Human Immunology 75 (2014) 1115–1119
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Foxp3 methylation status in children with primary immune thrombocytopenia Zhenping Chen a,b,c,⇑, Zhenxing Guo d, Jie Ma a,b,c, Jingyao Ma a,b,c, Fuhong Liu a,b,c, Runhui Wu a,b,c,⇑ a Beijing Key Laboratory of Pediatric Hematology Oncology, Hematology Oncology Center, Beijing Children’s Hospital, Capital Medical University, 56 Nanlishi Road, Beijing 100045, China b National Key Discipline of Pediatrics, Ministry of Education, Hematology Oncology Center, Beijing Children’s Hospital, Capital Medical University, 56 Nanlishi Road, Beijing 100045, China c Key Laboratory of Major Diseases in Children, Ministry of Education, Hematology Oncology Center, Beijing Children’s Hospital, Capital Medical University, 56 Nanlishi Road, Beijing 100045, China d Department of Hematology/Oncology, First Hospital of Tsinghua University, Beijing 100016, China
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Article history: Received 19 March 2014 Accepted 27 September 2014 Available online 7 October 2014 Keywords: Children Primary immune thrombocytopenia DNA methylation Foxp3
a b s t r a c t Aim: To investigate the status of DNA methylation in the Foxp3 promoter in pediatric ITP patients and assess the role of DNA methylation of Treg cells in the pathogenesis of ITP. Methods: Quantitative DNA methylation levels of Foxp3 promoter in pediatric ITP patients were detected by MassARRAY EpiTYPER. Methylation levels of Foxp3 promoter were analyzed in ITP patients and normal controls. Results: Significantly higher expression of CpG-2, CpG-3 and CpG-11.12 was observed in ITP patients compared to the controls. A subgroup analysis revealed that persistent and chronic ITP patients exhibited significantly higher CpG-6 expression than in the subgroup of newly diagnosed ITP patients. All patients who represented newly diagnosed ITP at admission were reclassified at later follow-up. In this follow-up subgroup analysis, there were significantly higher levels of CpG-6 in the persistent ITP group than that in the newly diagnosed ITP group. Conclusions: Our results indicate that defective Treg cell activity identified in ITP might be partially mediated through hypermethylation of CpG sites in the promoter region of Foxp3. Ó 2014 Published by Elsevier Inc. on behalf of American Society for Histocompatibility and Immunogenetics.
1. Introduction Primary immune thrombocytopenia (ITP) is an acquired autoimmune bleeding disorder characterized by antiplatelet autoantibody mediated destruction of platelet by the reticuloendothelial system (RES) [1–5]. Although autoreactive B lymphocytes secreting antiplatelet antibodies are considered as the primary immunologic defect in ITP, substantial evidence suggests that dysfunctional cellular immunity plays an important role in the etiology of ITP [6]. It is well known that Treg cells play a crucial role in maintaining selftolerance to prevent the development of autoimmunity [7]. Recently, several studies have shown that Treg cells play an important role in ITP patients. Treg cells in active ITP patients exhibit either defective function or are decreased in number [8–13]. ⇑ Corresponding authors at: Beijing Children’s Hospital, Capital Medical University, 56 Nanlishi Road, Beijing 100045, China. E-mail addresses: [email protected] (Z. Chen), runhuiwu@hotmail. com (R. Wu).
Moreover, B cell-targeted therapy with rituximab in ITP patients can restore the number and regulatory function of Treg cells, which may support a role for Tregs in the aetiology of ITP [14]. However, the precise action of Treg cells in ITP remains unclear. It is widely accepted that the interaction between environmental factors and the immune system plays crucial roles in the development of ITP. Epigenetic mechanisms are known to be important in the regulation of gene modification, and in recent years there has been an increasing focus on epigenetic changes in autoimmune diseases. DNA methylation is an epigenetic process involving in the regulation of many biological events including embryonic development, transcriptional regulation of gene expression, X chromosome inactivation, genomic imprinting, chromatin modification and silencing of endogenous retroviruses [15,16]. DNA methylation patterns are determined by the enzymatic processes of methylation and demethylation. Foxp3 is a member of winged helix/forehead transcription factors and identified as a key marker of Treg cells [17]. Several studies have established that Foxp3 plays an important role for the
http://dx.doi.org/10.1016/j.humimm.2014.09.018 0198-8859/Ó 2014 Published by Elsevier Inc. on behalf of American Society for Histocompatibility and Immunogenetics.
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maintenance and function of Treg cells [18–20]. Recent studies have suggested that epigenetic regulation of CpG methylation in the promoter region plays an essential role in the expression of Foxp3. Zorn et al. demonstrated that demethylation induced by azacytidine (Aza) in human natural killer (NK) cells results in increased Foxp3 expression [21]. In their study, NK cell line were treated with Aza at different doses for 3 days in vitro, and found the demethylating agent significantly increased the expression of Foxp3. Subsequent studies in Treg cells also showed that inhibition of methylation level by Aza induced Foxp3 expression. In contrast, methylation of CpG islands decreased the expression of Foxp3 [22]. Furthermore, the suppressive function of Treg cells was associated with increased methylation of CpG in the promoter region of Foxp3 [23]. Currently the role of Foxp3 methylation in ITP patients remains unclear. Therefore, in this study, we investigate the status of DNA methylation of Foxp3 in pediatric ITP patients to assess the possible role of DNA methylation of Treg cells in the pathogenesis of ITP. 2. Methods and materials 2.1. Study population Patients and control individuals were recruited from our hospital. The subjects included 82 pediatric primary ITP patients from our hospital and 19 normal controls (healthy children who attended our hospital for routine health examination). The diagnosis of primary ITP was based on previously reported criteria [24]. In China, a platelet count below 100 109/L is indicative of thrombocytopenia. Thrombocytopenia diagnosed within 3 months of presentation was defined as newly diagnosed ITP; Thrombocytopenia between 3 and 12 months from diagnosis was defined as persistent ITP; Lasting thrombocytopenia for longer than 12 months was defined as chronic ITP. Informed consents were obtained from the children’s parents or guardian of all subjects and controls. Our hospital-based ethics committee approved the study. 2.2. Isolation of PBMNCs PBMNCs were isolated from heparinized venous blood by density gradient centrifugation over Ficoll-Hypaque gradients and stored at 80 °C until used. 2.3. DNA extraction Genomic DNA of PBMNC cells was extracted using DNeasy Blood & Tissue Kit (QIAGEN, Hilden, Germany) according to the manufacturer’s protocol. DNA concentration and 260:280 absorbance ratios were calculated with a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Montchanin, DE, USA). 2.4. DNA methylation analysis Sequenom MassARRAY platform was used to perform the quantitative methylation analysis of erythroid specific genes [25]. This system uses matrix-assisted laser desorption/ionization time-offlight (MALDI-TOF) mass spectrometry in combination with RNA base-specific cleavage (MassCLEAVE). DNA methylation was analyzed by gene-specific amplification of bisulfite-treated DNA followed by in vitro transcription and MALDI-TOF analysis. DNA methylation standards (0%, 20%, 40%, 60%, 80% and 100%) were used to control the bias of PCR amplification. Correction algorithms based on the R statistical computing environment were used for data normalization.
2.5. Statistical analysis Data were reported as mean ± SD and statistical evaluations were performed using SPSS 16.0 software (SPSS Inc., Chicago, IL, USA). Student’s t-test analysis was used to compare the methylation level of CpG between ITP patients and normal controls as well as the difference between the subgroups of ITP patients. Correlation of DNA methylation levels and clinical parameters were assessed by Pearson correlation. For all statistical analysis, p < 0.05 was considered statistically significant. 3. Results 3.1. Clinical characteristic of ITP patients and controls Of the 82 ITP patients, 29 were girls and 53 boys, median age 52.5 months (range 3–168 months). 49 (59.76%) patients were newly diagnosed ITP, 18 (21.95%) persistent ITP and 15 (18.29%) chronic ITP. At the time of blood sample collection, the median platelet count of patients was 57 109/L (range 2 109/ L–89 109/L). 80 of the 82 patients were followed-up at a minimum of 1 year following first diagnosis, at which time the group was reclassified into 17(20.73%) patients with newly diagnosed ITP, 16(19.51%) patients with persistent ITP (15 from newly diagnosed ITP and 1 from persistent ITP at admission), and 47(57.32%) patients with chronic ITP (17 from newly diagnosed ITP and 15 from persistent ITP at admission). 2 (2.44%) patients in the group of persistent ITP were lost to follow-up. The characteristics of subjects and controls are recorded in Table 1. ITP stage at admission and follow-up is recorded in Table 2. 3.2. Higher methylation level of CpG sites in the Foxp3 promoter in ITP patients By using of MassARRAY EpiTYPER application, methylation levels of CpG sites in the Foxp3 gene were observed in both ITP patients and normal controls. For this study, we searched potential CpG island of the Foxp3 promoter via the http:// cpgislands.usc.edu/ website. However, we found no CpG island to meet our criteria. Consequently we chose a 450 bp transcription Table 1 Clinical characteristic of ITP patients and controls. Subjects
Patients with ITP
Normal controls
Number of cases Gender (M:F) Median age (months) PLT (median)
82 53:29 52.5 (3–168) 57 109/L (2–89 109/L)
19 10:9 84.0 (36–120) 291 (198–309 109/L)
ITP, primary immune thrombocytopenia; PLT, platelet counts at sample collection; M:F, male: female.
Table 2 Clinical staging of ITP patients at admission and at follow-up. At admission
Follow-up
Diagnosis
Number of cases
Diagnosis
Number of cases
Newly diagnosed ITP
49
Newly diagnosed ITP Persistent ITP Chronic ITP
17 15 17
Persistent ITP Chronic ITP Loss of follow-up
1 15 2
Persistent ITP
18
Chronic ITP
15
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Fig. 1. Schematic diagram of CpG sites in the Foxp3 promoter from CpG 1 to CpG11–12 (left to right). Sequenom MassARRAY was used to perform the analysis the quantitative methylation level in the Foxp3 promoter. Each CpG site of the target region and their positive sequences are shown. The CpG site labeled in red is not covered (CpG-5).
Fig. 2. Significantly higher expression of CpG-2, CpG-3 and CpG-11.12 in ITP patients compared to the control group. Error bars represent SD. p values were calculated by Student’s t-test analysis. ⁄p < 0.05, ⁄⁄p < 0.01.
initiation site near the Foxp3 promoter as a target sequence which included twelve different CpG sites labeled as CpG-1, CpG-2, CpG3, CpG-4, CpG-5, CpG-6, CpG-7, CpG-8.9, CpG-10, and CpG-11.12 (Fig. 1). Our data suggested that there was significant higher expression of CpG-2 in ITP patients compared to the control group (relative value 0.5940 ± 0.0729 vs. 0.5389 ± 0.0586, p < 0.01) (Fig. 2). Similar results were found for CpG-3 (relative value 0.4921 ± 0.1103 vs. 0.4211 ± 0.0825, p < 0.05) and CpG11.12 (0.7461 ± 0.0831 vs. 0.7016 ± 0.1004, p < 0.05) (Fig. 2). However, there was no significant difference in expression of the other CpG sites between the two groups (data not shown). 3.3. Follow-up: different methylation levels of Foxp3 in the subgroups of ITP patients In the subgroup analysis at follow-up, we found CpG-6 levels were expressed at significantly higher levels in the persistent ITP group (PITP) and the chronic ITP group (CITP) compared to the newly diagnosed ITP group (NITP) (methylation level PITP 0.9406 ± 0.0384 vs. NITP 0.8894 ± 0.0712; p < 0.05 and (CITP 0.9377 ± 0.0560 vs. NITP 0.8894 ± 0.0712; p < 0.01) (Fig. 3). However, there was no difference in expression of CpG-6 between persistent ITP group and chronic ITP group (relative value 0.9406 ± 0.0384 vs. 0.9377 ± 0.0560, p = 0.857). In addition, no significant difference in methylation at the other CpG sites was found among the three groups (data not shown). 3.4. Different methylation level of Foxp3 in newly diagnosed ITP patients at admission At follow-up, we divided the 49 newly diagnosed ITP patients at admission into three groups: newly diagnosed ITP (nNITP), persistent ITP (nPITP) and chronic ITP (nCITP). In the subgroup analysis,
Fig. 3. Different methylation level in the subgroup of ITP patients by follow-up. ‘‘NITP’’ = newly diagnosed ITP, ‘‘PITP’’ = persistent ITP, ‘‘CITP’’ = chronic ITP. p values were calculated by Student’s t-test analysis. ⁄p < 0.05, ⁄⁄p < 0.01, NS denotes no significant statistical difference.
we found there was significantly higher methylation of CpG-6 in the nPITP group than that in the nNITP group (methylation level 0.9380 ± 0.0382 vs. 0.8894 ± 0.0712, p < 0.05). However, no significant difference in CpG-6 methylation was observed between the nCITP and nNITP groups (relative value 0.9165 ± 0.0719 vs. 0.8894 ± 0.0712, p = 0.219) or nCITP and nPITP groups (relative value 0.9165 ± 0.0719 vs. 0.9380 ± 0.0382, p = 0.342) (Fig. 4). Also, no significant difference in methylation at the other CpG sites was found among the three groups (data not shown). 3.5. Methylation level of Foxp3 in chronic ITP patients At follow-up, we divided chronic ITP patients into three groups: nCITP (chronic ITP from newly diagnosed ITP at admission); pCITP (chronic ITP from persistent ITP at admission); cCITP (chronic ITP at admission). A greater tendency for CpG-6 methylation was observed in the pCITP group than that in nCITP group (relative value 0.9527 ± 0.0269 vs. 0.9165 ± 0.0719, p = 0.07), however this did not reach the statistical difference (Fig. 5). In addition, there was no significant difference in methylation level at the other Foxp3 CpG sites among the three groups (data not shown). 3.6. Correlation between methylation level in Foxp3 promoter and clinical parameters Because of higher expression of CpG in ITP patients, we investigated potential associations between degree of methylation at sites CpG-1–11.12 in the Foxp3 promoter with clinical parameters, such as platelet count at sample collection, number of CD4+ T cells,
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Fig. 4. Different methylation levels of Foxp3 in newly diagnosed ITP patients at admission. By follow-up, newly diagnosed ITP patients at admission were divided into three groups: newly diagnosed ITP (nNITP), persistent ITP (nPITP) and chronic ITP (nCITP). p values were calculated by Student’s t-test analysis. ⁄p < 0.05, NS denotes no significant statistical difference.
Fig. 5. Different methylation levels of Foxp3 in chronic ITP patients. nCITP, chronic ITP from newly diagnosed ITP at admission; pCITP, chronic ITP from persistent ITP at admission; cCITP, chronic ITP at admission. p values were calculated by Student’s t-test analysis.
CD8+ T cells, B lymphocytes and NK cells. No significant correlations were found (data not shown).
4. Discussion ITP is recognized as an acquired autoimmune disorder mediated by the RES and is mainly mediated by abnormal B lymphocytes. However, increasing evidence also suggests an important role for T lymphocytes in the pathophysiology of ITP. It is well recognized that although many studies support Th1 polarization of the immune response in ITP [26–31], others studies have found inconsistent [32]or contradictory [33] results. Subsequently, Treg cells are identified as important in the pathogenesis of ITP, albeit by a currently unknown mechanism. Increasing evidences show that genomic-wide hypomethylation can lead to inappropriate gene expression and contribute to the pathogenesis of some autoimmune diseases such as systemic lupus erythematosus (SLE), rheumatoid arthritis (RA) [34,35]. Our previous studies showed that increased plasma S-adenosylhomocysteine (SAH) concentration and decreased expression levels of DNMT3A, 3B, MBD2 and MBD4 might contribute to the pathophysiology of
ITP [36,37]. In addition, similar reductions in DNMT3A expression were identified in Egyptian patients with ITP [38]. These publications support the implications of our data, namely that DNA methylation is a potential mechanism in the pathophysiology of ITP. Although previous studies indicated an importance role for Treg cells in ITP, little is known about the role of DNA methylation of Treg cells in ITP patients. In this study, by using of MassARRAY EpiTYPER application, we found higher methylation levels of the CpG sites in the Foxp3 promoter in ITP patients at sites might play an important role in the pathogenesis of ITP. Mechanistically, DNA methylation change is thought to alter transcriptional regulation of gene expression. Hypermethylation usually leads to impaired gene expression. Our study firstly provides evidence to support a degree of hypermethylation of the Foxp3 promoter in ITP patients, which may explain previous reports of low expression of Foxp3 in ITP patients. Based on these results, it would be reasonable to hypothesize that impaired Treg cells result from hypermethylation of Foxp3, and this is closely related to the development of ITP. In order to investigate the potential role of CPG in different phases of ITP, we detected recorded methylation levels at CpG sites of the Foxp3 region in subgroups of ITP patients. Our results showed that significantly higher levels of CpG6 methylation were found in the persistent ITP and chronic ITP groups than that in newly diagnosed ITP group, which indicated CpG6 might be a focus for further research in an attempt to define markers to predict the outcome in newly diagnosed ITP. However, a rational explanation for the specific role of CpG6 remains elusive. As the largest children’s hospitals in China, we receive many ITP patients, especially those refractory to treatment elsewhere came to our hospital. At follow-up, we found newly diagnosed ITP patients at admission could be classified into three groups, including newly diagnosed ITP, persistent ITP and chronic ITP. Further subgroup analysis suggested significantly higher levels of methylated CpG-6 occurred in the persistent ITP group than that in the newly diagnosed ITP group, which is consistent with the of CPG6 results in different phases of ITP at follow-up. Our data suggested that CpG-6 might be considered as an important prognostics feature to predict whether newly diagnosed ITP patients at admission are at risk of developing into persistent ITP. Chronic ITP remains a major clinical problem. Because the majority of patients in our study became chronic ITP sufferers, we considered it important to analyse methylation levels of Foxp3 in chronic ITP patients. By the follow-up, we divided chronic ITP patients into three groups: chronic ITP from newly diagnosed ITP, chronic ITP from persistent ITP at admission and chronic ITP at admission. Although there was a greater tendency for increased methylation of CpG-6 in the pCITP group than that in nCITP group, no significant difference in Foxp3 CpG methylation was observed among the three groups, which indicated that methylation level of Foxp3 in chronic ITP was relatively stable over time. We recognize the need to increase the samples numbers for further study. Furthermore, hypermethylation of Foxp3 in ITP prompted us to investigate the relationship between CpG island of Foxp3 and clinical parameters. However, no significant correlations between the level of CpG and several clinical parameters including platelets counts at sample collection, number of CD4+ T cells, CD8+ T cells, B lymphocytes and NK cells was found. It is therefore likely that other mechanisms might be involved in determining the severity of ITP beyond Foxp3 methylation. In conclusion, our study demonstrated that pediatric ITP patients have a hypermethylation pattern in several CpG sites of the Foxp3 promoter, so that the Foxp3 promoter might be involved in the pathogenesis of ITP. In the future, further research remains necessary to investigate the roles of hypermethylation in CD4+ T cells in ITP patients.
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Authors’ contributions Z.C. performed and analyzed the experiments, analyzed data, and wrote the paper; Z.G. analyzed data and reviewed paper, J.M., J.M. and F.L. collected patient material, R.W. coordinated the study, analyzed and interpreted the data. Acknowledgments The authors would like to thank Dr. Daniel Edward Porter (Department of Orthopaedic Surgery, University of Edinburgh, Royal Hospital for Sick Children, UK) for critical review of the manuscript. This work was supported in part by grants from the National Natural Science Foundation of China – China (No. 81200351, No. 81000228), Beijing Natural Science Foundation of China – China (No. 7112050, No. 7122065). Beijing Municipal Administration of Hospitals Clinical medicine Development of special funding support – China, code ZY201404. References [1] McMillan R. Chronic idiopathic thrombocytopenic purpura. N Engl J Med 1981;304:1135–47. [2] Cines DB, Blanchette VS. Immune thrombocytopenic purpura. N Engl J Med 2002;346:995–1008. [3] Karpatkin S. Autoimmune thrombocytopenic purpura. Blood 1980;56:329–43. [4] Kelton JG, Gibbons S. Autoimmune platelet destruction: idiopathic thrombocytopenic purpura. Semin Thromb Hemost 1982;8:83–104. [5] Bussel JB, Cheng G, Saleh MN, Psaila B, Kovaleva L, Meddeb B, et al. Eltrombopag for the treatment of chronic idiopathic thrombocytopenic purpura. N Engl J Med 2007;357:2237–47. [6] Zhou B, Zhao H, Yang RC, Han ZC. Multi-dysfunctional pathophysiology in ITP. Crit Rev Oncol Hematol 2005;54:107–16. [7] Andre S, Tough DF, Lacroix-Desmazes S, Kaveri SV, Bayry J. Surveillance of antigen-presenting cells by CD4+ CD25+ regulatory T cells in autoimmunity: immunopathogenesis and therapeutic implications. Am J Pathol 2009;174:1575–87. [8] Liu B, Zhao H, Poon MC, Han Z, Gu D, Xu M, et al. Abnormality of CD4(+)CD25(+) regulatory T cells in idiopathic thrombocytopenic purpura. Eur J Haematol 2007;78:139–43. [9] Ling Y, Cao X, Yu Z, Ruan C. Circulating dendritic cells subsets and CD4+Foxp3+ regulatory T cells in adult patients with chronic ITP before and after treatment with high-dose dexamethasone. Eur J Haematol 2007;79:310–6. [10] Sakakura M, Wada H, Tawara I, Nobori T, Sugiyama T, Sagawa N, et al. Reduced Cd4+Cd25+ T cells in patients with idiopathic thrombocytopenic purpura. Thromb Res 2007;120:187–93. [11] Yu J, Heck S, Patel V, Levan J, Yu Y, Bussel JB, et al. Defective circulating CD25 regulatory T cells in patients with chronic immune thrombocytopenic purpura. Blood 2008;112:1325–8. [12] Aboul-Fotoh Lel M, Abdel Raheem MM, El-Deen MA, Osman AM. Role of T cells in children with idiopathic thrombocytopenic purpura. J Pediatr Hematol Oncol 2011;33:81–5. [13] Teke HU, Gunduz E, Akay OM, Gulbas Z. Abnormality of regulatory T-cells in remission and non-remission idiopathic thrombocytopaenic purpura patients. Platelets 2013;24:625–31. [14] Stasi R, Cooper N, Del Poeta G, Stipa E, Laura Evangelista M, Abruzzese E, et al. Analysis of regulatory T-cell changes in patients with idiopathic thrombocytopenic purpura receiving B cell-depleting therapy with rituximab. Blood 2008;112:1147–50. [15] Surani MA. Imprinting and the initiation of gene silencing in the germ line. Cell 1998;93:309–12. [16] Ng HH, Bird A. DNA methylation and chromatin modification. Curr Opin Genet Dev 1999;9:158–63.
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Foxp3 methylation status in children with primary immune thrombocytopenia Zhenping Chen a,b,c,⇑, Zhenxing Guo d, Jie Ma a,b,c, Jingyao Ma a,b,c, Fuhong Liu a,b,c, Runhui Wu a,b,c,⇑ a Beijing Key Laboratory of Pediatric Hematology Oncology, Hematology Oncology Center, Beijing Children’s Hospital, Capital Medical University, 56 Nanlishi Road, Beijing 100045, China b National Key Discipline of Pediatrics, Ministry of Education, Hematology Oncology Center, Beijing Children’s Hospital, Capital Medical University, 56 Nanlishi Road, Beijing 100045, China c Key Laboratory of Major Diseases in Children, Ministry of Education, Hematology Oncology Center, Beijing Children’s Hospital, Capital Medical University, 56 Nanlishi Road, Beijing 100045, China d Department of Hematology/Oncology, First Hospital of Tsinghua University, Beijing 100016, China
a r t i c l e
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Article history: Received 19 March 2014 Accepted 27 September 2014 Available online 7 October 2014 Keywords: Children Primary immune thrombocytopenia DNA methylation Foxp3
a b s t r a c t Aim: To investigate the status of DNA methylation in the Foxp3 promoter in pediatric ITP patients and assess the role of DNA methylation of Treg cells in the pathogenesis of ITP. Methods: Quantitative DNA methylation levels of Foxp3 promoter in pediatric ITP patients were detected by MassARRAY EpiTYPER. Methylation levels of Foxp3 promoter were analyzed in ITP patients and normal controls. Results: Significantly higher expression of CpG-2, CpG-3 and CpG-11.12 was observed in ITP patients compared to the controls. A subgroup analysis revealed that persistent and chronic ITP patients exhibited significantly higher CpG-6 expression than in the subgroup of newly diagnosed ITP patients. All patients who represented newly diagnosed ITP at admission were reclassified at later follow-up. In this follow-up subgroup analysis, there were significantly higher levels of CpG-6 in the persistent ITP group than that in the newly diagnosed ITP group. Conclusions: Our results indicate that defective Treg cell activity identified in ITP might be partially mediated through hypermethylation of CpG sites in the promoter region of Foxp3. Ó 2014 Published by Elsevier Inc. on behalf of American Society for Histocompatibility and Immunogenetics.
1. Introduction Primary immune thrombocytopenia (ITP) is an acquired autoimmune bleeding disorder characterized by antiplatelet autoantibody mediated destruction of platelet by the reticuloendothelial system (RES) [1–5]. Although autoreactive B lymphocytes secreting antiplatelet antibodies are considered as the primary immunologic defect in ITP, substantial evidence suggests that dysfunctional cellular immunity plays an important role in the etiology of ITP [6]. It is well known that Treg cells play a crucial role in maintaining selftolerance to prevent the development of autoimmunity [7]. Recently, several studies have shown that Treg cells play an important role in ITP patients. Treg cells in active ITP patients exhibit either defective function or are decreased in number [8–13]. ⇑ Corresponding authors at: Beijing Children’s Hospital, Capital Medical University, 56 Nanlishi Road, Beijing 100045, China. E-mail addresses: [email protected] (Z. Chen), runhuiwu@hotmail. com (R. Wu).
Moreover, B cell-targeted therapy with rituximab in ITP patients can restore the number and regulatory function of Treg cells, which may support a role for Tregs in the aetiology of ITP [14]. However, the precise action of Treg cells in ITP remains unclear. It is widely accepted that the interaction between environmental factors and the immune system plays crucial roles in the development of ITP. Epigenetic mechanisms are known to be important in the regulation of gene modification, and in recent years there has been an increasing focus on epigenetic changes in autoimmune diseases. DNA methylation is an epigenetic process involving in the regulation of many biological events including embryonic development, transcriptional regulation of gene expression, X chromosome inactivation, genomic imprinting, chromatin modification and silencing of endogenous retroviruses [15,16]. DNA methylation patterns are determined by the enzymatic processes of methylation and demethylation. Foxp3 is a member of winged helix/forehead transcription factors and identified as a key marker of Treg cells [17]. Several studies have established that Foxp3 plays an important role for the
http://dx.doi.org/10.1016/j.humimm.2014.09.018 0198-8859/Ó 2014 Published by Elsevier Inc. on behalf of American Society for Histocompatibility and Immunogenetics.
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maintenance and function of Treg cells [18–20]. Recent studies have suggested that epigenetic regulation of CpG methylation in the promoter region plays an essential role in the expression of Foxp3. Zorn et al. demonstrated that demethylation induced by azacytidine (Aza) in human natural killer (NK) cells results in increased Foxp3 expression [21]. In their study, NK cell line were treated with Aza at different doses for 3 days in vitro, and found the demethylating agent significantly increased the expression of Foxp3. Subsequent studies in Treg cells also showed that inhibition of methylation level by Aza induced Foxp3 expression. In contrast, methylation of CpG islands decreased the expression of Foxp3 [22]. Furthermore, the suppressive function of Treg cells was associated with increased methylation of CpG in the promoter region of Foxp3 [23]. Currently the role of Foxp3 methylation in ITP patients remains unclear. Therefore, in this study, we investigate the status of DNA methylation of Foxp3 in pediatric ITP patients to assess the possible role of DNA methylation of Treg cells in the pathogenesis of ITP. 2. Methods and materials 2.1. Study population Patients and control individuals were recruited from our hospital. The subjects included 82 pediatric primary ITP patients from our hospital and 19 normal controls (healthy children who attended our hospital for routine health examination). The diagnosis of primary ITP was based on previously reported criteria [24]. In China, a platelet count below 100 109/L is indicative of thrombocytopenia. Thrombocytopenia diagnosed within 3 months of presentation was defined as newly diagnosed ITP; Thrombocytopenia between 3 and 12 months from diagnosis was defined as persistent ITP; Lasting thrombocytopenia for longer than 12 months was defined as chronic ITP. Informed consents were obtained from the children’s parents or guardian of all subjects and controls. Our hospital-based ethics committee approved the study. 2.2. Isolation of PBMNCs PBMNCs were isolated from heparinized venous blood by density gradient centrifugation over Ficoll-Hypaque gradients and stored at 80 °C until used. 2.3. DNA extraction Genomic DNA of PBMNC cells was extracted using DNeasy Blood & Tissue Kit (QIAGEN, Hilden, Germany) according to the manufacturer’s protocol. DNA concentration and 260:280 absorbance ratios were calculated with a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Montchanin, DE, USA). 2.4. DNA methylation analysis Sequenom MassARRAY platform was used to perform the quantitative methylation analysis of erythroid specific genes [25]. This system uses matrix-assisted laser desorption/ionization time-offlight (MALDI-TOF) mass spectrometry in combination with RNA base-specific cleavage (MassCLEAVE). DNA methylation was analyzed by gene-specific amplification of bisulfite-treated DNA followed by in vitro transcription and MALDI-TOF analysis. DNA methylation standards (0%, 20%, 40%, 60%, 80% and 100%) were used to control the bias of PCR amplification. Correction algorithms based on the R statistical computing environment were used for data normalization.
2.5. Statistical analysis Data were reported as mean ± SD and statistical evaluations were performed using SPSS 16.0 software (SPSS Inc., Chicago, IL, USA). Student’s t-test analysis was used to compare the methylation level of CpG between ITP patients and normal controls as well as the difference between the subgroups of ITP patients. Correlation of DNA methylation levels and clinical parameters were assessed by Pearson correlation. For all statistical analysis, p < 0.05 was considered statistically significant. 3. Results 3.1. Clinical characteristic of ITP patients and controls Of the 82 ITP patients, 29 were girls and 53 boys, median age 52.5 months (range 3–168 months). 49 (59.76%) patients were newly diagnosed ITP, 18 (21.95%) persistent ITP and 15 (18.29%) chronic ITP. At the time of blood sample collection, the median platelet count of patients was 57 109/L (range 2 109/ L–89 109/L). 80 of the 82 patients were followed-up at a minimum of 1 year following first diagnosis, at which time the group was reclassified into 17(20.73%) patients with newly diagnosed ITP, 16(19.51%) patients with persistent ITP (15 from newly diagnosed ITP and 1 from persistent ITP at admission), and 47(57.32%) patients with chronic ITP (17 from newly diagnosed ITP and 15 from persistent ITP at admission). 2 (2.44%) patients in the group of persistent ITP were lost to follow-up. The characteristics of subjects and controls are recorded in Table 1. ITP stage at admission and follow-up is recorded in Table 2. 3.2. Higher methylation level of CpG sites in the Foxp3 promoter in ITP patients By using of MassARRAY EpiTYPER application, methylation levels of CpG sites in the Foxp3 gene were observed in both ITP patients and normal controls. For this study, we searched potential CpG island of the Foxp3 promoter via the http:// cpgislands.usc.edu/ website. However, we found no CpG island to meet our criteria. Consequently we chose a 450 bp transcription Table 1 Clinical characteristic of ITP patients and controls. Subjects
Patients with ITP
Normal controls
Number of cases Gender (M:F) Median age (months) PLT (median)
82 53:29 52.5 (3–168) 57 109/L (2–89 109/L)
19 10:9 84.0 (36–120) 291 (198–309 109/L)
ITP, primary immune thrombocytopenia; PLT, platelet counts at sample collection; M:F, male: female.
Table 2 Clinical staging of ITP patients at admission and at follow-up. At admission
Follow-up
Diagnosis
Number of cases
Diagnosis
Number of cases
Newly diagnosed ITP
49
Newly diagnosed ITP Persistent ITP Chronic ITP
17 15 17
Persistent ITP Chronic ITP Loss of follow-up
1 15 2
Persistent ITP
18
Chronic ITP
15
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Fig. 1. Schematic diagram of CpG sites in the Foxp3 promoter from CpG 1 to CpG11–12 (left to right). Sequenom MassARRAY was used to perform the analysis the quantitative methylation level in the Foxp3 promoter. Each CpG site of the target region and their positive sequences are shown. The CpG site labeled in red is not covered (CpG-5).
Fig. 2. Significantly higher expression of CpG-2, CpG-3 and CpG-11.12 in ITP patients compared to the control group. Error bars represent SD. p values were calculated by Student’s t-test analysis. ⁄p < 0.05, ⁄⁄p < 0.01.
initiation site near the Foxp3 promoter as a target sequence which included twelve different CpG sites labeled as CpG-1, CpG-2, CpG3, CpG-4, CpG-5, CpG-6, CpG-7, CpG-8.9, CpG-10, and CpG-11.12 (Fig. 1). Our data suggested that there was significant higher expression of CpG-2 in ITP patients compared to the control group (relative value 0.5940 ± 0.0729 vs. 0.5389 ± 0.0586, p < 0.01) (Fig. 2). Similar results were found for CpG-3 (relative value 0.4921 ± 0.1103 vs. 0.4211 ± 0.0825, p < 0.05) and CpG11.12 (0.7461 ± 0.0831 vs. 0.7016 ± 0.1004, p < 0.05) (Fig. 2). However, there was no significant difference in expression of the other CpG sites between the two groups (data not shown). 3.3. Follow-up: different methylation levels of Foxp3 in the subgroups of ITP patients In the subgroup analysis at follow-up, we found CpG-6 levels were expressed at significantly higher levels in the persistent ITP group (PITP) and the chronic ITP group (CITP) compared to the newly diagnosed ITP group (NITP) (methylation level PITP 0.9406 ± 0.0384 vs. NITP 0.8894 ± 0.0712; p < 0.05 and (CITP 0.9377 ± 0.0560 vs. NITP 0.8894 ± 0.0712; p < 0.01) (Fig. 3). However, there was no difference in expression of CpG-6 between persistent ITP group and chronic ITP group (relative value 0.9406 ± 0.0384 vs. 0.9377 ± 0.0560, p = 0.857). In addition, no significant difference in methylation at the other CpG sites was found among the three groups (data not shown). 3.4. Different methylation level of Foxp3 in newly diagnosed ITP patients at admission At follow-up, we divided the 49 newly diagnosed ITP patients at admission into three groups: newly diagnosed ITP (nNITP), persistent ITP (nPITP) and chronic ITP (nCITP). In the subgroup analysis,
Fig. 3. Different methylation level in the subgroup of ITP patients by follow-up. ‘‘NITP’’ = newly diagnosed ITP, ‘‘PITP’’ = persistent ITP, ‘‘CITP’’ = chronic ITP. p values were calculated by Student’s t-test analysis. ⁄p < 0.05, ⁄⁄p < 0.01, NS denotes no significant statistical difference.
we found there was significantly higher methylation of CpG-6 in the nPITP group than that in the nNITP group (methylation level 0.9380 ± 0.0382 vs. 0.8894 ± 0.0712, p < 0.05). However, no significant difference in CpG-6 methylation was observed between the nCITP and nNITP groups (relative value 0.9165 ± 0.0719 vs. 0.8894 ± 0.0712, p = 0.219) or nCITP and nPITP groups (relative value 0.9165 ± 0.0719 vs. 0.9380 ± 0.0382, p = 0.342) (Fig. 4). Also, no significant difference in methylation at the other CpG sites was found among the three groups (data not shown). 3.5. Methylation level of Foxp3 in chronic ITP patients At follow-up, we divided chronic ITP patients into three groups: nCITP (chronic ITP from newly diagnosed ITP at admission); pCITP (chronic ITP from persistent ITP at admission); cCITP (chronic ITP at admission). A greater tendency for CpG-6 methylation was observed in the pCITP group than that in nCITP group (relative value 0.9527 ± 0.0269 vs. 0.9165 ± 0.0719, p = 0.07), however this did not reach the statistical difference (Fig. 5). In addition, there was no significant difference in methylation level at the other Foxp3 CpG sites among the three groups (data not shown). 3.6. Correlation between methylation level in Foxp3 promoter and clinical parameters Because of higher expression of CpG in ITP patients, we investigated potential associations between degree of methylation at sites CpG-1–11.12 in the Foxp3 promoter with clinical parameters, such as platelet count at sample collection, number of CD4+ T cells,
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Fig. 4. Different methylation levels of Foxp3 in newly diagnosed ITP patients at admission. By follow-up, newly diagnosed ITP patients at admission were divided into three groups: newly diagnosed ITP (nNITP), persistent ITP (nPITP) and chronic ITP (nCITP). p values were calculated by Student’s t-test analysis. ⁄p < 0.05, NS denotes no significant statistical difference.
Fig. 5. Different methylation levels of Foxp3 in chronic ITP patients. nCITP, chronic ITP from newly diagnosed ITP at admission; pCITP, chronic ITP from persistent ITP at admission; cCITP, chronic ITP at admission. p values were calculated by Student’s t-test analysis.
CD8+ T cells, B lymphocytes and NK cells. No significant correlations were found (data not shown).
4. Discussion ITP is recognized as an acquired autoimmune disorder mediated by the RES and is mainly mediated by abnormal B lymphocytes. However, increasing evidence also suggests an important role for T lymphocytes in the pathophysiology of ITP. It is well recognized that although many studies support Th1 polarization of the immune response in ITP [26–31], others studies have found inconsistent [32]or contradictory [33] results. Subsequently, Treg cells are identified as important in the pathogenesis of ITP, albeit by a currently unknown mechanism. Increasing evidences show that genomic-wide hypomethylation can lead to inappropriate gene expression and contribute to the pathogenesis of some autoimmune diseases such as systemic lupus erythematosus (SLE), rheumatoid arthritis (RA) [34,35]. Our previous studies showed that increased plasma S-adenosylhomocysteine (SAH) concentration and decreased expression levels of DNMT3A, 3B, MBD2 and MBD4 might contribute to the pathophysiology of
ITP [36,37]. In addition, similar reductions in DNMT3A expression were identified in Egyptian patients with ITP [38]. These publications support the implications of our data, namely that DNA methylation is a potential mechanism in the pathophysiology of ITP. Although previous studies indicated an importance role for Treg cells in ITP, little is known about the role of DNA methylation of Treg cells in ITP patients. In this study, by using of MassARRAY EpiTYPER application, we found higher methylation levels of the CpG sites in the Foxp3 promoter in ITP patients at sites might play an important role in the pathogenesis of ITP. Mechanistically, DNA methylation change is thought to alter transcriptional regulation of gene expression. Hypermethylation usually leads to impaired gene expression. Our study firstly provides evidence to support a degree of hypermethylation of the Foxp3 promoter in ITP patients, which may explain previous reports of low expression of Foxp3 in ITP patients. Based on these results, it would be reasonable to hypothesize that impaired Treg cells result from hypermethylation of Foxp3, and this is closely related to the development of ITP. In order to investigate the potential role of CPG in different phases of ITP, we detected recorded methylation levels at CpG sites of the Foxp3 region in subgroups of ITP patients. Our results showed that significantly higher levels of CpG6 methylation were found in the persistent ITP and chronic ITP groups than that in newly diagnosed ITP group, which indicated CpG6 might be a focus for further research in an attempt to define markers to predict the outcome in newly diagnosed ITP. However, a rational explanation for the specific role of CpG6 remains elusive. As the largest children’s hospitals in China, we receive many ITP patients, especially those refractory to treatment elsewhere came to our hospital. At follow-up, we found newly diagnosed ITP patients at admission could be classified into three groups, including newly diagnosed ITP, persistent ITP and chronic ITP. Further subgroup analysis suggested significantly higher levels of methylated CpG-6 occurred in the persistent ITP group than that in the newly diagnosed ITP group, which is consistent with the of CPG6 results in different phases of ITP at follow-up. Our data suggested that CpG-6 might be considered as an important prognostics feature to predict whether newly diagnosed ITP patients at admission are at risk of developing into persistent ITP. Chronic ITP remains a major clinical problem. Because the majority of patients in our study became chronic ITP sufferers, we considered it important to analyse methylation levels of Foxp3 in chronic ITP patients. By the follow-up, we divided chronic ITP patients into three groups: chronic ITP from newly diagnosed ITP, chronic ITP from persistent ITP at admission and chronic ITP at admission. Although there was a greater tendency for increased methylation of CpG-6 in the pCITP group than that in nCITP group, no significant difference in Foxp3 CpG methylation was observed among the three groups, which indicated that methylation level of Foxp3 in chronic ITP was relatively stable over time. We recognize the need to increase the samples numbers for further study. Furthermore, hypermethylation of Foxp3 in ITP prompted us to investigate the relationship between CpG island of Foxp3 and clinical parameters. However, no significant correlations between the level of CpG and several clinical parameters including platelets counts at sample collection, number of CD4+ T cells, CD8+ T cells, B lymphocytes and NK cells was found. It is therefore likely that other mechanisms might be involved in determining the severity of ITP beyond Foxp3 methylation. In conclusion, our study demonstrated that pediatric ITP patients have a hypermethylation pattern in several CpG sites of the Foxp3 promoter, so that the Foxp3 promoter might be involved in the pathogenesis of ITP. In the future, further research remains necessary to investigate the roles of hypermethylation in CD4+ T cells in ITP patients.
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