Article Text
PDF
Abstract
Background Eosinophil-derived neurotoxin (EDN) and eosinophil cationic protein (ECP) are proteins released by activated eosinophils whose role in adult asthma remains unclear.
Objective To study associations between ECP, EDN and various asthma characteristics in adults from the Epidemiological Study on the Genetics and Environment of Asthma (EGEA).
Methods Plasma ECP and EDN levels were measured by ELISA. Cross-sectional analyses were performed in 941 adults (43±16 years old, 39% with asthma) at EGEA2 (2003–2007). Longitudinal analyses investigated the associations between EDN level at EGEA2 and changes in asthma characteristics between EGEA2 and EGEA3 (2011–2013, n=817). We used generalised estimated equations adjusted for age, sex, smoking status and body mass index to take into account familial dependence.
Results At EGEA2, both high ECP and EDN levels were associated with current asthma (adjusted OR (aOR) (95% CI): 1.69 (1.35–2.12) and 2.12 (1.76–2.57)). Among asthmatics, high EDN level was associated with asthma attacks (aOR: 1.50 (1.13–1.99)), wheezing and breathlessness (aOR: 1.38 (1.05–1.80)), use of asthma treatments (aOR: 1.91 (1.37–2.68)) and bronchial hyper-responsiveness (aOR: 2.03 (1.38–2.97)), even after further adjustment on ECP. High ECP level was associated with high neutrophil count and tended to be associated with chronic bronchitis. High EDN level at EGEA2 was associated with persistent asthma (aOR: 1.62 (1.04–2.52)), nocturnal symptoms (aOR from 2.19 to 3.57), worsening wheezing and breathlessness (aOR: 1.97 (1.36–2.85)) and nocturnal shortness of breath (aOR: 1.44 (1.04–1.98)) between EGEA2 and EGEA3.
Conclusions EDN and ECP were associated with different asthma expression in adults. EDN could be a potential biomarker to monitor asthma evolution in adults.
- asthma
- eosinophil biology
Data availability statement
No data are available. Due to third-party restrictions, EGEA data are not publicly available. Please see the following URL for more information: https://egeanet.vjf.inserm.fr/index.php/en/contacts-en. Interested researchers should contact egea.cohorte@inserm.fr with further questions regarding data access.
Statistics from Altmetric.com
Key messages
What is the key question?
Are eosinophil-derived neurotoxin (EDN) and eosinophil cationic protein (ECP) associated with the same asthma characteristics and with long-term asthma evolution in adults?
What is the bottom line?
By addressing for the first-time blood ECP and EDN in a large asthma cohort, this paper shows that ECP and EDN are associated with different asthma characteristics and highlights the potential interest of EDN to monitor asthma in adults.
Why read on?
This paper is not only the first study addressing both blood ECP and EDN levels in a large longitudinal cohort of adult asthmatics with extensive phenotypic characterisation; it is also the first to examine the associations between blood EDN and change in asthma characteristics over time.
Introduction
Asthma is a chronic inflammatory disease affecting around 270 million people worldwide.1 This heterogeneous disease is characterised by various clinical manifestations linked to distinct underlying biological mechanisms.2 3 Indeed, considerable efforts have been done to understand asthma pathophysiology in order to improve its management.
Among the well-recognised asthma phenotypes, eosinophilic asthma is the most studied one. It is characterised by an accumulation of eosinophils in the airways, which is the hallmark of type 2 (T2) inflammation.2 4 5 In adults, blood eosinophil count is considered as a reliable marker of airway eosinophilia,6 and high blood eosinophil count was associated with poor lung function, higher bronchial hyper-responsiveness (BHR) and total immunoglobulin E (IgE) level among adults with asthma from the Epidemiological Study on the Genetics and Environment of Asthma (EGEA).7 Eosinophils drive inflammation through a wide range of released mediators that can directly affect airway homeostasis.8 Among them, eosinophil-derived neurotoxin (EDN), also named RNase 2 and eosinophil cationic protein (ECP), also named RNase 3, are two basic proteins stored in granules which share 67% of structural homology.9 After release on eosinophil degranulation, they exhibit cytotoxic and anti-infectious properties through their ribonuclease activity,10 11 and are involved in lung epithelium damage, mucus hypersecretion, airways remodelling and inflammation.12
Among biomarkers of eosinophil activation, ECP is the most widely studied, and can be quantified in different biological fluids, especially in peripheral blood. Despite its lack of specificity for asthma diagnosis, among asthmatics, the usefulness of blood ECP in assessing airway inflammation, asthma control, as well as efficacy and compliance with classical anti-inflammatory and new biological targeted treatments has been underlined.12–14 Unlike ECP, evidence of clinical utility of EDN just begins to emerge in the literature. In young children, serum EDN level was more strongly correlated with symptom severity score than serum ECP level or total eosinophil count.14 15 Similar results were recently found in Korean adults.16 17 These findings led some authors to postulate that EDN could be a more reliable biomarker of eosinophilic inflammation and asthma activity than ECP, possibly because of its better stability.10 Recently, results from a cross-sectional study suggested that quantification of these two biomarkers could help to classify asthmatics since an increase in both serum ECP and urinary EDN was related to the highest likelihood of fixed airflow obstruction.18 However, to our knowledge, only four studies have simultaneously quantified ECP and EDN in adults with asthma,16 18–20 and only one has addressed both measurements in peripheral blood through a large-scale study focusing on asthma severity.16 Moreover, the need of follow-up studies to better characterise clinical significance of blood ECP and EDN and their relationship with long-term asthma characteristics has been highlighted in literature16 18 but was never conducted yet.
Taking advantage of the extensive clinical, functional and biological characterisation of adult participants from the EGEA cohort, we investigated and compared the associations between both ECP and EDN, and various asthma characteristics and their evolution.
Methods
Study design and population
EGEA (https://egeanet-vjf-inserm-fr.proxy.insermbiblio.inist.fr/) is a French cohort study based on an initial group of asthma cases, their first-degree relatives and population-based controls (EGEA1: 1991–1995, EGEA2: 2003–2007, EGEA3: 2011–2013). The protocol and descriptive characteristics have been published previously21 22 and are detailed in the online supplemental material.
Supplemental material
Cross-sectional analyses used data collected at EGEA2. Adult participants (aged ≥16 years) with available data for both ECP and EDN measurements were included. Participants with ever asthma but without current asthma were excluded from the analyses (n=75), as well as those for whom an analytical issue for ECP and/or EDN quantification was detected (n=39). At EGEA2, a total of 941 participants were included (567 never-asthmatics and 374 current-asthmatics, figure 1). Among the 941 participants, 817 were followed-up at EGEA3.
Flow chart of the participants included in the cross-sectional and longitudinal analyses. ECP, eosinophil cationic protein; EDN, eosinophil-derived neurotoxin; EGEA, Epidemiological Study of the Genetics and Environment of Asthma.
Definitions of ever asthma and current asthma
Participants with ever asthma were those who answered positively to at least one of the two following questions: ‘Have you ever had attacks of breathlessness at rest with wheezing?’ or ‘Have you ever had asthma attacks?’ or were recruited as asthmatic cases at EGEA1.
Among participants with ever asthma, current asthma was defined by the report of respiratory symptoms (wheeze, nocturnal chest tightness or attacks of breathlessness following strenuous activity, at rest or at night-time), or asthma attacks or use of inhaled and/or oral medicines because of breathing problems in the past 12 months.
In order to facilitate reading, participants with current asthma are called ‘asthmatics’ and those without asthma called ‘never-asthmatics’.
Clinical and biological characteristics (EGEA2 only)
Allergic sensitisation was defined by a positive skin prick test with a mean weal diameter ≥3 mm than the negative control for at least one of 12 common aeroallergens.
Plasma ECP and EDN levels were measured by commercially available ELISA kits: MBL International MESACUP 7618E and 7630, Nagoya, Japan, for human ECP and EDN, respectively. All samples were systematically diluted by 1:5 or 1:10 when needed, and assayed following manufacturer instructions. The assay range after dilution was 0.625–200 ng/mL for ECP and 3.0–200 ng/mL for EDN. All samples were analysed in duplicate at the Laboratory of Biochemistry Molecular Biology (CHRU of Lille) according to validated and standardised procedures. The minimum detection limits were 0.125 ng/mL for ECP and 0.62 ng/mL for EDN. Samples with an intra-assay coefficient of variation ≥20% for at least one biomarker were excluded from the analysis.23 24 ECP and EDN levels were log-transformed due to their skewed distribution and expressed as geometric mean and Q1–Q3.
Blood neutrophil and eosinophil counts and total serum IgE level were available in all participants. A panel of inflammatory mediators were also available in a subsample of the participants: serum interleukin (IL)-1Ra, IL-5, IL-6, IL-7, IL-8, IL-10, IL-13, tumour necrosis factor (TNF)-alpha, leptin, high-sensitivity C reactive protein (hs-CRP) and FENO.
Further details are provided in the online supplemental material.
Clinical characteristics available at EGEA2 and EGEA3
Asthma control has been assessed over 3-month period, using responses to survey questions to approximate as closely as possible the Global Initiative for Asthma 2015 definition as previously used.7 25 Participants were defined as having controlled, partly controlled and uncontrolled asthma if they had none, 1–2 or 3–4 of the following criteria, respectively: frequent daytime symptoms, any night-time symptoms, frequent use of reliever medication and any activity limitation.
Details of asthma control definition as well as exacerbations and chronic bronchitis are provided in the online supplemental material.
Evolution of asthma outcomes between EGEA2 and EGEA3
For each participant, changes in asthma characteristics (especially current asthma, asthma control, chronic bronchitis, nocturnal symptoms, treatments) between EGEA2 and EGEA3 were categorised as ‘persistent’ if the characteristic was present at both EGEA2 and EGEA3, ‘improved’ if the characteristic was present only at EGEA2, ‘worsened’ if the characteristic was present only at EGEA3 and ‘stable’ if the characteristic was not present at both surveys.
Statistical analyses
Standard univariate tests (t-test, analysis of variance test, Fisher’s exact test or χ2 test) were used to compare continuous and categorical variables between groups. Pearson’s correlation coefficients followed by Bonferroni’s multiple test correction were used to study correlations between inflammatory biomarkers. Associations between ECP, EDN and age, sex, smoking status and body mass index (BMI) were studied in participants without asthma in order to evaluate these associations independently of the disease.
We performed regression models to investigate the cross-sectional associations between each biomarker and current asthma in all participants, and between each biomarker and asthma characteristics, pulmonary function, allergic and inflammatory characteristics in asthmatics. Due to the small size of the uncontrolled group, ‘partly controlled’ and ‘uncontrolled’ were grouped together. To facilitate interpretation of the results, we rescaled the ECP and EDN levels using IQR, defined as the difference between the 25th and 75th percentiles. Log-transformed ECP and EDN values were divided by their IQR (0.44 for ECP and 0.26 for EDN), thus the resulting regression coefficient reflects an increase of one IQR (see online supplemental material for further details). Regression analyses were conducted using generalised estimated equations to take into account familial dependence between participants. Odds ratios were adjusted (aOR) for age, sex, smoking status (never-smokers, exsmokers or current smokers) and BMI (continuous). According to the Grubb’s test,26 two outliers were found among ECP measurements, and a sensitivity analysis was also performed after excluding them.
As ECP and EDN were correlated (r=0.45), and often associated with the same asthma characteristics, we performed supplementary analyses by including ECP and EDN in each model in order to disentangle the role of ECP from that of EDN in these associations. All cross-sectional associations between ECP and asthma characteristics did not remain significant after adjustment on EDN, and we decided to restrict the longitudinal analyses to EDN. We investigated the associations between EDN level at EGEA2 and changes in various asthma characteristics at EGEA3. We created two binary outcomes: (1) one that compared the ‘persistent’ to the ‘improved’ group (reference) among participants who have the characteristic at EGEA2; (2) one that compared the ‘worsened’ to the ‘stable’ group (reference) among those who did not have the characteristic at EGEA2.
Statistical analyses were performed using SAS software, V.9.4 (SAS Institute, Cary, North Carolina, USA). A p value <0.05 was considered statistically significant.
Results
Characteristics of the participants
In comparison to those included in the cross-sectional analyses (n=941), participants not included (n=505) have lower BHR. No differences were observed for age, sex, BMI, smoking or other asthma characteristics (see online supplemental table S1).
Table 1 shows the characteristics of the 941 participants. Their age was 43.5±16.5 years (mean±SD), 52% were women, 24% smokers and 374 had current asthma. In comparison to never-asthmatics, asthmatics were more often male (p=0.04), younger (p<0.0001), had higher blood eosinophil count, more often allergic sensitisation, lower lung function and higher BHR, and reported more often chronic bronchitis and dyspnoea (all p<0.0001). Among asthmatics, age of asthma onset was 10 years3–25 (median (Q1–Q3)), 55% reached full control of the disease; in the past 12 months, 44% reported having had asthma attacks, and 76% reported use of any asthma treatments.
Characteristics of the 941 participants, and according to asthma status
ECP and EDN levels, age, sex, BMI and smoking status
Among never-asthmatics, EDN level was significantly higher in men than in women, and ECP level increased significantly with BMI. No other associations were found (see online supplemental table S2). Among asthmatics, same associations were found (not shown) except between ECP and BMI (p=0.4).
Furthermore, ECP and EDN levels were positively correlated in all participants (r=0.45 (0.40–0.50), p<0.0001) and whatever the asthma status (see online supplemental figure S1).
ECP and EDN levels, current asthma, and asthma characteristics among asthmatics
Both ECP and EDN levels were significantly higher in asthmatics than in never-asthmatics (table 1 and figure 2), and high level of both biomarkers were positively associated with current asthma (table 2). Receiver operating characteristic curve analyses highlight better clinical performance of EDN for asthma diagnosis as compared with ECP (p=0.0003) (see online supplemental figure S2), in line with the stronger association found in regression models (table 2).
ECP and EDN levels according to current asthma status. Boxplots show the median (bar), the first and third quartiles (box), the 1st and 99th percentiles (whiskers). ECP, eosinophil cationic protein; EDN, eosinophil-derived neurotoxin. P values were adjusted on age, sex, smoking status and body mass index.
Cross-sectional associations between eosinophil mediator levels and various asthma characteristics
Among asthmatics, high ECP and EDN levels were positively associated with poor asthma control, reported chronic bronchitis, higher use of any asthma treatments or ICS in the past 12 months and higher BHR. High EDN level was also positively associated with reported asthma attacks, and wheezing and breathlessness in the past 12 months (table 2). No other significant associations were found. Associations with EDN remained after further adjustment on ECP, except for asthma control and chronic bronchitis. All associations between ECP and asthma characteristics did not remain significant after adjustment on EDN (see online supplemental table S3). Neither high ECP nor high EDN level was significantly associated with allergic sensitisation (table 2) and no association was found with any parameters of lung function (table 2 and data not shown).
After exclusion of the two ECP outliers, high ECP level was associated with chronic bronchitis (aOR: 2.00 (1.25–3.21)), even after adjustment on EDN (aOR: 1.82 (1.08–3.08)).
Overall, associations between blood eosinophil count and asthma outcomes were either weaker than those found with EDN level for current asthma, wheezing and breathlessness and BHR, or do not reach significance for asthma attacks, asthma control and chronic bronchitis. The only exception was the association found between high eosinophil count and poor lung function that was not evidenced with EDN or ECP level (see online supplemental table S4). After further adjustment on eosinophils, associations between EDN and current asthma or BHR remained significant (aOR=1 .53 (1.22–1.91) and aOR=1.63 (1.00–2.64) respectively).
ECP and EDN levels, eosinophil and neutrophil counts, total IgE level, and biomarkers of inflammation
Among asthmatics, high ECP and EDN levels were significantly associated with high eosinophil count and high total IgE level (figure 3).
Associations of ECP and EDN with blood neutrophilia and eosinophilia and total IgE in asthmatics. Forest plot show the 95% CI of OR (circle) and aOR (triangle). The vertical axis represents the significance threshold (OR=1). aORs were expressed for an increase corresponding to the value of the interquartile range (distance between the 25th and 75th percentile) of each biological marker; adjusted for age, sex, smoking status and body mass index. aOR, adjusted OR; ECP, eosinophil cationic protein; EDN, eosinophil-derived neurotoxin; IgE, immunoglobulin E.
The association between high ECP or EDN level and high eosinophil count was more evidenced for EDN level in line with the correlation analyses (r=0.52 (0.44–0.59), p<0.0001 for EDN; r=0.38 (0.29–0.47), p<0.0001 for ECP). High ECP level was also significantly associated with high neutrophil count (figure 3), consistently with the positive correlation between ECP level and neutrophil count (r=0.16 (0.06–0.26), p=0.004 for ECP; r=0.02 (−0.08 to –0.12), p=0.23 for EDN).
Similarly to eosinophil count, the association between high ECP or EDN level and high total IgE level was more evidenced for EDN (figure 3) in line with the correlation analyses (r=0.18 (0.08–0.28), p=0.008 for EDN; r=0.10 (0.0–0.20), p=0.01 for ECP).
Regarding biomarkers of inflammation, both ECP and EDN levels correlated with FENO. ECP level also positively correlated with hs-CRP level, and EDN level positively correlated with IL-13 level (table 3).
Correlations between eosinophil and inflammatory markers in asthmatics
Associations between EDN level at EGEA2 and asthma characteristics evolution between EGEA2 and EGEA3
In comparison to those lost to follow-up, participants included in longitudinal analyses (n=817) were more often women and non-smokers and reported less frequently chest tightness. The two groups did not differ for age, BMI, and other asthma characteristics (see online supplemental table S5).
High EDN level at EGEA2 was significantly associated with persistent current asthma and with the persistence of nocturnal shortness of breath and chest tightness. High EDN level at EGEA2 was also associated with worsening wheezing and breathlessness and worsening nocturnal chest tightness between EGEA2 and EGEA3 (table 4).
Longitudinal associations between EDN level at EGEA2 and evolution of asthma characteristics between EGEA2 and EGEA3
Discussion
The present study reported that EDN/RNase 2 and ECP/RNase 3 were associated with different asthma characteristics in adults. In cross-sectional analyses, ECP and EDN were associated with reported current asthma, poorly controlled asthma, chronic bronchitis, use of asthma treatments and BHR. Reported asthma attacks and wheezing and breathlessness were specifically associated with EDN. Associations with EDN remained after further adjustment on ECP, except for asthma control and chronic bronchitis. In contrast, associations with ECP, except neutrophil count and chronic bronchitis, did not remain significant after adjustment on EDN, suggesting that EDN was the biomarker driving the associations we found. High EDN level at EGEA2 was associated with persistent current asthma and two persistent nocturnal symptoms, and with worsening wheezing and breathlessness and nocturnal shortness of breath.
To our knowledge, this study is the first one studying blood ECP and EDN levels in a large longitudinal epidemiological study on asthma with extensive phenotypic characterisation of the participants. Among never-asthmatics, ECP level was positively associated with BMI, in accordance with a previous study.27 We also found a higher level of EDN in men than in women although EDN levels were not associated with gender in patients with COPD,28 and sex hormones seems neither to affect EDN release in vitro29 nor modify its urinary concentration.30 Further studies are warranted to investigate the gender EDN levels.
Neither high ECP nor high EDN level was significantly associated with allergic sensitisation among asthmatics. The associations with allergic sensitisation were mainly documented in early childhood,12 31–33 while being inconsistent in school-age children34–36 and poorly investigated in adults.19 20 Our results are not in accordance with previous findings in children with asthma,32 33 35 indicating that atopy severity was a major determinant of high ECP levels.32 Differences in asthma phenotypes prevalence between children and adults may partly explain these discrepancies. Among T2 asthma, allergy is much more frequent in childhood-onset versus adult-onset disease where innate lymphoid cells might be more important in maintaining eosinophilic response than T-helper 2 cells.37 Indeed, an increase in eosinophil mediator level could be closely related to atopic status in children but not in adults. To our knowledge, very few studies have addressed the association between atopy and eosinophil mediator levels in adults. Patelis et al showed that food but not airborne allergen sensitisation drive ECP level increase,20 in line with the lack of association between blood ECP or EDN level and house dust mite sensitisation evidenced by Gon et al.19 Consistently, we did not find any association between ECP or EDN level and airborne allergens classified as indoor/outdoor/moulds (not shown). Overall, these results suggest that ECP and EDN level are independent of airborne allergen sensitisation in adult asthmatics. However, we acknowledge that in the EGEA study, more than 80% of asthmatics had allergic sensitisation, and we were unable to unravel the role of atopy and asthma in the increase of ECP and EDN levels.
The present study helps to clarify the links between ECP, EDN and various asthma characteristics in adults. We identified significant associations between both high ECP and EDN levels and current asthma, asthma control, more frequent use of asthma treatment and higher BHR in asthmatics. Previously, Lee et al showed that EDN was an independent predictor of asthma severity in a large Korean adult cohort,16 and that in comparison to those with low EDN level, asthmatics with high EDN level had higher BHR. A recent study highlighted better performance of serum EDN level as compared with blood eosinophil count to assess asthma control status; this is in agreement with the present study showing a significant association between high EDN level but not high eosinophil count, and poor asthma control (see online supplemental table S4). In contrast with previous studies,12 16 19 38 we did not find any association between ECP or EDN and lung function. We acknowledge that as most of the asthmatics from the present study suffer from mild asthma with well-maintained lung function, leading to low variability in FEV1, our sample is probably not the most suitable to study such associations.
In our cross-sectional analyses, the majority of the associations were more evidenced with EDN and remained after further adjustment on ECP. In contrast, all associations with ECP did not remain significant after adjustment on EDN. Performance of ECP as asthma biomarker has been mainly addressed in children with asthma.12 14 We acknowledge that our choice of measuring ECP and EDN in plasma rather than in serum makes the comparisons with the available literature difficult because measurements are more frequent in serum. Indeed, serum ECP concentration is 5–10 fold higher than plasma one and reflects ex vivo eosinophil secretory activity whereas plasmatic one reflects ‘true’ blood concentration; the latter pretending being less correlated with asthma severity.9 31 Reliable serum concentration appeared more difficult to obtain than plasmatic ones since it is very sensitive to preanalytical conditions, and artefactual increase in serum ECP level has been demonstrated according to the type of needle, tube and time and temperature of sample storage.39 40 Moreover, among studies using plasma samples to measure ECP level in adults with asthma, none was performed with the same methodology as ours, making the comparison impossible since it is well-known that marker levels depend on both the type of blood sample and the ELISA manufacturer. To our knowledge, only one study measured plasma ECP level in healthy controls with the same method of ours, and reported results in the same range as the present study.41 Similarly, most of the previous studies measured EDN in serum rather than in plasma. Unlike to ECP, two studies used the same methodology to measure plasma EDN in adults with asthma, and their results are in good agreement with ours.42 Moreover, it has been shown that similar clinical performance was achieved for EDN, whatever the type of biological sample (serum, plasma, urine) and the methodology used.42–44
ECP/RNase 3 is characterised by an important molecular heterogeneity due to genetic polymorphisms and existence of several glycosylation patterns that affect both its blood concentration and functional properties.45 46 In an adult twin cohort, it has been shown that half of the blood ECP/RNase 3 variance was explained by genetic factors27; indeed, Noguchi et al identified three polymorphisms in ECP/RNase 3 promoter associated with three different baseline ECP concentrations.47 This genetic regulation could partly explain the discrepancies reported in the literature regarding its performance as clinical monitoring tool.10 Further, ECP measurement appeared trickier than that of EDN because of its higher cationic properties,10 suggesting that preanalytical issues could also take part in these discrepancies. Overall, this could explain the greater efficiency of EDN as asthma biomarker as previously reported in the literature.10 12 14 Our results need now to be validated in clinical practice.
Interestingly, we reported for the first time that EDN and ECP were associated with different asthma characteristics in adults: high EDN level was associated with higher asthma attacks and wheezing and breathlessness, whereas high ECP tended to be associated with chronic bronchitis. In the current state of knowledge, several hypotheses could be proposed to explain these specificities. First, the biological properties of ECP and EDN are different. The ability of EDN to induce airway remodelling and to increase T2 inflammation48 49 could partly explain its association with respiratory symptoms. Similarly, the ability of ECP to induce mucus secretion11 49 could partly explain its association with chronic bronchitis. Second, the selective secretory profile of eosinophil granule proteins according to stimulus9 suggests that some inflammatory mediators could promote ECP but not EDN release or conversely. This feature could both explain the moderate correlation between ECP and EDN and the distinct underlying inflammatory environment associated with each biomarker. Finally, high ECP versus high EDN level could reflect differences in eosinophil activation level or specific eosinophil subpopulation activation related to different asthma phenotypes. We found positive correlations between ECP and EDN and FENO, as well as between EDN and IL-13, that probably reflect the increase of T2 inflammation by EDN through dendritic cell activation.48 49 We also found a positive correlation between ECP and hs-CRP level and neutrophil count. The latter correlation is in accordance with the ability of neutrophils to synthetise and release ECP on activation.50 51 Overall, the associations between ECP, BMI, neutrophil count, hs-CRP and chronic bronchitis could be related to a distinct asthma phenotype. This hypothesis has been recently highlighted by Mogensen et al who have shown that serum ECP, unlike urinary EDN, was an independent risk factor of fixed air flow obstruction in a cohort of 403 adult asthmatics.18 Further studies are needed to confirm the link between ECP, EDN and distinct asthma phenotypes/endotypes, for example using clustering analyses.
Our study is the first to examine the associations between blood EDN and change in asthma characteristics over time. At EGEA2, high EDN level was associated with persistent current asthma and nocturnal shortness of breath and chest tightness, and with worsening wheezing and breathlessness and nocturnal chest tightness, suggesting that EDN could be a potential biomarker to monitor asthma evolution in adults. Our longitudinal analyses suffer from a lack of data that were not collected through self-administered questionnaire at EGEA3 such as the biological or functional data. We were also not able to study the association between EDN level evolution and asthma characteristic evolution which would have been more accurate to assess its clinical monitoring utility.
In conclusion, our results show that ECP and EDN seem to be associated with distinct asthma expression and different underlying inflammatory environment in adults. They also highlight the potential interest of EDN as asthma monitoring tool, especially in patients with high EDN level and active asthma. Finally, they suggest that high ECP versus high EDN level may be related to distinct asthma phenotypes or endotypes.
Data availability statement
No data are available. Due to third-party restrictions, EGEA data are not publicly available. Please see the following URL for more information: https://egeanet.vjf.inserm.fr/index.php/en/contacts-en. Interested researchers should contact egea.cohorte@inserm.fr with further questions regarding data access.
Ethics statements
Patient consent for publication
Ethics approval
Approvals were obtained from the relevant Ethics Committees and Institutional Review Board Committees (Cochin Port-Royal. Hospital and Necker-Enfants Malades Hospital, Paris): INSERM, RBM ‘Recherche BioMédicale’ RBM 91-005 and RBM 01-11; CNIL ‘Commission Nationale de l’Informatique et des Libertés’ no 109 427 (04/1990), no 900 198 (10/2000) and no 1 769 319 (2014); Institutional Review Board Committees (no 01-07-07, 04-05-03, 04-11-13 and 04-11-18); DGS ‘Direction Générale de la Santé’ no 2002/0106 and no 910 048. All participants signed a written informed consent.
Acknowledgments
The authors thank all those who participated to the setting of the study and on the various aspects of the examinations involved: interviewers, technicians for lung function testing and skin prick tests, blood sampling, IgE determinations, coders, those involved in quality control, data and sample management and all those who supervised the study in all centres. The authors are grateful to the three CIC-INSERM of Necker, Grenoble and Marseille who supported the study and in which participants were examined, and to the biobanks in Lille (CIC-INSERM), and at Annemasse (Etablissement français du sang) where biological samples are stored. They are indebted to all the participating individuals without whom the study would not have been possible. The authors also thank Laurent Orsi for his helpful support concerning statistical analysis.
References
Supplementary materials
Supplementary Data
This web only file has been produced by the BMJ Publishing Group from an electronic file supplied by the author(s) and has not been edited for content.
Footnotes
Contributors The authors’ contributions to the study were as follows: VG and RN were involved in the conception, hypotheses delineation and design of the analysis strategy of the study. FZ, PdN, AT and RM were involved in ECP and EDN measurement and interpretation. RM, RN and VS were involved in the acquisition of EGEA data and identification of asthma phenotypes. VG, JNA and RN analysed the data and performed statistical analyses. VG and RN wrote the paper. FZ, JA, VS, PdN, AT, RM and ZA reviewed the paper and revised it critically. All authors approved the final version of the manuscript.
Funding This work was funded by the French National Research Program for Environmental and Occupational Health of ANSES (EST/2017/1/158), the Région Hauts de France, National Hospital program of clinical research (PHRC-National 2012, EvAdA), ANR-CES-2009, and AGIR grants for chronical diseases.
Competing interests AT reports grant from Santelys, personal fees from ALK-Abello and non-financial support from AstraZeneca outside the submitted work.
Provenance and peer review Not commissioned; externally peer reviewed.
Supplemental material This content has been supplied by the author(s). It has not been vetted by BMJ Publishing Group Limited (BMJ) and may not have been peer-reviewed. Any opinions or recommendations discussed are solely those of the author(s) and are not endorsed by BMJ. BMJ disclaims all liability and responsibility arising from any reliance placed on the content. Where the content includes any translated material, BMJ does not warrant the accuracy and reliability of the translations (including but not limited to local regulations, clinical guidelines, terminology, drug names and drug dosages), and is not responsible for any error and/or omissions arising from translation and adaptation or otherwise.