Fibroblast-myofibroblast transition is differentially regulated by bronchial epithelial cells from asthmatic children

Abstract

BackgroundAirway remodeling is a proposed mechanism that underlies the persistent loss of lung function associated with childhood asthma. Previous studies have demonstrated that human lung fibroblasts (HLFs) co-cultured with primary human bronchial epithelial cells (BECs) from asthmatic children exhibit greater expression of extracellular matrix (ECM) components compared to co-culture with BECs derived from healthy children. Myofibroblasts represent a population of differentiated fibroblasts that have greater synthetic activity. We hypothesized co-culture with asthmatic BECs would lead to greater fibroblast to myofibroblast transition (FMT) compared to co-culture with healthy BECs.MethodsBECs were obtained from well-characterized asthmatic and healthy children and were proliferated and differentiated at an air-liquid interface (ALI). BEC-ALI cultures were co-cultured with HLFs for 96 hours. RT-PCR was performed in HLFs for alpha smooth muscle actin (α-SMA) and flow cytometry was used to assay for α-SMA antibody labeling of HLFs. RT-PCR was also preformed for the expression of tropomyosin-I as an additional marker of myofibroblast phenotype. In separate experiments, we investigated the role of TGFβ2 in BEC-HLF co-cultures using monoclonal antibody inhibition.ResultsExpression of α-SMA by HLFs alone was greater than by HLFs co-cultured with healthy BECs, but not different than α-SMA expression by HLFs co-cultured with asthmatic BECs. Flow cytometry also revealed significantly less α-SMA expression by healthy co-co-cultures compared to asthmatic co-cultures or HLF alone. Monoclonal antibody inhibition of TGFβ2 led to similar expression of α-SMA between healthy and asthmatic BEC-HLF co-cultures. Expression of topomyosin-I was also significantly increased in HLF co-cultured with asthmatic BECs compared to healthy BEC-HLF co-cultures or HLF cultured alone.ConclusionThese findings suggest dysregulation of FMT in HLF co-cultured with asthmatic as compared to healthy BECs. Our results suggest TGFβ2 may be involved in the differential regulation of FMT by asthmatic BECs. These findings further illustrate the importance of BEC-HLF cross-talk in asthmatic airway remodeling.

Interplay of extracellular matrix and leukocytes in lung inflammation

“…In vivo animal models of airway remodeling [147–149] and descriptive data from human bronchial biopsies [150–152] suggest the airway epithelium secretes proteins that regulate lung fibroblasts and airway remodeling with increased ECM deposition, including TGF-β [150, 153–155], VEGF [151, 156–159], periostin [160, 161], activin A [162–164], and follistatins [165, 166]. Furthermore, recent ex vivo investigations using primary bronchial epithelial cells (BECs) from asthmatic and healthy children demonstrated that when co-cultured with BECs from healthy children, lung fibroblast expression of types I and III collagen, hyaluronan, as well as expression of α-SMA, indicative of a myofibroblast phenotype, are downregulated [167, 168]. This downregulation is diminished in fibroblasts co-cultured with asthmatic BECs [167, 168], suggesting that, in addition to stimulatory signals, there must be epithelial-derived factors that inhibit fibroblasts and FMT, such that in normal airways, a balance of epithelial-secreted stimulatory and inhibitory factors that regulate fibroblasts and ECM deposition.…”

“…Furthermore, recent ex vivo investigations using primary bronchial epithelial cells (BECs) from asthmatic and healthy children demonstrated that when co-cultured with BECs from healthy children, lung fibroblast expression of types I and III collagen, hyaluronan, as well as expression of α-SMA, indicative of a myofibroblast phenotype, are downregulated [167, 168]. This downregulation is diminished in fibroblasts co-cultured with asthmatic BECs [167, 168], suggesting that, in addition to stimulatory signals, there must be epithelial-derived factors that inhibit fibroblasts and FMT, such that in normal airways, a balance of epithelial-secreted stimulatory and inhibitory factors that regulate fibroblasts and ECM deposition. Other studies have shown that airway epithelial cells synthesize hyaluronan and its degradative enzymes, the hyaluronidases (Hyals), in response to oxidative stress and other injurious agents [169–174] and that hyaluronan-enriched ECM synthesized by respiratory epithelial cells can impact monocyte adhesion [175].…”

Bronchial epithelium in children: a key player in asthma

“…It begins with a dedifferentiation period, then using an air-liquid interface culture allows a 21-28 day period for production of fully differentiated pseudostratified bronchial epithelium similar to the epithelium observed on the initial endobronchial biopsies (figure 2) [10]. These culture systems can also mimic cellular crosstalk through the air-blood barrier when co-culturing bronchial epithelial cells with inflammatory cells such as mast cells [20], fibroblasts [21] and macrophages [22]. The culture of bronchial epithelial cells allows the differentiation process in health and disease to be understood.…”

Asthma management in pediatric age group

“…Transformed deposition and composition of extracellular matrix proteins and hyperplasia of mucous gland is also noted. 15 One noteworthy characteristic of the epithelium is its ability to defend itself against oxidant injury, which is a characteristic enabling us to partially explain why asthmatic patients are hypersensitive to oxidant pollutants such as tobacco smoke. 16…”