|Year : 2020 | Volume
| Issue : 2 | Page : 138-144
Interplaying of regulatory T-cells and related chemokines in immune thrombocytopenic purpura patients
Zeyad A Shabeeb1, Yusur F Faraj1, Majed M Mahmood2, Baan Abdulatif Mtashar1
1 National Center of Hematology, Mustansiriyah University, Baghdad, Iraq
2 Department of Immunology, College of Science, Mustansiriyah University, Baghdad, Iraq
|Date of Submission||08-May-2020|
|Date of Acceptance||16-Sep-2020|
|Date of Web Publication||10-Nov-2020|
Dr. Zeyad A Shabeeb
National Center of Hematology, Mustansiriyah University, Baghdad
Source of Support: None, Conflict of Interest: None
BACKGROUND: Chronic immune thrombocytopenic purpura (ITP) is an immune-mediated bleeding disorder, in which platelets are opsonized by autoantibodies directed against platelet surface membrane glycoproteins, and prematurely cleared and destructed by Fc-receptors on the surface of macrophages in the reticuloendothelial system.
OBJECTIVES: This work is designed to show the contribution of lymphocyte subsets and platelet destruction in adult chronic ITP and role of infection.
MATERIALS AND METHODS: Transforming growth factor-β1 (TGF-β1) was measured using ELISA, and the frequency of regulatory T-cell (Treg) profile (CD4+CD25+CD127−) was investigated by FCM in blood samples of 50 Iraqi ITP patients (35 on-treatment ITP patients and 15 newly diagnosed) along with 20 age-matched healthy people that act as controls, as well as all patients were breath tested for detecting Helicobacter pylori using urea breath test. The study was carried out in the National Center of Hematology, Mustansiriyah University.
RESULTS: The results showed that although there was a significant reduction in Treg number in ITP patients compared with the control individuals (P < 0.001), the effect of treatment has shown a restored count of Tregs in comparison to the newly diagnosed ones (P = 0.002), while the assessment of cytokine serum level revealed that TGF-β1 was significantly increased (P = 0.001) in the on-treatment group of patients (TGF-β1 = 3.24 ± 0.3 ng/μl) in comparison with the nontreated group of patients (TGF-β1 = 1.75 ± 0.2 ng/μl). However, it was still significantly (P < 0.001) less than their values in the apparently healthy individuals (TGF-β1 = 9.0 ± 0.2 ng/μ). Moreover, 25 out of 50 (50%) showed positive results for the presence of H. pylori.
CONCLUSION: The present study revealed that Treg and its cytokines may play a fundamental role in the pathophysiology of adult chronic ITP since they contribute to the maintenance of peripheral immune tolerance. However, a causal link between H. pylori infection and ITP diseases is considerable.
Keywords: Flow cytometry, H. pylori, immune thrombocytopenic purpura, regulatory T-cells, transforming growth factor-β1
|How to cite this article:|
Shabeeb ZA, Faraj YF, Mahmood MM, Mtashar BA. Interplaying of regulatory T-cells and related chemokines in immune thrombocytopenic purpura patients. Iraqi J Hematol 2020;9:138-44
|How to cite this URL:|
Shabeeb ZA, Faraj YF, Mahmood MM, Mtashar BA. Interplaying of regulatory T-cells and related chemokines in immune thrombocytopenic purpura patients. Iraqi J Hematol [serial online] 2020 [cited 2021 Mar 6];9:138-44. Available from: https://www.ijhonline.org/text.asp?2020/9/2/138/300419
| Introduction|| |
Immune thrombocytopenic purpura (ITP) is an autoimmune bleeding disorder characterized by thrombocytopenia with platelet counts <100 × 109/L, in which patient's immune system is activated by platelet autoantigens resulting in immune-mediated platelet destruction and/or suppression of platelet production. ITP affects people of both genders at all ages. It is estimated to affect approximately 3.3/100,000 adults per year, higher in women than in men worldwide. Two distinct clinical syndromes manifest as an acute condition in children and a chronic condition in adults. The acute form often follows an infection and spontaneously resolves within 2 months. Chronic immune thrombocytopenia persists longer than 6 months, with a specific cause being unknown.,
Although ITP patients may be asymptomatic, clinical features of ITP, including skin petechiae and bleeding in the mucous membranes or internal organs, are easily manifested if the platelet count falls below (20 × 109/L). The diagnosis of ITP depends on clinical characteristics and the laboratory examinations conducted, as well as the ability of excluding other agents associated with thrombocytopenia.,
The mechanism of ITP is multifactorial. It has been found that the loss of tolerance resulting from a decreased number and defective function of regulatory T-cells (Tregs) plays an important part in the progression of the disease. Moreover, a role for cytotoxic T-cells in direct lysis of platelets and megakaryocytes in the bone marrow has been proposed., The high-level expression of the CD4 and CD25 surface markers of Tregs and the production of transforming growth factor-β1 (TGF-β1) suppress the proliferation of many immune cell types including T- and B-cells, either directly through cell contact or indirectly through secretion of cytokines, thereby dampening inappropriate immune activation and autoreactivity.
Since TGF-β1 is a critical regulator of thymic T-cell development and differentiation during the immune response as well as a crucial player in peripheral T-cell homeostasis and tolerance to self-antigens, therefore, its association in ITP is thought to be as a potent inhibitor of megakaryocyte maturation, and thus, its level has been inversely correlated with the disease activity.
In order to evaluate the role of immune cells and their cytokines in the pathogenesis of ITP, this study was designed to investigate the level of CD4+, CD25+, and CD127-profile markers to determine the percentage of regulatory T-lymphocyte using FCM technique, TGF-β1 cytokine to determine the activity of Tregs using ELISA assay. In addition, the present study aimed to evaluate the association of ITP disorder with some infectious diseases like Helicobacter pylori infection.
| Materials and Methods|| |
A control-based study has been carried out on Iraqi patients with ITP in the National Center of Hematology, Mustansiriyah University, Baghdad. Fifty patients are enrolled in this study (39 females and 11 males), with age ranging from 15 to 70 years; 35 of them were diagnosed as chronic ITP, with a history of disease from few months to several years, while 15 were newly diagnosed. Along with the patient group, 20 healthy controls with matched gender and age were involved and considered as a control group. This study is approved by the Ethical Committee of the National Center of Hematology, Mustansiriyah University, and appropriate patient and research study participant consent is obtained.
Parameters of study
patients were subjected to full medical history and complete clinical examination and clinical signs of ITP. All patients were investigated by complete blood picture (Convergence, Germany) and blood film, immunophenotyping profile of Tregs (CD4+CD25+CD127−) and serum level of TGF-β1, as well as the detection of H. pylori.
Blood samples were collected from all individuals (healthy controls and patients). About 5–10 ml of blood was aspirated using peripheral vein punctures and divided into 2 aliquots; the first one is transferred into EDTA tube for direct examination of CD markers, complete blood picture, and blood film. The second was dispensed in a nonheparinized plain tube and left for 15 min at 4°C to clot; then, it was centrifuged at 3000 rpm for 10 min to collect serum which stored in −80˚C until be used for determination of TGF-β1 cytokine. However, breath samples were obtained only from the (50) patients to identify infections by H. pylori.
In this study, immunophenotyping CD4+, CD25+, and CD127− (BD Biosciences, Germany) expression were investigated using fully equipped desktop four-color flow cytometry (FCM) (Partec, Germany). CyFlow Cube features a modular optical concept. This allows using different lasers as light sources. The CyFlow Cube allows easy optimization of the optics for any application by simple exchange of optical filters and mirrors.
One hundred microliters (μl) of whole blood or isolated leukocytes was mixed with 10 μl of conjugated antibodies in a test tube, mixed thoroughly, and then incubated for 15 min in the dark at room temperature.
From reagent A, 100 μl was mixed and incubated for 10 min in the dark at room temperature.
From reagent B, 2.5 ml were added and shaked gently and incubated for 20 min in dark at room temperature.
The sample was then analyzed by flow cytometer.
Calculation of results
Data acquisition, instrument control, and data analysis are controlled and performed by the CyView software (Cylab, USA).
Estimation of transforming growth factor-β1
It was performed according to TGF-β1 ELISA kit (Kombiotech, South Korea) using ELISA reader (Linear, China).
Determination of Helicobacter pylori
The test was estimated using H. pylori analyzer with breath card (Shenzhen Headway, China). the method as follows: Patients swallowed urea labelled with an uncommon isotope, non-radioactive carbon-13. In the subsequent 15 minutes, the detection of isotope-labelled carbon dioxide in exhaled breath indicates that the urea was split; this indicates that urease (the enzyme that H. pylori uses to metabolize urea) is present in the stomach, and hence that H. pylori bacteria are present. The device principle is based on the production of β ray by the nuclide radioactive decay on sample card when reaches the detector, it creates electrical pulse signal. The system processes the signal and then gives out the diagnostic results (negative or positive). In the meantime, the results are displayed on LCD display and are printed out through the printer.
| Results|| |
The two groups of patients and controls were matched based on age, gender, and their family history, as shown in [Table 1].
|Table 1: Characterization of controls and patients according to age, gender, family history, and treatment|
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Two immunological parameters were investigated in all healthy and patient participants; the immunophenotypic profile of Tregs and TGF-β1. Changes in concentration of these parameters in different settings and their significance were recorded and are shown in [Table 2].
|Table 2: Evaluation of immunological parameters (Tregs and TGF-β1) in all patent groups and controls|
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To reveal whether inefficient production of Tregs contributes to loss of peripheral tolerance among patients with chronic ITP, immunophenotyping profile represented by circulating CD4+CD25+CD127-cells were investigated, and the results came to state that the frequency of Tregs was diminished significantly in ITP patients compared to their counterparts of the control group (0.92 ± 0.1 vs. 4.80% ± 0.3%, P < 0.001). Blood samples obtained from the on-treatment patients have shown a significant higher Treg percentages (1.24% ± 0.2%) in comparison to the nontreated levels (0.23% ± 0.1%, P = 0.002) [Figure 1].
|Figure 1: Percentages of regulatory T-cells in treated and nontreated groups of patients compared to healthy control group|
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As the platelet counts reflect the disease activity and it is closely related to platelet destruction, the correlation between Tregs and the platelet counts was examined. The result showed a positive nonsignificant relationship in the control group (P = 0.13, r = +0.35) and patient group (P = 0.58, r = +0.08) [Figure 2].
|Figure 2: Correlative analysis of regulatory T-cell percentage and platelet counts (a) in control group (b) in patient group|
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To estimate the possible role of immune regulatory cytokine, TGF-β1, the level of this cytokine was evaluated in the sera of ITP patients as well as in healthy controls. The presented results indicated that TGF-β1 (2.75 ± 0. 25 vs. 9.00 ± 0.21) ng/ml was significantly decreased in ITP patients compared to the control values (P < 0.0001) which could not be achieved even in treated patients (TGF-β1 = 3.24 ± 0.33 ng/ml) in spite of the significant increase in its level in comparison to the nontreated patients (TGF-β1 = 1.75 ± 0.69 ng/ml, P = 0.001), as shown in [Figure 3].
|Figure 3: Levels of transforming growth factor-β1 in treated and nontreated groups of patients compared to healthy control group|
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All patients were breath tested to indicate the presence of H. pylori infection. Half of the patients (50%) were infected with bacteria and developed chronic ITP.
| Discussion|| |
The exact mechanism of the immune dysfunction in ITP is generally not well known, but a number of T-cell abnormalities have been demonstrated in patients with ITP. These T-cell abnormalities may be characterized by abnormal numbers and functions of Tregs., Tregs were expressing a panel of CD4+CD25+CD127−, and they secrete regulatory cytokines such as interleukin (IL)-10 and TGF-β1 to induce hemostasis and maintain peripheral immune tolerance.
In the current study, the results have demonstrated that Tregs were reduced in number in ITP patients compared to healthy individuals, while the on-treatment group of patients has shown higher levels in comparison to the nontreated (newly diagnosed) individuals. Consistent with these results, there are many reports describing reduced numbers of Tregs in adult ITP patients.,,, Furthermore, many studies reported a highly significant decrease in the percentage of Tregs in children with acute ITP compared with controls., Possible reasons for decreased Treg numbers can be due to impaired development, survival, proliferation, and/or stability of Tregs, while Wu et al.'s study has shown that the percentages of CD4+CD25+CD127− cells were almost stable when determined by flow cytometry between ITP patients and healthy controls.
Moreover, Yu et al. also found a comparable frequency of circulating CD4+CD25+Foxp3 + Tregs between the patients and the controls, so they suggested that functional defects, not the frequency, in Tregs contribute to the breakdown of self-tolerance in patients with chronic ITP., The defects in Treg function may be explained by failed cell contact-dependent suppression or reduced secretion of cytokines including IL-10, TGF-β1, or IL-35 that mediate suppression. Reduced Treg activity may also be due to increased resistance of effector T-cells to suppression. Whereas many other studies concluded that both Treg frequency and their functional characteristics were defective in ITP patients and this might be responsible for loss of self-tolerance and subsequently destructive immune responses observed in ITP patients. Meanwhile, researchers  mentioned that impaired regulatory compartment, including Tregs and Bregs, has been reported leading to immune dysregulation in ITP patients. In response to dexamethasone therapy, a similar increase in Treg percentages was highlighted by others. The same treatment-induced upregulation was found by Chun-Yan et al. but with a higher level than the healthy controls, whereas using another protocol of treatment (rituximab), another study recorded that the elevation was not significantly different between patients in remission and controls. All these results were contradicted with what was studied by Wu et al. who did not reach a significant change among the three groups (pretreatment patients, posttreatment patients, and healthy control).
In contrast to this, Bakara et al. have found a significant positive correlation between Treg percentage and platelet counts in acute ITP patients indicating a close association between Treg percentage and the parameters known to reflect the degree of platelet destruction. Meanwhile, defective Treg function and number may be explained by reduced secretion of cytokines that mediate suppression including IL-10, TGF-β, or IL-35. In the current study, TGF-β1 was not significantly correlated with platelet counts, and then, it could affect the relation between Tregs and platelet counts, as shown in [Figure 3]. These findings raise the possibility that Tregs may regulate the disease phenotype, particularly in relation to the degree of thrombocytopenia. Furthermore, Zhang et al. found that the percentage of circulating Tregs may be decreased during active disease and the extent of this decrease correlates with the severity of the disease.
TGF-β1 is a central player in maintaining the immune response balance, which belongs to regulator T-cell cytokine. TGF-β1 was found to be an important inhibitor of B-cell proliferation and autoantibody production. It also suppresses some Th1 and Th2 cell-mediated autoimmune diseases., The dominant function of TGF-β1 is to regulate peripheral immune homeostasis. This cytokine is considered as an additional mechanism responsible for peripheral tolerance. Accordingly, it seems that abnormal production of TGF-β1 by Tregs may represent additional mechanisms responsible for deleterious immune reactions occurring in ITP patients.
This result may suggest that TGF-β1 low levels might be inversely correlated with the disease progression, and its protective effects against ITP development cannot be ignored, this might give a hope for a new strategy in the ITP treatment since it has no cure, and relapses may occur years after seemingly successful medical or surgical management.
These findings were in concordance with several previous reports which have shown that the levels of TGF-β1 cytokine were reduced in ITP patients,,,, while Panitsas et al. showed that although patients tended to have lower circulating TGF-β1 levels, the difference was not significant.
In respect to the response to treatment, treated patients show significantly higher levels of TGF-β1 compared to nontreated ones, yet they were significantly lower than the healthy controls. Such findings support some other results found by Guo et al. who recorded similar patterns of response of TGF-β1 levels, yet Li et al. have stated that the treatment-induced increment was significantly higher than the healthy group., This inversely related relationship between TGF-β1 and the disease progression might provide an idea of producing drugs that stimulate TGF-β1 secretion for ITP treatment.
This study showed that circulating TGF-β1 levels are strongly correlated with the platelet counts in the healthy control group (P = 0.01, r = +0.59), and this correlation turned out to be weak and nonsignificant in patient groups (newly diagnosed and on-treatment patients) (P = 0.95, r = +1.52; P = 0.7, r = 0.07), respectively [Figure 4].
|Figure 4: Correlative analysis of transforming growth factor-β1 levels and platelet counts (a) in control group (b) in nontreated patients (c) in the on-treatment group of patients|
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While, in a study done by Bao et al., the correlative analysis indicated a strong positive correlation between the levels of TGF-β1 and the degree of improvement in platelet counts.
| Conclusion|| |
The present study revealed that Treg and its cytokines may play a fundamental role in the pathophysiology of adult chronic ITP since they contribute to the maintenance of peripheral immune tolerance. However, because of the high ratio of patients revealing positive urea breath test, a causal link between H. pylori infection and ITP can be suggested.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4]
[Table 1], [Table 2]