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Elevated eosinophils, IL5 and IL8 in induced sputum in asthma patients with accelerated FEV1 decline

Open ArchivePublished:January 10, 2020DOI:https://doi.org/10.1016/j.rmed.2020.105875

      Highlights

      • Significant part of patients with mild asthma present with accelerated FEV1 decline.
      • Accelerated FEV1 decline was associated with eosinophils in induced sputum.
      • Patients with accelerated FEV1 decline had increased IL5 and IL8 in induced sputum.
      • Eosinophils, IL5, IL8 have potential to directly influence airway remodelling.
      • Eosinophils, IL5, IL8 potential biomarkers and therapeutic targets of remodelling.

      Abstract

      Background

      Some patients with asthma present with accelerated lung function decline. This phenomenon is mostly associated with severe exacerbations and with poor asthma control.

      Objective

      Our aim was to detect the extent of FEV1 decline in patients with mild asthma and to discriminate clinical, functional and inflammatory factors associated with accelerated FEV1 decline.

      Methods

      We recruited 50 patients with mild asthma for pulmonary function testing and induced sputum sampling 12–15 years after the initial diagnosis. In 33 patients, from whom sputum of a good quality was obtained, inflammatory cells were counted and concentrations of cytokines IL-2, IL-4, IL-5, IL-8, IL-10, IFN-γ, angiogenin and VEGF in the sputum were measured by cytometric bead array.

      Results

      Eighteen of 33 patients presented with accelerated FEV1 decline of more than 30 ml/year, with a mean (SEM) of 43.2 (3.9) ml/year, compared to 15 control patients with a FEV1 decline of 14.4 (2.1) ml/year. In the accelerated FEV1 decline group, we found elevated sputum levels of IL5 with a median (IQR) of 1.8 (0.4–3.2) pg/ml vs. 0.2 (0.1–1.2) pg/ml, p = 0.04; IL8 with a mean (SEM) of 1503 (194) pg/ml vs. 938 (177) pg/ml, p = 0.04; and eosinophils with a median (IQR) of 223 (41–1020) cells/μl vs. 39 (1–190) cells/μl, p = 0.03. No significant differences in other measured parameters were detected between the two groups.

      Conclusion

      Elevated sputum eosinophils, IL5 and IL8, which have a potential to stimulate airway remodelling, might be a useful non-invasive biomarkers and therapeutic targets of accelerated FEV1 decline in asthma patients.

      Keywords

      Abbreviations:

      FEV1 (forced expiratory volume in first second), FVC (forced vital capacity), TI (Tiffeneau index), FeNO (exhaled nitric oxide), PD20 (provocative dose causing 20% drop), post BD (post bronchodilation), IL (interleukin), IL5-R (interleukin 5 receptor), VEGF (vascular endothelial growth factor), IFNγ (interferon gamma), TGFβ (transforming growth factor beta), BAL (broncho-alveolar lavage), ASM (airway smooth muscle), TNNC (total number of non-squamous cells), ICS (inhaled corticosteroids), LABA (long acting beta agonists), SABA (short acting beta agonist), ACT (asthma control test), BMI (body mass index), SEM (standard error of the mean), IQR (interquartile range), p (probability)

      1. Introduction

      Some patients with asthma present with accelerated lung function decline. Lung volumes (FEV1) physiologically show a decline with age after full development of the lungs is reached around the age of 25 years, but the rate of decline normally does not exceed 20 ml per year [
      • Lange P.
      • Parner J.
      • Vestbo J.
      • Schnohr P.
      • Jensen G.A.
      15-year follow-up study of ventilatory function in adults with asthma.
      ]. Observing longitudinal FEV1 decline in asthma patients, researchers found FEV1 decreases with ageing in each subject, with a median decline slope of 40.9 ml/year and a wide range from 2 to 200 ml/year [
      • Cibella F.
      • Cuttitta G.
      • Bellia V.
      • Bucchieri S.
      • D'Anna S.
      • Guerrera D.
      • et al.
      Lung function decline in bronchial asthma.
      ]. Great variability in FEV1 decline among patients with asthma was also shown by other studies [
      • Lange P.
      • Parner J.
      • Vestbo J.
      • Schnohr P.
      • Jensen G.A.
      15-year follow-up study of ventilatory function in adults with asthma.
      ,
      • Tracey M.
      • Villar A.
      • Dow L.
      • Coggon D.
      • Lampe F.C.
      • Holgate S.T.
      The influence of increased bronchial responsiveness, atopy, and serum IgE on decline in FEV1. A longitudinal study in the elderly.
      ,
      • Graff S.
      • Demarche S.
      • Henket M.
      • Paulus V.
      • Louis R.
      • Schleich F.
      Increase in blood eosinophils during follow-up is associated with lung function decline in adult asthma.
      ,
      • Coumou H.
      • Westerhof G.A.
      • de Nijs S.B.
      • Zwinderman A.H.
      • Bel E.H.
      Predictors of accelerated decline in lung function in adult-onset asthma.
      ,
      • Grol M.H.
      • Gerritsen J.
      • Vonk J.M.
      • Schouten J.P.
      • Koëter G.H.
      • Rijcken B.
      • et al.
      Risk factors for growth and decline of lung function in asthmatic individuals up to age 42 years. A 30-year follow-up study.
      ]. In some patients, this decline was comparable to the decline in healthy subjects, while in some it exceeded normal values.
      Accelerated lung function decline is probably a functional reflection of airway remodelling [
      • Pascual R.M.
      • Peters S.P.
      Airway remodeling contributes to the progressive loss of lung function in asthma: an overview.
      ,
      • Kasahara K.
      • Shiba K.
      • Ozawa T.
      • Okuda K.
      • Adachi M.
      Correlation between the bronchial subepithelial layer and whole airway wall thickness in patients with asthma.
      ]. Airway remodelling is a consequence of structural and functional changes in the airways due to injury and repair processes of the airway tissue [
      • Keglowich L.F.
      • Borger P.
      The three A's in asthma - airway smooth muscle, airway remodeling & angiogenesis.
      ]. The frequency of exacerbations (that cause injury to airway tissue) was shown to be associated with the loss of lung function in patients with asthma of different severity [
      • Bai T.R.
      • Vonk J.M.
      • Postma D.S.
      • Boezen H.M.
      Severe exacerbations predict excess lung function decline in asthma.
      ,
      • O'Byrne P.M.
      • Pedersen S.
      • Lamm C.J.
      • Tan W.C.
      • Busse W.W.
      Severe exacerbations and decline in lung function in asthma.
      ,
      • Matsunaga K.
      • Ichikawa T.
      • Oka A.
      • Morishita Y.
      • Kanai K.
      • Hiramatsu M.
      • et al.
      Changes in forced expiratory volume in 1 second over time in patients with controlled asthma at baseline.
      ]. Other factors associated with accelerated FEV1 decline are features of uncontrolled asthma (frequent asthma symptoms, high FEV1 variability, poorer baseline FEV1/FVC) or exposure to cigarette smoke [
      • Peat J.K.
      • Woolcock A.J.
      • Cullen K.
      Rate of decline of lung function in subjects with asthma.
      ,
      • Ulrik C.S.
      • Backer V.
      • Dirksen A.
      A 10 year follow up of 180 adults with bronchial asthma: factors important for the decline in lung function.
      ]. Apart from specific clinical and functional factors, some studies have shown a correlation between greater FEV1 decline and elevated levels of inflammatory cells (sputum or blood eosinophils, and neutrophils, bronchial CD8+ T cells), cytokines (serum periostin, IL5 and IFNγ), Clamidophillia pneumonia infection, or genetic predisposition [
      • Graff S.
      • Demarche S.
      • Henket M.
      • Paulus V.
      • Louis R.
      • Schleich F.
      Increase in blood eosinophils during follow-up is associated with lung function decline in adult asthma.
      ,
      • Coumou H.
      • Westerhof G.A.
      • de Nijs S.B.
      • Zwinderman A.H.
      • Bel E.H.
      Predictors of accelerated decline in lung function in adult-onset asthma.
      ,
      • Sakagami T.
      • Hasegawa T.
      • Koya T.
      • Furukawa T.
      • Kawakami H.
      • Kimura Y.
      • et al.
      Cluster analysis identifies characteristic phenotypes of asthma with accelerated lung function decline.
      ,
      • Broekema M.
      • Volbeda F.
      • Timens W.
      • Dijkstra A.
      • Lee N.A.
      • Lee J.J.
      • et al.
      Airway eosinophilia in remission and progression of asthma: accumulation with a fast decline of FEV1.
      ,
      • den Otter I.
      • Willems L.N.
      • van Schadewijk A.
      • van Wijngaarden S.
      • Janssen K.
      • de Jeu R.C.
      • et al.
      Lung function decline in asthma patients with elevated bronchial CD8, CD4 and CD3 cells.
      ,
      • Kanemitsu Y.
      • Matsumoto H.
      • Izuhara K.
      • Tohda Y.
      • Kita H.
      • Horiguchi T.
      • et al.
      Increased periostin associates with greater airflow limitation in patients receiving inhaled corticosteroids.
      ,
      • Pasternack R.
      • Huhtala H.
      • Karjalainen J.
      Chlamydophila (Chlamydia) pneumoniae serology and asthma in adults: a longitudinal analysis.
      ,
      • Jongepier H.
      • Boezen H.M.
      • Dijkstra A.
      • Howard T.D.
      • Vonk J.M.
      • Koppelman G.H.
      • et al.
      Polymorphisms of the ADAM33 gene are associated with accelerated lung function decline in asthma.
      ,
      • Litonjua A.A.
      • Sparrow D.
      • Guevarra L.
      • O'Connor G.T.
      • Weiss S.T.
      • Tollerud D.J.
      Serum interferon-gamma is associated with longitudinal decline in lung function among asthmatic patients: the Normative Aging Study.
      ]. Lung function decline has also been observed in patients with mild asthma [
      • Broekema M.
      • Volbeda F.
      • Timens W.
      • Dijkstra A.
      • Lee N.A.
      • Lee J.J.
      • et al.
      Airway eosinophilia in remission and progression of asthma: accumulation with a fast decline of FEV1.
      ]. However, there is limited data on the long-term influence of these changes, and which are major clinical and immunologic biomarkers for recognition of such patients. Furthermore, we need to explore potential strategies to prevent remodelling during the early phase of this process.
      Therefore, we designed a long-term study in which we wanted to evaluate patients with mild asthma, who presented with accelerated FEV1 decline after 12–15 years of clinical follow-up. We wanted to discriminate the possible clinical, functional and inflammatory factors or biomarkers linked to accelerated FEV1 decline in these subjects.

      2. Methods

      2.1 Study design and patients

      We recruited 50 patients with mild asthma of age between 34 and 70 years, in whom the diagnosis of asthma was given 12–15 years ago at referral respiratory clinic with either a positive methacholine or reversibility test. Asthma severity was defined according to GINA guidelines (mild asthma: asthma that can be controlled by step 1 or 2 treatment defined in GINA guidelines - as needed or daily low-dose ICS, as needed low dose ICS/LABA, leukotriene receptor antagonist), and asthma control was assessed using consensus-based GINA symptom control tool [
      GINA Global Initiative for Asthma
      Global Strategy for Asthma Management and Prevention.
      ]. Exclusion criteria were FEV1 <80%, other chronic lung diseases, important diseases of other organs, or autoimmune diseases. Participants informed consent was provided by all patients on central IEC approved protocol designed to study asthma (KME95/06/13).
      To find the right patients, we first identified patients with asthma diagnosis through retrospective medical records review. Selection of candidates with mild asthma was assessed through accessible medical documentation and by patients’ data obtained by mail and phone call.
      From the first visit clinical data, the results of atopy test and lung function tests with spirometry and a reversibility test or methacholine provocation were collected. After the diagnosis, the patients were managed by their physicians through primary medical care. We invited the patients to our clinic 12–15 years after their first visit for further investigation. At this second study time point, the patients performed spirometry (pre- and post-bronchodilator inhalation), FeNO measurements, induced sputum and clinical data were collected. Measurements were performed when asthma was stable and well controlled. Patients had not experienced an asthma exacerbation or acute illness for at least 4 weeks prior to investigation and were otherwise healthy. Patients were instructed to skip the morning dose of inhaled medications on the study test day.
      Of the 50 candidates, we collected induced sputum of a good quality from 33 patients, whose data were further analysed. We divided the patients into two groups according to FEV1 decline: one group had accelerated FEV1 decline (a decline of more than 30 ml per year), and the control group had normal FEV1 decline (a decline of less than 30 ml per year).

      2.2 Clinical data

      Patients’ data were assessed by a self-administered questionnaire concerning asthma symptoms, exacerbations, adherence to the prescribed inhalation medication, onset and duration of asthma, somatic diseases, socioeconomic status, smoking status, and other clinical data. The prescribed asthma treatment regimen was additionally obtained through medical records of primary care physicians. All patients had prescribed anti-inflammatory inhalation medications, of whom 29 were instructed to receive daily low dose ICS and four ICS/LABA in as needed regimen. More detailed characteristics of the patients are shown in Table 1. Standardized Asthma Control Test (ACT) was used as a numerical asthma control tool [
      • Nathan R.A.
      • Sorkness C.A.
      • Kosinski M.
      • Schatz M.
      • Li J.T.
      • Marcus P.
      • et al.
      Development of the asthma control test: a survey for assessing asthma control.
      ].
      Table 1Patients’ characteristics.
      Accelerated (n = 18)Normal (n = 15)
      Age (years)51.3 (2.4)48.0 (2.5)
      Gender (female/male)12/68/7
      Age at asthma diagnosis (years)27.5 (4.0)30.6 (3.1)
      FVC baseline (%)99.6 (2.3)100.1 (2.5)
      PostBD FEV1, baseline (%)97.5 (2.8)97.3 (3.5)
      TI, baseline (%)78 (1.4)77.9 (1.8)
      Positive reversibility test, baseline (No. of patients)53
      Positive metacholine test, baseline (No of patients)1312
      PD 20, baseline (mg)0.4 (0.2–1.8)0.6 (0.3–2.1)
      Disease duration (years)19 (15–29)15.5 (14–26)
      Active/ex-smokers (No (%))1/1 (5.5/5.5)2/0 (13.3/0)
      BMI (kg/m2)25.7 (22.5–28.1)26.0 (23.4–28.6)
      Allergic asthma (No (%))10 (55.5)9 (60)
      Adult onset asthma (No (%))12 (66%)8 (53%)
      ICS use (No (%))11 (61)13 (87)
      FVC, at control (%)95.7 (2.5)107.7 (2.7) *
      PostBD FEV1, at control (%)87.7 (2.7)97.8 (2.9) *
      TI, at control (%)72.6 (1.3)74.3 (1.1)
      ACT, at control (points)20 (19–24)20 (20–23)
      FeNO, at control (ppB)31 (18–57)30 (12–38)
      No of self-reported mild exacerbations per year2 (0–4)1 (0–2)
      Hospitalization due to asthma (No per year)00
      Emergency visit (No per year)00
      PostBD: post-bronchodilation, FVC: forced vital capacity, FEV1: forced expiratory volume in first second, TI: Tiffenou index, ACT: asthma control test, FeNO: exhaled nitric oxide, BMI: body mass index, ICS: inhaled corticosteroid. *p < 0.01.
      To determine atopy status a skin prick tests were performed with a Pan-European standard prick test panel: Cat, Dog, Dermatophagoides pteronyssinus, Dermatophagoides farinae, Blatella, Birch, Hazel, Alder, Olive, Cypress, Plane, Grass mix, Artemisia, Ambrosia, Parietaria, Alternaria, Cladosporium, Aspergillus (HAL Allergy, Leiden, Netherlands).

      2.3 Lung function tests

      Forced expiratory volume in first second (FEV1) and forced vital capacity (FVC) were measured in the same respiratory laboratory with two different spirometry systems (at the first visit Sensormedic Vmax was used, and at the second visit Viasys Vmax Encore pneumotachograph - model 2005, Viasys, Wuerzburg, DE, was used). At the time of spirometry system change, one-week period was dedicated to compare the results among different systems. Intraindividual repeatibility of FVC and FEV1 was the same if calculated within the same spirometer or within the two different devices. Spirometers were calibrated daily with a 3-L syringe. ERS criteria for acceptability and reproducibility of the blows were respected (ERS Spirometry Driving Licence curriculum). The best two of at least three reproducible tests were chosen with the largest sum of FEV1 plus FVC. The two best blows were within 150 ml of both the FVC and FEV1 measurements. The highest FEV1 was used in analyses. FEV1 data are reported both as absolute values and as percentages of predicted values. Annual FEV1 decline (ml/year) was calculated by subtracting FEV1 (post-bronchodilator) at the first and second measurements, divided by the number of years between the two measurements. Post-bronchodilator FEV1 was chosen rather than pre-bronchodilator FEV1 to avoid the influence of variable smooth muscle contraction that could overestimate FEV1 decline.
      The reversibility test was defined as positive if FEV1 increased by 12% (200 ml) or more after the administration of 400 mg of salbutamol. Bronchial hyper-responsiveness was tested using a dosimeter nebulisation method with a maximal cumulative dose of 4 mg of methacholine. The provocative dose causing a 20% fall in FEV1 (PD20) was calculated.
      Levels of exhaled nitric oxide (FeNO) were measured by an EcoMedics model CLD sp with the DENOX 88 module for measurement of exhaled NO concentration (FeNO). Flow and volume calibration were performed every morning, and the detection range was set from 0 to 100 ppb of NO. The exhaled breath was collected during a single exhalation at a pre-set flow rate. At least three measurements were performed in the same session, and the largest result was reported as the final value. An expiratory resistor was used to exclude NO contamination of exhaled breath from the nasal passages. Levels of FeNO above 50 ppb were considered elevated [
      • Dweik R.A.
      • Boggs P.B.
      • Erzurum S.C.
      • Irvin C.G.
      • Leigh M.W.
      • Lundberg J.O.
      • et al.
      American Thoracic Society Committee on Interpretation of Exhaled Nitric Oxide Levels (FENO) for Clinical Applications. An official ATS clinical practice guideline: interpretation of exhaled nitric oxide levels (FENO) for clinical applications.
      ].

      2.4 Sputum induction and processing

      Sputum was induced and processed according to previously described methods [
      • Cataldo D.
      • Foidart Lau L.
      • Bartsch P.
      • Djukanovic R.
      • Louis R.
      Induced sputum: comparison between isotonic and hypertonic saline solution inhalation in patients with asthma.
      ]. Briefly, subjects inhaled 4.5% hypertonic saline, nebulized via an ultrasonic nebulizer (PARI MASTER Type 84.0100, PARI GmbH, Starnberg, Germany), for three 5-min periods. To minimize saliva contamination, we asked the subjects to mouthwash thoroughly and expectorate saliva into a separate container before producing sputum. At least 2 ml of sputum had to be collected into a sterile container in order to proceed with sputum processing. The collected sputum was immediately processed. The volume of the entire sputum sample was determined, and an equal volume of 0.1% dithiothreitol (Sputolysin R, Calbiochem, San Diego, CA) was added. The samples were then mixed gently with a vortex mixer and incubated for 15 min at room temperature to ensure complete homogenization. After filtration and centrifugation, cell-free supernatants were frozen at −80 °C until subsequent analysis. The total number of non-squamous cells (TNNCs) per ml of sputum sample was assessed using a haemocytometer. Cytospins were stained according to the May-Grünwald-Giemsa and Papnicolaou methods. Differential cell counts (eosinophils, neutrophils, mononuclear cells, macrophages, and basophils) were performed by one observer, who counted 200 non-epithelial cells. The quality of the induced sputum was assessed according to the recommendations of Pizzichini E et al. Only samples with a score of 7 or higher were used for further analysis [
      • Pizzichini E.
      • Pizzichini M.M.
      • Efthimiadis A.
      • Evans S.
      • Morris M.M.
      • Squillace D.
      • et al.
      Indices of airway inflammation in induced sputum: reproducibility and validity of cell and fluid-phase measurements.
      ,
      • Marc M.M.
      • Korosec P.
      • Kosnik M.
      • Kern I.
      • Flezar M.
      • Suskovic S.
      • et al.
      Complement factors C3a, C4a, and C5a in chronic obstructive pulmonary disease and asthma.
      ].

      2.5 Detection of mediators

      The detection of cytokines or angiogenic mediators was performed as previously reported. IL2, IL5, IL8, IL10, IFNγ, VEGF and angiogenin concentrations were measured with cytometric bead arrays (BD Biosciences, San Diego, CA, US), which contain microparticles that are dyed to different fluorescence intensities. Each particle is coupled with an antibody against IL2, IL5, IL8, IL10, IFNγ, VEGF or angiogenin and represents a discrete population that is unique in its FL-3/FL-4 intensity. The capture beads were incubated with recombinant standards or test samples (sputum supernatant) with phycoerythrin-conjugated detection antibodies to form sandwich complexes. Flow cytometric analysis was performed using a FACSCalibur flow cytometer (BD Biosciences). Data were acquired and analysed using Becton Dickinson Cytometric Bead Array CBA software, and concentrations were extrapolated from the standard curves by plotting the recombinant calibrator concentration against the FL-2 mean fluorescence intensity [
      • Chen R.
      • Lowe L.
      • Wilson J.D.
      • Crowther E.
      • Tzeggai K.
      • Bishop J.E.
      • et al.
      Simultaneous quantification of six human cytokines in a single sample using microparticle-based flow cytometric technology.
      ,
      • Petrovic M.G.
      • Korosec P.
      • Kosnik M.
      • Osredkar J.
      • Hawlina M.
      • Peterlin B.
      • et al.
      Local and genetic determinants of vascular endothelial growth factor expression in advanced proliferative diabetic retinopathy.
      ].

      2.6 Statistical analysis

      All statistical analyses were performed in the GraphPad Prism programme. Quantitative data are represented as medians and interquartile ranges or means and standard errors, as appropriate (the normality of the distribution was evaluated using the Shapiro-Wilk test, and the assumption of normality was rejected if p < 0.05). Parameters that failed normality were PD20, FeNO, BMI, ACT, number of exacerbations, number of inflammatory cells (total, eosinophils, neutrophils, mononuclear cells), IL2, IL5, IL10, IFNγ, VEGF, and angiogenins. Qualitative data are represented as sample proportions. The data were compared between groups using the Mann-Whitney U test or unpaired t-test, as appropriate. Two tailed p-values of <0.05 were considered significant.

      3. Results

      3.1 Patient characteristics and spirometry

      Among 33 asthma patients who were included in the analyses, 18 patients presented with accelerated FEV1 decline of more than 30 ml/year over a 12-15-year period, with a mean (SEM) of 43.2 (3.9) ml/year. In 15 patients included in the normal FEV1 decline group, the average FEV1 decline was less than 30 ml/year with a mean (SEM) of 14.4 (2.1) ml/year. The initial FEV1 was similar between the groups (3480 (186) ml and 3610 (216) ml), but the FEV1 measured after 12–15 years was significantly lower in the accelerated FEV1 decline group (2830 (152) ml) compared to that of the control group with normal FEV1 decline (3450 (247) ml). Individual data of FEV1 change from baseline to control measurements are presented graphicaly (Fig. 1) . When we compared the presence of atopy, initial PD 20, ACT scores, exhaled NO, smoking history (active smoking/pack years), or BMI, these values did not significantly differ between the two groups. The accelerated FEV1 decline group reported more mild or moderate asthma exacerbations of 2 (0–4) per year in comparison to the normal FEV1 decline group with 1 (0–2) exacerbations per year, but the trend was not significant (Table 1).
      Fig. 1
      Fig. 1Individual data of FEV1 change in the time-period of 12–15 years in asthma patients with A: accelerated, and B: normal FEV1 decline.
      Among 29 patients with prescribed daily doses of ICS 14 patients reported adherence with the prescribed regimen of more than 80%, the reported adherence in six patients was between 50 and 80%. The adherence was zero in nine patients, who stopped taking the prescribed low dose ICS within few months after the first visit. Later on they used only SABA when they experienced symptoms. When we compared the patients who received the prescribed ICS to those who stopped receiving ICS, there was a statistically non-significant trend towards a higher rate of FEV1 annual decline in the non-treated patients. We did not detect any differences in the reported exacerbation rate, levels of eosinophils or other inflammatory cells, cytokines or angiogenic factors between treated and non-treated patients.

      3.2 Inflammatory cells

      We found significantly increased numbers of eosinophils, 223 (41–1020)/μl, in the induced sputum of the accelerated FEV1 decline group compared to 39 (1–190)/μl eosinophils in the normal FEV1 decline group (p = 0.03) (Fig. 2B ). We further found a correlation between numbers of eosinophils and FEV1 decline (R = 0.40, p = 0.02). There was a trend toward increased percentages of eosinophils in induced sputum in patients with accelerated FEV1 decline 4 (1–18)% vs. 1 (1–4)%, although it didn't reach statistical significance. Sputum eosinophilia (defined as proportion of eosinophils of 3% or more) was detected in 11 out of 18 (61%) patients with accelerated FEV1 decline and in 5 of 15 (33%) of patients with normal FEV1 decline. There were no differences between the accelerated FEV1 group and the normal FEV1 decline group in terms of the total numbers of inflammatory cells (3080 (1830–8060)/μl vs. 3330 (1850–4770)/μl, respectively), neutrophils (1070 (430–2750)/μl vs. 1070 (370–2100)/μl, respectively) or mononuclear cells (89 (49–290)/μl vs. 116 (42–199)/μl, respectively) (Fig. 2A, C, D).
      Fig. 2
      Fig. 2Numbers of inflammatory cells. A: Total number of non-squamous cells (TNNC), B: eosinophils, C: neutrophils, and D: mono-nucleated cells in induced sputum of asthma patients with accelerated or normal FEV1 decline.

      3.3 Cytokines and angiogenetic factors

      We measured significantly higher induced sputum levels of IL5 (1.8 (0.4–3.2) pg/ml, p = 0.04) and IL8 (1503 (194) pg/ml, p = 0.04) in the accelerated FEV1 decline group compared to the levels of IL5 (0.2 (0.1–1.2) pg/ml) and IL8 (938 (177) pg/ml) in the normal FEV1 decline group (Fig. 3B and C). Levels of other measured cytokines, specifically IL2 (17.3 (11.7–18) pg/ml), IL10 (0.6 (0.2–4.3) pg/ml), and IFNγ (2.6 (2.4–4.1) pg/ml), in the accelerated FEV1 decline group were similar to the levels of IL2 (17.8 (14.9–38.5) pg/ml), IL10 (0.3 (0.2–13.5) pg/ml) and IFNγ (2.5 (2.4–2.7) pg/ml) in patients with normal FEV1 decline (Fig. 3A, D, G). We further measured levels of angiogenic factors. Although there was a trend towards higher levels of angiogenin (4277 (812–8803) pg/ml vs. 894 (275–8470) pg/ml) and VEGF (752 (243–1813) pg/ml vs. 513 (228–1120) pg/ml) in patients with accelerated FEV1 decline, this trend did not reach statistical significance (Fig. 3E and F).
      Fig. 3
      Fig. 3Levels of cytokines. A: IL2, B: IL5, C: IL8, D: IL 10, E: angiogenin, F: VEGF, and G: IFN γ in induced sputum of asthma patients with accelerated and normal FEV1 decline.
      Additionally, we also performed a correlation analysis of FeNO, inflammatory cells and cytokines in induced sputum. We found a significant correlation between FeNO and eosinophils (r = 0.35, p < 0.05), IL8 and neutrophils (r = 0.55, p < 0.005) and between IL8 and total numbers of inflammatory cells (r = 0.72, p < 0.005). There were no other significant correlations.

      4. Discussion

      In our study, we observed significantly accelerated FEV1 decline 12–15 years after asthma diagnosis confirmation (mean 43.2 vs. 14.4 ml per year) in more than half of the patients with mild asthma who were followed by their primary physicians. Furthermore, accelerated FEV1 decline was significantly associated with increased induced sputum eosinophils and IL5 and IL8 levels. Taken together, these data suggest that some patients with mild asthma should be further recognized and treated, and it is important to define which characteristics, potential risk factors, and underlying mechanisms are related to long-term FEV1 decline.
      Previous studies demonstrated that airway remodelling, which consists of epithelial injury, goblet cell hyperplasia, sub-epithelial layer thickening, airway smooth muscle hyperplasia and angiogenesis, might be the leading cause of accelerated FEV1 decline [
      • Pascual R.M.
      • Peters S.P.
      Airway remodeling contributes to the progressive loss of lung function in asthma: an overview.
      ,
      • Kasahara K.
      • Shiba K.
      • Ozawa T.
      • Okuda K.
      • Adachi M.
      Correlation between the bronchial subepithelial layer and whole airway wall thickness in patients with asthma.
      ]. Thus, airway remodelling might play a major role in asthma progression and might be present in children at early stages of the disease (4 years of age) [
      • Turato G.
      • Barbato A.
      • Baraldo S.
      • Zanin M.E.
      • Bazzan E.
      • Lokar-Oliani K.
      • et al.
      Nonatopic children with multitrigger wheezing have airway pathology comparable to atopic asthma.
      ]. The triggers of airway remodelling are not completely understood, and some studies have suggested that asthma-related inflammation is not responsible per se for remodelling and that the two processes can occur as two separate features of asthma [
      • Keglowich L.F.
      • Borger P.
      The three A's in asthma - airway smooth muscle, airway remodeling & angiogenesis.
      ].
      Overall, it was suggested that important risk factors for accelerated FEV1 decline are smoking, uncontrolled asthma with severe exacerbations, frequent asthma symptoms, airway hyper-responsiveness, and FEV1 variability. Apart from smoking and severe exacerbations, genetic predisposition, BMI, some inflammatory cells (eosinophils, neutrophils and CD8+ T cells), and cytokines (IFN-γ, periostin) have also been suggested to be associated with accelerated FEV1 decline [
      • Graff S.
      • Demarche S.
      • Henket M.
      • Paulus V.
      • Louis R.
      • Schleich F.
      Increase in blood eosinophils during follow-up is associated with lung function decline in adult asthma.
      ,
      • Coumou H.
      • Westerhof G.A.
      • de Nijs S.B.
      • Zwinderman A.H.
      • Bel E.H.
      Predictors of accelerated decline in lung function in adult-onset asthma.
      ,
      • Ulrik C.S.
      • Backer V.
      • Dirksen A.
      A 10 year follow up of 180 adults with bronchial asthma: factors important for the decline in lung function.
      ,
      • Sakagami T.
      • Hasegawa T.
      • Koya T.
      • Furukawa T.
      • Kawakami H.
      • Kimura Y.
      • et al.
      Cluster analysis identifies characteristic phenotypes of asthma with accelerated lung function decline.
      ,
      • Broekema M.
      • Volbeda F.
      • Timens W.
      • Dijkstra A.
      • Lee N.A.
      • Lee J.J.
      • et al.
      Airway eosinophilia in remission and progression of asthma: accumulation with a fast decline of FEV1.
      ,
      • den Otter I.
      • Willems L.N.
      • van Schadewijk A.
      • van Wijngaarden S.
      • Janssen K.
      • de Jeu R.C.
      • et al.
      Lung function decline in asthma patients with elevated bronchial CD8, CD4 and CD3 cells.
      ,
      • Kanemitsu Y.
      • Matsumoto H.
      • Izuhara K.
      • Tohda Y.
      • Kita H.
      • Horiguchi T.
      • et al.
      Increased periostin associates with greater airflow limitation in patients receiving inhaled corticosteroids.
      ,
      • Pasternack R.
      • Huhtala H.
      • Karjalainen J.
      Chlamydophila (Chlamydia) pneumoniae serology and asthma in adults: a longitudinal analysis.
      ,
      • Jongepier H.
      • Boezen H.M.
      • Dijkstra A.
      • Howard T.D.
      • Vonk J.M.
      • Koppelman G.H.
      • et al.
      Polymorphisms of the ADAM33 gene are associated with accelerated lung function decline in asthma.
      ,
      • Matsumoto H.
      Serum periostin: a novel biomarker for asthma management.
      ].
      Currently, it is not clear whether accelerated FEV1 decline is also important for patients with mild asthma. The results of our study suggest that a considerable number of patients with mild asthma present with significant acceleration of FEV1 decline in the long term – we detected accelerated FEV1 decline of more than 30 ml/year in 55% of patients after 12–15 years. This is consistent with a previous study by Broekema et al. who demonstrated that 36% (17 of 47) of mild asthmatic patients showed accelerated FEV1 decline after at least 5 years of follow-up. Moreover, both the current study and the Broekema study showed similar levels of FEV1 change with 43.2 and 50.3 ml per year for the accelerated mild asthmatic subgroup, and 14.4 and 18.6 for the normal mild asthmatic subgroup, respectively [
      • Broekema M.
      • Volbeda F.
      • Timens W.
      • Dijkstra A.
      • Lee N.A.
      • Lee J.J.
      • et al.
      Airway eosinophilia in remission and progression of asthma: accumulation with a fast decline of FEV1.
      ]. These similar levels of FEV1 decline were detected despite the fact that majority of our patients were treated with ICS, in contrast in Broekema study anti-inflammatory treatment was one of the exclusion criteria. This finding is somehow consistent with previous studies, which reported a lack of influence of ICS treatment change on FEV1 decline, and a lack of impact of regular ICS use on the progression of airway remodelling in patients with asthma [
      • Graff S.
      • Demarche S.
      • Henket M.
      • Paulus V.
      • Louis R.
      • Schleich F.
      Increase in blood eosinophils during follow-up is associated with lung function decline in adult asthma.
      ,
      • Girodet P.O.
      • Ozier A.
      • Bara I.
      • Tunon de Lara J.M.
      • Marthan R.
      • Berger P.
      Airway remodeling in asthma: new mechanisms and potential for pharmacological intervention.
      ].
      Comparable levels of FEV1 decline were reported for patients with moderate and severe asthma by other groups [
      • Graff S.
      • Demarche S.
      • Henket M.
      • Paulus V.
      • Louis R.
      • Schleich F.
      Increase in blood eosinophils during follow-up is associated with lung function decline in adult asthma.
      ,
      • Coumou H.
      • Westerhof G.A.
      • de Nijs S.B.
      • Zwinderman A.H.
      • Bel E.H.
      Predictors of accelerated decline in lung function in adult-onset asthma.
      ].
      Further, we tried to discriminate the factors associated with accelerated FEV1 decline. We could not identify any baseline clinical or functional characteristics of patients, which would be helpful in recognizing patients at risk. Most patients were non-smokers, and among three active smokers, only one presented with accelerated FEV1 decline. The proportion of female gender was non-significantly higher (66% vs. 53%). There were no differences in age, BMI, and asthma disease duration between the two groups. Baseline values of lung volume (FVC and FEV1) and levels of hyper-responsiveness measured by metacholine provocation were similar between the groups. We did not measure levels of inflammatory cells or cytokines at the first visit; thus, we lack information on these baseline levels.
      Among follow-up factors, we stress that the observed higher rate of self-reported mild to moderate exacerbations, although it did not reach statistical significance, suggests that patients with more frequent asthma exacerbations (even if they are mild) should be followed-up more regularly by a pulmonologist. Patients with accelerated FEV1 decline reported good asthma control comparable to that of the control group, with only few symptoms and a median ACT of 20 points. Lower as expected ACT values in some patients are probably a result of the miss-interpretation of the questions 2 and 4 about shortness of breath and the use of SABA (many patients were not familiar with this questionnaire). Regarding treatment with ICS, there was a higher proportion of patients who did not receive ICS in the accelerated FEV1 decline group (39% vs. 13%). However, the fact that a majority of patients had accelerated FEV1 decline despite reported regular use of ICS either points to insufficient doses of ICS or supports previous findings describing the development of ICS-independent irreversible airway obstruction [
      • Goleva E.
      • Hauk P.J.
      • Hall C.F.
      • Liu A.H.
      • Riches D.W.
      • Martin R.J.
      • et al.
      Corticosteroid-resistant asthma is associated with classical antimicrobial activation of airway macrophages.
      ]. We also did not detect any inflammation-suppressing effects resulting in lower numbers of eosinophils and other inflammatory cells or lower cytokine levels in induced sputum of patients receiving ICS. Opposite to previous findings in a group of difficult-to-treat asthma patients, we didn't confirm the associations between FEV1 decline and FeNO levels, the reason might be in different asthma populations (mild vs. difficult-to-treat asthma). FeNO values in our patients with mild asthma were low, exceeding 50ppB only in 7 patients – 5 (27%) in accelerated and 2 (13%) in normal FEV1 decline group [
      • van Veen I.H.
      • ten Brinke A.
      • Sterk P.J.
      • Sont J.K.
      • Gauw S.A.
      • Rabe K.F.
      • Bel E.H.
      Exhaled nitric oxide predicts lung function decline in difficult-to-treat asthma.
      ].
      The most important findings in our asthma patients with accelerated FEV1 decline were significantly elevated levels of sputum eosinophils and the cytokines IL5 and IL8, which might play a role in pathogenesis and seem to have a good potential as biomarkers of accelerated FEV1 decline.
      Eosinophils play an important role in asthma pathogenesis: they are promotors of the inflammatory response and are directly and indirectly linked to airway remodelling. Cytotoxic proteins secreted by eosinophils can directly damage airway epithelial cells. Eosinophils further produce TGFβ1, which promotes fibroblast proliferation, the maturation of myo-fibroblasts and collagen synthesis. They may also help direct angiogenesis in the asthmatic submucosa by producing several angiogenetic factors, including VEGF [
      • Chetta A.
      • Zanini A.
      • Foresi A.
      • D'Ippolito R.
      • Tipa A.
      • Castagnaro A.
      • et al.
      Vascular endothelial growth factor up-regulation and bronchial wall remodelling in asthma.
      ,
      • Halwani R.
      • Al-Muhsen S.
      • Al-Jahdali H.
      • Hamid Q.
      Role of transforming growth factor–β in airway remodeling in asthma.
      ,
      • Hoshino M.
      • Takahashi M.
      • Aoike N.
      Expression of vascular endothelial growth factor, basic fibroblast growth factor, and angiogenin immunoreactivity in asthmatic airways and its relationship to angiogenesis.
      ], which was non-significantly increased in our patients with accelerated FEV1 decline. The association of sub-epithelial fibrosis and submucosal eosinophils has been documented in animal models of asthma and in patients with asthma [
      • Wenzel S.E.
      • Schwartz L.B.
      • Langmack E.L.
      • Halliday J.L.
      • Trudeau J.B.
      • Gibbs R.L.
      • et al.
      Evidence that severe asthma can Be divided pathologically into two inflammatory subtypes with distinct physiologic and clinical characteristics.
      ,
      • Motojima S.
      • Frigas E.
      • Loegering D.A.
      • Gleich G.J.
      Toxicity of eosinophil cationic proteins for Guinea pig tracheal epithelium in vitro.
      ]. Some previous studies have already shown that increased sputum eosinophils are a risk factor for persistent airway limitation in patients with asthma, are associated with lower predicted FEV values and higher FEV1 decline and with thickening of the sub-epithelial basement membrane [
      • Coumou H.
      • Westerhof G.A.
      • de Nijs S.B.
      • Zwinderman A.H.
      • Bel E.H.
      Predictors of accelerated decline in lung function in adult-onset asthma.
      ,
      • Ulrik C.S.
      • Backer V.
      • Dirksen A.
      A 10 year follow up of 180 adults with bronchial asthma: factors important for the decline in lung function.
      ,
      • Broekema M.
      • Volbeda F.
      • Timens W.
      • Dijkstra A.
      • Lee N.A.
      • Lee J.J.
      • et al.
      Airway eosinophilia in remission and progression of asthma: accumulation with a fast decline of FEV1.
      ,
      • ten Brinke A.
      • Zwonderman A.H.
      • Sterk P.J.
      • Rabe K.F.
      • Bel E.H.
      Factors associated with persistent airflow limitation in severe asthma.
      ]. Interestingly, although there was a trend toward increased percentages of eosinophils in induced sputum of our patients, the difference between the groups was higher and also statistically significant when we compared absolute numbers of eosinophils. Furthermore, numbers of eosinophils significantly correlated to FEV1 decline. Similar better correlation was reported by a group of Broekema [
      • Broekema M.
      • Volbeda F.
      • Timens W.
      • Dijkstra A.
      • Lee N.A.
      • Lee J.J.
      • et al.
      Airway eosinophilia in remission and progression of asthma: accumulation with a fast decline of FEV1.
      ]. We think that absolute numbers of eosinophils quantitatively might better express the possible impact on accelerated FEV1 decline.
      Cytokines IL5 and IL8, which were elevated in induced sputum of our patients with accelerated FEV1 decline, seem to be important in asthma inflammation as well as in remodelling. Although we couldn't detect a significant correlation between sputum IL5 and eosinophils, IL5 is a very important systemic regulator of eosinophil dynamics, and is also involved in the local recruitment of mast cells. In animal model of atopic asthma, prophylactic treatment with anti–IL-5 significantly reduced the subepithelial fibrosis produced by repeated allergen challenge [
      • Blyth D.I.
      • Wharton T.F.
      • Pedrick M.S.
      • Savage T.J.
      • Sanjar S.
      Airway subepithelial fibrosis in a murine model of atopic asthma: suppression by dexamethasone or anti-interleukin-5 antibody.
      ]. In another chronic exposure model BAL profibrotic TGFβ1 levels, collagen content and fibrotic changes were all decreased in IL5Ra-deficient mice [
      • Tanaka H.
      • Komai M.
      • Nagao K.
      • Ishizaki M.
      • Kajiwara D.
      • Takatsu K.
      • et al.
      Role of interleukin-5 and eosinophils in allergen-induced airway remodeling in mice.
      ]. In humans, anti–IL-5 treatment in asthma patients was associated with a significant reduction in the numbers and percentage of airway eosinophils expressing mRNA for TGF-β1 and the concentration of TGF-β1 in BAL fluid [
      • Flood-Page P.
      • Menzies-Gow A.
      • Phipps S.
      • Ying S.
      • Wangoo A.
      • Ludwig M.S.
      • et al.
      Anti-IL-5 treatment reduces deposition of ECM proteins in the bronchial subepithelial basement membrane of mild atopic asthmatics.
      ]. Prolonged, 12-month treatment with anti-IL5 resulted in improvements in exacerbations, systemic and local eosinophil counts, and reductions in total airway area and airway wall area on CT compared to findings in the placebo group [
      • Haldar P.
      • Brightling C.E.
      • Singapuri A.
      • Hargadon B.
      • Gupta S.
      • Monteiro W.
      • et al.
      Outcomes after cessation of mepolizumab therapy in severe eosinophilic asthma: a 12-month follow-up analysis.
      ]. Additionally to increased levels of IL8 in induced sputum in our patients with accelerated FEV1 decline, we also detected a positive correlation of IL8 and neutrophil numbers, which is consistent with its primary function to attract neutrophils to sites of inflammation. Apart from chemo-attractive function, YKL-40-induced IL8 expression and production of human bronchial epithelial cells were found to stimulate the proliferation and migration of bronchial smooth muscle cells [
      • Tang H.
      • Sun Y.
      • Shi Z.
      • Huang H.
      • Fang Z.
      • Chen J.
      • et al.
      YKL-40 induces IL-8 expression from bronchial epithelium via MAPK (JNK and ERK) and NF- B pathways, causing bronchial smooth muscle proliferation and migration.
      ]. Further, IL8 has been reported to promote submucosal angiogenesis in asthmatics [
      • Keglowich L.
      • Roth M.
      • Philippova M.
      • Resink T.
      • Tjin G.
      • Oliver B.
      • et al.
      Bronchial smooth muscle cells of asthmatics promote angiogenesis through elevated secretion of CXC-chemokines (ENA-78, GRO-α, and IL-8).
      ]. Together with increased permeability, higher density of the submucosal vasculature results in oedema and is also a predisposition for tissue inflammation. Increased vascularization has been shown to correlate with airflow limitation and bronchial hyper-responsiveness [
      • Hoshino M.
      • Takahashi M.
      • Aoike N.
      Expression of vascular endothelial growth factor, basic fibroblast growth factor, and angiogenin immunoreactivity in asthmatic airways and its relationship to angiogenesis.
      ,
      • Li X.
      • Wilson J.W.
      Increased vascularity of the bronchial mucosa in mild asthma.
      ,
      • Kanazawa H.
      • Hirata K.
      • Yoshikawa J.
      Involvement of vascular endothelial growth factor in exercise induced bronchoconstriction in asthmatic patients.
      ]. Elevated serum IL5, IFNγ and periostin levels were detected in patients with rapid lung function decline [
      • Kanemitsu Y.
      • Matsumoto H.
      • Izuhara K.
      • Tohda Y.
      • Kita H.
      • Horiguchi T.
      • et al.
      Increased periostin associates with greater airflow limitation in patients receiving inhaled corticosteroids.
      ,
      • Litonjua A.A.
      • Sparrow D.
      • Guevarra L.
      • O'Connor G.T.
      • Weiss S.T.
      • Tollerud D.J.
      Serum interferon-gamma is associated with longitudinal decline in lung function among asthmatic patients: the Normative Aging Study.
      ]. There are no previous reports of associations of local IL5 or IL8 levels with lung function decline.
      A variability of detected eosinophils, IL5 and IL8 values in induced sputum, which are not consistently high in patients with accelerated FEV1 decline, is probably due to interference of other factors in this process. Further studies to address this issue are needed.
      Data listed above suggest that eosinophils, IL5 and IL8 do not act only as modifiers of the inflammatory response but also have the potential to directly influence processes in airway remodelling, either through the enhancement of collagen content and fibrotic changes and the stimulation of proliferation and migration of bronchial smooth muscle cells or by promoting submucosal angioneogenesis. For cases with a high eosinophilic component, a targeted anti-IL5 treatment was developed, which could also potentially be highly beneficial for patients with accelerated FEV1 decline and high IL5-associated remodelling.
      On the other hand, elevated eosinophils and cytokines may be only bystanders - the consequence of an ongoing remodelling process. ASM cells are highly productive cells; among many other inflammatory and angiogenetic mediators, they produce IL2, IL5, IL6, IL8, IL10, VEGF, angiogenin, and βFGF [
      • Hirst S.J.
      Regulation of airway smooth muscle cell immunomodulatory function: role in asthma.
      ]. Thus, IL5 and IL8 might serve as non-invasive biomarkers of remodelling and accelerated FEV1 decline, and there might be a role for treatments resulting in a reduction in ASM (bronchial thermoplasty), but larger studies are needed to confirm this possibility.
      The limitation of our study is the small number of patients. Although the patients were carefully selected and shared similar clinical and functional characteristics, we believe that some correlations would be more evident with larger number of patients. Another limitation is the lack of an objective control over the actual use of inhaled medications or the frequency of exacerbations; instead, we had to rely on patient reports from questionnaires. Further, sputum collection and analyses at baseline could give us additional valuable data about inflammation and cytokines in naive patients at the point of confirmation diagnosis and enable us to evaluate their dynamics.
      In conclusion, the results of our study showed accelerated FEV1 decline of more than 30 ml/year in more than half of patients who actually had only mild asthma with few symptoms. It is important to recognize and more precisely treat and follow up this group of patients. Sputum eosinophils, IL5 and IL8, which might interfere in airway remodelling either directly or indirectly by stimulating the inflammatory response, are potential non-invasive biomarkers of accelerated FEV1 decline in asthma patients and potential therapeutic targets. To confirm these assumptions, additional larger in vivo and in vitro studies are required.

      Funding source

      None.

      CRediT authorship contribution statement

      Mateja Marc-Malovrh: Conceptualization, Methodology, Formal analysis, Writing - original draft. Luka Camlek: Conceptualization, Methodology, Investigation. Sabina Škrgat: Investigation. Izidor Kern: Resources. Matjaž Fležar: Resources. Manca Dežman: Investigation. Peter Korošec: Conceptualization, Methodology, Resources, Supervision.

      Declaration of competing interest

      None.

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