Foxp3 Enhancer Research Articles

Lactoferrin-derived Chimeric Peptide (LFch) boosts TGFβ1-induced Treg Differentiation by diminishing of TCR Signal intensity via TβRIII

Abstract Regulatory T cells (Tregs) are a special subset of T cells to suppress excessive immune response and maintain immunologic tolerance. It is known that lesser TCR signal strength is favorable for Foxp3+ Treg differentiation. We recently reported that LF substantially promoted Foxp3 expression by activated CD4+ T cells and this activity was further enhanced by TGF-β1. In the present study, we explored the effect of lactoferrin chimera (LFch, synthetic LF-derived chimeric peptides) along with TGFβ1 on Foxp3+ Treg differentiation. LFch itself did not induce Foxp3 expression. However, LFch dramatically enhanced TGFβ1-induced Foxp3 expression. LFch little affected the phosphorylation of Smad3, which is a canonical TGF-β1 mediator. Instead, LFch decreased the phosphorylation of S6, IkB, and PI3 kinase (an activation hallmark of TCR signaling). These effects of LFch were completely abolished by pretreatment of LFch with soluble TβRIII. Consistently, the enhancement of TGFβ1-induced Foxp3 by LFch was also abrogated by treatment of soluble TβRIII. Finally, TGFβ1/LFch-induced T cell population substantially suppressed the proliferation of responder CD4+ T cells. These results indicate that LFch potently enhances TGFβ1-induced Foxp3+ Treg differentiation by diminishing TCR signal intensity through TβRIII.

IL-6 and TNFα Drive Extensive Proliferation of Human Tregs Without Compromising Their Lineage Stability or Function.

Treg therapies are being tested in clinical trials in transplantation and autoimmune diseases, however, the impact of inflammation on Tregs remains controversial. We challenged human Tregs ex-vivo with pro-inflammatory cytokines IL-6 and TNFα and observed greatly enhanced proliferation stimulated by anti-CD3 and anti-CD28 (aCD3/28) beads or CD28 superagonist (CD28SA). The cytokine-exposed Tregs maintained high expression of FOXP3 and HELIOS, demethylated FOXP3 enhancer, and low IFNγ, IL-4, and IL-17 secretion. Blocking TNF receptor using etanercept or deletion of TNF receptor 2 using CRISPR/Cas9 blunted Treg proliferation and attenuated FOXP3 and HELIOS expression. These results prompted us to consider using CD28SA together with IL-6 and TNFα without aCD3/28 beads (beadless) as an alternative protocol for therapeutic Treg manufacturing. Metabolomics profiling revealed more active glycolysis and oxidative phosphorylation, increased energy production, and higher antioxidant potential during beadless Treg expansion. Finally, beadless expanded Tregs maintained suppressive functions in vitro and in vivo. These results demonstrate that human Tregs positively respond to proinflammatory cytokines with enhanced proliferation without compromising their lineage identity or function. This property can be harnessed for therapeutic Treg manufacturing.

Epigenetic Control of Foxp3, CAMTA1 and FUT7 Genes in Human Regulatory T Cells

Regulatory T cells (Treg) are essential for immune tolerance and play a key role in autoimmune diseases, allergy and cancer. Foxp3 (Forkhead Box Transcription factor), encoded on Xp11.23 chromosome, is critical for the development, function and lineage commitment of Tregs, and has been widely used as a Treg specific marker. However, human CD4+CD25+Foxp3+ T cells are heterogeneous and include resting and activated Tregs, as well as non-suppressive Treg-like cells. The ability to distinguish between these subtypes may be critical in the development of Treg-based cell therapies. CD15s (sialyl Lewis x) was identified as a highly specific marker of activated and most suppressive effector Treg (eTreg), and the use of both CD45RA and CD15s enables the isolation of several CD4+CD25+Foxp3+ T cell subtypes with diverse functional profiles.Foxp3 is mainly regulated by epigenetic processes such as histone acetylation and methylation of CpG islands. Three regions on Foxp3 locus - enhancer, promoter and CNS 2 (also known as Treg Specific Demethylated Region, TSDR) contribute to Foxp3 expression via demethylation of their CpG islands and can be used as molecular markers that distinguish Treg from conventional CD4+ T lymphocytes (Tcon) and different Treg maturation stages.In this study, we characterised the epigenetic profile of four populations of CD4+CD25+ T cells: CD45RA+ CD15s- Foxp3low (naïve nTregs), CD45RA-15s-Foxp3low (non-suppressive Treg-like cells), CD45RA-CD15s+Foxp3high (eTregs) and Tcons, and CD34+ cells, all isolated from the peripheral blood of healthy male and female donors. Methylation patterns of Foxp3 locus enhancer, promoter, pre-TSDR (located upstream of TSDR) and TSDR were investigated together with those of calmodulin binding transcription activator (CAMTA 1), and fucosyltransferase 7 (FUT 7) promoters. CAMTA 1 was previously identified as a potential second molecular marker that could differentiate Treg from Tcon as this gene is encoded on chromosome 1 and is not affected by X-chromosome inactivation. FUT 7 promoter was chosen as FUT 7 mediates synthesis of CD15s expressed in both eTregs and CD34+ cells and, therefore, it was expected to be demethylated in these cell populations to allow for protein expression.Genomic DNA, isolated from the 5 cell populations, was subjected to bisulphite conversion followed by PCR-amplification of each gene region with bisulphite DNA specific primers and cloning into pGEM-T vector. Up to 24 clones (20 sequences fully covering each amplicon) were analysed and methylation status of each CpG position within all 6 gene regions was determined. As these gene regions were amplified from the same DNA sample, the methylation differences reflect the average status of the cell population.We demonstrate the intricate methylation patterns of FUT7 and CAMTA1 promoters and Foxp3 enhancer, promoter, pre-TSDR and TSDR for 10 male and female donors. Overall, Treg cell populations from donors of both genders displayed high demethylation levels in Foxp3 promoter and TSDR while only certain CpGs (4, 9, 11, 20-30 in Foxp3 enhancer and 1, 5, 7-9 in pre-TSDR) were more demethylated in Treg subtypes compared to Tcon. CD34+, CD45RA-CD15s- Foxp3low and eTreg populations had high demethylation levels in CpGs 5-16 of FUT7 promoter (highest in CpGs 5, 8-10) compared to nTreg and Tcon, suggesting that this regulatory element can be used as an additional molecular marker to distinguish eTreg from nTreg and Tcon. CAMTA 1 promoter (especially CpGs 2 and 11) was more demethylated in CD34+ and Treg cell populations compared to Tcon, with CD45RA-CD15s- Foxp3low cells displaying the highest demethylation levels. In conclusion, the findings in this study suggest that Treg-specific epigenetic changes are evident not only in several regions of Foxp3 locus, but also in CAMTA1 and FUT7 promoters. Interestingly, certain CpG positions in these regions are consistently more demethylated in Treg subtypes, which suggests their importance in epigenetic control, most probably as part of the recognition sequence for methylation-sensitive transcription factors. DisclosuresNo relevant conflicts of interest to declare.

A Comparison of Ex Vivo Expanded Human Regulatory T Cells Using Allogeneic Stimulated B Cells or Monocyte-Derived Dendritic Cells.

Alloreactive regulatory T cells (arTregs) are more potent than polyclonal Tregs at suppressing immune responses to transplant antigens. Human arTregs can be expanded with allogeneic CD40L-stimulated B cells (sBcs) or stimulated-matured monocyte-derived dendritic cells (sDCs). Here, we compared the expansion efficiency and properties of arTregs stimulated ex vivo using these two types of antigen-presenting cells. Compared to sBcs, sDCs stimulated Tregs to expand two times more in number. The superior expansion-inducing capacity of sDCs correlated with their higher expression of CD80, CD86, and T cell-attracting chemokines. sBc- and sDC-arTregs expressed comparable levels of FOXP3, HELIOS, CD25, CD27, and CD62L, demethylated FOXP3 enhancer and in vitro suppressive function. sBc- and sDCs-arTregs had similar gene expression profiles that were distinct from primary Tregs. sBc- and sDC-arTregs exhibited similar low frequencies of IFN-γ, IL-4, and IL-17A-producing cells, and the cytokine-producing arTregs expressed high levels of FOXP3. Almost all sBc- and sDC-arTregs expressed CXCR3, which may enable them traffic to inflammatory sites. Thus, sDCs-arTregs that expand more readily, are phenotypically similar to sBc-arTregs, supporting sDCs as a viable alternative for arTreg production for clinical evaluation.

Distinct Foxp3 enhancer elements coordinate development, maintenance, and function of regulatory T cells

The transcription factor Foxp3 plays crucial roles for Treg cell development and function. Conserved non-coding sequences (CNSs) at the Foxp3 locus control Foxp3 transcription, but how they developmentally contribute to Treg cell lineage specification remains obscure. Here, we show that among Foxp3 CNSs, the promoter-upstream CNS0 and the intergenic CNS3, which bind distinct transcription factors, were activated at early stages of thymocyte differentiation prior to Foxp3 promoter activation, with sequential genomic looping bridging these regions and the promoter. While deletion of either CNS0 or CNS3 partially compromised thymic Treg cell generation, deletion of both completely abrogated the generation and impaired the stability of Foxp3 expression in residual Treg cells. As a result, CNS0 and CNS3 double-deleted mice succumbed to lethal systemic autoimmunity and inflammation. Thus, hierarchical and coordinated activation of Foxp3 CNS0 and CNS3 initiates and stabilizes Foxp3 gene expression, thereby crucially controlling Treg cell development, maintenance, and consequently immunological self-tolerance.

Lactoferrin modulates BMDCs to be tolerogenic leading to enhancement of Foxp3+iTreg differentiation as well as suppression of T cell proliferation

Abstract DCs are professional APCs for initiating both immune response and immune tolerance. Tolerogenic DCs are generally known as regulatory DCs that suppress immune responses through regulation of T cells such as induction of T cell anergy/apoptosis and Treg. Since we have demonstrated that lactoferrin (LF) induces functional Tregs and has a protective effect against IBD, we explored the tolerogenic effect of LF on mouse bone marrow-derived DC (BMDC) and its underlying mechanisms. LF enabled BMDCs to suppress the proliferation of allogenic CD4+ T cells, and elevated levels of Arg-1, IDO-1, and CD39/73 (known as key T cell suppressor molecules). Consistently, the suppressive activity of LF-treated DCs (LF-DCs) was disappeared by inhibitors of Arg-1 and CD39. Finally, LF-DCs enhanced TGFβ1-induced OVA-specific Foxp3+ T cell population. Collectively, these results suggest that lactoferrin effectively causes DCs to be tolerogenic by both direct suppression of T cell proliferation and indirect enhancement of Treg differentiation.

Bile acid metabolites control Th17 and Treg cell differentiation

Abstract Bile acids are abundantly present in the mammalian gut, where they undergo bacteria-mediated transformation, generating a large pool of bioactive molecules. While they have been shown to affect host metabolism, cancer progression and innate immunity, it is unknown whether bile acids affect the function of adaptive immune cells such as T cells expressing IL-17a (Th17 cells) and regulatory T cells (Tregs) that mediate inflammatory and anti-inflammatory responses, respectively. By screening a small-molecule library primarily composed of bile acid metabolites, we identified two distinct derivatives of lithocholic acid (LCA), 3-oxoLCA and isoalloLCA, as specific regulators of Th17 and Treg cells. While 3-oxoLCA inhibited Th17 cell differentiation by directly binding to its key transcription factor RORγt (retinoid-related orphan receptor γt), isoalloLCA enhanced Treg differentiation through the production of mitochondrial reactive oxygen species (mitoROS), leading to increased FoxP3 expression. IsoalloLCA-mediated Treg enhancement required an intronic FoxP3 enhancer the conserved noncoding sequence 3 (CNS3), suggesting a distinct mode of action from other previously identified Treg enhancing metabolites that require CNS1. Lastly, oral administration of 3-oxoLCA and isoalloLCA to mice led to reduced Th17 and increased Treg cell differentiation in the intestinal lamina propria. Altogether, our data suggest novel mechanisms by which bile acid metabolites control host immune responses by directly modulating the Th17 and Treg balance.

Bacterial metabolism of bile acids promotes generation of peripheral regulatory T cells.

Intestinal health relies on the immunosuppressive activity of CD4+ regulatory T (Treg) cells1. Foxp3 expression defines this lineage and can be induced extrathymically by dietary or commensal-derived antigens in a process assisted by the Foxp3 enhancer CNS1 (conserved non-coding sequence 1)2–4. Microbial fermentation products including butyrate facilitate the generation of peripherally-induced Treg (pTreg) cells5–7, indicating that metabolites shape colonic immune cell composition. In addition to dietary components, bacteria modify host-derived molecules, generating a number of bioactive substances. This is epitomized by transformation of bile acids (BAs), which creates a complex pool of steroids8 presenting a range of physiological functions9. Here, we screened the major species of deconjugated BAs for their ability to potentiate pTreg cell differentiation. We found that the secondary BA 3β-hydroxydeoxycholic acid (isoDCA) increased Foxp3 induction by acting on dendritic cells (DCs) to diminish their immunostimulatory properties. Farnesoid X receptor ablation in DCs enhanced Treg cell generation and imposed a transcriptional profile similar to isoDCA, suggesting interaction between this BA/nuclear receptor pair. To investigate isoDCA in vivo, we took a synthetic biology approach and designed minimal microbial consortia containing engineered Bacteroides strains. IsoDCA-producing consortia increased colonic RORγt+ Treg cells in a CNS1-dependent manner, indicative of enhanced extrathymic differentiation.

Epigenetic and transcriptional analysis supports human regulatory T cell commitment at the CD4+CD8+ thymocyte stage

The natural CD25+ FOXP3+ regulatory T cell (Treg) population is generated as a distinct lineage in the thymus, but the details of Treg development in humans remain unclear, and the timing of Treg commitment is also contested. Here we have analyzed the emergence of CD25+ cells at the CD4+CD8+ double positive (DP) stage in the human thymus. We show that these cells share T cell receptor repertoire with CD25+ CD4 single-positive thymocytes, believed to be committed Tregs. They already have a fully demethylated FOXP3 enhancer region and thus display stable expression of FOXP3 and the associated Treg phenotype. Transcriptome analysis also grouped the DP CD25+ and CD4 CD25+ thymocytes apart from the CD25− subsets. Together with earlier studies, our data are consistent with human Treg commitment already at the DP thymocyte stage. We suggest that the most important antigens and signals necessary for human Treg differentiation may be found in the thymic cortex.

Bile acid metabolites control TH17 and Treg cell differentiation.

Bile acids are abundant in the mammalian gut where they undergo bacteria-mediated transformation, generating a large pool of bioactive molecules. Although bile acids are known to affect host metabolism, cancer progression and innate immunity, it is unknown whether they affect adaptive immune cells such as T helper cells expressing IL-17a (Th17 cells) and regulatory T cells (Tregs). By screening a library of bile acid metabolites, we identified two distinct derivatives of lithocholic acid (LCA), 3-oxoLCA and isoalloLCA, as T cell regulators. 3-oxoLCA inhibited Th17 cell differentiation by directly binding to its key transcription factor RORγt (retinoid-related orphan receptor γt) and isoalloLCA enhanced Treg differentiation through the production of mitochondrial reactive oxygen species (mitoROS), leading to increased FoxP3 expression. IsoalloLCA-mediated Treg enhancement required an intronic FoxP3 enhancer, the conserved noncoding sequence 3 (CNS3), a distinct mode of action from previously-identified Treg enhancing metabolites that require CNS1. Administration of 3-oxoLCA and isoalloLCA to mice reduced Th17 and increased Treg cell differentiation in the intestinal lamina propria. Our data suggest novel mechanisms by which bile acid metabolites control host immune responses by directly modulating the Th17 and Treg balance.

Peripherally induced regulatory T cells contribute to the control of autoimmune diabetes in the NOD mouse model

Type 1 diabetes (T1D) results from the autoimmune destruction of pancreatic beta cells and is partly caused by deficiencies in the Foxp3+ regulatory T-cell (Treg) compartment. Conversely, therapies that increase Treg function can prevent autoimmune diabetes in animal models. The majority of Tregs develop in the thymus (tTregs), but a proportion of Foxp3+ Tregs is generated in the periphery (pTregs) from Foxp3- CD4+ T-cell precursors. Whether pTregs play a distinct role in T1D has not yet been explored. We report here that pTregs are a key modifier of disease in the nonobese diabetic (NOD) mouse model for T1D. We generated NOD mice deficient for the Foxp3 enhancer CNS1 involved in pTreg induction. We show that CNS1 knockout decreased the frequency of pTregs and increased the risk of diabetes. Our results show that pTregs fulfill an important non-redundant function in the prevention of beta cell autoimmunity that causes T1D.

Histone/protein deacetylase inhibitor therapy for enhancement of Foxp3+ T-regulatory cell function posttransplantation.

T-regulatory (Treg) cells are like other cells present throughout the body in being subject to biochemical modifications in response to extracellular signals. An important component of these responses involves changes in posttranslational modifications (PTMs) of histones and many nonhistone proteins, including phosphorylation/dephosphorylation, ubiquitination/deubiquitination, and acetylation/deacetylation. Foxp3, the key transcription factor of Tregs, is constantly being rapidly turned over, and a number of these PTMs determine its level of expression and activity. Of interest in the transplant setting, modulation of the acetylation or deacetylation of key lysine residues in Foxp3 can promote the stability and function, leading to increased Treg production and increased Treg suppressive activity. This mini-review focuses on recent data concerning the roles that histone/protein deacetylases (HDACs) play in control of Treg function, and how small molecule HDAC inhibitors can be used to promote Treg-dependent allograft survival in experimental models. These data are discussed in the light of increasing interest in the identification and clinical evaluation of isoform-selective HDAC inhibitors, and their potential application as tools to modulate Foxp3+ Treg cell numbers and function in transplant recipients.

The mechanisms shaping the repertoire of CD4+ Foxp3+ regulatory T cells.

Regulatory T (Treg) cells expressing Foxp3 transcription factor control homeostasis of the immune system, antigenic responses to commensal and pathogenic microbiota, and immune responses to self and tumour antigens. The Treg cells differentiate in the thymus, along with conventional CD4+ T cells, in processes of positive and negative selection. Another class of Treg cells is generated in peripheral tissues by inducing Foxp3 expression in conventional CD4+ T cells in response to antigenic stimulation. Both thymic and peripheral generation of Treg cells depends on recognition of peptide/MHC ligands by the T-cell receptors (TCR) expressed on thymic Treg precursors or peripheral conventional CD4+ T cells. This review surveys reports describing how thymus Treg cell generation depends on the selecting peptide/MHC ligands and how this process impacts the TCR repertoire expressed by Treg cells. We also describe how Treg cells depend on sustained signalling through the TCR and how they are further regulated by Foxp3 enhancer sequences. Finally, we review the impact of microbiota-derived antigens on the maintenance and functionality of the peripheral pool of Treg cells.

Increased proportions of functionally impaired regulatory T cell subsets in systemic sclerosis

Treg abnormalities have been implicated in the pathogenesis of systemic sclerosis (SSc). Treg subpopulations and their cytokines, IL-10 and TGF-β in the peripheral blood of early stage SSc patients were investigated. We hypothesized that epigenetically regulated methylation of the FOXP3 promoter and enhancer regions are altered in Tregs of SSc patients, which might be involved in the T cell imbalance. CD4+CD25+Foxp3+CD127– Treg cells were significantly elevated in patients with diffuse cutaneous SSc and in patients with anti-Scl-70/RNA-Pol-III autoantibody positivity and with lung fibrosis. Increased CD62L+ Treg cells were present in all SSc subgroups. The production of immunosuppressive cytokines by both CD127– and CD62L+ Tregs was diminished. We observed reduced methylation of Treg specific FOXP3 enhancer regions, and elevated FOXP3 gene expression in active SSc cases with negative correlation in the frequency of CD62L+IL-10+ Tregs. Our data indicate an inappropriate distribution and cytokine production of Treg cells in early form SSc.

Vitamin C enhances the expression of IL17 in a Jmjd2-dependent manner.

Previously, we reported that vitamin C facilitates the CpG demethylation of Foxp3 enhancer in CD4+Foxp3+ regulatory T cells (Tregs) by enhancing the activity of a DNA demethylase ten-eleven-translocation (Tet). However, it is not clear whether vitamin C affects other helper T cell lineages like T helper type 17 (Th17) cells which are related with Tregs. Here, we show that the expression of interleukin-17A (IL17) increases with the treatment of vitamin C but not with other antioxidants. Interestingly, the upregulation of IL17 was not accompanied by DNA demethylation in Il17 promoter and was independent of Tet enzymes. Rather, vitamin C reduced the trimethylation of histone H3 lysine 9 (H3K9me3) in the regulatory elements of the Il17 locus, and the effects of vitamin C were abrogated by knockdown of jumonji-C domain-containing protein 2 (jmjd2). These results suggest that vitamin C can affect the expression of IL17 by modulating the histone demethylase activity.

DNA Demethylation of the Foxp3 Enhancer Is Maintained through Modulation of Ten-Eleven-Translocation and DNA Methyltransferases.

Stable expression of Foxp3 is ensured by demethylation of CpG motifs in the Foxp3 intronic element, the conserved non-coding sequence 2 (CNS2), which persists throughout the lifespan of regulatory T cells (Tregs). However, little is known about the mechanisms on how CNS2 demethylation is sustained. In this study, we found that Ten-Eleven-Translocation (Tet) DNA dioxygenase protects the CpG motifs of CNS2 from re-methylation by DNA methyltransferases (Dnmts) and prevents Tregs from losing Foxp3 expression under inflammatory conditions. Upon stimulation of Tregs by interleukin-6 (IL6), Dnmt1 was recruited to CNS2 and induced methylation, which was inhibited by Tet2 recruited by IL2. Tet2 prevented CNS2 re-methylation by not only the occupancy of the CNS2 locus but also by its enzymatic activity. These results show that the CNS2 methylation status is dynamically regulated by a balance between Tets and Dnmts which influences the expression of Foxp3 in Tregs.

CD200R/Foxp3-mediated signalling regulates microglial activation.

The heterogeneity of microglial functions have either beneficial or detrimental roles in specific physiological or pathological environments. However, the details of what transcriptional mechanisms induce microglia to take beneficial phenotypes remain unknown. Here, we report that Foxp3 is essential for beneficial outcome of the microglial response and depends upon signalling by the immunoglobulin CD200 through its receptor (CD200R). Foxp3 expression was up-regulated in microglia activated by excitotoxicity-induced hippocampal neuroinflammation. Suppression of CD200R prevented anti-inflammatory phenotype of microglia, but over-expression of Foxp3 enhanced it. Phosphorylation of STAT6, a downstream effector of CD200R, modulated transcription of Foxp3. Finally, CD200R/Foxp3-mediated signalling enhanced hippocampal neuronal viability and conferred a degree of neuroprotection, presumably by counteracting inducible nitric oxide synthase. We conclude that enhancement of Foxp3 through CD200R could be neuroprotective by targeting the microglia.

Azacytidine mitigates experimental sclerodermic chronic graft-versus-host disease.

BackgroundPrevious studies have demonstrated that regulatory T cells (Tregs) play a protective role in the pathogenesis of chronic graft-versus-host disease (cGVHD). Tregs constitutively express the gene of the transcription factor Foxp3 whose CNS2 region is heavily methylated in conventional CD4+ T cells (CD4+Tconvs) but demethylated in Tregs.MethodsHere, we assessed the impact of azacytidine (AZA) on cGVHD in a well-established murine model of sclerodermic cGVHD (B10.D2 (H-2d) → BALB/cJ (H-2d)).ResultsThe administration of AZA every 48 h from day +10 to day +30 at the dose of 0.5 mg/kg or 2 mg/kg mitigated chronic GVHD. Further, AZA-treated mice exhibited higher blood and thymic Treg frequencies on day +35, as well as higher demethylation levels of the Foxp3 enhancer and the IL-2 promoter in splenocytes at day +52. Interestingly, Tregs from AZA-treated mice expressed more frequently the activation marker CD103 on day +52. AZA-treated mice had also lower counts of CD4+Tconvs and CD8+ T cells from day +21 to day +35 after transplantation, as well as a lower proportion of CD4+Tconvs expressing the Ki67 antigen on day +21 demonstrating an anti-proliferating effect of the drug on T cells.ConclusionsOur results indicate that AZA prevented sclerodermic cGVHD in a well-established murine model of cGVHD. These data might serve as the basis for a pilot study of AZA administration for cGVHD prevention in patients at high risk for cGVHD.Electronic supplementary materialThe online version of this article (doi:10.1186/s13045-016-0281-2) contains supplementary material, which is available to authorized users.

Vitamin C Facilitates Demethylation of the Foxp3 Enhancer in a Tet-Dependent Manner.

Demethylation of CpG motifs in the Foxp3 intronic element, conserved noncoding sequence 2 (CNS2), is indispensable for the stable expression of Foxp3 in regulatory T cells (Tregs). In this study, we found that vitamin C induces CNS2 demethylation in Tregs in a ten-eleven-translocation 2 (Tet2)-dependent manner. The CpG motifs of CNS2 in Tregs generated in vitro by TGF-β (iTregs), which were methylated originally, became demethylated after vitamin C treatment. The conversion of 5-methylcytosin into 5-hydroxymethylcytosin was more efficient, and the methyl group from the CpG motifs of Foxp3 CNS2 was erased rapidly in iTregs treated with vitamin C. The effect of vitamin C disappeared in Tet2(-/-) iTregs. Furthermore, CNS2 in peripheral Tregs in vivo, which were demethylated originally, became methylated after treatment with a sodium-dependent vitamin C transporter inhibitor, sulfinpyrazone. Finally, CNS2 demethylation in thymic Tregs was also impaired in Tet2(-/-) mice, but not in wild type mice, when they were treated with sulfinpyrazone. Collectively, vitamin C was required for the CNS2 demethylation mediated by Tet proteins, which was essential for Foxp3 expression. Our findings indicate that environmental factors, such as nutrients, could bring about changes in immune homeostasis through epigenetic mechanisms.

Therapeutic Treatment of Arthritic Mice with 15-Deoxy Δ12,14-Prostaglandin J2 (15d-PGJ2) Ameliorates Disease through the Suppression of Th17 Cells and the Induction of CD4+CD25-FOXP3+ Cells.

The prostaglandin, 15-deoxy Δ12,14-prostaglandin J2 (15d-PGJ2), is a lipid mediator that plays an important role in the control of chronic inflammatory disease. However, the role of prostanoid in rheumatoid arthritis (RA) is not well determined. We demonstrated the therapeutic effect of 15d-PGJ2 in an experimental model of arthritis. Daily administration of 15d-PGJ2 attenuated the severity of CIA, reducing the clinical score, pain, and edema. 15d-PGJ2 treatment was associated with a marked reduction in joint levels of proinflammatory cytokines. Although the mRNA expression of ROR-γt was profoundly reduced, FOXP3 was enhanced in draining lymph node cells from 15d-PGJ2-treated arthritic mice. The specific and polyclonal CD4+ Th17 cell responses were limited during the addition of prostaglandin to cell culture. Moreover, in vitro 15d-PGJ2 increased the expression of FOXP3, GITR, and CTLA-4 in the CD4+CD25− population, suggesting the induction of Tregs on conventional T cells. Prostanoid addition to CD4+CD25− cells selectively suppressed Th17 differentiation and promoted the enhancement of FOXP3 under polarization conditions. Thus, 15d-PGJ2 ameliorated symptoms of collagen-induced arthritis by regulating Th17 differentiation, concomitant with the induction of Tregs, and, consequently, protected mice from diseases aggravation. Altogether, these results indicate that 15d-PGJ2 may represent a potential therapeutic strategy in RA.