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The Journal of Immunology, 2008, 181, 3897 -3905
Copyright © 2008 by The American Association of Immunologists, Inc.
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The Production of IL-10 by Human Regulatory T Cells Is Enhanced by IL-2 through a STAT5-Responsive Intronic Enhancer in the IL-10 Locus

Kazue Tsuji-Takayama*, Motoyuki Suzuki*, Mayuko Yamamoto*, Akira Harashima*, Ayumi Okochi*, Takeshi Otani*, Toshiya Inoue*, Akira Sugimoto*, Terumasa Toraya*, Makoto Takeuchi*, Fumiyuki Yamasaki{dagger}, Shuji Nakamura1,* and Masayoshi Kibata*

* Cell Biology Institute, Research Center, Hayashibara Biochemical Laboratories, Fujisaki, Okayama, Japan; and {dagger} Kurashiki Medical Center, Bakuro-cho, Kurashiki, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
STAT5 molecules are key components of the IL-2 signaling pathway, the deficiency of which often results in autoimmune pathology due to a reduced number of CD4+CD25+ naturally occurring regulatory T (Treg) cells. One of the consequences of the IL-2-STAT5 signaling axis is up-regulation of FOXP3, a master control gene for naturally occurring Treg cells. However, the roles of STAT5 in other Treg subsets have not yet been elucidated. We recently demonstrated that IL-2 enhanced IL-10 production through STAT5 activation. This occurred in two types of human Treg cells: a novel type of umbilical cord blood-derived Treg cell, termed HOZOT, and Tr1-like Treg cells, IL-10-Treg. In this study, we examined the regulatory mechanisms of IL-10 production in these Treg cells, focusing specifically on the roles of STAT5. By performing bioinformatic analysis on the IL-10 locus, we identified one STAT-responsive element within intron 4, designated I-SRE-4, as an interspecies-conserved sequence. We found that I-SRE-4 acted as an enhancer element, and clustered CpGs around the I-SRE-4 were hypomethylated in IL-10-producing Treg cells, but not in other T cells. A gel-shift analysis using a nuclear extract from IL-2-stimulated HOZOT confirmed that CpG DNA methylation around I-SRE-4 reduced STAT5 binding to the element. Chromatin immunoprecipitation analysis revealed the in situ binding of IL-2-activated STAT5 to I-SRE-4. Thus, we provide molecular evidence for the involvement of an IL-2-STAT5 signaling axis in the expression of IL-10 by human Treg cells, an axis that is regulated by the intronic enhancer, I-SRE-4, and epigenetic modification of this element.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The STAT molecules play key roles in intracellular signal transduction after cellular stimulation by cytokines, growth factors, and hormones. Among seven mammalian family members, two highly related STAT5 gene products, STAT5a and STAT5b, have been of particular interest because their activation can be induced by a wide spectrum of cytokines. Recently, especially in regulatory T (Treg)2 biology, STAT5 proteins have been recognized as particularly important due to their essential downstream roles in the IL-2/IL-2R signaling pathway (1, 2, 3, 4). Naturally occurring Treg (nTreg) cells are generated and maintained in an IL-2-dependent manner. This was demonstrated in studies of mice deficient for IL-2, IL-2R{alpha}, and IL-2Rβ, in which autoimmune diseases developed due to lower numbers of nTregs (5, 6). Also, studies of STAT5a and STAT5b double-knockout mice revealed the contribution of STAT5 molecules to nTreg development because a subset of these mice exhibited autoimmune pathology very similar to IL-2 or IL-2R knockout mice (7). A human patient with a STAT5b mutation displayed immune dysregulation associated with decreased numbers and impaired suppressive ability of CD4+CD25high cells, indicating both developmental and functional defects of nTreg cells (8). An essential role of STAT5 molecules in the IL-2/IL-2R signaling pathway was confirmed by experiments using mice with an IL-2Rβ mutation, which exclusively activates STAT5 (4). Therefore, the IL-2-STAT5 signaling axis contributes to some of the essential properties of nTreg cells. Furthermore, one result of the IL-2-STAT5 signaling axis is the enhancement of nTreg-specific gene expression, the transcription factor FOXP3 (4). It has been shown that STAT5 molecules directly control FOXP3 gene expression, and STAT consensus sequences were identified within promoter or intron regions (4, 9). Although roles of IL-2/IL-2R signaling were firmly established in nTreg cells, its roles in other types of Treg cells are poorly understood.

IL-10 is an inhibitory cytokine mediating suppression by regulatory or suppressor T cells (10). Despite its essential roles in immune responses, the mechanisms regulating IL-10 production are not well understood. We previously reported that IL-2 was effective for enhancement of IL-10 production in certain types of Treg cells (11). Such cells are categorized as IL-10-producing Treg cells, including Tr1-like cells, IL-10-Treg, and a newly characterized Treg cell line (designated HOZOT) with a phenotype of CD4+CD8+. In that study, we analyzed the mechanisms of high IL-10 production by these Treg cells in comparison with other T cells, especially Th2 cells, which are typical high producers of IL-10 among non-Treg cells. For Th2 cells, both transcriptional and epigenetic mechanisms have been reported in the regulation of IL-10 production. At the transcriptional level, transcriptional activators, such as c-jun, jun B (12), and NF-AT (13), have been documented as essential molecules for IL-10 production. At an epigenetic level, a Th2-specific transcription factor, GATA-3, has been described as a chromatin remodeling molecule. GATA3 can bind to DNase I hypersensitivity (HS) sites residing on both the 5'-proximal region and intron 4, and then induce remodeling of chromatin structure by increasing histone acetylation (14). In this situation, GATA-3 acts as a stabilizing factor, and not a transcriptional activator, keeping an open chromatin configuration for the IL-10 gene. Our previous study indicated that, in contrast to Th2 cells, GATA-3 plays a less important role in IL-10-producing Treg cells (IL-10-Treg and HOZOT) because GATA-3 expression in these cells is relatively low compared with Th2 cells. We further demonstrated that STAT5 molecules activated by IL-2/IL-2R signaling were involved in the mechanism enhancing IL-10 production in HOZOT, suggesting that, in addition to nTreg cells, the IL-2-STAT5 signaling axis defines another type of Treg cells, IL-10-producing Treg.

Recent studies of cytokine gene expression have focused on epigenetic changes, because the DNA methylation status at CpG sites is often associated with cytokine expression (15). In particular, its status in the region of conserved noncoding sequences (CNS) is relevant for a cell’s differentiation or activation. Hypomethylation is associated with chromatin’s open status, whereas hypermethylation corresponds to chromatin’s closed status (16). Therefore, the extent to which CpG sites are methylated correlates with the level of cytokine production. Representative cases were reported for Th1-type cytokines, IL-2 (17, 18) and IFN-{gamma} (19, 20), and Th2-type cytokines, IL-4 (21, 22), IL-5, and IL-13 (23). With regard to IL-10, however, there are only a few reports of CpG methylation, and no significant correlation was found between IL-10 production levels and CpG methylation status.

In this study, we used HOZOT cells to determine how IL-2 signaling enhanced the production of IL-10 by Treg cells. We first investigated whether STAT5 molecules directly bound to the IL-10 locus and whether binding elements acted as enhancers. We also examined the possibility of epigenetic regulation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Generation of HOZOTs and other types of T cell lines from umbilical cord blood (UCB)

All T cells used in this study were generated from mononuclear cells of UCB, as previously reported (24). UCB was obtained from the Kurashiki Medical Center in compliance with the institutional review board and with informed consent of the donors according to the Declaration of Helsinki. The mononuclear cells from the UCB were prepared by gradient centrifugation using Ficoll-Paque (GE Healthcare).

Human regulatory T cell lines, HOZOTs, were generated by coculture with murine stromal cell lines, as previously reported (24). HOZOT-1 and -4, representative HOZOT cell lines, were used in this study. Briefly, to generate HOZOTs, CD34– mononuclear cells were enriched by negative selection using a MACS CD34+ isolation kit (Miltenyi Biotec) and miniMACS column, according to the manufacturer’s instructions. The cells were cultured over stromal cells in RPMI 1640 medium supplemented with 10% heat-inactivated FBS, 100 U/ml penicillin, and 50 µg/ml streptomycin at 37°C in 5% CO2. HOZOT-1 and -4 were established by coculture with murine stromal cell lines, MS-5 and ST2, respectively. Once established as cell lines, HOZOTs were expanded in medium containing 10 ng/ml IL-2 (PeproTech). HOZOTs were purified by Ficoll-Paque to deplete debris from mouse stromal cell lines killed by HOZOTs before experiments.

The nTreg cells were obtained by cultivation of CD25+ cells from UCB mononuclear cells by modification of previously reported methods (25, 26). The CD25+ cells were cultured on dendritic cells (DC), which were induced from GM-CSF- and IL-4-stimulated CD14+ cells.

Conventional T (conT) cells were obtained by at least 1-wk cultivation of CD4+CD25– cells on plates coated with anti-CD3 mAb (UCHT1; R&D Systems) and CD28 mAb (37407.111; R&D Systems) (CD3/CD28) in the presence of 10 ng/ml IL-2.

The IL-10-producing Treg (IL-10-Treg) cell line was also obtained from CD4+CD25– mononuclear cells, as previously described (27). The cells were cultured on immature DC derived from UCB in the presence of 1 nM vitamin D3 (Sigma-Aldrich) and 50 nM dexamethasone (Sigma-Aldrich). The cells were expanded for another 2 wk in the presence of vitamin D3/dexamethasone and IL-2.

The Th2 cell line was obtained from CD4+CD25– mononuclear cells, as described previously (28). The cells were cultured with plate-bound CD3/CD28 in the presence of 10 ng/ml human rIL-4 (PeproTech) and 4 µg/ml anti-human IFN-{gamma} mAb (Hayashibara Biochemical Laboratories) for 48 h. Then, the cells were expanded by addition of 10 ng/ml IL-2. After 6 days, the production of IL-10, IFN-{gamma}, and IL-4 was assessed in the supernatants from the CD3/CD28-treated cells.

Naive T cells were prepared as CD4+CD25– mononuclear cells. The percentage of CD45RA+ cells was >90%. Without cultivation, genomic DNA was isolated for methylation analysis.

IL-10 measurements by ELISA

Cells (2 x 105 cells/ml) were cultivated for 16 h in the presence or absence of IL-2 in 24-well trays, which were precoated with CD3/CD28. The supernatants were collected and then used for IL-10 measurements by human IL-10 ELISA kit (eBioscience).

Bioinformatics

Alignment between the mouse and human IL-10 loci was performed, and the extent of DNA sequence homology was computed with a web-based program called Regulatory Visualization Tools for Alignment (rVISTA; www.gsd.lbl.gov/vista) (29). The plot of the percentage of sequence identity referred to the human sequence. Regions with a length of at least 100 bp, which showed at least 75% sequence identity at each segment of the alignment between successive gaps, are identified as CNS and are shown in red in Fig. 1.


Figure 1
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  		FIGURE 1. Bioinformatic analysis of the conserved SRE on the IL-10 locus. The locations of the 5'-proximal region, exons, and introns of the IL-10 locus are illustrated at the top. Yellow boxes show exons (E) 1–5, and the blue lines show the 5'-proximal region and introns (I) 1–4 in the IL-10 locus. Corresponding sequences between human and mouse were aligned by rVISTA 2.0. The degree of interspecies conservation of the DNA within this segment is represented by the histogram. CNS (interspecies conservation more than 75%) are emphasized in red. SREs on the human IL-10 locus were identified using the TRANSFAC database, and are displayed with red stripes on a gray line under the histograms. The detailed sequences spanning P-SRE and I-SRE-4 are depicted at the bottom. Blue and red squares show positions of CpG dinucleotides and STAT consensus sequences, respectively.

 
Reporter gene construction

Starting with genomic DNA from a human T cell leukemia line, HUT-78, the 5'-proximal region (1.5 kbp upstream of the transcription starting site (TSS) on the IL-10 locus) and a part of intron 4 (a 162-bp fragment including STAT-responsive element (SRE) within intron 4 (I-SRE-4) on the IL-10 locus) were amplified by AccuPrime Tac (Invitrogen) using the following sets of specific primers. For the IL-10 5'-proximal region, the sense primer was 5'-GATGAAAACAGACACAGGGAGGATGAGTG and the antisense primer, 5'-GTCTGTCTTGTGGTTTGGTTTTGC. For intron 4 (I-SRE-4 wild type), the sense primer was 5'-GATTCTCACTTAACCTGGAGTTGGTTCAA and the antisense primer was 5'-CATAGGCCGCACGGTTTCTGGGAAATCAG. For intron 4 (I-SRE-4 mutant), the sense primer was 5'-GATTCTCACTTAACCTGGAGTTGGTTCAA and the antisense primer was 5'-CATAGGCCGCACGGTTTCTGAAGGATCAG. Restriction endonuclease linkers were added to the amplification by the second PCR. The amplified fragments were ligated into a pT7Blue TA vector (Merck). These fragments from the 5'-proximal region and intron 4 on the IL-10 locus were digested by XhoI/BglII and SalI/BamHI, respectively, and then gel purified on β-agarose (Lonza). The fragment of the 5'-proximal region was ligated into the XhoI/BglII site of the 5'-multicloning site in the luciferase expression vector, pGL4.10[luc2] (Promega), and the fragment of intron 4 was ligated into the SalI/BamHI site at the 3'-multicloning site in pGL4.10[luc2] or pGL4.23[luc2/minP] (Promega).

Transient transfection and reporter gene assays

ConT cells (1 x 106 cells) were transfected with 10 µg of a firefly luciferase vector and 0.5 µg of a Renilla luciferase vector (pGL4.75; Promega) by electroporation using Gene Pulser Xcell (Bio-Rad). After transfection, cells were stimulated with or without 10 ng/ml IL-2 in the presence or absence of CD3/CD28 for 16 h. To circumvent the effects of endogenous IL-2 production, anti-IL-2 polyclonal Ab (pAb) (AB-202-NA; R&D Systems) and anti-IL-2R mAb (22722.2; R&D Systems) (both at final concentrations of 5 µg/ml) were added to the culture in the absence of exogenous IL-2. Cell extracts were prepared, and the luciferase activity was measured with the Dual Luciferase Reporter assay kit (Promega), according to the manufacturer’s instruction. All firefly luciferase activity was normalized to Renilla luciferase.

Bisulfite PCR for methylation analysis

Genomic DNA from T cells was isolated using DNeasy (Qiagen), according to the manufacturer’s instructions. Bisulfite PCR was performed, as described previously (30). Briefly, the genomic DNA (5 µg) was digested with EcoRI and then incubated in 3.2 M sodium bisulfite (Sigma-Aldrich) and 500 nM hydroquinone (Sigma-Aldrich) for 16 h at 55°C and then desalted using a DNA cleanup column (Promega). The bisulfite-converted DNA was treated with 300 mM NaOH for 20 min at 37°C and then stored at –80°C. Using the treated DNA, hot-start PCR for IL-10 5'-proximal region and intron 4 was performed with AccuPrime TaqDNA polymerase (Invitrogen). The primer sets for their amplification are as follows: IL-10 5'-proximal region, 5'-GTATAGTTGGGGTGGGGGATAGTTGAAGAG and 5'-CTCTATCCCCCTTTTATATTATAAACTCAC; IL-10 intron 4, 5'-GGTTTAGTTAGTGAGGAGTTGTTGTTTTG and 5'-CTATACATACCTTCTTTTACAAATC. The PCR products were cloned into a pT7Blue TA vector, and 20 clones from each sample were picked up for DNA sequencing.

Gel-shift and competition assays

HOZOT-1 was treated with 10 ng/ml IL-2 for 2 h. Nuclear extracts were prepared from the cells, as described previously (31). The sequences of the unmethylated and methylated oligonucleotides used in this study are shown in Fig. 5, A and B. These oligonucleotides were purchased from Sigma-Aldrich. The double-stranded oligonucleotide probes were end labeled with [{gamma}32P]ATP using T4 polynucleotide kinase (Invitrogen), according to the manufacturer’s instruction. Nuclear extracts (5–10 µg) were incubated with 20 fmol double-stranded probes in binding solution (1 mM EDTA, 30 mM NaCl, 5% glycerol, 0.1% Nonidet P-40, 1 mg/ml BSA, and 2 µg/ml poly(dI-dC)) at room temperature for 30 min. Unlabeled probes were added with a 100-fold excess as competitors. Anti-STAT5 pAb (C-17), anti-STAT3 pAb (H-190), or control rabbit pAb (DakoCytomation) were added to the binding reaction for supershift experiments. The reactions were separated on 4% nondenaturing polyacrylamide gels and exposed to autoradiographic film for 24 h.


Figure 5
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  		FIGURE 5. ChIP analysis to measure acetylation levels of histone H3/H4 at the sites of P-SRE and I-SRE-4. Locations of primers for ChIP-quantitative real-time PCR (ChIP-qPCR) (arrows) at the 5'-proximal (A) and the intron 4 (B) regions. C, ChIP experiments using IL-2-treated HOZOT-1 and Th2 cells. Proteins and DNA were cross-linked with formaldehyde, cells were lysed, and DNA was sheared. ChIP was performed using either rabbit pAb or anti-acetyl-histone H3 pAb. Quantification of immunoprecipitated DNA fragments was performed by real-time PCR using primer for P-SRE or I-SRE-4. Values were normalized to corresponding input control. **, p < 0.01 compared with the value of lane 5. The data are given as means ± SD for triplicate samples and are representatives of three independent experiments.

 
Western blot analysis

Western blotting was performed using whole cell extracts from HOZOT-1 cells treated with or without IL-2, as described previously (32, 33). The membranes were probed with anti-phospho-STAT5 mAb (8-5-2; Upstate Biotechnology), anti-phospho-STAT3 mAb (B-7; Santa Cruz Biotechnology), or anti-phospho-STAT1 pAb (Tyr701; Santa Cruz Biotechnology), and then assessed with a chemiluminescent system (SuperSignal West Pico Chemiluminescent Substrate; Pierce). After treatment with a deprobing solution containing 62.5 nM Tris-HCl (pH 6.8), 2% SDS, and 100 nM 2-ME for 60 min at 55°C, the membrane was used for second detection with anti-STAT5b pAb (C-17; Santa Cruz Biotechnology), anti-STAT3 pAb (H-190; Santa Cruz Biotechnology), or anti-STAT1 pAb (E-23; Santa Cruz Biotechnology). All of the data were representative of more than three independent experiments.

Chromatin immunoprecipitation (ChIP) assays

HOZOT-1 cells (2 x 107) were treated with IL-2 (10 ng/ml) for 4 h. The cells were fixed with 1% paraformaldehyde for 10 min at room temperature, and then the fixation was stopped with 1.25 M glycine. Fixed cells were washed with cold PBS. Cells were treated with lysis buffer (Santa Cruz Biotechnology) and sonicated five times (30 s each) to prepare chromatin extracts. The extracts were pretreated with salmon sperm DNA-blocked protein G beads (GE Healthcare) and then immunoprecipitated with anti-acetyl histone H3 pAb (Upstate Biotechnology), anti-acetyl histone H4 pAb (Upstate Biotechnology), anti-STAT5 pAb (C-17; Santa Cruz Biotechnology), anti-STAT3 pAb (H-190; Santa Cruz), or control rabbit pAb (DakoCytomation) for 1 h at 4°C. The immune complexes were further incubated with protein G beads at 4°C overnight. After washing the immunoprecipitates with washing buffer (Santa Cruz Biotechnology), DNA was recovered by incubation with 1% SDS and 0.1 M NaHCO3 at 67°C overnight. The DNA samples were purified and used as templates for real-time PCR, which was performed using sets of specific primers as follows: IL-10 5'-proximal region, 5'-GACCCAATTATTTCTCAATCCC and 5'-GAGCTCCTCCTTCTCTAACC; IL-10 intron 4, 5'-AGTCTGATTTCCCAGAAACC and 5'-GTGCATTGACCTTCATCTCC; IL-2 5'-proximal region, 5'-GTTTACTCTTGCTCTTGTC and 5'-CCTCTTTGTTACATTAGCCC. Real-time PCR was performed using a LightCycler 480 Real-Time PCR System (Roche Applied Science), and the cycle threshold values for each immunoprecipitate were normalized to the input cycle threshold value as relative amounts.

Statistics

All data are expressed as the means ± SD. Statistical differences between groups were analyzed using one-way ANOVA, followed by post hoc Bonferroni/Dunn’s tests. Values of p < 0.01 were considered to be statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Bioinformatic approach to the identification of SREs in the IL-10 locus

We previously reported that IL-2 treatment could enhance IL-10 production in certain types of Treg cells, so-called IL-10-producing Treg, including IL-10-Treg and HOZOT (24). We also demonstrated that the enhancement was mediated through STAT5 activation (11). To examine whether STAT5 molecules could directly bind to the IL-10 locus, we performed a bioinformatic search for SREs on the IL-10 locus using web-based software, rVISTA. This program can identify interspecies-conserved sequences for specific transcription factors by linking to the most widely used database, TRANSFAC (29). Comparative sequence analyses among vertebrates is quite effective for finding functional coding and noncoding elements (34). As shown in Fig. 1, SREs located in noncoding or coding regions were distributed over the entire human IL-10 locus, including the 5'-proximal region, introns 2 and 4, and exon 5. A total of six SREs were found, five of which resided in the noncoding region. Among them, the interspecies-conserved SRE was found only within intron 4 (at position +3200 bp relative to the TSS), whereas the other five SREs were not conserved between human and mouse. This suggested that the SRE on intron 4, designated as I-SRE-4, was particularly important for the regulation of IL-10 gene expression.

Intronic SRE acts as an enhancer for gene expression

To determine the functional roles of I-SRE-4, we constructed a luciferase reporter vector, consisting of a promoter sequence derived from the 5'-proximal IL-10 region (from –1.5 to –0.02 kbp relative to the TSS) and an enhancer sequence derived from I-SRE-4 (from +3082 to +3243 bp relative to the TSS). This 162-bp fragment with the enhancer sequence includes no other responsive elements for a transcription factor except for I-SRE-4. Mutation at I-SRE-4 was also introduced into the STAT consensus sequence. As target cells for the transient assay, we used conT cells because the cells are responsive to CD3/CD28 stimulation and are more readily transfected than HOZOT cells. After transfection with the vectors, the cells were treated with IL-2 and CD3/CD28 stimulated for 16 h, and then luciferase activity was measured. As shown in Fig. 2A, the luciferase reporter vector with 5'-proximal region (lane 2) showed a 1.4-fold induction compared with a control vector without promoter/enhancer (lane 1). Addition of the wild-type I-SRE-4 at the 3' site (lane 3) resulted in a 2.6-fold induction, whereas addition of a mutant I-SRE-4 (lane 4) yielded only a 1.8-fold induction. When the minimal promoter TATA was used as a promoter, no enhancer activity of I-SRE-4 was observed (lane 5). These results suggested that I-SRE-4 could function as a cis-acting enhancer element on the IL-10 locus.


Figure 2
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  		FIGURE 2. Reporter assay demonstrates I-SRE-4 enhancer activity. A, Reporter constructs were generated by combining a firefly luciferase gene with a promoter from the IL-10 5'-proximal region or TATA-containing sequence (TATA), and with an enhancer driven from I-SRE-4-containing sequence with or without mutation (I-SRE-4 mutant or wild, respectively). ConT cells were transfected with these constructs and a Renilla luciferase construct, and then treated with 10 ng/ml IL-2 and CD3/CD28 stimulation. After overnight cultivation, cells were harvested and assayed for luciferase activity. The amount of luciferase activity was shown by normalization to Renilla luciferase activity for each transfection. **, p < 0.01 compared with the value of lane 3. B, Effects of endogenous IL-2 were examined in the same reporter assay. To exclude endogenous IL-2, Abs for IL-2/IL-2R were added for 2 h before stimulation. Subsequently, conT cells were transfected with the indicated constructs or null vector. The transfected cells were treated with the indicated combination of IL-2 and/or CD3/CD28 stimulation. **, p < 0.01 compared with the value of lane 3. The data are given as means ± SD for triplicate cultures and are representative of three independent experiments.

 
We further examined the effects of endogenous IL-2 on the enhancer activity of I-SRE-4. Because conT produced endogenous IL-2 following CD3/CD28 stimulation, we used anti-IL-2 pAb and anti-IL-2R mAb to block its effect. Using the vector containing both the 5'-proximal region and I-SRE-4, the treatment with exogenous IL-2 alone did not enhance luciferase activity (Fig. 2B, lane 2) compared with no stimulation. In combination with CD3/CD28 stimulation, exogenous IL-2 induced a 2.8-fold increase in activity (lane 3). Treatment with anti-IL-2/IL-2R Abs resulted in a 30% reduction (lane 4), indicating a role of endogenous IL-2 produced by CD3/CD28 stimulation. Experiments using a vector containing a mutant I-SRE-4 showed no significant effect of blocking Abs (lanes 7 and 8), confirming the involvement of I-SRE-4 in the endogenous IL-2 response.

Specific CpG demethylation around I-SRE-4 in human IL-10-producing Treg cells

Because it has been reported that differential pattern of cytokine gene expression is also regulated by epigenetic modification as well as transcription factor binding on the locus in various T cell subsets, we next focused on the possible epigenetic modifications. As a first step, we analyzed CpG methylation patterns among a variety of T cells in relation to their expression of the IL-10 gene. We compared a total of six normal T cell lines together with naive T cells and one leukemic B cell line, Daudi. The six T cell lines included two HOZOTs (HOZOT-1 and -4), one IL-10-Treg, one nTreg, one Th2, and one conT, which produced graded levels of IL-10, as shown in Table I. Upon CD3/CD28 stimulation, HOZOT-1, HOZOT-4, and IL-10-Treg produced IL-10 in a range of 120-1500 ng/ml in the absence of IL-2. In the presence of exogenous IL-2, production increased to a range of 1560–2300 ng/ml. Th2 cells also produced a higher level (2020 ng/ml) of IL-10 with CD3/CD28 stimulation, but no IL-2 enhancement was observed. ConT and nTreg cells produced only low levels (14–44 ng/ml) of IL-10, even in the presence of IL-2.


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  		Table I. IL-10 production (pg/ml)a

 
Next, we performed bisulfate genomic sequencing analysis over the intron 4 region spanning from +2.8 to +3.7 kbp downstream of TSS. CpG methylation over the 5'-proximal region was also examined to evaluate a human-specific SRE in this region, designated SRE within proximal region (P-SRE), an effective element for STAT3 binding in LPS/IFN-{alpha}- or IL-10-stimulated macrophages. We sequenced 20 individual DNA clones derived from each T cell line and Daudi (which failed to produce IL-10). The results of the methylation/demethylation analyses are depicted in Fig. 3 as a pie chart. We determined the methylation status of 16 CpG sites within intron 4 (labeled 1–16 in Fig. 3) and eight CpG sites within the 5'-proximal region (labeled 1–8 in Fig. 3). In Fig. 3, we indicated the IL-10-producing ability as "High" for >1000 pg/ml, "Low" for <100 pg/ml, and "No" for those below detection.


Figure 3
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  		FIGURE 3. Analysis of the methylation patterns at CpG sites in the 5'-proximal and intron 4 regions among different T and B cells. Locations of CpG positions (small numbered open circles with vertical lines) in the 5'-proximal or intron 4 regions are indicated on the map with a base pair scale originating at the TSS. The demethylated/methylated ratio at each CpG position is depicted by a pie chart. The number of the pie charts corresponds to the number of the CpG positions on the map. Short horizontal bars above the map show positions of P-SRE and I-SRE-4.

 
In the region around I-SRE-4, five CpG sites (positions 8, 9, 10, 11, and 12) were hypomethylated in HOZOT-1, HOZOT-4, and IL-10-Treg (2, 20, and 10% of an average methylation rate of five sites, respectively). In contrast, these sites were hypermethylated in nTreg (70%), conT (40%), and naive T (52%). Th2 also showed hypermethylation of CpGs at positions 9 and 10 (60%). Daudi exhibited almost complete methylation (95%) at these sites. CpG sites 1–4 within intron 4 were hypomethylated (0–42%) in all T cells, except at position 4 of nTreg (90%), in contrast to Daudi (84%). These data suggest that the CpG hypomethylation pattern in the I-SRE-4 region is associated with high levels of IL-10 production in IL-2-responsive Tregs such as HOZOTs and IL-10-Treg.

In the 5'-proximal region, CpG site 1, located at P-SRE, was moderately methylated in HOZOT-1 (15%) and HOZOT-4 (30%), but highly (90%) methylated in IL-10-Treg. In Th2 cells, CpG site 1 was 45% methylated. The methylation level of this CpG site did not correlate well with the level of IL-10 production. Therefore, the CpG hypomethylation status of I-SRE-4 is a better indicator of high IL-10 production than that of P-SRE.

In vitro methylation of SRE inhibits STAT binding

To examine the effects of CpG methylation of I-SRE-4 on STAT binding, we performed a gel-shift analysis using oligonucleotide probes containing either I-SRE-4 (Fig. 4A) or P-SRE (Fig. 4B). The nuclear extracts were prepared from IL-2-treated or untreated HOZOT-1. As shown in Fig. 4C, when an unmethylated probe (P1) was used for the analysis, the band was shifted when the extract came from IL-2-treated cells, but not untreated cells. We also used three types of methylated probes, as follows: one with a methylated CpG site 9 (P2); another methylated at CpG sites 9, 10, and 11 (P3); and the third methylated at CpG sites 10 and 11 (P4). We found that the intensity of the shifted bands with P2, P3, and P4 was significantly reduced (30–38% reduction in their intensity determined by densitometry analysis; data not shown). Furthermore, when the mutant probe (Mu) on a STAT consensus sequence was used, no shifting of the band was observed (Fig. 4C). Use of a probe containing a repeated STAT consensus sequence (Rep) resulted in dense, double-shifted bands, which are probably STAT tetramer. As shown in Fig. 4D, experiments using unmethylated (P1), mutant (Mu), or repeat (Rep) probes as competitors revealed the specificity of factor binding to the STAT consensus sequence. Supershift experiments with anti-STAT5 pAb or anti-STAT3 pAb showed that the factors that bound to P1 included primarily STAT5 and, to a lesser extent, STAT3 (Fig. 4E). In summary, these in vitro experiments showed that I-SRE-4 could bind directly to STAT-containing nuclear factors induced by IL-2, and that binding was dependent upon CpG methylation.


Figure 4
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  		FIGURE 4. Gel-shift analyses using oligonucleotide probes corresponding to I-SRE-4 or P-SRE. Nuclear extracts from HOZOT-1 were prepared after treatment with or without IL-2 and used for gel-shift analysis by incubating with different types of oligonucleotide probes containing SRE. The probes used for the experiments are listed below the maps of intron 4 (A) and the 5'-proximal (B) regions. Probes containing I-SRE-4 are either unmethylated (P1) or methylated (P2, P3, and P4). Probes with mutant (Mu) or repeated (Rep) sequences of I-SRE-4 were also used. C, Gel-shift experiments using I-SRE-4-containing probes. Arrows indicate shifted bands. D, Competition experiments performed by incubating extracts with a 100-fold molar excess of unlabeled oligonucleotides before addition of labeled P1 probe. E, Supershift experiments performed by incubating extracts with anti-STAT5 or anti-STAT3 pAbs for 2 h before addition of labeled P1 probe. F, Gel-shift analysis performed using an unmethylated probe for P-SRE. The data are representatives of three independent experiments.

 
In contrast to I-SRE-4, a P-SRE-containing probe (P5) produced only a weak shifting of the band even under demethylated conditions (Fig. 4F). This result indicates a lower in vitro affinity of the STAT-containing factors for P-SRE than I-SRE-4.

Histone acetylation levels at SREs

As another indicator of epigenetic control, we assessed the histone acetylation status in the regions of P-SRE and I-SRE-4, which reflects chromatin accessibility of the genes. We performed ChIP with anti-acetyl histone H3 pAb using HOZOT-1 and Th2 cells. DNA fragments in the immunoprecipitates were amplified by real-time PCR with PCR primer sets corresponding to the P-SRE and I-SRE-4 region (Fig. 5, A and B). As shown in Fig. 5C, acetylation of histone H3 in the I-SRE-4 regions was observed in both HOZOT-1 and Th2 cells, but its acetylation level was markedly higher in HOZOT-1 than in Th2 cells. In the P-SRE region, acetylation levels were comparable between the two cells. These results confirmed that chromatin in the I-SRE-4 region was kept open in HOZOT-1, but was closed in Th2 cells.

In situ binding of activated STAT5

To further examine whether STAT5 was actually bound to SRE in HOZOT-1 cells in situ, we next performed a ChIP analysis with anti-STAT5 pAb. Because STAT5 was phosphorylated by IL-2 exposure during a 240-min treatment of HOZOT-1 (Fig. 6A), we used chromatin from the cells with or without IL-2 treatment for 120 min for ChIP analysis. As a negative control, we used a set of PCR primers for Ag receptor-responsive elements (ARRE) of IL-2 gene (17), in which the STAT consensus sequence is not included. The ChIP analysis revealed that IL-2-activated STAT5 bound to both P-SRE and I-SRE-4 regions on the IL-10 locus (Fig. 6B). The level of binding to I-SRE-4 was higher than that to P-SRE. Almost no binding was observed to ARRE, a negative control region in the IL-2 locus. These results suggest that STAT5 molecules, upon activation by IL-2, bind primarily to I-SRE-4 in situ in IL-2-responsive Treg cells.


Figure 6
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  		FIGURE 6. In situ detection of IL-2-activated STAT5 bound to I-SRE-4. A, Western blot detection of phospho-STAT5 in HOZOT-1 after IL-2 (10 ng/ml) treatment. B, ChIP analysis of STAT5 binding to SREs. Cross-linked chromatin complexes from IL-2-treated or untreated HOZOT-1 were immunoprecipitated with anti-STAT5 pAb or a control pAb. ChIP-quantitative real-time PCR (ChIP-qPCR) was performed using three sets of primers for P-SRE (as in Fig. 5A), I-SRE-4 (as in Fig. 5B), and, as a negative control, ARRE from the IL-2 promoter. Relative amounts of PCR product were normalized to the amount of input chromatin. **, p < 0.01 compared with the value in the absence of IL-2. The data are representative of three independent experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
IL-10 is an immunosuppressive cytokine regulating growth and/or differentiation of a variety of immune cells, including Th1 cells, CTLs, NK cells, B cells, and DC (10). Therefore, deficiency or dysregulated expression of IL-10 production is often a main cause of autoimmune diseases or exaggerated inflammatory responses. IL-10-deficient mice as well as patients with Crohn’s disease display severe chronic enterocolitis, in which the up-regulation of inflammatory cytokines such as IFN-{gamma}, IL-12, TNF-{alpha}, IL-1, and IL-18 is observed, demonstrating the protective role of IL-10 in inflammatory conditions (35). Systemic lupus erythematosus is associated with the high production of IL-10, which leads to impaired T cell responses and dysregulation of B cell activation (36). Administration of anti-IL-10 Ab resulted in a long-lasting reduction of symptoms in the systemic lupus erythematosus patient. Although the control of IL-10 production is key to immune homeostasis in vivo, molecular mechanisms regulating IL-10 production are poorly understood.

IL-10 production by human Treg cells was enhanced by exogenous IL-2 through the activation of STAT5 molecules, as we previously reported (11). This response to IL-2 was found only in IL-10-producing Treg cells, such as HOZOT and IL-10-Treg, but not in nTreg or Th2. In the case of nTreg, the IL-2-STAT5 signaling axis leads to FOXP3 up-regulation. Therefore, we asked whether HOZOT or IL-10-Treg cells also use the IL-2-STAT5 signaling axis for regulation of IL-10. In this study, we first addressed the question as to whether STAT5 could bind to the IL-10 locus. Bioinformatic analysis using rVISTA 2.0 (29) showed that several SREs were spread across the entire IL-10 locus in both the 5'-proximal (promoter) and intronic regions. One SRE within the 5'-proximal region was not conserved among five species. However, it was reported to be a STAT3 binding site following exposure of human B cells and macrophages to IFN-{alpha} (37), LPS (38), or IL-10 (39). Other SREs within intron 2, reportedly STAT1 binding sites following IFN-{gamma} treatment (40), were also not conserved. One SRE found within intron 4, designated as I-SRE-4, was conserved across species. We speculated that I-SRE-4 might be critical for IL-10 gene expression because interspecies-conserved regions often correspond to DNase I HS sites (41), which could become targets for binding of transcription factors. Therefore, we focused our analysis on I-SRE-4.

We next demonstrated that I-SRE-4 indeed acted as an enhancer element within the IL-10 locus. This enhancer worked efficiently in combination with a promoter derived from the 5'-proximal region, but not with the minimal promoter TATA. Because the 5'-proximal region includes responsive elements for NF-AT (41) and NF-{kappa}B (42), I-SRE-4 may cooperate with these elements to exert its enhancing effects.

One intriguing finding of our studies is that the STAT5 enhancer element was localized in the intronic region on the IL-10 locus. It has been reported that STAT5 binds to intronic elements on a number of other gene loci. Nelson et al. (43) performed comprehensive ChIP analysis with anti-STAT5 Ab using an IL-3-dependent cell line, BaF/3, and demonstrated that a large proportion of functional STAT5 binding sites was located within introns. A number of functional intronic SREs have been found for immunologically important genes. Among them, the FOXP3 gene contains a conserved SRE within intron 1. This intronic SRE is a downstream target of the IL-2-STAT5 signaling axis in nTreg cells, leading to up-regulation of FOXP3 expression (4, 9, 44). Its functional property was confirmed by luciferase assay, in which 293 cells were cotransfected with a SRE-containing reporter construct and a constitutive activated STAT5 expression construct. As another example, intron 2 of the IL-4 gene contains a SRE that bound STAT5 and enhanced IL-4 gene transcription in mast cells (45). Although accumulating evidence revealed obvious roles of intronic enhancers for STAT5, it remains unclear how these enhancers regulate cytokine gene transcription. Further studies are needed to understand the molecular interaction with the 5'-proximal promoter.

We next asked whether epigenetic regulation was also involved in determining the level of IL-10 expression. Different types of T cells, including HOZOTs, conT, Th2, and nTreg, produce different amounts of IL-10 when the IL-2-STAT5 signaling axis is activated to the same extent. Interestingly, around I-SRE-4, there is a cluster of CpGs, suggesting the possibility of epigenetic regulation of IL-10 gene expression through CpG methylation. Many cytokine loci are targets for such control, including IL-2 (17, 18), IL-4, IL-13 (22), and IFN-{gamma} (46) loci in various types of T cells. For example, in naive T cells, TCR stimulation causes the promoter of the IL-2 locus to undergo immediate CpG demethylation. As a consequence, the promoter region of activated T cells becomes permissive for transcriptional activation of NF-AT and AP-1 (18). CpG hypomethylation at ARRE of the IL-2 gene correlates with positive regulation of IL-2 expression in both stimulated and resting CD4+ T cells (17). Examples of epigenetic regulation (demethylation of specific CpG tracks) in Th1 and Th2 cells include the NF-AT-responsive element in the IFN-{gamma} loci (46) and Th2-specific HS sites on IL-4 and IL-13 loci (22).

As for IL-10 expression, Dong et al. (47) analyzed the CpG methylation pattern of the IL-10 locus in different types of human CD4+ memory T cells, which were categorized by IL-10 and IFN-{gamma} expression. They concluded that there was no good correlation between methylation pattern and IL-10 expression. Curiously, they did not specify I-SRE-4 as a relevant CpG site, although they included this element in their study. In contrast, our study revealed an epigenetic significance of I-SRE-4 for IL-10 production. One characteristic of this element is species specificity, namely clustered CpG dinucleotides around I-SRE-4 were found in human cells, but not in mouse. There is another CpG site within the promoter region, designated P-SRE, but this CpG was methylated even in IL-10-Treg cells, typically high producers of IL-10. Therefore, I-SRE-4 contributes more to IL-10 gene expression than P-SRE.

In general, DNA methylation regulates gene expression through two distinct mechanisms. First, it inhibits the binding of transcription factors by imposing direct physical constraints (48). In our system, methylation-specific gel-shift analyses demonstrated that a methylated probe containing I-SRE-4 physically inhibited STAT binding. Second, methylated DNA recruits regulatory proteins (such as MBD2), which contain a methyl-CpG binding domain. These proteins generate an inaccessible chromatin structure by assembling multisubunit complexes containing corepressors and histone deacetylases (49). We examined chromatin accessibility by ChIP analysis with anti-acetyl-histone H3/H4 Abs. The ChIP analysis revealed that IL-2-activated STAT5 bound to I-SRE-4, but did not increase histone acetylation above that observed with the unstimulated control.

One intriguing question is how the epigenetic modification observed in our study occurred during IL-10-Treg cell differentiation, in particular, the CpG demethylation around I-SRE-4. There are several reports that the binding of STATs acts as an inducer of chromatin remodeling in immune systems. For example, during TCR{gamma}{delta} T cell differentiation, IL-7-activated STAT5 can interact with p300/CREB-binding protein and enhance the accessibility of regulatory molecules to the TCR{gamma} locus (50). Th1 and Th2 cells provide other examples. During Th1 cell differentiation, IL-12-activated STAT4 directly binds to the Il18r1 locus, transiently increases histone acetylation, and subsequently decreases DNA methyltransferase association on the locus, resulting in higher expression of IL-18R in Th1 cells (51). In contrast, during Th2 cell differentiation, intronic STAT5 binding induces chromatin remodeling on the IL-4 locus (45). Therefore, in our IL-10 system, there is a possibility that during induction of HOZOT in primary culture, STAT5 mediates chromatin alteration by binding to I-SRE-4. Further analysis is required to test this hypothesis.

In conclusion, our results, which are summarized in a model shown in Fig. 7, provide new insights into the importance of both epigenetic and transcriptional regulation for high IL-10 production in human Treg cells. Moreover, we have provided the first molecular evidence for the functional relevance of the IL-2-STAT5-IL-10 signaling axis in induced Treg cells, in contrast to the IL-2-STAT5-FOXP3 signaling axis.


Figure 7
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  		FIGURE 7. Hypothetical models showing the relationship between transcription factor binding and CpG methylation on the IL-10 locus. A, Models for high IL-10 production by HOZOTs, IL-10-Treg, and Th2 cells. B, A model for low IL-10 production by nTreg, conT, and naive T cells. C, A model for no IL-10 production by Daudi. In each, the IL-10 locus is shown as a bold line consisting of a 5'-proximal region, exons 1–4 (E1–E4), intron 4, and exon 5. CpG sites located in noncoding regions in potential transcription binding sites are depicted as circles with vertical lines. Shading of a circle indicates methylation levels. STAT can bind to hypomethylated, but not hypermethylated SREs. In addition to STAT, TCR-stimulated transcription factors, NF-AT and NF-{kappa}B, are shown.

 

   Acknowledgments
 
We express our sincere appreciation to Dr. Koichi Ikuta (Laboratory of Biological Protection, Department of Biological Responses, Institute for Virus Research, Kyoto University), Dr. Noriko M. Tsuji (Age Dimension Research Center, National Institute for Advanced Industrial Science and Technology), Dr. Kunnzo Orita (Hayashibara), and Dr. Tsunetaka Ohta (Biomedical Institute, Research Center, Hayashibara Biochemical Laboratories) for helpful discussions. We also thank Dr. Kenji Akita (Biomedical Institute, Research Center, Hayashibara Biochemical Laboratories) for technical support using radioisotopic materials.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Address correspondence and reprint requests to Dr. Shuji Nakamura, Cell Biology Institute, Research Center, Hayashibara Biochemical Laboratories, 675-1 Fujisaki, Okayama 702-8006, Japan. E-mail address: shnakamu{at}hayashibara.co.jp Back

2 Abbreviations used in this paper: Treg, regulatory T; ARRE, Ag receptor-responsive elements; ChIP, chromatin immunoprecipitation; CNS, conserved noncoding sequences; conT, conventional T; DC, dendritic cell; HS, hypersensitivity; I-SRE-4, STAT-responsive element within intron 4; nTreg, naturally occurring regulatory T; P-SRE, STAT-responsive element within proximal region; pAb, polyclonal Ab; rVISTA, Regulatory Visualization Tools for Alignment; SRE, STAT-responsive element; TSS, transcription start site; UCB, umbilical cord blood. Back

Received for publication April 15, 2008. Accepted for publication July 10, 2008.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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