Cigarette Smoke Exposure and Glucocorticoid Insensitivity of Airway Smooth Muscle Cells

Abstract ID 93099 Poster Board 548 Background/Objective: Chronic obstructive pulmonary disease (COPD) is a leading cause of morbidity and mortality, whereas over 300 million individuals suffer from asthma worldwide. Cigarette smoking is recognized as the major risk factor in COPD and asthma patients who smoke often have poorly controlled disease. Glucocorticoids (GC) are the most effective anti-inflammatory drugs available. However, COPD and refractory severe asthma are frequently resistant to glucocorticoid therapy, thus posing an unmet need for better therapies. Although immune cells have been used to investigate glucocorticoid insensitivity (GCI), airway smooth muscle (ASM) cells have emerged as key contributors not only to airway hyperresponsiveness (AHR) but also to airway inflammation in patients with COPD and severe asthma. The objective of this study was to investigate effects of cigarette smoke exposure on glucocorticoid sensitivity of ASM cells. Hypothesis: Cigarette smoking exposure renders ASM cells insensitive to glucocorticoid transactivation. Methods: Primary human ASM cells derived from healthy controls and asthma patients were treated with various concentrations of cigarette smoking extract (CSE) for 24 h or two weeks. GC transactivation was measured using a Glucocorticoid Response Element (GRE)-driven luciferase reporter assay and measuring GC-inducible gene expression by qRT-PCR. Single-cell RNA sequencing was performed to identify differentially expressed genes (DEGs) in HASM cells and confirmed by qRT-PCR. Twenty normal nonsmoking subjects were enrolled as controls, with screening FEV1> 80% predicted. Forty current smokers were also enrolled, divided into subjects who demonstrated AHR, with a positive methacholine challenge (n = 20) versus those without AHR (n = 20). DEGs expression from peripheral blood mononuclear cells (PBMC) in chronic smokers, with and without AHR were determined by western blot assays. Data are presented as the mean ± SEM. P Results: CSE treatment for 24 h impaired GRE activation and the GC induction of several anti-inflammatory genes in multiple HASM cell lines in a dose-dependent manner with 70% reduction in 5% CSE-treated HASM cells. Chronic treatment with 2.5% CSE caused transcriptomic changes in HASM cells including Regulator of G Protein Signaling 2 (RGS2), a key regulator of ASM hypercontraction and AHR in asthma. Importantly, the GC dexamethasone enhanced β2-agonist albuterol-induced RGS2 expression in HASM cells, which was also impaired in CSE-treated HASM cells. In addition, RGS2 protein expression was significantly reduced in PBMC from chronic smokers without AHR, and to a greater degree in smokers with AHR, versus non-smokers. Conclusions: Our results indicate that CSE exposure not only profoundly impairs GC transactivation but also abolished dexamethasone and β2-agonist-induced RGS2 protein expression in HASM cells. Since RGS2 upregulation is a genomic mechanism of β2-agonist-induced bronchoprotection that is enhanced by GC, our study suggests ASM as an important target for glucocorticoid therapy and may contribute to cigarette smoking-related GC insensitivity seen in patients with COPD and severe asthma. Funding Source: Supported by National Institutes of Health R01 HL164593, the Allergy, Asthma & Immunology Education and Research Trust and State of Nebraska LB595 Cancer & Smoking Disease Research Development Program.