Abstract
This study demonstrates that bronchial thermoplasty reduces smooth muscle and neural innervation of the airway up to 12 months post-therapy, whereas the airway epithelium is relatively resistant to thermal damage https://bit.ly/3wlQOJX
Introduction
Although patients with severe asthma comprise only 5–10% of the total asthma population, they account for a disproportionate amount of healthcare expenditure, owing to relatively frequent asthma exacerbations and lung function decline [1, 2]. Characteristic structural changes in the airway wall, termed airway remodelling, contribute to airway hyperresponsiveness (AHR) and exacerbations [3–5] and are relatively resistant to present treatment strategies [6]. The changes associated with airway remodelling in the large airways include goblet cell hyperplasia, increased deposition of extracellular matrix proteins and collagen, sub-basement membrane thickening, myofibroblast infiltration, airway smooth muscle (ASM) hyperplasia and/or hypertrophy, increased angiogenesis and increased neural innervation [5, 7, 8]. Compared to patients with mild–moderate asthma, those with severe asthma have increased myofibroblast accumulation, subepithelial fibrosis, submucosal collagen and increased area of smooth muscle and submucosal glands [3, 4].
Bronchial thermoplasty is a mechanical therapeutic intervention approved for the treatment of patients with moderate or severe asthma [9]. Bronchial thermoplasty generates radiofrequency energy, which produces a transient increase in temperature within the airway wall. Although several studies have confirmed a reduction in ASM 6 months [10–15] and up to 2 years post-bronchial thermoplasty [16], the underlying mechanism of action is not clear [17, 18]. It is hypothesised that reduced ASM results in a decreased ability of airways to constrict, and a subsequent decrease in airway hyperresponsiveness, which in turn has been correlated with improved asthma control [10, 13], reduced frequency of asthmatic exacerbations and hospitalisations [19–21]. Recent studies describe reduced neural innervation post-bronchial thermoplasty [13, 22]. Studies have reported reduced collagen within the sub-basement membrane (the prominent eosinophilic hyaline layer beneath the true epithelial basement membrane, which is characteristic of asthma) 6 weeks after bronchial thermoplasty [11] and increased collagen in this layer 3 months following bronchial thermoplasty [10]. However, the effect of bronchial thermoplasty on collagen deposition within the submucosa itself has not been studied. Since there is some concern that bronchial thermoplasty may trigger compensatory remodelling and scarring, our aim was to do a comprehensive assessment of airway remodelling up to 1 year post-treatment.
The hyperthermia produced by bronchial thermoplasty affects the fluidity and stability of cellular membranes and impairs the function of transmembrane transport proteins and surface extracellular surface receptors [23, 24], which are susceptible to further apoptotic and/or necrotic signalling as proteins involved in DNA replication and stability are damaged [17]. However, studies examining the heat profile and short-term effects of bronchial thermoplasty are few and incomplete [17, 18], and few safety data associated with the thermal profile of the device are available. Therefore, we initially assessed the temperature profile of the bronchial thermoplasty device in an animal model and then went on to replicate similar conditions in cell culture. Finally, we evaluated the effect of bronchial thermoplasty on indices of airway remodelling in a cohort of well-characterised patients with severe asthma who were prescribed bronchial thermoplasty, over a 12-month period.
Methods
See the supplementary material for detailed methods.
Animal studies
To determine the thermal profile of bronchial thermoplasty in the airway wall, a 6-month-old (∼40 kg) White-Landrace piglet bronchiole, with relatively similar airway anatomy to the adult human lung, was used [25]. The piglet was treated in compliance with the Canadian Council for Animal Care and the protocol received approval from the University of Calgary animal care and use committee (AC14-0135). The thermal profile of bronchial thermoplasty was captured using a thermal camera (FLIR SC660; Wilsonville, OR, USA) in 3–5-mm diameter bronchioles in three different lobes. The temperature was recorded every 1.0 ms starting immediately before activation of the bronchial thermoplasty instrument, the length of the activation time (10 s) and 8 s after the bronchial thermoplasty instrument shut off. The results were used to inform the cell culture temperature profiles.
Cell culture studies
Primary human ASM and bronchial epithelial (HBE) cells were obtained [26]. Cells were treated with media heated to 37°C (control), 65°C or 85°C for 10 s and cultured further for 24 h at 37°C. ASM cell viability was determined 0.5, 3 and 24 h post-treatment.
Study participants
Participants (aged 18–65 years) diagnosed with asthma and prescribed regular maintenance inhaled corticosteroid (>1000 mg·day−1 beclomethasone or equivalent) and long-acting β-agonist (>100 μg·day−1 salmeterol or equivalent) were recruited from the investigators’ tertiary-care asthma clinics for observational study (table 1). The University of Calgary conjoint health research ethics board approved the protocol (REB14-1100). All participants provided written informed consent prior to study participation. Following baseline bronchoscopy and biopsies, participants underwent bronchial thermoplasty (Alair; Boston Scientific, Natick, MA, USA) and were subsequently assessed at 1, 3, 6, 9 and 12 months post-procedure. Repeat bronchoscopy and biopsies were performed at 6 weeks and 12 months (figure 1). Nine participants completed the study.
Histology and morphometric analyses
Routine histological examination using haematoxylin and eosin (H&E) was performed, with additional stains used to identify goblet cells (periodic acid schiff) and collagen (picrosirius red (PSR) with the aid of polarised light). Goblet cells were normalised to the sub-basement membrane length. The thickness of the sub-basement membrane area, the prominent eosinophilic hyaline layer beneath the true epithelial basement membrane (also variably referred to as the “reticular basement membrane”), was measured by multiple point counts [27]. It consists of multiple components other than just collagen, such as matrix proteins [28–32]. The submucosal collagen deposition was analysed using the natural birefringent properties of collagen when stained with PSR, normalised to the total submucosal area of the biopsy. This method is regarded as highly sensitive in the assessment of collagen [33, 34]. Immunohistochemistry using antibodies for α-smooth muscle actin (SMA), CD31 and S100 was used to identify smooth muscle, vascular endothelial cells and nerves, respectively. Sections stained with H&E, α-SMA and CD31 were assessed using stereology (Stereo Investigator; MBF Bioscience, Williston, VT, USA). ASM, neural bundles, submucosal glands and submucosal collagen were normalised to submucosal area; the percentage area of red and green birefringent collagen, representative of type I and III collagen, respectively, was normalised to total collagen area. Investigators performing the analysis were blinded to patients and biopsy schedules, and a subset of biopsies were recounted to ensure repeatability.
Statistical analysis
All data were normally distributed, as assessed by the Shapiro–Wilks test, with equal variance, as tested by the Brown–Forsythe test. All data are expressed as mean±sem or 95% CI. Data were analysed by two-tailed mixed model repeated-measures ANOVA with the Student–Newman–Keul post hoc analysis, paired t-test, Chi-squared test or Spearman correlation using Sigmaplot 14.0 (Systat, San Jose, CA, USA) for analysis and plotted in GraphPad Prism 6.0. Significance was set a priori at p<0.05.
Results
Thermal profile of the bronchial thermoplasty procedure
Thermal mapping of the effect of bronchial thermoplasty in the piglet bronchiole was performed using a thermal camera (figure 2a,b and supplementary video), replicated three times in three bronchioles. A maximal temperature of 82.9±8.3°C was reached ∼7 s after initiating bronchial thermoplasty at the four points of contact. At these contact points, the average temperature approximated 64°C during 18 s of recording, decreasing to ∼50°C at the end. The airway between the points of contact did not reach this maximal temperature, but increased to a maximum of ∼60°C and then decreased to ∼55°C during 18 s of recording.
Effects of hyperthermia on airway smooth muscle and epithelial cells
To determine the effect of hyperthermia on individual structural cell types in vitro, growth medium was heated to various temperatures equating to bronchial thermoplasty, as discerned from the thermal profiling studies, and transferred to chamber slides containing cultured ASM cells or HBE cells. By testing different media temperatures and observing the thermal profiles, we determined that media heated to 85°C and added to a cell chamber incubated at 37°C best replicated the thermal profile seen at the bronchial thermoplasty contact points, referred to as the “maximal temperature profile”. The cell chamber temperature spiked to 72°C, dropped rapidly to 65°C and declined more slowly with an average temperature of 61°C, compared to an average temperature of 64°C in the pig airway (figure 2c). Medium heated to 65°C and added to the cell chamber best replicated the thermal profile seen in between the bronchial thermoplasty contact points, referred to as the “intermediate temperature profile”. The cell chamber temperature spiked to 60°C and to 52°C (compared to an average of 55°C in the pig airway; figure 2d). Medium heated to 37°C was added to the cell chambers as control.
Exposure of ASM cells to the intermediate temperature profile had no effect on cell morphology at 24 h post-exposure when compared to control (figure 3a–d). However, exposure to the maximal temperature profile produced marked cytoplasmic disruption, condensation of nuclei and loss of cellular adhesion at 24 h post-exposure (figure 3e, f). In contrast, exposure to heated media had no effect on HBE cell morphology (data not shown).
There was a significant reduction in ASM cell viability in response to both the maximal and intermediate temperature conditions 0.5 h post-exposure; viability recovered after 3 and 24 h of cell culture in the intermediate temperature group, but remained submaximal at 3 and 24 h in the maximal temperature group (figure 4).
The effect of bronchial thermoplasty on airway remodelling
68 biopsy samples obtained from the nine study participants (baseline n=24, 6 weeks n=22, 12 months n=22) were adequate for analysis (figure 1). Each biopsy was analysed independently, and the results averaged for each patient at each time point to achieve a single data point. ASM expressed as a percentage of the submucosal area was significantly reduced at 6 weeks and 12 months (baseline 12.05±1.03%, 6 weeks 4.40±1.30%, 12 months 5.07±1.66%; figure 5a and 6a). Collagen deposition, defined as collagen present within the submucosa, beneath and excluding the sub-basement membrane [32, 35, 36], expressed as a percentage of total submucosal area, was significantly, but transiently reduced at 6 weeks following bronchial thermoplasty (baseline 2.47±0.33%, 6 weeks 1.16±0.21%, 12 months 1.63±0.30%; figure 5b and 7a). The dominant red birefringent collagen present at baseline was replaced by increased green birefringent collagen at 6 weeks post-bronchial thermoplasty, but reverted to the baseline pattern at 12 months post-bronchial thermoplasty (figure 5b and 7b, c). Average vascular area and vascular density, representing size and numbers of vessels, respectively, expressed as a percentage of submucosal area, were increased at 6 weeks following bronchial thermoplasty, but subsequently reverted to baseline levels at 12 months (vascular area: baseline 27 600±7672 μm2, 6 weeks 66 664±15083 μm2, 12 months 45 921±11819 μm2; vascular density: baseline 3.54±0.48%, 6 weeks 5.85±0.61%, 12 months 3.75±0.77%; figure 7d, e). Bronchial thermoplasty significantly reduced nerve bundles identified in the submucosa at 6 weeks post-bronchial thermoplasty and 12 months (baseline 9.457±3.405, 6 weeks 3.585±1.821, 12 months 2.460±0.803; figure 5c and 6b). There was no significant difference in goblet cell metaplasia, sub-basement membrane thickness or area of submucosal glands (supplementary figure S1b and c) following bronchial thermoplasty at any time point.
The effect of bronchial thermoplasty on clinical measurements
Patient demographics and baseline measurements prior to bronchial thermoplasty are in table 1. Clinical follow-up did not reveal significant changes in Asthma Control Questionnaire (ACQ), Asthma Quality-of-Life Questionnaire (AQLQ), forced expiratory volume in 1 s (FEV1) or forced vital capacity (FVC) following bronchial thermoplasty (figure 8). However, using the Chi-squared test of proportion to compare 12 months to baseline, six out of nine participants had an improvement in the ACQ score greater than the minimal clinically important difference of 0.5, while only one had a >0.5 deterioration (difference 55%, 95% CI 10.5–78.7; p=0.02). A similar trend was seen for AQLQ improvement in four out of nine versus deterioration in one out of nine, but this was not statistically significant. This resulted in a significant correlation between ASM and ACQ (using all data points, thereby accounting for change; r=0.408, p=0.048; no other significant correlations between clinical metrics and airway remodelling were demonstrated). No significant differences were noted for minimal clinically important difference changes in FEV1 or FVC at 12 months (defined as a change of ≥12% and 200 mL).
Discussion
In order to determine the temperature profiles appropriate to use in our cell culture experiments, we examined the thermal profile of bronchial thermoplasty in a porcine model using a thermal camera. The porcine tracheobronchial system shares morphological characteristics with that of the human, including similar tracheal length, bronchiolar segmentation, and bronchiolar diameter. Moreover, both porcine and human bronchioles have a highly differentiated pseudostratified epithelial lining and submucosa with submucosal glands and airway smooth muscle [25, 37, 38] making the pig lung the favoured animal model to train bronchoscopists [39–41] and therefore the best choice to model bronchial thermoplasty in the human. We found the maximal temperature of the airway wall was ∼83°C at the bronchial thermoplasty contact points; significantly higher than the intervening wall which reached a maximum of only 60°C. It is possible that some variations in morphology in the pig compared to the human bronchioles may alter heat distribution. Additionally, it is possible that the spike in temperature in a live animal (or human) is likely to be more transient as blood flow in an intact lung would redistribute the applied heat. However, using excised airways was a necessary adaptation to allow imaging. The average temperatures at the contact points and in the intervening wall were 64°C and 55°C, respectively, and are consistent with the findings of Chernyavsky et al. [42], who found that the temperatures were ≥65°C and ≤55°C at and between the contact points, respectively. The heterogeneous heating by bronchial thermoplasty may explain, at least in part, why a circumferential reduction in ASM of only ∼50% has been demonstrated [43] and the variability in other histological measures that have been reported to date.
High-frequency electrical energy, without heat generation (rheoplasty), has been shown to affect bronchial epithelium, with a reduction in goblet cell metaplasia [44]. In the case of bronchial thermoplasty, the radiofrequency energy translates to thermal energy in tissues. Hyperthermia affects cell membrane stability and impairs transmembrane transport protein and extracellular surface receptor function as well as proteins involved in DNA replication and stability [23, 24]. Therefore, cells exposed to heat are susceptible to apoptotic and/or necrotic signalling [17]. Previously, ASM cells have been shown to be susceptible to heat with a loss of myofilaments being observed at >48°C [45] and a loss of ASM contractility at >55°C [46]. Cell cultures were exposed to maximal and intermediate temperature profiles, to represent the situation at and between bronchial thermoplasty contact points, respectively. Similar to our study, in which we found that 61°C for 10 s disrupted ASM cells, Chernyavsky et al. [42] found irreversible loss of ASM cell viability with 59°C for 10 s. In addition, we found that HBE cells were relatively resistant to thermal injury when analysed 24 h after heat application, consistent with Chernyavsky et al. [42].
A characteristic of airway remodelling, and a major distinguishing feature of severe asthma, is increased ASM [3–5]; therefore, any reduction of ASM is potentially of clinical benefit. Previous studies have demonstrated a reduction of ASM in severe asthmatic patients after bronchial thermoplasty in the short term [10, 11, 13–15], which are now substantiated by the Bronchial Thermoplasty Induced Airway Smooth Muscle Reduction and Clinical Response in Severe Asthma (TASMA) trial, which, to date, is the only bronchial thermoplasty trial demonstrating ASM reduction 6 months post-bronchial thermoplasty in comparison to standard care [15]. We found a similar reduction in ASM in the short-term, but extend these findings and confirm the report by others [10, 15, 16], by demonstrating a sustained and significant reduction in ASM at >12 months post-bronchial thermoplasty.
Our findings support the safety of bronchial thermoplasty with regard to the airway epithelium [19, 21, 47]. Although our in vitro studies with HBE cells suggest that bronchial thermoplasty does not adversely affect the bronchial epithelium, we cannot rule out an early, short-term effect. Using optical coherence tomography (OCT) to examine the airways immediately after bronchial thermoplasty, Goorsenberg et al. [43] reported that bronchial and peribronchial oedema and sloughing of the epithelium were seen in the majority of the treated airways. Oedema was also seen in distal nontreated airways. On repeating OCT 6 weeks later, the mucosa was essentially re-epithelialised, and the oedema had mostly receded. This is consistent with the demonstration of increased epithelial integrity in biopsies attained 3 months post-bronchial thermoplasty [42]. Interestingly, culture of epithelial cells harvested from patients have demonstrated increased proliferation post-bronchial thermoplasty, where the authors suggested that effects on remodelling post-bronchial thermoplasty relate to reduced epithelial stimulation of fibroblasts [48]. Although we did not identify any alterations in airway epithelial cell morphology nor increase in goblet cell metaplasia (supplementary figure S1), it is plausible that bronchial thermoplasty affects the cross-talk between epithelial cells and underlying fibroblasts and smooth muscle. We did not find an effect on sub-basement membrane thickness, in contrast to other studies [10, 11].
We were concerned that thermal injury to the airway wall could give rise to scar formation and therefore assessed the effect of bronchial thermoplasty on submucosal collagen. We found a significant reduction in collagen deposition in the submucosa at 6 weeks post-bronchial thermoplasty, which then returned to baseline levels by 12 months post-bronchial thermoplasty (figure 7a). We are the first investigators to assess submucosal collagen after bronchial thermoplasty. We speculate that the apparent reduction in submucosal collagen can be explained by the presence of a wound repair response with oedema fluid pushing apart the collagen fibres. Interestingly, there was also a transient but significant increase in vascular density at 6 weeks which may represent new, “leaky” vessels as seen in wound repair (figure 7d) [49]. Collagen staining with PSR exhibits different colours under polarised light, including red, green and yellow. There is debate whether these different colours represent different types of collagen, as proposed by Junqueira et al. [50]. who claimed that type I collagen shows red birefringence whereas type III collagen appears green [33, 50, 51]. However, other studies dispute this claim, suggesting that these variations in birefringence are related to the thickness of the collagen fibrils only [51]. Whether or not the colours represent specific types of collagen, it is apparent that there was a significant change at 6 weeks compared to baseline, coinciding with the other changes which suggest a wound repair response [33, 34]; this appeared to have resolved at 12 months post-bronchial thermoplasty (figure 7b, c). A previous study showed a reduction in vessel number at 6 weeks post-bronchial thermoplasty which did not reach significance, while another showed no difference at 3 months post-bronchial thermoplasty [10, 13]. The vessels at the 12-month time point showed no changes compared to baseline, in contrast to the fact that a significant increase in the number of vessels and/or percentage of vascular area in the airways of subjects with asthma has been observed in comparison to healthy controls [52] and correlates with bronchial hyperresponsiveness [53].
The nerves in the airway submucosa are part of the parasympathetic nervous system and are critical to the regulation of smooth muscle tone and contribute to the pathophysiology of bronchospasm [54]; they have also been implicated in the regulation of epithelial mucus production [55]. We found significantly decreased numbers of nerve bundles at 6 weeks and 12 months post-bronchial thermoplasty in comparison to baseline (figure 6b), a finding that is in keeping with other authors who found similar reduction in nerves at 12 months post-bronchial thermoplasty [10, 22].
The morphological changes which occur as a result of bronchial thermoplasty are key structural features responsible for AHR [5, 7, 8]. The attenuation of airway remodelling is consistent with preclinical data which demonstrate reduction of airway hyperresponsiveness in dogs [56]. The airway dysfunction that results from airway hyperresponsiveness is important in the pathophysiology of asthma morbidity and occasional asthma mortality [57, 58], and previous clinical reports have demonstrated reduced hospitalisations and exacerbations following bronchial thermoplasty [19–21]. Although we did not specifically assess airway hyperresponsiveness in this study, we found no change in pre-bronchodilator FEV1 and FVC, consistent with previous results [19–21] that showed no significant alteration of lung function following bronchial thermoplasty.
This study was not powered to identify clinical improvement and we did not detect either a statistically significant or clinically meaningful change in the AQLQ score, but did demonstrate a clinically relevant (>0.5-unit reduction) long-term improvement in ACQ, in accord with the Asthma Intervention Research (AIR), AIR2, Research in Severe Asthma and TASMA trials [15, 19–21, 47]. These data complement previous reports that the number of symptom-free days increased and severe exacerbations decreased following bronchial thermoplasty treatment [15, 19]. ASM reduction is significantly correlated with asthma control, demonstrating the clinical significance of the effect of bronchial thermoplasty on ASM [13]. However, in contrast, the change of ACQ attributed to bronchial thermoplasty in the TASMA trial was correlated with baseline plasma eosinophils, not ASM [15], suggesting that inflammation and signals to stimulate ASM contractility may be more indicative of exacerbations than ASM itself. Similarly, yet another study demonstrated clinical improvement in both ACQ and AQLQ 12 months following bronchial thermoplasty along with a reduction of lung neural innervation; however, a clear relationship between clinical improvement and reduced lung nerves was not assessed [22]. Although we did not find statistically significant differences in our clinical findings, we did demonstrate that ASM and ACQ were significantly correlated (r=0.408, p=0.048) in line with Ichikawa et al. [13], as well as clinically meaningful improvements in six out of nine patients with ACQ.
A limitation to our study is the lack of a control/sham group. Incorporation of a sham group was not feasible, as the prospect of withholding treatment would reduce enthusiasm for enrolment and provide additional ethical concerns. However, the main objective of our study was to examine airway remodelling and not clinical improvement, and follow-up assessment of airway hyperresponsiveness was not part of this study.
In conclusion, this is the first study to systematically examine the temperature profile of bronchial thermoplasty on the airway wall in situ and observe the effect of this heat profile on smooth muscle and epithelial cells. We found that bronchial thermoplasty heats the airway wall unevenly, depending on the proximity to the bronchial thermoplasty catheter wires, and that peak temperatures were higher than expected. Heat had a significant deleterious effect on ASM morphology and viability, while bronchial epithelial cells were relatively resistant. We also observed a significant reduction in ASM and numbers of submucosal nerve bundles, which were sustained up to 12 months following bronchial thermoplasty with no observed changes in goblet cell metaplasia, the thickness of the sub-basement membrane or the submucosal glands. We did find a transient decrease in submucosal collagen, which had different birefringent properties, and an increase in vessel density which we speculate may represent a wound repair response at 6 weeks post-bronchial thermoplasty. However, we found no evidence of scarring at the 1 year post-bronchial thermoplasty time point.
Presently, bronchial thermoplasty is the only therapy which has been shown to reverse certain indices of airway remodelling, namely smooth muscle volume and numbers of nerve bundles, in severe asthma. Our study confirmed this and identified that, although there may be a wound repair response 6 weeks after bronchial thermoplasty, this is transient, with no evidence of scarring at 12 months post-bronchial thermoplasty, a reassuring finding with regard to safety with this treatment.
Supplementary material
Supplementary Material
Please note: supplementary material is not edited by the Editorial Office, and is uploaded as it has been supplied by the author.
Supplementary methods ERJ-00622-2021.Supplement
Supplementary figure S1. Histological evaluation of bronchial thermoplasty. a) Biopsy area was similar between all timepoints. b) Sub-basement membrane thickness was similar between baseline, 6-week and 12-month follow-up. c) Submucosal gland area normalised to total submucosal area and baseline, 6-week and 12-month follow-up showed no significant differences at any timepoint. d) Goblet cells normalised to subbasement membrane length were not significantly altered following bronchial thermoplasty at any timepoint. ERJ-00622-2021.Figure_S1
Supplementary video. Thermal profile of bronchial thermoplasty applied to excised porcine airways. The Alair Bronchial Thermoplasty device was used in excised porcine lungs and the thermal profile was captured by the FLIR SC660 camera. ERJ-00622-2021.Video
Shareable PDF
Supplementary Material
This one-page PDF can be shared freely online.
Shareable PDF ERJ-00622-2021.Shareable
Footnotes
This article has an editorial commentary: https://doi.org/10.1183/13993003.02018-2021
All de-identified data are available from the corresponding authors upon reasonable request.
Author contributions: N. Jendzjowsky conducted morphometry and analysis, prepared figures and the manuscript; A. Laing conceptualised the study, conducted morphometry, experiments, analysis and, prepared figures; M. Malig conducted morphometry and analysis; J. Matyas assisted with morphometry; E. de Heuvel prepared samples, helped with experiments and contributed to manuscript preparation; C. Dumonceaux recruited participants and collected clinical data; E. Dumoulin conducted bronchial thermoplasty and collected samples; A. Tremblay conceptualised the study, conducted bronchial thermoplasty, collected samples, coordinated patient selection and contributed to manuscript preparation; R. Leigh conceptualised the study, coordinated patient recruitment, and contributed to manuscript preparation; A. Chee conceptualised the study, obtained funding, conducted bronchial thermoplasty and collected samples; M.M. Kelly conceptualised the study, coordinated histology, morphometry, experiments and immunohistochemistry and contributed to manuscript preparation. All authors approved the final version of the manuscript.
This article has supplementary material available from erj.ersjournals.com
Conflict of interest: All authors have no conflict of interest to disclose.
Support statement: This study was funded by the Lung Association Grant in Aid program and the American Association of Bronchology and Interventional Pulmonology. N. Jendzjowsky is a Parker B. Francis Fellowship Recipient. R. Leigh holds the GSK Professorship in Inflammatory Lung Disease. M.M. Kelly holds the Endowed Chair of Pediatric Respirology, Alberta Children's Hospital Foundation, Cumming School of Medicine and the Alberta Lung Association. Funding information for this article has been deposited with the Crossref Funder Registry.
- Received March 3, 2021.
- Accepted May 16, 2021.
- Copyright ©The authors 2022. For reproduction rights and permissions contact permissions{at}ersnet.org