Alshehri, Wael
(2019)
Effect of cigarette smoke extract and electronic cigarette vapour extract on the production of cytokines and chemokines in human airway smooth muscle cells.
MSc(Res) thesis, University of Nottingham.
Abstract
Introduction:Chronic obstructive pulmonary disease (COPD) is characterised by an inflammatory response in the airways to inhaled substances like cigarette smoke. It is known that cigarette smoke extract (CSE) stimulates the production of a number of inflammatory cytokines and chemokines in human airway smooth muscle cells (HASMCs). E-cigarettes are used to aid smoking cessation, but they may also have pro-inflammatory effects. The effect of e-cigarette vapour extract (ECVE) on the production of cytokines and chemokines in HASMCs is unknown. The transcription factor NF-κB regulates immune functions and serves as a mediator of inflammatory responses. It is widely implicated in cigarette smoke-induced airway inflammation. We aim to compare the effect of CSE and ECVE on the production of inflammatory cytokines and chemokines in HASMCS, and investigate whether NF-κB is involved in mediating CSE- and ECVE-induce cytokines and chemokines.
Methods: CSE (from 2 cigarettes) and ECVE with and without nicotine (N+/N- respectively) (from 6ml e-liquid, 0.24mg/ml nicotine) were prepared by bubbling cigarette smoke or e-cigarette vapour into 20ml of cell culture medium. Confluent and growth-arrested HASMCs were treated with CSE and ECVE for 24 hours and cell viability was analysed by MTT assay. Enzyme-linked immunosorbent assays (ELISA) were used to measure the secretion of cytokines and chemokines in to the cell culture medium. In addition, NF-κB was inhibited by using BAY11-7082 to evaluate its involvement in the production of cytokines and chemokines.
Results: 5% CSE and 10% ECVE (N+/N-) reduced HASMC cell viability, whereas CSE (1,2, and 3.5%) and ECVE (1, 2.5 and 5%) did not affect cell viability. CSE (1, 2 and 3.5%) concentration-dependently induced the production of IL-5, IL-6, IL-8, IL-13, vascular endothelial growth factor (VEGF) and CCL2, but not CXCL-10 and RANTES. ECVE (N+/N-) (1, 2.5 and 5%) only induced the production of IL-13, but not the others. BAY11- 7082 (a NF-κB inhibitor) partially inhibited the production of CSE-induced IL-6 and IL-8 at the highest concentration tested (100µM), while causing complete inhibition of VEGF production at all investigated concentration (0.1-100µM). But it had no effect on CSE-induced IL-5, MCP-1 and IL-13 and ECVE-induced IL-13.
Conclusion: Our results suggest that CSE has a wider pro-inflammatory effect in the airway than ECVE. However, the ability of ECVE without nicotine (N-) to induce cytotoxicity and IL-13 production in HASMCs in a similar manner to ECVE with nicotine (N+) suggests that e-cigarette vapor in itself is harmful to the airway and induces pro-inflammatory response independently of the presence of nicotine. We have shown that NF-κB is involved in mediating CSE-induced IL-6, IL-8 and VEGF production but is not involved in CSE- and ECVE-induced IL-13 from HASMCs. This suggests that the signalling pathways involved in CSE and ECVE-induced pro-inflammatory responses in HASMCs differ from one another.
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