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. 2019 Sep;61(3):322–331. doi: 10.1165/rcmb.2018-0326OC

Figure 3.

Figure 3.

Inhibition of bone morphogenetic protein (BMP)/transforming growth factor (TGF)-β/SMAD signaling amplifies IL-13– and IL-17A–induced goblet cell metaplasia and hyperplasia (GCMH) in human airway epithelium. (A) Human airway basal cells derived from healthy donors (n = 4) were cultured on ALI for 16 days to establish differentiated airway epithelium. IL-13 (20 ng/ml) alone or together with BMP4 (100 ng/ml), TGF-β (10 ng/ml), DMH-1 (1 μM), or A-8301 (1 μM) were added to the ALI medium in the basolateral chamber and ALIs were cultured for another 4 days. (B) ALI membranes were fixed and stained for MUC5AC. Representative images shown here were from one representative experiment. Scale bars: 100 μm. (C) Quantification of MUC5AC production in ALI membranes generated from the experiment shown in B. MUC5AC index was scored using ImageJ software (National Institutes of Health) based on the total area of MUC5AC immunopositivity out of the imaging area normalized to the control without IL-13 stimulation (mean ± SD; n = 3 independent imaging fields; **P ≤ 0.001 and ***P ≤ 0.0001). (D) Immunoblot analysis of FOXA3 and SAM pointed domain containing ETS transcription factor (SPDEF) of ALI cultures generated as described in B. β-actin was used as loading control. (E) Human airway basal cells from donors with asthma (n = 2), cystic fibrosis (CF) (n = 3), and chronic obstructive pulmonary disease (COPD; n = 2) were cultured on ALI for 16 days and then treated with IL-13 (20 ng/ml), IL-13 with BMP (100 ng/ml) and TGF-β (10 ng/ml), or IL-13 with DMH-1 (1 μM) and A8301 (1 μM) for 4 days. After treatment, ALI membranes were fixed and stained for MUC5AC. Representative images shown here were from one representative experiment on representative donors. Scale bars: 100 μm. (F) Quantification of MUC5AC in the experiment described in E. A total of three independent areas were imaged for each sample and quantified (mean ± SD; n = 3 independent imaging fields; **P ≤ 0.001 and ***P ≤ 0.0001) (G) Human airway basal cells derived from a healthy donor and a donor with asthma were cultured on ALI for 16 days and then treated with IL-17A (50 ng/ml), IL-17A with BMP (100 ng/ml) and TGF-β (10 ng/ml), or IL-17A with DMH-1 (1 μM) and A8301 (1 μM) for 4 days. ALI membranes were fixed and stained with MUC5AC. Representative images shown here are from one representative experiment on representative donors. Scale bars: 100 μm. (H) Quantification of MUC5AC in the experiment described in G. MUC5AC index was scored as described previously here. A total of three independent areas were imaged and quantified (mean ± SD; n = 3 independent imaging fields; ***P ≤ 0.0001 and **P ≤ 0.001). DMH-1 = a BMP-SMAD pathway inhibitor.