Correlation between serum IL-32 concentration and clinical parameters of stable COPD: a retrospective clinical analysis

This study was to investigate the association between serum interleukin 32 (IL-32) concentration and clinical parameters in patients with stable chronic obstructive pulmonary disease (COPD). One hundred and sixteen patients with stable COPD and 70 healthy subjects were included in the study. The serum concentration of IL-32 was detected by enzyme-linked immunosorbent assay. The correlation between serum IL-32 and clinical parameters of patients with COPD was analyzed by T-test, one-way analysis of variance, multiple linear regression and receiver operating characteristic curve. The serum concentration of IL-32 in patients with stable COPD was higher than that in healthy control group (p < 0.001) and increased serum IL-32 was positively correlated with GOLD grading (p = 0.026), mMRC score (p = 0.004) and clinical medical history (p = 0.005), but negatively related to FEV1/FVC (p = 0.001) and FEV1% predicted (p = 0.001). Patient's COPD grading (p = 0.001), clinical medical history (p < 0.001) and FEV1/FVC (p = 0.001) exerted a significant impact on serum IL-32. The sensitivity and specificity of serum IL-32 for discerning COPD patients from healthy individuals were 85.34% and 64.29%, and the area under the curve was 0.808 (p < 0.001). Increased IL-32 is involved in the chronic disease progression of COPD, suggesting that IL-32 may be a molecular biomarker that reflects the severity of COPD and contributes to the disease diagnosis.

Serum IL-32 concentration in patients with stable COPD positively correlates with GOLD grade, mMRC score and clinical medical history. The results of the T test showed that serum IL-32 concentration in patients with stable COPD was not associated with gender, smoking and BMI (p > 0.05) ( Table 1). The data variance test for the correlation between serum IL-32 and GOLD grade, mMRC score and clinical medical history of patients showed that the overall variance of each group was equal (F = 3.295 and p = 0.073 for GOLD grade; F = 2.169 and p = 0.120 for mMRC score; F = 0.922 and p = 0.401 for clinical medical history) ( Table 1). The statistical results showed that serum IL-32 concentration in stable COPD patients with GOLD-3 to 4 (186.5 ± 50.4 pg/mL), mMRC score of 4 (189.8 ± 48.2 pg/mL) and longer clinical medical history (187.6 ± 45.8 pg/mL for ≧ 20 years; 169.9 ± 51.1 pg/mL for history ≧ 10 < 20 years) was higher than that in patients with GOLD-1 to 2 (149.6 ± 52.3 pg/mL) (p = 0.026; Fig. 1B), mMRC score of 2 (154.1 ± 55.3 pg/mL) and 3 (155.3 ± 52.8 pg/mL) (p = 0.004; Fig. 1C) and shorter medical history (147.7 ± 48.9 pg/mL for history < 10 years) (p = 0.005; Fig. 1D).
Serum IL-32 concentration in patients with stable COPD negatively correlates with pulmonary function. The Table 3). The influence of FEV1/FVC (partial regression analysis = − 110.15) on IL-32 was greater than that of COPD grading (partial regression analysis = 16.62) and clinical medical history (partial regression analysis = 10.53).
Diagnostic efficacy test of serum IL-32 in distinguishing patients with COPD from healthy individuals. The ROC analysis suggested that the threshold value of serum IL-32 concentration for distinguishing patients with COPD from healthy individuals was 105 pg/mL ( Fig. 4A; Table 4), the value showed a sensitivity of 85.34% and a specificity of 64.29% (Fig. 4B). Taking the value as a critical point, the area under the curve (AUC) was 0.808, the standard error was 0.0315, and the 95% confidence interval was 0.746-0.862 (Z = 9.77; p < 0.001) (Fig. 4C).

Discussion
Interleukin-32 (IL-32) is originally cloned from natural killer cells by interleukin-18 (IL-18) induction, it was later discovered that its biological role is similar to cytokines and plays a key role in chronic inflammation 8,10-12 . Previous studies show that IL-32 can induce immune cells to produce a variety of other cytokines and chemokines, involving in the specific immune response and inflammatory response of COPD 8,10,12,13 . We investigated the concentration of serum IL-32 in stable COPD patients and found that the serum concentration of IL-32 in patients with stable COPD was higher than that in healthy individuals, indicating that there was a correlation between increased serum concentration of IL-32 and COPD. Previous studies show that IL-32 can activate the phosphorylation of the nuclear transcription factor kappa B (NF-κB) and p38 mitogen-activated protein kinase (p38MAPK), inducing the production of inflammatory factors such as tumor necrosis factor-α (TNF-α), IL-1β, IL-6 and IL-8, these cytokines play an important role in the pathogenesis of COPD 10,14 .
The GOLD score of COPD patients can reflect objective severity of the disease (the higher the score, the more serious the disease). Our study showed that there was a strong positive correlation between serum IL-32 concentration and GOLD score, which suggested that IL-32 might be a molecular biomarker that reflects the severity of COPD. Previous studies show that down-regulating the concentration of IL-32 leads to the increase of some inflammatory factors, including TNF-α, IL-6, IL-8 and cell adhesion factor (ICAM)-1, this phenomenon indicates that IL-32 is an important pro-inflammatory cytokine 8,11 . The GOLD score is only an indicator of respiratory function, and there is still a certain degree of limitation for evaluating COPD as a systemic disease. The mMRC score is another indicator to evaluate the quality of life of patients with COPD. It has been widely used in the evaluation of the condition and therapeutic efficacy of patients with COPD and the prediction of the risk of death in patients 15 . Our study showed that serum IL-32 concentration in patients with stable COPD was positively correlated with mMRC score, suggesting that IL-32 was related to the severity of patients with COPD. However, the clinical evaluation of serum IL-32 to COPD still needs to be confirmed by further research. Elevated www.nature.com/scientificreports/ serum IL-32 concentration in patients with long clinical medical history of COPD also reflects that IL-32 could be associated with the chronic development of COPD. Because IL-32 is involved in the COPD immune response, researchers have proposed to treat COPD by inhibiting the secretion of IL-32 7 . Controlling the inflammatory response mediated by IL-32 may be another theoretical basis for the development of new drugs for the treatment of COPD in the future. It is well known that FEV1% and FEV1/FVC are important indicators for evaluating respiratory function in patients with COPD, and the decrease in both indicates the progression of COPD and the increase risk in mortality 16 . In our study, the linear correlation analysis showed that serum IL-32 concentration in patients with stable COPD was negatively correlated with FEV1% and the FEV1/FVC, suggesting that changes in serum IL-32 can reflect the severity of COPD disease progression. The theory of airway inflammation is an important mechanism of the pathogenesis of COPD, and excessive production of inflammatory factors induces immune damage, leading to deterioration of lung function. Previous studies shows that increased IL-32 was negatively correlated with decreased FEV1, FEV1/FVC and oxygenation index in patients with acute exacerbation of COPD, confirming that IL-32 has a direct pro-inflammatory effect and also is the cause of chronic airway inflammation persisting and progressively worsening 7,17,18 . We did a multiple linear regression and found that COPD grading, clinical medical history and FEV1/FVC value in stable COPD affected the serum concentration of IL-32, and the FEV1/FVC had the most obvious influence on IL-32. As a pro-inflammatory cytokine, IL-32 not only involves in the continuous chronic inflammation of the airway but also reflects the severity of patients with COPD, and therefore it can be used as an evaluation indicator of disease progression and treatment effect in patients with COPD.
To further understand whether IL-32 has a reference value in the diagnosis of COPD, we conducted further diagnostic analysis. Through a ROC analysis, we found that the sensitivity and specificity for distinguishing patients with COPD from healthy individuals were 85.34% and 64.29%, and the area under the curve reached 0.808, indicating that IL-32 can reflect the disease occurrence of COPD and has a good value for diagnosis of COPD. It is reported that IL-32 is significantly elevated in patients with chronic inflammatory diseases of the airways, and IL-32 has the effect of inhibiting airway remodeling 7,8,10,[17][18][19] . However, the effect of IL-32 on the inhibition of airway remodeling and the promotion of airway inflammatory response remains to be confirmed. Our findings may suggest a hypothesis that serum IL-32 may be involved in the inflammatory process of COPD, inhibition of IL-32 may cut off IL-32 mediated inflammatory response, thereby reducing airway remodeling and airflow limitation in patients with COPD, and alleviating symptoms in patients. This will be a new direction for clinical treatment of COPD.
In summary, serum IL-32 concentration is higher in patients with stable COPD than in healthy individuals, and increased IL-32 is positively correlated with GOLD grade, mMRC score and clinical medical history of patients but negatively with FEV1/FVC and FEV1% predicted, suggesting that IL-32 is involved in the chronic Table 1. Relationship between serum concentration of IL-32 and clinical features in stable COPD patients (n = 116). N number, COPD chronic obstructive pulmonary disease, IL-32 interleukin-32, M ± SD mean ± standard deviation, BMI body mass index, GOLD Global Initiative for Chronic Obstructive Lung Disease, mMRC modified british medical research council. a The patients with mMRC score of 4 showed a higher concentration of serum IL-32 than those with mMRC score of 2 and 3. b The concentration of serum IL-32 in the patients with a longer history was up-regulated compared to those with relatively short history.

Parameter
Group N www.nature.com/scientificreports/ www.nature.com/scientificreports/ disease process of COPD and changes in IL-32 can be used to assess the severity of COPD and contributes to the diagnosis of COPD. However, there are also some shortcomings in this study. Firstly, the study only included Chinese patients and might have a geographical and ethnographic bias. Secondly, this study did not involve patients with acute exacerbation. Thirdly, this study did not investigate the specific intrinsic molecular mechanism of IL-32 elevation in COPD. Fourthly, this study did not involve the phenotype matter for example bronchitic vs. other phenotypes, nor did it give a definitive conclusion about the relationship between BMI and IL-32. Fifthly, there were less smokers in the control arm and non-smokers are more likely to have healthier habits like exercise and diet that may positively affect their health thus introduce a bias. In the future, the in-depth research to explain these issues should be conducted.

Methods
Ethics statement. This is an observational retrospective analysis (on patient's blood specimen for test program  The demographic characteristics between the observation group and the control group, including gender, age, and smoking status, were not statistically significant (p > 0.05) ( Table 5).

Objects. Between
Diagnostic criteria for patients enrolled and severity rating of COPD. The diagnosis and severity evaluation of COPD were performed by the diagnostic criteria of the Global Initiative for Chronic Obstructive Pulmonary Disease (GOLD) 1,3,6 . Objective criteria: after inhalation of 300 to 400 μg of salbutamol, the ratio of forced expiratory volume (FEV1) in the first second to forced vital capacity (FVC) is less than 0.70. The severity of COPD: GOLD-1, FEV1% predicted is more or equal to 80%; GOLD-2, the FEV1% predicted is more or equal to 50%, but less than 80%; GOLD-3, the FEV1% predicted is more or equal to 30%, but less than 50%; and GOLD-4, the FEV1% predicted is less than 30%.
Inclusion criteria. Inclusion criteria: (1) must meet the diagnostic criteria of COPD and be a stable COPD (the patient's cough, expectoration and shortness of breath are in stable condition or just show a mild symptoms or the condition is basically restored to the state before acute exacerbation) 1,3 ; (2) must have a lung function test within 3 days before enrollment; (3) no antibiotics, glucocorticoids and theophylline were used within 2 weeks before enrollment; (4) without laboratory and imaging evidences of pulmonary infection and pneumonia. www.nature.com/scientificreports/ www.nature.com/scientificreports/ Exclusion criteria. Exclusion criteria: (1) patients who used immunosuppressive drugs 1 week before blood collection; (2) acute infection occurred within 1 month before enrollment; (3) known etiology or pathological manifestations of airflow-limited diseases such as bronchiectasis, tuberculosis, lung cystic fibrosis and tumors; and (4) combined with serious heart, liver, kidney, hematopoietic system, endocrine system and other primary diseases, or mental illness and skin disease.
Collection and processing of blood specimens. On the next day after the patient was enrolled, 5 mL of fasting elbow venous blood was taken in the morning, centrifuged (centrifugal radius 5 cm, rotation speed 3,000 r/min) for 10 min, and the supernatant was aspirated and stored in a − 70 °C refrigerator.  Objective evaluation. All included patients must undergo three lung function tests before collecting the specimens and cannot be performed on the same day (three days before including into the study, the lung function test was performed once a day). The average of the three tests was calculated as statistical data. This study selected two data directly related to the diagnosis and classification of COPD: FEV1/FVC and FEV1% predicted.

Enzyme
Statement for strengthening the reporting of observational studies in epidemiology (STROBE). This study was a retrospective case-control study and was divided into seven sections: headings, abstracts, background presentations, methods, results, discussion, and other information. The study clearly described the grouping of the study and the sample size, study design, and test indicators. The statistical methods were described in detail, and the systematic errors and data offsets of the variables were analyzed. The data of the study were detailed in the results section, and the main findings of the study were explained in discussion. The keywords and terminology used in this study help to ensure the correct indexing of article in the electronic database.
Statistical processing of research data. The statistical software used in this study included IBM SPSS Statistics and MedCalc statistical software. The count data was expressed in terms of rate and analyzed by chi-square test and Fisher exact probability method. The measurement data was expressed in the form of mean ± standard deviation, and the comparison between groups was performed by analysis of variance and T test. The receiver operating characteristic (ROC) curve was used to determine the threshold of observed indicator and evaluate the diagnostic efficacy. Levene's Test for Equality of Variances was performed to determine whether the data is from a normal distribution or not. A statistical p value of less than 0.05 was considered to be statistically significant. Table 5. Clinical information and general data of included patients (COPD = 116 cases; Control = 70 cases). 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%; 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.