Imbalance between Subpopulations of Regulatory T Cells in Patients with Acute Exacerbation of COPD

ABSTRACT

Human regulatory T cells (Tregs) have been reported to be not significantly different in the peripheral blood of patients with chronic obstructive pulmonary disease (COPD) and healthy controls. Recent research has identified some new markers for Tregs and indicated that Tregs are composed of distinct subpopulations. The aim of the study was to describe the changing patterns of circulating Treg subpopulations in patients with acute exacerbation of COPD (AECOPD) and healthy controls, and to explore their potential roles in AECOPD pathogenesis. Blood samples were obtained from 30 never-smokers with normal lung function and 30 patients with COPD before and after they had an exacerbation. The proportions of Treg subpopulations were evaluated using flow cytometry. In the peripheral blood, decreased proportions of CD4⁺CD25⁺CD127^low Tregs, CD4⁺CD25⁺CD45RA⁺ Tregs, and CD4⁺CD25⁺CD62L⁺ Tregs and an increased proportion of CD4⁺CD25⁺CD45RO⁺ Tregs were found in patients with stable COPD compared with non-smokers with normal lung function. The patients showed further changes in Treg subpopulations when they had an AECOPD, with an overall decrease in a suppressive subset, indicating that the immune negative regulatory population of Tregs did not play an effective role. Immune homeostasis favored inflammation, and a negative correlation between the circulating tumor necrosis factor-alpha and the proportions of CD4⁺CD25⁺CD62L⁺ cells (r = −0.698, p < 0.05) in patients with AECOPD was found. The imbalance between the suppressive subsets and the proinflammatory subset of Tregs and the decline of Treg subpopulations with immunosuppressive activity may play important roles in AECOPD progression.

KEYWORDS:

• COPD
• regulatory T cell
• subpopulation
• inflammation

Introduction

Chronic obstructive pulmonary disease (COPD) is a common disease that severely threatens human health. The social and economic burden of COPD will rise to the 5th worldwide, and COPD will become the 3rd leading cause of death worldwide by 2020 (1). The prevention, early detection, and treatment of acute exacerbation of COPD (AECOPD) are clinically significant because of its increasing social burden and negative impact on the quality of life, pulmonary function, and disease progression.

Recently, accumulated evidence has indicated that patients with COPD exhibit many of the characteristics of a classical autoimmune response (2,3). T lymphocytes are believed to be the key cells in regulating airway inflammation in COPD (4). Human regulatory T cells (Tregs), a critical subset of T cells that use a variety of mechanisms to suppress the immune response, have gained considerable attention (5–9) and Treg abnormalities have been described in adaptive immunity of COPD. In patients with COPD, Treg proportions fluctuated in bronchoalveolar lavage (BAL) or in different types of pulmonary tissues. However, the frequencies of Tregs in the peripheral blood do not differ significantly among patients with COPD, healthy smokers, and never-smokers (10–13). In most previous studies, Tregs have only been considered as a single population. However, the phenotypic and functional heterogeneity of human Tregs is a major obstacle for understanding their clinical relevance. Dissecting Tregs into subsets, especially defining the suppressive population, may contribute to the striking findings of the studies in the adaptive immunity of COPD (14–16).

The aim of this study was to explore whether there were abnormal distributions in the suppressive phenotype of Tregs in the blood of patients with AECOPD. The findings showed their potential roles in AECOPD and provided new insight into the potential mechanisms underlying acute inflammation and adaptive immunity in AECOPD.

Materials and methods

Study subjects

Patients^{Table 1} with COPD were recruited in the First Affiliated Hospital of Guangxi Medical University, China. Never-smokers with normal lung function were also recruited as controls. Written informed consent was obtained from all the volunteers. The study was approved by the local Ethics Review Board at Guangxi Medical University, China (number 2014KY-C-008), and performed according to the Declaration of Helsinki. COPD diagnosis was established according to the definition supplied by the Global Initiative for Chronic Obstructive Lung Disease (GOLD) document (17). Patients with COPD had a forced expiratory volume in 1 second (FEV¹)/forced vital capacity ratio <70%, and FEV¹ was <80% of the predicted value post bronchodilators. All patients with COPD were clinically stable and had not experienced any exacerbations for ≥3 months preceding inclusion in the study. Participants with neoplasm, interstitial lung disease, autoimmune disease, cardiovascular disease, cerebrovascular disease, diabetes, allergic disease, infection, or other immune-related diseases were excluded. Patients receiving systematic corticosteroids were excluded.

We aimed to explore whether there were abnormal distributions in the subpopulations of Tregs in the blood of COPD patients in exacerbated and stable phases, and whether the frequency of different Treg subsets was correlated with disease severity as defined by FEV¹, the inflammatory agent in the blood, oxygen pressure (PO₂), and partial pressure of carbon dioxide (PCO₂).

The diagnosis of AECOPD was primarily determined by the clinical presentations of the patients complaining of an acute change in symptoms (baseline dyspnea, cough, and/or sputum production) that is beyond normal day-to-day variation (18). Other medical conditions such as pneumonia, congestive heart failure, pneumothorax, pleural effusion, PE, or arrhythmia were ruled out before a patient was considered to have AECOPD. Acute dyspnea of congestive heart failure can be differentiated by the increase in plasma B-type natriuretic peptides associated with other clinical manifestations (1,18,19). Full lung function including gas transfer at the time of exacerbation was obtained. Subject baseline characteristics are summarized in Table 1.

Table 1. Baseline characteristics of all subjects.

		Patients with AECOPD (n = 30)^a
	Healthy controls (n = 30)	Exacerbated state	Stable state
Age (years)	63.4 ± 7.9	65.7 ± 6.3
Male/female	24/6	25/5
Current/ex‐smokers	0/0	18/22
Smoking history (pack-years)	0	23.8 ± 3.7
FEV^1 (% predicted)	93.6 ± 3.7	50.0 ± 5.2^*	57.9 ± 6.5^*
FEV^1/FVC (%)	78.2 ± 3.0	55.1 ± 9.2^*	60.3 ± 2.5^*
FVC (L)	2.9 ± 0.3	1.1 ± 0.1^*	1.3 ± 0.4^*^,^**
RV/TLC	26.6 ± 3.8	43.2 ± 9.3^*	36.0 ± 7.7^*
RV (% predicted)	87.6 ± 5.5	117.0 ± 6.1^*	113.4 ± 7.1^*
DLCO (% predicted)	87.4 ± 3.3	61.2 ± 7.5^*	67.3 ± 8.5^*
BMI (kg/m^2)	27.7 ± 1.7	24.8 ± 2.6	25.4 ± 2.4^*

Data are presented as the median (IQR) for smoking history and the mean ± SD for all others.

AECOPD, acute exacerbations of chronic obstructive pulmonary disease; FEV¹, forced expiratory volume in 1 second; FVC, forced vital capacity; RV, residual volume; TLC, total lung capacity; DLCO, diffusion capacity for carbon monoxide of the lung; BMI, body mass index.

^a The COPD patients were the same in stable and exacerbated states.

^* p < 0.05 versus healthy controls.

^** p < 0.05 between before and after acute exacerbations.

Spirometry and lung function

The COPD patients did not receive any treatment with inhaled corticosteroids or oral anti-inflammatory drugs for at least 4 weeks prior to the start of the study, and neither regular long-acting β2-agonists nor long-acting anti-cholinergic drugs were allowed within 2 weeks. Short-acting β2-agonists and/or anti-cholinergic drugs were used on demand. All COPD patients had a post bronchodilator FEV¹/FVC of less than 70% that were not reversible. Dynamic spirometry (FVC and FEV¹) was performed post-bronchodilatation using a Vitalograph spirometer (Vitalograph Ltd., Buckingham, UK), as outlined previously (20). Pulmonary function tests were completed by patients when they were recruited. COPD was confirmed by using a 5th percentile as a lower limit of normal as estimated by the global lung initiative (GLI) predicting equations (21). The quality of spirometry measurement was ensured by looking at the length of expiration (22).

Laboratory assays

Peripheral venous blood samples of all patients with COPD were collected to carry out routine blood tests, blood gas analysis, and C reactive protein (CRP), fibrinogen, interleukin (IL)-8, tumor necrosis factor-alpha (TNF-α), IL-35, and Treg subset analyses. Peripheral venous blood samples (30 mL) from each patient were collected in heparin-coated anticoagulant tubes. Plasma was separated by centrifugation at 1,800 g for 20 minutes and stored at −80°C. All AEOCD patients' peripheral venous blood samples were collected on the first day after hospitalization. The concentrations of TNF-α, IL-8, and IL-35 in the peripheral venous blood samples were measured using a multiplex ELISA system (Lincoplex Systems, St. Charles, MO, USA). Fibrinogen and CRP were measured in peripheral blood samples using a radioimmunoassay (RIA) as per the manufacturer's instructions (RIA Kits, Beijing North Institute of Biological Technology, China).

Cell collection

Peripheral blood samples from each subject were collected in ethylenediaminetetraacetic acid-treated tubes and were processed to measure peripheral blood mononuclear cells (PBMCs) for flow cytometry setup procedures. Blood samples were layered onto Ficoll-Paque Plus tubes (Amersham Biosciences, Amersham, Bucks, UK) and centrifuged (400 g for 20 minutes at 21°C); PBMCs were then harvested. Cells were washed twice in divalent cation-free Hanks balanced salt solution at 300 g for 5 minutes at 4°C. PBMCs were resuspended, and viable counts were obtained.

Flow cytometry of Treg subsets

Flow cytometry freshly obtained human PBMC samples were stained as previously described (23) with anti-hCD4-FITC (e-Bioscience, San Diego, CA, USA), anti-hCD25-PE-Cy5 (BD Biosciences, San Jose, CA, USA), and anti-hCD45RA-APC (BD Biosciences). Anti-hCD45RO-APC, anti-hCD26L-APC, and anti-hCD127-APC (BD Biosciences) antibodies were used for surface marker staining of each subpopulation. We calculated the ratio of CD127^low, CD45RA⁺, CD45RO⁺, and CD62L⁺ Tregs from the peripheral blood.

Statistical analysis

The results are presented as the mean ± SEM. The Shapiro–Wilk test was used to evaluate the distribution of variance. For data distributed normally, comparisons between three groups were analyzed by one-way analysis of variance (ANOVA). The Wilcoxon signed-rank test was used to compare data with a non-normal distribution (24). Correlations were assessed by Spearman's rank correlation coefficient. Statistical analysis was performed using SPSS for Windows V.16.0 (Chicago, IL, USA). Here, a value of p < 0.05 was considered significant in all statistical tests.

Results

Demographic characteristics of the study population

A total of 30 COPD patients and 30 healthy controls were included in the study. The characteristics of the population are summarized in Table 1. There was no difference between the groups in terms of age and sex. There were significantly more number of smokers or ex-smokers in the COPD group than the health control group. Lung functions of COPD patients were examined at stable (SCOPD) and AECOPD phases. As expected, the lung function was significantly better in the stable phase than that in the exacerbated phase (Table 1).

Varied systemic inflammatory media in AECOPD patients

Figure 1 shows the main clinical characteristics of participants. Compared with the healthy control group, blood gas analysis in the COPD group showed whether AECOPD or SCOPD had statistically significant differences, and PaCO₂ values were higher in the AECOPD group than those in the SCOPD group; PaO₂ values were lower in AECOPD group than those in the SCOPD group.

Figure 1. Laboratory data for patients with AECOPD and healthy controls. Results are expressed as the (mean ± SD). n = 30; ^*p < 0.05; ^**p < 0.001.

Compared with the healthy control group, IL-8, CRP, fibrinogen, and TNF-α were significantly higher in the AECOPD group. Fibrinogen, IL-8, CRP, and TNF-α in the AECOPD group were higher than those in the SCOPD group, and there was a significant difference.

IL-35, a newly identified inhibitory cytokine, is preferentially expressed by Tregs and is required for their maximum suppressive activity (25). We observed that IL-35 levels were persistently lower in the peripheral blood from COPD patients than the healthy controls.

Varied subpopulation frequencies of circulating Tregs in patients with AECOPD, SCOPD, and healthy controls

In line with previous studies (10,11), Treg identification using CD4⁺CD25⁺Foxp3⁺ showed no difference in the circulating Treg number in patients with COPD compared with smokers and never-smokers. However, as shown in Figure 2, patients with SCOPD showed significantly increased proportions of CD4⁺CD25⁺CD45RO⁺ Tregs but decreased proportions of CD4⁺CD25⁺CD127^low Tregs, CD4⁺CD25⁺CD45RA⁺ Tregs, and CD4⁺CD25⁺CD62L⁺ Tregs. This indicated that the imbalance between immunosuppressive subsets and immunoenhancing subsets in CD4⁺CD25⁺ T cells shifted toward immune tolerance in patients with COPD responses. Strikingly, in comparison to the SCOPD group, patients with AECOPD showed the same trend of variation as the proportion of each subset. Varied subpopulation frequencies suggested that the Treg subsets with low immunosuppressive activity and low expression of Foxp3 increased and the Treg subsets with high immunosuppressive activity or/and high expression of Foxp3 decreased in patients with AECOPD responses, which may lead to the development of inflammation and the disorder of acute exacerbation.

Figure 2. Flow cytometry analysis of Treg cell subpopulations in patients with AECOPD. (A) Flow cytometry analysis of CD62L expression in CD4⁺CD25⁺ Treg cells. (B) The percentages of each circulating T-regulatory subset among CD4⁺ T cells in patients with AECOPD, SCOPD, and healthy controls. The results are expressed as the (mean ± SD). n = 30; ^*p < 0.05; ^**p < 0.001.

Correlation between the suppressive phenotype of circulating Tregs, systemic inflammatory media, and disease severity

To further explore the role of phenotypes of circulating Tregs in AECOPD, we next analyzed the correlation between suppressive phenotypes of circulating Tregs and systemic inflammatory media; i.e., PaO₂, PaCO₂, and spirometry. TNF-α was negatively correlated with PaO₂ (r = 0.666, p < 0.05) and the proportions of CD4⁺CD25⁺CD62L⁺ cells in patients with AECOPD (r = 0.698, p < 0.05; Figure 3).

Figure 3. Correlations between suppressive phenotypes of circulating Tregs in AECOPD patients and systemic inflammatory media. (A) TNF-alpha and the frequency of CD4⁺CD25⁺CD62L⁺ Treg cells. There was a correlation between TNF-alpha and PO₂. (B) TNF-alpha and PaO₂ in AECOPD patients. There was a correlation between TNF-alpha and CD62. Data were determined by Spearman's rank correlation coefficients.

Discussion

AECOPD is an acute event characterized by worsening of the patient's respiratory symptoms (typically dyspnea, cough, increased sputum volume, and/or sputum purulence) that is beyond normal day-to-day variations and leads to a change in medication (1). On average, each COPD patient suffers from 0.5 to 3.5 episodes of acute exacerbations per year, and AECOPD is a major cause of death with associated large medical expenditures. The prevention, early detection, and treatment of AECOPD are clinically significant because of its increasing social burden and negative impact on quality of life, pulmonary function, and disease progression. Previous studies that identified Tregs as a whole did not find any significant differences in the frequencies of circulating Tregs in patients with COPD. According to an epidemiological study, the cost of AECOPD for an inpatient in the People's Republic of China was 11,598 RMB a year (26). Especially for patients whose hospital stay ends in death, their expenses, which are related to the high cost of life-support, increased remarkably (27). Here, based on this new definition of Treg subsets, we revealed significantly decreased proportions of Treg subpopulations with high immunosuppressive activity and an increased proportion of the Treg subpopulation with low immunosuppressive activity in patients with COPD compared with healthy controls with normal lung function. More importantly, we found an imbalance between these subsets in the inflammatory media in patients with AECOPD. Investigations of these markers in COPD are rare, and to the best of our knowledge, this is the first study addressing CD4⁺CD25⁺CD62L⁺ expression on blood cells from subjects with COPD and AECOPD.

Tregs with a suppressive function play a major role in maintaining self-tolerance. It has been reported that only a minority of human CD4 T cells expressing the highest levels of CD25 (called CD25^bright cells) have suppressor activity (28), and in humans, up to 40% of the peripheral-blood CD4 T cells express CD25 to some extent. Therefore, many recent studies have focused on this population (29–32). However, the proportion of cells in the CD25^bright population in peripheral blood does not differ (32) significantly among patients with COPD, healthy smokers, and never-smokers (10–13). Recently, some new markers for Tregs have been reported, suggesting that better markers or combinations of markers are, therefore, clearly required to accurately identify Tregs in humans. We screened some markers, in combination with CD4 and CD25, with the aim of finding suppressive activity.

CD127 expression was shown to be inversely associated with FoxP3 and a suppressive function of human CD4⁺ Tregs in the peripheral blood (23). CD127 expressing cells have been studied in allergic asthma, gastric cancer, and glioma (33–35). As indicated above, CD25^bright expression on CD4⁺ cells usually implies Tregs. The present study showed that a decreased percentage of CD4⁺CD25⁺CD127^low cells was associated with both COPD patients and those patients who had COPD acute exacerbation (Figure 2). Although the number of patients in the present study was rather small, the data are consistent with previously published results (20,36). As described previously (37,38), human CD4⁺Foxp3⁺ T cells are phenotypically heterogeneous and include the CD45RO⁺ and CD45RA⁺ T cell subtypes. CD4⁺CD25⁺CD45RA⁺ Tregs possess a suppressive capacity and continuously express Foxp3 in vitro, and CD4⁺CD25⁺CD45RO⁺ Tregs lack suppressive activity or have limited suppressive activity, but can produce IL-2, IL-17, and interferon (IFN)-γ (14–16,39,40), which are important for T-cell activation and pulmonary inflammation (41,42). Our study showed that an increased percentage of CD4⁺CD25⁺CD45RO⁺ Treg cells and a decreased percentage of CD4⁺CD25⁺CD45RA⁺ Treg cells were associated with both COPD patients and those patients who had COPD acute exacerbation (Figure 2). One report showed that human memory CD4⁺CD25⁻ T cells could be efficiently differentiated into Foxp3⁺ T cells via TCR activation with TGF-β, but this phenomenon was restricted to CD62L⁺CCR7⁺ memory CD4⁺ T cells. Furthermore, the phenomenon demonstrated that such CD4⁺CD62L⁺ central memory T cell-derived Foxp3⁺ T cells were functional in suppressing the proliferation of T effector cells in mice (43). Our study showed that a decreased percentage of the memory CD4⁺CD25⁺CD62L⁺ cells was associated with both COPD patients and those patients who had COPD acute exacerbation.

Based on this new definition of Treg subsets, we revealed significantly decreased proportions of CD4⁺CD25⁺CD127^low Tregs, CD4⁺CD25⁺CD45RA⁺ Tregs, and CD4⁺CD25⁺CD62L⁺ Tregs and an increased proportion of the CD4⁺CD25⁺CD45RO⁺ Tregs in patients with SCOPD compared with non-smokers with normal lung function. The present data implied that the expression of Treg subsets with suppressive activity in the blood appears to decline, but the expression of Treg subsets that lack suppressive activity in the blood appears to increase in patients with COPD, and those appear to further decline in patients with AECOPD, which suggested that an imbalance between anti-inflammatory and pro-inflammatory subsets may induce prolonged immune activation. More importantly, we found a negative correlation between circulating TNF-α and PaO₂ and a stronger negative correlation between the circulating TNF-α and the proportions of CD4⁺CD25⁺CD62L⁺ cells in patients with AECOPD, which further linked the immune imbalance with inflammatory and disease severity in patients with AECOPD. We did not find any correlation between the circulating Treg subsets and airflow limitations or lung function. Therefore, from these findings, we hypothesize that a decrease in the immunosuppressive Treg populations, together with enhanced pro-inflammatory responses, induced AECOPD. This persistent systemic inflammation is believed to be involved in the progressive acute exacerbation characteristic of COPD.

The COPD subjects included in this study were clinically stable with no history of recurrent infectious exacerbations and in no need of regular medications, apart from short-acting bronchodilators on demand. Additionally, our data indicated that there was a negative correlation between the circulating TNF-α and the proportions of CD4⁺CD25⁺CD62L⁺ cells in patients with AECOPD. However, the detailed mechanism of this correlation is still unclear and requires further study. We focused on the characteristics of the frequency of different Treg subsets for AECOPD. We hypothesize that it is related to the intrinsic constraints of the cells and not due to their similarity to the Treg phenotype. A limitation of the study was that we did not investigate the control of response to infection/inflammation by regulating the balance between Th17 and Treg. The balance between Th17 and Treg has been shown to be important for the control of response to inflammation/infection (44). Another limitation of the study was that the types of AECOPD (e.g. viral versus bacterial versus non-infective) were not analyzed separately. In real clinical practice, it was difficult to definitively determine whether an episode of AECOPD was caused by bacteria or virus.

In summary, this study provided further evidence for the role of adaptive immunity in COPD pathogenesis and demonstrated that the imbalance between the subpopulations of Tregs may contribute to the progression of inflammation in patients with AECOPD. Of note, the correlations between the CD4⁺CD25⁺CD62L⁺ cells in the peripheral blood and circulating TNF-α indicated a closer correlation between systemic immune activation and systemic inflammation activation, which might facilitate our understanding of the underlying mechanisms of systemic inflammation and adaptive immunity in AECOPD. The clinical implication of the present study may be that the exacerbation of COPD can be ameliorated by modulating different subpopulations of Tregs to restore immune homeostasis.

Authors' contributions

XY and BH contributed to recruiting the patients, performing all data collection, collecting and processing samples, and writing the manuscript. XZ contributed as primary investigator and was responsible for designing the study and writing the manuscript. WS and WL performed laboratory-based assays. ZH, JD, and JZ recruited the patients and collected clinical data. JB contributed as lead investigator and was responsible for designing the study, analyzing the data, and writing the manuscript. All authors read and approved the final manuscript.

Declaration of interest

None.

Ethical approval

This study is approved by the Ethics Committee of the First Affiliated Hospital of Guangxi Medical University.

Acknowledgments

The authors thank Guorong Liang, Haijuan Tang, ZhixiuRan and Yi Liang (First Affiliated Hospital of Guangxi Medical University) for their support with screening the participating subjects throughout the study.

Funding

This work was supported by the National Natural Science Foundation of China (Nos. 81260011 and 30860106) and the Guangxi Natural Science Foundation (No. 2013GXNSFAA019136).