Promoter Hypermethylation of GATA3, IL-4, and TGF-β Confers Susceptibility to Vogt-Koyanagi-Harada Disease in Han Chinese

Abstract

Purpose: We investigated the role of promoter methylation of transcriptional and inflammatory factors, including TBX21, GATA3, RORγt, FOXP3, IFN-γ, IL-4, IL-17A, and TGF-β in the development of Vogt-Koyanagi-Harada (VKH) disease.

Methods: The promoter methylation levels were detected by the Sequenom MassARRAY system in CD4⁺ T cells that were separated from 20 healthy individuals and 32 VKH patients (20 in the active stage without medication, 12 in inactive stage with medication). The mRNA expression level of GATA3, IL-4, and TGF-β in CD4⁺ T cells was analyzed by real-time RT-PCR.

Results: The promoter methylation levels of GATA3, IL-4, and TGF-β were significantly higher in active VKH patients than in healthy individuals (P < 0.05). A decreased mRNA expression of GATA3 and TGF-β was found in active VKH patients, which was correlated negatively with the DNA methylation of these factors. Treatment with systemic corticosteroid and cyclosporin A (CsA) decreased the methylation level of GATA3 and TGF-β in association with an increased mRNA expression of molecules and reduced disease activity.

Conclusions: Our findings suggest that promoter hypermethylation of GATA3 and TGF-β in CD4⁺ T cells confers risk to VKH disease in Han Chinese.

Uveitis, an inflammatory disease of the uvea, is considered to be one of the important causes of blindness in the world.^1,2 Vogt-Koyanagi-Harada (VKH) disease is a common uveitis entity in Asians.^3,4 It is a multisystemic disease characterized by bilateral panuveitis and other systemic involvements, such as vitiligo, and auditory and central nervous system damage.^5,6 Even though the etiology and pathogenesis of VKH disease remain unclear, T-cell–driven cellular immune responses have been recognized to have an important role in its pathogenesis.^7–9 

Various studies have found that cytokines are crucial in the occurrence and development of VKH disease.^7,10,11 Upregulated expression of IFN-γ, IL-17, TBX21, and RORγt in peripheral blood mononuclear cells (PBMCs) was detected in active VKH patients,^10,12–14 suggesting that Th1 and Th17 lymphocytes are associated with this disease. 

Cyclosporin A, which is an immunosuppressive drug, and corticosteroids are extensively used in the treatment of VKH disease.^15,16 After treatment with CsA and corticosteroids, decreased expression of IFN-γ, IL-17, TBX21, and RORγt, and fewer Th1 and Th17 cells were observed in these VKH patients.¹⁴ Correspondingly, the immunoregulatory cytokine TGF-β was increased in VKH patients during an inactive phase of the disease.¹⁷ Furthermore, it was shown that the regulatory T cells were diminished in frequency and function in patients with active VKH uveitis.¹⁸ 

DNA methylation is an important epigenetic mechanism, which has been proven to encode inheritable information that can affect gene function without changing the DNA sequence.^19,20 DNA methylation is known to be crucial in the regulation of gene expression, and is involved in the regulation of the immune response²¹ and cytokine expression during T-cell differentiation.^22,23 Early studies have shown that as a naïve CD4⁺ T-cell differentiated into a Th1 cell, increased IFN-γ expression was associated with reduced DNA methylation level of the IFN-γ promoter region, and that IL-4 silencing was related to DNA methylation of GATA3 and IL-4 genes.^24,25 

As methylation of CpG dinucleotides is a widely accepted phenomenon occurring during the immune response, attention recently has focused on the role of epigenetic alterations in the development of autoimmune disease.^26–29 A growing body of literature has demonstrated the relationship between impaired T-cell DNA methylation and some forms of autoimmune disease.^30–33 A significant hypomethylation of T-cell DNA has been demonstrated in patients with active systematic lupus erythematosus (SLE) and rheumatoid arthritis (RA), suggesting that T-cell mediated autoimmunity contributes to the pathogenesis of these diseases.³⁴ Other studies demonstrated that the global methylation levels of CD4⁺T cells were significantly upregulated in patients with psoriasis, particularly in the promoter regions.³⁵ Whether DNA methylation at the promoter region of inflammatory and transcriptional factors in CD4⁺T cells is associated with the pathogenesis of uveitis has not yet been addressed and, therefore, was the theme of this study. Vogt-Koyanagi-Harada disease was chosen as a model of uveitis, since it is a well-defined uveitis entity and because it often is observed in a Chinese uveitis clinic, allowing sufficiently large patient sample sizes to be obtained. The results showed that DNA hypermethylation of the GATA3, IL-4, and TGF-β locus may confer risk to VKH disease. 

Materials and Methods

Study Population

To measure the methylation status of the transcription and inflammatory factors of CD4⁺T cells, we recruited 20 untreated VKH patients with active uveitis (10 male, 10 female; 38.65 ± 18.3), 12 inactive VKH patients who were on treatment with CsA and corticosteroids (4 male, 8 female; 35.33 ± 13.1) and 20 healthy controls (10 male, 10 female; 36.85 ± 13.4). The detailed medications of the included inactive patients are shown in Supplementary Table S1. All participants in this study were enrolled at the First Affiliated Hospital of Chongqing Medical University (Chongqing, China) between April 2015 and December 2016 and were Chinese Han. The patients were diagnosed according to the criteria of the First International Workshop for VKH disease.³⁶ All participants signed a written informed consent. All procedures were performed in accordance with the Declaration of Helsinki and were approved by the Clinical Research Ethics Committee of the First Affiliated Hospital of Chongqing Medical University (Permit Number: 2009-201008). 

CD4⁺ T-Cell Isolation

Venous blood was collected, stored in heparinized syringes, and then used for PBMC isolation by Ficoll-Hypaque density gradient centrifugation. Human CD4 mAb-conjugated magnetic microbeads and magnetic sorting columns (Miltenyi Biotec, Bergisch Gladbach, Germany) were used for CD4⁺ T-cell separation from PBMCs. The CD4⁺ T-cell purity was determined by flow cytometry and confirmed to be above 95%. 

DNA Extraction

Extraction of genomic DNA from CD4⁺ T cells was performed using the QIAamp DNA Blood Mini Kit (Qiagen, Valencia, CA, USA). The concentration and quality of all DNA samples were analyzed by a Nanodrop 2000 apparatus (Thermo Fisher Scientific, Wilmington, DE, USA). 

DNA Methylation Analysis by MassARRAY

DNA sequences of each target gene promoter region were determined from the University of California, Santa Cruz (UCSC; Santa Cruz, CA, USA) website (available in the public domain at http://www.genome.ucsc.edu). Polymerase chain reaction primers for target genes were designed through the EpiDesigner online application (available in the public domain at http://www.epidesigner.com) on the basis of the obtained DNA sequence. A T7 promoter tag and a 10-mer tag were added to every reverse primer and forward primer, respectively, for in vitro transcription. The specific primer sequences of each target gene are shown in Table 1. 

Table 1

 

Primer Sequence for Amplifying Target Genes

 

Bisulfate conversion of genomic DNA (1 μg) was performed using the EZ DNA Methylation Kit (Agena Bioscience, San Diego, CA, USA) according to the instruction manual. Quantitative methylation analysis of bisulfite-treated genomic DNA was conducted through the Sequenom's MassARRAY EpiTYPER system (Sequenom, Inc., San Diego, CA, USA), which depends on matrix-assisted laser desorption/ionization-time of flight mass spectrometer (MALDI-TOF-MS), according to the instruction manual. Bisulfite-treated DNA was amplified as follows: 95°C for 4 minutes; 95°C for 20 seconds, 56 °C for 30 seconds, and 72°C for 60 seconds (DNA was amplified for 45 cycles by repeating the 3 steps); 72°C for 3 minutes. Shrimp alkaline phosphatase (SAP; Sequenom, Inc.) was used to dephosphorylate the unincorporated dNTPs at 37°C for 20 minutes, 85°C for 5 minutes. Subsequently, the transcription and cleavage were conducted by RNase A (Sequenom, Inc.) and T7 RNA&DNA polymerase (Sequenom, Inc.) at 37°C for 3 hours. The cleavage reactants were desalted using the clean resin (Sequenom, Inc.) and dispensed on a 384 SpectroCHIP (Sequenom, Inc.). The mass spectrums were obtained by MassARRAY MALDI-TOF-MS and analyzed by EpiTYPER software v1.2 (Sequenom, Inc.). Duplicate independent analyses of each bisulfite-treated sample were performed. Data of poor quality for the quantitative methylation detection of each CpG site were excluded. 

Real-Time PCR

TRIzol Reagent (Invitrogen, San Diego, CA, USA) was used for RNA exaction from CD4⁺T cells, and then a reverse transcriptase kit (Takara, Dalian, China) was used to obtain cDNA. Real-time PCR was performed with the Applied Biosystems 7500 System with the SYBR-Green method. Polymerase chain reaction primer sequences of the target gene and the internal reference gene β-actin were as follows: β-actin: forward: 5′-GGATGCAGAAGGAGATCAC TG-3′, reverse: 5′-CGATCCACACGGAGTACTTG-3′; GATA3: forward: 5′-GCGGGCTCTATCACAAAATGA-3′, reverse: 5′-GCTCTCCTGGCTGCAGACAGC-3′; IL-4: forward: 5′-CACAACTGAGAAGGAAACCTTCTG-3′, reverse: 5′-CTCTCTCATGATCGTCTTTAGCCTTTC-3′; TGF-β: forward: 5′-GGACACCAACTATTGCTTCAG-3′, reverse: 5′-TCCAGGCTCCAAATGTAGG-3′. The 2^−ΔΔCt method was applied to analyze the quantitative PCR (qPCR) data. 

Statistical Analysis

The Mann-Whitney test, independent-sample t-test, and Pearson correlation test used in this study were performed using SPSS 17.0 (SPSS, Inc., Chicago, IL, USA). A P value < 0.05 was regarded as significant. 

Results

Promoter Hypermethylation of GATA3, IL-4 and TGF-β Was Detected in CD4⁺T cells From Active VKH Patients

To assess whether epigenetic differences in CD4⁺T cells are related to VKH disease, we measured the promoter methylation levels of TBX21, GATA3, RORγt, FOXP3, IFN-γ, IL-4, IL-17A, and TGF-β using MassARRAY spectrometry and compared the values to those obtained in healthy individuals. The lengths of the target regions and number of covered CpG sites are shown in Table 1. Due to the limitation of the used method, the methylation level could be assessed only if the target fragments had a length between 100 and 500 base pairs. In addition, not every CpG site of the target regions could be measured, as a number of PCR products were either too short or too long (<1500 or >7000 Da). 

The results demonstrated that only the methylation level of CpG-7.8.9 in GATA3 was markedly higher in active VKH patients when compared to healthy individuals (P = 1.429 × 10⁻⁴; Fig. 1a), whereas the methylation status of the rest of CpG units in GATA3 showed no significant difference between the cases and controls (Table 2). Similar to the result of GATA3, we also found a significantly increased methylation level of the CpG-2 site in the IL-4 target region in active VKH patients (P = 0.010; Fig. 1b). Additionally, 3 CpG (CpG-6, CpG-10.11 and CpG-13.14) units of TGF-β exhibited significantly increased methylation levels in active VKH patients as compared to healthy individuals (P = 0.013, P = 3.385 × 10⁻⁵, P = 0.023, respectively; Figs. 1c–d). 

Figure 1

 

GATA3, IL-4, and TGF-β methylation levels in CD4⁺ T cells from active VKH patients (n = 20) and healthy controls (n = 20). Methylation levels of the CpG-7.8.9 unit of GATA3 (a), CpG-2 of IL-4 (b), as well as CpG-6 (c), CpG-10.11(d), and CpG-13.14 (e) of TGF-β were all significantly increased in VKH patients compared to that observed in healthy controls. Data represent mean ± SEM. *P < 0.05, ***P < 0.001.

Table 2

 

Methylation Levels Each CG Units of TBX21, GATA3, RORγt, FOXP3, IFN-γ, IL-4, IL-17A, TGF-β in CD4⁺ T-cell of VKH Patients and Healthy Controls

 

No significant differences concerning methylation levels of TBX21, RORC, FOXP3, IFN-γ, and IL-17A could be detected between the cases and controls. The methylation data of all the CpG units assessed in our study are shown in Table 2. 

Correlation Between Promoter Methylation and mRNA Expression of GATA3, IL-4, TGF-β

To investigate the association between the methylation level of promoters and the gene expression, we analyzed the mRNA expression levels of GATA3, IL-4, and TGF-β in CD4⁺ T cells. The mRNA expression levels of GATA3 and TGF-β were markedly decreased in active VKH patients than healthy individuals (P = 7.136 × 10⁻⁴, P = 1.5×10⁻³, respectively; Fig. 2). However, no significant difference in the IL-4 expression between the cases and controls was observed (data not shown). 

Figure 2

 

The mRNA expression levels of GATA3 and TGF-β in CD4⁺ T cell from VKH patients and healthy controls. The GATA3 (a) and TGF-β (b) mRNA expression were markedly reduced in VKH patients compared to healthy controls. (a) VKH, n = 7; controls = 5. (b) VKH: n = 8; controls = 7. *P < 0.05. ***P < 0.001.

When analyzing the relationship between the methylation of the CpG-7.8.9 unit with the mRNA expression level of GATA3, we found that the DNA methylation showed a negative correlation with mRNA expression (P = 0.035, r = −0.611; Fig. 3a). In addition, the methylation percentage of each CG unit of CpG-6, CpG-10.11, and CpG-13.14 in TGF-β also was negatively correlated with mRNA expression (P = 0.082, r = −0.464; P = 0.031, r = −0.556; P = 0.013, r = −0.622; Figs. 3b–d). 

Figure 3

 

Correlation between DNA methylation and respective mRNA expression level. The DNA methylation level of CpG-7.8.9 (a) unit in GATA3 as well as CpG-6 (b), CpG-10.11 (c), and CpG-13.14 (d) units in TGF-β were negatively correlated with their mRNA expression.

Methylation Level and mRNA Expression of GATA3 and TGF-β in CD4⁺ T-Cell From Inactive VKH Patients With Medication

To investigate the effect of conventional treatment on methylation changes of GATA3 and TGF-β, we detected the methylation level of GATA3 and TGF-β in CD4⁺ T cells from inactive VKH patients who were on treatment with CsA and corticosteroids. The methylation level of CpG-7.8.9 in GATA3 was markedly decreased in inactive VKH patients when compared to active VKH patients (P = 3.738 × 10⁻⁴; Fig. 4a). Simultaneously, we found that the methylation levels of 3 CpG (CpG-6, CpG-10.11, and CpG-13.14) units of TGF-β were significantly reduced in inactive VKH patients (P = 0.003, P = 1.714 × 10⁻⁴; P = 0.013, respectively; Figs. 4b–d). Moreover, the results showed that the mRNA expression of GATA3 and TGF-β were notably increased in inactive VKH patients compared to the active VKH patients (P = 0.007, P = 0.005, respectively; Figs. 4e–f). 

Figure 4

 

The DNA methylation and mRNA expression levels of GATA3 and TGF-β in CD4⁺ T cell from active and inactive VKH patients. Methylation levels of the CpG-7.8.9 unit of GATA3 (a) as well as CpG-6 (b), CpG-10.11(c), and CpG-13.14 (d) of TGF-β were all significantly decreased in inactive VKH patients compared to that observed in active. The GATA3 (e) and TGF-β (f) mRNA expression were markedly increased in inactive VKH patients compared to active. (a–d) active VKH, n = 20; inactive VKH, n = 12. (e, f) active VKH, n = 7; inactive VKH, n = 7). Data represent mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001.

Discussion

As far as we know, our study is the first to investigate the association between promoter methylation of IFN-γ, IL-4, IL-17A, TGF-β, TBX21, GATA3, RORγt, as well as FOXP3 with VKH disease. Our findings showed that the CpG-7.8.9 unit of GATA3, CpG-2 site of IL-4 and 3 CG units (CpG-6, CpG-10.11, and CpG-13.14) of TGF-β were hypermethylated in active VKH disease patients. Furthermore, we have found that the mRNA expression of GATA3 and TGF-β was markedly decreased in active VKH patients, and that this was correlated negatively with their promoter DNA methylation levels. Our results also showed that the methylation levels of the CpG-7.8.9 unit of GATA3 and 3 CG units (CpG-6, CpG-10.11, and CpG-13.14) of TGF-β were reduced in inactive VKH patients. 

The Th1 cell–secreted IFN-γ and Th2 cell–secreted IL-4 have important roles in the pathogenesis of autoimmune disease.^37,38 The transcriptional factors of TBX21 and GATA3 are indispensable to the induction of IFN-γ and IL-4 gene expression.^39,40 GATA3 can block Th1 differentiation by inhibiting TBX21 and β chain of the IL-12 receptor expression.^41,42 A recent study demonstrated that GATA3 is significantly hypermethylated in ulcerative colitis patients as compared to healthy individuals.⁴³ These findings suggest that hypermethylation of the CpG-7.8.9 unit at GATA3 may contribute to the pathogenesis of an autoimmune disease, such as VKH. 

It has been shown that significant demethylation of the IL-4 gene promoter occurs in a mouse model of childhood allergic asthma.⁴⁴ Earlier reports showed that loss of GATA3 expression resulted in a decreased production of Th2 cytokines and was associated with an increased DNA methylation at the IL-4 gene locus.⁴⁵ In this study, we also found an increased DNA methylation of the CpG-2 site at the IL-4 promoter region in VKH. However, no significant difference in the mRNA expression of IL-4 between cases and controls was detected. It is possible that the decreased GATA3 results in the hypermethylation of IL-4, but that small amounts of residual GATA3 are sufficient for the control of IL-4 expression.⁴⁵ 

Transforming growth factor–β has a critical role during an inflammatory response, including the initiation, progression, and resolution processes.⁴⁶ Early studies have shown that TGF-β is a potent immunosuppressive agent that limits clonal T lymphocyte expansion and downregulation of inflammation.^47–49 A recent study showed an increased expression of TGF-β in PBMCs obtained from inactive VKH patients, suggesting that the immunoregulatory cytokine TGF-β may be associated with the resolution of VKH disease.¹⁷ Hypermethylation of the TGF-β gene promoter is associated with various cancers, such as lung, prostate, ovarian, and gastric cancers.^50–52 It has been reported that among 100 cancer samples, 44.0% (22/50) and 82.0% (41/50) harbored methylated CpG sites in the TGF-β promoter of lung and prostate cancer samples, respectively.⁵⁰ The latter study also revealed that hypermethylation of the TGF-β promoter was related to a metastatic phenotype.⁵⁰ In addition, these studies also showed that hypermethylation of the TGF-β gene promoter is correlated negatively with its mRNA expression in human tumor cell lines.^50–52 Our study showed similar results, whereby three different CG units (CpG-6, CpG-10.11, and CpG-13.14) of the TGF-β gene promoter were significantly hypermethylated in VKH patients and that the methylation level of each unit was correlated negatively with TGF-β mRNA expression. Our data thus provide evidence that hypermethylation of the TGF-β gene promoter is involved in the development of VKH disease. 

Cyclosporin A and corticosteroids have been used in the treatment of VKH disease,^15,16 but whether they will affect DNA methylation still is not known. A recent study showed that another immunosuppressive drug, methotrexate can significantly reduce the methylation level of FOXP3 upstream enhancer that leads to increased FOXP3 expression and reduces disease activity in rheumatoid arthritis patients.⁵³ In our study, we found that the methylation levels of GATA3 and TGF-β promoter were decreased and the mRNA expressions were increased in the inactive VKH patients who were on treatment with CsA and corticosteroids. These results suggested that the therapeutic effect of CsA and corticosteroids may be associated with the reduction of the methylation level of GATA3 and TGF-β promoter and increase of the mRNA expression. 

It is worthwhile to point out that whether the increased methylation levels of GATA3 and TGF-β promoter are specific for active VKH patients is not known. More studies on other uveitis entities may address this problem. 

Our study has some limitations. Firstly, our study was conducted in a relatively small sample and the results should be validated in a larger case-control study. Secondly, we only evaluated the methylation levels in promoter regions of eight genes, and we cannot exclude the association between the aberrant methylation status in other regions and VKH disease. Thirdly, although we have found that hypermethylation of GATA3, TGF-β, and IL-4 gene promoter is associated with VKH disease, further researches are needed to elucidate the exact mechanisms whereby methylation of these genes affects the risk of acquiring VKH. 

In conclusion, our findings showed that increased promoter methylation of GATA3, TGF-β, and IL-4 confers risk to VKH disease in Han Chinese and the promoter hypermethylation of GATA3 and TGF-β is negatively associated with their mRNA expression. Moreover, our results supported the notion that the methylation changes of GATA3 and TGF-β are related to the activity of VKH disease. Our findings may provide an insight into how these factors are involved in VKH pathogenesis, which hopefully will offer new perspectives for the management of these patients. 

Acknowledgments

The authors thank all donors enrolled in the present study. 

Supported by Natural Science Foundation Major International (Regional) Joint Research Project (81320108009), Chongqing Key Laboratory of Ophthalmology (CSTC, 2008CA5003), National Key Clinical Specialties Construction Program of China, Chongqing Science & Technology Platform and Base Construction Program (cstc2014pt-sy10002), and the Major Research Development Program of China (2016YFC0904000). 

Disclosure: Y. Zhu, None; H. Yu, None; Y. Qiu, None; Z. Ye, None; W. Su, None; J. Deng, None; Q. Cao, None; G. Yuan, None; A. Kijlstra, None; P. Yang, None