Introduction
Chronic obstructive pulmonary disease (COPD) is a highly prevalent condition that is characterized by persistent airflow limitation and is a major cause of morbidity and mortality worldwide [
1,
2]. In addition to parenchymal destruction, chronic inflammation can lead to airway remodeling and narrowing, which contributes to the characteristic reduction in airflow and symptoms of cough, shortness of breath and excess sputum production present in COPD [
3]. The clinical course is frequently punctuated by periods of acute symptom worsening, which are called exacerbations. These exacerbations are responsible for a large number of hospitalizations each year that exact heavy economic and societal burdens on the healthcare system [
4]. The most common triggers for acute exacerbations of COPD are viral respiratory tract infections, which can also initiate chronic airway inflammation by inducing innate immune responses that activate pro-inflammatory mediators [
5‐
7]. One of the more common pathogens is respiratory syncytial virus (RSV), which directly infects the airway epithelium [
1,
5,
7].
Popular symptomatic management for patients with COPD include bronchodilators such as the long-acting β2-agonists (LABAs) which target airway epithelia and smooth muscle cells [
2,
8,
9]. First approved in 2013, olodaterol (Striverdi “Respimat”) is a novel inhaled ultra-LABA with over 24 h of bronchodilatory effect [
2,
10‐
12]. It is a potent and selective β2-adrenergic receptor (β2AR) agonist with a rapid onset of action [
8,
9,
13]. By forming a stable complex with the β2ARs located in airway smooth muscles cells, olodaterol not only relaxes airway smooth muscles by stimulating adenylyl cyclase to upregulate cyclic AMP production, but also maintains broncho-protection for 24 h and therefore enables its once-daily dosing regimen [
8,
14,
15]. Usage of LABAs (such as salmeterol) has been shown to decrease the risk of exacerbations in COPD patients by up to 20% compared to a placebo [
16], but this is not well understood. It has been suggested that this may be due to synergy with inhaled corticosteroids, which result in an amplified anti-inflammatory effect [
17], but a meta-analysis showed that LABAs were able to inhibit exacerbations in the absence of ICS as well [
18]. The airway epithelium plays a key role in initiaing immune responses [
19]. Therefore, we hypothesize that LABAs have their own intrinsic anti-inflammatory effects when applied to the airways of COPD patients via the airway epithelium.
Materials and methods
Reagents and supplies
Olodaterol hydrochloride [BI 1744 Cl] was provided by Boehringer Ingelheim Pharma GmbH & Co. KG, Germany. Interleukin (IL-8) ELISA kit [Cat# CHC1303] was purchased from Life Technologies (Carlsbad, CA, USA). Anti-Muc5AC antibody [Cat# ab24071] for immunohistochemistry was purchased from Abcam (Cambridge, United Kingdom). ICI 118,551 hydrochloride [Cat# I127-5MG] was purchased from Sigma Aldrich (St. Louis, MO, US). Butaxamine hydrochloride [B1385-50MG] was also purchased from Sigma Aldrich.
Respiratory syncytial virus (RSV) preparation
Human Long strain, type A RSV (American Type Culture Collection (ATCC), Rockville, MD, USA) was propagated on Hep-2 (ATCC) cell monolayers as previously described [
20]. The culture medium consisted of Dulbecco’s Minimum Eagle Medium (DMEM) containing heat inactivated 10% fetal bovine serum (FBS), 100 U/mL penicillin/streptomycin, non-essential amino acids and sodium pyruvate was incubated at 37 °C in a 5% CO
2 incubator. After 4 days of culture, the condition media containing free virus was harvested and centrifuged at 10,000×
g for 10 min at 4 °C to remove cellular debris. The clear supernatants were then pooled and concentrated by ultrafiltration through a 100 kDa cut-off membrane [Cat# UFC910008] from Millipore (Burlington, MA, USA) according to the manufacturer’s instructions. RSV infection was performed at multiplicity of infection 1 (MOI
1) for 90 min as per previously published protocols [
21]. Following viral infection, cultures were washed with PBS to remove unbound virus and exposed to air for 16 h.
Cell culture
A total of 10 subjects (5 COPD and 5 controls) were grouped based on spirometry results according to recommendations by the Global Initiative for Chronic Obstructive Lung Disease (GOLD) [
22] whereby subjects with normal lung function were considered controls. Of the five subjects in the COPD cohort and the five subjects in the non-COPD cohort, one was a non-smoker, two were former smokers and two were current smokers. The patient demographics information is shown in Table
1. Three out of the five COPD subjects were using pulmonary medications (LAMA/SABA/Prednisone, LABA/SABA, budesonide). Non-COPD subjects were not using any pulmonary medication. All subjects underwent bronchoscopy for a variety of different clinical indications including evaluation of lung nodules/masses and chronic cough. Prior to bronchoscopy, all subjects underwent thoracic computed tomography (CT) as per protocol. Using cytology brushes [Cat# 4206] from Primed Canada, human primary bronchial epithelial cells (BEC) were collected in small airways (< 2 mm in diameter), generally in the right upper or left upper lobes, far away from any nodules or masses as noted on chest CTs (usually in the contralateral lung). All experiments in this study were approved by the Research Ethics Board of University of British Columbia/Providence Healthcare (REB# H11-02713 and REB# H15-01778). Patient-derived bronchial epithelial cells (BECs) were cultured in Pneumacult-Ex medium [Cat# 05008] from STEMCELL Technologies (Vancouver, BC, Canada). Cells were seeded at 80,000 per well in a 24-well plate and differentiation was induced once the cells reached 100% confluency.
Table 1
BEC subject demographics information
Age (years ± SD) | 66 ± 8.8 | 64.5 ± 9.9 |
Sex (male: female) | 1:4 | 3:2 |
FEV1/FVC (Mean % ± SD) | 76.5 ± 3.3 | 55.9 ± 11.4 |
NCI-H292 lung epithelial cells were purchased from the ATCC and grown in Roswell Park Memorial Institute medium (RPMI) supplemented with 10% heat-inactivated FBS. The NCI-H292 cells were cultured at 37 °C in a 5% CO2 humidified incubator. The media was changed three times per week and the cells were passaged once they reached 90–100% confluence. Cells were used experimentally between passages 75–90.
ALI cell culture
Air–liquid interface (ALI) cultures were grown on 24-well Corning cell culture inserts [Cat# 3470] using Pneumacult-ALI media [Cat# 0051] from STEMCELL Technologies and protocol as previously detailed [
23]. Cultures were maintained as ALIs for 21–28 days until pseudostratification of the epithelium was visualized via microscopy to contain various epithelial cell types. Medium replaced 3 times per week and the apical surface rinsed with warm phosphate buffered saline (PBS) weekly to study the effects of olodaterol treatment on the apical surface with and without RSV infections.
10 µM olodaterol treatment was administered on the apical side for 8 h, followed by aspiration of the olodaterol-containing media and exposing the cultures to air for 16 h. This pre-treatment procedure was repeated for 3 days for a total of 24 h drug exposure, to better mimic the in vivo patient regiment. ALI cultures were not exposed to olodaterol treatment on the apical side for 24 continuous hours as this would lead to de-differentiation of the pseudostratified epithelium.
Immunohistochemical (IHC) analysis
ALI cultures with or without olodaterol and RSV treatments were processed as formalin-fixed-paraffin-embedded (FFPE) blocks, and cut into 4 μm sections. Sections were de-paraffinized in CitriSolv (Fisher Scientific, Toronto, ON, 22-143-975) and rehydrated. Expression level of mucin 5AC (Muc5AC) was detected via a Leica autostainer at 1:1000 primary anti-Muc5AC antibody dilution (AB24071, AbCam, Cambridge, MA, USA) and using the Bond Polymer Refine Red Detection kit on the Leica Bond Autostainer according to the manufacturer’s protocol.
Production of RSV viral particles in infected ALI cultures were also quantified. ALI sections were de-waxed, rehydrated, and subjected to heat-induced antigen retrieval in citrate buffer (pH 6.0, Invitrogen) for 22 min. All sections were blocked in universal protein block (Dako, Burlington, ON, Canada) at room temperature for 30 min before incubating with a 1:100 dilution of an RSV-specific antibody (NCL-RSV3, NovoCastra Labs), in Tris-buffered saline (TBS) overnight at 4 °C. The biotin-free MACH4 AP-Polymer Detection kit containing alkaline phosphatase (M4U536, Biocare Medical, Markham, ON) was then used to detect proteins of interest following manufacturer’s protocols with Warp Red Chromogen (WR806, Biocare Medical) as the substrate.
Negative controls were performed using a matched isotype antibody. Hematoxylin was used for counter-staining of the nuclei of all IHC. Colour segmentation via ImagePro Plus (Media Cybernetics, Silver Spring, MD, USA) was performed for quantification; the area of positivity was normalized to the total epithelial surface.
Olodaterol and LPS treatment in NCI-H292 cells
NCI-H292 cells were incubated with complete media containing olodaterol for 8 h. Following pre-treatment with olodaterol, LPS was added directly into the media to a final concentration of 2 µg/mL and the culture was allowed to incubate overnight at 37 °C for 16 h before the cell-free supernatant was collected.
siRNA transfections with olodaterol and RSV infection or lipopolysaccharide (LPS) treatment
Specific siRNAs targeting the β2AR mRNA transcript (Silencer Select s1121, s1122, s1223, s531994), scrambled non-targeting siRNA (Silencer Select 4390844) and GAPDH-targeting siRNA (Silencer Select 4390849) were purchased from Life Technologies. The β2AR siRNAs were combined in equal proportions to create a custom pool. All transfections were performed using lipofectamine RNAiMax (Life Technologies) according to the manufacturer’s instructions. Cells were plated in 24-well plates (CORNING, NY, USA) at 60,000 cells per well. Two days later, the cells were transfected with siRNA at 10 nM and incubated with the transfection complex for 24 h, after which the media was replaced with fresh complete media. After 48 h, media in the LABA treatment groups were replaced with complete media containing 10 µM olodaterol and allowed to incubate for 8 h. At 56 h, LPS was added to achieve a final concentration of 2 µg/mL and then incubated overnight. Alternatively for RSV infection, at 56 h media from all wells were removed and saved. Fresh complete media or RSV-containing media at MOI1 was added and allowed to incubate with the cells in a 37 °C humidified incubator for 90 min with hand shaking every 20 min to disperse the virus. After 90 min, cells were washed with PBS and aspirated before the original media was returned and the cultures allowed to incubate overnight. At 72 h, media and cell lysates were collected and centrifuged at 6000×g for 5 min to remove cellular debris. Total RNA was isolated using the RNeasy mini kit (Qiagen, Hilden, Germany).
Blocking of β2AR with ICI 118,551 and butaxamine
Blocking of the β2AR in NCI-H292 cells was performed using the β2AR-selective antagonists ICI 118,551 and butaxamine. ICI 118,551 was utilized at a final concentration of 30 nM and butaxamine was utilized at a final concentration 100 nM. Cells were incubated in complete media containing the respective antagonist for 1 h prior to other treatments.
Reverse cDNA synthesis and real-time quantitative polymerase chain reaction
cDNA was synthesized from total RNA isolates using the iScript cDNA Synthesis Kit (BioRad Laboratories, Hercules, CA, USA) and quantitative real-time PCR was performed using iTaq Universal Sybr Green Supermix (BioRad Laboratories). Subsequent data were normalized to the housekeeping gene hypoxanthine–guanine phosphoribosyltransferase (HPRT). Glycerol-3-phosphate dehydrogenase (GAPDH) was used to confirm the efficacy of the knockdown. Custom DNA primers targeting β2AR, GAPDH and HPRT cDNA transcripts were designed via PrimerBlast based on the sequence obtained from GeneBank (NM_000024.6) and are as follows: β2AR forward: 5′-ATGGGCACTTTCACCCTCTG-3′; β2AR reverse: 5′-GCTCCGGCAGTAGATAAG-GG-3′; GAPDH forward: 5′-AAGAAGGTGGTGAAGCA-GGC-3′; GAPDH reverse: 5′-CGTCAAAGGTGGAGGAGTGG-3′; HPRT forward: 5′-TGACACTGGCAAAACAATGCA-3′; HPRT reverse: 5′-GGTCCTTTTCACCAGCAAGCT-3′. Knockdown efficiency of the target genes were analyzed via the ddCt method.
Enzyme-linked immunosorbent (ELISA) assay
Concentrations of IL-8 in supernatants collected from primary ALI cultures and NCI-H292 cells were measured by ELISA as per manufacturer’s instructions.
Statistical analysis and data presentation
Results were analyzed using GraphPad Prism 5.0 software (GraphPad Software, San Diego, CA, USA). Graphical values are represented as the mean ± standard error of mean. Two-tailed unpaired Student t-tests were used to compare the means between two groups while ANOVA with Bonferroni correction was used for multiple group comparisons.
Where possible, data generated via primary ALI cultures were normalized to internal experiment controls to maintain consistency across experiments. This is a result of primary cells having inherent genetic variability due to being sourced from different subjects. Hence, data are presented as “fold change” in place of absolute values. NCI-H292-generated data was not normalized as cell lines lack the heterogeneity of primary cultures.
Discussion
The most important and novel finding of our study was that olodaterol, an ultra-LABA, which is commonly used in COPD for bronchodilation, has significant anti-inflammatory effects in the airways of COPD patients. We showed that olodaterol was able to suppress inflammation related to RSV, a common respiratory pathogen that leads to COPD exacerbations, including suppression of IL-8 and airway mucin production/secretion and RSV viral particle production. We also showed that this anti-inflammatory effect was mediated through the β2-adrenergic receptor, the classic receptor for LABAs, as demonstrated through both siRNA gene silencing and receptor blocking experiments.
Some previously published literature have offered evidence that β2-adrenergic receptor agonists may have additional anti-inflammatory properties. This has been demonstrated previously with the SABA salbutamol, which was shown to inhibit p38 MAPK phosphorylation [
28]. However, in regards to LABAs, they were first proposed to be anti-inflammatory based on the increased effectiveness of LABA/ICS combination therapy to attenuate airway inflammation compared to ICS monotherapy [
29]. A popular theory was that it enhanced the anti-inflammatory effects of ICS by facilitating translocation of the glucocorticoid receptors into the nucleus [
17]. However, a more recent study examining the molecular mechanisms of LABA/ICS treatment refuted this concept [
30]. Several groups have noted that LABAs on their own (in the absence of glucocorticoids) demonstrate anti-inflammatory effects by inducing anti-inflammatory gene expression [
30‐
32]. Consistent with these observations, in animal models, olodaterol has been shown to inhibit the recruitment of neutrophils to the lungs and the secretion of inflammatory mediators such as TNF-α in response to an inflammatory stimuli in the airways [
27]. Furthermore, a study of 32 healthy volunteers found that salmeterol was able to reduce neutrophil recruitment and degranulation post LPS-inhalation [
33]. Our findings extend these observations by demonstrating that LABAs have direct anti-inflammatory effects on COPD airway epithelial cells, which are the primary sites of disease in COPD.
We also extend previous findings by demonstrating that the anti-inflammatory effects in the airways treated with LABAs are mediated by the β2AR. While the exact downstream mechanism for interaction with inflammatory pathways is unclear, we postulate that olodaterol exerts an effect on NF-κB, a transcription factor for many cytokines including IL-8. LPS and RSV trigger an innate inflammatory response via different mechanisms but both lead to activation of NF-κB. In lung epithelial cells, RSV drives production of IL-8 through intracellular activation of Toll-like receptor 3 (via MyD88-independent pathway for later NF-κB activation) and retinoic acid-inducible gene I receptor (involved in translocation of NF-κB) [
34]. LPS, meanwhile, is able to induce NF-κB mediated secretion of IL-8 through activation of TLR4 via the MyD88-dependent and MyD88-independent pathways [
35]. Olodaterol may therefore play a role in stabilizing the inhibition of cytosolic NF-κB (through IκB proteins) and preventing translocation into the nucleus, leading to an inhibition of cytokine production.
Clinically, patients are prescribed olodaterol at a daily dose of 5 µg aerosolized in 22.1 µL of carrier fluid [
36]. The experimental concentration of 10 µM olodaterol was chosen based on calculations of this aerosolized dose of olodaterol into the average patient’s airways’ periciliary fluid volume. The calculation for this concentration was derived from the calculations used by Dorscheid et al. to approximate the concentration of the ICS dexamethasone in the sol layer in one inhalation, assuming 10–30% deposition [
37]. The resulting concentration was approximately 10 µM. To test dose-concentration, we evaluated various doses of olodaterol in the primary ALI cultures and observed that 3 µM demonstrated sub-optimal IL-8 suppression and concentrations above 10 µM were associated with notable toxicity to the cells. As the drug concentration used in this study mimics that of in vivo applications, the current study results suggest clinical relevance. It is also notable that the anti-inflammatory effect of 10 µM of olodaterol on LPS-induced IL-8 production in the airway cells is similar to that achieved by 1 µM of dexamethasone on TNF-α induced IL-8 secretion (approximately 50%) [
38]. The 1 µM dose of dexamethasone approximates the dissolved concentration of one inhalation of aerosolized dexamethasone observed in the periciliary fluid of the airways [
37]. In addition, it is worth noting that there is previously published work detailing the ability of the LABA formoterol in inhibiting coronavirus (HCoV-229E) infection-induced IL-8 secretion in human nasal epithelial cells [
39]. Thus, there is precedence for the ability of LABAs to inhibit viral infection-induced inflammation, as was also shown in this paper. Together, the data suggest that LABAs independently exert an anti-inflammatory effect in the airways of COPD patients and may explain their salutary effects on the risk of exacerbation in COPD patients when used alone or in combination with ICS.
There were some limitations to the current study. Firstly, olodaterol appeared to also suppress the extent of RSV infection. Olodaterol may therefore have an effect in inhibiting viral replication or viral entry into the cell but the scope of this paper did not explore these potential effects. Secondly, this project used IL-8 as the biomarker of choice to monitor the anti-inflammatory effects of olodaterol. IL-8 is a known neutrophil chemoattractant and has been implicated in COPD pathogenesis. However, inflammation encompasses a wide variety of cytokines and inflammatory mediators and the effect of olodaterol on these other markers warrants further exploration. For example, it has been demonstrated that the airway epithelium constitutively secretes IL-10 [
40], which exhibits a direct modulating effect on inflammation through inhibiting IL-8 gene expression [
41]. Recent data from Ağaç et. al. showed that activation of the β2AR can up-regulate IL-10 [
42]. Thus, olodaterol may modulate IL-8 suppression through integration with an IL-10 secreting pathway via β2AR. Thirdly, this study focused solely on the anti-inflammatory effects of olodaterol. It would also be worthwhile to explore whether this same effect of olodaterol can be extrapolated to other LABAs (eg. formoterol, salmeterol).
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