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Severity of chronic obstructive pulmonary disease with ‘exacerbator with emphysema phenotype’ is associated with potential biomarkers
  1. Wendong Hao1,2,
  2. Manxiang Li2,
  3. Yunqing Zhang1,
  4. Cailian Zhang1,
  5. Ping Wang1
  1. 1 Department of Respiratory and Critical Care Medicine, Yan'an University Affiliated Hospital, Yan'an, China
  2. 2 Department of Respiratory and Critical Care Medicine, The First Affiliated Hospital of Xi'an Jiaotong University, Xi'an, China
  1. Correspondence to Wendong Hao, Department of Respiratory and Critical Care Medicine, Yan'an University, Yan'an, Shaanxi 716099, China; hwdokgood{at}hotmail.com

Abstract

Objective The present study was designed to investigate the biomarkers levels of fractalkine (FKN), neutrophil elastase (NE) and matrix metalloproteinase-12 (MMP-12) in chronic obstructive pulmonary disease (COPD) with ‘exacerbator with emphysema phenotype’ and to evaluate the associations between the biomarkers levels and the severity of disease by spirometric measurements.

Methods A total of 84 COPD patients and 49 healthy controls were enrolled in our study. ELISA were utilised to detect the FKN, MMP-12 and NE in serum from all subjects.

Results FKN (p<0.001), NE (p=0.039) and MMP-12 (p<0.001) in serum of COPD patients showed higher levels than that of healthy control subjects. Serum FKN (p<0.001), MMP-12 (p<0.001) and NE (p=0.043) levels were significantly higher in severe and very severe COPD patients than that in mild and moderate COPD patients. Circulating FKN, MMP-12 and NE expression levels were significantly elevated (p<0.001) in COPD smokers compared with COPD non-smokers. The smoke pack years were negatively correlated with FEV1%pred (r=−0.5036), FEV1/FVC ratio (r=−0.2847) (FEV, forced expiratory volume; FVC, forced vital capacity). Similarly, we observed a strong positive correlation between the smoke pack years and serum levels of FKN (r=0.4971), MMP-12 (r=0.4315) and NE (r=0.2754). FEV1%pred was strongly negatively correlated with cytokine levels of FKN (r=−0.4367), MMP-12 (r=−0.3295) and NE (r=−0.2684). Likewise, FEV1/FVC ratio was negatively correlated with mediators of inflammation levels of FKN (r=−0.3867), MMP-12 (r=−0.2941) and NE (r=−0.2153).

Conclusion Serum FKN, MMP-12 and NE concentrations in COPD patients are directly associated with the severity of COPD with ‘exacerbator with emphysema phenotype’. This finding suggests that FKN, MMP-12 and NE might play an important role in the pathophysiology of COPD.

  • FKN
  • exacerbator with emphysema phenotype
  • chronic obstructive pulmonary disease
  • neutrophil elastase
  • matrix metalloproteinase-12

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Introduction

Chronic obstructive pulmonary disease (COPD) is a heterogeneous disease characterised by chronic inflammation of the airways and lungs. Its morbidity and mortality are high, which seriously endangers public health and brings a heavy economic burden to families and society.1 Therefore, studies on biomarkers that can evaluate the severity of airflow limitation of COPD have increased year by year.2 3 However, there is currently no universal biomarker utilised in the development and progression of COPD. Nowadays, the pathogenesis of COPD includes chronic inflammation, protease–antiprotease imbalance and oxidative stress. Chronic inflammation and protease–antiprotease imbalance are considered to be essential pathogenesis of COPD.4

CX3CL1 (fractalkine, FKN) has two forms of the membrane: attached form and the shed form. The membrane-attached FKN form mediates firm adhesion of CX3CR1-expressing cells (the only receptor for FKN) to the endothelial cells, and soluble FKN form recruits CX3CR1-expressing cells in physiological and pathological processes. There are many studies showing that FKN plays an important role in the physiological and pathological processes of various inflammatory diseases, such as atherosclerosis and cardiovascular diseases, cerebral infarction, ischaemia/reperfusion injury, dermatological diseases and lung cancer.5–9 One study pointed out that cigarette smoke (CS) induces functional FKN expression in arterial endothelium and leucocytes from patients with COPD show increased FKN-dependent adhesiveness, suggesting that targeting the FKN/CX3CR1 axis might prevent COPD-associated cardiovascular disorders.10 Another study have shown that FKN level was significantly increased in lung tissue of COPD smokers compared with COPD non-smokers, and FKN was the only chemokine that was found to be upregulated.11 In addition, our previous study3 found that serum CX3CL1 is elevated in COPD patients, but the study did not distinguish the specific phenotype of COPD. Therefore, the present study was designed to investigate the biomarker levels of FKN in COPD with ‘exacerbator with emphysema phenotype’.

Neutrophil elastase (NE) degrades elastase and also stimulates the production and secretion of mucin, which eventually leads to high airway mucus secretion and airway obstruction in COPD patients.12 NE has been shown elevated in induced sputum and plasma of COPD patients.13 14 Matrix metalloproteinases (MMPs) are a family of similar structures that can degrade extracellular matrices, leading to structural damage and airway remodelling in COPD patients.15 An animal experiment16 has shown that MMP-9/MMP-12 inhibitors can substantially improve morphologic emphysema, small airway remodelling and functional consequences of these lesions in non-mouse species. MMP-9, MMP-12 is an essential mediator of emphysema formation, and suggests that MMP-9 and MMP-12 may be potential intervention targets.

COPD is a heterogeneous disease and it is currently believed that the same phenotype may have similar clinical outcomes and therapeutic responses. According to the Spanish COPD phenotype classification,17 the ‘exacerbator with emphysema phenotype’ is a phenotype of the common phenotype of COPD with poor prognosis and poor treatment response. Therefore, the aim of this paper was to investigate the biomarkers levels of FKN, NE andMMP-12 in COPD ‘exacerbator with emphysema phenotype’ and to evaluate the associations between biomarkers levels and the severity of disease by spirometric measurements.

Materials and methods

Patients and grouping

The study population was selected from all patients who underwent pulmonary function between 1 January 2016 and 31 December 2018 at the respiratory function laboratory of Yan'an University Affiliated Hospital. According to the 2017 revision of the Global Initiative for Chronic Obstructive Lung Disease (GOLD) report,18 a ratio of forced expiratory volume in 1 s (FEV1) to forced vital capacity (FVC) of less than 0.7 after inhaled bronchodilators was considered to be incompletely reversible in airflow limitation. COPD was staged in accordance with the GOLD guidelines: GOLD I (mild), FEV1 ≥80% predicted; GOLD II (moderate), FEV1 <80% and ≥50% predicted; GOLD III (severe), FEV1 <50% and ≥30% predicted and GOLD IV (very severe), FEV1 <30% predicted.

Patients with COPD who have experienced acute exacerbations ≥2 times in the past year were defined as frequent acute exacerbations. Emphysema can be clarified by the ratio of residual volume/total lung capacity, carbon monoxide diffusion and chest high-resolution CT. Exclusion criteria were as follows: 1. Interstitial lung disease, lung cancer, allergic bronchial asthma, tuberculosis, pleural effusion and heart, liver and kidney failure. 2. Type 2 diabetes, inflammatory bowel disease, rheumatoid arthritis, systemic lupus erythematosus and other autoimmune diseases.

Sample collection and measurement of cytokines

Venous blood samples were taken from each participant . Blood samples were drawn into anticoagulant-free tubes and serum was separated by centrifugation at 3000 rpm for 20 min . Serum samples were stored at −80°C for further analysis. Serum levels of FKN, MMP-12 and NE were measured using ELISA according to the manufacturer’s instructions.

Symptom assessments

The Chronic Obstructive Pulmonary Disease Assessment Test (CAT) questionnaire consists of eight items designed to comprehensively assess the health status of patients with COPD.19 Eight items assess the best and worst case scenarios of cough, phlegm, chest tightness, dyspnoea, activity limitations at home, confidence, sleep quality and energy levels. Each item is scored from 0 to 5, so the CAT questionnaire score ranged from 0 to 40.

Statistical analysis

Continuous variables with normal distribution were presented as mean±SD and categorical variables were represented as percentages. Bivariate correlation analysis was used to analyse the relations of inflammatory cytokines versus the severity of COPD ‘exacerbator with emphysema phenotype’. All data are processed using GraphPad Prism 5 and SPSS V.13.0 statistical software package. Statistical significance was accepted as p<0.05.

Results

Subject characteristics

Eighty-four COPD patients and forty-nine healthy controls were enrolled in this cross-sectional study. Characteristics of the subjects are shown in table 1. There were considerable differences in body mass index (BMI), pack years and pulmonary function between the two groups. COPD patients had a significantly lower BMI (p<0.001), FEV1%predicted (p<0.001), FEV1/FVC ratio (p<0.001) compared with healthy controls. However, smoke pack years of COPD patients were significantly higher than that in healthy controls (p<0.001).

Table 1

Clinical characteristics of participants

Expression levels of serum FKN, NE and MMP-12 in subjects

Circulating FKN (p<0.001, figure 1A), NE (p=0.039, figure 1B) and MMP-12 (p<0.001, figure 1C) in COPD patients showed higher levels than that of healthy controls. Figure 2 depicts the distribution of FKN, MMP-12, NE (pg/mL) serum levels in COPD patients in relation to GOLD grading. Serum FKN and MMP-12 (p<0.001) levels were significantly greater in severe and very severe COPD patients than that in mild and moderate COPD patients. Similarly, circulating NE (p=0.043) level was higher in severe and very severe COPD patients than that in mild and severe COPD patients. Figure 3 shows the distribution of FKN, MMP-12 and NE levels in COPD smoker patients and COPD non-smoker patients. Circulating cytokine expression levels were significantly elevated (p<0.001) in COPD smokers compared with COPD non-smokers.

Figure 1

Comparison of FKN (A), MMP-12 (B), NE (C) in COPD patients and healthy subjects. COPD, chronic obstructive pulmonary disease; FKN, fractalkine; MMP-12, matrix metalloproteinase-12; NE, neutrophil elastase.

Figure 2

Levels of serum FKN, MMP-12,NE among the studied groups in COPD based on GOLD severity stages. # P<0.001, compared with mild (Gold I) COPD patients. *P<0.05, compared with mild (GOLD I) COPD patients. COPD, chronic obstructive pulmonary disease; FKN, fractalkine; GOLD, Global Initiative for Chronic Obstructive Lung Disease; MMP-12, matrix metalloproteinase-12; NE, neutrophil elastase.

Figure 3

Comparison of cytokines between COPD smoker, COPD non-smoker, healthy smoker and healthy non-smoker. CN, COPD non-smoker; COPD, chronic obstructive pulmonary disease; CS, COPD smoker; FKN, fractalkine; HN, healthy non-smoker; HS, healthy smoker; MMP-12, matrix metalloproteinase-12; NE, neutrophil elastase. # P<0.001, compared with healthy smoker and healthy non-smoker. *P<0.05, compared with COPD non-smoker.

Correlation among serum biomarkers and pack years and pulmonary function in COPD patients

The smoke pack years was negatively correlated with FEV1%pred (r=−0.5036), FEV1/FVC ratio (r=−0.2847). Similarly, we observed a strong positive correlation between the smoke pack years and serum levels of FKN (r=0.4971), MMP-12 (r=0.4315)and NE (r=0.2754) (table 2). FEV1%pred was strongly negatively correlated with cytokine levels of FKN (r=−0.4367), MMP-12 (r=−0.3295) and NE (r=−0.2684). Likewise, FEV1/FVC ratio was negatively correlated with mediators of inflammation levels of FKN (r=−0.3867), MMP-12 (r=−0.2941) and NE (r=−0.2153) (table 3).

Table 2

Correlation among pack year and cytokines and pulmonary function in COPD patients

Table 3

Correlation between biomarkers and pulmonary function in COPD patients

Discussion

The Spanish Guidelines for COPD (GesEPOC) describe four clinical phenotypes. The ‘exacerbator with emphysema phenotype’ is an important one of the four clinical phenotypes.17 The objective of this study was to evaluate the relationship between serum biomarkers level and severity of COPD patients. First, our results showed that COPD patients have higher levels of serum FKN, MMP-12 and NE compared with healthy subjects. Serum cytokine levels of FKN, MMP-12 and NE were positively correlated with the severity of COPD. Second, the results also indicated that the serum levels of FKN, MMP-12 and NE in COPD smokers are significantly higher than those of COPD non-smokers. Therefore, we can also say that smoking plays a critical role in the pathophysiology of COPD. Third, our findings also revealed that smoking is associated with the severity of COPD and may also be associated with higher serum levels of FKN, MMP-12 and NE-mediated inflammation of the airways and lungs in patients with COPD. The study also showed a strong positive correlation between smoke pack years and serum levels of FKN, MMP-12 and NE. There is an important negative correlation between smoke pack years and FEV1/FVC(%) and FEV1% predicted. A significant negative correlation between serum levels of FKN, MMP-12 and NE and FEV1% predicted was observed. Population-based evidence suggests that COPD patients have a proinflammatory state in which serum levels of many inflammatory cytokines are elevated.

In our present study, serum FKN protein level was significantly higher in COPD ‘exacerbator with emphysema phenotype’ patients compared with healthy subjects. The results of this work are consistent with our previous research.3 Furthermore, the serum FKN level of severe–very severe patients (GOLD 3&4) was significantly higher than that of mild–moderate patients (GOLD 1&2). McComb and his colleagues reported that upregulation of CX3CL1 and its receptors in lung endothelium and leucocytes in a mice model of cigarette smoke-induced emphysema.11 In addition, soluble CX3CL1 stimulates CX3CR1 + leucocyte chemotaxis,20 tobacco smoke-induced CX3CR1 + leucocyte recruitment and accumulation can lead to alveolar wall destruction,21 and extravasation of inflammatory cells was found in alveolus.22 The above studies are basic research and animal experiments. These studies indicate increased expression of CX3CL1 in cigarette smoke-induced animal models, and also CX3CL1-mediated chemotaxis of CX3CR1 + leucocytes can promote the accumulation of inflammatory cells and the development of COPD in the lung. However, our study is a clinical experiment and the results suggested that serum levels of FKN in COPD ‘exacerbator with emphysema’ phenotype patients are associated with disease severity. Therefore, clinically, our study also showed that the CX3CL1–CX3CR1 axis plays a major role in the pathophysiological process of COPD.

In the present study, the serum NE level was significantly higher in COPD smokers compared with COPD non-smokers. Moreover, serum NE protein level was significantly higher in COPD ‘exacerbator with emphysema phenotype’ patients compared with healthy subjects. This is consistent with previous studies,23 24 which indicated that NE levels in serum and peripheral blood mononuclear cells of COPD patients are much higher than in healthy subjects. In addition, the serum NE level of severe–very severe patients (GOLD 3&4) was significantly higher than that of mild–moderate patients (GOLD 1&2). The results of this study agree with previous works, which have suggested that FEV1 was significantly decreased in subjects with elevated NE and alpha-1 antitrypsin in bronchoalveolar lavage fluid and plasma.25 Another study reported that NE levels in saliva were significantly higher in COPD patients than in healthy subjects, and NE could reflect smoking status in COPD patients.26

In our current study, it was reported that serum MMP-12 level is significantly elevated in COPD patients compared with healthy subjects. Moreover, serum levels of MMP-12 were higher in COPD smokers compared with COPD non-smokers. The finding of our study agrees with Butsch et al,27 who found that concentrations of serum MMP-12 were significantly elevated in COPD patients compared with healthy non-smokers. In addition, our finding showed that the concentrations of circulating MMP-12 of severe–very severe patients (GOLD 3&4) were significantly higher than that of mild–moderate patients (GOLD 1&2). In contrast to our results, one study28 reported that there was no relationship between MMP-12 gene expression and the disease severity in COPD patients. Another small sample study29 showed an increase expression in MMP-1 RNA, protein and activity in the lung parenchyma of patients with emphysema, however, no MMP-12 expression was found in these samples. COPD is a highly heterogeneous disease. Therefore, there are numerous studies on the phenotype of COPD. The ‘clinical phenotype’ is defined as a characteristic or set of characteristics of a disease that can be used to distinguish individuals with COPD and are related to significant clinical outcomes, such as symptoms, exacerbations, treatment response, disease progression or death.30 The results of these studies are inconsistent with our findings. The probable reason for this is that COPD is very complicated and has various clinical manifestations, and these published literature reports did not distinguish the clinical phenotype of COPD.

In this study, due to strict inclusion and exclusion criteria, the sample size of our study was quite small. Simultaneously, we assessed the association between inflammatory cytokines expression levels and disease severity only based on the correlation analysis with five indicators: FKN, MMP-12, NE, FEV1/FVC and FEV1/pred. In addition, our study did not investigate whether FKN, NE and MMP-12 protein levels are differentially expressed in serum, induced sputum, bronchoalveolar lavage fluid and their association with disease severity. To avoid these limitations, our further study will recruit more participants and detect more indicators such as high-resolution CT, bronchoscopy alveolar lavage fluid, airway epithelial cells, airway smooth muscle cells or other direct indicators.

Main messages

  • Expression of fractalkine (FKN), neutrophil elastase (NE) and matrix metalloproteinase-12 (MMP-12) in serum was elevated in chronic obstructive pulmonary disease (COPD) patients.

  • Circulating FKN, NE and MMP-12 levels were increased with the severity of COPD stages.

  • Cytokine levels of FKN, MMP-12 and NE were substantially increased in COPD patients and well correlated with disease severity (FEV1% pred).

Current research questions

  • Patients were all from a single-centre, which may introduce a potential selection bias.

  • The sample size of this study in subgroup was relatively small.

  • This is a clinical study that requires further confirmation from animal experiments and basic research.

References

Footnotes

  • Contributors M-XL and C-LZ jointly supervised the design of this study, and are both guarantors of the article. W-DH, Y-QZ and PW were involved in data collection and helped with the literature review. W-DH analysed the data and wrote the majority of the paper. All authors approved the final version of the manuscript.

  • Funding The study was supported by the Yan'an University Affiliated Hospital Cultivation Fund (2018PT-06).

  • Competing interests None declared.

  • Patient consent for publication Patients provided written informed consent to take part in the study.

  • Ethics approval The study complies with the Declaration of Helsinki and was approved by the Yan'an University Affiliated Hospital Ethics Committee.

  • Provenance and peer review Not commissioned; externally peer reviewed.

  • Data availability statement Data are available upon reasonable request.