Clinic Rev Allerg Immunol DOI 10.1007/s12016-016-8590-3

Epigenetic Variability of CD4+CD25+ Tregs Contributes to the Pathogenesis of Autoimmune Diseases

Ye Shu1,2 & Qinghua Hu3 & Hai Long1 & Christopher Chang4 & Qianjin Lu1 & Rong Xiao1

# Springer Science+Business Media New York 2016

Abstract Autoimmune diseases are characterized by aberrant andtrimethylationlevelsofhistoneH3andH4whencompared immune responses against healthy cells and tissues. However, with effector T cells, leading to an open chromatin structure. the exact mechanisms underlying the development of these MicroRNAs such as miR-155, miR-126, and miR-10a also conditions remain unknown. CD4+CD25+ regulatory T cells exert an important influence on the differentiation, develop- (Tregs) are a subset of mature T cells which have an important ment, and immunological functions of Tregs. Aberrant epige- role in maintaining immune homeostasis and preventing au- netic modifications affecting Foxp3 and other key in toimmune diseases. Forkhead box p3 (Foxp3), a member of Tregs contribute to disease activity and tissue inflammation in the fork head factor family, is recognized as a autoimmune diseases, which holds great potential for providing marker of CD4+CD25+ Tregs. The decreased number and/ novel targets for epigenetic therapies. Advances in research into or function of CD4+CD25+ Tregs in peripheral blood and the epigenetic regulation of CD4+CD25+ Tregs may also lead related tissues has been demonstrated in systemic lupus to the identification of new epigenetic biomarkers for diagnosis erythematosus, systemic sclerosis, and other autoimmune and prognosis. diseases, which are at least partially regulated by epigenetic mechanisms. Epigenetics refers to the study of potentially Keywords Tregs . Foxp3 . Epigenetic mechanism . heritable alterations in expression without underlying Autoimmune diseases . DNA methylation . Histone changes of the nucleotide sequence, mainly including DNA deacetylation . MicroRNA methylation, histone modification, and microRNAs (miRNAs). For example, DNA methylation status of CpG islands on the Foxp3 gene, which may be affected by normal aging and regulated by environmental factors, plays an impor- Abbreviations tant role in modulating the homeostasis of Foxp3 expression 5-Aza-2′- 5-Aza-2′-deoxycytidine in Tregs. Foxp3 gene in Tregs also shows distinct acetylation dC ATF Activating ATRA All-trans retinoic acid * Rong Xiao [email protected] CNS Conserved noncoding DNA sequence CREB cAMP response element binding 1 Department of Dermatology, The Second Xiangya Hospital, Central CTLA-4 Cytotoxic T lymphocyte-associated antigen-4 South University, Hunan Key Laboratory of Medical Epigenetics, CXCR4 Chemokine 4 Changsha, China DEP Diesel exhaust particles 2 Department of Dermatology, Hunan Province Children’sHospital, DMR Differentially methylated region Changsha, China Foxp3 Forkhead box p3 3 Department of Cardiovascular Surgery, Xiangya Hospital, Central HDACi Histone deacetylase inhibitor South University, Changsha, China IL-2 Interleukin-2 4 Division of Rheumatology, Allergy and Clinical Immunology, KLF Kruppel-like factor University of California at Davis, Davis, CA, USA MSP Methylation-specific PCR Clinic Rev Allerg Immunol

NFAT Nuclear factor of activated T cells Forkhead box p3 (Foxp3) is a member of the fork head NF-kB Nuclear factor-kB transcription factor family and is exclusively expressed in PAH Polycyclic aromatic hydrocarbons CD4+CD25+ Treg cells [1]. Foxp3 plays an important role PD-1 Programmed death-1 in the growth, differentiation, and function of Tregs and is SLE Systemic lupus erythematosus recognized as a marker of CD4+CD25+ Treg cells. In 2001, SOCS1 Suppressorofcytokinesignaling1 Brunkow et al. [2] were the first to report Foxp3 in a study on STAT5 Signal transducer and activator of transcription 5 the Scurfy mouse. Because of a mutation in the Foxp3 gene, TGF-β Transforming growth factor-β the Scurfy mice suffered from CD4+ T cell-mediated lympho- TGP Total glucosides of paeony cyte proliferative diseases and showed cachexia and lympho- TNFR Tumor necrosis factor receptor cyte infiltration in multiple organs. Interestingly, the Scurfy TRAIL- Tumor necrosis factor-related apoptosis-induc- mice recovered from the lymphoproliferative disorders after DR5 ing ligand-death receptor 5 adoptive transfer of normal CD4+CD25+ Tregs. Furthermore, Tregs CD4+CD25+ regulatory T cells FOXP3 transgenic mice had an increased number of CD4+ TSA Trichostatin A CD25+ Tregs, whereas FOXP3 knockout mice lacked CD4+ TSDR Treg-specific demethylated region CD25+ Tregs.Wildin et al. [3] and Bennett et al. [4] found that human and murine Foxp3 genes are homologous and that mutations in FOXP3 caused human IPEX syndrome, which is characterized by immune dysfunction, multiple endocrine Introduction diseases, intestinal disease, and X linkage syndrome. Patients with an autoimmune disease have an abnormal acti- vation of immunocompetent cells, which is associated with Signaling Pathways Inducing the Expression the production of large amounts of autoantibodies and damage of CD4+CD25+ Tregs to multiple organs. However, the exact pathogenesis of auto- immune disease remains unclear. CD4+CD25+ regulatory T The TCR signaling pathway plays a key role in the differen- cells (Tregs) are a subset of mature T cells which has an im- tiation of CD4+CD25+ Tregs [5]. Numerous transcription fac- portant role in maintaining immune homeostasis and tors in the downstream TCR signaling pathway, such as nu- preventing autoimmunity. The development of autoimmune clear factor of activated T cells (NFAT), cAMP response ele- diseases is often associated with a decreased number of ment binding protein (CREB), and activating transcription CD4+CD25+ Tregs or a functional deficiency of these cells. factor (ATF), combine with the Foxp3 promoter or enhancer Several factors influence the CD4+CD25+ Treg number and and participate in Foxp3 regulation. The nuclear factor-kB function. Recent studies suggest that epigenetic mechanisms (NF-kB) signaling pathway induced by the TCR signaling may play an important role in regulating CD4+CD25+ Tregs. pathway is also involved in Treg differentiation. Themis1 and Vav1, newly identified members of the TCR signaling pathway, have been found to interact with each other and Characteristics and Functions of CD4+CD25+ Tregs impair Tregs suppressive functions, and modulate the occur- rence and development of inflammatory bowel disease [6]. In Classification and Markers of CD4+CD25+ Tregs addition, the CD28 signaling pathway plays an important role in the generation and maintenance of Tregs. Previous studies CD4+CD25+ Tregs account for 1 to 3 % of CD4+ T cells in have shown that the number of Foxp3+ T cells in the thymus human peripheral blood and 5 to 10 % of CD4+ T cells in and periphery is decreased to a large extent in the absence of murine lymph node and spleen. Currently, there are two rec- CD28 or its ligands, CD80 and CD86 (Fig. 1). ognized classes of CD4+CD25+ Treg cells, natural Tregs The interleukin-2 (IL-2) signaling pathway also plays an (nTregs), and induced Tregs (iTregs). nTregs develop in the important role in Treg development and immune function. It thymus and experience both positive and negative selection, has been demonstrated that IL-2 is obligatory for the differen- whereas iTregs are derived from the periphery. tiation, expression, and immunosuppressive function of Tregs CD4+CD25+ Tregs express a variety of , such as [7]. Tregs exert suppressive effects on Th1, Th2, and Th17 cytotoxic T lymphocyte-associated antigen-4 (CTLA-4), tumor cells and contribute to the imbalance of the immunologic ho- necrosis factor receptor (TNFR) superfamily member, Toll-like meostasis between Tregs and effector Th cells, thus inhibiting receptor 4, C-X-C chemokine receptor type 4 (CXCR4), pro- the development of allergic and autoimmune reactions. TCR grammed death-1 (PD-1), and lymphocyte antigen complex 6. and CD28 signaling pathways alone do not sufficiently induce However, these proteins lack specificity because they are also Foxp3 expression. This indirect evidence comes from the ob- commonly expressed by activated effector T cells. servation of delayed Foxp3+ thymocytes after birth, while the Clinic Rev Allerg Immunol

Fig. 1 Signaling pathways inducing the expression of Foxp3 in CD4+CD25+ Tregs. Signaling pathways play a key role in the differentiation of CD4+CD25+ Tregs through provoking Foxp3 induction and generation, which involve the TCR and CD28 signaling, interleukin-2 signaling, transforming growth factor-β signaling, and beta2-adrenergic receptor signaling

CD4+CD25+Foxp3− thymocytes were easily detected. This cells and macrophages. IL-10 can inhibit the activation of may be because that Foxp3 induction and generation needs a monocytes and NK cells, IL-2 synthesis by Th1 cells, MHC- secondary signaling, namely, the IL-2 receptor (CD25). Lio II expression in dendritic cells and macrophages, and func- and Hsieh [8] presented a hypothesis on Treg differentiation: tions of antigen-presenting cells. Fibrinogen-like protein 2 is firstly, enhanced TCR signaling leads to increased expression another important inhibitory cytokine secreted by Tregs with of CD25 (alpha chain of IL-2 receptor), then the Treg precur- receptor-mediated immune suppression effects (Fig. 2). sors gain the ability to receive IL-2 signals, and finally induce the expression of Foxp3 through the action of STAT5. Cell to Cell Contact The transforming growth factor-β (TGF-β) signaling path- way is also involved in the induction of Foxp3, as well as the CTLA-4 in CD4+CD25+ Tregs can bind the surface receptors generation of peripheral T cells and thymocytes. Transcription of a target cell and inhibit their activation and proliferation. factors Smad3 and NFAT are required for maintaining the In vitro experiments showed that anti-CTLA-4 antibodies can enhancer activity of FOXP3, and histidine acetylation of partly block the inhibitory function of Tregs. CD25 expressed Smad3 and NFAT in the enhancer region plays an important on CD4+CD25+ Tregs can competitively bind IL-2, which role in the derivation of FOXP3. It was demonstrated that plays a key role in T cell proliferation, thereby inhibiting T rapamycin can stimulate the expression of Foxp3 and genera- cell proliferation and differentiation. PD-1 combining with its tion and development of induced Tregs via CD39 and Runt- ligand can block PI3K pathway mediated by a T cell signaling related transcription factor 1 (Runx1) pathway in the presence molecule CD28 and inhibit T cell proliferation and of IL-2 and TGF-β [9]. Beta2-adrenergic receptor signaling differentiation. also plays an important role in the regulation of Tregs suppres- sive functions by increasing cAMP and CTLA-4 expression Effects on Apoptosis and Cell Necrosis and protein kinase A-dependent CREB phosphorylation [10]. CD4+CD25+ Tregs can directly destroy effective T cells by Mechanisms of Immunosuppressive Action releasing granzyme A/B and perforin or through tumor necro- of CD4+CD25+ Tregs sis factor-related apoptosis-inducing ligand (TRAIL)-death receptor 5 (TRAIL-DR5) pathway. Granzyme is a serine Secretion of Inhibitory Cytokines esterase that can enter the cytoplasm, activate protease direct- ly, and cause cell apoptosis. Tregs exert suppressive effects on CD4+CD25+ Tregs secrete many immunoinhibitory cyto- effector T cells by PD-1 signaling, and the PD-1 antibody was kines, such as TGF-β and IL-10. TGF-β can inhibit IL-1 reported to block the therapeutic effects of Tregs in allergic and IL-2 secretion and T cell proliferation, render NK cells asthma. A recent study demonstrated that Foxp3 downregu- dysfunctional, and inhibit the immune function of dendritic lated cell growth but upregulated cell apoptosis by inducing Clinic Rev Allerg Immunol

Fig. 2 Mechanisms of immunosuppressive action of CD4+CD25+ Tregs. The immune suppression functions of CD4+ CD25+ Tregs have an important role in maintaining immune homeostasis, which involve secreting inhibitory cytokines such as TGF-β, IL-10, and fibrinogen-like protein 2; adjusting cell-cell contact; inducing apoptosis and cell necrosis via granzyme, perforin, caspases, etc; and interfering cell metabolisms

the expression of proapoptotic molecules, caspases-3 and Foxp3 expression and Treg numbers. In addition, older mice caspases-7 [11]. Another study showed that Foxp3 can induce produced higher levels of IL-10, and dendritic cells expressed the expression of poly(ADP-ribose) polymerase-1, caspase-3, lower levels of CD86 compared with younger mice, suggestive and caspase-9, and reduce the expression of antiapoptotic of a stronger inhibitory function of Tregs (Fig. 3). molecules, thus inhibiting cell proliferation and inducing cell Nadeau et al. [20] found that a high intake of environmental apoptosis [12]. airborne polycyclic aromatic hydrocarbons (PAH) resulted in FOXP3 hypermethylation and decreased Foxp3 expression in Interference with Cell Metabolism children. Exposure to diesel exhaust particles (DEP) was also related to increased Foxp3 methylation and decreased FOXP3 CD4+CD25+ Tregs can interfere with the stability and the expression in children [21]. The relationship between aging metabolic pathways of target cells and thereby inhibit the and Treg cell function is apparent but the mechanisms are still immune response. The second messenger cAMP is involved unclear. in these processes via its consumption of IL-2, eventually Methylation status of CpG islands on the Foxp3 gene may inducing apoptosis of the target cells. be a potential biomarker for certain diseases. Ngalamika et al. [22] noted that the TSDR on FOXP3 was highly conserved. TSDR was demethylated in nTregs while highly methylated in Epigenetic Mechanisms Are Involved effector T cells. It has been reported that the levels of FOXP3 in the Regulation of CD4+CD25+ Tregs TSDR methylation in peripheral blood Tregs were significant- ly different between active systemic lupus erythematosus DNA Methylation and CD4+CD25+ Tregs (SLE) and healthy controls. This phenomenon was also ob- served in psoriasis patients and patients with chronic bacterial The methylation state of Treg-specific demethylated region infections compared with healthy controls. FOXP3 TSDR (TSDR) plays an important role in maintaining the stable methylation status can be a potential marker to assess severe expression of Foxp3 in Tregs [13–15]. Except a conserved autoimmune diseases and chronic bacterial infections. promoter region, the Foxp3 gene has three adjacent conserved Wiencke et al. [23] utilized methylation-specific PCR (MSP) noncoding intronic sequences—CNS1, CNS2, and CNS3 [16, methodology to detect DNA methylation of the Foxp3 gene 17]. Analysis of the DNA methylation status of the FOXP3 and immunohistochemical method to detect infiltrated Tregs showed hypomethylation of the CpG islands in the CNS2 in human glioma. The analysis revealed a highly positive cor- region of nTregs, incomplete hypomethylation in iTregs, and relation between demethylation and infiltrated Tregs and a complete methylation in other Th cells [16]. Lal et al. [18] negative correlation between infiltrated Tregs and patient sur- reported that another CpG island in the conserved upstream vival rates. enhancer region was hypomethylated in nTregs while methyl- Methylation status of CpG islands on the Foxp3 gene may ated in iTregs. Garg et al. [19] studied C57BL mice of different also be a predictor of immune tolerance. Syed et al. [24]found age groups and found that with advanced age, hypomethyla- significant differences in Treg function and the demethylation tion of CpG sites on FOXP3 was associated with increased status of the Foxp3 promoter region between patients with and Clinic Rev Allerg Immunol

Fig. 3 Epigenetic mechanisms involved in the regulation of CD4+CD25+ Tregs. Epigenetic mechanisms play an important role in the regulation of CD4+ CD25+ Tregs by DNA methylation, histone modification, and microRNA regulation. Epigenetic regulators, such as Uhrf1, KLF10, and KLF14, affect the number and/or functions of Tregs. Various medicines and environmental factors exert simulating or inhibitory effects on the number and/or functions of Tregs by epigenetic mechanisms. miR-155, miR-126, and miR-10a also contribute to the stimulating effect on Tregs

without immune tolerance after peanut oral immunotherapy. versus-host disease patients who received Foxp3+ Tregs When the patients stopped the peanut immunotherapy and pretreated with all-trans retinoic acid experienced alleviation regained the allergy to peanuts, the methylation level of the of the disease. Obata et al. [29] found that Uhrf1, a DNA meth- Foxp3 promoter region increased correspondingly. Swamy ylation regulator, accelerated intestinal Treg proliferation and et al. [25] reported that timothy grass and dust mite dual sub- maturation, whereas intestinal Tregs in Uhrf1 gene knockout lingual immunotherapy induced immune tolerance by epigenet- mice failed to proliferate or mature normally. The Uhrf1 defect ic modifications. The sublingual immunotherapy induced led to hypomethylation of cell cycle-dependent kinase inhibitor demethylation of Foxp3 CpG sites, promoted allergen- gene CDKN1A and high expression of P21, which then influ- specific CD4+CD25+Foxp3+ Tregs, reduced antigen-specific enced Treg differentiation and development. IgE levels, and increased the levels of specific IgG4 to timothy grass and dust mite. Histone Modification and CD4+CD25+ Tregs Many pharmaceuticals act by altering the methylation status of Foxp3 CpG sites in Tregs. Singer et al. [26] discovered that Histone modification is an important mechanism to regulate CD4+CD25+Foxp3+ Treg expression, activation, and sup- differentiation and development of Tregs. ChIP technology pressor function in the lungs of wild-type mice with acute showed that Foxp3 gene in Tregs has an open euchromosome respiratory distress syndrome improved significantly after 5- structure and different histone H3 and H4 acetylation and aza-2′-deoxycytidine (5-aza-2′-dC) treatment, and the mice trimethylation levels compared with the effector T cells [11]. recovered rapidly from the disease. Moreover, mice with Xiong et al. [30] revealed that the Foxp3 promoter comprised Treg-depleted (diphtheria toxin-treated Foxp3) and two completely different areas: one in the disconnected state recombinase activating gene-1-null mice with lymphocyte or regulated by polycomb histone methyltransferase complex Treg clearance had no recovery after treatment with 5-aza-2′- and the other in the connected state regulated by histone ace- deoxycytidine, whereas Treg-deficient mice that received tyltransferase. Kruppel-like factor (KLF) that is involved in exogenous Tregs after 5-aza-2′-deoxycytidine treatment were the polycomb element can alter this state. The expression of saved from lung inflammation. Foxp3 and the differentiation of iTregs were influenced both Mangano et al. [27]reportedthat5-aza-2′-deoxycytidine in vivo and in vitro when the Foxp3 promoter in KLF10(−/−) treatment strengthened hypomethylation of Foxp3 CpG mice was silenced by the EZH2 gene through trimethylation islands, increased the percentage of Tregs in the circulatory of histone H3 lysine 27 (H3K27me3). Using chromatin pre- system, and improved clinical symptoms in a mice model of cipitation and genome-integrated reporter assays, Xiong et al. allergic encephalomyelitis. Lu et al. [28] demonstrated that [31] demonstrated that KLF10 protein could act as a toggle all-trans retinoic acid (ATRA) strengthened histone acetyla- switch between two contrary histone modification pathways, tion and hypomethylation of the Foxp3 promoter region, Sin3-HDAC and PCAF, to regulate Foxp3 expression. maintained Foxp3 expression and suppressor function of Sarmento et al. [32] found that KLF14-knockout T cells easily Foxp3+ Tregs, and prevented the conversion of Tregs into differentiated into adaptive Tregs with stronger suppressive Th1 and Th17 cells in vitro and in vivo. Furthermore, graft- function in vivo and in vitro. They suggested that KLF14 Clinic Rev Allerg Immunol integrated into the Foxp3 TSDR enhancer region, remodeled [40]. Tselios et al. [41] reported a similar negative correlation the chromatin structure at the FOXP3 TSDR region, and between the number of CD4+CD25+ Tregs and SLE disease inhibited Foxp3 expression at the messenger RNA (mRNA) activity. CD4+CD25+ Tregs are therefore considered as a po- and protein levels. tential cellular marker to evaluate the disease activity in SLE CD4+CD25+ Tregs require lysine acetylation in the Foxp3 patients. Xing et al. [42] found that patients with lupus nephri- region to maintain their Treg functions. Acetylation of Foxp3 tis showed an increased level of Th17/Treg ratio and SLEDAI region facilitates its binding to the IL-2 promoter gene and scores, with reduced peripheral CD4+CD25+Foxp3+ Tregs inhibits endogenous IL-2 production. Histone deacetylase in- compared to lupus patients without renal damage. hibits the transcription of the Foxp3 gene to some extent [33]. Lin et al. [43] reported that active SLE patients had a lower Histone deacetylase inhibitor (HDACi) treatment enhances Foxp3 expression rate in CD4+CD25+ Tregs than SLE pa- the homeostasis mediated by Treg proliferation and is thought tients in remission and healthy normal controls, while SLE to be used as an effective method of increasing Treg numbers patients in remission had lower Foxp3 expression rates than and suppressor function. Liesz et al. [34] administered the healthy controls. The ratio of Treg/Th17 cells in patients with histone deacetylase inhibitor Trichostatin A (TSA) to mice active SLE was decreased significantly when compared with with experimental stroke and found that TSA not only in- inactive SLE patients and normal controls, while the ratio of creased Treg numbers and their immune suppressive function, Treg/Th17 cells in patients with active SLE increased after but also boosted IL-10 expression, reduced infarct size, and glucocorticoid therapy, and the ratio correlated negatively controlled brain inflammation. The HDAC6 inhibitor, with severity of the disease [44]. Decreased Tregs and tubastatin, displayed similar effects. Li et al. [35]proposed Foxp3 gene expression were observed in multiple organs of that the histone methyltransferase Jmjd3 plays an important lupus-prone BXSB mice [45]. Purified CD4+CD25+ Treg role in regulating T cell differentiation. Jmjd3 defects inhibit treatment following remission raised the survival rates and Tregs from differentiating into Th1 cells. extended the disease remission period in NZB/NZW F1 lupus mice compared to lupus mice without administration of CD4+ MicroRNA Regulation and CD4+CD25+ Tregs CD25+ Tregs [46]. It is possible that Treg-based immunother- apy may eventually be used to improve the prognosis of SLE. MicroRNA regulation has an important impact on Treg differ- Epigenetic regulation of FOXP3 and other related genes in entiation and development, particularly through regulation by SLE Tregs has been documented and considered important in microRNA-155. The number of Tregs in the thymus and the pathogenesis of lupus autoimmunity. Zhao et al. [47] periphery of microRNA-155-deficient mice is decreased sig- reported that the promoter region of Tregs in SLE patients nificantly, while the suppressor function of Tregs remains maintained a hypermethylation status, and had decreased per- unaffected [36]. Lu et al. [37] demonstrated that Foxp3- centage of CD4+CD25+ Tregs in the peripheral blood com- dependent microRNA-155 sustained IL-2 amounts by pared to healthy controls. Several studies [48]haveutilized targeting suppressor of cytokine signaling 1 (SOCS1) expres- genome-wide analytic methods to detect the DNA methylome sion and activating transcription factor signal transducer and in different immune cells from SLE patients. CD4+ T cells, activator of transcription 5 (STAT5). The study suggested a CD19+ B cells, and CD14+ monocytes showed severe hypo- positive role of microRNA-155 in sustaining Treg prolifera- methylation in interferon (type I) signaling related genes in tive activity and numbers via its inhibition on SOCS1. Qin both acute and stable phases of SLE. Notably, this epigeneti- et al. [38] reported that miR-126 could upregulate Tregs cally poised hypersensitivity to interferon also existed in expression and the suppressive function of Tregs by acting Tregs. Total glucosides of paeony (TGP) is a traditional on the PI3K-Akt pathway. Jeker et al. [39] suggested that Chinese medicine with anti-inflammatory and immune regu- miR-10a may be indispensable for Tregs stabilization and latory functions and is used to treat rheumatoid arthritis and function. other autoimmune diseases. Zhao et al. [47] treated CD4+ T cells of SLE patients and healthy controls with TGP and found decreased methylation levels on the Foxp3 promoter region, Epigenetic Variability of CD4+CD25+ Tregs significantly increased Foxp3 expression, and a higher per- in the Pathogenesis of Autoimmune Diseases centage of CD4+CD25+ Tregs in TGP-treated CD4+ T cells from SLE patients compared to CD4+ T cells before treat- Systemic Lupus Erythematosus ment. TGP-treated CD4+ T cells of healthy controls demon- strated no such difference. Moreover, IFN-γ and IL-2 produc- A study found a reduced number of CD4+CD25+Foxp3+ tion of TGP-treated CD4+ T cells in SLE patients both Tregs in the peripheral blood of SLE patients compared to increased significantly compared to the production before healthy controls, and a negative correlation between Treg treatment. Regna et al. [49] administered ITF2357, a specific numbers and the SLE disease activity index (SLEDAI) score class I and II histone deacetylase inhibitor, to NZB/W lupus Clinic Rev Allerg Immunol mice and found increased Foxp3 acetylation levels and Using bisulfite sequencing, Wang et al. [60] reported percentage of Tregs, as well as reduced serum inflammatory hypermethylation of Foxp3 promoter region in CD4+ T cells cytokines and kidney damage. of systemic sclerosis patients, which was accompanied by Clinical research has confirmed that many other medica- reduced Foxp3 expression and percentage of CD4+CD25+ tions can restore the quantity of CD4+CD25+ Tregs, as well Foxp3+ Tregs in CD4+ T cells. Moreover, when CD4+ T cells as their suppressor function, and thereby can potentially re- of scleroderma patients were treated with 5-aza, a methylation build the immune tolerance in SLE patients and animal inhibitor in cell culture, the methylation levels of the Foxp3 models, although the mechanisms remain unclear. Several promoter reduced greatly, followed by increased Foxp3 studies have administered cyclophosphamide, methylprednis- expression and percentage of CD4+CD25+Foxp3+ Tregs in olone, and intravenous gamma immunoglobulins to SLE CD4+ T cells. Taken together, these suggest that the hyperme- patients and observed that the peripheral CD4+CD25+ Tregs thylation status in the Foxp3 promoter region results in increased significantly during remission [50, 51]. Treatment decreased CD4+CD25+Foxp3+ Tregs in systemic sclerosis with A771726, an active metabolite of leflunomide, and KN- patients, and influences disease severity of systemic sclerosis. 93, an inhibitor of calmodulin-dependent protein kinase IV, increased the number of Foxp3+ Tregs and reduced kidney Rheumatoid Arthritis damage in MRL/lpr lupus mice [52, 53]. It is a fascinating question whether these medications increase Foxp3 expres- Several researchers have reported a reduced percentage and sion and/or the number of Foxp3+ Tregs through epigenetic impaired suppressive function of CD4+CD25+ Tregs in the mechanisms. peripheral blood of rheumatoid arthritis patients. Niu et al. [61] found that the percentage of Th17 cells and the levels Systemic Sclerosis of related cytokines IL-17, IL-23, IL-6, and TNF-α were sig- nificantly higher in the peripheral blood of rheumatoid arthri- Many studies have reported significantly decreased CD4+ tis patients compared to normal individuals. Furthermore, the CD25+Foxp3+ Tregs in patients with systemic sclerosis. percentage of CD4+CD25+ Tregs and TNF-β1 levels was Klein et al. [54] demonstrated that the frequency of CD4+ significant lower in the peripheral blood of rheumatoid arthri- CD25+Foxp3+ Tregs in skin lesions but not in peripheral tis patients compared to normal individuals. These results blood in patients with limited cutaneous systemic sclerosis suggest that Th17/Treg imbalance is involved in the pathogen- or diffuse cutaneous systemic sclerosis was reduced signifi- esis of rheumatoid arthritis. The percentage of CD4+ cantly compared to patients with other inflammatory skin dis- CD25(high)CD127(low/−) Tregs in the peripheral blood of eases. Antiga et al. [55] found lower number of CD4+CD25+ active rheumatoid arthritis patients was reduced compared to Foxp3+ Tregs, and reduced expression of IL-10 and TGF-β in healthy controls and negatively correlated with rheumatoid peripheral blood and skin lesions of patients with limited arthritis disease activity score and CRP and ESR levels in cutaneous systemic sclerosis or diffuse cutaneous systemic these patients [62]. sclerosis compared with healthy controls by cytofluorometry, Kennedy et al. [63] detected a differentially methylated immunohistochemistry, and enzyme-linked immunosorbent region (DMR) upstream of the Foxp3 promoter in rheumatoid assays. Kataoka et al. [56] found a decreased percentage of arthritis patients and healthy controls. The authors found that CD4+CD25+Foxp3+ Tregs in patients with systemic sclerosis the methylation level of DMR was negatively correlated to the when compared with healthy subjects. The expression of the expression of the Foxp3 gene. Furthermore, lower DMR transcription factor Runx1 in Tregs was also reduced in methylation level and higher Foxp3 gene expression were patients with systemic sclerosis compared to healthy subjects. found in the Tregs from patients with negative rheumatoid Mathian et al. [57] found lower proportions of active and factor compared to Tregs from patients with positive rheuma- resting Tregs in the blood and skin of systemic sclerosis toid factor. Using cell culture experiments, Cribbs et al. [64] patients compared to healthy subjects. Baraut et al. [58] found that methotrexate treatment induced hypomethylation reported reduced frequency and immune inhibitory function of a Foxp3 upstream enhancer in Tregs of rheumatoid arthritis of peripheral CD4+CD25+Foxp3+ Tregs in patients with dif- patients. Also, increased Foxp3 and CTLA-4 expression and fuse cutaneous systemic sclerosis compared to healthy con- recovered immune inhibitory Treg function were observed in trols. Autologous hematopoietic stem cell transplantation rheumatoid arthritis patients following methotrexate resulted in the recovery of the decreased frequency and treatment. immune suppressive function of peripheral CD4+CD25+ Lin et al. [65] explored kaempferol, a common flavonoid in Foxp3+ Tregs. Another study found a decreased number of vegetables and fruits, as treatment for collagen-induced arthri- circulating CD4+CD25+Foxp3+ Tregs in systemic sclerosis tis in rats. The study found a reduced phosphorylation level of patients compared with healthy controls and increased Tregs Foxp3 and enhanced expression of Foxp3 in Tregs, along with after clinical treatment [59]. improved immune suppressor function of Tregs and alleviated Clinic Rev Allerg Immunol arthritis symptoms in collagen-induced arthritis rats following numbers in clinical remission and after treatment [59, 74]. kaempferol treatment. Richetta et al. [74] further found that increased Tregs Rossetti et al. [66] found that rapamycin treatment induced were associated with reduced PASI scores in clinical re- the expansion of Tregs in rheumatoid arthritis patients and mission of psoriasis patients. restored Treg-mediated immune suppression by demethyla- Zhao et al. [75] reported significantly higher miR-210 tion of a Foxp3-specific demethylation region. A study [67] in CD4+ T cells of psoriasis patients than in normal con- found decreased levels of both Foxp3 mRNA and microRNA- trols. Furthermore, Foxp3 was confirmed to be a target 21 in rheumatoid arthritis patients, as well as an increased gene regulated by microRNA-210. CD4+ T cells from proportion of Th17 cells and decreased CD4+CD25+ healthy controls had reduced Foxp3 expression and im- Foxp3+ Treg proportion in peripheral blood mononuclear paired Treg immune suppressor function following over- cells from rheumatoid arthritis patients compared to healthy expression of microRNA-210. Contrarily, CD4+ T cells of controls. MicroRNA-21 serves as a negative feedback and can psoriasis patients demonstrated increased Foxp3 expres- affect the balance of Treg and Th17 cells in rheumatoid arthri- sion and restored Treg immune suppressor function fol- tis patients. Zhou et al. [68] also observed that reduced expres- lowing microRNA-210 inhibition. sion of miR-146a in Tregs of patients with rheumatoid arthritis Although Tregs serve as a suppressor of autoimmune re- might be correlated with a proinflammatory phenotype of sponse, they can also differentiate into Th17 cells that induce Tregs and disease activity. inflammatory reactions under proinflammatory circum- Various side effects of immunosuppressive drugs have lim- stances. Tregs in patients with severe psoriasis had a higher ited their clinical application in the treatment of rheumatoid tendency of differentiating into Th17 cells compared to arthritis, but research on other medications with less toxicity healthy controls. Treatment with a histone deacetylase inhib- have made considerable progress both in clinical trials and in itor, Trichostatin A, inhibited this abnormal conversion of animal experiments in collagen-induced arthritis rodent Tregs in severe psoriasis patients [76]. models. Treatment with 99Tc-methylenediphosphonate, dual- Treatment of psoriasis patients with cyclosporine and the specificity phosphatase 5, rebamipide, and poly(ADP-ribose) antitumor necrosis factor alpha antibody, etanercept, inhibited polymerase-1 inhibitors has led to increases in the number of the activation of Th17 cells and increased CD4+CD25+ Foxp3+ Tregs and a reduced clinical score of arthritis in Foxp3+ Treg numbers in peripheral blood [77, 78]. Other human subjects and arthritis mice [69–73]. Further investiga- studies have shown that both CD4+CD25+Foxp3+ Treg num- tion into the therapeutic mechanisms, including the epigenetic ber and function were augmented in psoriasis patients and aspect, of these medicines and their roles in modulating CD4+ psoriasis-like mice after PUVA therapy [79, 80]. These drugs CD25+Foxp3+ Tregs is warranted. and treatments restored the quantity or function of Tregs in psoriasis patients and psoriasis-like mice, and further studies Psoriasis are required to explore the relationship between this mecha- nism and epigenetics. Psoriasis is an autoimmune skin disease, and a growing number of studies have reported an imbalance of Th17 Other Autoimmune Diseases and Treg cells and impaired Treg numbers and/or func- tions in psoriasis. Flow cytometry demonstrated signifi- Ferraro et al. [81] found an unbalanced state characterized by cantly decreased CD4+CD25+Foxp3+ Treg numbers in downregulated Treg and upregulated Th17 immunity in the the peripheral blood from psoriasis patients compared to pancreatic-draining lymph nodes of type 1 diabetes, which healthy controls, and restored CD4+CD25+Foxp3+ Treg was not observed in the peripheral blood. Wang et al. [82]

Table 1 Targets or biomarkers for DNA methylation of CD4+ Change of Tregs DNA Methylation References CD25+ Tregs in autoimmune diseases SLE Peripheral blood ↓ (N) Hypermethylation in Foxp3 promoter [40–45, 47] Mice multiple organ ↓ (N) TGP → Tregs ↑ (N) SSc Peripheral blood ↓ (N and F) Hypermethylation in Foxp3 promoter [54–60] Human skin lesions ↓ (N and F) 5-AZA → Tregs ↑ (N) RA Peripheral blood ↓ (N and F) MTX → Tregs ↑ (N and F) [61–64, 66] Rapamycin → Tregs ↑ (N and F) Diabetes Lymph nodes ↓ (F) Hypermethylation in Foxp3 promoter [81, 82]

N number of Tregs, F function of Tregs, TGP total glucosides of paeony, 5-AZA 5-aza-2′-deoxycytidine, MTX methotrexate Clinic Rev Allerg Immunol

Table 2 Targets or biomarkers for histone modification of CD4+ Change of Tregs Histone modification References CD25+ Tregs in autoimmune a diseases SLE Peripheral blood ↓ (N) ITF2357 → Tregs ↑ (N) [40–45, 49] Mice multiple organ ↓ (N) Psoriasis Peripheral blood ↓ (N) Trichostatin A → Tregs ↑ (N) [59, 74, 76]

N number of Tregs, F function of Tregs a ITF2357 is a histone deacetylase inhibitor

suggested the presence of a hypermethylation status in the Discussion Foxp3 promoter in patients with fulminant type 1 diabetes, which blocks IRF-7 binding to the Foxp3 promoter and re- CD4+CD25+ Tregs play important roles in maintaining duces Foxp3 expression, and thus interferes with the develop- immune homeostasis. Many autoimmune diseases have ment of Tregs. Hezova et al. [83] found that miRNA-510 was decreased number and/or impaired immune suppressor func- upregulated and both miRNA-342 and miRNA-191 were tion of CD4+CD25+ Tregs in periphery and/or target organs. downregulated in Tregs of patients with type 1 diabetes. The interaction and balance between Th17 cells and Treg cells In Tregs of ulcerative colitis patients, Mohammadnia- appear to be particularly relevant in autoimmunity. The Afrouzi et al. [84] found significantly increased expression of etiology and pathogenesis of autoimmune diseases are very miR-31 level and decreased expression of miR-21, miR-146a, complex, involving genetic factors, multiple autoantigens, and miR-155 levels. Singh et al. [85] observed that miR-15b/ cross-reacting antigens caused by bacterial and viral infection, 16 upregulated Treg development and alleviated the severity of immune regulation abnormalities, environmental factors, and autoimmune colitis by the mammalian target of rapamycin so on. For autoimmune diseases, the genetic susceptibility (mTOR) signaling pathway. defines the underlying propensity to develop disease, with De Santis et al. [86] found that decreased miR-106b each autoimmune disease containing its own specific sets of and miR-25 expression may influence Treg activity in genetic variants. For example, susceptibility genes of systemic patients with multiple sclerosis by regulating the TGF-β lupus erythematosus include IRF5, STAT4, and TNFSF4, signaling pathway. miR26a was observed to increase while susceptibility genes of systemic sclerosis include Tregs, decrease Th17-related cytokines, and alleviate dis- HLA-A23, HLA-B18, and HLA-DR11. ease severity by repressing IL-6 in experimental autoim- Environmental factors represent external triggers for auto- mune encephalomyelitis (EAE) mice, a well-established immune diseases and they exert their effects in genetically animal model of human multiple sclerosis [87]. miR-17- susceptible individuals. Similar to genetic factors, each auto- 92 cluster was proved to be important for differentiation immune disease has its own set of identified inducing factors. of Treg through induction of IL-10 expression in EAE For example, systemic lupus erythematosus can be affected by mice [88]. These findings suggest a potentially crucial factors such as pregnancy, ultraviolet light, and viral infec- role of epigenetics in regulating the number and function tions, while systemic sclerosis can be influenced by exposure of Tregs and in the pathogenesis of autoimmune diseases. to silicon.

Table 3 Targets or biomarkers for miRNA regulation of CD4+ Change of Tregs miRNA regulation References CD25+ Tregs in autoimmune diseases RA Peripheral blood ↓ (N and miR-21 ↓ [61, 62, 67, F) miR-146a ↓ 68] Psoriasis Peripheral blood ↓ (N) miR-210 ↓→Tregs ↑ (N and F) [59, 74, 75] Diabetes Lymph nodes ↓ (F) miR-510 ↑ miR-342 ↓ miR-191 ↓ [81, 83] Autoimmune miR-31 ↑ miR-21 ↓ miR-146a ↓ miR- colitis 155 ↓ miR-15b/16 → Tregs ↑ (N) [84, 85] Multiple sclerosis miR-106b ↓ miR-25 ↓ [86–88] miR26a → Tregs ↑ (N) miR-17-92 → Tregs ↑ (N and F)

N number of Tregs, F function of Tregs Clinic Rev Allerg Immunol

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