Abstract
BackgroundInflammatory cytokines (e.g. IL-13) and mechanical perturbations (e.g. scrape injury) to the epithelium release profibrotic factors such as TGF-β2, which may, in turn, stimulate subepithelial fibrosis in asthma. We hypothesized that prolonged IL-13 exposure creates a plastic epithelial phenotype that is profibrotic through continuous secretion of soluble mediators at levels that stimulate subepithelial fibrosis.MethodsNormal human bronchial epithelial cells (NHBE) were treated with IL-13 (0, 0.1, 1, or 10 ng/ml) for 14 days (day 7 to day 21 following seeding) at an air-liquid interface during differentiation, and then withdrawn for 1 or 7 days. Pre-treated and untreated NHBE were co-cultured for 3 days with normal human lung fibroblasts (NHLF) embedded in rat-tail collagen gels during days 22–25 or days 28–31.ResultsIL-13 induced increasing levels of MUC5AC protein, and TGF-β2, while decreasing β-Tubulin IV at day 22 and 28 in the NHBE. TGF-β2, soluble collagen in the media, salt soluble collagen in the matrix, and second harmonic generation (SHG) signal from fibrillar collagen in the matrix were elevated in the IL-13 pre-treated NHBE co-cultures at day 25, but not at day 31. A TGF-β2 neutralizing antibody reversed the increase in collagen content and SHG signal.ConclusionProlonged IL-13 exposure followed by withdrawal creates an epithelial phenotype, which continuously secretes TGF-β2 at levels that increase collagen secretion and alters the bulk optical properties of an underlying fibroblast-embedded collagen matrix. Extended withdrawal of IL-13 from the epithelium followed by co-culture does not stimulate fibrosis, indicating plasticity of the cultured airway epithelium and an ability to return to a baseline. Hence, IL-13 may contribute to subepithelial fibrosis in asthma by stimulating biologically significant TGF-β2 secretion from the airway epithelium.
Highlights
Inflammatory cytokines (e.g. IL-13) and mechanical perturbations to the epithelium release profibrotic factors such as TGF-β2, which may, in turn, stimulate subepithelial fibrosis in asthma
Using a co-culture model of fully mucociliary-differentiated normal human bronchial epithelial cells and normal human lung fibroblasts embedded in a collagen gel [27,28,29], we found that prolonged (14 days) exposure to IL-13 during the differentiation phase induced an increase in MUC5AC expression which persisted for up to seven days following withdrawal of IL-13
IL-13 treatment and plasticity of the Normal human bronchial epithelial cells (NHBE) At day 22, after 14 days treatment with 1 or 10 ng/ml of IL-13 followed by 1-day withdrawal of IL-13, the NHBE demonstrate a increase in MUC5AC protein as detected by immunofluorescence (Fig. 2A)
Summary
Inflammatory cytokines (e.g. IL-13) and mechanical perturbations (e.g. scrape injury) to the epithelium release profibrotic factors such as TGF-β2, which may, in turn, stimulate subepithelial fibrosis in asthma. Therapies using corticosteroids and β2 agonists alleviate inflammation and improve pulmonary airflow in mild to moderate asthma; their efficacy in reversing structural remodeling in the airways of chronic asthmatics has been limited leading to an impaired quality of life, significant airflow obstruction, bronchial hyperresponsiveness, and decline in lung function [2,3,4,5,6,7,8,9,10]. The mechanisms underlying these airway structural changes are complex, and only partially understood
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