Association between serum CCL-18 and IL-23 concentrations and disease progression of chronic obstructive pulmonary disease

This study aimed to investigate the association between serum concentrations of chemokine (C–C Motif) ligand 18 (CCL-18) and interleukin 23 (IL-23) and clinical parameters of chronic obstructive pulmonary disease (COPD). The serum concentrations of CCL-18 and IL-23 were tested by enzyme linked immunosorbent assay (ELISA). The association between their concentrations and clinical parameters of COPD patients were analyzed by linear regression, logistic regression and ROC curve. The results showed that the serum concentrations of CCL-18 and IL-23 in COPD patients were increased compared with healthy people (P < 0.001) and that patients with acute exacerbation of COPD (AECOPD) had higher serum CCL-18 and IL-23 concentrations than stable patients (P < 0.001). Synergistic increase of CCL-18 and IL-23 in COPD patients was positively correlated with COPD patients' higher GOLD grade (P < 0.001), higher mMRC score (P < 0.001) and longer medical history (P < 0.001), but negatively correlated with the forced expiratory volume in one second (FEV1)/forced vital capacity (FVC) (P < 0.001) and FEV1% predicted (P < 0.001). The serum concentrations of CCL-18 and IL-23 were most related to the GOLD grade (OR = 2.764 for CCL-18 and OR = 4.215 for IL-23) and detection of both showed considerable sensitivity (72.57% for CCL-18 and 76.92% for IL-23) and specificity (92.50% for CCL-18 and 77.5% for IL-23) in identifying COPD. Increased serum concentrations of CCL-18 and IL-23 correlated with the disease progression of COPD and they could be used as biomarkers for disease evaluation of COPD.

Sensitivity and specificity of serum CCL-18 and IL-23 to distinguish COPD from healthy people. The ROC analysis showed that a serum CCL-18 concentration of 168.3 ng/mL could be used as a cutoff value to distinguish COPD from healthy people (Supp.  Figure 2A). In addition, a serum IL-23 concentration of 563.3 ng/L (Supp. Table 4) was found to be a cutoff value to distinguish COPD from healthy people (Supp.  Figure 2B). Comparing the CCL-18 and IL-23, the sensitivity and specificity of the two to identify COPD did not show a significant differences (difference between areas = 0.0393, Z = 1.341, Figure 2C).  Table 6).

Discussion
It is generally believed that COPD is characterized by chronic inflammation of the airways, lung parenchyma, and pulmonary blood vessels. Alveolar macrophages, T lymphocytes (especially CD) and neutrophils are increased in different parts of the lung 3,12,13 . It has been found that the activation of various inflammatory mediators and cytokines runs through the entire chronic inflammatory process, including leukotriene B4 (LTB4), IL-8, tumor necrosis factor-α (TNF-α), and other mediators 3,9,[12][13][14][15] . In this study, we tested the changes in serum CCL18 and IL-23 concentrations in patients with COPD and found that the serum CCL-18 and IL-23 concentrations in COPD patients were higher than those in healthy people, which means that these two proteins are related to the occurrence and development of COPD. In addition, we found that their concentrations in AECOPD were significantly higher than that in stable COPD, indicating that they can respond quickly to the disease progression of COPD. Studies have shown that CCL-18 can induce the aggregation of inflammatory cells, participate in the activation of many inflammatory signaling pathways, and thus involve in the enlargement and chronicity of inflammation [16][17][18] . IL-23 has been reported to be an important chaperone in the proliferation and stability of Th17 cells, and it promotes the production of IL-17A, IL-17F, IL-22, thus involves in the development of inflammatory diseases 10,11 . IL-23 mediates lung inflammation and emphysema formation through the IL-23/IL-17 pathway 19 . A potential mechanism by which erythromycin reduces airway inflammation in patients with COPD may involve inhibition of IL-17/IL-23-mediated signaling pathways 20 . The grade of COPD severity (GOLD grading) is based on two clinical indices, FEV1/FVC and FEV1% predicted, which are sensitive indicators for evaluating airflow limitation 21,22 . The GOLD recommends the mMRC Dyspnea Scale as a tool for respiratory function assessment 23 . In our study, we found that serum concentrations of CCL-18 and IL-23 were positively correlated with COPD patients' higher GOLD grade and higher mMRC score, which means that the increase of the two is related to the deterioration of COPD. The persistently high levels of CCL-18 in the lung can upregulate the expressions of TNF-α, INF-γ, matrix metalloproteinase (MMP)-2 and MMP-9, thereby aggravating the pulmonary inflammation response 24 . Increased CCL-18 in COPD is significantly correlated with the proportion of FEV1 decline and the risk of future acute exacerbations 17,18 . Interestingly, we found a positive linear association between the serum CCL-18 and IL-23 concentrations in patients with COPD, which suggests that they may exert a synergistic effect in the progression of COPD. We also noticed that the serum CCL-18 and IL-23 concentrations were negatively correlated with the FEV1/FVC and FEV1% predicted in COPD patients, indicating that the inflammatory response mediated by CCL-18 and IL-23 is closely related to the degree of lung function damage in COPD patients. A previous study shows that the CCL-18 is highly expressed in the lungs, and exerts the chemotactic effects on lymphocytes, but has also been shown to stimulate fibrinogen activity and collagen production in lung fibroblasts 18 . Respiratory airflow limitation in COPD patients is associated with increased mucus secretion in the airways, and excessive mucus secretion is associated with decreased FEV1. IL-23 can stimulate bronchial epithelial goblet cells and submucosal glands to increase mucus secretion [9][10][11] , which has been shown to be facilitated by elastase-induced pulmonary inflammation and emphysema formation mediated by the IL-23/IL-17 pathway 7-11,19 . www.nature.com/scientificreports/ www.nature.com/scientificreports/ The ROC curve analysis in our study showed that CCL-18 (sensitivity of 72.57% and specificity of 92.50%) and IL-23 (sensitivity of 76.92% and specificity of 77.50%) could be used to distinguish COPD from healthy people. The Logistic Regression analysis in our study showed that the GOLD grade (OR = 2.764) and clinical medical history (OR = 7.358) were primarily responsible for the increase in CCL-18 concentrations and that the GOLD grade (OR = 4.215) affected the serum concentrations of IL-23. Our findings indicate that CCL-18 and IL-23 participate in the inflammatory process of COPD and are correlated with irreversible airway inflammation and airflow restriction, so they may be used as inflammatory markers for disease evaluation and efficacy evaluation of COPD. We speculate that there may be a vicious cycle of positive feedback regulation. The deterioration of COPD disease promotes the release of pro-inflammatory factors such as CCL-18 and IL-23 in the body, leading to accumulation of macrophages and neutrophils in the lung, further aggravating COPD chronic inflammation of the airway, vice versa 7,11,[16][17][18][19] . Our findings will provide some references for further explanation of the etiology and pathogenesis of COPD.
The shortcomings of this study may have the following points. These patients included in the study may not reflect the characteristics of different geographical conditions and ethnicities. This study is only a nonintervention observational study and did not perform testing for changes on CCL-18 and IL-23 in patients with COPD after any treatment. The study did not focus on the molecular mechanisms of CCL-18 and IL-23 changes in COPD. The subjects of this study are mainly stable COPD, and AECOPD is only a small part of it. The study would have been more robust had it considered "stable COPD" and "AECOPD" as two separate groups. In future research, we should pay attention to the above topics. The conclusions of this study suggest that CCL-18 and IL-23 are involved in the occurrence and development of COPD, and have certain clinical value in assessing the severity of the disease and judging the prognosis of patients. They are expected to become biomarkers of COPD, and interventions for them are expected to become new therapeutic targets for COPD.

Conclusion
COPD patients had higher serum CCL-18 and IL-23 concentrations than healthy subjects, and the patients with AECOPD showed higher concentrations than those with stable patients. Increased CCL-18 and IL-23 were positively correlated with COPD patients' higher GOLD grade, higher mMRC score, but negatively related to the FEV1/FVC and FEV1% predicted. The testing of serum concentrations for CCL-18 and IL-23 contributed to diagnosis and evaluation of COPD, and their concentrations were most affected by the GOLD grading.

Methods
Ethics statement. This study was a retrospective observational study and did not involve any form of therapeutic intervention. Written informed consent was obtained from all patients recruited into the study. Xi'an Medical University, Xi'an, China) were included in this observational study. The order in which patients and healthy controls were enrolled in the group was determined based on their visit time to each hospital. There were 92 cases in stable COPD group and 21 cases in AECOPD group, including 61 males and 52 females, with an average age of (68.85 ± 12.4) years. The 80 healthy controls were from the outpatient medical examination department, including 35 males and 45 females with an average age of (69.43 ± 11.7) years. There were no significant differences in demographic characteristics such as gender, age, and smoking between the two groups (p > 0.05) ( Table 1).

Pulmonary function test. All stable COPD patients and healthy controls received lung function test
before collecting blood samples. AECOPD patients were unable to cooperate with this test because of severity of the disease, the test was not required mandatorily (the reports of previous pulmonary function testing are needed to confirm the diagnosis of COPD). Pulmonary function test was performed using a pulmonary function meter, the forced expiratory volume in one second (FEV1)/forced vital capacity (FVC) and FEV1% predicted were recorded. The test was performed three times at different times; an average value was taken for statistical analysis 25 .
Inclusion and exclusion criteria for COPD patients. Inclusion criteria are as follows: (1) met diagnostic criteria for COPD 22,25 ; (2) had a pulmonary function test report within 3 days (AECOPD patients did not make this requirement); (3) stable COPD patients had a stable period of more than 2 weeks; and (4) no treatment with oxygen, antibiotics, glucocorticoids, and theophylline within the last 1 month. Exclusion criteria are as follows: (1) treated with immunosuppressive drugs in the past month; (2) other airflow-limited diseases, including bronchiectasis, tuberculosis, lung cystic fibrosis and tumors and so on; (3) with severe heart, brain, liver, kidney, hematopoietic, endocrine system and skin diseases; (4) with mental illness, mental disorder, and cognitive impairment; (5) except for AECOPD, patients with stable COPD must not have respiratory infections, and (6) infectious diseases other than the respiratory system.
Specimen preparation. Early morning fasting, 5 mL of venous blood without anticoagulants was collected from the elbow vein of the subjects, which was stored in a centrifuge tube and left to coagulate at room temperature. The coagulated blood was equilibrated and centrifuged (3000 rpm, 5 to 10 min) to obtain supernatants. The supernatants were carefully sucked out of the centrifuge tube and put into new EP tubes, and stored in a − 20 °C refrigerator.
Enzyme-linked immunosorbent assay (ELISA). The concentrations of CCL-18 (Jinma Biotechnology Co., Ltd., Shanghai, China) and IL-23 (Fangchen Biotechnology Co., Ltd., Beijing, China) in serum were measured by sandwich-type ELISA. Briefly, the microtiter plates were coated with purified CCL-18 and IL-23 Table 1. Demographic characteristics of included populations (COPD = 113; Control = 80). BMI body mass index, mMRC modified british medical research council for dyspnea scale for symptom classification of COPD, COPD chronic obstructive pulmonary disease, GOLD global initiative for chronic obstructive lung disease. a "Yes" in the smoking category refers to current smokers; "No" refers to those who have never smoked or have quit smoking for more than 3 months. GOLD grade: 1 = the forced expiratory volume in one second (FEV1) % predicted is more or equal to 80%, 2 = the FEV1% predicted is more or equal to 50%, but less than 80%, 3 = the FEV1% predicted is more or equal to 30%, but less than 50%, and 4 = the FEV1% predicted is less than 30%. b "Medical history" refers to the time span (years) from the diagnosis of COPD to the patient being enrolled in this study. www.nature.com/scientificreports/ antibodies to make solid-phase antibodies. The smples were sequentially added to the microwells coated with the monoclonal antibody, and combined with HRP-labeled CCL-18 and IL-23 antibodies to form an antibodyantigen-enzyme-labeled antibody complex. After thorough washing, the substrate TMB was used to develop the color. TMB converted to blue under the catalysis of HRP enzyme, and turned into the final yellow under the action of acid. The absorbance (OD value) was measured with a microplate reader at a wavelength of 450 nm, and the concentrations of CCL-18 and IL-23 in the sample were calculated by a standard curve. Intra-group variation analysis: the same sample was tested for 3 times on the same microplate; between-group variation analysis: the same sample was tested repeatedly three times at different times; and data analysis showed that the variation did not affect the detection results.
Modified british medical research council (mMRC) score. Dyspnea of patients was evaluated by the modified british medical research council (mMRC) scale 23 . Grade 0, I only get breathless with strenuous exercise; Grade 1, I get short of breath when hurrying on level ground or walking up a slight hill; Grade 2, on level ground, I walk slower than people of the same age because of breathlessness, or I have to stop for breath when walking at my own pace on the level; Grade 3, I stop for breath after walking about 100 yards or after a few minutes on level ground; Grade 4, I am too breathless to leave the house or I am breathless when dressing.
Statistics of data. The statistical ideas applied are as follows: (1) the statistical software used in the analysis was the SPSS 20.0; (2) the measurement data are expressed by mean ± standard deviation (M ± SD) and the comparison of the two sample means was analyzed by t test; (3) before the analysis, the Levene' s Test for Equality of Variances test for the data was performed; Student's t test was used to analyze normally distributed data with unknown population standard deviation σ, and infer the probability of the difference through t distribution theory; the results of t test was taken for uniform variance and the corrected result of t test was taken for uneven variance; (4) the percentage was used to represent the count data, and the chi-square test was used to compare the two sample rates; (5) Pearson Correlation Test was used to analyze the relationship between the indicators; (6) receiver operating characteristic curve (ROC) analysis was used to determine the cutoff value, sensitivity, and specificity of observations; the ROC curve analysis was applied in our study design according to The Standards for Reporting of Diagnostic Accuracy (STARD) Initiative guidelines 26 ; and (7) P < 0.05 was considered statistically significant.

Data availability
The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.