Yang et al. Respir Res (2021) 22:65 https://doi.org/10.1186/s12931-021-01659-2 RESEARCH Open Access Olodaterol exerts anti-infammatory efects on COPD airway epithelial cells Nan Yang1† , Gurpreet K. Singhera1†, Yi Xuan Yan1, Michael P. Pieper2, Janice M. Leung1, Don D. Sin1 and Delbert R. Dorscheid1* Abstract Background: Airway infammation is a key feature of chronic obstructive pulmonary disease (COPD) and inhaled corticosteroids (ICS) remain the main treatment for airway infammation. Studies have noted the increased efcacy of ICS and long-acting beta 2 agonist (LABA) combination therapy in controlling exacerbations and improving airway infammation than either monotherapy. Further studies have suggested that LABAs may have inherent anti-infamma- tory potential, but this has not been well-studied. Objective: We hypothesize that the LABA olodaterol can inhibit airway infammation resulting from exposure to respiratory syncytial virus (RSV) via its binding receptor, the β2-adrenergic receptor. Methods: Human bronchial epithelial brushing from patients with and without COPD were cultured into air–liquid interface (ALI) cultures and treated with or without olodaterol and RSV infection to examine the efect on markers of infammation including interleukin-8 (IL-8) and mucus secretion. The cell line NCI-H292 was utilized for gene silencing of the β2-adrenergic receptor via siRNA as well as receptor blocking via ICI 118,551 and butaxamine. Results: At baseline, COPD-ALIs produced greater amounts of IL-8 than control ALIs. Olodaterol reduced RSV- mediated IL-8 secretion in both COPD and control ALIs and also signifcantly reduced Muc5AC staining in COPD-ALIs infected with RSV. A non-signifcant reduction was seen in control ALIs. Gene silencing of the β2-adrenergic receptor in NCI-H292 negated the ability of olodaterol to inhibit IL-8 secretion from both RSV infection and lipopolysaccharide stimulus, as did blocking of the receptor with ICI 118,551 and butaxamine. Conclusions: Olodaterol exhibits inherent anti-infammatory properties on the airway epithelium, in addition to its bronchodilation properties, that is mediated through the β2-adrenergic receptor and independent of ICS usage. Keywords: Airway epithelium, Interleukin-8, Airway infammation, Olodaterol, Long-acting beta agonist, COPD, Beta 2-adrenergic receptor Introduction parenchymal destruction, chronic infammation can lead Chronic obstructive pulmonary disease (COPD) is a to airway remodeling and narrowing, which contributes highly prevalent condition that is characterized by per- to the characteristic reduction in airfow and symptoms sistent airfow limitation and is a major cause of mor- of cough, shortness of breath and excess sputum produc- bidity and mortality worldwide [1, 2]. In addition to tion present in COPD [3]. Te clinical course is frequently punctuated by periods of acute symptom worsening, which are called exacerbations. Tese exacerbations are *Correspondence: [email protected] responsible for a large number of hospitalizations each †Nan Yang and Gurpreet K. Singhera contributed equally to this work 1 Center for Heart Lung Innovation, St. Paul’s Hospital, University of British year that exact heavy economic and societal burdens on Columbia, Vancouver, BC, Canada the healthcare system [4]. Te most common triggers for Full list of author information is available at the end of the article acute exacerbations of COPD are viral respiratory tract © The Author(s) 2021. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creat iveco mmons .org/publi cdoma in/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Yang et al. Respir Res (2021) 22:65 Page 2 of 11 infections, which can also initiate chronic airway infam- and sodium pyruvate was incubated at 37 °C in a 5% CO 2 mation by inducing innate immune responses that acti- incubator. After 4 days of culture, the condition media vate pro-infammatory mediators [5–7]. One of the more containing free virus was harvested and centrifuged at common pathogens is respiratory syncytial virus (RSV), 10,000×g for 10 min at 4 °C to remove cellular debris. which directly infects the airway epithelium [1, 5, 7]. Te clear supernatants were then pooled and concen- Popular symptomatic management for patients with trated by ultrafltration through a 100 kDa cut-of mem- COPD include bronchodilators such as the long-acting brane [Cat# UFC910008] from Millipore (Burlington, β2-agonists (LABAs) which target airway epithelia and MA, USA) according to the manufacturer’s instructions. smooth muscle cells [2, 8, 9]. First approved in 2013, RSV infection was performed at multiplicity of infection olodaterol (Striverdi “Respimat”) is a novel inhaled 1 (MOI1) for 90 min as per previously published proto- ultra-LABA with over 24 h of bronchodilatory efect [2, cols [21]. Following viral infection, cultures were washed 10–12]. It is a potent and selective β2-adrenergic recep- with PBS to remove unbound virus and exposed to air for tor (β2AR) agonist with a rapid onset of action [8, 9, 13]. 16 h. By forming a stable complex with the β2ARs located in airway smooth muscles cells, olodaterol not only relaxes Cell culture airway smooth muscles by stimulating adenylyl cyclase A total of 10 subjects (5 COPD and 5 controls) were to upregulate cyclic AMP production, but also maintains grouped based on spirometry results according to rec- broncho-protection for 24 h and therefore enables its ommendations by the Global Initiative for Chronic once-daily dosing regimen [8, 14, 15]. Usage of LABAs Obstructive Lung Disease (GOLD) [22] whereby subjects (such as salmeterol) has been shown to decrease the risk with normal lung function were considered controls. Of of exacerbations in COPD patients by up to 20% com- the fve subjects in the COPD cohort and the fve subjects pared to a placebo [16], but this is not well understood. It in the non-COPD cohort, one was a non-smoker, two has been suggested that this may be due to synergy with were former smokers and two were current smokers. Te inhaled corticosteroids, which result in an amplifed anti- patient demographics information is shown in Table 1. infammatory efect [17], but a meta-analysis showed that Tree out of the fve COPD subjects were using pulmo- LABAs were able to inhibit exacerbations in the absence nary medications (LAMA/SABA/Prednisone, LABA/ of ICS as well [18]. Te airway epithelium plays a key SABA, budesonide). Non-COPD subjects were not using role in initiaing immune responses [19]. Terefore, we any pulmonary medication. All subjects underwent bron- hypothesize that LABAs have their own intrinsic anti- choscopy for a variety of diferent clinical indications infammatory efects when applied to the airways of including evaluation of lung nodules/masses and chronic COPD patients via the airway epithelium. cough. Prior to bronchoscopy, all subjects underwent thoracic computed tomography (CT) as per protocol. Materials and methods Using cytology brushes [Cat# 4206] from Primed Can- Reagents and supplies ada, human primary bronchial epithelial cells (BEC) were Olodaterol hydrochloride [BI 1744 Cl] was provided by collected in small airways (< 2 mm in diameter), gener- Boehringer Ingelheim Pharma GmbH & Co. KG, Ger- ally in the right upper or left upper lobes, far away from many. Interleukin (IL-8) ELISA kit [Cat# CHC1303] was any nodules or masses as noted on chest CTs (usually in purchased from Life Technologies (Carlsbad, CA, USA). the contralateral lung). All experiments in this study were Anti-Muc5AC antibody [Cat# ab24071] for immunohis- approved by the Research Ethics Board of University of tochemistry was purchased from Abcam (Cambridge, British Columbia/Providence Healthcare (REB# H11- United Kingdom). ICI 118,551 hydrochloride [Cat# I127- 02713 and REB# H15-01778). Patient-derived bronchial 5MG] was purchased from Sigma Aldrich (St. Louis, MO, epithelial cells (BECs) were cultured in Pneumacult-Ex US). Butaxamine hydrochloride [B1385-50MG] was also medium [Cat# 05008] from STEMCELL Technologies purchased from Sigma Aldrich. (Vancouver, BC, Canada). Cells were seeded at 80,000 per Respiratory syncytial virus (RSV) preparation Human Long strain, type A RSV (American Type Cul- Table 1 BEC subject demographics information ture Collection (ATCC), Rockville, MD, USA) was propa- gated on Hep-2 (ATCC) cell monolayers as previously Non-COPD COPD described [20]. Te culture medium consisted of Dul- Age (years SD) 66 8.8 64.5 9.9 ± ± ± becco’s Minimum Eagle Medium (DMEM) containing Sex (male: female) 1:4 3:2 heat inactivated 10% fetal bovine serum (FBS), 100 U/ FEV /FVC (Mean % SD) 76.5 3.3 55.9 11.4 mL penicillin/streptomycin, non-essential amino acids 1 ± ± ± Yang et al. Respir Res (2021) 22:65 Page 3 of 11 well in a 24-well plate and diferentiation was induced Tris-bufered saline (TBS) overnight at 4 °C. Te biotin- once the cells reached 100% confuency. free MACH4 AP-Polymer Detection kit containing alka- NCI-H292 lung epithelial cells were purchased from line phosphatase (M4U536, Biocare Medical, Markham, the ATCC and grown in Roswell Park Memorial Institute ON) was then used to detect proteins of interest follow- medium (RPMI) supplemented with 10% heat-inacti- ing manufacturer’s protocols with Warp Red Chromogen vated FBS.