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 Table of Contents  
ORIGINAL ARTICLE
Year : 2017  |  Volume : 30  |  Issue : 3  |  Page : 906-911

Assessment of T-regulatory cells in type 1 diabetes mellitus


1 Department of Clinical Pathology, Faculty of Medicine, Menoufia University, Menoufia, Egypt
2 Department of Pediatrics, Faculty of Medicine, Menoufia University, Menoufia, Egypt

Date of Submission21-Aug-2016
Date of Acceptance06-Nov-2016
Date of Web Publication15-Nov-2017

Correspondence Address:
Samar S Salman
Shibin El-Kom, Menoufia, 32511
Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/1110-2098.218280

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  Abstract 

Objective
The aim of the study was to evaluate T-regulatory cells (Tregs) expression in the peripheral blood of children with type 1 diabetes mellitus (T1D).
Background
T1D is mainly a T-cell-mediated autoimmune disease characterized by the destruction of pancreatic β cells leading to insulin deficiency. It is a common autoimmune disorder in childhood, but the disease may become manifest at any age, even in adults. Tregs are subsets of T cells that have an essential role in maintaining tolerance; thus, these cells may play an important role in the pathogenesis of T1D.
Patients and methods
This study was carried out in the Clinical Pathology Department, Faculty of Medicine, Menoufia University, between August 2014 and November 2015. The study included 50 children, who included 30 children diagnosed as T1D and 20 age-matched and sex-matched apparently healthy children as controls. All children were subjected to complete blood count, glycated hemoglobin evaluation, and surface and cytoplasmic detection of Tregs by flow cytometry.
Results
This study showed that Tregs (CD4+ CD25+ FoxP3+) decreased in diabetic children in comparison with normal controls (P < 0.001). It also showed a higher decrease in the percentage of Tregs (CD4+ CD25+ FoxP3+) in uncontrolled diabetic children (hemoglobin A1c > 7.0%) in comparison with controlled diabetic children (hemoglobin A1c < 7.0%) (P < 0.001).
Conclusion
Diminished Tregs proved that breakdown of immune tolerance often leads to the development of autoimmune diseases including T1D, which confirms the essential role of Tregs in the pathogenesis of T1D.

Keywords: autoimmune disease, immunotherapy, tolerance, T-regulatory cells, type. 1 diabetes mellitus


How to cite this article:
El-Edel RH, Kamal Eldein SM, Abo El Fotoh WM, Salman SS. Assessment of T-regulatory cells in type 1 diabetes mellitus. Menoufia Med J 2017;30:906-11

How to cite this URL:
El-Edel RH, Kamal Eldein SM, Abo El Fotoh WM, Salman SS. Assessment of T-regulatory cells in type 1 diabetes mellitus. Menoufia Med J [serial online] 2017 [cited 2019 Nov 21];30:906-11. Available from: http://www.mmj.eg.net/text.asp?2017/30/3/906/218280




  Introduction Top


Diabetes mellitus is a common chronic, metabolic syndrome that results in hyperglycemia as a cardinal biochemical feature. Type 1 diabetes mellitus (T1D) is the most common type of diabetes in children and adolescents. T1D is caused by deficiency of insulin secretion due to pancreatic β-cell damage. Most cases of T1D are primarily due to T-cell-mediated pancreatic islet β-cell destruction, which occurs at a variable rate [1].

The highest annual increase in the incidence of T1D in children and aggressive process of β-cell destruction indicate the need to know the different causes of the pathogenesis of the disease [2].

The immune system has developed different mechanisms for maintaining self-tolerance known as 'recessive and dominant tolerance'. 'Recessive tolerance' is performed by negative selection of high-affinity clones in the thymus, whereas 'dominant tolerance' is mediated by specialized cells in the periphery, called 'regulatory T-cells' (Tregs) [3].

Several subtypes of Tregs have been identified, the most prominent among them being CD4+, CD25+, and FoxP3+ Tregs, also called natural Tregs (nTregs). The nTregs develop in the thymus and have a central role in the regulation of the immune response in various conditions like allergic diseases, human malignancies, infections, and autoimmune disorders. Several regulatory mechanisms have been attributed to Tregs, in which both direct cell-to-cell contact as well as production of soluble cytokines has been highlighted [4].

Extensive studies have shown that Tregs are crucial for controlling T1D development. Although the exact pathogenic mechanisms leading to T1D still remain largely unclear, two different hypotheses have been proposed. First, it has been hypothesized that the presence of a defective Treg population in T1D patients contributes to diabetes development. In contrast, the second hypothesis suggests that effector T cells (Teffs) in T1D patients are more resistant to Treg suppression, which may contribute to the onset of T1D [5]. In this study we tried to assess Tregs in T1D to identify their role in the disease as several studies have found significantly decreased Tregs in children with T1D.


  Participants and Methods Top


This study was carried out in the Clinical Pathology Department, Faculty of Medicine, Menoufia University, between August 2014 and November 2015. The age of the children ranged from 3 to 9 years. The study included 50 children who were divided into two groups:

  • Group 1 included 30 children who were diagnosed with T1D. They were divided into two subgroups according to the value of glycated hemoglobin: group 1a, the controlled group (HbA1c < 7.0%) and group 1b, the uncontrolled group (HbA1c > 7.0%)
  • Group 2 included 20 age-matched and sex-matched apparently healthy controls.


The children were subjected to the following after taking consent from their legal guardians: (a) estimation of random blood glucose levels; (b) evaluation of glycated hemoglobin (HbA1c) (for diabetic patients); (c) complete blood count; and (d) detection of CD4+ CD25+ FoxP3+ Tregs by flow cytometry. The methodology adopted was as follows:

  • Five milliliters of venous blood samples were taken by sterile venipuncture after minimal venous stasis using sterile disposable syringes. The samples were divided into two tubes: 2 ml was taken in a plain tube for evaluation of random blood glucose levels and 3 ml was taken in another tube containing K-EDTA salt as the anticoagulant for HbA1c, complete blood count, and CD markers
  • Complete blood profile was determined using CELL DYN Ruby (Abbott Laboratories, Santa Clara, California, USA)
  • Random blood glucose was evaluated using Synchron CX9 Autoanalyser (Beckman, Fullerton, California, USA)
  • Glycated hemoglobin was evaluated using ion exchange resin method (Milipore, Bedford, MA, U.S.A)
  • Flow cytometric analysis of Tregs was carried out using the following reagents:


CD3 PE mouse (antihuman) antibodies (BD Bioscience, New Jersey, USA; catalog no. 345766), CD4 fluorescein isothiocyanate (FITC) mouse (antihuman) antibodies (BD Bioscience; catalog no. A10997), CD8 FITC mouse (antihuman) antibodies (BD Bioscience; catalog no. A07757), CD25 FITC mouse (antihuman) antibodies (BD Bioscience; catalog no. 13009), FoxP3 PE mouse (antihuman) antibodies (IPEX, JM2, scurfin) (BD Bioscience; catalog no. 560046), CD4 PerCP mouse (antihuman) antibodies (BD Bioscience; catalog no. 345770), IntraPrep (leukocytic permeabilization reagent (BD Bioscience; catalog no. B61411AA).

A set of tubes was assigned to every patient and control. Five microliter of both CD3 and CD4 FITC monoclonal antibody were added into the first tube. In the second tube 5 μl of both CD3 and CD8 FITC monoclonal antibody was added; 5 μl of isotope control (Ig) was added into the third tube; the fourth tube was an empty tube for negative control. The fifth tube contained 5 μl of both CD4 PerCP plus CD25 FITC monoclonal antibody. A volume of 100 μl of peripheral blood was added to every tube (after adjusting the total leukocyte count to 10 000/cm). The tubes were vortexed gently and incubated for 30 min in the dark at room temperature (18–25°C). The cells were subjected to red blood cell lysis with 2 ml of lysing solution for 3 min. Centrifugation at 300g for 5 min was performed and the supernatant was removed. The cells were washed three times with PBS.

The fifth tube was then fixed with 100 μl of Cell Fix 1× (BD Biosciences) and kept at 4°C in the dark for 15 min. The cells were washed three times with PBS. A volume of 0.5 μl of FoxP3 PE monoclonal antibody and 100 μl of perm B2× (BD Biosciences) were added to the same tubes and they were incubated for 15 min in the dark at room temperature (18–25°C). The cells were washed three times with PBS. The samples were run in FACS (Becton Dickinson TriTEST immunofluorescence reagent package insert; Becton Dickinson, San Jose, CA) Caliber and then gating was performed on CD4. The coexpression of CD25 and FoxP3 (Treg cells) was then analyzed. In the gated population, the percentage of positive cells was ascertained by dual platform technique using cell quest as shown in [Figure 1] and [Figure 2].
Figure 1: Flow cytometric analysis of CD3+, CD4+, and CD8+ cells. (a) forward-scatter/side-scatter analysis of peripheral blood gates around the lymphocyte population (R1). (b) Analysis of lymphocyte population (R1) as regards CD3 PE and CD4 FITC. (c) Analysis of lymphocyte population (R1) as regards CD3 PE and CD8 FITC. FITC, fluorescein isothiocyanate.

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Figure 2: Flow cytometric analysis of CD4+, CD25+, and Fox3+ cells (Treg cells). (a) FSC/SSC analysis of peripheral blood gates around the lymphocyte population (R1). (b) Analysis of lymphocyte population (R1) as regards CD4 PerCP+ cells (R2). (c) Analysis of CD4 PerCP+ cells (R2) as regards CD25 FITC and FoxP3 PE (R3) represent CD4+ CD25+ cells and (R4) represent CD4+ CD25+ FoxP3+. FITC, fluorescein isothiocyanate.

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Statistical analysis

Data were collected and analyzed using SPSS V.20 (SPSS Inc., Chicago, Illinois, USA). Both Student's t-test and the Mann–Whitney test were conducted for quantitative variables and the c2-test for qualitative variables with a significance level of P value less than 0.05. Spearman's correlation was used when these data were not normally distributed.


  Results Top


In this study the mean age of diabetic children was 6.45 ± 3.1 years. They consisted of 14 boys and 16 girls. The mean age of controls was 6.35 ± 2.85 years. They consisted of 10 boys and 10 girls [Table 1].
Table 1: Sociodemographic data and anthropometric measurements of the studied groups

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There was no significant difference between cases and controls with respect to hemoglobin, platelets, white blood cells (WBCs), and lymph%. Results showed that the number of T-helper cells (CD4+ CD25+%) was lower in diabetic children than in controls, with highly significant difference between them (P < 0.001). Comparison between diabetic patients and controls regarding CD3+% and CD4+% showed significant difference. CD4+ CD25+ FoxP3+ (Tregs) was also decreased in diabetic children compared with controls, with highly significant difference (P < 0.001) [Table 2] and [Figure 3]).
Table 2: Comparison between diabetic children and controls as regards their laboratory investigation and CD markers

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Figure 3: Comparison between CD4+ CD25+ FoxP3+% in diabetic children and that in the healthy group. FITC, fluorescein isothiocyanate.

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No significant difference was observed between controlled diabetic and uncontrolled diabetic patients as regards platelets, WBC counts, and hemoglobin level. As regards CD3+%, CD4+%, and T-helper% there was significant difference between the controlled and uncontrolled groups, as shown in [Table 3]. The mean Tregs% in controlled patients was higher (1.7 ± 0.23) in comparison with that in uncontrolled patients (0.49 ± 0.26), with highly significant difference (P = 0.0001) [Table 3]. The result showed positive correlation between lymph%, T-helper cells, and Tregs. It also showed negative correlation between random blood glucose level, HbA1C, WBCs, and Tregs, as shown in [Table 4].
Table 3: Comparison between controlled and uncontrolled diabetic patients as regards their laboratory investigation and CD markers

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Table 4: Spearman's correlation between Treg cells (CD4+CD25+FoxP3+%) and other studied parameters

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  Discussion Top


Insulin-dependent diabetes mellitus is an autoimmune disease in which pancreatic beta cells are destroyed by autoreactive T lymphocytes. It is a multifactorial disorder, and a close interaction of genetic and environmental factors underlies the disease pathogenesis [5]. The present study included 30 diabetic children; their mean age was 6.45 ± 3.1 years (14 boys and 16 girls). They were compared with 20 healthy age-matched children (10 boys and 10 girls). In this study the anthropometric measures of diabetic patients were in the average percentiles (5th–95th). In contrast, Khadilkar et al. [6] stated that in Indian T1D patients, particularly in those diagnosed at a younger age, growth is likely to be affected, and they suggested that intensive insulin therapy may improve the growth of these children over a period of time. The percentage of consanguinity in diabetic patients in this study was 21.4%. This is in agreement with the results of Abd Elaziz et al. [7], who found that consanguinity rate was 27.3% among diabetic children. The majority of uncontrolled diabetic children presented clinically with diabetic ketoacidosis (coma) (87.5% of the diabetic group). This was in agreement with the results of Ghali and El-Dayem [8], who reported that diabetic ketoacidosis was prevalent in 80% of Egyptian T1D patients.

In this study significant decrease was found in cases more than in controls as regards T-helper cells%. Lindley et al. [9] agreed with the results of this study. They attributed this to the reduced ability to inhibit T-cell proliferation in adults with newly diagnosed T1D. In contrast, Abd Elaziz et al. [7] showed no significant difference. He attributed his finding to the increased CD4+ CD25+ expression in inflammatory and autoimmune diseases by activated Teff. Factors related to these differences among studies include the definition of CD25+ cells, disease duration, and, most importantly, appropriate matching for age and HLA type in the control population. Another reason is that CD25 is expressed by both activated and Tregs.

Assessment of Tregs showed highly significant difference between cases and controls (P < 0.001) with more decreased Tregs% in cases than in controls, as Tregs have a crucial rule in the suppression of autoimmune response. Glisic-Milosavljevic et al. [10] showed similar results, which were attributed to the enhanced Tregs apoptosis by the effect of autoantibodies. Bart and Timothy [11] made a similar observation. They stated that as the disease develops the number of FoxP3+ Treg cells decreases and their function is progressively lost at the site of inflammation and a concomitant rise in the number and function of pathogenic TH1 and TH17 autoreactive T cells occurs. Moreover, Brusko et al. [12] showed that the frequency of Tregs defined by the frequency of total FoxP3+ T cells, CD25+ FoxP3+ T cells, or the absolute number of FoxP3+ T cells did not differ significantly as a function of T1D state. In contrast, Szypowska et al. [13] showed that there was no difference in the frequency of apoptotic CD4+ CD25 high FoxP3+ cells between diabetic children under the age of 5 and age-matched healthy controls. They suggested that the loss of function is mainly attributable to the emerging Teffs being resistant to suppression.

This study also showed decreased T-helper cells% and Tregs% in the uncontrolled group compared with the controlled group, with highly significant difference (P < 0.001). This is in agreement with the findings of Ibrahim et al. [14], who attributed it to greater suppression of tolerance and aggressive autoimmune mechanism in the uncontrolled group. No significant correlation between age and Tregs (CD4+ CD25+ FoxP3+%) was detected in this study. This is in agreement with the results of Szypowska et al. [13], who attributed it to the fact that the process of autoimmunity starts before the appearance of symptoms. The significant negative correlation found in the present study between HbA1c and Tregs is confirmed by the study conducted by Farres et al. [15], denoting the occurrence of more autoimmune response in uncontrolled diabetic patients.

The positive correlation found between T-helper cells% and Tregs% can be explained by the fact that Tregs are subsets of T-helper cells.


  Conclusion Top


In this study, we found that the percentage of Tregs is lower in cases than in controls. Also, the percentage of Tregs was lower in uncontrolled diabetic children than in controlled diabetic children. This proved that Treg cells have a crucial role in the pathogenesis of the disease. The next challenge is to expand these cells and use them as immunotherapy to address patient and disease heterogeneity and tissue specificity of immune modulation.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

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Abd Elaziz DS, Hafez MH, Galal NM, Meshaal SS, El Marsafy MA. CD4+ CD25+ cells in type 1 diabetic patients with other autoimmune manifestations. J Adv Res 2013; 5:647–655.  Back to cited text no. 7
    
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Glisic-Milosavljevic S, Waukau J, Jailwala P, Jana S, Khoo H-J, Albertz H, et al. At risk and recent-onset type 1 diabetic subjects have increased apoptosis in the CD4+ CD25+high T cell fraction. PLoS One 2007; 2:e146.  Back to cited text no. 10
    
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Bart OR, Timothy IM. Immune modulation in humans: implications for type 1 diabetes mellitus. Nat Rev Endocrinol 2014; 10:229–242.  Back to cited text no. 11
    
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Brusko T, Wasserfall C, Mcgrail K, Schatz R. No alterations in the frequency of FOXP3+ regulatory T-cells in type 1 diabetes. Diabetes 2007; 56:604–612.  Back to cited text no. 12
    
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Szypowska A, Stelmaszczyk-Emmel A, Demkow U, Łuczyński W. Low frequency of regulatory T cells in the peripheral blood of children with type 1 diabetes diagnosed under the age of five, Arch Immunol Ther Exp (Warsz) 2012; 60:307-313.  Back to cited text no. 13
    
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Ibrahim WE, Mohamed HG, Ali HH, El-Sharnoby EE. Cell-mediated immunity in recent-onset type 1 diabetic children. Egypt J Pediatr Allergy Immunol 2008; 6:69-76.  Back to cited text no. 14
    
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Farres MN, Abo-Ali FH, Shahin RY, Abdel-Rehim AS, Eid Y, Mohamed NA, Gendi MG Regulatory T cells in type 2 diabetes mellitus: are they playmakers? Int J Adv Res Biol Sci 2015; 2:116–123.  Back to cited text no. 15
    


    Figures

  [Figure 1], [Figure 2], [Figure 3]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4]



 

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