The association between airway eosinophilic inflammation and IL-33 in stable non-atopic COPD

The association between airway eosinophilic inflammation and IL-33 in stable non-atopic COPD Background: Interleukin(IL)-33 is an epithelial alarmin important for eosinophil maturation, activation and survival. The aim of this study was to examine the association between IL-33, its receptor expression and airway eosinophilic inflammation in non-atopic COPD. Methods: IL-33 concentrations were measured in exhaled breath condensate (EBC) collected from healthy non- smokers, asthmatics and non-atopic COPD subjects using ELISA. Serum and sputum samples were collected from healthy non-smokers, healthy smokers and non-atopic COPD patients. Based on sputum eosinophil count, COPD subjects were divided into subgroups with airway eosinophilic inflammation (sputum eosinophils > 3%) or without (sputum eosinophils ≤3%). IL-33 and soluble form of IL-33 receptor (sST2) protein concentrations were measured in serum and sputum supernatants using ELISA. ST2 mRNA expression was measured in peripheral mononuclear cells and sputum cells by qPCR. Hemopoietic progenitor cells (HPC) expressing ST2 and intracellular IL-5 were enumerated in blood and induced sputum by means of flow cytometry. Results: IL-33 levels in EBC were increased in COPD patients to a similar extent as in asthma and correlated with blood eosinophil count. Furthermore, serum and sputum IL-33 levels were higher in COPD subjects with sputum eosinophilia than in those with a sputum eosinophil count ≤3% (p < 0.001 for both). ST2 mRNA was overexpressed in sputum cells obtained from COPD patients with airway eosinophilic inflammation compared to those without sputum eosinophilia (p < 0.01). Similarly, ST2 + IL-5+ HPC numbers were increased in the sputum of COPD patients with airway eosinophilia (p < 0.001). Conclusions: Our results indicate that IL-33 is involved in the development of eosinophilic airway inflammation in non-atopic COPD patients. Keywords: IL-33, Eosinophils, COPD Background of evidence indicates that eosinophilic COPD is a dis- Chronic Obstructive Pulmonary Disease (COPD) is one tinct phenotype of the disease, and one that is character- the most common chronic diseases in adults, and one of ized by a higher risk of exacerbations but better the main causes of morbidity and mortality [1]. The response to treatment with inhaled corticosteroids [3, 4]. prevalence of COPD at Global Initiative for Chronic COPD subjects have higher percentages of eosinophils Obstructive Lung Disease (GOLD) stage 2 or higher has in sputum and higher concentrations of eosinophil cat- been estimated at 10.1% overall, although the precise fig- ionic protein than healthy controls, and their airway eo- ure varies according to the region [2]. A growing body sinophil numbers further increase during disease exacerbation [5, 6]. Data from the ECLIPSE (Evaluation of COPD Longitudinally to Identify Predictive Surrogate * Correspondence: damian.tworek@umed.lodz.pl End-points) study showed that over three years of obser- Department of General and Oncological Pulmonology, Medical University of Lodz, Kopcinskiego 22, 90-153 Lodz, Poland vation, 37.4% of COPD patients demonstrated a Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Tworek et al. Respiratory Research (2018) 19:108 Page 2 of 11 persistent blood eosinophil count greater than or equal least 10 pack-years) without COPD and 20 healthy to 2% [7]. Non-allergic COPD patients with blood eo- non-smokers were included in the second phase examin- sinophil counts > 250 cells/μL are characterized by ing the association between IL-33 and sputum eosino- higher airway eosinophil numbers, higher sputum inter- philia (Sputum study). COPD was diagnosed according leukin(IL)-5 and haptoglobin levels, greater CCL20 and to GOLD criteria by demonstrating irreversible broncho- CCL24 concentrations in bronchoalveolar lavage (BAL) constriction following inhalation of salbutamol and more pronounced airway remodeling [8]. (www.goldcopd.org). Asthma was diagnosed according IL-33 is one of the epithelial alarmins generated in re- to Global Initiative for Asthma (GINA) criteria based on sponse to danger signals, and is primarily produced by the history of typical respiratory symptoms and positive epithelial cells [9]. Recent evidence has demonstrated result of either bronchodilator reversibility testing or that IL-33 precedes IL-5 in regulating eosinophil com- metacholine challenge (www.ginasthma.org). mitment and is required for eosinophil homeostasis [10]. All participants, except for several asthmatic subjects, One such result of IL-33 activity is that activates and were non-atopic as determined by negative results of augments the maturation of hemopoietic progenitor skin prick testing with common aeroallergens. COPD cells (HPC). HPC are considered to be innate effector patients were treated with bronchodilators only. Subjects cells in allergic asthma [11, 12]: They produce an abun- with a history of infection, or who had used antibiotics dance of pro-inflammatory cytokines and differentiate or systemic/inhaled steroids within the previous four into mature eosinophils in the process of “in situ weeks were excluded. All subjects gave their written hemopoiesis” at sites of inflammation [13]. informed consent, and the study was approved by the The aim of the present study was to determine the Ethics Board of the Medical University of Lodz. The de- presence of an association between IL-33 level, the ex- tailed characteristics of the subjects are presented in pression of its receptor and the degree of eosinophilic Table 1 and Table 2. Patients with COPD were evaluated inflammation in non-atopic COPD. using COPD Assessment Test (CAT), modified Medical Research Council dyspnea scale (mMRC), six-minute Methods walking test (6MWT) and BODE index (see Subjects Additional file 1). Sixteen non-smoking healthy subjects, 23 non-smoking asthmatics and 25 non-atopic COPD subjects (smokers Biological samples collection and measurements and ex-smokers with a history of at least 10 pack-years) The EBC was collected according to the recommenda- were included in the first part of the study assessing the tions of the European Respiratory Society [14]. Sputum association between IL-33 in breath condensate (EBC) samples were induced using hypertonic saline and proc- with blood eosinophilia (EBC study). Forty patients with essed as described previously [15]. The COPD subjects COPD (smokers and ex-smokers with a history of at were then divided into subgroups based on sputum eo- least 10 pack-years), 20 smokers (with a history of at sinophil count: one group with airway eosinophilic Table 1 Characteristics of subjects included in the exhaled breath condensate (EBC) study Healthy Non-smokers Asthma COPD Number of subjects 16 23 25 Sex (M/F) 11/5 11/12 11/14 ll** Age (years) 56.75 ± 2.71 54.22 ± 3.05 66.96 ± 1.76 Pack-years 0 0 39.10±3.70 Time since diagnosis (years) – 10.13 ± 2.34 4.46 ± 0.84 *† PB FEV % of predicted (%) 107.90 ± 3.23 82.59 ± 4.56 62.32 ± 2.76 *† PB FEV /FVC (%) 76.32 ± 1.46 68.14 ± 2.38 54.03 ± 1.89 Current smokers (%) 0 0 32 Use of ICS (%) 0 0 0 Atopic subjects (%) 0 56.52 0 Blood eosinophil number (cells/μl) 181 ± 35 315 ± 35 242 ± 31 Blood eosinophil percentage (%) 3.01 ± 0.54 4.74 ± 0.59 3.16 ± 0.41 PB post bronchodilator, FEV forced expiratory volume in first second, FVC forced vital capacity, ICS inhaled corticosteroids * † ‡ § ll COPD vs Asthma p < 0.001; COPD vs Healthy Non-smokers p < 0.001; COPD vs Asthma p < 0.01; Asthma vs Healthy Non-smokers p < 0.05; COPD vs Healthy ** Non-smokers p < 0.05; COPD vs Asthma p < 0.01 Tworek et al. Respiratory Research (2018) 19:108 Page 3 of 11 Table 2 Characteristics of subjects included in the sputum study Healthy Non-smokers Healthy Smokers COPD Number of subjects 20 20 40 Sex (M/F) 10/10 11/9 22/18 a† Age (years) 56.84±2.14 57.85±2.00 67.40±1.12 Pack-years 0 32.14±2.96 41.99±2.78 Time since diagnosis (years) –– 7.05±1.00 a† PB FEV % of predicted 101.9±2.92 96.88±6.26 61.98±2.76 a† PB FEV %FVC 77.69±1.43 74.39±1.18 52.49±1.57 BMI (kg/m ) 27.23 ± 0.80 27.72 ± 0.68 28.39 ± 1.05 CAT score –– 15.8 ± 1.18 6MWT (m) –– 388.6 ± 11.08 mMRC score –– 1(0–3) BODE index –– 1(0–6) Current smokers (%) 0 100 42.5 Use of ICS (%) 0 0 0 No. of atopic subjects (No) 0 0 0 Sputum eosinophils (%) 0.89 ± 0.28 1.43 ± 0.28 2.93 ± 0.95 Sputum neutrophils (%) 18.89 ± 3.77 23.28 ± 2.23 37.40 ± 4.93 a# Sputum macrophages (%) 65.69 ± 3.76 56.85 ± 1.99 44.70 ± 41 Sputum lymphocytes (%) 6.43 ± 0.84 4.26 ± 0.72 6.60 ± 1.99 Sputum eosinophils (No of cells/ g of sputum × 10 ) 1.60 ± 0.77 1.55 ± 0.73 2.70 ± 1.33 Sputum neutrophils (No of cells/ g of sputum ×10 ) 14.95 ± 3.12 19.40 ± 10.01 19.50 ± 7.36 Sputum macrophages (No of cells/ g of sputum ×10 ) 16.10 ± 2.79 31.00 ± 5.62 41.98 ± 12.55 Sputum lymphocytes (No of cells/ g of sputum ×10 ) 2.60 ± 0.47 2.10 ± 0.45 5.92 ± 3.25 PB post bronchodilator, FEV forced expiratory volume in first second, FVC forced vital capacity, CAT COPD assessment test, 6MWT six-minute walk test, mMRC modified Medical Research Council dyspnea scale, ICS inhaled corticosteroids a † ‡ COPD vs Healthy Non-smokers, p < 0.05; COPD vs Healthy Smokers p < 0.05; median (min-max) inflammation (i.e. sputum eosinophil level > 3%) and the Comparisons between healthy non-smokers, asthmatics other without (i.e. sputum eosinophil level ≤ 3%). The and COPD subjects, as well as between healthy 3% cut-off value for sputum eosinophilia was used based non-smokers, healthy smokers and COPD subjects, were on previous studies on eosinophilic airway inflammation analyzed with one-way ANOVA and the post hoc in COPD [16, 17]. The serum and sputum supernatants Tukey’s test, or with the Kruskal-Wallis test and the post were tested for IL-33 and sST2 concentrations by means hoc Dunn’s test, where appropriate. of enzyme-linked immunosorbent assays, while the EBC Differences between COPD subjects with and without was tested for IL-33 only. eosinophilia were analyzed using the Student’st-testor ST2 mRNA expression in peripheral mononuclear U-Mann Whitney test, where appropriate. Correlation ana- cells (PBMC) and sputum cells was measured using lysis was conducted using either Pearson’s or Spearman’s qPCR. Hemopoietic progenitor cells expressing ST2 and correlation coefficient according to the distribution of vari- intracellular IL-5 were enumerated in blood and induced ables. Significance was accepted at P <0.05. sputum obtained from COPD subjects with and without airway eosinophilia by means of flow cytometry (see gating Results strategy in Additional file 2). Detailed information on the Exhaled breath condensate study study procedures can be found in the Additional file 1. Absolute blood eosinophil number and percentage were elevated in asthmatics compared to healthy non-smokers Statistical analysis (p < 0.05). No significant difference in blood eosinophil The results were analyzed using GraphPad Prism 6 counts was observed between COPD patients, healthy (GraphPad Software, San Diego, CA). For clarity, all re- non-smokers and asthmatic subjects (Table 1). sults are presented as means ± SEMs. The normality of EBC IL-33 levels were significantly higher in asthmatic data distribution was tested with the Shapiro-Wilk test. (3.57 ± 0.81 pg/ml) and COPD (2.50 ± 0.33 pg/ml) Tworek et al. Respiratory Research (2018) 19:108 Page 4 of 11 patients compared to healthy controls (1.27 ± 0.57 pg/ml), Similarly to the serum findings, IL-33 sputum levels with no significant difference observed between the first were significantly higher in COPD subjects with sputum two groups (Fig. 1a). IL-33 levels in EBC correlated signifi- eosinophilia (22.72 ± 7.42 pg/ml) than in those without cantly with blood eosinophil numbers and percentages in (1.13 ± 0.56 pg/ml; p < 0.001) (Fig. 2d). the asthmatic (see Additional file 3) and COPD subjects sST2 was elevated in the serum (p < 0.05) of COPD (Fig. 1b and c). patients compared with the healthy controls (Fig. 3a). A similar trend was observed for sputum sST2 levels Sputum study (p = 0.059) (Fig. 3b). No significant difference was ob- Sputum differential counts are presented in Table 2.It served in serum and sputum sST2 levels in COPD was found that 25% of COPD subjects had sputum eo- subjects with regard to sputum eosinophil counts sinophilia > 3% (mean sputum eosinophil percentage (Fig. 3c and d). 8.89 ± 3.18%). ST2 mRNA was overexpressed in PBMC and sputum IL-33 serum concentrations were significantly elevated in cells from healthy smokers and COPD subjects com- COPD subjects (32.86±1.74 pg/ml) compared with healthy pared with healthy non-smokers (Fig. 4a and b). ST2 non-smokers (22.12±1.45 pg/ml; p < 0.001) and smokers mRNA expression was significantly higher in sputum without COPD (24.45±1.33 pg/ml; p < 0.01) (Fig. 2a). In cells from COPD patients with eosinophil airway inflam- addition, serum IL-33 levels were found to be significantly mation (Relative quantification(RQ) = 1.20 ± 0.85) than higher in the subgroup of COPD patients with sputum eo- in those without airway eosinophilia (RQ = 0.05 ± 0.01; sinophilia (43.98±4.76 pg/ml) compared to those with a p < 0.01) (Fig. 4d). sputum eosinophil level ≤3% (29.15±2.01 pg/ml; p < 0.001) The significant correlations between IL-33, sST2, ST2 (Fig. 2c). mRNA and sputum eosinophil content and clinical pa- Sputum IL-33 levels were significantly increased in rameters in COPD are presented in Fig. 5. All analyzed COPD subjects (6.52 ± 2.37 pg/ml) compared with correlations are presented in Additional file 4. healthy non-smokers (0.0 ± 0.0 pg/ml; p < 0.05) and No significant difference in circulating HPC count was healthy smokers (2.92 ± 2.45 pg/ml; p < 0.05) (Fig. 2b). found between COPD subjects with sputum eosinophilia a * bc 8 8 r=0.48; p<0.05 r=0.44; p<0.05 6 6 4 4 2 2 0 0 0 200 400 600 800 0 5 10 15 Eos [cells/µl] Eos [%] Fig. 1 IL-33 concentrations in exhaled breath condensate (EBC) in healthy non-smokers, asthmatics and non-atopic COPD subjects (a). Data are presented as mean ± SEM. Correlations between EBC IL-33 and blood eosinophil numbers (b) and percentage (c) in COPD patients. *p < 0.05 Healthy Non-Smokers Asthma COPD IL-33 [pg/ml] IL-33 [pg/ml] IL-33 [pg/ml] Tworek et al. Respiratory Research (2018) 19:108 Page 5 of 11 Serum Sputum *** * ab ** *** *** cd 60 40 0 0 Fig. 2 Serum (a)and sputum (b) IL-33 concentrations in healthy non-smokers, healthy smokers and patients with COPD. Comparison of serum (c)and sputum (d) IL-33 between COPD subjects with and without sputum eosinophilia. Data are presented as mean ± SEM. *p < 0.05; **p < 0.01; ***p <0.001 2 2 and those without (422.90 ± 97.65/1 mln PBMC vs (16.83 ± 14.65 × 10 /gram of sputum vs 0.19 ± 0.07 × 10 553.70 ± 138.70/1 mln PBMC, respectively; p = 0.97). /gram of sputum; p < 0.01) (Fig. 6d and e). However, COPD subjects with sputum eosinophilia No difference was observed in circulating ST2 + IL-5+ demonstrated significantly higher numbers of sputum HPC between the two subgroups of COPD patients (see HPC than those without eosinophilia (35.64 ± 27.39 × Additional file 6). However, the absolute numbers of 2 2 10 /gram of sputum vs 1.19 ± 0.34 × 10 /gram of ST2 + IL-5+ HPC, but not their percentage, were signifi- sputum, respectively; p < 0.001) (see Additional file 5). cantly higher in the sputum of eosinophilic COPD pa- Similarly, no differences were found between the two tients than in those with normal COPD (1.58 ± 1.38 × 2 2 groups with regard to percentage or absolute number of 10 /gram of sputum vs 0.009 ± 0.009 × 10 /gram of circulating ST2 + HPC (see Additional file 6). However, sputum; p < 0.001) (Fig. 6g and h). both the percentage of ST2 expression on HPC and the No correlation was found between the percentages absolute number of ST2 + HPC in sputum were higher in (r = 0.24; p = 0.28) or absolute numbers (r =0.30; p=0.38) patients with a sputum eosinophil count > 3% compared to of ST2+ and IL-5+ HPC in sputum. those without (7.80 ± 1.58% vs 2.20 ± 2.22%; p <0.01 and 2 2 2.36 ± 1.83 × 10 /gram of sputum vs 0.018 ± 0.01 × 10 Discussion /gram of sputum; p < 0.001, respectively) (Fig. 6a and b). Our findings show for the first time that increased ex- No differences regarding the percentages and absolute pression of IL-33 and its receptor ST2 is associated with numbers of circulating IL-5 + HPC were observed be- airway eosinophilia in non-atopic COPD patients. tween COPD patients with sputum eosinophilia and IL-33 has been extensively studied in allergic asthma those without (see Additional file 6). In sputum, absolute and other allergic conditions. Some studies suggest that IL-5 + HPC counts, but not percentages, were elevated IL-33 may be also implicated in the pathogenesis of in COPD patients with sputum eosinophilia COPD. IL-33 and sST2 serum levels are elevated in Healthy Non-Smokers e th Smok H al y ers COPD Sputum Eos 3% Healthy Non-Smokers Healthy Smokers Sputum Eos >3% COPD Sputum Eos 3% Sputum Eos >3% Serum IL-33 [pg/ml] Serum IL-33 [pg/ml] Sputum IL-33 [pg/ml] Sputum IL-33 [pg/ml] Tworek et al. Respiratory Research (2018) 19:108 Page 6 of 11 Serum Sputum ab 1500 100 0 0 cd 1500 150 1000 100 500 50 0 0 Fig. 3 Serum (a) and sputum (b) sST2 concentrations in healthy non-smokers, healthy smokers and patients with COPD. Comparison of serum (c) and sputum (d) sST2 between COPD subjects with and without sputum eosinophilia. Data are presented as mean ± SEM. *p < 0.05 COPD patients compared to healthy smokers [18]. To further explore these findings, the second part of Furthermore, immunofluorescence staining showed that our study investigated the expression of IL-33, its recep- the expression of IL-33 was increased in the lung tissues tor and its soluble form in the periphery and the airways of patients with COPD, indicating that IL-33 of patients with and without airway eosinophilia. Our re- upregulation was probably related to systemic and sults showed that the level of IL-33 protein is markedly airway inflammation in COPD [18–20]. It has also been increased in the serum and sputum of patients with eo- reported that cigarette smoke markedly enhanced the sinophilic COPD. expression of IL-33 and ST2 in the lung tissue of mice, sST2 is a decoy receptor regulating the activity of which was accompanied by neutrophil and macrophage IL-33. As sST2 is not detected in EBC, its levels were infiltration and elevated expression of pro-inflammatory not assessed in the EBC obtained from COPD patients cytokines and chemokines in the airways [19]. [21]. However, our findings confirm those of previous Interestingly, Kim et al. found blood eosinophil counts reports showing increased levels of serum sST2 in to be correlated with plasma IL-33 levels in COPD COPD [18]. These findings also suggest a trend toward patients [20]. overexpression of sputum sST2 in the whole COPD Recently, Gluck et al. showed that IL-33 protein is de- group. Animal studies have shown that sST2 may exert tectable at low levels in EBC, and its level is elevated in a protective function in acute lung injury and asthmatics compared with healthy controls [21]. Our re- ovalbumin-induced allergic asthma [22, 23], and Hacker sults demonstrate that IL-33 is elevated in EBC collected et al. suggest that sST2 plays a critical role in the from non-atopic COPD subjects to the same extent as in anti-inflammatory regulatory mechanism in early stages asthma. Furthermore, EBC IL-33 levels correlated posi- of COPD [24]. Although our findings do not indicate tively with blood eosinophil numbers and percentages, significant differences in sST2 concentrations between not only in asthma but also in COPD. COPD subjects with and without sputum eosinophilia, Healthy Non-Smokers Healthy Smokers COPD Healthy Non-Smokers Sputum Eos 3% Healthy Smokers Sputum Eos >3% COPD Sputum Eos 3% Sputum Eos >3% Serum sST2 [pg/ml] Serum sST2 [pg/ml] Sputum sST2 [pg/ml] Sputum sST2 [pg/ml] Tworek et al. Respiratory Research (2018) 19:108 Page 7 of 11 PBMC Sputum ab ** **** 0.25 0.6 *** *** 0.20 0.4 0.15 0.10 0.2 0.05 0.00 0.0 cd ** 0.25 2.5 0.20 2.0 0.15 1.5 0.10 1.0 0.05 0.5 0.00 0.0 Fig. 4 ST2 mRNA expression in peripheral blood mononuclear cells (PBMC) (a) and sputum cells (b) in n healthy non-smokers, healthy smokers and patients with COPD. Comparison of ST2 mRNA expression in PBMC (c) and sputum cells (d) between COPD subjects with and without sputum eosinophilia. Data are presented as mean ± SEM. **p < 0.01; ***p < 0.001; ****p < 0.0001 sputum sST2 levels nevertheless correlated negatively are known to act as pro-inflammatory effector cells of al- with sputum eosinophil counts, suggesting it plays a lergic inflammation [11]. HPC express ST2 and IL-33 as protective role against airway eosinophilic inflammation. potent activators of HPC, leading to the release of a num- On the contrary, sputum cells obtained from COPD ber of cytokines and chemokines, including IL-5. HPC can patients with airway eosinophilia overexpressed ST2 differentiate into mature eosinophils at the site of inflam- mRNA. ST2 is expressed by many types of cells that mation in a process called “in situ eosinophilopoiesis”. may be present in the airways, including eosinophils. IL-33 accelerates the maturation of HPC and modulates The activation of eosinophils by IL-33 through ST2 leads their migration into airways in allergic asthma [26, 27]. to the production of superoxide and IL-8 and increases Little is known of the role of HPC in COPD. Some eosinophil survival [25]. It is important to note that studies have reported reduced numbers of circulating IL-33 activates eosinophils at least to the same degree as HPC in COPD [28], while others report similar circulat- IL-5 [25]. The significant correlations between IL-33 and ing and sputum HPC numbers between COPD and ST2 and sputum eosinophil counts found in our study healthy non-atopic subjects [29]. Our present findings support the hypothesis that IL-33 may be involved in the do not suggest any differences in circulating HPC num- development and maintenance of eosinophilic airway in- bers between COPD patients with sputum eosinophilia flammation in non-atopic COPD subjects. and those without. However, a striking difference was Finally, the HPC count as well as ST2 and intracellular observed in the number of sputum HPC between the IL-5 expression by these cells were measured in the pa- two groups of COPD patients, with significantly elevated tients with sputum eosinophilia and in those without. HPC HPC numbers found in those with sputum eosinophils Healthy Non-Smokers Heal hy Smokers COPD Healthy Non-Smokers Sputum Eos 3% Healthy Smokers Sputum Eos >3% COPD Sputum Eos 3% Sputum Eos >3% PBMC ST2 mRNA expression [RQ] PBMC ST2 mRNA expression [RQ] Sputum ST2 mRNA expression [RQ] Sputum ST2 mRNA expression [RQ] Tworek et al. Respiratory Research (2018) 19:108 Page 8 of 11 Fig. 5 Significant correlations between serum and sputum IL-33 and sST2, ST2 mRNA and clinical parameters in COPD > 3%. This was accompanied by overexpression of intra- from activated cells. Measuring IL-33 protein content is cellular IL-5 and ST2 by sputum HPC indicating in- challenging and previous studies give varying results for creased activation of these cells in eosinophilic COPD, serum and sputum [30, 31]. Nonetheless, our findings on analogously to allergic asthma. ST2 expression confirm the IL-33 measurements and sup- As IL-33 modulates the trafficking of HPC, it is possible port the association between IL-33 and eosinophilic that increased IL-33 levels may be at least partially respon- phenotype of COPD. The best way to determine IL-33 ex- sible for the augmented influx of HPC into airways ob- pression would be to measure it directly in the main served in COPD patients with eosinophilic inflammation. source of the cytokine, i.e. the airway epithelium; however, In addition, increased numbers of ST2 + IL-5 + HPC were studies comparing IL-33 expression in eosinophilic COPD seen in the sputum of patients with airway eosinophilia. involving invasive methods are warranted. In addition, the This finding suggests that IL-33 activates HPC in eosino- results may have been affected by the fact that our group philic COPD. Therefore, in those subjects, HPC may act of COPD subjects was older than those of the other two as effector cells in an analogous way to allergic asthma, by groups. However, as no correlation has been found be- fostering the development of a local IL-5 rich environ- tween IL-33 and ST2 expression and the age of partici- ment independent of the IgE pathway. pant, it is unlikely that this may be the case. There are several limitations to our study. First, the IL-33 protein levels were low in a significant number of Conclusions exhaled breath and sputum specimens. This could be due In conclusion, our results suggest that increased IL-33 is to the rapid neutralization of IL-33 following its release associated with airway eosinophilia in non-atopic COPD. Tworek et al. Respiratory Research (2018) 19:108 Page 9 of 11 a b de f gh i Fig. 6 Expression of ST2 and intracellular IL-5 by sputum hemopoietic progenitor cells (HPC) in COPD subjects with sputum eosinophil count ≤3% (n = 10) and > 3% (n = 10). The percentage of sputum HPC expressing ST2, IL-5 and double positive cells for ST2 and IL-5 are presented in sub-figures a, d and g, respectively. Absolute numbers of HPC expressing ST2, IL-5 and double positive cells for ST2 and IL-5 per gram of sputum are presented in sub-figures b, e and h, respectively. Representative example of flow cytometry acquisition of sputum ST2+ (c), IL-5+ (f) and ST2 + IL-5+ (i) HPC. Data are presented as mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001 It is tempting to speculate that IL-33 is involved in the forced expiratory volume in first second; FVC – forced vital capacity; CAT recruitment and activation of HPC into the airways. This – COPD assessment test; 6MWT – six-minute walk test; mMRC – modified Medical Research Council dyspnea scale. (PDF 39 kb) may result in the creation of a local, IL-5 rich inflamma- Additional file 5: Figure E2. Circulating (A) and sputum (B) tory state similar to that observed in allergic asthma. hemopoietic progenitor cells (HPC) in patients with and without sputum Thus, IL-33 may be a potential therapeutic target in the eosinophilia. **p < 0.01. (PDF 56 kb) subgroup of COPD patients characterized by eosino- Additional file 6: Figure E3. The percentage and absolute numbers of philic inflammation. circulating hemopoietic progenitor cells (HPC) expressing ST2 (A and B, respectively), intracellular IL-5 (C and D, respectively) and double positive for ST2 and IL-5 (E and F, respectively) in COPD patients with (sputum eosinophils Additional files > 3%) and without (sputum eosinophils ≤3%) sputum eosinophilia. (PDF 51 kb) Additional file 1: Detailed Method description. (PDF 118 kb) Additional file 2: Figure E4. Hemopoietic progenitor cells gating strategy. (PDF 88 kb) Abbreviations 6MWT: Six-minute walking test; CAT: COPD Assessment Test; COPD: Chronic Additional file 3: Figure E1. Correlations between IL-33 concentrations Obstructive Pulmonary Disease; EBC: Exhaled breath condensate; in exhaled breath condensate and blood eosinophil numbers (A) and GINA: Global Initiative for Asthma; GOLD: Global Initiative for Chronic percentage (B) in asthmatic patients. (PDF 49 kb) Obstructive Lung Disease; HPC: Hemopoietic progenitor cells; IL: Interleukin; Additional file 4: Table E1. Correlations between serum and sputum mMRC: Modified Medical Research Council; PBMC: Peripheral mononuclear IL-33 and sST2, ST2 mRNA and clinical parameters in COPD. FEV1 – cells; RQ: Relative quantification; sST2: Soluble ST2; ST2: IL-33 receptor Tworek et al. Respiratory Research (2018) 19:108 Page 10 of 11 Acknowledgements 5. Balzano G, Stefanelli F, Iorio C, De Felice A, Melillo EM, Martucci M, Melillo G. The authors would like to thank Dr. Jacek Szymański for his assistance in the Eosinophilic inflammation in stable chronic obstructive pulmonary disease. flow cytometry acquisition. Am J Respir Crit Care Med. 1999;160:1486–92. 6. Saetta M, Di Stefano A, Maestrelli P, Turato G, Ruggieri MP, Roggeri A, Calcagni P, Mapp CE, Ciaccia A, Fabbri LM. Airway eosinophilia in chronic bronchitis Funding during exacerbations. Am J Respir Crit Care Med. 1994;150:1646–52. This work was supported by Medical University of Lodz. 7. Singh D, Kolsum U, Brightling CE, Locantore N, Agusti A, Tal-Singer R. Eosinophilic inflammation in COPD: prevalence and clinical characteristics. Availability of data and materials Eur Respir J. 2014;44:1697–700. The datasets used and/or analysed during the current study are available 8. Kolsum U, Damera G, Pham T-H, Southworth T, Mason S, Karur P, Newbold from the corresponding author on reasonable request. P, Singh D. Pulmonary inflammation in COPD patients with higher blood eosinophil counts. J Allergy Clin Immunol. 2017; https://doi.org/10.1016/j. Authors’ contributions jaci.2017.04.027. DT is the primary author and has made significant contributions to the 9. Mitchell PD, O’Byrne PM. Biologics and the lung: TSLP and other epithelial conception, and the acquisition, analysis, and interpretation of the data; and cell-derived cytokines in asthma. Pharmacol Ther. 2016; https://doi.org/10. drafted, reviewed, and approved the manuscript for publication. SM has 1016/j.pharmthera.2016.06.009. recruited subjects participating in the study; and has reviewed and approved 10. Johnston LK, Hsu C-L, Krier-Burris RA, Chhiba KD, Chien KB, McKenzie A, the manuscript for publication. KS, JK, ZK, assisted with the acquisition of the Berdnikovs S, Bryce PJ. IL-33 precedes IL-5 in regulating eosinophil commitment data; and have reviewed and approved the manuscript for publication. PG, and is required for eosinophil homeostasis. J Immunol. 2016;197:3445–53. EB-L, PK have made significant contributions to the design and interpretation 11. Allakhverdi Z, Comeau MR, Smith DE, Toy D, Endam LM, Desrosiers M, Liu Y- of the data; and have reviewed, revised, and approved the manuscript for J, Howie KJ, Denburg JA, Gauvreau GM, Delespesse G. CD34+ hemopoietic publication. AA has made significant and substantial contributions to the progenitor cells are potent effectors of allergic inflammation. J Allergy Clin conception, design, and interpretation of the data; and has reviewed, revised, Immunol. 2009;123:472–8. and approved the manuscript for publication. 12. Tworek D, Heroux D, O’Byrne SN, Mitchell P, O’Byrne PM, Denburg JA. Toll- like receptor-induced expression of epithelial cytokine receptors on Ethics approval and consent to participate haemopoietic progenitors is altered in allergic asthma. Clin Exp Allergy. All subjects gave their written informed consent, and the study was 2017;47:900–8. approved by the Ethics Board of the Medical University of Lodz. 13. Hui CCK, McNagny KM, Denburg JA, Siracusa MC. In situ hematopoiesis: a regulator of TH2 cytokine-mediated immunity and inflammation at mucosal Competing interests surfaces. Mucosal Immunol. 2015;8:701–11. The authors declare that they have no competing interests. 14. Horváth I, Hunt J, Barnes PJ, Alving K, Antczak A, Baraldi E, Becher G, van Beurden WJC, Corradi M, Dekhuijzen R, Dweik RA, Dwyer T, Effros R, Erzurum S, Gaston B, Gessner C, Greening A, Ho LP, Hohlfeld J, Jöbsis Q, Publisher’sNote Laskowski D, Loukides S, Marlin D, Montuschi P, Olin AC, Redington AE, Springer Nature remains neutral with regard to jurisdictional claims in Reinhold P, van Rensen ELJ, Rubinstein I, et al. Exhaled breath condensate: published maps and institutional affiliations. methodological recommendations and unresolved questions. Eur Respir J. 2005;26:523–48. Author details 15. Bafadhel M, McCormick M, Saha S, McKenna S, Shelley M, Hargadon B, Mistry V, Department of General and Oncological Pulmonology, Medical University of Reid C, Parker D, Dodson P, Jenkins M, Lloyd A, Rugman P, Newbold P, Lodz, Kopcinskiego 22, 90-153 Lodz, Poland. Department of Pulmonology Brightling CE. Profiling of sputum inflammatory mediators in asthma and and Allergy, Medical University of Lodz, Kopcinskiego 22, 90-153 Lodz, chronic obstructive pulmonary disease. Respiration. 2012;83:36–44. Poland. Department of Biomedicine and Genetics, Medical University of 16. Brightling CE, Bleecker ER, Panettieri RA, Bafadhel M, She D, Ward CK, Xu X, Birrell Lodz, Pomorska 251, 92-213 Lodz, Poland. Department of Internal Medicine, C, van der Merwe R, van der Merwe R. Benralizumab for chronic obstructive Asthma and Allergy, Medical University of Lodz, Kopcinskiego 22, 90-153 pulmonary disease and sputum eosinophilia: a randomised, double-blind, Lodz, Poland. placebo-controlled, phase 2a study. Lancet Respir Med. 2014;2:891–901. 17. Siva R, Green RH, Brightling CE, Shelley M, Hargadon B, McKenna S, Received: 5 March 2018 Accepted: 7 May 2018 Monteiro W, Berry M, Parker D, Wardlaw AJ, Pavord ID. Eosinophilic airway inflammation and exacerbations of COPD: a randomised controlled trial. Eur Respir J. 2007;29:906–13. References 18. Xia J, Zhao J, Shang J, Li M, Zeng Z, Zhao J, Wang J, Xu Y, Xie J. Increased 1. Soriano JB, Abajobir AA, Abate KH, Abera SF, Agrawal A, Ahmed MB, IL-33 expression in chronic obstructive pulmonary disease. Am J Physiol Aichour AN, Aichour I, MTE A, Alam K, Alam N, Alkaabi JM, Al-Maskari F, Lung Cell Mol Physiol. 2015;308:L619–27. Alvis-Guzman N, Amberbir A, Amoako YA, Ansha MG, Antó JM, Asayesh H, 19. Qiu C, Li Y, Li M, Li M, Liu X, McSharry C, Xu D. Anti-interleukin-33 inhibits Atey TM, EFGA A, Barac A, Basu S, Bedi N, Bensenor IM, Berhane A, Beyene cigarette smoke-induced lung inflammation in mice. 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Blood eosinophils: a biomarker of response to Extrafine beclomethasone/formoterol in chronic obstructive pulmonary disease. Am J 24. Hacker S, Lambers C, Pollreisz A, Hoetzenecker K, Lichtenauer M, Mangold A, Respir Crit Care Med. 2015;192:523–5. Niederpold T, Hacker A, Lang G, Dworschak M, Vukovich T, Gerner C, Tworek et al. Respiratory Research (2018) 19:108 Page 11 of 11 Klepetko W, Ankersmit HJ. Increased soluble serum markers caspase-cleaved cytokeratin-18, histones, and ST2 indicate apoptotic turnover and chronic immune response in COPD. J Clin Lab Anal. 2009;23:372–9. 25. Cherry WB, Yoon J, Bartemes KR, Iijima K, Kita H. A novel IL-1 family cytokine, IL-33, potently activates human eosinophils. J Allergy Clin Immunol. 2008;121:1484–90. 26. Allakhverdi Z, Smith DE, Comeau MR, Delespesse G. Cutting edge: the ST2 ligand IL-33 potently activates and drives maturation of human mast cells. J Immunol. 2007;179:2051–4. 27. 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Eosinophilic and neutrophilic airway inflammation in the phenotyping of mild-to-moderate asthma and chronic obstructive pulmonary disease. COPD J Chronic Obstr Pulm Dis. 2017;14:181–9. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Respiratory Research Springer Journals

The association between airway eosinophilic inflammation and IL-33 in stable non-atopic COPD

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Abstract

Background: Interleukin(IL)-33 is an epithelial alarmin important for eosinophil maturation, activation and survival. The aim of this study was to examine the association between IL-33, its receptor expression and airway eosinophilic inflammation in non-atopic COPD. Methods: IL-33 concentrations were measured in exhaled breath condensate (EBC) collected from healthy non- smokers, asthmatics and non-atopic COPD subjects using ELISA. Serum and sputum samples were collected from healthy non-smokers, healthy smokers and non-atopic COPD patients. Based on sputum eosinophil count, COPD subjects were divided into subgroups with airway eosinophilic inflammation (sputum eosinophils > 3%) or without (sputum eosinophils ≤3%). IL-33 and soluble form of IL-33 receptor (sST2) protein concentrations were measured in serum and sputum supernatants using ELISA. ST2 mRNA expression was measured in peripheral mononuclear cells and sputum cells by qPCR. Hemopoietic progenitor cells (HPC) expressing ST2 and intracellular IL-5 were enumerated in blood and induced sputum by means of flow cytometry. Results: IL-33 levels in EBC were increased in COPD patients to a similar extent as in asthma and correlated with blood eosinophil count. Furthermore, serum and sputum IL-33 levels were higher in COPD subjects with sputum eosinophilia than in those with a sputum eosinophil count ≤3% (p < 0.001 for both). ST2 mRNA was overexpressed in sputum cells obtained from COPD patients with airway eosinophilic inflammation compared to those without sputum eosinophilia (p < 0.01). Similarly, ST2 + IL-5+ HPC numbers were increased in the sputum of COPD patients with airway eosinophilia (p < 0.001). Conclusions: Our results indicate that IL-33 is involved in the development of eosinophilic airway inflammation in non-atopic COPD patients. Keywords: IL-33, Eosinophils, COPD Background of evidence indicates that eosinophilic COPD is a dis- Chronic Obstructive Pulmonary Disease (COPD) is one tinct phenotype of the disease, and one that is character- the most common chronic diseases in adults, and one of ized by a higher risk of exacerbations but better the main causes of morbidity and mortality [1]. The response to treatment with inhaled corticosteroids [3, 4]. prevalence of COPD at Global Initiative for Chronic COPD subjects have higher percentages of eosinophils Obstructive Lung Disease (GOLD) stage 2 or higher has in sputum and higher concentrations of eosinophil cat- been estimated at 10.1% overall, although the precise fig- ionic protein than healthy controls, and their airway eo- ure varies according to the region [2]. A growing body sinophil numbers further increase during disease exacerbation [5, 6]. Data from the ECLIPSE (Evaluation of COPD Longitudinally to Identify Predictive Surrogate * Correspondence: damian.tworek@umed.lodz.pl End-points) study showed that over three years of obser- Department of General and Oncological Pulmonology, Medical University of Lodz, Kopcinskiego 22, 90-153 Lodz, Poland vation, 37.4% of COPD patients demonstrated a Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Tworek et al. Respiratory Research (2018) 19:108 Page 2 of 11 persistent blood eosinophil count greater than or equal least 10 pack-years) without COPD and 20 healthy to 2% [7]. Non-allergic COPD patients with blood eo- non-smokers were included in the second phase examin- sinophil counts > 250 cells/μL are characterized by ing the association between IL-33 and sputum eosino- higher airway eosinophil numbers, higher sputum inter- philia (Sputum study). COPD was diagnosed according leukin(IL)-5 and haptoglobin levels, greater CCL20 and to GOLD criteria by demonstrating irreversible broncho- CCL24 concentrations in bronchoalveolar lavage (BAL) constriction following inhalation of salbutamol and more pronounced airway remodeling [8]. (www.goldcopd.org). Asthma was diagnosed according IL-33 is one of the epithelial alarmins generated in re- to Global Initiative for Asthma (GINA) criteria based on sponse to danger signals, and is primarily produced by the history of typical respiratory symptoms and positive epithelial cells [9]. Recent evidence has demonstrated result of either bronchodilator reversibility testing or that IL-33 precedes IL-5 in regulating eosinophil com- metacholine challenge (www.ginasthma.org). mitment and is required for eosinophil homeostasis [10]. All participants, except for several asthmatic subjects, One such result of IL-33 activity is that activates and were non-atopic as determined by negative results of augments the maturation of hemopoietic progenitor skin prick testing with common aeroallergens. COPD cells (HPC). HPC are considered to be innate effector patients were treated with bronchodilators only. Subjects cells in allergic asthma [11, 12]: They produce an abun- with a history of infection, or who had used antibiotics dance of pro-inflammatory cytokines and differentiate or systemic/inhaled steroids within the previous four into mature eosinophils in the process of “in situ weeks were excluded. All subjects gave their written hemopoiesis” at sites of inflammation [13]. informed consent, and the study was approved by the The aim of the present study was to determine the Ethics Board of the Medical University of Lodz. The de- presence of an association between IL-33 level, the ex- tailed characteristics of the subjects are presented in pression of its receptor and the degree of eosinophilic Table 1 and Table 2. Patients with COPD were evaluated inflammation in non-atopic COPD. using COPD Assessment Test (CAT), modified Medical Research Council dyspnea scale (mMRC), six-minute Methods walking test (6MWT) and BODE index (see Subjects Additional file 1). Sixteen non-smoking healthy subjects, 23 non-smoking asthmatics and 25 non-atopic COPD subjects (smokers Biological samples collection and measurements and ex-smokers with a history of at least 10 pack-years) The EBC was collected according to the recommenda- were included in the first part of the study assessing the tions of the European Respiratory Society [14]. Sputum association between IL-33 in breath condensate (EBC) samples were induced using hypertonic saline and proc- with blood eosinophilia (EBC study). Forty patients with essed as described previously [15]. The COPD subjects COPD (smokers and ex-smokers with a history of at were then divided into subgroups based on sputum eo- least 10 pack-years), 20 smokers (with a history of at sinophil count: one group with airway eosinophilic Table 1 Characteristics of subjects included in the exhaled breath condensate (EBC) study Healthy Non-smokers Asthma COPD Number of subjects 16 23 25 Sex (M/F) 11/5 11/12 11/14 ll** Age (years) 56.75 ± 2.71 54.22 ± 3.05 66.96 ± 1.76 Pack-years 0 0 39.10±3.70 Time since diagnosis (years) – 10.13 ± 2.34 4.46 ± 0.84 *† PB FEV % of predicted (%) 107.90 ± 3.23 82.59 ± 4.56 62.32 ± 2.76 *† PB FEV /FVC (%) 76.32 ± 1.46 68.14 ± 2.38 54.03 ± 1.89 Current smokers (%) 0 0 32 Use of ICS (%) 0 0 0 Atopic subjects (%) 0 56.52 0 Blood eosinophil number (cells/μl) 181 ± 35 315 ± 35 242 ± 31 Blood eosinophil percentage (%) 3.01 ± 0.54 4.74 ± 0.59 3.16 ± 0.41 PB post bronchodilator, FEV forced expiratory volume in first second, FVC forced vital capacity, ICS inhaled corticosteroids * † ‡ § ll COPD vs Asthma p < 0.001; COPD vs Healthy Non-smokers p < 0.001; COPD vs Asthma p < 0.01; Asthma vs Healthy Non-smokers p < 0.05; COPD vs Healthy ** Non-smokers p < 0.05; COPD vs Asthma p < 0.01 Tworek et al. Respiratory Research (2018) 19:108 Page 3 of 11 Table 2 Characteristics of subjects included in the sputum study Healthy Non-smokers Healthy Smokers COPD Number of subjects 20 20 40 Sex (M/F) 10/10 11/9 22/18 a† Age (years) 56.84±2.14 57.85±2.00 67.40±1.12 Pack-years 0 32.14±2.96 41.99±2.78 Time since diagnosis (years) –– 7.05±1.00 a† PB FEV % of predicted 101.9±2.92 96.88±6.26 61.98±2.76 a† PB FEV %FVC 77.69±1.43 74.39±1.18 52.49±1.57 BMI (kg/m ) 27.23 ± 0.80 27.72 ± 0.68 28.39 ± 1.05 CAT score –– 15.8 ± 1.18 6MWT (m) –– 388.6 ± 11.08 mMRC score –– 1(0–3) BODE index –– 1(0–6) Current smokers (%) 0 100 42.5 Use of ICS (%) 0 0 0 No. of atopic subjects (No) 0 0 0 Sputum eosinophils (%) 0.89 ± 0.28 1.43 ± 0.28 2.93 ± 0.95 Sputum neutrophils (%) 18.89 ± 3.77 23.28 ± 2.23 37.40 ± 4.93 a# Sputum macrophages (%) 65.69 ± 3.76 56.85 ± 1.99 44.70 ± 41 Sputum lymphocytes (%) 6.43 ± 0.84 4.26 ± 0.72 6.60 ± 1.99 Sputum eosinophils (No of cells/ g of sputum × 10 ) 1.60 ± 0.77 1.55 ± 0.73 2.70 ± 1.33 Sputum neutrophils (No of cells/ g of sputum ×10 ) 14.95 ± 3.12 19.40 ± 10.01 19.50 ± 7.36 Sputum macrophages (No of cells/ g of sputum ×10 ) 16.10 ± 2.79 31.00 ± 5.62 41.98 ± 12.55 Sputum lymphocytes (No of cells/ g of sputum ×10 ) 2.60 ± 0.47 2.10 ± 0.45 5.92 ± 3.25 PB post bronchodilator, FEV forced expiratory volume in first second, FVC forced vital capacity, CAT COPD assessment test, 6MWT six-minute walk test, mMRC modified Medical Research Council dyspnea scale, ICS inhaled corticosteroids a † ‡ COPD vs Healthy Non-smokers, p < 0.05; COPD vs Healthy Smokers p < 0.05; median (min-max) inflammation (i.e. sputum eosinophil level > 3%) and the Comparisons between healthy non-smokers, asthmatics other without (i.e. sputum eosinophil level ≤ 3%). The and COPD subjects, as well as between healthy 3% cut-off value for sputum eosinophilia was used based non-smokers, healthy smokers and COPD subjects, were on previous studies on eosinophilic airway inflammation analyzed with one-way ANOVA and the post hoc in COPD [16, 17]. The serum and sputum supernatants Tukey’s test, or with the Kruskal-Wallis test and the post were tested for IL-33 and sST2 concentrations by means hoc Dunn’s test, where appropriate. of enzyme-linked immunosorbent assays, while the EBC Differences between COPD subjects with and without was tested for IL-33 only. eosinophilia were analyzed using the Student’st-testor ST2 mRNA expression in peripheral mononuclear U-Mann Whitney test, where appropriate. Correlation ana- cells (PBMC) and sputum cells was measured using lysis was conducted using either Pearson’s or Spearman’s qPCR. Hemopoietic progenitor cells expressing ST2 and correlation coefficient according to the distribution of vari- intracellular IL-5 were enumerated in blood and induced ables. Significance was accepted at P <0.05. sputum obtained from COPD subjects with and without airway eosinophilia by means of flow cytometry (see gating Results strategy in Additional file 2). Detailed information on the Exhaled breath condensate study study procedures can be found in the Additional file 1. Absolute blood eosinophil number and percentage were elevated in asthmatics compared to healthy non-smokers Statistical analysis (p < 0.05). No significant difference in blood eosinophil The results were analyzed using GraphPad Prism 6 counts was observed between COPD patients, healthy (GraphPad Software, San Diego, CA). For clarity, all re- non-smokers and asthmatic subjects (Table 1). sults are presented as means ± SEMs. The normality of EBC IL-33 levels were significantly higher in asthmatic data distribution was tested with the Shapiro-Wilk test. (3.57 ± 0.81 pg/ml) and COPD (2.50 ± 0.33 pg/ml) Tworek et al. Respiratory Research (2018) 19:108 Page 4 of 11 patients compared to healthy controls (1.27 ± 0.57 pg/ml), Similarly to the serum findings, IL-33 sputum levels with no significant difference observed between the first were significantly higher in COPD subjects with sputum two groups (Fig. 1a). IL-33 levels in EBC correlated signifi- eosinophilia (22.72 ± 7.42 pg/ml) than in those without cantly with blood eosinophil numbers and percentages in (1.13 ± 0.56 pg/ml; p < 0.001) (Fig. 2d). the asthmatic (see Additional file 3) and COPD subjects sST2 was elevated in the serum (p < 0.05) of COPD (Fig. 1b and c). patients compared with the healthy controls (Fig. 3a). A similar trend was observed for sputum sST2 levels Sputum study (p = 0.059) (Fig. 3b). No significant difference was ob- Sputum differential counts are presented in Table 2.It served in serum and sputum sST2 levels in COPD was found that 25% of COPD subjects had sputum eo- subjects with regard to sputum eosinophil counts sinophilia > 3% (mean sputum eosinophil percentage (Fig. 3c and d). 8.89 ± 3.18%). ST2 mRNA was overexpressed in PBMC and sputum IL-33 serum concentrations were significantly elevated in cells from healthy smokers and COPD subjects com- COPD subjects (32.86±1.74 pg/ml) compared with healthy pared with healthy non-smokers (Fig. 4a and b). ST2 non-smokers (22.12±1.45 pg/ml; p < 0.001) and smokers mRNA expression was significantly higher in sputum without COPD (24.45±1.33 pg/ml; p < 0.01) (Fig. 2a). In cells from COPD patients with eosinophil airway inflam- addition, serum IL-33 levels were found to be significantly mation (Relative quantification(RQ) = 1.20 ± 0.85) than higher in the subgroup of COPD patients with sputum eo- in those without airway eosinophilia (RQ = 0.05 ± 0.01; sinophilia (43.98±4.76 pg/ml) compared to those with a p < 0.01) (Fig. 4d). sputum eosinophil level ≤3% (29.15±2.01 pg/ml; p < 0.001) The significant correlations between IL-33, sST2, ST2 (Fig. 2c). mRNA and sputum eosinophil content and clinical pa- Sputum IL-33 levels were significantly increased in rameters in COPD are presented in Fig. 5. All analyzed COPD subjects (6.52 ± 2.37 pg/ml) compared with correlations are presented in Additional file 4. healthy non-smokers (0.0 ± 0.0 pg/ml; p < 0.05) and No significant difference in circulating HPC count was healthy smokers (2.92 ± 2.45 pg/ml; p < 0.05) (Fig. 2b). found between COPD subjects with sputum eosinophilia a * bc 8 8 r=0.48; p<0.05 r=0.44; p<0.05 6 6 4 4 2 2 0 0 0 200 400 600 800 0 5 10 15 Eos [cells/µl] Eos [%] Fig. 1 IL-33 concentrations in exhaled breath condensate (EBC) in healthy non-smokers, asthmatics and non-atopic COPD subjects (a). Data are presented as mean ± SEM. Correlations between EBC IL-33 and blood eosinophil numbers (b) and percentage (c) in COPD patients. *p < 0.05 Healthy Non-Smokers Asthma COPD IL-33 [pg/ml] IL-33 [pg/ml] IL-33 [pg/ml] Tworek et al. Respiratory Research (2018) 19:108 Page 5 of 11 Serum Sputum *** * ab ** *** *** cd 60 40 0 0 Fig. 2 Serum (a)and sputum (b) IL-33 concentrations in healthy non-smokers, healthy smokers and patients with COPD. Comparison of serum (c)and sputum (d) IL-33 between COPD subjects with and without sputum eosinophilia. Data are presented as mean ± SEM. *p < 0.05; **p < 0.01; ***p <0.001 2 2 and those without (422.90 ± 97.65/1 mln PBMC vs (16.83 ± 14.65 × 10 /gram of sputum vs 0.19 ± 0.07 × 10 553.70 ± 138.70/1 mln PBMC, respectively; p = 0.97). /gram of sputum; p < 0.01) (Fig. 6d and e). However, COPD subjects with sputum eosinophilia No difference was observed in circulating ST2 + IL-5+ demonstrated significantly higher numbers of sputum HPC between the two subgroups of COPD patients (see HPC than those without eosinophilia (35.64 ± 27.39 × Additional file 6). However, the absolute numbers of 2 2 10 /gram of sputum vs 1.19 ± 0.34 × 10 /gram of ST2 + IL-5+ HPC, but not their percentage, were signifi- sputum, respectively; p < 0.001) (see Additional file 5). cantly higher in the sputum of eosinophilic COPD pa- Similarly, no differences were found between the two tients than in those with normal COPD (1.58 ± 1.38 × 2 2 groups with regard to percentage or absolute number of 10 /gram of sputum vs 0.009 ± 0.009 × 10 /gram of circulating ST2 + HPC (see Additional file 6). However, sputum; p < 0.001) (Fig. 6g and h). both the percentage of ST2 expression on HPC and the No correlation was found between the percentages absolute number of ST2 + HPC in sputum were higher in (r = 0.24; p = 0.28) or absolute numbers (r =0.30; p=0.38) patients with a sputum eosinophil count > 3% compared to of ST2+ and IL-5+ HPC in sputum. those without (7.80 ± 1.58% vs 2.20 ± 2.22%; p <0.01 and 2 2 2.36 ± 1.83 × 10 /gram of sputum vs 0.018 ± 0.01 × 10 Discussion /gram of sputum; p < 0.001, respectively) (Fig. 6a and b). Our findings show for the first time that increased ex- No differences regarding the percentages and absolute pression of IL-33 and its receptor ST2 is associated with numbers of circulating IL-5 + HPC were observed be- airway eosinophilia in non-atopic COPD patients. tween COPD patients with sputum eosinophilia and IL-33 has been extensively studied in allergic asthma those without (see Additional file 6). In sputum, absolute and other allergic conditions. Some studies suggest that IL-5 + HPC counts, but not percentages, were elevated IL-33 may be also implicated in the pathogenesis of in COPD patients with sputum eosinophilia COPD. IL-33 and sST2 serum levels are elevated in Healthy Non-Smokers e th Smok H al y ers COPD Sputum Eos 3% Healthy Non-Smokers Healthy Smokers Sputum Eos >3% COPD Sputum Eos 3% Sputum Eos >3% Serum IL-33 [pg/ml] Serum IL-33 [pg/ml] Sputum IL-33 [pg/ml] Sputum IL-33 [pg/ml] Tworek et al. Respiratory Research (2018) 19:108 Page 6 of 11 Serum Sputum ab 1500 100 0 0 cd 1500 150 1000 100 500 50 0 0 Fig. 3 Serum (a) and sputum (b) sST2 concentrations in healthy non-smokers, healthy smokers and patients with COPD. Comparison of serum (c) and sputum (d) sST2 between COPD subjects with and without sputum eosinophilia. Data are presented as mean ± SEM. *p < 0.05 COPD patients compared to healthy smokers [18]. To further explore these findings, the second part of Furthermore, immunofluorescence staining showed that our study investigated the expression of IL-33, its recep- the expression of IL-33 was increased in the lung tissues tor and its soluble form in the periphery and the airways of patients with COPD, indicating that IL-33 of patients with and without airway eosinophilia. Our re- upregulation was probably related to systemic and sults showed that the level of IL-33 protein is markedly airway inflammation in COPD [18–20]. It has also been increased in the serum and sputum of patients with eo- reported that cigarette smoke markedly enhanced the sinophilic COPD. expression of IL-33 and ST2 in the lung tissue of mice, sST2 is a decoy receptor regulating the activity of which was accompanied by neutrophil and macrophage IL-33. As sST2 is not detected in EBC, its levels were infiltration and elevated expression of pro-inflammatory not assessed in the EBC obtained from COPD patients cytokines and chemokines in the airways [19]. [21]. However, our findings confirm those of previous Interestingly, Kim et al. found blood eosinophil counts reports showing increased levels of serum sST2 in to be correlated with plasma IL-33 levels in COPD COPD [18]. These findings also suggest a trend toward patients [20]. overexpression of sputum sST2 in the whole COPD Recently, Gluck et al. showed that IL-33 protein is de- group. Animal studies have shown that sST2 may exert tectable at low levels in EBC, and its level is elevated in a protective function in acute lung injury and asthmatics compared with healthy controls [21]. Our re- ovalbumin-induced allergic asthma [22, 23], and Hacker sults demonstrate that IL-33 is elevated in EBC collected et al. suggest that sST2 plays a critical role in the from non-atopic COPD subjects to the same extent as in anti-inflammatory regulatory mechanism in early stages asthma. Furthermore, EBC IL-33 levels correlated posi- of COPD [24]. Although our findings do not indicate tively with blood eosinophil numbers and percentages, significant differences in sST2 concentrations between not only in asthma but also in COPD. COPD subjects with and without sputum eosinophilia, Healthy Non-Smokers Healthy Smokers COPD Healthy Non-Smokers Sputum Eos 3% Healthy Smokers Sputum Eos >3% COPD Sputum Eos 3% Sputum Eos >3% Serum sST2 [pg/ml] Serum sST2 [pg/ml] Sputum sST2 [pg/ml] Sputum sST2 [pg/ml] Tworek et al. Respiratory Research (2018) 19:108 Page 7 of 11 PBMC Sputum ab ** **** 0.25 0.6 *** *** 0.20 0.4 0.15 0.10 0.2 0.05 0.00 0.0 cd ** 0.25 2.5 0.20 2.0 0.15 1.5 0.10 1.0 0.05 0.5 0.00 0.0 Fig. 4 ST2 mRNA expression in peripheral blood mononuclear cells (PBMC) (a) and sputum cells (b) in n healthy non-smokers, healthy smokers and patients with COPD. Comparison of ST2 mRNA expression in PBMC (c) and sputum cells (d) between COPD subjects with and without sputum eosinophilia. Data are presented as mean ± SEM. **p < 0.01; ***p < 0.001; ****p < 0.0001 sputum sST2 levels nevertheless correlated negatively are known to act as pro-inflammatory effector cells of al- with sputum eosinophil counts, suggesting it plays a lergic inflammation [11]. HPC express ST2 and IL-33 as protective role against airway eosinophilic inflammation. potent activators of HPC, leading to the release of a num- On the contrary, sputum cells obtained from COPD ber of cytokines and chemokines, including IL-5. HPC can patients with airway eosinophilia overexpressed ST2 differentiate into mature eosinophils at the site of inflam- mRNA. ST2 is expressed by many types of cells that mation in a process called “in situ eosinophilopoiesis”. may be present in the airways, including eosinophils. IL-33 accelerates the maturation of HPC and modulates The activation of eosinophils by IL-33 through ST2 leads their migration into airways in allergic asthma [26, 27]. to the production of superoxide and IL-8 and increases Little is known of the role of HPC in COPD. Some eosinophil survival [25]. It is important to note that studies have reported reduced numbers of circulating IL-33 activates eosinophils at least to the same degree as HPC in COPD [28], while others report similar circulat- IL-5 [25]. The significant correlations between IL-33 and ing and sputum HPC numbers between COPD and ST2 and sputum eosinophil counts found in our study healthy non-atopic subjects [29]. Our present findings support the hypothesis that IL-33 may be involved in the do not suggest any differences in circulating HPC num- development and maintenance of eosinophilic airway in- bers between COPD patients with sputum eosinophilia flammation in non-atopic COPD subjects. and those without. However, a striking difference was Finally, the HPC count as well as ST2 and intracellular observed in the number of sputum HPC between the IL-5 expression by these cells were measured in the pa- two groups of COPD patients, with significantly elevated tients with sputum eosinophilia and in those without. HPC HPC numbers found in those with sputum eosinophils Healthy Non-Smokers Heal hy Smokers COPD Healthy Non-Smokers Sputum Eos 3% Healthy Smokers Sputum Eos >3% COPD Sputum Eos 3% Sputum Eos >3% PBMC ST2 mRNA expression [RQ] PBMC ST2 mRNA expression [RQ] Sputum ST2 mRNA expression [RQ] Sputum ST2 mRNA expression [RQ] Tworek et al. Respiratory Research (2018) 19:108 Page 8 of 11 Fig. 5 Significant correlations between serum and sputum IL-33 and sST2, ST2 mRNA and clinical parameters in COPD > 3%. This was accompanied by overexpression of intra- from activated cells. Measuring IL-33 protein content is cellular IL-5 and ST2 by sputum HPC indicating in- challenging and previous studies give varying results for creased activation of these cells in eosinophilic COPD, serum and sputum [30, 31]. Nonetheless, our findings on analogously to allergic asthma. ST2 expression confirm the IL-33 measurements and sup- As IL-33 modulates the trafficking of HPC, it is possible port the association between IL-33 and eosinophilic that increased IL-33 levels may be at least partially respon- phenotype of COPD. The best way to determine IL-33 ex- sible for the augmented influx of HPC into airways ob- pression would be to measure it directly in the main served in COPD patients with eosinophilic inflammation. source of the cytokine, i.e. the airway epithelium; however, In addition, increased numbers of ST2 + IL-5 + HPC were studies comparing IL-33 expression in eosinophilic COPD seen in the sputum of patients with airway eosinophilia. involving invasive methods are warranted. In addition, the This finding suggests that IL-33 activates HPC in eosino- results may have been affected by the fact that our group philic COPD. Therefore, in those subjects, HPC may act of COPD subjects was older than those of the other two as effector cells in an analogous way to allergic asthma, by groups. However, as no correlation has been found be- fostering the development of a local IL-5 rich environ- tween IL-33 and ST2 expression and the age of partici- ment independent of the IgE pathway. pant, it is unlikely that this may be the case. There are several limitations to our study. First, the IL-33 protein levels were low in a significant number of Conclusions exhaled breath and sputum specimens. This could be due In conclusion, our results suggest that increased IL-33 is to the rapid neutralization of IL-33 following its release associated with airway eosinophilia in non-atopic COPD. Tworek et al. Respiratory Research (2018) 19:108 Page 9 of 11 a b de f gh i Fig. 6 Expression of ST2 and intracellular IL-5 by sputum hemopoietic progenitor cells (HPC) in COPD subjects with sputum eosinophil count ≤3% (n = 10) and > 3% (n = 10). The percentage of sputum HPC expressing ST2, IL-5 and double positive cells for ST2 and IL-5 are presented in sub-figures a, d and g, respectively. Absolute numbers of HPC expressing ST2, IL-5 and double positive cells for ST2 and IL-5 per gram of sputum are presented in sub-figures b, e and h, respectively. Representative example of flow cytometry acquisition of sputum ST2+ (c), IL-5+ (f) and ST2 + IL-5+ (i) HPC. Data are presented as mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001 It is tempting to speculate that IL-33 is involved in the forced expiratory volume in first second; FVC – forced vital capacity; CAT recruitment and activation of HPC into the airways. This – COPD assessment test; 6MWT – six-minute walk test; mMRC – modified Medical Research Council dyspnea scale. (PDF 39 kb) may result in the creation of a local, IL-5 rich inflamma- Additional file 5: Figure E2. Circulating (A) and sputum (B) tory state similar to that observed in allergic asthma. hemopoietic progenitor cells (HPC) in patients with and without sputum Thus, IL-33 may be a potential therapeutic target in the eosinophilia. **p < 0.01. (PDF 56 kb) subgroup of COPD patients characterized by eosino- Additional file 6: Figure E3. The percentage and absolute numbers of philic inflammation. circulating hemopoietic progenitor cells (HPC) expressing ST2 (A and B, respectively), intracellular IL-5 (C and D, respectively) and double positive for ST2 and IL-5 (E and F, respectively) in COPD patients with (sputum eosinophils Additional files > 3%) and without (sputum eosinophils ≤3%) sputum eosinophilia. (PDF 51 kb) Additional file 1: Detailed Method description. (PDF 118 kb) Additional file 2: Figure E4. Hemopoietic progenitor cells gating strategy. (PDF 88 kb) Abbreviations 6MWT: Six-minute walking test; CAT: COPD Assessment Test; COPD: Chronic Additional file 3: Figure E1. Correlations between IL-33 concentrations Obstructive Pulmonary Disease; EBC: Exhaled breath condensate; in exhaled breath condensate and blood eosinophil numbers (A) and GINA: Global Initiative for Asthma; GOLD: Global Initiative for Chronic percentage (B) in asthmatic patients. (PDF 49 kb) Obstructive Lung Disease; HPC: Hemopoietic progenitor cells; IL: Interleukin; Additional file 4: Table E1. Correlations between serum and sputum mMRC: Modified Medical Research Council; PBMC: Peripheral mononuclear IL-33 and sST2, ST2 mRNA and clinical parameters in COPD. FEV1 – cells; RQ: Relative quantification; sST2: Soluble ST2; ST2: IL-33 receptor Tworek et al. Respiratory Research (2018) 19:108 Page 10 of 11 Acknowledgements 5. Balzano G, Stefanelli F, Iorio C, De Felice A, Melillo EM, Martucci M, Melillo G. The authors would like to thank Dr. Jacek Szymański for his assistance in the Eosinophilic inflammation in stable chronic obstructive pulmonary disease. flow cytometry acquisition. Am J Respir Crit Care Med. 1999;160:1486–92. 6. Saetta M, Di Stefano A, Maestrelli P, Turato G, Ruggieri MP, Roggeri A, Calcagni P, Mapp CE, Ciaccia A, Fabbri LM. Airway eosinophilia in chronic bronchitis Funding during exacerbations. Am J Respir Crit Care Med. 1994;150:1646–52. This work was supported by Medical University of Lodz. 7. Singh D, Kolsum U, Brightling CE, Locantore N, Agusti A, Tal-Singer R. Eosinophilic inflammation in COPD: prevalence and clinical characteristics. Availability of data and materials Eur Respir J. 2014;44:1697–700. The datasets used and/or analysed during the current study are available 8. Kolsum U, Damera G, Pham T-H, Southworth T, Mason S, Karur P, Newbold from the corresponding author on reasonable request. P, Singh D. Pulmonary inflammation in COPD patients with higher blood eosinophil counts. J Allergy Clin Immunol. 2017; https://doi.org/10.1016/j. Authors’ contributions jaci.2017.04.027. DT is the primary author and has made significant contributions to the 9. Mitchell PD, O’Byrne PM. Biologics and the lung: TSLP and other epithelial conception, and the acquisition, analysis, and interpretation of the data; and cell-derived cytokines in asthma. Pharmacol Ther. 2016; https://doi.org/10. drafted, reviewed, and approved the manuscript for publication. SM has 1016/j.pharmthera.2016.06.009. recruited subjects participating in the study; and has reviewed and approved 10. Johnston LK, Hsu C-L, Krier-Burris RA, Chhiba KD, Chien KB, McKenzie A, the manuscript for publication. KS, JK, ZK, assisted with the acquisition of the Berdnikovs S, Bryce PJ. IL-33 precedes IL-5 in regulating eosinophil commitment data; and have reviewed and approved the manuscript for publication. PG, and is required for eosinophil homeostasis. J Immunol. 2016;197:3445–53. EB-L, PK have made significant contributions to the design and interpretation 11. Allakhverdi Z, Comeau MR, Smith DE, Toy D, Endam LM, Desrosiers M, Liu Y- of the data; and have reviewed, revised, and approved the manuscript for J, Howie KJ, Denburg JA, Gauvreau GM, Delespesse G. CD34+ hemopoietic publication. AA has made significant and substantial contributions to the progenitor cells are potent effectors of allergic inflammation. J Allergy Clin conception, design, and interpretation of the data; and has reviewed, revised, Immunol. 2009;123:472–8. and approved the manuscript for publication. 12. Tworek D, Heroux D, O’Byrne SN, Mitchell P, O’Byrne PM, Denburg JA. Toll- like receptor-induced expression of epithelial cytokine receptors on Ethics approval and consent to participate haemopoietic progenitors is altered in allergic asthma. Clin Exp Allergy. All subjects gave their written informed consent, and the study was 2017;47:900–8. approved by the Ethics Board of the Medical University of Lodz. 13. Hui CCK, McNagny KM, Denburg JA, Siracusa MC. In situ hematopoiesis: a regulator of TH2 cytokine-mediated immunity and inflammation at mucosal Competing interests surfaces. Mucosal Immunol. 2015;8:701–11. The authors declare that they have no competing interests. 14. Horváth I, Hunt J, Barnes PJ, Alving K, Antczak A, Baraldi E, Becher G, van Beurden WJC, Corradi M, Dekhuijzen R, Dweik RA, Dwyer T, Effros R, Erzurum S, Gaston B, Gessner C, Greening A, Ho LP, Hohlfeld J, Jöbsis Q, Publisher’sNote Laskowski D, Loukides S, Marlin D, Montuschi P, Olin AC, Redington AE, Springer Nature remains neutral with regard to jurisdictional claims in Reinhold P, van Rensen ELJ, Rubinstein I, et al. Exhaled breath condensate: published maps and institutional affiliations. methodological recommendations and unresolved questions. Eur Respir J. 2005;26:523–48. Author details 15. 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Respiratory ResearchSpringer Journals

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