Fibroblast gene expression following asthmatic bronchial epithelial cell conditioning correlates with epithelial donor lung function and exacerbation history

www.nature.com/scientificreports OPEN Received: 22 May 2018 Accepted: 6 October 2018 Published: xx xx xxxx Fibroblast gene expression following asthmatic bronchial epithelial cell conditioning correlates with epithelial donor lung function and exacerbation history Stephen R. Reeves1,2,3, Kaitlyn A. Barrow2, Tessa K. Kolstad2, Maria P. White2, Lucille M. Rich2, Thomas N. Wight4 & Jason S. Debley 1,2,3 Airway remodeling may contribute to decreased lung function in asthmatic children. Bronchial epithelial cells (BECs) may regulate fibroblast expression of extracellular matrix (ECM) constituents and fibroblast-to-myofibroblast transition (FMT). Our objective was to determine if human lung fibroblast (HLF) expression of collagen I (COL1A1), hyaluronan synthase 2 (HAS2), and the FMT marker alpha-smooth muscle actin (α-SMA) by HLFs conditioned by BECs from asthmatic and healthy children correlate with lung function measures and exacerbation history among BEC donors. BECs from asthmatic (n = 23) and healthy children (n = 15) were differentiated at an air-liquid interface (ALI) and then co-cultured with HLFs for 96 hours. Expression of COL1A1, HAS2, and α-SMA by HLFs was determined by quantitative polymerase chain reaction (qPCR). FMT was quantified by measuring HLF cytoskeletal α-SMA by flow cytometry. Pro-collagen Iα1, hyaluronan (HA), and PGE2 were measured in BEC-HLF supernatant. Correlations between lung function measures of BEC donors, and COL1A1, HAS2, and α-SMA gene expression, as well as supernatant concentrations of HA, pro-collagen Iα1, hyaluronan (HA), and PGE2 were assessed. We observed that expression of α-SMA and COL1A1 by HLFs co-cultured with asthmatic BECs was negatively correlated with BEC donor lung function. BEC-HLF supernatant concentrations of pro-collagen Iα1 were negatively correlated, and PGE2 concentrations positively correlated, with asthmatic BEC donor lung function. Expression of HAS2, but not α-SMA or COL1A1, was greater by HLFs co-cultured with asthmatic BECs from donors with a history of severe exacerbations than by HLFs co-cultured with BECs from donors who lacked a history of severe exacerbations. In conclusion, α-SMA and COL1A1 expression by HLFs co-cultured with BECs from asthmatic children were negatively correlated with lung function measures, supporting our hypothesis that epithelial regulation of HLFs and airway deposition of ECM constituents by HLFs contributes to lung function deficits among asthmatic children. Furthermore, epithelial regulation of airway HAS2 may influence the susceptibility of children with asthma to experience severe exacerbations. Finally, epithelial-derived PGE2 is a potential regulator of airway FMT and HLF production of collagen I that should be investigated further in future studies. Asthma is the most prevalent chronic lung disease of childhood affecting an estimated 14% of the world’s pediatric population1. Longitudinal studies in asthmatic children have demonstrated lung function deficits that persist 1 Division of Pulmonary and Sleep Medicine, Seattle Children’s Hospital, Seattle, WA, USA. 2Center for Immunity and Immunotherapies, Seattle Children’s Research Institute, Seattle, WA, USA. 3Department of Pediatrics, University of Washington, Seattle, WA, USA. 4Matrix Biology Program, Benaroya Research Institute, Seattle, WA, USA. Correspondence and requests for materials should be addressed to J.S.D. (email: ) SCIentIfIC REportS | (2018) 8:15768 | DOI:10.1038/s41598-018-34021-6 1 www.nature.com/scientificreports/ Asthmatic Subjects Healthy Subjects n = 23 n = 15 P Value Age yrs (mean ± SD) 12.2 (3.0) 11.5 (3.3) 0.5 Sex (% male) 57% 47% 0.6 Currently using inhaled steroids (%) 13 (57%) History of Allergic Rhinitis, n; (%) 19 (83%) History of Eczema, n; (%) 11 (48%) Positive RAST, n; (%) 18 (78%) IgE IU/mL (median ± SD) 273.6 (421.2) 15.3 (11.3) <0.001 FVC % predicted (mean ± SD) 100.3 (12.9) 104.7 (12.9) 0.2 FEV1% predicted (mean ± SD) 87.7 (13.0) 103.9 (11.9) 0.001 FEV1/FVC Ratio (mean ± SD) 0.79 (0.06) 0.90 (0.1) <0.001 FEF25–75% predicted (mean ± SD) 68.5 (24.6) 98.0 (20.8) <0.001 FENO ppb (mean ± SD) 28.1 (35.7) 10.5 (6.2) 0.03 Table 1. Subject Characteristics. RAST, Radioallergosorbent testing; FVC, Forced vital capacity; FEV1, Forced expiratory volume in one second; FEF25–75, Forced expiratory flow between 25% and 75% of expiration. into adulthood2,3. One possible mechanism explaining differences in lung function observed between asthmatic and healthy individuals is airway remodeling. Airway remodeling encompasses multiple pathologic changes that have been observed in asthmatic airways4. In adult asthma, basement membrane thickening has been well studied and is considered pathognomonic of the disease. Fewer studies of biopsy specimens exist in children; however, both qualitative5,6 and quantitative7,8 data have demonstrated increased airway basement membrane thickness in children with asthma. Additional studies have shown that basement membrane thickness at infancy does not predict subsequent asthma9. Taken together, data from both epidemiologic and pathologic studies support the premise that airway remodeling in asthmatic individuals is not present early in life, but evolves during childhood and persists into adulthood. There has been increasing focus on the role of the airway epithelium as a driver of asthma pathogenesis given that bronchial epithelial cells (BECs) are the initial point of contact between the environment and the host10. Prior work from our laboratory has demonstrated increased expression of pro-remodeling signaling mediators by primary BECs obtained from asthmatic children in well-differentiated air-liquid interface (ALI) cultures11. Further studies have demonstrated that healthy human lung fibroblasts (HLFs) co-cultured with differentiated BECs display greater production of extracellular matrix (ECM) components including type I and III collagen, hyaluronan (HA), and fibronectin when co-cultured with primary BECs obtained from asthmatic donors12. Separate studies have also confirmed increased expression of alpha smooth muscle actin (α-SMA) and tropomyosin-I from HLFs co-cultured with asthmatic BECs compared to healthy BECs indicative of a greater fibroblast to myofibroblast transition (FMT)13. To investigate potential associations between ex vivo BEC regulation of HLFs, and the lung function and exacerbation history of asthmatic BEC donors, we utilized our primary differentiated BEC/HLF co-culture model and medical history and spirometry data from healthy and asthmatic children who donated BECs. We tested the hypothesis that lung function and/or exacerbation history of BEC donors would be associated with the expression of genes related to airway remodeling by HLFs conditioned by BECs from children with asthma. Specifically, we hypothesized that expression of genes related to FMT (α-SMA) and ECM production [collagen I (COL1A1) and hy (...truncated)