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Abstract

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. REFERENCES

Objective

Systemic sclerosis (SSc) is an autoimmune disease with a predilection for women. The interaction between CD40 and CD154 (CD40L) is known to be involved in the development of SSc. Although CD40L is overexpressed in patients with SSc, the mechanisms leading to this overexpression are not well understood. We previously demonstrated that DNA demethylation reactivates the silent X chromosome, resulting in CD40L overexpression in healthy women. We hypothesized that CD40L up-regulation by DNA demethylation and subsequent reactivation of the silent X chromosome in female patients with SSc explain the susceptibility of women to SSc. The aim of this study was to investigate the effect of DNA demethylation on CD40L expression in CD4+ T cells from female patients with SSc.

Methods

CD40L expression in CD4+ T cells from patients with SSc and healthy control subjects was measured by flow cytometry and real-time reverse transcription–polymerase chain reaction. Bisulfite sequencing was performed to determine the methylation status of the CD40L regulatory region.

Results

CD40L expression was significantly elevated in female patients with SSc. The methylation levels of the DNA regulatory sequences were reduced in female patients with SSc compared with healthy women, and there was a significant inverse correlation between the average methylation level and CD40L mRNA expression in female patients with SSc. In contrast, no significant difference was observed in the expression of CD40L between male patients with SSc and male control subjects. The DNA regulatory regions in both male patients and male control subjects were largely unmethylated.

Conclusion

Demethylation of CD40L regulatory elements on the inactive X chromosome contributes to CD40L overexpression in CD4+ T cells from female patients with SSc.

Systemic sclerosis (SSc) is an autoimmune connective tissue disease characterized by immune activation, including autoantibody production and excessive extracellular matrix deposition (1). More than 80% of patients with SSc are female (2). The following 5 major factors are involved in scleroderma: T cells, B cells, fibroblasts, vascular endothelial cells, and cytokines/chemokines; T cells play a central role in the development of SSc (3–5). An important T cell and B cell collaboration is the interaction of the costimulatory receptor CD40 and its ligand CD40L (6). CD40L (also known as CD154), which is mainly expressed on the surface of activated CD4+ T cells, is a type II membrane protein that belongs to the tumor necrosis factor family (7). Elevated CD40L expression is observed in patients with SSc and in TSK mice (8–10). Blockade of the CD40–CD40L interaction by anti-CD40L monoclonal antibody significantly reduced cutaneous fibrosis and anti–topoisomerase I autoantibody production (10). Thus, CD40L plays an important role in SSc. However, little is known about the mechanisms underlying the overexpression of CD40L in SSc.

CD40L is encoded on the X chromosome (7). To maintain the balance of genes encoded on the X chromosome in males and females, one X chromosome in females is normally inactivated (11), but the mechanisms of its inactivation have not been fully clarified. Epigenetic modifications, such as DNA methylation, that suppress gene expression play an important role in the initiation and maintenance of X chromosome inactivation (12–15). We previously demonstrated that DNA methyltransferase inhibitors demethylate certain X-linked genes, including CD40L, and double their expression in healthy women but not men (16). It is not known whether CD40L overexpression is caused by the demethylation of CD40L regulatory sequences and reactivation of the inactive X chromosome in patients with SSc. A sex difference in the DNA demethylation status of CD40L may account for the female predisposition to SSc.

We compared the expression of CD40L and its methylation status in the regulatory region of CD4+ T cells from patients with SSc and healthy control subjects. Both the level of CD40L protein and messenger RNA (mRNA) expression were significantly elevated, and the DNA methylation level of the CD40L gene regulatory domains was reduced in female patients with SSc compared with female control subjects. Moreover, there was a significant inverse correlation between the average methylation level and CD40L mRNA expression in female patients with SSc. In contrast, we observed no significant increase in CD40L expression in male patients with SSc compared with male control subjects. The CD40L regulatory regions were largely unmethylated in both male patients and male control subjects.

PATIENTS AND METHODS

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. REFERENCES

Subjects.

We enrolled 26 patients with SSc (16 women, mean ± SD age 40.5 ± 13.2 years; 10 men, mean ± SD age 39.7 ± 12.0 years) who were seen at the outpatient dermatology clinic and the inpatient ward of the Second Xiangya Hospital, Central South University. The patients had no family history of SSc, and all patients met the criteria for SSc established by the American College of Rheumatology (17). The clinical profiles of the patients with SSc are shown in Table 1. The control subjects were 15 healthy women (mean ± SD age 36.6 ± 11.0 years) and 10 healthy men (mean ± SD age 36.8 ± 7.4 years) recruited from the medical staff at the Second Xiangya Hospital. None of the control subjects had a family history of autoimmune disorders. The study was approved by the human ethics committee of the Central South University Xiangya Medical College. Patients and control subjects were matched for age and sex in all experiments; T cell samples from each group were paired and studied in parallel.

Table 1. Characteristics of the 26 patients with systemic sclerosis (SSc)
Patient/ age/sexSSc subtypeDisease duration, yearsTreatment regimen
1/32/FDiffuse2Prednisone, 10 mg/day
2/49/FDiffuse0.5None
3/23/FLimited2Retinoic acid, 20 mg/day
4/28/FDiffuse1.5Prednisone, 7.5 mg/day
5/29/FLimited1Chinese herbal medicines
6/58/FDiffuse1Chinese herbal medicines; prednisone, 5 mg/day
7/45/FLimited2Chinese herbal medicines
8/23/FDiffuse1Prednisone, 7.5 mg/day
9/53/FDiffuse0.3None
10/48/FDiffuse3Chinese herbal medicines
11/22/FDiffuse0.5Methylprednisone, 8 mg/day
12/57/FLimited1.5None
13/49/FLimited0.5Chinese herbal medicines
14/55/FLimited1.5Prednisone, 10 mg/day
15/46/FDiffuse0.3None
16/31/FDiffuse2Prednisone, 12.5 mg/day
17/33/MLimited3Prednisone, 7.5 mg/day
18/25/MDiffuse0.5None
19/59/MDiffuse1Chinese herbal medicines
20/30/MDiffuse0.5None
21/35/MLimited1None
22/44/MLimited2Chinese herbal medicines
23/46/MDiffuse1.5Retinoic acid, 20 mg/day
24/24/MDiffuse1Prednisone, 10 mg/day
25/53/MDiffuse1Chinese herbal medicines
26/48/MDiffuse0.5None

Isolation of peripheral blood mononuclear cells (PBMCs) and CD4+ T cells.

Venous peripheral blood (60 ml) was collected from all study subjects and preserved with heparin. PBMCs were isolated by density-gradient centrifugation (Ficoll-Hypaque; Shanghai Hengxin Chemical Reagent). CD4+ T cells were then isolated by positive selection, using magnetic beads according to the manufacturer's protocol (Miltenyi Biotec). The purity of enriched CD4+ T cell isolates as evaluated by flow cytometry was generally >95%.

Flow cytometric analysis.

Aliquots of the PBMC suspension (5 × 105 cells in 100 μl) were incubated with fluorescein isothiocyanate (FITC)–conjugated anti-human CD154 (CD40L) and phycoerythrin (PE)–conjugated anti-human CD4 (Becton Dickinson) for 20 minutes at room temperature, washed in 2 ml of stain buffer (pH 7.4) containing 0.2% (weight/volume) bovine serum albumin (BSA) (Becton Dickinson), and centrifuged at 400g for 5 minutes. Cell pellets were resuspended in 0.5 ml of phosphate buffered saline/BSA. The cells were then sorted on a FACSCalibur flow cytometer (Becton Dickinson), and the data were analyzed using CellQuest software (Becton Dickinson). Detector sensitivity and off-line compensation of FITC and PE emission spectra overlaps were set using unstained and single color–stained samples and the automatic compensation software utility.

RNA isolation and CD40L mRNA quantification.

Total RNA was isolated from CD4+ T cells, using RNeasy Mini Kits (Qiagen). First-strand complementary DNA (cDNA) synthesis was performed with the RevertAid First Strand cDNA Synthesis Kit (Fermentas). CD40L and β- actin transcripts were assayed by quantitative real-time reverse transcription–polymerase chain reaction (RT-PCR) in a Rotor-Gene 3000 Thermal Cycler (Corbett Research). The primers used were as follows: for CD40L, forward 5′- CACCCCCTGTTAACTGCCTA-3′ and reverse 5′-CTGGATGTCTGCATCAGTGG-3′; for β-actin, forward 5′-GCACCACACCTTCTACAATGAGC-3′ and reverse 5′-GGATAGCACAGCCTGGATAGCAAC-3′. A dilution series of sample cDNA was used to generate a standard curve to estimate the relative concentration of transcript in each sample. Water in place of cDNA was run as a negative control, and β-actin was used as a loading control.

Genomic DNA extraction and bisulfite sequencing.

Genomic DNA was isolated from CD4+ T cells, using a TIANamp Genomic DNA Kit (Tiangen Biotech). Bisulfite conversion was performed with an EpiTect Bisulfite Kit (Qiagen). A 553-bp fragment from the promoter or upstream enhancer (−402 to +150) and a 761-bp fragment from the downstream enhancer (+11498 to +12259) of the CD40L locus were amplified in two rounds of nested PCR assays, using the designed primers shown in Table 2. The amplified products were then cloned into pGEM T vectors (Promega), and 8 independent clones were sequenced. Our previous studies showed that demethylation of these promoter and enhancer regions is correlated with CD40L overexpression on CD4+ T cells from women but not men with lupus (16).

Table 2. Primers used for bisulfite sequencing in 2 rounds of nested polymerase chain reaction assays
FragmentRoundPrimer sequenceLocation, bp
553 bp, −402 to +1501Forward 5′-GAAGAATTCAGTTGATGGGATATTAGTTATAAAATTAATTT-3′−448 to −407
  Reverse 5′-AAATCTAGACCCAATCATCTAAATAATAAAAAAAACAA-3′+194 to +153
 2Forward 5′-TTTGAATTCATGTGTTTTTTTTTTTATATATTAGGTTTT-3′−402 to −366
  Reverse 5′-AATTCTAGAAAATTTTCATACTAATAAACAATCCAATAA-3′+150 to +116
761 bp, +11498 to +122591Forward 5′-TTTGAATTCAAGATAAGGTTATTATGTATAGGTTGAATTT-3′+11466 to +11506
  Reverse 5′-ATTTCTAGAAAAACAAAATTTATAAAAACATCACAAAAA-3′+12311 to +12271
 2Forward 5′-TTTGAATTCGAGTAAATAGTAGATAATTTGTTAAGTTT-3′+11498 to +11536
  Reverse 5′-CCCTCTAGATACTTCCTCTTTCCCCAACCTAACTAAAAA-3′+12259 to +12221

Statistical analysis.

Results are expressed as the mean ± SD. Student's t-test was used to test the equality of means. All analyses were performed with SPSS software version 16.0. P values less than 0.05 were considered significant.

RESULTS

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. REFERENCES

Increased CD40L expression in CD4+ T cells from female patients with SSc.

The expression of CD40L on the surface of CD4+ T cells from SSc patients and healthy control subjects was assessed by flow cytometry. The proportion of CD4+ T cells expressing CD40L was significantly higher in female patients with SSc compared with female control subjects (P < 0.05) (Figure 1A). No significant difference in the expression of CD40L was observed between male patients with SSc and male control subjects (Figure 1B). To confirm these results, we assessed the CD40L mRNA level in CD4+ T cells, using real-time RT-PCR. The mean level of CD40L mRNA relative to β-actin was significantly higher in female patients with SSc compared with female control subjects (P < 0.05) (Figure 1C). Among men, CD40L mRNA expression in CD4+ T cells was not significantly different between patients and control subjects (Figure 1D).

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Figure 1. Expression of CD40L in CD4+ T cells from patients with systemic sclerosis (SSc). A and C, The proportion of CD4+ T cells expressing CD40L (A) and the level of CD40L mRNA expression in CD4+ T cells (C) were significantly higher in female patients with SSc compared with female control subjects. B and D, The proportion of CD4+ T cells expressing CD40L (B) and the level of CD40L mRNA expression in CD4+ T cells (D) were not significantly different in male patients with SSc and male control subjects. Bars show the mean ± SD. ∗ = P < 0.05.

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CD40L promoter and enhancer methylation in CD4+ T cells from healthy men and women.

CD40L is regulated by a core promoter and upstream and downstream enhancers. The fragment from −402 to +150 contains the transcription initiation site and the binding sites of transcription factor NF-AT, and the fragment from +11498 to +12259 contains the binding sites of transcription factor NF-κB (18). We previously observed that DNA methyltransferase inhibitors such as 5-azacytidine increase CD40L expression via demethylation of the promoter and enhancer regions in healthy women, and methylation of these regions in reporter constructs suppresses function by cassette methylation, indicating that changes in methylation are relevant to transcription (16).

CD40L promoter and enhancer methylation patterns were compared in CD4+ T cells from healthy men and women. The methylation patterns of the 10 CG pairs in the promoter region of 8 cloned fragments from a representative woman are shown in Figure 2A. Half of the fragments were unmethylated, while the other half were methylated. In contrast, all of the cloned fragments from a representative man were largely unmethylated (Figure 2B). These results are consistent with the fact that one allele is inactivated in women, and that men have one active gene. The average methylation patterns of the 10 CG pairs in the CD40L promoter in CD4+ T cells from 10 healthy women and 10 healthy men are shown in Figures 2C and D, respectively. The mean methylation level of the CD40L promoter was significantly lower in men. Figures 2E and F show the results of a similar analysis of the CD40L enhancer in CD4+ T cells from the same female and male subjects. The mean methylation level of the CD40L enhancer was much lower in men.

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Figure 2. CD40L promoter and enhancer methylation in CD4+ T cells from healthy men and women. CD4+ T cells were isolated from 10 healthy women and 10 healthy men, and 8 fragments per subject were cloned and sequenced as described in Patients and Methods. A and B, Methylation patterns of the 10 CG pairs in the promoter region in 8 cloned fragments from a representative female subject (A) and a representative male subject (B). The location of each CG pair is shown on the x-axis, and the fragment numbers are shown on the y-axis. ○ = unmethylated cytosines; • = methylated cytosines. C–F, Methylation levels of the CD40L promoter in women (C) and men (D) and of the enhancer in women (E) and men (F). The amplified CG regions are shown on the x-axis, and the mean methylation levels of each CG pair are shown on the y-axis.

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CD40L promoter and enhancer methylation in CD4+ T cells from female patients with SSc.

To determine whether changes in DNA methylation contribute to CD40L overexpression in CD4+ T cells from female patients with SSc, we analyzed the methylation status of the 10 CG pairs in the 553-bp region (−402 to +150) and the 8 CG pairs in the 761-bp region (+11498 to +12259), using bisulfite genomic DNA sequencing. Figures 3A–C show that the methylation levels of the CG pairs in the CD40L promoter and enhancer in 16 female patients with SSc were significantly lower than those in 15 healthy female control subjects (P < 0.001). Furthermore, the expression of CD40L mRNA was inversely correlated with the mean methylation level of the promoter (r = −0.629, P < 0.01) and the enhancer (r = −0.727, P < 0.01) (Figures 3D and E).

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Figure 3. CD40L promoter and enhancer methylation patterns in CD4+ T cells from female patients with systemic sclerosis (SSc) (n = 16) and healthy female control subjects (n = 15). CD4+ T cells were isolated, and fragments of the promoter and enhancer of the CD40L locus were amplified by nested polymerase chain reaction. Amplified fragments were cloned, and 8 clones from each amplified fragment were sequenced. A and B, The mean methylation levels of 10 CG pairs in the promoter (A) and 8 CG pairs in the enhancer (B) were much lower in female patients with SSc compared with female control subjects. C, The mean ± SD methylation levels of the CD40L promoter and enhancer were significantly lower in female patients compared with controls. ∗∗∗ = P < 0.001. D and E, The mean DNA methylation levels of the CD40L promoter (D) and enhancer (E) were inversely correlated with the relative mRNA level in female patients with SSc.

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CD40L promoter and enhancer methylation in CD4+ T cells from male patients with SSc.

Next, we investigated the methylation status of CD40L regulatory domains in male subjects. The CG pairs from both male patients with SSc and healthy male control subjects were largely unmethylated, and the mean methylation levels were very low (Figure 4). In male patients with SSc and male control subjects, there was no significant difference in the methylation levels of the promoter and the enhancer.

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Figure 4. CD40L promoter and enhancer methylation patterns in CD4+ T cells from male patients with systemic sclerosis (SSc) (n = 10) and healthy male control subjects (n = 10). The mean methylation levels of 10 CG pairs in the promoter (A) and 8 CG pairs in the enhancer (B) were low and were not significantly different between patients and controls.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. REFERENCES

The interaction between CD40L and CD40 plays a pivotal role in various autoimmune diseases, including SSc (19–21). The combination of CD40L on T cells and CD40 on B cells provides an essential signal for the initiation of immune responses, including B cell activation and differentiation, and the production of pathogenic autoantibodies (22). CD40 is also expressed on fibroblasts. In cultured fibroblasts, CD40 expression was induced by stimulation with soluble CD40L, and CD40 expression was increased on fibroblasts from SSc lesions (20, 21). Blocking the CD40–CD40L interaction using anti-CD40L monoclonal antibody significantly reduced cutaneous fibrosis and anti–topoisomerase I autoantibody production (10). Thus, CD40L plays an important role in the development of SSc by stimulating B cells and fibroblasts via the CD40–CD40L interaction, resulting in the production of autoantibodies and tissue fibrosis. CD40L expression is increased in patients with SSc and in TSK mice (8–10). Here, we demonstrated that the expression of CD40L protein and mRNA was significantly elevated in CD4+ T cells from female patients with SSc compared with female control subjects. In contrast, the expression of CD40L was not significantly different in male patients with SSc and male control subjects. Our search of the literature revealed no studies comparing the expression of CD40L in male and female patients with SSc. To our knowledge, this report represents the first documentation that elevated CD40L expression is specific to female patients with SSc.

There is a growing body of evidence indicating that epigenetic alterations including DNA methylation are strongly involved in the pathogenesis of autoimmune diseases (23–25). We previously reported that immunity-related genes such as ITGAL/CD11a, TNFSF7/CD70, and perforin are hypomethylated and overexpressed in CD4+ T cells from patients with systemic lupus erythematosus, and that this contributes to the development of autoimmunity (26–28). We also documented that CD4+ T cell DNA is hypomethylated, and that the level of DNA methyltransferase 1 is significantly decreased in patients with SSc (29).

In the present study, we observed hypomethylation and overexpression of CD40L in CD4+ T cells from female patients with SSc. We analyzed the methylation status of 10 CG pairs located at position −402 to +150 and that of 8 CG pairs located at position +11498 to +12259, a core promoter and upstream and downstream enhancers of CD40L that affect CD40L transcription. In female patients with SSc, both the promoter and enhancer regions were demethylated, and an inverse correlation between the mean methylation level and CD40L expression was documented. Hypomethylation of the regulatory regions is characteristic for a transcriptionally permissive chromatin configuration, and active promoters are typically hypomethylated. In contrast, CD40L expression was increased only slightly in male patients with SSc, and no significant difference was observed in the methylation level of CD40L regulatory domains between male patients and male control subjects.

Why is there a difference in the methylation level of the CD40L regulatory regions between men and women? CD40L is encoded on the X chromosome; females have two alleles, and males have one allele. Normally, one X chromosome allele is inactivated in females to maintain the balance of genes encoded on the X chromosome between males and females (11). Bisulfite sequencing of DNA from CD4+ T cells revealed that healthy women have one methylated and one unmethylated CD40L gene; the single CD40L gene in healthy men was largely unmethylated (Figure 2). Because one X chromosome is inactivated in females by processes that include DNA methylation (30), the methylated gene derives from the inactive X chromosome. Previously, we demonstrated that DNA methyltransferase inhibitors such as 5-azacytidine reactivated the silent X chromosome and doubled CD40L expression in healthy women but not in healthy men (16).

In the present study, we observed that the methylation level of CD40L regulatory sequences in female patients with SSc was significantly decreased compared with that in control subjects, and that there was an inverse correlation with CD40L expression. We posit that DNA hypomethylation in CD4+ T cells leads to reactivation of the silent X chromosome and to an increase in CD40L expression in female patients with SSc. We also showed that the methylation level of CD40L regulatory sequences and CD40L expression were not significantly different in male patients with SSc and male control subjects. The CD40L regulatory sequences on the single X chromosome were largely unmethylated in both male patients and male controls, because their single X chromosome is constitutively active. It is possible that T cells interact with B cells and fibroblasts via pathways other than CD40L–CD40 in male patients with SSc. Our findings indicate that demethylation of the CD40L regulatory sequences on the inactive X chromosome in CD4+ T cells from female patients with SSc contributes to the development of scleroderma through the overexpression of CD40L. Our previous studies demonstrated that CD40L is demethylated and overexpressed in CD4+ T cells from women but not men with lupus (16). The demethylation of CD40L and possibly of other genes on the inactive X chromosome may contribute to the striking female susceptibility to these diseases.

In summary, CD40L overexpression in CD4+ T cells from patients with SSc is associated with demethylation of specific CpG-rich regions within regulatory regions exclusively in women. Reactivation of the silent X chromosome attributable to DNA demethylation may contribute to the female susceptibility to SSc.

AUTHOR CONTRIBUTIONS

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. REFERENCES

All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. Dr. Xiao had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

Study conception and design. Lian, Xiao, Hu, Kanekura, Lu.

Acquisition of data. Lian, Hu, Jiang, Wang, Yang.

Analysis and interpretation of data. Lian, Xiao, Hu, Jiang, Li, Zhao.

REFERENCES

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. REFERENCES