Clinical and molecular phenotyping of asthma and COPD
Boudewijn, Ilse Maria
IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from it. Please check the document version below.
Document Version
Publisher's PDF, also known as Version of record
Publication date: 2019
Link to publication in University of Groningen/UMCG research database
Citation for published version (APA):
Boudewijn, I. M. (2019). Clinical and molecular phenotyping of asthma and COPD. University of Groningen.
Copyright
Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).
Take-down policy
If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim.
Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons the number of authors shown on this cover page is limited to 10 maximum.
1
Asthma and COPD are highly prevalent, obstructive airways diseases. Asthma is a chronic inflammatory airways disease, affecting approximately 300 million people worldwide(1). The global prevalence of asthma is estimated to be around 4.3% but varies widely across countries, highest prevalences being reported in developed countries (e.g. 15.3% in The Netherlands) and lowest prevalences in developing countries (e.g. 2.9% in Bangladesh)(1). The disease is characterized by recurrent attacks of dyspnea, cough and wheeze. Asthma leads to decreased quality of life and contributes significantly to overall healthcare costs(2,3). COPD is the third leading cause of death worldwide according to the World Health Organization(4). Important characteristics of the disease are irreversible and progressive airflow obstruction due to airway inflammation, airway narrowing and loss of lung tissue. Cigarette smoking is the major cause of COPD, but other noxious particles such as air pollution and biomass fuel used for cooking are currently acknowledged as important risk factors(5). Both asthma and COPD are considered heterogeneous diseases in terms of clinical presentation, underlying inflammatory profiles and environmental factors influencing disease severity. Disentangling the heterogeneity of obstructive airways diseases is an important field of research, which eventually can lead to development of tailored treatment for each individual patient. In this thesis, we aimed to investigate the heterogeneity of asthma and COPD by focusing on clinical and molecular phenotyping.
CLINICAL PHENOTYPING
Airway hyperresponsiveness; a hallmark of asthma?
Prevalence of AHR
Airway hyperresponsiveness (AHR) is a key feature of asthma. It is defined as exaggerated airway narrowing upon exposure to non-specific stimuli, such as cigarette smoke, cold air or perfumes. Although AHR is usually accompanied by respiratory symptoms, it can also be present in asymptomatic subjects, so-called asymptomatic AHR. It is estimated that the prevalence of asymptomatic AHR varies between 2.2 to 14.3% in the general population(6). In the past decades, several explanations for asymptomatic AHR have been put forward such as increased airway inflammation, airway remodeling and altered perception of symptoms(7–9). Of importance, the presence of asymptomatic AHR is not an innocent feature, but is associated with the development of asthma later in life(10,11).
Measurements of AHR
The first report describing the use of inhaled acetylcholine to measure AHR was published by Robert Tiffeneau in 1945(12). Since then, techniques to measure AHR have been
improved. Nowadays, measurement of AHR is performed using a provocation test during which increasing, doubling concentrations of a stimulus are administered by inhalation, each step being followed by measurement of the forced expiratory volume in 1 second (FEV1). A 20% fall in FEV1 after administering a concentration of a stimulus (PC20) less than a certain threshold concentration, which differs for each stimulus inhaled, indicates the presence of AHR. Traditionally, two types of stimuli are being used for provocation testing: direct and indirect stimuli. Direct stimuli, such as histamine and methacholine (an acetylcholine analogue), act directly on the airway smooth muscle(13). Indirect stimuli, such as adenosine, act through adenosine receptors on inflammatory and neuronal cells which release mediators (e.g. histamine, leukotrienes and acetylcholine) upon stimulation leading to airway constriction(14). Of interest, the PC20 adenosine 5’ monophosphate (AMP) is more strongly associated with eosinophilic airway inflammation than the PC20 methacholine(15). This indicates that provocation testing with AMP can serve as a surrogate marker of airway inflammation. This hypothesis is strengthened by the observation that improvement in PC20 AMP after treatment with inhaled corticosteroids, is closely related to decreased eosinophilic airway inflammation(16). A drawback of an AMP provocation test is that a substantial proportion of asthma patients does not reach a 20% fall in FEV1 in response to the maximum concentration of AMP that can be nebulized(17,18). To address this disadvantage, our group has developed a dry powder adenosine formulation, which enables us to administer higher doses of inhaled adenosine. In two previous studies in asthma, the adenosine dry powder provocation test was shown to feasible, safe and well-tolerated(19,20).
Small airways dysfunction
Prevalence of small airways dysfunction in asthma
Small airways are defined as airways with an internal diameter less than 2mm generally starting at the 8th generation of the bronchial tree. As small airways only account for a
maximum of 15% of total airway resistance in the normal situation(21), the importance of small airways dysfunction in respiratory diseases has been ignored for decades. Nowadays, small airways are increasingly recognized as important contributors to clinical features of asthma, such as asthma control, frequency of exacerbations and nocturnal symptoms(22). Additionally, small airways dysfunction contributes to the severity of airway hyperresponsiveness. This was among others demonstrated by Telenga et al, who showed that more severe small airway obstruction, indicated by a lower forced expiratory flow at 50% (FEF50) of the forced vital capacity (FVC), was significantly associated with more severe hyperresponsiveness in asthma patients, independently from the FEV1(23). Also, Verbanck et al demonstrated that ventilation heterogeneity of the small conducting airways (Scond) was a major determinant of
1
airway hyperresponsiveness in asthma, independently of airway inflammation(24). The prevalence of small airways dysfunction in asthma is estimated to be 50-60% and can be present across all degrees of asthma severity, even in those patients with a normal FEV1(25). Table 1 provides an overview of the prevalence of small airways dysfunction in asthma according to multiple measurement techniques.
Measurements of small airways dysfunction
Several techniques to measure small airways dysfunction are currently available, but, unfortunately, a golden standard has not yet been established. Traditionally, forced expiratory flow rates at 50% or between 25 and 75% of the FVC provide information on airflow obstruction originating from the small airways. In addition, Slow Vital Capacity (SVC) minus FVC or the FVC/SVC ratio reflects obstruction of the small airways during a forced expiratory maneuver. Hyperinflation is a result of small airways obstruction and can be measured with body plethysmography that provides information on residual volume (RV), functional residual capacity (FRC) and RV/total lung capacity (RV/TLC). Airway resistance and reactance can be measured with Impulse Oscillometry (IOS), as reflected by small airway resistance (R5-R20) or reactance (X5). In addition, ventilation heterogeneity of the small conducting (Scond) and acinar (Sacin) airways is measured with multiple breath nitrogen washout (MBNW). Finally, air trapping as a result of small airways obstruction can be quantified on computed tomopgraphy (CT) – scans by comparing in- and expiratory density of the lung parenchyma: decreased expiratory attenuation of the lung parenchyma indicates air trapping.
Treatment of asthma: extrafine and non-extrafine inhaled corticosteroids
Inhaled corticosteroids (ICS) have been the mainstay of asthma treatment since the introduction of beclomethasone dipropionate in pressurized aerosols in the 1970s. Years of extensive research have taught us that ICS reduce airway inflammation and improve airway hyperresponsiveness to both direct and indirect stimuli in asthma patients(16,28–30). Ward and colleagues showed that 12-month treatment with fluticasone significantly decreases airway hyperresponsiveness compared to placebo, with a maximum effect after continued treatment, in this case 12 months(30). Of interest, they showed that the number of eosinophils and mast cells in bronchoalveolar lavage (BAL) fluid significantly decreases after 3-month treatment with fluticasone compared to placebo, with no further changes after 12-month treatment. Thus, ICS continuously decrease airway hyperresponsiveness, while the maximal diminishing effect of ICS on airway inflammation is reached after 3 months. This suggests that other beneficial effects of ICS, next to a reduction in inflammation, contribute to the ongoing improvement in airway hyperresponsiveness in this population.
Table 1. Prevalence of small airways dysfunction in asthma
Small airways
measurement Cut-off for SAD Prevalence Characteristics asthma population
Spirometry
FEF25-75% < 60% predicted 54% 442 asthma patients (step 1-4 BTS asthma guidelines) #
< LLN 12% 222 asthma patients without proximal airways obstruction‡
FEF50% < LLN 36% 94 mild-to-moderate asthma patients
SVC-FVC > 10% 10% 222 asthma patients without proximal airways obstruction‡
Body Plethysmography
RV > 100% predicted 52% 321 predominantly mild asthma patients$
> ULN 31% 222 asthma patients without proximal airways obstruction‡
RV/TLC > 35% 57% 321 predominantly mild asthma patients$
> ULN 24% 222 asthma patients without proximal airways obstruction‡
> ULN 48% 305 poorly controlled asthma patients†
FRC > 120% predicted 26% 222 asthma patients without proximal airways obstruction‡
> 120% predicted 48% 305 poorly controlled asthma patients†
Impulse Oscillometry
R5-R20 > 0.03 kPa s L-1 48% 63 mild-to-moderate asthma patients
> 0.03 kPa s L-1 65% 192 asthma patients (step 2 BTS asthma guidelines)¶
> 0.03 kPa s L-1 64% 63 asthma patients (step 3 BTS asthma guidelines)¶
> 0.03 kPa s L-1 70% 123 asthma patients (step 4 BTS asthma guidelines)¶
> 0.075 kPa s L-1 33% 33 asthma patients with FEV
1 ≥ 80% predicted
> 0.1 kPa s L-1 42% 442 asthma patients (step 1-4 BTS asthma guidelines)#
Multiple Breath Nitrogen Washout
Sacin > ULN* 46% 37 asthma patients (step 3-5 GINA guidelines)
> 0.12 L-1 53% 66 well-controlled asthma patients
> 0.12 L-1 53% 30 stable unselected asthma patients##
> ULN** 61% 18 asthma patients admitted for severe exacerbation§
> ULN** 74% 19 stable asthma patients§
Scond > 0.042 100% 30 stable unselected asthma patients ##
> ULN** 56% 18 asthma patients admitted for a severe exacerbation§
> ULN** 11% 19 stable asthma patients§
CT scan
Air trapping Air trapping index*** 56% 45 uncontrolled mild or moderate asthma patients
Prevalences adapted from Usmani et al(25); an equal symbol indicates the same study; *as defined as mean +1.64SD in the control group; **adapted from Verbanck et al(26); ***according to the definition of the Fleischner Society(27); ‡defined as FEV1% predicted>80% and FEV1/FVC≥0.7; SAD= small airways dysfunction; LLN= lower limit of normal; ULN= upper limit of
normal; BTS= British Thoracic Society; GINA= Global Initiative for Asthma; FEF25-75=forced expiratory flow between 25 and
75% of the FVC; FEF50= forced expiratory flow at 50% of the FVC; SVC= slow vital capacity; FVC= forced vital capacity; RV=
residual volume; TLC= total lung capacity; FRC= functional residual capacity; R5-R20: resistance between 5 and 20Hz; Sacin=
1
Additionally, ICS effectively reduce respiratory symptoms, improve asthma control and protect against exacerbations and asthma-related mortality(31–33). The latter was investigated in a nested case-control study of 66 subjects who died of asthma and 2681 matched asthma controls(31). The authors concluded that subjects who died of asthma had used significantly less canisters of low-dose ICS compared to their matched controls, irrespective of age, gender, prior hospitalization for asthma and the use of other asthma medication. In conclusion, these findings underline the beneficial effects of ICS in the treatment of asthma.
Extrafine and non-extrafine particle ICS
Extrafine particle ICS have a mass median aerodynamic diameter (MMAD) smaller than 2 µm, in contrast to non-extrafine particle ICS, which have a MMAD between 2 and 4 µm(34,35). It has been shown that peripheral lung deposition is higher after administration of monodisperse particles sized 1.5 µm (25%) compared to particles sized 3 µm (17%) and 6 µm (11%)(36). In the latter study, lower oropharyngeal deposition was observed for 1.5 µm-sized particles (15%) compared to particles with an MMAD of 3 µm (31%) and 6 µm (43%). These results confirm the hypothesis that higher peripheral lung deposition and lower oropharyngeal deposition is achieved with extrafine particle ICS. Many, but not all, clinical studies comparing extrafine and non-extrafine particle ICS show beneficial effects of non-extrafine particle treatment in terms of asthma control, improvement of small airways function and hospitalization(37–40). For example, Yamaguchi et al showed that asthma patients treated for 12 weeks with extrafine HFA-beclomethasone 200 µg b.i.d. (n=26) had a significant higher decrease in R5-R20 compared to asthma patients treated for 12 weeks with non-extrafine CFC-beclomethasone 400 µg b.i.d. (n=12) (37). Of interest, no significant difference in the change of FEV1 was observed between the two treatments. In addition, Hoshino et al showed that 14 asthma patients randomly assigned to receive extrafine ciclesonide 200 µg q.d. for 8 weeks had a significant improvement in small airways function as measured with R5-R20 and X5 while small airways function in 16 asthma patients receiving non-extrafine fluticasone 100 µg b.i.d. for 8 weeks remained unchanged(38). Again, FEV1 was not affected by either extrafine ciclesonide or non-extrafine fluticasone in this study. Finally, a real-life comparison study based on a retrospective cohort of ~20,000 primary care asthma patients compared treatment with extrafine HFA-beclomethasone (Qvar) and non-extrafine HFA-beclomethasone (Clenil). The authors show that better asthma control was achieved with Qvar, as well as less respiratory-related hospitalizations(39). These findings further support the notion that extrafine particle ICS are beneficial for asthma patients, when compared to non-extrafine particle ICS. However, large double-blind prospective studies that compare the same drugs in the same devices with the
only difference being particle size are lacking to confirm these observations. In addition to this, it could be speculated that certain subgroups of asthma patients would benefit in particular from extrafine ICS treatment, but studies addressing this need, i.e. the identification of a so-called ‘extrafine particle sensitive phenotype’, are not available. Future studies are necessary to explore which asthma phenotype specifically benefits from extrafine or non-extrafine ICS.
Remission of asthma
Currently, no cure for asthma exists. Of interest, a considerable number of asthma patients experiences ‘clinical remission’, which is defined as the absence of respiratory symptoms and no use of asthma medication at a certain moment in life. It is estimated that clinical remission rates vary between 30% and 52%(41). A decade ago, the term ‘complete remission’ was introduced, which specifies remission by adding extra criteria to the definition: a normalized pulmonary function and absence of airway hyperresponsiveness. Vonk et al applied this new definition to assess remission rates in a cohort of 119 allergic asthmatic children with a mean follow-up of 30 years, i.e. up to adult life(42). In total, 52% of subjects experienced remission of which 22% were in complete remission and 30% were in clinical remission. In another study, 209 asthmatic children with proven airway hyperresponsiveness were followed-up at age 25 and 49 years(43). The prevalence of complete remission was 7% at age 25 and 10% at age 49. These studies emphasize that remission of asthma occurs in a small percentage, but such cases still represent a substantial number of asthma patients. Knowledge on underlying mechanisms that drive asthma remission can aid in developing new asthma therapies that might even lead to a cure of asthma. In this respect, especially the phenotype complete remission is of interest. This phenotype comprises patients with a normalized pulmonary function and absence of hyperresponsiveness, despite not using any asthma treatment, which is suggestive of a reversible biological factor driving remission of asthma. On the other hand, subjects in clinical remission still exhibit an aberrant pulmonary function, which raises the question whether biological processes or an altered perception of symptoms play a role in this population.
Studies investigating the underlying pathophysiology of complete asthma remission mechanisms are scarce. Broekema et al studied subjects with either current asthma, clinical remission or complete remission(44). They showed that the number of sputum eosinophils was not statistically different between the groups but eosinophilic activation in the airway wall, measured with eosinophilic peroxidase (EPX) staining, was significantly lower in complete remission versus both clinical remission and current asthma (Figure 1A-B). Of interest, basement membrane thickening, a feature
1
of airway remodeling, was similar between complete remission and current asthma(Figure 1C). Boulet et al investigated subjects in complete remission, clinical remission and current asthma(45). Their findings support the observations of Broekema et al in that inflammatory cells in sputum are similar across the three groups. They additionally showed that the suppressive function of regulatory T-cells is decreased in both complete remission and asthma compared to healthy controls. These findings raise the hypothesis that an altered activation of eosinophilic inflammation, but not necessarily reversed remodeling or normalization of regulatory T-cell function, contribute to complete remission of asthma.
Figure 1. Eosinophilic inflammation and basement membrane thickening in complete remission, clinical remission, asthma patients not using inhaled corticosteroids (no ICS) and asthma patients using inhaled corticosteroids (ICS) (reprinted with permission from reference 44); A) percentage of sputum
eosinophils, B) eosinophilic peroxidase (EPX) immunopositivity (pixels) in bronchial biopsies and C) basement membrane thickness. P-values are adjusted for age, sex and current smoking.
Smoking and respiratory health
Tobacco smoking accounts for more than 6 million deaths per year, according to the World Health Organization(46). It is a major cause of respiratory diseases, such as Chronic Obstructive Pulmonary Disease (COPD) and lung cancer. Next to this, smoking negatively influences the clinical expression of asthma(47). In addition, even in subjects without respiratory diseases smoking is associated with increased airway inflammation and more small airways dysfunction(48,49).
COPD
The effects of smoking on the respiratory tract that contribute to the development of COPD are numerous(50,51). First, smoking decreases the integrity of the respiratory epithelium and impairs cilia function, causing a diminished barrier function leading to an impaired defense against toxins and microorganisms. Second, airway inflammation is enhanced by smoking through proliferation of macrophages in the alveolar space, which produce pro-inflammatory cytokines leading to recruitment of neutrophils, eosinophils and lymphocytes. Also, the phagocytic properties of macrophages are impaired, which causes accumulation of dead cells and, subsequently, increased inflammation. Of importance, macrophages produce reactive oxygen species and proteases, which induce tissue destruction leading to emphysema. Third, cigarette smoke also has immunosuppressive effects, amongst others illustrated by less airway natural-killer cells and inhibition of the memory T-cell response in smokers compared to non-smokers, leading to an increased susceptibility for respiratory infections. Of interest, the majority of smokers does not develop COPD, which implies that next to exposure to noxious particles, other factors such as genetic susceptibility are of great importance. It remains a major challenge to identify the so-called ‘susceptible smoker’, i.e. those smokers that will develop COPD upon exposure to risk factors.
Asthma and smoking
Surprisingly, smoking is as prevalent among asthma patients as in the general population(52). Asthma patients who smoke experience more respiratory symptoms and have more frequent exacerbations(53). Furthermore, smoking is associated with an accelerated lung function decline in asthma patients(54,55). In a population study of 234 subjects with asthma performed in Denmark, Lange et al found heavy smokers (15g tobacco/day) to have an additional decline in FEV1 of 22.5ml/year compared to non-smokers(54). These findings were confirmed by O’Byrne et al, who showed that the 3-year decline in FEV1 in 492 asthmatic smokers and 2432 asthmatic non-smokers was 264ml versus 181mL, respectively(55). Of interest, smokers with asthma show a
1
diminished response to inhaled corticosteroids, although clinical trials addressing ICS treatment specifically in asthmatic smokers are limited(55–59). Telenga et al showed that current smokers (n=30) with asthma had a lower increase in FEV1 compared to never-smokers (n=55) after 2-week ICS treatment, although this effect was not observed after 1-year treatment(56). Short-term effects of ICS were also studied by Chalmers et al, comparing 3-week treatment with 1000µg fluticasone daily in smokers (n=17) and never-smokers (n=21) with asthma(57). They showed that in never-smokers, ICS decreased peak expiratory flow (PEF) variability, increased FEV1 and improved bronchial hyperresponsiveness while in smokers, no significant change in any of these parameters was observed. Long-term studies on ICS effects are scarce. Pederson et al showed that 9-month treatment with either high (1600µg/d) or low (400µg/d) dose budesonide did not improve FEV1 or hyperresponsiveness in asthmatic smokers (n=37), while asthmatic never- and ex-smokers (n=47) did significantly improve in both parameters(58). On the other hand, in a large randomized trial with 3-year follow-up in which asthma patients were randomized between 400µg budesonide daily and placebo, smokers (n=492) and non-smokers (n=2432) were equally responsive to budesonide in terms of decline in FEV1(55). Tomlinson et al treated 28 non-smokers and 19 smokers with asthma for 12 weeks with 400µg beclomethasone daily and showed a significant decrease in PEF variability after treatment in non-smokers, when compared to smokers(59). However, after 12-week treatment with a higher dose of ICS, i.e. 1200µg daily, no difference in change in PEF variability between smokers (n=21) and non-smokers (n=27) was observed. Findings from these studies emphasize that: 1) studies on ICS treatment in smokers with asthma are scarce, 2) study outcomes in individual studies (i.e. PEF variability, FEV1, hyperresponsiveness) are variable, which hampers the direct comparison of these studies, 3) short term effects of ICS seems to be impaired in smokers with asthma but 4) longer treatment duration or higher doses of ICS might be more effective. Based on these findings, the exact role of ICS in asthma patients who smoke is not exactly clear. In conclusion, smoking is prevalent in asthma and contributes to more severe disease. Although smoking cessation is essential for these patients, achieving this appears difficult. Studies targeting this specific group of asthma patients are needed to improve treatment and eventually clinical outcome.
Small airways dysfunction and smoking
Smoking is known to induce small airways dysfunction. The latter was demonstrated by Verbanck et al, who showed that smokers with more than 10 pack-years showed increased ventilation heterogeneity (i.e. higher Scond and Sacin) of the small airways compared to never-smokers(48). These findings can potentially be explained by higher numbers of inflammatory cells and higher levels of inflammatory mediators present in
the small airways of smokers compared to non-smokers(60,61), which in turn may lead to persistent airway remodeling and narrowing. As mentioned previously, the majority of studies investigating ICS-treatment in smokers with asthma have used non-extrafine particle ICS, which mainly deposit in the central airways. It could be speculated that extrafine particle ICS, which show a higher preference for deposition in the peripheral airways, are more beneficial in smokers with asthma.
MOLECULAR PHENOTYPING
Genomic research in obstructive lung diseases
In the past decades, new techniques have become available to study underlying pathophysiological mechanisms of obstructive lung diseases, such as genome wide association (GWA) studies and transcriptomics. GWA studies have identified genomic regions containing DNA variants that possibly play a role in asthma development. These genomic regions contain genes such as SMAD3 and ORMDL3 for asthma(62) and HHIP and FAM13A for COPD(63). Transcriptomics is defined as ‘the study of the transcriptome’, which comprises the complete set of RNA transcripts present in a cell, varying from protein-coding messenger RNA transcripts to non-coding RNA transcripts such as microRNAs and long non-coding RNAs. Analysis of RNA expression levels in obstructive lung diseases is nowadays a topic of wide interest and can aid in understanding mechanisms driving a disease. For example, a study investigating the bronchial epithelium in COPD has shown that COPD patients exhibit a distinct gene expression profile compared to controls which resembles COPD-induced gene expression changes occurring in lung tissue(64). In addition, bronchial epithelial gene expression in COPD changes after treatment with ICS and this altered gene expression profile is enriched in genes associated with smokers without COPD versus smokers with COPD, suggesting that ICS can reverse disease activity(65). Results of these studies investigating the transcriptome in COPD are of interest in two ways: 1) they provide information on transcriptional mechanisms driving a disease or a treatment and 2) transcriptional alterations can potentially serve as a biomarker to aid in e.g. diagnosing a disease or monitoring response to treatment. In this respect, studies on bronchial gene expression in asthma are also of interest. Woodruff and colleagues found 22 genes to be differentially expressed in the airways of asthma patients compared to controls, of which 3 genes were also affected by 1-week ICS treatment: POSTN, CLCA1 and SERPINB2(66). They additionally showed that higher baseline expression of these 3 genes correlated with improvement in FEV1. Further, a study comparing airway gene expression between patients with asthma and COPD, identified overlapping gene expression changes suggesting changes in biological processes that are shared between asthma and COPD(67). These findings
1
are illustrative of the role of transcriptomics as a potential biomarker and as a tool to explore disease-mechanisms. Although transcriptional findings in the airway wall are of great interest, extensive research is hampered by the difficulty to obtain these samples, which requires an invasive bronchoscopy. Alternatives are currently explored, such as less invasive sampling from the nasal epithelium. It has been shown that the smoking-associated nasal epithelial gene expression profile shows considerable overlap with the smoking-associated bronchial gene expression profile(68,69). This implies that the nasal epithelium is a promising site for future transcriptomic research in asthma and COPD.
OUTLINE OF THE THESIS
In Chapter 2 we investigate subjects with proven airway hyperresponsiveness who do not experience any respiratory symptoms, so-called ‘asymptomatic airway hyperresponsiveness (AHR)’. Since small airways dysfunction is associated with respiratory symptoms and with AHR, we hypothesized that subjects with asymptomatic AHR have less small airways dysfunction than subjects with current asthma. We performed a cross-sectional study including subjects with asthma, subjects with asymptomatic AHR and healthy controls, in which we especially focused on small airways function measured with impulse oscillometry at baseline and during a methacholine provocation test.
Chapter 3 investigates the presence and extent of small airways dysfunction,
emphysema and parenchymal disease measured with CT-scans in association with ageing and smoking in a healthy population. We applied a new technique to assess CT-scans, ‘parametric response mapping’, which is based on a voxel-by-voxel comparison of lung attenuation on in- and expiratory CT-scans, which has the advantage that it allows discrimination between emphysema and small airways dysfunction.
In Chapter 4 we describe results of the OLiVIA study: a randomized controlled trial in which we compared extrafine with non-extrafine particle ICS treatment in smokers and ex-smokers with asthma. Since smoking induces small airways dysfunction, we hypothesized that smokers and ex-smokers with asthma would benefit more from extrafine compared to non-extrafine ICS. In Chapter 5, we performed a post-hoc analysis of this study in which we investigated whether subjects having small airways dysfunction at baseline, benefit more from extrafine compared to non-extrafine ICS. We additionally explored which other clinical variables predict a favorable response to either extrafine or non-extrafine ICS.
Chapter 6 describes the comparison of two formulations of adenosine used for bronchial
provocation testing in asthma: wet nebulized adenosine 5’ monophosphate (AMP) and the recently developed dry powder adenosine. Since not all asthma patients experience a 20% fall in FEV1 after administering the highest possible dose of AMP, and dry powder
adenosine can be produced in higher doses, we hypothesized that dry powder adenosine would be a more sensitive test to measure airway hyperresponsiveness in asthma.
In Chapter 7 we aimed to define a nasal epithelial gene expression profile that distinguishes smoking COPD patients from smoking controls. We additionally explored whether this gene expression profile was overlapping with COPD-associated gene expression in the bronchial epithelium in two independent cohorts.
Chapter 8 describes our investigations to assess whether ICS treatment in smokers and
ex-smokers with asthma is reflected by changes in nasal epithelial gene expression in 2 cohorts of asthma patients treated for 2 weeks and 3 months with ICS. We furthermore explored whether these ICS-induced nasal gene expression changes overlap with corticosteroid-induced bronchial gene expression changes in 2 independent cohorts of asthma patients and in air-liquid interface cultures of human bronchial epithelial cells.
Chapter 9 describes a study in which we aim to unravel underlying pathophysiological
mechanisms driving remission of asthma by analysis of microRNA expression in bronchial biopsies of subjects with complete remission of asthma, subjects with persistent asthma and healthy controls. We integrated microRNA expression with protein-coding- and long non-coding RNA expression by performing Bayesian network modeling, with the aim to gain insight in the complex interactions of transcripts contributing to remission of asthma.
1
REFERENCES
(1) To T, Stanojevic S, Moores G, Gershon AS, Bateman ED, Cruz A a, et al. Global asthma prevalence in adults: findings from the cross-sectional world health survey. BMC Public Health. 2012 Jan;12(1):204. (2) Bahadori K, Doyle-Waters MM, Marra C, Lynd L, Alasaly K, Swiston J, et al. Economic burden of asthma: a
systematic review. BMC Pulm Med. 2009 Dec 19;9(1):24.
(3) Juniper EF. Quality of life in adults and children with asthma and rhinitis. Allergy. 1997;52(10):971–7. (4) World Health Organization. Global Healthy Observatory data: Top 10 causes of death [Internet]. Available
from: http://www.who.int/gho/mortality_burden_disease/causes_death/top_10/en/
(5) Eisner MD, Anthonisen N, Coultas D, Kuenzli N, Perez-Padilla R, Postma D, et al. An official American Thoracic Society public policy statement: Novel risk factors and the global burden of chronic obstructive pulmonary disease. Am J Respir Crit Care Med. 2010;182(5):693–718.
(6) Jansen DF, Timens W, Kraan J, Rijcken B, Postma DS. (A)Symptomatic bronchial hyper-responsiveness and asthma. Respir Med. 1997;91(3):121–34.
(7) Laprise C, Laviolette M, Boutet M, Boulet LP. Asymptomatic airway hyperresponsiveness: Relationships with airway inflammation and remodelling. Eur Respir J. 1999;14(1):63–73.
(8) Betz R, Kohlhaufl M, Kassner G, Mullinger B, Maier K, Brand P, et al. Increased sputum IL-8 and IL-5 in asymptomatic nonspecific airway hyperresponsiveness. Lung. 2001;179(2):119–33.
(9) Boulet LP, Turcotte H. Lung hyperinflation, perception of bronchoconstriction and airway hyperresponsiveness. Clin Investig Med. 2007;30(1):2–11.
(10) Brutsche MH, Downs SH, Schindler C, Gerbase MW, Schwartz J, Frey M, et al. Bronchial hyperresponsiveness and the development of asthma and COPD in asymptomatic individuals: SAPALDIA Cohort Study. Thorax. 2006;61(8):671–7.
(11) Xu X, Rijcken B, Schouten JP, Weiss ST. Airways responsiveness and development and remission of chronic respiratory symptoms in adults. Lancet. 1997;350(9089):1431–4.
(12) Tiffeneau R, Beauvallet M. Epreuve de bronchoconstriction et de bronchodilation par aérosols. Emploi pour la dépistage, la mesure et le contrôle des insuffisances respiratoires chroniques. Bull l’Académie R Médicine. 1945;129:165−8.
(13) Cockcroft DW, Davis BE. Mechanisms of airway hyperresponsiveness. J Allergy Clin Immunol. 2006 Sep;118(3):551-9-1.
(14) Van Schoor J, Joos GF, Pauwels RA. Indirect bronchial hyperresponsiveness in asthma: Mechanisms, pharmacology and implications for clinical research. Eur Respir J. 2000;16(3):514–33.
(15) Van den Berge M, Meijer RJ, Kerstjens HAM, De Reus DM, Koëter GH, Kauffman HF, et al. PC 20 Adenosine 5’-Monophosphate Is More Closely Associated with Airway Inflammation in Asthma Than PC 20 Methacholine. Am J Respir Crit Care Med. 2001 Jun;163(7):1546–50.
(16) van den Berge M, Kerstjens HA, Meijer RJ, de Reus DM, Koëter GH, Kauffman HF, et al. Corticosteroid-induced improvement in the PC20 of adenosine monophosphate is more closely associated with reduction in airway inflammation than improvement in the PC20 of methacholine. Am J Respir Crit Care Med. 2001 Oct 1;164(7):1127–32.
(17) Cohen J, Postma DS, Douma WR, Vonk JM, De Boer AH, ten Hacken NHT. Particle size matters: diagnostics and treatment of small airways involvement in asthma. Eur Respir J. 2011 Mar;37(3):532–40.
(18) Prieto L, Reig I, Rojas R, Ferrer A, Domenech J. The effect of challenge method on sensitivity and reactivity to adenosine 5’-monophosphate in subjects with suspected asthma. Chest. 2006;130(5):1448–53. (19) Lexmond AJ, Hagedoorn P, Van Der Wiel E, Ten Hacken NHT, Frijlink HW, De Boer AH. Adenosine dry
powder inhalation for bronchial challenge testing, part 1: Inhaler and formulation development and in vitro performance testing. Eur J Pharm Biopharm. 2014;86(1):105–14.
(20) Lexmond AJ, van der Wiel E, Hagedoorn P, Bult W, Frijlink HW, ten Hacken NHT, et al. Adenosine dry powder inhalation for bronchial challenge testing, part 2: proof of concept in asthmatic subjects. Eur J Pharm Biopharm. 2014;88(1):148–52.
(21) Macklem PT, Mead J. Resistance of central and peripheral airways measured by a retrograde catheter. J Appl Physiol. 1967;22(3):395–401.
(22) van der Wiel E, ten Hacken NHT, Postma DS, van den Berge M. Small-airways dysfunction associates with respiratory symptoms and clinical features of asthma: a systematic review. J Allergy Clin Immunol. 2013 Mar;131(3):646–57.
(23) Telenga ED, van den Berge M, Ten Hacken NHT, Riemersma R a, van der Molen T, Postma DS. Small airways in asthma: their independent contribution to the severity of hyperresponsiveness. Eur Respir J. 2013 Mar;41(3):752–4.
(24) Downie SR, Salome CM, Verbanck S, Thompson B, Berend N, King GG. Ventilation heterogeneity is a major determinant of airway hyperresponsiveness in asthma, independent of airway inflammation. Thorax. 2007 Aug;62(8):684–9.
(25) Usmani OS, Singh D, Spinola M, Bizzi A, Barnes PJ. The prevalence of small airways disease in adult asthma: A systematic literature review. Respir Med. 2016;116:19–27.
(26) Verbanck S, Thompson BR, Schuermans D, Kalsi H, Biddiscombe M, Stuart-Andrews C, et al. Ventilation heterogeneity in the acinar and conductive zones of the normal ageing lung. Thorax. 2012 Sep;67(9):789– 95.
(27) Austin JH, Müller NL, Friedman PJ, Hansell DM, Naidich DP, Remy-Jardin M, et al. Glossary of terms for CT of the lungs: recommendations of the Nomenclature Committee of the Fleischner Society. Radiology. 1996;200(2):327−31.
(28) Childhood Asthma Management Program Research Group, Szefler S, Weiss S, Tonascia J, Adkinson NF, Bender B, et al. Long-term effects of budesonide or nedocromil in children with asthma. N Engl J Med. 2000;343(15):1054–63.
(29) Boulet LP, Turcotte H, Prince P, Lemière C, Olivenstein R, Laprise C, et al. Benefits of low-dose inhaled fluticasone on airway response and inflammation in mild asthma. Respir Med. 2009;103(10):1554–63. (30) Ward C, Pais M, Bish R, Reid D, Feltis B, Johns D, et al. Airway inflammation, basement membrane
thickening and bronchial hyperresponsiveness in asthma. Thorax. 2002;57(4):309–16.
(31) Suissa S, Ernst P, Benayoun S, Baltzan M, Cai B. Low-dose inhaled corticosteroids and the prevention of death from asthma. N Engl J Med. 2000 Aug 3;343(5):332–6.
(32) Pauwels RA, Pedersen S, Busse WW, Tan WC, Chen Y-Z, Ohlsson S V, et al. Early intervention with budesonide in mild persistent asthma: a randomised, double-blind trial. Lancet. 2003;361(9363):1071– 6.
(33) Donahue JG, Weiss ST, Livingston JM, Goetsch M a, Greineder DK, Platt R. Inhaled steroids and the risk of hospitalization for asthma. Jama. 1997;277(11):887–91.
(34) de Vries TW, Rottier BL, Gjaltema D, Hagedoorn P, Frijlink HW, de Boer AH. Comparative in vitro evaluation of four corticosteroid metered dose inhalers: Consistency of delivered dose and particle size distribution. Respir Med. 2009;103(8):1167–73.
(35) Leach CL, Davidson PJ, Hasselquist BE, Boudreau RJ. Influence of Particle Size and Patient Dosing Technique on Lung Deposition of HFA-Beclomethasone from a Metered Dose Inhaler. J Aerosol Med. 2005;18(4):379–85.
(36) Usmani OS, Biddiscombe MF, Barnes PJ. Regional lung deposition and bronchodilator response as a function of β2-agonist particle size. Am J Respir Crit Care Med. 2005;172(12):1497–504.
(37) Yamaguchi M, Niimi A, Ueda T, Takemura M, Matsuoka H, Jinnai M, et al. Effect of inhaled corticosteroids on small airways in asthma: Investigation using impulse oscillometry. Pulm Pharmacol Ther. 2009 Aug;22(4):326–32.
(38) Hoshino M. Comparison of Effectiveness in Ciclesonide and Fluticasone Propionate on Small Airway Function in Mild Asthma. Allergol Int. 2010;59(1):59–66.
(39) Price D, Thomas M, Haughney J, Lewis RA, Burden A, Von Ziegenweidt J, et al. Real-life comparison of beclometasone dipropionate as an extrafine- or larger-particle formulation for asthma. Respir Med. 2013;107(7):987–1000.
1
(40) van der Molen T, Postma DS, Martin RJ, Herings RMC, Overbeek JA, Thomas V, et al. Effectiveness of initiating extrafine-particle versus fine-particle inhaled corticosteroids as asthma therapy in the Netherlands. BMC Pulm Med. 2016 Dec 17;16(1):80.
(41) Upham JW, James AL. Remission of asthma: The next therapeutic frontier? Pharmacol Ther. 2011;130(1):38–45.
(42) Vonk JM, Postma DS, Boezen HM, Grol MH, Schouten JP, Koëter GH, et al. Childhood factors associated with asthma remission after 30 year follow up. Thorax. 2004 Nov 1;59(11):925–9.
(43) Carpaij OA, Nieuwenhuis MAE, Koppelman GH, van den Berge M, Postma DS, Vonk JM. Childhood factors associated with complete and clinical asthma remission at 25 and 49 years. Eur Respir J. 2017;49(6):1601974.
(44) Broekema M, Timens W, Vonk JM, Volbeda F, Lodewijk ME, Hylkema MN, et al. Persisting remodeling and less airway wall eosinophil activation in complete remission of asthma. Am J Respir Crit Care Med. 2011;183(3):310–6.
(45) Boulet LP, Turcotte H, Plante S, Chakir J. Airway function, inflammation and regulatory T cell function in subjects in asthma remission. Can Respir J. 2012;19(1):19–25.
(46) World Health Organization. WHO global report on trends in prevalence of tobacco smoking 2015. WHO Mag. 2015;1–359.
(47) Polosa R, Thomson NC. Smoking and asthma: Dangerous liaisons. Eur Respir J. 2013;41(3):716–25. (48) Verbanck S, Schuermans D, Meysman M, Paiva M, Vincken W. Noninvasive assessment of airway
alterations in smokers: the small airways revisited. Am J Respir Crit Care Med. 2004 Aug 15;170(4):414–9. (49) Roth MD, Arora A, Barsky SH, Kleerup EC, Simmons M, Tashkin DP. Airway Inflammation in Young
Marijuana and Tobacco Smokers. Am J Respir Crit Care Med. 1998;157:928–37.
(50) Stämpfli MR, Anderson GP. How cigarette smoke skews immune responses to promote infection, lung disease and cancer. Nat Rev Immunol. 2009;9(5):377−84.
(51) Alexander LEC, Shin S, Hwang JH. Inflammatory diseases of the lung induced by conventional cigarette smoke: a review. Chest. 2015;148(5):1307–22.
(52) Livingston E, Thomson NC, Chalmers GW. Impact of smoking on asthma therapy: A critical review of clinical evidence. Drugs. 2005;65(11):1521–36.
(53) Thomson NC. Asthma and smoking-induced airway disease without spirometric COPD. Eur Respir J. 2017;49(5):1–14.
(54) Lange P, Scharling H, Ulrik CS, Vestbo J. Inhaled corticosteroids and decline of lung function in community residents with asthma. Thorax. 2006;61(2):100–4.
(55) O’Byrne PM, Lamm CJ, Busse WW, Tan WC, Pedersen S. The effects of inhaled budesonide on lung function in smokers and nonsmokers with mild persistent asthma. Chest. 2009;136(6):1514–20. (56) Telenga ED, Kerstjens HAM, ten Hacken NHT, Postma DS, Van den Berge M. Inflammation and
corticosteroid responsiveness in ex-, current- and never-smoking asthmatics. BMC Pulm Med. 2013;13(1). (57) Chalmers GW, Macleod KJ, Little S a, Thomson LJ, McSharry CP, Thomson NC. Influence of cigarette
smoking on inhaled corticosteroid treatment in mild asthma. Thorax. 2002 Mar;57(3):226–30. (58) Pederson B, Dahl R, Karlström R, Christer P, Venge P. Eosinophil and Neutrophil activity in asthma in a
one-year trial with inhaled budesonide. Am J Respir Crit Care Med. 1996;153:1519−29.
(59) Tomlinson JEM, McMahon AD, Chaudhuri R, Thompson JM, Wood SF, Thomson NC. Efficacy of low and high dose inhaled corticosteroid in smokers versus non-smokers with mild asthma. Thorax. 2005;60(4):282–7.
(60) Takizawa H, Tanaka M, Takami K, Ohtoshi T, Ito K, Satoh M, et al. Increased expression of inflammatory mediators in small-airway epithelium from tobacco smokers. Am J Physiol Lung Cell Mol Physiol. 2000;278(5):L906-13.
(61) Lams BEA, Sousa AR, Rees PJ, Lee TH. Subepithelial immunopathology of the large airways in smokers with and without chronic obstructive pulmonary disease. Eur Respir J. 2000;15(3):512–6.
(62) Vicente CT, Revez JA, Ferreira MAR. Lessons from ten years of genome-wide association studies of asthma. Clin Transl Immunol. 2017;6(12):e165.
(63) Yuan C, Chang D, Lu G, Deng X. Genetic polymorphism and chronic obstructive pulmonary disease. Int J Chron Obstruct Pulmon Dis. 2017;12:1385–93.
(64) Steiling K, van den Berge M, Hijazi K, Florido R, Campbell J, Liu G, et al. A dynamic bronchial airway gene expression signature of chronic obstructive pulmonary disease and lung function impairment. Am J Respir Crit Care Med. 2013 May 1;187(9):933–42.
(65) van den Berge M, Steiling K, Timens W, Hiemstra PS, Sterk PJ, Heijink IH, et al. Airway gene expression in COPD is dynamic with inhaled corticosteroid treatment and reflects biological pathways associated with disease activity. Thorax. 2014 Jan 7;69(1):14–23.
(66) Woodruff PG, Boushey H a, Dolganov GM, Barker CS, Yang YH, Donnelly S, et al. Genome-wide profiling identifies epithelial cell genes associated with asthma and with treatment response to corticosteroids. Proc Natl Acad Sci USA. 2007;104(40):15858–63.
(67) Christenson SA, Steiling K, Van Den Berge M, Hijazi K, Hiemstra PS, Postma DS, et al. Asthma-COPD overlap: Clinical relevance of genomic signatures of type 2 inflammation in chronic obstructive pulmonary disease. Am J Respir Crit Care Med. 2015;191(7):758–66.
(68) Zhang X, Sebastiani P, Liu G, Schembri F, Zhang X, Dumas YM, et al. Similarities and differences between smoking-related gene expression in nasal and bronchial epithelium. Physiol Genomics. 2010;41(1):1–8. (69) Imkamp K, Berg M, Vermeulen CJ, Heijink IH, Guryev V, Kerstjens HA, et al. Nasal epithelium as a proxy for bronchial epithelium for smoking-induced gene expression and expression Quantitative Trait Loci. J Allergy Clin Immunol. 2018 Jul;142(1):314-317.e15.
