Cigarette and IL-17A synergistically induce bronchial epithelial-mesenchymal transition via activating IL-17R/NF-κB signaling

Abstract

Background: IL-17A directly induces epithelial-mesenchymal transition (EMT) in alveolar epithelial cells. It could coordinate with cigarette smoke extract (CSE) to promote proliferation of bronchial epithelial cells. In this study, we aim to explore the direct effect of IL-17A and CSE on EMT in bronchial epithelial cells.

Methods: Bronchial epithelial cells were isolated from C57BL/6 mice, and cocultured with CSE or/and IL-17A. E-cadherin and Vimentin expressions in cells were detected using immunofluorescence staining. IL-17R expression was detected using immunohistochemistry staining. NF-κB expression was assessed using western blotting. When NF-κB was inhibited by BAY 11-7821, expressions of NF-κB, E-cadherin and Vimentin were measured.

Results: The protein expression of E-cadherin in bronchial epithelial cells was lowest in CSE + IL-17A group, followed by CSE group. In contrast, the protein expression of Vimentin was highest in CSE + IL-17A group, followed by CSE group. Similarly, IL-17R and NF-κB expressions were highest in CSE + IL-17A group, followed by CSE group and IL-17A group. NF-κB inhibitor could inhibit the expressions of E-cadherin and Vimentin.

Conclusions: Cigarette and IL-17A could synergistically induce EMT in bronchial epithelial cells through activating IL17R/NF-κB signaling. Our findings contribute to a better understanding in airway EMT and pathogenesis of respiratory diseases, which are involved IL-17A and cigarette smoking. Those will provide novel avenues in the immunotherapy of lung diseases.

Keywords: Bronchial epithelial cell; Cigarette smoke extract; Epithelial-mesenchymal transition; IL-17; NF-κB.

Fig. 1

Cells identification. When bronchial epithelial cells were isolated and cultured, cells were identified by immunofluorescence staining of CK-18. Cells were mainly CK-18+ staining. (× 400 magnification)

Fig. 2

IL-17R expression in bronchial epithelial cells. When bronchial epithelial cells were stimulated by cigarette smoke extract (CSE) or/and IL-17A, IL-17R expression in cells were detected using immunohistochemistry staining. In CSE group and IL-17A group, IL-17R expression was increased when compared with controls. IL-17R expression was highest in CSE + IL-17A group. a control group. b CSE group. c IL-17A group. d CSE + IL-17A group. (× 400 magnification)

Fig. 3

The protein expression of NF-κB in bronchial epithelial cells. Bronchial epithelial cells were inhibited NF-κB, and then stimulated by cigarette smoke extract (CSE) or/and IL-17A. NF-κB expression was measured using Western blotting. In CSE group and IL-17A group, NF-κB expression was increased when compared with controls. NF-κB expression was highest in CSE + IL-17A group. When NF-κB was inhibited, NF-κB expressions in all group were significantly reduced. a Western blotting. b Quantitation of protein bands

Fig. 4

E-cadherin expression in bronchial epithelial cells. When bronchial epithelial cells were stimulated with cigarette smoke extract (CSE) or/and IL-17A, E-cadherin expression in cells was detected using immunofluorescence staining. E-cadherin expression in CSE group was lower than that in controls, and was lowest in CSE + IL-17A group. When NF-κB was inhibited, E-cadherin expression was increased in cells stimulated with CSE and CSE + IL-17A compared to those without inhibition. a E-cadherin expression in control group without NF-κB inhibition. b E-cadherin expression in CSE group without NF-κB inhibition. c E-cadherin expression in IL-17A group without NF-κB inhibition. d E-cadherin expression in CSE + IL-17A group without NF-κB inhibition. e E-cadherin expression in control group with NF-κB inhibition. f E-cadherin expression in CSE group with NF-κB inhibition. g E-cadherin expression in IL-17A group with NF-κB inhibition. h E-cadherin expression in CSE + IL-17A group with NF-κB inhibition. (× 400 magnification)

Fig. 5

Vimentin expression in bronchial epithelial cells. When bronchial epithelial cells were stimulated with cigarette smoke extract (CSE) or/and IL-17A, Vimentin expression in cells was detected using immunofluorescence staining. Vimentin expression in CSE group was higher than that in controls, and was highest in CSE + IL-17A group. When NF-κB was inhibited, Vimentin expression was decreased in cells stimulated with CSE and CSE + IL-17A after NF-κB inhibited compared to those without inhibition. a Vimentin expression in control group without NF-κB inhibition. b Vimentin expression in CSE group without NF-κB inhibition. c Vimentin expression in IL-17A group without NF-κB inhibition. d Vimentin expression in CSE + IL-17A group without NF-κB inhibition. e Vimentin expression in control group with NF-κB inhibition. f Vimentin expression in CSE group with NF-κB inhibition. g Vimentin expression in IL-17A group with NF-κB inhibition. h Vimentin expression in CSE + IL-17A group with NF-κB inhibition. (× 400 magnification)