MCP-4 and Eotaxin-3 Are Novel Biomarkers for Chronic Obstructive Pulmonary Disease

The aim of our study was to examine the production of monocyte chemoattractant protein (MCP-4) and eotaxin-3 during the onset and progression of COPD. The expression levels of MCP-4 and eotaxin-3 were evaluated in COPD samples and healthy controls using immunostaining and ELISA. The relationship between the clinic pathological features in the participants and the expression of MCP-4 and eotaxin-3 were evaluated. The association of MCP-4/eotaxin-3 production in COPD patients was also determined. The results revealed enhanced production of MCP-4 and eotaxin-3 in COPD patients especially the cases with AECOPD in both bronchial biopsies and bronchial washing fluid samples. Furthermore, the expression signatures of MCP-4/eotaxin-3 show high AUC values in distinguishing COPD patients and healthy volunteers and AECOPD and stable COPD cases, respectively. Additionally, the number of MCP-4/eotaxin-3 positive cases was notably increased in AECOPD patients compared to those with stable COPD. Moreover, the expression of MCP-4 and eotaxin-3 was positively correlated in COPD and AECOPD cases. In addition, the levels of MCP-4 and eotaxin-3 could be increased in HBEs stimulated with LPS, which is a risk factor of COPD. Moreover, MCP-4 and eotaxin-3 may exert their regulatory functions in COPD by regulating CCR2, 3, and 5. These data indicated that MCP-4 and eotaxin-3 were potential markers for the clinical course of COPD, which could provide guidance for accurate diagnosis and treatment for this disease in future clinical practice.

Eotaxin-3 could promote local eosinophilic infammation and is the most efcient eotaxin to induce the migration or transmigration of eosinophils [12,13]. However, the expression profle and detailed regulatory function of these cytokines in COPD remain largely unknown.
Te aim of this study was to evaluate the expression levels of MCP-4 and eotaxin-3 in patients with COPD and investigate whether the production of these cytokines was associated with the infammation and development of this disease. Terefore, MCP-4 and eotaxin-3 could be novel markers of the disease progression, and targeted therapy against these cytokines may also be developed for the treatment of COPD patients in future clinical studies.

Clinical Specimens.
A total of 47 COPD patients and 28 healthy volunteers were enrolled in this study between Jan 2012 and Oct 2015. Written informed consent forms were signed by all participants, and our protocol was approved by the Ethics Committee of the First People's Hospital of Chongqing Liang Jiang New Area, China. Airfow limitation was evaluated using spirometry and defned as postbronchodilator FEV1/FVC < 0.70, and COPD was classifed according to Global Initiative for Chronic Obstructive Lung Disease criteria [15]. Dyspnoea, cough, or sputum production was observed in all patients. Additionally, patients diagnosed with respiratory disorders other than COPD, such as asthma or bronchiectasis, were excluded in this study. All participants had stopped smoking for more than 2 months prior to this study. Patients with conditions known to be associated with increased cytokines in bronchial washing fuid, such as ALI/ARDS1 [16], were also excluded. Te production of MCP-4 and eotaxin-3 was evaluated in all participants.

Criteria of Acute Exacerbation.
Acute exacerbation was confrmed according to the existence of any two essential symptoms (dyspnoea, sputum purulence, or sputum quantity), or one major as well as one minor symptom (wheezing, sore throat, cough, or nasal congestion/discharge) for at least two days. Acute exacerbation was defned as deterioration in respiratory symptoms that requires antibiotics and/or systemic corticosteroid treatment, which are not required for the treatment of stable COPD.

Collection of Bronchial Biopsies and Bronchial Washing.
Bronchoscopies were carried out in accordance with the relevant guidelines [17]. Bronchoscopy was performed on stable COPD patients following blood test and lung function test after 12 h fasting. Bronchoscopy was performed on AECOPD patients one day following admission before corticosteroids were given. Only moderate/mild exacerbation patients were included in the study. Patients did not receive any other medication before bronchoscopy. Te upper airways were anaesthetized using lignocaine (Xylocaine ® ; Sweden), and then fberoptic bronchoscopy (Olympus, Japan) and bronchial washing were performed nasally by wedging the bronchoscope into the segmental bronchus of the middle lobe. A total of 20 ml of 0.9% of sterile NaCl was injected and immediately aspirated. In order to remove any mucus and big cell debris, 5 ml of bronchial washing fuid was centrifuged at 400g for 10 min and 1500g for 30 min. Small cell debris was removed through ultracentrifugation at 12,000g for 2 min. Subsequently, the supernatant was collected and aliquoted. Te samples were stored at −80°C until further use.

Immunohistochemistry Analysis.
Immunostaining of MCP-4 and eotaxin-3 was performed on parafn-embedded sections. Te bronchial biopsies were dewaxed using xylene and rehydrated through a graded ethanol series and water. Antigen retrieval was carried out using microwave treatment in 10 mM sodium citrate for 15 min. Tissues were then incubated with FCS at room temperature for 30 min and with corresponding primary antibody against MCP-4 (1 : 100; cat. No. SC-271124, Santa Cruz Biotechnology) or eotaxin-3 (1 : 200; cat. No. MA5-23858, Termo Fisher Scientifc) in a humid chamber at 4°C overnight. Te following day, the sections were washed using PBS and incubated with biotin labeled secondary antibody (1 : 50; Dako, UK). Subsequently, antigen was visualized by streptavidinbiotin-peroxidase system (ABC kit; Dako, UK). Quantifcation of staining intensity was performed using ImageJ software (version 1.46; NIH, Bethesda, MD, USA). Te stained biopsies were visualized under microscope and digital images were captured with fxed settings. In brief, by comparing to the control group (secondary antibody alone), threshold of positive staining was defned using the eyedropper tool of ImageJ. Color signal above the threshold was considered as positive staining, and vice versa. For automated counting, the optical counts of positive staining were determined using the defned threshold. Te staining results were presented as mean density (integrated optical density/ area). Staining intensities in normal vs. COPD or stable COPD vs. AECOPD samples were analyzed using Student's t-test. Te diferences were considered statistically signifcant at p < 0.05.

Cell Culture and Treatments.
Human bronchial epithelial cells (HBEs) were purchased from ATCC (Manassas, VA). Cells were cultured with RPMI-1640 medium (Life Technologies) supplemented with penicillin and streptomycin in a humid incubator at 37°C supplied with 5% CO 2 . When cells were ∼80% confuent, they were they were seeded onto a six-well plate at the density of 5 × 10 5 cells/well. Cells were treated with LPS (Sigma-Aldrich, St. Louis, MO), and nontreated cells were used as control. For the dose-efect study, cells were treated with LPS at indicated concentrations (0, 0.01, 0.1, and 1.0 µg/mL) and incubated for 24 h. For the time-efect study, LPS (0.1 µg/mL) was added to the culture supernatant and cells were collected after 0, 2, 6, and 24 h. Bonferroni correction was used for multiple comparison. Ten, culture supernatants were collected and stored at −20°C for ELISA, and cell pellets were stored at −80°C for mRNA extraction.

Measurement of Cytokine Production.
Te levels of MCP-4 and eotaxin-3 were determined in bronchial washing fuid samples/culture supernatants. In brief, the samples were centrifuged at 1500 rpm at 4°C for 5 min. Expression of MCP-4 and eotaxin-3 was measured using the abovementioned antibodies (1 : 50 for MCP-4 and 1 : 100 for eotaxin-3). Serial dilutions of recombinant standards (MCP-4: cat. No. 327-P4; eotaxin-3: cat. No. 653-E3, R&D Systems) and samples were conducted according to the protocol of manufacturers. Te standard curves were generated using the recombinant standards. Te levels of cytokines were quantifed using the serial dilutions of recombinant falling within the linear range.

Statistical
Analysis. Data were presented as mean ± SEM based on at least three independent experiments. Diference in distribution of the two groups was analyzed using the χ 2 test. Comparisons of diference were performed using Student's t-test or Mann-Whitney U test using GraphPad Prism Software (version 7; San Diego, CA).
Receiver operating characteristic (ROC) curve analysis was used to evaluate the power of MCP-4/eotaxin-3 expression signature in distinguishing COPD from healthy or AECOPD from stable COPD cases. To examine linear correlations between two variables, Pearson's correlation test was conducted when appropriate. p < 0.05 was considered to indicate a statistically signifcant diference.

Expression Levels of MCP-4 and Eotaxin-3 Were Increased in COPD Patients.
Te efects of infammatory cytokines on chronic infammatory airway diseases such as COPD have been emphasized recently [18][19][20]. Te numbers of T lymphocytes and production of proinfammatory cytokines are increased in airway during the development of COPD [5]. Although previous reports have elucidated the roles of MCP-4 and eotaxin-3 in COPD [21][22][23], the detailed mechanisms remained largely unknown. Terefore, we hypothesized that the production of infammatory cytokines MCP-4 and eotaxin-3 is associated with the progression of COPD. In order to investigate this, bronchial biopsies and bronchial washing fuid samples from COPD patients and healthy donors were collected, and the expression levels of MCP-4 and eotaxin-3 were determined.
Te characteristics of COPD patients and healthy volunteers are presented in Table 1. No signifcant diference in gender was observed between healthy volunteers and COPD patients. However, average age was remarkably increased in COPD group (p < 0.05). Body mass index (BMI) of all participants was within normal range, while that of COPD patients was relatively lower, but the diference was not statistically signifcant. Fourteen out of 47 COPD patients had no history of smoking, and their medical histories were reviewed. All fourteen patients were passive smokers. In addition, years of smoking and quitting time were remarkably increased in COPD patients (p < 0.05). In the results of immunostaining on bronchial biopsies, COPD patients exhibited signifcantly elevated production of MCP-4 and eotaxin-3 (Figures 1(a) and 1(b)). Furthermore, the levels of MCP-4 and eotaxin-3 were measured in bronchial washing fuid collected from COPD and control groups.
Consistent with the fndings of immunostaining, the results of ELISA suggested that production of MCP-4 and eotaxin-3 was remarkably enhanced in bronchial washing fuid samples from COPD patients (Figures 1(c) and 1(d)). ROC curve analyses indicated that MCP-4/eotaxin-3 expression signature exhibited high AUC value in distinguishing COPD patients and healthy controls, with sensitivity of 79.22/ 82.64% and specifcity of 71.29/69.43%, respectively (Figures 1(e) and 1(f )).

Production of MCP-4 and Eotaxin-3 Was Upregulated in
Patients with AECOPD Compared to Stable COPD. To study whether the production of MCP-4 and eotaxin-3 is correlated with the development of COPD, the production of these two cytokines was evaluated in bronchial biopsies and bronchial washing fuid samples from patients with AECOPD (n � 35) and those with stable COPD (n � 12). Tirty exacerbations were bacterial-associated and fve were virus-related. A total of thirty patients were treated with antibiotics, and ten were treated with systemic corticosteroids. Twenty-two patients were supplied with oxygen during acute exacerbation. Duration of acute exacerbations ranged from 3 to 15 days. Te clinical features of the participants are summarized in Table 2. Tere was no signifcant diference in gender between AECOPD and stable COPD groups. In addition, average age showed no diference in patients with AECOPD compared with stable COPD group. BMI of all participants was within normal range, and no signifcant diference was observed between the two experimental groups. Years of smoking exhibited no diference between AECOPD and stable COPD groups. Furthermore, there was no diference in respect to WBC, RBC, percentages of lymphocytes/neutrophils/eosinophils, and ESR, but CRP/ IL-6 levels were remarkably increased in patients with AECOPD (p < 0.05). Te data of immunostaining revealed elevated levels of MCP-4 and eotaxin-3 in AECOPD patients compared to those with stable COPD (Figures 2(a) and 2(b)). In addition, the results of ELISA were in agreement with these fndings, as AECOPD patients exhibited increased production of MCP-4 and eotaxin-3 compared to stable COPD group (Figures 2(c) and 2(d)). Furthermore, MCP-4/eotaxin-3 expression signature also showed AUC values of 0.6182/0.6002 in difering AECOPD from stable COPD patients, with sensitivity of 38.26/39.33% and specifcity of 76.42/78.66% (Figures 2(e) and 2(f )).

Association of MCP-4 and Eotaxin-3 Levels with Clinicopathological Features of COPD Patients.
Te correlations between MCP-4 and eotaxin-3 production and the characteristics of patients with COPD were analyzed, and the results are presented in Table 3. No signifcant diference in age, gender, and smoking history was observed between MCP-4/eotaxin-3 positive and negative groups. In addition, the number of MCP-4/eotaxin-3 positive cases was significantly increased in AECOPD compared to stable COPD group (p < 0.05). Moreover, positive correlation between MCP-4 and eotaxin-3 production was revealed in the bronchial washing fuid samples of COPD patients, and the association was also observed in AECOPD cases (Figure 3; r � 0.679, p < 0.05; r � 0.625, p < 0.05), suggesting the correlation between MCP-4 and eotaxin-3 levels in the airway of COPD patients.

LPS Stimulated the Production of MCP-4 and Eotaxin-3 in
HBEs. In order to confrm that MCP-4 and eotaxin-3 are putative biomarkers for COPD, further function studies were performed on HBEs. Te protein levels of MCP-4 and eotaxin-3 in the culture medium of HBEs were increased following the treatment with LPS (0.01−1 µg/mL) for 24 h in a dose-dependent manner (Figures 4(a) and 4(b)). In addition, LPS-stimulated production of MCP-4 and eotaxin-3 in HBEs also exhibited a time-dependent manner (Figures 4(c) and 4(d)). Consistent with these fndings, the mRNA levels of MCP-4 and eotaxin-3 were also elevated in HBEs stimulated with LPS (Figures 5(a) and 5(b)). In further doseand time-efect studies, LPS-induced increase of MCP-4 and eotaxin-3 mRNAs also exhibited dose-and time-dependent manner (Figures 5(c)-5(f )).

Discussion
COPD is a type of infammatory disorder in lung, and the infammatory process is characterized with aberrant activation of both innate and adaptive immune responses in airway tracts [1][2][3]. Te essential roles of cytokines including MCP-4 and eotaxin-3 in the pathogenesis of COPD have been previously revealed [4,[6][7][8]; however, the detailed function and underlying mechanisms are not completely understood. MCP-4 is involved in the recruitment of eosinophils, monocytes, and T-cells in mucosal infammatory disorders such as asthma [23]. Enhanced production of MCP-4 has been detected in asthma samples especially in those with acute asthma exacerbations [23]. Eotaxin-3 is one of the eosinophil chemotactic factors released by bronchial and infammatory cells, and it is involved in the recruitment and activation of eosinophils [13,24,25]. Recent study has   revealed increased mRNA levels of eotaxin-3 in airway epithelial brushings of asthmatic patients [26]. Tis study was conducted to evaluate the expression levels of MCP-4 and eotaxin-3 in COPD patients and healthy controls. Te association between the expression signatures of MCP-4/ eotaxin-3 and clinicopathological features of patients was also investigated. Our data indicated that the production of MCP-4 and eotaxin-3 was promoted in both bronchial biopsies and bronchial washing fuid samples obtained from COPD patients especially from those with AECOPD. Te results suggested that the levels of MCP-4 and eotaxin-3 were signifcantly upregulated in the airway of AECOPD patients, indicating that the production of these two cytokines might predict the progression of COPD. In summary, upregulated MCP-4 and eotaxin-3 expression could be novel biomarkers for the development of COPD. Consistent with our fndings, previous reports also revealed increased production of systemic MCP-4 in the serum samples from COPD patients [11,14]. Similarly, the expression levels of eotaxin-3 were elevated in respiratory infammatory diseases such as patients with asthma and AECOPD [12,13,27]. Furthermore, ROC curve analyses also suggested that MCP-4/eotaxin-3 expression signature exhibited high AUC value in distinguishing COPD patients and healthy controls. Additionally, the number of MCP-4/eotaxin-3 positive cases was remarkably elevated in AECOPD patients compared to those with stable COPD. Furthermore, the expression levels of MCP-4 and eotaxin-3 were positively correlated in both COPD and AECOPD groups. In addition, the production of MCP-4 and eotaxin-3 in HBEs could be stimulated by LPS, which is a key risk factor for COPD. Moreover, MCP-4 and eotaxin-3 may exert their regulatory functions in COPD through CCR2, 3, and 5. However, there were some limitations in this study. For example, the sample size was limited and large-scale study is required in future to confrm       these fndings. Additionally, previous study indicated that age might afect the production of cytokines/chemokines [28], which was not observed in this study. Te correlation between age and MCP-4/eotaxin-3 production should be explored in future study using increased sample size and broadened age range. In summary, our results suggested that the production of MCP-4 and eotaxin-3 could be associated with the development of COPD, and these cytokines could be putative indicator of the onset and progression of COPD. Tese fndings may provide evidence to guide the diagnosis/ treatment of this disease in future clinical practice.

Data Availability
Te datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Ethical Approval
Our protocol was approved by the Ethics Committee of the First People's Hospital of Chongqing Liang Jiang New Area.

Consent
Written informed consent was obtained from patients.

Conflicts of Interest
Te authors declare that they have no conficts of interest.

Authors' Contributions
TL initiated this study. FC, KB, ZP, RJ, WL, ZL, and LH conducted the experiments and data analyses. All authors drafted the manuscript and approved the fnal version.