from both asthmatic and healthy donors the expression of specific differentiation-associated genes from P2-P4 compared to expression at P1, with signifi- or markers of epithelial basal cells between asthmatic and cantly increased expression at P4 and P5 compared to P1 healthy BECs, no significant differences were observed at by asthmatic BECs and significantly increased expression P1 (Additional file 2:FigureS2). at P5 by healthy BECs (Fig. 3a), however, there was not a In order to assure that differences in gene expression difference in the overall pattern of CXCL10 expression patterns were not related to differential expression of house- with increasing cell passage between asthmatic and keeping genes over multiple passages we compared the ex- healthy donors (p = 0.9). Expression of IFIH1 was signifi- pression of GAPDH by BECs across the five passages and cantly elevated at P4 and P5 compared to expression at P1 found no differences in mRNA Ct values (Additional file 3: for BECs from both asthmatic and healthy donors (p < Figure S1). We also assessed mRNA expression for each of 0.05, Fig. 3b), without pattern differences between the the genes studied prior to normalization by GAPDH to subject groups (p = 0.4), but was not significantly different show the natural variation in mRNA levels across asthmatic at P2 or P3. In contrast, Expression of IL-33 was signifi- and healthy donors. For TGFβ1, TGFβ2, MUC5AC, FSTL3, cantly decreased at P4 and P5 compared to P1 by BECs and activin A, mRNA Ct values were significantly lower from both asthmatic and healthy donors (p <0.01; Fig. 3c); (greater un-normalized gene expression) at P4 and P5 com- however, expression of IL-33 at P2 and P3 were not pared to P1 (p < 0.05; Additional file 4:Figure S3) by BECs significantly different compared to P1, and there were no from both asthmatic and healthy donors, and there were significant differences in IL-33 gene expression patterns not significant pattern differences in mRNA Ct values with increasing cell passage between BECs from asthmatic between the two subject groups with increasing cell passage and healthy donors (p = 0.4). Gene expression of TSLP for TGFβ1(p =0.4), FSTL3 (p = 0.4), and activin A (p =0.6). remained stable throughout all 5 successive passages and However, mRNA Ct values were significantly lower (greater was not significantly different compared to P1 by BECs gene expression prior to normalization) by BECs from asth- from both asthmatic and health donors (Fig. 3d). Of note, matic as compared to healthy donors for TGFβ2(p = 0.004) at P1 expression of TSLP, normalized to GAPDH, was sig- and MUC5AC (p = 0.04). Un-normalized mRNA Ct values nificantly greater by asthmatic as compared to healthy for CXCL10 and IFIH1 were significantly lower at P4 and BECs (Additional file 2:Figure S2). P5 compared to P1 among asthmatic and healthy BECs (p < To assess the stability of expression of airway epithelial 0.05; Additional file 5:FigureS4),whereas IL-33mRNA Ct differentiation-associated genes and markers of epithelial values were significant higher at P4 and P5 compared to P1 basal cells over serial BEC passages, we measured the ex- among asthmatic and healthy BECs (p < 0.05). For TP63 and pression of the basal cell-associated gene TP63, the epithe- KRT5, there were no significant differences in mRNA Ct lial marker cytokeratin 5 (KRT5), ciliogenesis-associated values among or between asthmatic and healthy BECs with genes TUBB4A and FOXJ1, and genes coding for the tight increasing cell passage (Additional file 6:FigureS5),whereas junctional proteins zona occluden-1 (TJP1) and occludin mRNA Ct values were significantly lower at P4 and P5 (OCLN). Expression of all of these genes was stable compared to P1 for asthmatic and healthy BECs for through at least P3 (Fig. 4). Expression of TP63 and KRT5 TUBB4A (p = 0.03), TPJ1 (p < 0.01), and OCLN (p < 0.05) at passages 4 and 5 were not significantly different from without pattern differences between asthmatic and healthy expression at P1 by BECs from both asthmatic and healthy BECs. Finally, FOXJ1 mRNA Ct values were significantly donors, but was more variable between individual cell higher at P4 and P5 compared to P1 for asthmatic and lines at later passages (Fig. 4a, b). Among BECs from both healthy BECs, with a similar pattern between asthmatic and asthmatic and healthy subjects expression of TJP1 was healthy BECs. Reeves et al. BMC Pulmonary Medicine (2018) 18:91 Page 7 of 11 a b IFIH1 CXCL10 40 * P1 P2 P3 P4 P5 P1 P2 P3 P4 P5 P1 P2 P3 P4 P5 P1 P2 P3 P4 P5 Asthma Healthy Asthma Healthy * Expression significantly different than P1 (p<0.05) * Expression significantly different than P1 (p<0.01) TSLP IL-33 P1 P2 P3 P4 P5 P1 P2 P3 P4 P5 P1 P2 P3 P4 P5 P1 P2 P3 P4 P5 Asthma Healthy Asthma Healthy * Expression significantly different than P1 (p<0.01) Fig. 3 Expression of innate immunity and immunomodulatory genes by primary BECs. Expression of CXCL10 (a), IFIH1 (b), IL-33 (c), and TSLP (d) by BECs at P1 (n = 6 asthma donors, n = 6 healthy donors), P2 (n = 6 asthma donors, n = 6 healthy donors), P3 (n = 4 asthma donors, n = 6 healthy donors), P4 (n = 6 asthma donors, n = 6 healthy donors), and P5 (n = 6 asthma donors, n = 6 healthy donors) are presented as box-and-whisker plots which depict the interquartile range and median (the ends of each box represent the upper and lower quartiles, error bars represent the maximum and minimum, and the horizontal line within the box represents the median). To compare expression of genes at P2-P5 to expression at P1, and to compare patterns of gene expression between asthmatic and healthy donors, ordinary two-way ANOVA with Dunnett’s multiple comparisons test was used for normally distributed data, and Kruskal-Wallis ANOVA with Dunn’s multiple comparisons test was used for non- normally distributed data Discussion up to passage 3 has been reported previously , those In the present study, we have demonstrated that primary observations were based on a review of the available lit- differentiated BECs obtained from children with or with- erature. In the present study, we present for the first out atopic asthma maintain stable expression of a panel time a study designed to compare the stability of gene of genes related to airway remodeling, innate immunity, expression of multiple airway remodeling, innate im- immunomodulation, epithelial differentiation, and epi- munity, immunomodulation, epithelial differentiation, thelial basal cells through passage 3 in ex vivo ALI cell and epithelial basal cells genes by BECs over successive cultures. We further report that in primary BECs beyond passages. Our findings further support the use of primary passage 3 expression of the studied genes became signifi- BECs obtained from pediatric donors at passage ≤3. cantly more variable with expression of most genes in- Primary ex vivo cell cultures of BECs obtained by bron- creasing (TGFβ1, TGFβ2, activin A, FSTL3, MUC5AC, chial brushings have been used successfully in children and CXCL10, IFIH1, TUBB4A, TJP1, OCLN) and expression adults for more than a decade and have become an attract- of other genes decreasing (IL-33, FOXJ1). Some genes ive model to study the airway epithelium in various diseases. were also found to be stable throughout the five pas- This is especially true in the pediatric population where sages studied (TSLP, TP63, KRT5). While the assump- obtaining cells via airway biopsies is problematic given that tion that primary BECs retain their original phenotype performing a sedated bronchoscopy in a pediatric subject Relative expression compared to Passage 1 Relative expression compared to Passage 1 (normalized to GAPDH) (normalized to GAPDH) Relative expression compared to Passage 1 Relative expression compared to Passage 1 (normalized to GAPDH) (normalized to GAPDH) Reeves et al. BMC Pulmonary Medicine (2018) 18:91 Page 8 of 11 b c TP63 KRT5 TUBB4A 4 10 20 1 5 0 0 P1 P2 P3 P4 P5 P1 P2 P3 P4 P5 P1 P2 P3 P4 P5 P1 P2 P3 P4 P5 P1 P2 P3 P4 P5 P1 P2 P3 P4 P5 Asthma Healthy Asthma Healthy Asthma Healthy * Expression significantly different than P1 (p=0.05) e f FOXJ1 TJP1 OCLN 10 10 8 8 6 6 * * 4 4 2 2 * * 0 0 P1 P2 P3 P4 P5 P1 P2 P3 P4 P5 P1 P2 P3 P4 P5 P1 P2 P3 P4 P5 P1 P2 P3 P4 P5 P1 P2 P3 P4 P5 Asthma Healthy Asthma Healthy Asthma Healthy * Expression significantly different than P1 (p<0.01) * Expression significantly different than P1 (p=0.05) * Expression significantly different than P1 (p<0.01) Fig. 4 Expression of genes associated with airway epithelial basal cells, ciliogenesis, and epithelial tight junctions. Expression of TP63 (a), KRT5 (b), TUBB4A (c), TJP1 (d), OCLN (e), and FOXJ1 (f)by BECsatP1 (n =6 asthma donors, n = 6 healthy donors), P2 (n = 6 asthma donors, n = 6 healthy donors), P3 (n = 4 asthma donors, n = 6 healthy donors), P4 (n = 6 asthma donors, n = 6 healthy donors), and P5 (n =6 asthma donors, n = 6 healthy donors) are presented as box-and-whisker plots which depict the interquartile range and median (the ends of each box represent the upper and lower quartiles, error bars represent the maximum and minimum, and the horizontal line within the box represents the median). To compare expression of genes at P2-P5 to expression at P1, and to compare patterns of gene expression between asthmatic and healthy donors, ordinary two-way ANOVA with Dunnett’s multiple comparisons test was used for normally distributed data, and Kruskal-Wallis ANOVA with Dunn’smultiple comparisons test was used for non-normally distributed data for research purposes is ethically challenging. The initial children was both effective and safe. In a separate study, description of the procedure to obtain primary BECs for Lane and colleagues reported similar results in a cohort of research purposes from children already under anesthesia children with and without mild asthma . In that study, for a clinical indication was published by Doherty et al. in the authors included an additional control group of 2003 . In that study, the authors compared the yield and children who did not undergo bronchial brushings and viability of cells obtained via non-bronchoscopic airway compared post-operative symptoms between the groups. brushings through an endotracheal tube under general No significant risk of adverse symptoms was reported with anesthesia in 63 pediatric subjects to brushings obtained the most frequent symptom reported being a mild cough from a control population of adult patients undergoing in less than half of the participants that underwent the bronchoscopy. Doherty and colleagues reported that a simi- bronchial brushings. Similar to the results reported by lar number of cells were obtained via the blind bronchial Doherty et al., Lane and colleagues found that the brushing as compared to the brushings obtained during non-bronchoscopic brushings provided sufficient cells to bronchoscopy. Furthermore, the success rate of cell cultures carry out studies of RNA and protein-based assays, but obtained using this methodology was reported to be 82% also contained a sufficient population of basal cells to despite a trend to lower viability of the cells obtained via the propagate in ex vivo cell cultures over multiple cell blind bronchial brushings, suggesting that this method har- passages. Importantly, the subjects with mild asthma vested a sufficient amount of viable basal cells to establish displayed no greater risk of adverse outcomes compared the cell cultures . Importantly, no adverse events where to the healthy control subjects further demonstrating the reported following the bronchial brushings and the authors usefulness of this model in studying the role of BECs in concluded that this method of harvesting primary BECs in pediatric asthma. Relative expression compared to Passage 1 Relative expression compared to Passage 1 (normalized to GAPDH) (normalized to GAPDH) Relative expression compared to Passage 1 Relative expression compared to Passage 1 (normalized to GAPDH) (normalized to GAPDH) Relative expression compared to Passage 1 Relative expression compared to Passage 1 (normalized to GAPDH) (normalized to GAPDH) Reeves et al. BMC Pulmonary Medicine (2018) 18:91 Page 9 of 11 Optimal growth conditions for primary BECs have been other studies have compared primary BECs differentiated extensively studied and have been reported elsewhere in at an ALI to existing transformed or immortalized cell the literature (reviewed by Gruenert et al. ). Most pro- lines. In one such study, Stewart and colleagues compared tocols utilize a defined, serum-free media that has been two different donor-derived primary BECs with three optimized to exclude contaminating cells such as fibro- different available bronchial cell lines. In that study, the blasts. In addition to factors related to the growth media, primary BECs expressed several markers of BEC differen- the phenotype of the BECs grown in cell culture is critic- tiation and developed measurable trans-epithelial elec- ally dependent on the culture conditions. For example, trical resistance (TEER), albeit with intra-donor and cells can either be grown as a confluent monolayer in sub- intra-experimental variability . Measurements of TEER merged cell culture or grown in a semi-permeable trans- were more consistent in Calu-3 cells; however, these cells well insert at an air-liquid interface . Cells grown at an also displayed distinct disparities in their expression of ALI differentiate into a mucociliary phenotype that more several markers of epithelial differentiation when com- closely resembles the native human airway epithelium pared to the primary BECs. Another cell line examined in than submerged cultures . Recent studies have empha- that study (BEAS-2B) failed to differentiate at an ALI. sized differences in epithelial responses during stimulation These findings underscore the importance of choosing an experiments based on whether a submerged vs. differenti- appropriate model system for a given experimental ques- ated ALI culture model was used. For example, Kikuchi tion. Details regarding characteristics of available trans- and colleagues compared the responses of BECs grown at formed cell lines have been reviewed elsewhere by an ALI to submerged BECs cultures. Following stimula- Papazian et al. . tion with IL-4 or IL-13 no differences in STAT6 phos- In this study we assessed over serial cell passages in ALI phorylation were observed between BECs grown in cultures the stability of expression of genes associated with submerged culture or at an ALI . Conversely, the airway epithelial basal cells and structural features unique downstream effects of GM-CSF and TGFβ2 secretion in to differentiated airway epithelium. Interestingly, expres- these models were found to be markedly different leading sion of the airway epithelial basal cell associated gene p63 the authors to conclude that responses to IL-4 or IL-13 [18, 19] and epithelial marker cytokeratin 5 were are critically dependent on the cell culture model system overall stable in ALI cultures over 5 consecutive cell pas- utilized in the study. In a separate series of experiments sages, although there was a non-significant modest trend reported by Pezzulo et al., the authors examined the gene toward decreased and more variable p63 expression at P5 expression profile of primary BECs grown in either sub- and increased variability of cytokeratin 5 expression at P4 merged conditions or differentiated at an ALI and com- and P5. Expression of the cillogenesis-associated genes pared their findings to both the in vivo condition as well TUBB4A and FOXJ1 [21, 22] as well as the tight as to a BEC cell line using genome-wide transcriptional junctional-associated genes TJP1 and OCLN were profiling . The authors of that study demonstrated stable through P3, however, expression of both of these that BECs differentiated at an ALI not only displayed mor- genes become significantly more variable at passages be- phological characteristics most similar to the in vivo con- yond P3. In summary, similar to our observations for the dition (goblet cells, the presence of cilia, etc.), but also remodeling-associated genes, innate immune response most closely recapitulated the transcriptional profile of the genes, and immunomodulatory genes studied, expression native airway epithelium. Thus, the authors concluded of several genes associated with airway epithelial differen- that the primary BECs differentiated at an ALI most tiation were stable through P3, however, for later passages closely represented the biology of the airway epithelium. became significantly more variable. While primary BECs obtained via bronchial brushes or The present study design includes several important bronchoscopic biopsy differentiated at an ALI most strengths, including the use of primary BECs obtained closely resemble the in vivo airway epithelium, there are from asthmatic and non-asthmatic children that are several caveats that must also be taken into account that differentiated at an ALI. Additionally, our cohort is may limit their utility in some experimental models. carefully phenotyped based on medical history and Given that primary BECs are derived from individual hu- clinical features such as lung function and allergy test- man donors there is a significant degree of variability be- ing. Despite these strengths our study also has several tween BECs from different donors. Furthermore, the inherent limitations. In this study, our main outcome is findings in the present study would also suggest that cell the stability of gene expression over 5 successive pas- passage number also contributes to increased phenotypic sages in cell culture. We included cells obtained from variability. The need to utilize greater replicates during both asthmatic and healthy donors in order to ensure experiments significantly increases the timeline and ex- that stability of gene expression was generalizable to pense and may make primary cells less attractive for both groups. Indeed, we have demonstrated that BECs high-throughput screening studies. With this in mind, obtained from both asthmatic and non-asthmatic donors Reeves et al. BMC Pulmonary Medicine (2018) 18:91 Page 10 of 11 similarly display stable gene expression through passage Additional files 3. Variability in gene expression beyond passage 3 was Additional file 1 Primary quantitative PCR datasets. (XLSX 37 kb) also observed in BECs derived from asthmatic and Additional file 2: Figure S2. Comparison of gene expression between healthy donors to a similar degree. Although we did not asthmatic and healthy BECs at passage 1 (P1). Expression of genes related observe statistically significant differences in the stability to airway remodeling (panel A.; TGFβ1, TGFβ2, MUC5AC, activin A, and and variability of gene expression between BECs from FSTL3), innate immunity and immunomodulatory genes (panel B.; CXCL10, IFIH1, IL-33, and TSLP), and expression of genes associated with airway asthmatic and healthy donors, our sample size was insuf- epithelial basal cells, ciliogenesis, and epithelial tight junctions (panel C.; ficient to detect subtle differences in patterns of gene ex- TP63, KRT5, TUBB4A, TJP1, OCLN, and FOXJ1) by primary asthmatic (grey pression with increasing passage between BECs from plots) and healthy (white plots) BECs at P1 (n = 6 asthma donors, n =6 healthy donors). Expression of each gene (normalized to GAPDH) relative to asthmatic and healthy children. Furthermore, this study the median of healthy BECs are presented as box-and-whisker plots which is also underpowered to perform subgroup analysis of depict the interquartile range and median (the ends of each box represent the data such as gender differences. Lung function data the upper and lower quartiles, error bars represent the maximum and minimum, and the horizontal line within the box represents the median). demonstrates that our cohort of asthmatic donors have The Wilcoxon signed rank test was used to test differences in expression of a mild degree of airflow limitation signifying a relatively specific genes between asthmatic and healthy BECs. (EMF 109 kb) mild asthma phenotype in our cohort. Additional file 3: Figure S1. Expression of GAPDH as a reference gene. Additional limitations of our study include that we did Ct values for GAPDH were compared for each cell passage. No significant differences were observed from P1 through P5. (TIF 73 kb) not perform TEER measurements over serial passages Additional file 4: Figure S3. Un-normalized mRNA expression of genes and did not perform histological sections and/or immu- related to airway remodeling by primary BECs. Un-normalized mRNA Ct nostaining of our ALI cultures for basal cell markers or values for TGFβ1 (A.), TGFβ2 (B.), MUC5AC (C.), activin A (D.), and FSTL3 proteins associated with airway epithelial cell differenti- (E.) by BECs at P1 (n = 6 asthma donors, n = 6 healthy donors), P2 (n =6 asthma donors, n = 6 healthy donors), P3 (n = 4 asthma donors, n =6 ation. Although beyond the scope of the current study, healthy donors), P4 (n = 6 asthma donors, n = 6 healthy donors), and P5 such outcome measures would be of interest in future (n = 6 asthma donors, n = 6 healthy donors) are presented as individual studies of primary airway epithelial cells over serial cul- data points for each donor cell line. To compare expression of genes at P2-P5 to expression at P1, and to compare patterns of gene expression ture passages. We did however study the expression sta- between asthmatic and healthy donors, ordinary two-way ANOVA with bility of genes associated with airway epithelial basal Dunnett’s multiple comparisons test was used for normally distributed cells, ciliogenesis, and epithelial tight junctions. Expres- data, and Kruskal-Wallis ANOVA with Dunn’s multiple comparisons test was used for non-normally distributed data. (EMF 195 kb) sion of the basal cell-associated gene TP63 as well as Additional file 5: Figure S4. Un-normalized mRNA expression of innate cytokeratin 5 were stable through P5, whereas expres- immunity and immunomodulatory genes by primary BECs. Un- sion of genes associated with ciliogenesis and tight junc- normalized mRNA Ct values for CXCL10 (A.), IFIH1 (B.), IL-33 (C.), and TSLP tions were less stable beyond P3. Of note, several groups (D.) by BECs at P1 (n = 6 asthma donors, n = 6 healthy donors), P2 (n =6 asthma donors, n = 6 healthy donors), P3 (n = 4 asthma donors, n =6 have demonstrated over the past several years that air- healthy donors), P4 (n = 6 asthma donors, n = 6 healthy donors), and P5 way epithelial basal cells can be expanded in culture and (n = 6 asthma donors, n = 6 healthy donors) are presented as individual retain their ability to differentiate at the ALI many pas- data points for each donor cell line. To compare expression of genes at P2-P5 to expression at P1, and to compare patterns of gene expression sages removed from the host [21, 24]. A final limitation between asthmatic and healthy donors, ordinary two-way ANOVA with of our study is that we did not analyze airway epithelial Dunnett’s multiple comparisons test was used for normally distributed gene expression across serial passages from submerged data, and Kruskal-Wallis ANOVA with Dunn’s multiple comparisons test was used for non-normally distributed data. (EMF 155 kb) undifferentiated cultures. Additional file 6: Figure S5. Un-normalized mRNA expression of genes associated with airway epithelial basal cells, ciliogenesis, and epithelial tight Conclusions junctions by primary BECs. Un-normalized mRNA Ct values for TP63 (A.), KRT5 While ex vivo primary BECs differentiated at an ALI (B.), TUBB4A (C.), TPJ1 (D.), FOXJ1 (E.), and OCLN (F.) by BECs at P1 (n =6 asthma donors, n = 6 healthy donors), P2 (n = 6 asthma donors, n =6 healthy represent one of the best available models to study the donors), P3 (n = 4 asthma donors, n = 6 healthy donors), P4 (n =6 asthma role of the airway epithelium in disease processes such donors, n = 6 healthy donors), and P5 (n = 6 asthma donors, n =6 healthy as asthma in children, care must be taken to ensure that donors) are presented as individual data points for each donor cell line. To compare expression of genes at P2-P5 to expression at P1, and to compare cell phenotype and gene expression patterns are pre- patterns of gene expression between asthmatic and healthy donors, ordinary served such that ex vivo studies reflect the in vivo condi- two-way ANOVA with Dunnett’s multiple comparisons test was used for tion as closely as possible. We have provided new normally distributed data, and Kruskal-Wallis ANOVA with Dunn’smultiple comparisons test was used for non-normally distributed data. (EMF 224 kb) evidence that primary BECs from children differentiated at an ALI display stable gene expression patterns over 3 successive passages; however, we have also shown that Abbreviations gene expression becomes significantly more variable at ALI: Air-liquid interface; BDR: Bronchodilator responsive; BEC: Bronchial epithelial later passages, which could potentially affect study out- cell; BEGM: Bronchial epithelial growth medium; FEF : Forced expiratory flow 25-75 between 25 and 75% of FVC; FENO: Fraction of exhaled nitric oxide; FEV1: Forced comes if later passages are used in experiments. These expiratory volume in 1s;FVC:Forcedvital capacity; GAPDH:Glyceraldehyde3- findings should be carefully considered in future study phosphate dehydrogenase; ICS: Inhaled corticosteroid; IgE: Immunoglobulin E; designs using primary BECs in ex vivo model systems. RAST: Radioallergosorbent testing; RNA: Ribonucleic acid Reeves et al. BMC Pulmonary Medicine (2018) 18:91 Page 11 of 11 Funding 14. Karp PH, Moninger TO, Weber SP, Nesselhauf TS, Launspach JL, National Heart, Lung, and Blood Institute (JD: R01HL128361, SR: K08HL135266), Zabner J, Welsh MJ. An in vitro model of differentiated human airway National Institute of Allergy and Infectious Diseases (JD: U19 AI125378), Parker B. epithelia. Methods for establishing primary cultures. Methods Mol Biol. Francis Foundation Fellowship (SR). 2002;188:115–37. 15. Kikuchi T, Shively JD, Foley JS, Drazen JM, Tschumperlin DJ. Differentiation- Availability of data and materials dependent responsiveness of bronchial epithelial cells to IL-4/13 The primary quantitative PCR datasets used and/or analyzed for this study are stimulation. Am J Physiol Lung Cell Mol Physiol. 2004;287:L119–26. provided in Additional file 1: Appendix I. 16. Pezzulo AA, Starner TD, Scheetz TE, Traver GL, Tilley AE, Harvey BG, Crystal RG, McCray PB Jr, Zabner J. The air-liquid interface and use of primary cell Authors’ contributions cultures are important to recapitulate the transcriptional profile of in vivo Conception and Design: SR, JD, KB; Conducted Experiments: KB, MW, MN, LR; airway epithelia. Am J Physiol Lung Cell Mol Physiol. 2011;300:L25–31. Drafted the Manuscript, Critical Revision: KB, SR, MW, MN, JD; All authors 17. Papazian D, Wurtzen PA, Hansen SW. Polarized airway epithelial models for have read and approved the manuscript. immunological co-culture studies. Int Arch Allergy Immunol. 2016;170:1–21. 18. Hackett TL, Singhera GK, Shaheen F, Hayden P, Jackson GR, Hegele RG, Van Ethics approval and consent to participate Eeden S, Bai TR, Dorscheid DR, Knight DA. Intrinsic phenotypic differences Written consent was obtained from parents of subjects and assent was obtained of asthmatic epithelium and its inflammatory responses to respiratory for children ≥ age10years to participateinthis. Theworkpresented in this study syncytial virus and air pollution. Am J Respir Cell Mol Biol. 2011;45:1090- was approved by the Seattle Children’s Hospital Institutional Review Board. 1100. 19. Warner SM, Hackett TL, Shaheen F, Hallstrand TS, Kicic A, Stick SM, Knight Competing interests DA. Transcription factor p63 regulates key genes and wound repair in The authors declare that they have no competing interests, financial or non- human airway epithelial basal cells. Am J Respir Cell Mol Biol. 2013;49:978- financial. 988. 20. Hackett TL, Shaheen F, Johnson A, Wadsworth S, Pechkovsky DV, Jacoby DB, Kicic A, Stick SM, Knight DA. Characterization of side population cells from Publisher’sNote human airway epithelium. Stem Cells. 2008;26:2576-2585. Springer Nature remains neutral with regard to jurisdictional claims in published 21. Walters MS, Gomi K, Ashbridge B, Moore MA, Arbelaez V, Heldrich J, Ding maps and institutional affiliations. BS, Rafii S, Staudt MR, Crystal RG. 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Doherty GM, Christie SN, Skibinski G, Puddicombe SM, Warke TJ, de Courcey F, Cross AL, Lyons JD, Ennis M, Shields MD, Heaney LG. Non-bronchoscopic sampling and culture of bronchial epithelial cells in children. Clin Exp Allergy. 2003;33:1221–5. 8. Lane C, Burgess S, Kicic A, Knight D, Stick S. The use of non-bronchoscopic brushings to study the paediatric airway. Respir Res. 2005;6:53. 9. Pierrou S, Broberg P, O'Donnell RA, Pawlowski K, Virtala R, Lindqvist E, Richter A,WilsonSJ, Angco G,MollerS, etal. Expression of genes involved in oxidative stress responses in airway epithelial cells of smokers with chronic obstructive pulmonary disease. Am J Respir Crit Care Med. 2007;175:577–86. 10. Gras D, Petit A, Charriot J, Knabe L, Alagha K, Gamez AS, Garulli C, Bourdin A, Chanez P, Molinari N, Vachier I. Epithelial ciliated beating cells essential for ex vivo ALI culture growth. BMC Pulm Med. 2017;17:80. 11. 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BMC Pulmonary Medicine – Springer Journals
Published: May 29, 2018
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