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 Table of Contents  
ORIGINAL ARTICLE
Year : 2019  |  Volume : 3  |  Issue : 3  |  Page : 596-604

Role of regulatory T-cells in chronic obstructive pulmonary disease


1 Department of Chest Diseases, Al-Azhar University, Cairo, Egypt
2 Department of Clinical Pathology, Al-Azhar University, Cairo, Egypt
3 Department of Internal Medicine, Al-Azhar University, Cairo, Egypt

Date of Submission18-Jun-2019
Date of Decision18-Jun-2019
Date of Acceptance01-Jul-2019
Date of Web Publication10-Feb-2020

Correspondence Address:
MD Heba H Eltrawy
Lecturer of Chest Diseases, Faculty of Medicine for Girls, AlAzher University; Chest Department, Al-Zahraa University Hospital, 11517 Al-Abbaseya, Cairo
Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/sjamf.sjamf_58_19

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  Abstract 


Background Chronic obstructive pulmonary disease (COPD) is a gradually progressive disease, it might have a subordinate autoimmune pathogenesis. The CD4+CD25+FOXP3+T-regulatory cells (Treg) have an important role in controlling immune and allergic reactions.
Objective To determine if Treg is involved in COPD and its value in the predilection of COPD development.
Patients and methods This case–control study was conducted on 30 COPD patients and 20 age-matched and sex-matched healthy controls. Spirometry and flow cytometry were done.
Results The Treg% was significantly lower in COPD than controls (P<0.05). CD4+ nonsignificantly differed between COPD patients and controls. Forced expiratory volume in the first second/forced vital capacity ratio, forced expiratory volume in the first second %, and forced vital capacity % were significantly decreased, while COPD duration and occupational and/or biomass exposure were significantly increased in the COPD subgroup with lower Treg frequencies. All of them are predictive factors for lower Treg frequencies. Patients with lower Treg frequencies have a 2.8-fold increased risk of COPD development (odds ratio=2.8, confidence interval 0.86–9.0).
Conclusion Lower frequencies of Treg (autoimmunity) might be a risk factor for COPD development.

Keywords: CD25+ T-regulatory cells, CD4+ T-regulatory cells, chronic obstructive pulmonary disease, FOXP3, pulmonary function


How to cite this article:
Eltrawy HH, Elshennawy S, Abozaid SY, Mostafa S. Role of regulatory T-cells in chronic obstructive pulmonary disease. Sci J Al-Azhar Med Fac Girls 2019;3:596-604

How to cite this URL:
Eltrawy HH, Elshennawy S, Abozaid SY, Mostafa S. Role of regulatory T-cells in chronic obstructive pulmonary disease. Sci J Al-Azhar Med Fac Girls [serial online] 2019 [cited 2020 Feb 29];3:596-604. Available from: http://www.sjamf.eg.net/text.asp?2019/3/3/596/278036




  Introduction Top


The role of autoimmunity in the pathogenesis of chronic obstructive pulmonary disease (COPD) is recently recognized. Regulatory T-cells (Tregs) are a subset of CD4 T-cells that play an important role in regulating autoimmune responses [1].

T-lymphocytes are believed to be key cells in regulating airway inflammation in COPD. Activated T-cells can cause a different kind of tissue injuries that lead to COPD by direct cellular effects, release of diverse proinflammatory and deleterious mediators, and/or recruitment and activation of other immune and parenchymal effector cells [2].

Many studies have reported that the activation of T-cells is tightly controlled by active mechanisms and negative regulatory mechanisms. In the past, CD4+ CD25+ FOXP3+ Tregs, a critical subpopulation of T-cells that use different mechanisms to suppress the immune response, have gained considerable attention, and Treg abnormalities have been noted in many chronic inflammatory and autoimmune disorders [3]. Recently, accumulating evidence indicates that patients with COPD express a variety of the characteristics of a classical autoimmune response. Thus, a variety of the studies have evaluated the role of Tregs in adaptive immunity of COPD [4].

Tregs are a specialized, phenotypically, and functionally distinct, subset of T-cells that regulate immune response, thereby maintaining homeostasis and self-tolerance. It has been proved that Tregs are able to prevent T-cell proliferation and cytokine production and play an important critical role in the inhibition of autoimmunity [5].

It was demonstrated in many studies that lack of functional FOXP3 (and therefore Tregs) in humans developes a variety of profound autoimmune symptoms so FOXP3 is important in immune tolerance therefore it is critical for controlling otherwise self-reactive T-cells in our bodies [6].

Therefore, in many inflammatory diseases, the balance between Tregs and effector T-cells may be critical. While Tregs are able to act as regulatory cells immediately on leaving the thymus, naïve CD4 T-cells can also, under appropriate conditions, be converted into adaptive Tregs. Other T-cells with regulatory capacity have also been described that express regulatory cytokines such as interleukin-10 and transforming growth factor β [6].

Additionally, the ability to stimulate the differentiation of Tregs on demand is an area of great therapeutic relevance and active research. The hallmark of natural Tregs in the resting immune system is the expression of high levels of the surface marker, CD25. Rather inconveniently, this cannot be reliably used to identify Tregs, since CD25 is also expressed on activated effector T-cells, making identification in inflamed tissues difficult. The most widely accepted marker of natural Tregs is therefore the expression of FOXP3, which is essential for their development, function, and homeostasis [6].

Low levels of circulating CD4+CD25+ T-cells also correlate with a higher disease activity or poor prognosis. It has been proposed that downregulation of Treg cells may be caused by suppressed proliferation of peripheral CD4+ CD25+ T-cells, as noticed in vitro. Thereby, the balance between proinflammatory and Tregs could be disturbed, leading to the breakdown of self-tolerance [7].


  Objective Top


To determine if Treg is involved in COPD and its value in the predilection of COPD development.


  Patients and methods Top


Type, time, and place of the study

This case–control study was conducted at the Chest Diseases Department, Al-Zahraa University Hospital, Cairo, Egypt during the period from August 2017 to December 2018.

Ethical approval

This study was done after approval by the ethics review committee of Al-Azhar University. All data were anonymous and coded to assure confidentiality of participants. Each participant signs informed written consent before enrollment into the study.

Selection of study participants

  1. Thirty known COPD patients attended the chest outpatient clinic for regular follow-up. They were diagnosed based on the GOLD, 2016 criteria [postbronchodilator forced expiratory volume in the first second (FEV1)/ forced vital capacity (FVC) ratio <0.7] and an increase in FEV1 less than 200 ml, or less than 12% of the baseline value 20 min after 200 µg puffs of inhaled Salbutamol (given via a metered-dose inhaler at the time of diagnosis).
  2. Twenty age-matched and sex-matched healthy volunteers serve as a control group. Their spirometric indices and arterial blood gas (ABG) parameters within the normal range with no history of chronic chest diseases.


Exclusion criteria

Patients with known collagen vascular diseases and other chest diseases were excluded from the study.


  Methods Top


The following data were reported age, sex, smoking (status and index), family history of COPD, frequent upper respiratory tract infection, and occupational and/or biomass exposure. Additionally, data regarding frequent use of antibiotic, systemic steroids oral corticosteroids (OCS), and inhaled corticosteroids were collected.

Spirometry was carried out on a Medisoft-Hyperair compact+flow meter pulmonary function testing device. The test procedure was explained in full detail to patients and controls. Short and long-acting β2-agonists and xanthine were withheld before the test for 6, 12, and 24 h, respectively. Exercise, heavy meals, and smoking were avoided 6 h before the test. The FEV1%, FVC%, FEV1/FVC ratio, and forced expiratory flow rate 25–75% were calculated. Spirometric parameters were calculated using the best of three technically acceptable performances in accordance with the recommendations of the European Respiratory Society [8].

ABG: was performed after a 15-min resting period in ambient room air using Gem Premier 3000, blood gas analyzer (Instrumentation Laboratory Company, Lexington, Kentucky, USA) and on 384 (Siemens, Munich, Germany) for detection of oxygen saturation %, PaO2 (mmHg), PaCO2 (mmHg), HCO3 (mEq/L), and pH.

Absolute and percentage lymphocyte count was performed using KX-21N hematology analyzer (Sysmex, Kobe, Japan), an automated hematology cell counter.

Detection of CD4+CD25+ and FOXP3+ T-regulatory cells in peripheral blood by flow cytometry assay

EDTA blood samples were processed within 2 h of collection. A measure of 50 μl of blood was mixed with the following monoclonal antibodies: fluorescein isothiocyanate-conjugated antihuman CD4 (catalog number: AO7750, lot number 100; Immunotech, Beckman Coulter, Marsellia, France) and allo phycoerythrin-conjugated antihuman CD25 (catalog number: FAB1020A, lot number: LXJ0215071; Minneapolis, Minnesota, USA). All monoclonal antibodies were incubated in the dark for 20 min. Then the samples were washed with phosphate-buffered saline (PBS), incubated with a lysing solution for 8 min in the dark and washed again with PBS before detection of nuclear phycoerythrin-conjugated antihuman FOXP3 (catalog number: FC012; R&D systems, Bio-Techne, Minneapolis, USA). All samples were washed two times with cold 1X PBS, resuspended in fresh 1X FOXP3 fixation buffer, incubated at 2–8°C for 30 min. Then all the samples were washed two times with fresh, cold 1X FOXP3 permeabilization and wash buffer; FOXP3 monoclonal antibodies were added to all samples and incubated for 30 min at 2–8°C. Then the samples were washed once with cold 1X FOXP3 permeabilization and wash buffer and resuspended in 200–500 μl PBS. The optimal concentration for each antibody was determined by titration experiment.

Flow cytometry assay

Flow cytometry was conducted at the Clinical Pathology Department of Al-Zahraa Hospital, Al-Azhar University on four colors FACSCalibur (BD, Biosciences, San Jose, California, USA). CellQuest Pro Software (BD Biosciences) was used for data analysis. Compensation setting was established before acquiring the samples using color calibrite beads (lot number 5093879; BD, Biosciences) for unlabeled, FITC, PE color compensation, and (lot number 70689; BD, Biosciences) for APC color compensation. Isotype controls, IgG1a FITC/IgG2 PE, and IgG APC were obtained for the detection of nonspecific binding.

Gating strategy of the proportion of T-regulatory cells

Expression of the proportion of Tregs in CD4+ lymphocyte gate was considered as the region of coexpression of positive staining of surface marker CD25 and intracellular staining of FOXP3, after acquisition was set to 500 000 cells ([Figure 1]).
Figure 1 Detection results of flow cytometry: (a) gating of lymphocytes, (b) gating of CD4+ lymphocytes, (c) healthy control, (d) COPD patient. COPD, chronic obstructive pulmonary disease.

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Statistical analysis

Data were coded and entered using SPSS (the Statistical Package for the Social Sciences), version 25. Data were summarized using mean±SD, and range in quantitative data and using frequency (count) and relative frequency (percentage) for categorical data. Comparisons between quantitative variables were done using the nonparametric Mann–Whitney test. For comparing categorical data, χ2 test was performed. Exact test was used instead when the expected frequency is less than 5. Correlations between quantitative variables were done using the Spearman correlation coefficient. Lymphocytes % of total lymphocyte count (20–40%) was used as a cutoff value for normal lymphocytes % [9]. CD4% of lymphocytes 25–49% for men and 27–54% for women were used as a cutoff value for normal CD4 frequencies [10]. The 1.2% was used as a cutoff value for normal CD4+, 25+FOXP3 (Treg) frequencies [11]. Accordingly, COPD patients were divided into two subgroups; COPD patients with normal Treg frequencies (≥1.2%) and COPD patients with lower Treg frequencies (<1.2%). The odds ratio (OR) was calculated to measure the association between the risk factors and the outcome. Multivariate logistic regression analysis was used to identify the most relevant risk factors affecting Treg frequencies among patients with COPD. The strength of relevance between the risk factors and the outcome was determined according to the value of the beta coefficient (B), and significance according to the Wald χ2 test; also, the OR was calculated. P values less than 0.05 were considered as statistically significant.


  Results Top


[Table 1] shows that in the COPD group there was significantly higher frequency of smokers, family history of COPD, history of recurrent, occupational and/or biomass exposure, higher mean±SD of smoking index, PaCO2, HCO3, and erythrocyte sedimentation rate first hour with significant decrease mean±SD of all spirometric indices, PaO2 and O2 saturation compared with controls.
Table 1 Comparison of all studied variables between chronic obstructive pulmonary disease group and control group

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[Table 2] showed that there was no significant difference between COPD patients and controls in absolute lymphocytic count/cmm3, CD4% of lymphocytes (P>0.05), and CD4% frequencies (P>0.05). However, there was significant decrease lymphocytes % of total lymphocyte count in COPD compared with controls (21.11±7.96 vs. 32.6±7.3, P=0.012), and CD4+, 25+FOXP3 (Treg) (1.10±0.64 vs. 1.9±1.07, P=0.002). There were significantly higher frequencies of lymphopenia in COPD compared with controls (26.7 vs. 0.0%) with lower frequencies of CD4+, CD25+, FOXP3 (Treg) in COPD compared with controls (60.0 vs. 25.0%, P=0.031).
Table 2 Comparison of lymphocytes (absolute count, %, and status), CD4 (% of lymphocytes and status), and CD4+CD24+FOXP3 (T-regulatory cells) between chronic obstructive pulmonary disease group and control group

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[Table 3] shows that COPD duration/year and occupational or biomass exposure were significantly increased in the COPD subgroup with lower Treg frequencies compared with the COPD subgroup with normal Treg frequencies. Additionally, the COPD subgroup with lower frequency of Treg have nonsignificant increase of use of antibiotic and OCS, with similar use of inhaled corticosteroids compared with the COPD subgroup with normal Treg frequency.
Table 3 Comparison of all studied variables between chronic obstructive pulmonary disease subgroup with normal T-regulatory cells frequencies and chronic obstructive pulmonary disease subgroup with low T-regulatory cells frequencies

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[Table 4] shows that the lymphocytes (absolute count, %, and status), CD4 (% of lymphocytes and frequencies), and CD4+CD24+FOXP3 (Treg) did not differ between the COPD subgroup with normal Treg frequencies and COPD subgroup with lower Treg frequencies (P>0.05).
Table 4 Comparison of lymphocytes (absolute count, %, and status), CD4 (% of lymphocytes and status), and CD4+CD24+FOXP3 (T-regulatory cells) between chronic obstructive pulmonary disease subgroup with normal T-regulatory cells frequencies and chronic obstructive pulmonary disease subgroup with low T-regulatory cells frequencies

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[Table 5] shows that the CD4+, 25+FOXP3 Treg was negatively correlated with COPD duration/year and positively correlated with FVC%, FEV1, and FEV1/FVC ratio ([Figure 2]).
Table 5 Correlation of CD4+, 25+FOXP3 T-regulatory cells with all parametric variables among the chronic obstructive pulmonary disease group

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Figure 2 Correlation of CD4+, 25+FOXP3 Treg with COPD duration FEV1/FVC ratio and FVC%, FEV1%. COPD, chronic obstructive pulmonary disease; FEV1, forced expiratory volume in the first second; FVC, forced vital capacity; Treg, T-regulatory cells.

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[Table 6] multivariate logistic regression analysis revealed that the most significant risk factors relevant to lower frequency of Treg in COPD patients was FEV1% (walid=10.85, P=0.001 with OR=2.14), FEV1/FVC ratio (walid=10.06, P=0.001, with OR=2.01), FVC% (walid=9.02, P=0.002 with OR=1.91) COPD duration (walid=4.9, P=0.026 with OR=1.3) followed by occupational or biomass exposure (walid=4.8, P=0.028 with OR=8.0). Additionally, [Table 7] demonstrates that those with lower Treg frequencies have a 2.8-fold risk for the development of COPD compared with those with normal Treg Frequencies (OR=2.8, CI 0.86–9.0)
Table 6 Logistic regression analysis for lower T-regulatory cells cell frequencies in patients with chronic obstructive pulmonary disease

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Table 7 Lower frequencies of CD4+CD25+FOXP3 (T-regulatory cells) as a risk factor for chronic obstructive pulmonary disease

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  Discussion Top


The role of autoimmunity in the pathogenesis of COPD is recently recognized [1]. CD4+CD25+ Treg are critical immunomodulatory cells that maintain balance of the immune system [12].

The main finding of the present study is that the Treg (CD4+CD25+ and FOXP3+) were decreased in COPD patients compared with controls ([Table 2]). Our results are in concordance with Lee et al. [13] who found that CD4+CD25+ Treg and FOXP3 expression decreased in patients with COPD and emphysema. On the other hand, Smyth et al. [14] found that CD4+CD25+ Treg cells increased in patients with COPD and in healthy smokers. The different results refer to different numbers of cases between two studies.

Our results indicate that the CD4+CD25+ Treg cells may protect the lungs from the effect of tobacco smoke components in healthy smokers whereas patients with COPD lack this protecting mechanism. The mechanism by which Treg lymphocytes undergo upregulation in normal smokers and do not in patients with COPD is partially understood [14]. Another important finding of our study is that those with lower Treg frequencies have a 2.8-fold risk for the development of COPD compared with those with normal Treg frequencies (OR=2.8, CI 0.86–9.0) ([Table 7]). With immune anergy and suppressive immunity, CD4+CD25+ Treg cells produce memory cells in the presence of infection signal and inhibit the occurrence of pathological immune response. Our study showed that comparing COPD patients with healthy controls, the expression of Treg FOXP3 was significantly decreased in COPD and there is no significant difference in the percentage of CD4+CD25+ between two groups. The population of Treg cells’ functioning suppressive immunity broke homeostasis which might promote the occurrence and development of COPD. The possible explanation of these findings is that the role of CD4+CD25+ Treg cells in COPD pathogenesis involves the shift from innate immunity to adaptive immunity which mimics autoimmune processes (uncontrolled immune reaction), with increased formation of CXCR3, CXCL10, and interferon gamma. Interaction between these mediators will lead to the induction and activation of MMR2, MMR9 to MMR12, and in turn leads to the degradation of elastin and collagen and subsequently development of emphysema [15].Our study has shown that COPD patients have higher frequencies of lymphopenia with lower frequencies of Treg compared with controls with normal CD4+ ([Table 2]). These findings point out that we cannot rely on the lymphocytic count or CD4+ count as indicators of lower Treg frequencies in patients with COPD. Although the impact of lymphopenia on chronic inflammatory disease has not been well known, lymphopenia was found to be related to all-cause mortality in patients with COPD [16].

Another finding of the current study is that COPD patients with lower Treg frequencies have significant increase of COPD duration and it is a predictive factor of lower Treg frequencies. Moreover, Treg was negatively correlated with COPD duration. This finding points out that patients with lower Treg frequencies (potential autoimmunity) develop COPD earlier in life than those with normal Treg frequencies. Additionally, the higher prevalence of occupational and/or biomass exposure among our patients with lower Treg frequencies indicates that both occupational and/or biomass exposure and lower Treg frequencies could have a synergistic effect for COPD development.

The nonsignificant increase of antibiotics and OCS use among our patients with lower Treg frequencies than those with normal Treg frequencies ([Table 2]) denotes that the potential autoimmunity among these patients could warrant systemic steroids to control underlying inflammation which is further supported by a positive correlation detected between Treg and spirometric indices (FEV1/FVC ratio, FEV1%, and FVC%) in our patients ([Table 5]).

In the current study both COPD subgroups have nearly similar age, sex, smoking index, PFT, and ABG indices so the underlying autoimmunity could not have deleterious effects on COPD patients.

This study is just a snapshot in addressing the issue of underlying autoimmune hypothesis in COPD. However, this study has limitations that needs to be mentioned, first is the small number of study participants. Second, the CD4+, CD4+CD25+ FOXP3 was measured once although the lymphocyte trend is more accurate than single measurement. Third, samples for the assessment of Treg cells obtained from blood and not from the site of inflammation including bronchoalveolar lavage or biopsy. If we believe that COPD has an autoimmune component, then the function of Tregs in the lungs of COPD patients is a crucial issue. Studies so far have focused on Treg expression, rather than function. Lymphoid follicles may be the most physiologically relevant location for Treg function to be studied as follicles are likely to be the major location of antigen presentation. The ethical and technical complexities of applying immunological and molecular techniques to human lung tissue research are not to be underestimated, but the potential rewards in terms of increasing our understanding of autoimmunity in COPD could be high.


  Conclusion Top


Lower frequencies of Treg cells (autoimmunity) might be a risk factor for COPD development in some patients. It was related to longer COPD duration, occupational and/or biomass exposure, and spirometric indices. Further study conducted on a large number of COPD is highly recommended for more declaration of the issue of autoimmunity in COPD.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

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Celli BR, MacNee W, Agusti A, Anzueto A, Berg B, Buist AS et al. Standards for the diagnosis and treatment of patients with COPD: a summary of the ATS/ERS position paper. Eur Respir J 2004; 23:932–946.  Back to cited text no. 1
    
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Curtis JL. Cell-mediated adaptive immune defense of the lungs. Proc Am Thorac Soc 2005; 2:412–416.  Back to cited text no. 2
    
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Bonarius HP, Brandsma CA, Kerstjens HA, Koerts JA, Kerkhof M, Nizankowska-Mogilnicka E et al. Antinuclear autoantibodies are more prevalent in COPD in association with low body mass index but not with smoking history. Thorax 2011; 66:101–107.  Back to cited text no. 4
    
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Margherita M, Campanelli R, Fois G, Villani L, Bonetti E, Catarsi P et al. Reduced frequency of circulating CD41CD25brightCD127lowFOXP31 regulatory T cells in primary myelofibrosis. Blood. 2016; 128:1660–1662.  Back to cited text no. 11
    
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Rabe KF, Hurd S, Anzueto A, Barnes PJ, Buist SA, Calverley P et al. Global Initiativefor Chronic Obstructive Lung Disease. Global strategy for the diagnosis, management, and prevention of chronic obstructive pulmonary disease: GOLD executive summary. Am J Respir Crit Care Med 2007; 176:532–555.  Back to cited text no. 12
    
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Lee SH, Goswami S, Grudo A, Song LZ, Bandi V, Goodnight-White S et al. Antielastin autoimmunity in tobacco smoking-induced emphysema. Nat Med 2007; 13:567.  Back to cited text no. 13
    
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Smyth LJ, Starkey C, Vestbo J, Singh D. CD4-regulatory cells in COPD patients. Chest 2007; 132:156–663.  Back to cited text no. 14
    
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    Figures

  [Figure 1], [Figure 2]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6], [Table 7]



 

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