Studies on the pathophysiology of bile acid-induced inflammatory airways disease

Abstract

Background: Gastro-oesophageal reflux and aspiration may be associated with airway disease. Bile acid aspiration has been shown to be prevalent among patients with advanced airway disease, which raises the concern that recurrent microaspiration of bile acids may cause airway epithelial injury which may lead to inflammatory airway disease. Objectives: To investigate the possible links between bile acids and lung injury which may cause pro-inflammatory cytokine release using BEAS- 2B, NCI-H292, 16HBE14o- and Calu-3 cell lines. Moreover, this study aimed at exploring whether primary bronchial epithelial cell cultures taken from lung transplant patients (PHBECs), tracheal epithelial cells (upper airway cells) from the subglottic area cells, BEAS-2B and 16HBE14o- cell lines undergo EMT like processes after stimulation with primary and secondary bile acid. Finally, the role of duodeno-gastro-oesophageal refluxate in mucus production from NCI-H292 and Calu-3 cell lines was investigated. Methods: BEAS-2, 16HBE14o-, NCI-H292 and Calu-3 cell lines were cultured and stimulated with Bile acid (BAs). Pro-inflammatory markers including IL-8, IL-6 and GMCSF were measured. Primary bronchial epithelial cell cultures taken from lung transplant patients and tracheal epithelial cells from the subglottic area cells were cultured. The effect of individual primary and secondary bile acids were evaluated by 48 hour challenge. Post-challenge TGFβ1, MMP9 and pro-collagen concentrations were measured using commercial ELISAs. RNA was isolated to determine the changes in expression of an epithelial marker E-cadherin and the mesenchymal marker fibronectin at a molecular level using a quantitative real-time PCR. The NCI-H292 and Calu-3 cell lines were also used as an in vitro model system to study MUC gene regulation. Cells were stimulated with bile acids and mucin content of the culture medium measured using an indirect ELISA and quantitative real-time PCR was used to evaluate MUC5AC and MUC5B expression in NCI-H292 and Calu-3 cell lines. ii Results: The cells used in this study elevated levels of IL-8, IL-6 and GMCSF released after treatment by primary and secondary bile acids. IL-8, IL-6 and GM-CSF are common finding in airways and lung disease where reflux and aspiration have been implicated as a possible injury. Significantly greater concentration of TGFβ1, MMP9 and pro-collagen were measured in cultures of PHBEs, tracheal epithelial cells, BEAS-2B and 16HBE14o- cell lines treated with BAs. E-cadherin expression significantly decreased while fibronectin was significantly increased. MUC5AC and MUC5B levels were induced in response to stimulation of Calu-3 and NCI H292 cells with BAs. Conclusion: The NCI-H292, Calu-3, BEAS-2B and 16HBE14-o cell lines are sensitive and useful models to study human respiratory processes and diseases related to BAs aspiration-induced lung injury. Aspiration of bile acids may cause cell injury, inflammation and cell death relevant to the pathophysiology of chronic airways disease. Furthermore, Bile acids stimulated EMT related processed in PHBEC, tracheal epithelial cells, BEAS-2B and 16HBE14O-cell lines. The Calu-3 and NCI H292 cells can be used as an in vitro model system to study MUC5AC and MUC5B gene regulation.