Identification of proteomic changes in primary human bronchial epithelial cells in response to chronic formoterol treatment

F. S. Vyas, C. Coveney, D. J. Boocock, C. P. Nelson. Biosciences, Nottingham Trent University, Nottingham, United Kingdom.

Introduction: Bronchial asthma is characterised by bronchoconstriction, airway hyper-responsiveness and elevated mucus secretion. Pathophysiological changes observed in asthma involve altered expression of inflammatory mediators and reflect genetic heterogeneity and environmental influences (1). Current therapies include long-acting β₂-adrenoceptor (β₂-AR) agonists (LABAs), which exploit the bronchodilatory action of β₂-ARs on airway smooth muscle cells (2). Short-term use of these treatments causes beneficial airway relaxation, but chronic use exacerbates asthma symptoms (2-3). Identification of proteomic changes in response to chronic LABA treatment could therefore provide insight into the molecular mechanisms involved in LABA-induced exacerbations and might allow the design of more effective asthma therapies.

Method: Primary human bronchial epithelial cells (HBEC) were subjected to the long-acting β₂-AR agonist formoterol (100 nM) for 48h. Cells were lysed and subjected to SWATH-MS (label free quantification by data independent acquisition; 4) to identify differentially expressed proteins. The identified proteins were processed for network analysis using MetaCore^TM pathway analysis suite and the formoterol-mediated changes in expression of proteins present in the network were confirmed by western blotting. Furthermore, to investigate changes in protein expression following more chronic β₂-AR stimulation, HBECs were subjected to formoterol (100 nM) for 7 days and protein expression evaluated by western blotting. One-way ANOVA followed by Dunnett's post-hoc test was employed to assess statistical significance.

Results: SWATH-MS analysis identified 9 differentially expressed proteins in response to formoterol treatment in HBECs. Network analysis revealed that the top ranked network included calreticulin, peroxiredoxin-1 and macrophage migration inhibitory factor (MIF). Expression of these three proteins under control and formoterol-stimulated conditions at 48 h and 7 days were confirmed using western blotting (Table 1).

Table 1: Calreticulin, peroxiredoxin-1 and MIF expression in HBECs under control and formoterol (100 nM; 48h and 7 day)-stimulated conditions. *p<0.05 and **p<0.01 versus control cells. Data represent means ± S.E.M. obtained from three independent experiments.

Conclusion: Calreticulin and Peroxiredoxin-1 are both up-regulated in HBECs following 48h exposure to formoterol, but return to control levels within 7 days. In contrast, MIF is down-regulated at 48h, but substantially up-regulated upon more chronic exposure to formoterol. These changes in MIF levels could contribute to asthma exacerbations observed upon chronic use of formoterol.

References:

1. Koczulla AR et al. (2017). Drug Discov Today 2: 388-396.

2. Billington CK et al. (2017). Handbook of Experimental Pharmacology 237: 23-40.

3. Walker J et al. (2011). British Journal of Pharmacology 163: 18-28.

4. Huang Q et al. (2015). Proteomics 15:1215-23.