Eosinophils promote epithelial to mesenchymal transition of bronchial epithelial cells

Abstract

Eosinophilic inflammation and remodeling of the airways including subepithelial fibrosis and myofibroblast hyperplasia are characteristic pathological findings of bronchial asthma. Epithelial to mesenchymal transition (EMT) plays a critical role in airway remodelling. In this study, we hypothesized that infiltrating eosinophils promote airway remodelling in bronchial asthma. To demonstrate this hypothesis we evaluated the effect of eosinophils on EMT by in vitro and in vivo studies. EMT was assessed in mice that received intra-tracheal instillation of mouse bone marrow derived eosinophils and in human bronchial epithelial cells co-cultured with eosinophils freshly purified from healthy individuals or with eosinophilic leukemia cell lines. Intra-tracheal instillation of eosinophils was associated with enhanced bronchial inflammation and fibrosis and increased lung concentration of growth factors. Mice instilled with eosinophils pre-treated with transforming growth factor(TGF)-β1 siRNA had decreased bronchial wall fibrosis compared to controls. EMT was induced in bronchial epithelial cells co-cultured with human eosinophils and it was associated with increased expression of TGF-β1 and Smad3 phosphorylation in the bronchial epithelial cells. Treatment with anti-TGF-β1 antibody blocked EMT in bronchial epithelial cells. Eosinophils induced EMT in bronchial epithelial cells, suggesting their contribution to the pathogenesis of airway remodelling.

Figure 1. Inflammatory cells and elevation of cytokines in eosinophil-treated mice.

(A) The total cell count in BALF was significantly increased in the group receiving instillation of eosinophils compared to control mice (each group, n = 4). (B) Differential cell count showed significantly increased number of macrophages and lymphocytes in eosinophil-instilled mice compared to controls. (C) Mice intratracheally instilled with eosinophils had significantly elevated levels of MCP-1/CCL2 and TGF-β1 in BALF compared to controls. IFN-γ and IL-13 showed no significant change. Data are expressed as means ± SEM. *P<0.05; ^§P = 0.05.

Figure 2. Inflammation and fibrosis of the airway walls induced by eosinophils instillation in vivo.

(A–F) Representative lung sections from control (n = 3; A, C, E) and eosinophil-instilled mice (n = 4; B, D, F). Specimens were stained with Hematoxylin-Eosin (A, B), Masson trichrome (C, D) and immunostained for type I collagen (E, F) and examined by light microscopy. Mice receiving intra-tracheal eosinophils had increased infiltration of inflammatory cells around the bronchi (B), peribronchial fibrosis (D) and deposition of type I collagen (F) in lung tissue compared to controls (A, C and E). (G, H) Collagen deposition (G) and immunoreactive areas for type I collagen (H) were quantified using WinROOF software. Lung tissue of eosinophil-instilled mice showed significant deposition of collagen as compared to control. The data are expressed as the mean percentage of the total lung field area ± S.E.M. The scale bars indicate 50 µm. Data are expressed as means ± SEM. *P<0.05.

Figure 3. Induction of epithelial–mesenchymal transition in the airways by eosinophils in vivo.

(A, B) Representative immunofluorescence staining of E-cadherin (green), α-SMA (red) and nuclei (blue) in the airways of mice instilled with saline (A) or eosinophils (B). (C, D) Quantification by densitometry disclosed low expression of E-cadherin (C) and increased expression of α-SMA (D) in mice after eosinophil instillation (n = 4) compared to control (n = 3). The data are expressed as the mean percentage of the total lung field area ± SEM. The scale bars indicate 50 µm. Data are expressed as means ± SEM. *P<0.05.

Figure 4. Human eosinophils induced EMT morphological changes in BEAS-2B cells.

(A, D) control, (B, E) BEAS-2B cells co-cultured with eosinophils. (C, F) BEAS-2B cells cultured in the presence of TGF-β1. BEAS-2B cells were stained with Diff-Quick after washing out eosinophils (A, B, C). (D, E, F) Immunofluorescence staining was performed with phalloidin (red) and DAPI for nuclei staining (blue) after washing out eosinophils. The scale bars indicate 50 µm. (G, H, I) RNA expression of E-cadherin and vimentin in BEAS-2B cells co-cultured with or without eosinophils and in the presence of TGF-β1 as assessed by RT-PCR and densitometry analysis. (K, L, M) Protein expression of E-cadherin and αSMA in BEAS-2B cells co-cultured with or without eosinophils as assessed by Western blotting and densitometry analysis. Data are expressed as means ± SEM. **P<0.01; *P<0.05.

Figure 5. Eosinophils induced secretion of TGF-β1 from BEAS-2B cells.

(A) TGF-β1 levels in the supernatant from each cell condition. (B) Representative Western blotting of Smad3 and p-Smad3. (C) The ratio of p-Smad3 to Smad3 analyzed by densitometry. (D, E, F) Morphological changes in each cell condition. (G, H, I, J) Effect of anti-TGF-β1 antibody on the protein level of TGF-β1 and RNA expression of E-cadherin and vimentin during each cell condition. BEAS-2B cells were stained with DIFF-QUICK. The scale bars indicate 50 µm. Data are expressed as means ± SEM. *P<0.05.

Figure 6. Relevant role of TGF-β1 in airway remodeling and the need of direct contact of eosinophils for TGF-β1 production.

(A, B, C) Mice receiving instillation of eosinophils pre-treated with TGF-β1 siRNA have less peribronchial fibrosis than those receiving eosinophils pre-treated with scrambled RNA as assessed by Masson staining. (D) Quantification of Masson positive areas disclosed significant difference. (E, F) TGFβ1 levels in the supernatant in Boyden chamber assay and using 2% paraformaldehyde-fixed eosinophils. Culture supernatants were used to measure TGF-β1 by ELISA. Data are expressed as means ± SEM. **P<0.05. The scale bars indicate 100 µm. Data are expressed as means ± SEM. **P<0.05.

Figure 7. Eol-1 cell lines induce EMT in BEAS-2B cells by increasing TGF-β1 expression.

(A, B) Control and BEAS-2B cells co-cultured with EoL-1 cells. (C) BEAS-2B cells co-cultured with EoL-1 cells in the presence of anti-TGF-β1 neutralizing antibody. BEAS-2B cells lysates were subjected to RT-PCR analysis. The expression level of E-cadherin (D) and vimentin (E) mRNA was measured by RT-PCR and normalized to that of GAPDH mRNA. TGF-β1 (F) levels in the supernatant from each culture condition, and the ratio of p-Smad3 to Smad3 (G) in BEAS-2B cells as analyzed by densitometry. The scale bars indicate 50 µm. Data are expressed as means ± SEM. *P<0.05.

Figure 8. Eol-1 cells induce EMT in BEAS-2B cells via the JNK/Smad3 pathway.

(A) The concentration of TGF-β1 in the supernatant of BEAS-2B in the presence of PI3 kinase (LY294002), JNK (SP600125), MEK1/2 (MUO126) or p38 kinase (SB203580) inhibitor. Inhibitors of both PI3 and JNK kinases suppress the morphological features (B, C, D, E) and molecular markers (F, G, H) of EMT. Data are expressed as means ± SEM. *P<0.05; **P<0.01.