http://informahealthcare.com/aut ISSN: 0891-6934 (print), 1607-842X (electronic) Autoimmunity, 2014; 47(4): 234–241 ! 2014 Informa UK Ltd. DOI: 10.3109/08916934.2013.801462

REVIEW ARTICLE

Epigenetic regulation of cytokine expression in systemic lupus erythematosus with special focus on T cells Christian M. Hedrich, Jose C. Crispin, and George C. Tsokos

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Division of Rheumatology, Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts, USA

Abstract

Keywords

Epigenetic events play a central role in the priming, differentiation and subset determination of T lymphocytes. Through their influence on chromatin conformation and DNA-accessibility to transcription factors and RNA polymerases, epigenetic marks allow or prevent gene expression and control cellular functions including cytokine expression. CpG-DNA methylation and posttranslational modifications to histone tails are the two most well accepted epigenetic mechanisms. The involvement of epigenetic mechanisms in the pathogenesis of systemic lupus erythematosus (SLE) has been suggested by the development of lupus-like symptoms by individuals who received procainamide or hydralazine treatment resulting in a reduction of CpG-DNA methylation. To date, a growing body of literature indicates that the deregulation of cytokine expression through epigenetic disturbances can result in altered immune responses and autoimmune reactions. Over the past decade, various global and regional epigenetic alterations have been reported in immune cells from patients with SLE and other autoimmune disorders. More recently, the molecular mechanisms that result in epigenetic disturbances have been addressed, and deregulated transcription factor networks have been demonstrated to mediate epigenetic alterations in B and T lymphocytes from SLE patients. A better understanding of the molecular events that contribute to epigenetic alterations and subsequent immune imbalance is essential for the establishment of disease biomarkers and identification of potential therapeutic targets.

Autoimmune disease, chromatin, epigenetic, inflammation, SLE

Introduction Systemic lupus erythematosus (SLE) is a severe autoimmune disease that can affect every organ in the human body and is facilitated by an aberrant immune response [1]. Despite multiple aims for discovering the pathogenic origin of SLE, a definitive understanding of the molecular mechanisms resulting in the clinical picture of SLE still remains to be determined. Genetic factors confer a predisposition to the development of SLE. However, only a small subset of patients exhibit disease-causing mutations in single genes (e.g. the complement components C1q, C2 or C4) [1,2]. Thus, SLE is believed to result from the combined effects of genomic variants in a number of susceptibility genes and hormonal, environmental and epigenetic changes. Risk alleles and nongenetic modifying factors together contribute to a different extent to the development, progression and outcome of SLE [3]. Epigenetic mechanisms of gene regulation represent events that affect gene expression without affecting the DNA sequence [3,4]. Epigenetic marks of genes and gene loci Correspondence: Christian M. Hedrich, MD, Division of Rheumatology, Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02115, USA. E-mail: chedrichbidmc.harvard.edu

History Received 25 January 2013 Revised 9 April 2013 Accepted 28 April 2013 Published online 24 April 2014

determine signal- and tissue-specific expression patterns. The addition of methyl groups to the 50 carbon position of cytosine within cytosine-phosphate-guanosine (CpG-)dinucleotides reduces DNA accessibility to transcription factors and RNA polymerases. CpG-DNA methylation is mediated by ‘‘maintenance’’ or ‘‘de novo’’ DNA methyltransferases (DNMTs) [5–7]. Maintenance enzymes are responsible for the remethylation of the second strand during cell division (e.g. DNMT1), yet de novo enzymes mediate CpG-DNA methylation independent of the cell cycle (e.g. DNMT3). However, more recently, it became clear that this classical grouping is somewhat oversimplified and that maintenance enzymes can also have de novo methyltransferase activity [6,8,9]. Nucleosomes are comprised of 146 base pairs of genomic DNA coiled around a histone octamer consisting of two copies of each of the histone proteins: H2A, H2B, H3 and H4. Post-translational modifications to histone tails control the organization of nucleosomes. The most common histone modifications include acetylation, methylation, ubiquitination, phosphorylation, sumoylation, citrullination, ADP ribosylation and proline isomerisation. Single histone tail markers as well as the combination of multiple histone modifications mediate changes in nucleosome arrangement and chromatin structure [3,10–13]. CpG-DNA methylation and the histone code usually reflect each other. Transcriptionally inactive

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(hetero-)chromatin is characterized by high levels of CpGDNA methylation and repressive histone modifications. Actively transcribed genes exhibit low levels of CpG-DNA and histone methylation but high levels of permissive histone acetylation [6,14]. Epigenetic patterns are generally variable, allowing cells and tissues to differentiate and/or adapt to environmental changes. The observation that otherwise healthy individuals can develop lupus-like symptoms in response to the inhibition of CpG-DNA methylation by medications such as hydralazine or procainamide lead to the conclusion that epigenetic mechanisms play a central role in the pathophysiology of SLE [3,5,6,14–16].

Epigenetic control and autoimmunity

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CpG-DNA methylation Over the past decade, the impact of disrupted epigenetic patterns on immune homeostasis has been documented. Environmentally triggered epigenetic mechanisms appear to be the ‘‘missing link’’ between genomic predispositions for SLE and disease development, progression and outcome. Epigenetic mechanisms play a key role during the priming, differentiation and subset determination of T cells. The expression of lineage defining cytokines, including interleukin (IL-)4 and interferon gamma (IFN-g) are under tight epigenetic control [17]. Although the Il4 and Ifn genes are both partially silenced in naı¨ve CD4þ T lymphocytes, Ifng is accessible and Il4 is silenced in Th1 cells. Conversely, Il4 is accessible and Ifn is silenced in Th2 cells. Similar patterns can be seen in cytokine genes within the IL10 cluster on chromosome 1q31-32 [4,18]. T cells exhibit reduced CpGDNA methylation in a regulatory region in the 4th intron of Il10 in response to co-culture with IL-27, a strong inducer of IL-10 expression [19]. IL-19 is silenced in T lymphocytes through CpG-DNA methylation while in macrophages that express IL-19, the Il19 gene undergoes site and tissue specific de-methylation in several regions [20]. Epigenetic alterations of cytokine genes have been associated with various autoimmune/inflammatory disorders. In SLE, global CpG-DNA hypo-methylation correlates with disease activity. A number of cytokine genes are overexpressed in CD4þ T lymphocytes from SLE patients in a chromatin-dependent manner, including IL4 [21], IL6 [22], IL10, IL13 [23] and IL17A [24]. Increased expression of IL-6 contributes to the differentiation of neutrophils, B and T lymphocytes and the induction of IgG production [25]. IL-10 exerts pro-inflammatory functions through the induction of B cell activation and differentiation and IgG production [4]. Increased IL-17 production instigates inflammation and promotes autoantibody responses [26–28]. Together with the adjacent IL17A gene, IL17F is subject to demethylation in T cells from SLE patients [24,29]. However, IL-17 F fails to be expressed in T cells from SLE patients in response to TCR stimulation secondary to trans-repressive activity of the transcription factor cAMP response element modulator (CREM)a on the IL17F promoter. This may result in reduced levels of IL-17 F homo- and IL-17 A/F heterodimers which both have reduced inflammatory potential when compared to IL-17 A [29].

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In contrast to the aforementioned effector cytokines, IL-2 fails to be expressed in SLE T lymphocytes [30]. The failure to express IL-2 contributes to reduced numbers and impaired function of regulatory T cells, affects activation-induced cell death, and attenuates cytotoxic CD8þ T cell function [31]. The failure to express IL-2 and the resulting imbalance between IL-2 and pro-inflammatory effector cytokine IL-17 A appears to be central in the pathophysiology of SLE [26]. Recently, we demonstrated that CpG-DNA methylation contributes to subset-specific IL-2 and IL-17 A expression in naı¨ve, central memory and effector memory CD4þ T lymphocytes [32]. We linked subset-specific cytokine expression patterns with the abundance of the transcription factor CREMa, which in turn induces de novo CpG-DNA methylation of the IL2 promoter through the recruitment of DNMT3a and trans-activates IL17A (Figure 1). Because SLE patients have increased numbers of effector memory T cells, this could be a central step in the pathogenesis of SLE. In addition to direct epigenetic events in cytokine genes, cytokine expression in SLE can be deregulated indirectly IL2

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Figure 1. CREMa-mediated chromatin structure of the IL2 and IL17A genes in T cells from SLE patients. (A) The compact chromatin conformation of the IL2 gene in SLE T cells is achieved by CREMamediated repressive histone modifications filled brown circles: histone H3K27 tri-methylation), and CpG-DNA methylation filled red circles. CREMa recruitment the DNA methyltransferase DNMT)3 a to the IL2 promoter, mediates histone methylation though the recruitment of histone methyltransferase G9 a, and histone deacetylation through histone deacetylase HDAC1. (B) Conversely, CREMa favors permissive chromatin conformation of IL17A with increased H3K18 acetylation filled blue circles and reduced H3K27 tri-methylation open brown circles. The ‘‘open’’ chromatin conformation around the IL17A gene in CREMa-expressing T lymphocytes could be achieved through the recruitment of histone acetyltransferases HAT, resulting in H3K18 acetylation and reduced of CpG-DNA methylation open red circles. In contrast to the IL2 gene, CREMa does not recruit DNMT3a or HDAC1 to the IL17A gene in SLE T cells.

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through inappropriate expression of signaling molecules that are subject to CpG-DNA hypomethylation, including CD70/ CD26L [33,34], CD6 [35], CD11A [36,37], CD40L/CD154 [38] in T cells and CD5 [39,40] in B cells (Table 1). Similarly to SLE T lymphocytes, rheumatoid arthritis synovial fibroblasts exhibit a general hypo-methylation pattern [3]. De-methylated CpG-elements within the IL6 promoter are responsible for monocyte activation and inflammatory responses [41]. Hypo-methylation and subsequent activation of LINE-1, a human endogenous retroviral element, contribute to the autoimmune phenotype of rheumatoid arthritis [42]. In line with our findings in T cells from SLE patients, we demonstrated reduced CpG-DNA methylation of the IL17A promoter and increased expression of IL-17 A in T cells from rheumatoid arthritis patients. However, IL2 promoter methylation and gene expression in T cells from rheumatoid arthritis patients was comparable to healthy controls [32]. Histone modifications Alterations of the histone code appear to play a key role in the pathophysiology of various autoimmune disorders, including SLE. However, histone modifications are complex and remain poorly understood [3]. Region specific histone acetylation in some tissues is associated with increased disease activity, whereas histone acetylation in other regions has protective effects. In SLE, acetylation of the TNF promoter in monocytes is associated with increased monocyte maturation and cytokine expression [43]. In the IL17 locus of SLE T cells, spanning the IL17A and IL17F genes, histone tails are hypomethylated and hyperacetylated. This contributes to increased expression of pro-inflammatory IL-17 A, but also causes decreased expression of IL-17 F through the aforementioned mechanism [24,29]. Conversely, and as a

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reflection of CpG-DNA methylation patterns, histone acetylation of IL2 is reduced and histone methylation is increased in T lymphocytes from SLE patients [30] (Figure 1). The importance of a tight balance between activating and repressing epigenetic marks is further emphasized by the disruption of the histone code in other autoimmune/inflammatory disorders. Fibroblasts from patients with rheumatoid arthritis exhibit general histone hyperacetylation that likely results in increased expression of pro-inflammatory genes [32]. Monocytes from patients with the autoinflammatory disorder chronic recurrent multifocal osteomyelitis (CRMO) fail to produce IL-10 [44]. Next to its aforementioned activating effects on B cells that play a role in SLE, IL-10 exerts immune-regulatory functions on antigen-presenting cells and limits the expression of pro-inflammatory cytokines, such as IL-6 and TNF-a [4,18]. The expression of the proinflammatory cytokines IL-6 and TNF-a is not affected by the underlying molecular defect, resulting in an imbalance between pro- and anti-inflammatory cytokine expression by CRMO monocytes rather than increased expression of proinflammatory cytokines [45]. Disturbances in the histone code of cytokine genes have been established central to the pathogenesis of many autoimmune disorders. However, patterns are complex with areas of tissue-specific hyper- or hypo-acetylation and warrant further study in order to be completely understood (Table 1).

Epidemiological contributors to autoimmunity Age and autoimmunity Epigenetic changes accumulate with age and contribute to the growing incidence of autoimmune disorders in the elderly [11]. Age-related epigenetic changes have been documented to be caused by reduced DNMT1-activity and particularly

Table 1. Central epigenetic modifications in SLE. CpG-DNA methylation Reduction CpG-DNA hypomethylation in B and T lymphocytes [3,10,13] In vivo and in vitro CpG-DNA demethylation results in SLE-like symptoms [3,10,13] Altered DNA methyltransferase expression in SLE T lymphoctes [3,10,13] CpG-DNA demethylation in SLE is mediated by GADD45a [3,10,12,13,46] CpG-DNA hypomethylation and increased cytokine expression: IL4 [19], IL6 [20], IL10 [21], IL13 [21], IL17 [22,27] CpG-DNA demethylation of co-stimulatory molecules: CD6 [32], CD11A [33], CD40L [34], CD70 [31], CD5 [35,36] CpG-DNA demethylation of perforin-1 [PRF1] mediates lysis of monocytes [3,10,13] CpG-DNA demethylation and increased expression of the protein phosphatase 2 A [PP2A] CpG-DNA demethylated DNA induces anti-DNA antibody production [3,10,13] CREMa contributes to CpG-DNA demethylation of IL17A in effector memory CD4þ T cells [30] Increase CpG-DNA hypermethylation of cytokine genes in SLE T lymphocytes in mediated by CREMa: IL2 [28,30] Histone modifications Activating histone marks Globally increased histone acetylation Tumor necrosis factor [TNF] promoter [39]: – increased TNF-a expression – increased monocyte maturation Increased H3K18 acetylation of the IL17A and Il17F genes in SLE T lymphocytes [22,27] Lupus-prone MRL/lpr mice express histone acetyltransferases p300, p300/CBP-associated factor and HDAC7 at reduced levels [3,10] Repressive histone marks Reduced H3K18 acetylation at the IL2 promoter in SLE T lymphocytes [28] Increased histone deacetylase 1 [HDAC1] recruitment to the IL2 promoter in SLE T cells [28] Lupus-prone MRL/lpr mice express the histone deacetylase sirtuin-1 at increased levels [3,10] Increased H3K27 tri-methylation at the IL2 promoter in SLE T lymphocytes [28] Histone modifications and autoantibody production SLE auto-antibodies react to apoptosis-related histone modifications [3,10,12,13]

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accumulate in genes that are involved in cell differentiation. The degree of reduced CpG-DNA methylation even reflects life expectancy [10,11,46]. The result of progressive CpGDNA demethylation is the enrichment of so-called ‘‘senescent’’ T lymphocytes in the elderly but also in patients with autoimmune disorders. Senescent T lymphocytes are characterized by reduced CD28 surface expression, shortening of telomere DNA, and the expression of genes that are usually suppressed in CD4þ T cells of young and healthy individuals, including killer cell immunoglobulin-like receptors (KIR), perforin, and the signaling molecule CD70. The activation of these genes contributes to autoimmune phenomena in autoimmune disorders [10,21,47] (Table 1).

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Hormonal effects and X chromsome inactivation in autoimmunity Female predominance in most autoimmune disorders, including SLE (9-10:1), primary Sjo¨gren’s syndrome (9:1), rheumatoid arthritis (3:1) and multiple sclerosis (2:1) has been proposed to (at least partially) result from hormonal differences and the presence of two X chromosomes in women [1,3,10,48]. Ovarian hormones include estrogens, progestins and androgens, which all undergo cyclic changes during the menstrual cycle and during pregnancy [49]. In the context of autoimmunity, estrogens have been most widely studied and have been proposed involved in the pathophysiology of several autoimmune disorders [49,50]. Several distinct differences in the absolute and relative numbers of T lymphocytes between women and men have been demonstrated. Healthy women exhibit increased number of circulating total CD4þ T cells when compared to men [49,50]. Furthermore, CD4þ T cell subset distribution shows several distinct differences between genders: Females have enriched Th2 lymphocyte populations and promote B cell activation, while men exhibit a relative predominance of Th1 and CD8þ T cells over other lymphocyte subsets. This is reflected by hormonal changes during the menstrual cyle, where low estrogen doses promote Th1-mediated immune responses, whereas higher estrogen levels (during the follicular phase) invoke Th2-mediated immunity [49,51]. Dosedependent estrogen effects have particularly been reported for interferon (IFN)-g expression by Th1 cells. Although lowdose estrogen treatment increase IFN-g production by Th1 cells through increased responsiveness to IL-12 and subsequent activation of the transcription factor Stat4, high doses of estrogens result in reduced IFN-g expression [52,53]. These dose-dependent estrogen effects are reflected by increased disease activity during pregnancy with reduced Th1 responses and impaired IFN-g expression [49]. Because T helper lymphocyte subset determination and signature cytokine expression are largely controlled by epigenetic mechanisms [54], it appears likely that estrogens affect chromatin composition of lineage-defining genes in T cells. In agreement with this assumption, we recently demonstrated that estrogens increase the expression of the transcription factor and epigenetic modulator CREMa [55] that has been demonstrated to govern cytokine expression and T cell subset distribution in SLE, favoring effector memory

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CD4 T cell phenotypes [32]. Estrogens furthermore affect the innate immune system. They positively influence the number of circulating neutrophils and delay their apoptosis [56,57]. Estrogens furthermore enhance the expression of Toll-like receptors (TLR-2, -3 and -4) on monocytes, which may play a direct role in the pathophysiology of SLE or affect the differentiation of T helper subsets [58]. The sex chromosomes are derived from a common ancestor autosome and still comprise similar genes in pseudo-autosomal regions. Most X-linked genes are not gender-specific and exhibit equal expression patterns between females and males. This is achieved by inactivation of one X chromosome in women through a complicated epigenetic event, including all three major mechanisms: CpG-DNA methylation, repressive histone modifications, and micro-(mi)RNAs (not discussed in this overview). Xlinked genes that require transcriptional control can potentially contribute to disease pathogenesis. This can be caused by an abnormal number of X chromosomes or incomplete silencing, resulting in over-expression of single genes [3,10,48]. It has been demonstrated that reduced CpGDNA methylation of the CD40L gene contributes to the female predominance in SLE and rheumatoid arthritis [10,38,59]. In both disorders, CD40L expression is increased in a chromatin-dependent manner. The CD40 molecule is expressed on the surface of B cells and other antigenpresenting cells, and the CD40 ligand (CD40L) is expressed on T cells. CD40:CD40L interactions are central for immunoglobulin class-switch, memory B cell development and germinal center formation. Thus, the CD40:CD40L axis centrally contributes to immune activation and if overactivated, can contribute to autoimmune pathology [10,60]. Interestingly, women who lack one X chromosome (Turner’s syndrome; 45, X0) develop SLE incidences that are comparable to those in men. However, the fragmentation or absence of one X chromosome results in an increased risk to develop other autoimmune disorders, including type 1 diabetes [3,10,12,13]. Environmental factors contributing to gender differences One of the most common chemical exposures among women is the use of cosmetic products. Several case-control studies and one case series document a weak association between the use of hair dye with the development of SLE [50,61–67]. This is of special interest, since hair products frequently contain acrylamides that share chemical similarities with medications that have been linked with the onset of drug-induced lupus, including hydralazine or procainamide [50]. Further associations have been shown between the frequent use of lipstick [68] or nail polish [69,70] exposure with the onset of autoimmune disease. However, control studies in male populations are lacking secondary to the reduced exposure of men to the aforementioned substances [50].

Mechanisms of epigenetic deregulation Regardless of recent advances in the field, the underlying mechanisms contributing to disturbed CpG-DNA methylation

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patterns and histone modifications in autoimmune diseases remain largely unknown.

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Disrupted MAPK signaling and loss of inhibition Impaired extracellular signal-regulated kinase (ERK) pathways have been suggested to contribute to cytokine deregulation in SLE lymphocytes [15,16,71,72]. In vitro treatment of B and T cells with mitogen-activated protein kinase (MAPK) inhibitors results in down-regulation of DNMT activity and the expression of methylation-sensitive genes, suggesting that extracellular signal-regulated kinase (ERK) pathways are essential for the maintenance of CpG-DNA methylation through DNMT1. This is in agreement with decreased DNMT expression and impaired ERK signaling in T cells from SLE patients with active disease [3,10,12,13]. Studies, targeting the reduced abundance of DNA methyltransferases as a potential cause of CpG-DNA demethylation in SLE, identified noncoding RNAs. MiRNAs act as posttranscriptional regulators of gene expression through their binding to target messenger RNAs [3]. In lupus-prone mice, miR-21 and miR-148 a have been proposed to inhibit DNMT1 expression, thus contributing to DNA demethylation of lupus-relevant genes [73,74]. In T lymphocytes from SLE patients, miR-126 and miR-29 b are expressed at increased levels, negatively regulating DNMT1 [75,76]. However, studies investigating DNMT expression in SLE patients have produced conflicting results with both increased and reduced levels. This may be the result of variability in disease activity, variable pathophysiological mechanisms in single individuals with the diagnosis SLE, discrepancies between DNMT mRNA and protein expression, and varying biological activity of DNMTs in the context of different genes, cells, tissues and diseases [3,10,13]. The hypothesis that MAPK pathways may be essential for the determination and maintenance of epigenetic patterns, including histone modifications, has recently been supported by findings in monocytes from patients with the autoinflammatory disorder CRMO. In monocytes from CRMO patients, the failure to express IL-10 is caused by an impairment of TLR4-mediated activation of the MAP-kinases ERK1 and ERK2, resulting in attenuated recruitment of the transcription factor Sp1 to the IL10 promoter and reduced histone phosphorylation [18,44,45]. ‘‘Active’’ CpG-DNA demethylation and CpG-DNA hydroxymethylation More recently, further molecules have been suggested to be involved in DNA demethylation of T cells from SLE patients, including: DNA damage inducible protein alpha (GADD45a), activation-inducible deaminase (AID), and the methyl-CpG binding domain 4 (MBD-4) related G:T glycosilase [3,10,12,13,77]. GADD45a is expressed at increased levels in CD4þ T lymphocytes from SLE patients and has been suggested to promote CpG-DNA demethylation in a two-step enzymatic process. After deamination of CpG nucleotides by AID, generating a G:T mismatch, MBD-4 removes thymine bases, resulting in the replacement of the missing base with an unmethylated cytosine group, mediating gradually progressing CpG-DNA demethylation [3].

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CpG-DNA hydroxymethylation (hmCpG) has been proposed a demethylation intermediate, offering a novel measure of CpG-DNA methylation. During this recently proposed path of CpG-DNA demethylation, 10–11 translocation (TET) family proteins oxidate methyl-cytosine into 5-hydroxymethylcytosine [78]. TET proteins and 5-hmCpG have been demonstrated to play important roles in deregulated CpG-DNA methylation during embryonic development, leukemia and autoimmune disease [79–82]. In SLE patients, high levels of the TET family proteins may be responsible for increased 5-hmCpG in CD4þ T lymphocytes, offering an additional mechanism of CpG-DNA demethylation [79] (Table 1). Transcription factors orchestrating epigenetic remodeling in SLE Recent data from our group indicates that CREMa contributes to epigenetic remodeling of cytokine genes during the priming and subset determination of CD4þ T cells. CREMa reciprocally affects the expression of IL2 and IL17A by the interaction with epigenetic mediators (Figure 1). CREMa recruits DNMT3a to the IL2 promoter of effector memory T cells, resulting in epigenetic silencing while it mediates demethylation of IL17A in the same cells in a yet to be determined mechanism. This mechanism could be central for the pathophysiology of SLE. T cells from SLE patients exhibit increased CREMa expression which affects IL-2 and IL-17 A expression in the aforementioned manner, favoring effector memory T cell phenotypes [24,30,32]. Our knowledge of mechanisms contributing to histone modifications in autoimmune disorders is even more limited. Recently, reduced acetylation of histone 3 lysine 18 (H3K18ac) and increased histone 3 lysine 27 trimethylation (H3K27me3) of the IL2 gene in SLE T lymphocytes has been linked to the recruitment of CREMa to the IL2 proximal promoter (Figure 1A) [30]. In addition to the aforementioned DNMT3a, CREMa recruits histone deacetylase (HDAC)1 and the histone methyltransferase G9a to the IL2 promoter mediating changes to the histone code [83,84]. Conversely, CREMa increased expression in SLE T cells has been linked to increased H3K18 acetylation and reduced H3K27 tri-methylation, resulting in an ‘‘opening’’ of the IL17 locus and increased expression of IL17 A [24] (Figure 1B). However, the molecular mechanisms by which CREMa contributes to the remodeling of the IL17A and IL17F genes remain to be elucidated. More evidence for the involvement of transcription factors in the establishment of epigenetic marks in immune cells has recently been generated, investigating B lymphocyte maturation protein (Blimp-)1 [85]. Blimp-1-deficient mice have been documented to produce increased amounts of follicular helper T cells, which play a role in the generation of germinal centers responses and the development and maintenance of autoimmune responses [85]. Furthermore, Blimp-1 plays a role in T cell homeostasis and Blimp-deficient animals, in agreement with patients with SLE, display increased numbers of effector memory T cell phenotypes [85–89]. One proposed mechanism is the disruption of epigenetic patterns in Blimpdeficient immune cells. Blimp-1 has been demonstrated to recruit histone deacetylases (HDACs) and histone methyltrasferases (HMTs) to B cell-specific genes, thus potentially

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DOI: 10.3109/08916934.2013.801462

contributing to immune cell biology by the maintenance of epigenetic patterns [90]. Further evidence suggesting an involvement of deregulated histone marks in SLE was provided by studies in lupus-prone MRL/lpr mice. Overexpression of the HDAC Sirtuin-1 in concert with reduced expression of the E1A binding protein p300, p300/ CBP-associated factor, and HDAC7 in T lymphocytes from lupus prone MRL/lpr mice suggest an involvement of these factors in the disruption of the epigenome in autoimmune disorders [3,10]. However, the molecular mechanisms resulting in the impaired expression and activity of these histone modifiers remain to be elucidated. In a series of elegant experiments, regulatory factor X1 (RFX1) has been demonstrated involved in the demethylation of lupus-relevant genes, namely CD11A (also ITGAL) and CD70 (also TNFSF7). RFX1 is a transcription factor that specifically recruits to highly conserved sequences in X-box promoters of MHC class II genes [91] where it exerts either activating or repressing effects [92]. In CD4þ T cells from SLE patients, both the expression and activity of RFX1 have been documented reduced, contributing to an impaired recruitment of DNMT1 and HDAC1 to the promoters of CD11A and CD70 resulting in increased gene expression [37].

Epigenetic patterns as disease biomarkers and potential future therapeutic target In light of the central involvement of cell-cycle, cell-subset and tissue specific epigenetic marks in the regulation of cytokine expression and immune homeostasis, attenuated epigenetic patterns in autoimmune disease appear to be promising biomarkers for disease activity and/or outcome and potential therapeutic targets. We recently demonstrated that epigenetic patterns of cytokine genes follow disease activity in SLE. Experimental evidence documenting the efficiency of HDAC inhibitors is growing. However, currently available ‘‘epigenetic drugs’’ (including HDACs and DNMT inhibitors) mediate untargeted genome-wide epigenetic changes which are likely to result in the deregulation of previously unaffected genes [3,10,12,13]. Target-directed epigenetic modifiers may reverse epigenetic alterations of single genes or genomic regions. The transcription factor CREMa that reciprocally mediates trans-repression and epigenetic silencing of IL-2 while trans-activating IL-17 A could be a promising candidate [24,30,32]. Such target-directed therapeutic approaches may be used as future treatment of autoimmune diseases and cancer.

Conclusions In light of most recent reports, the central involvement of epigenetic mechanisms during lymphocyte differentiation and immune programming is widely accepted. Altered epigenetic marks of immune cells contribute to the development of autoimmune disorders, including SLE. Recent reports provide insight into the involvement of the transcription factors CREMa and Blimp-1 in epigenetic processes in immune cells. Deregulated expression of these transcription factors in SLE favors effector T cell phenotypes. However, our current knowledge is limited to common epigenetic patterns in single autoimmune diseases, and we lack a global understanding for

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the molecular mechanisms that result in epigenetic deregulation and inflammation. Specific epigenetic patterns promise to be of predictive, diagnostic and prognostic value in multiple autoimmune disorders. Furthermore, epigenetic changes could prove to be therapeutic targets. However, current treatment options are non-specific and mediate genome-wide epigenetic changes that harbor the risk of severe treatment-associated side effects. In the future, mediators of epigenetic remodeling such as CREMa may be viable therapeutic options, preventing or reversing epigenetic alterations in autoimmune disease.

Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper. References 1. Tsokos, G. C. 2011. Systemic lupus erythematosus. N Engl J Med. 365: 2110–2121. 2. Truedsson, L., A. A. Bengtsson, and G. Sturfelt. 2007. Complement deficiencies and systemic lupus erythematosus. Autoimmunity. 40: 560–566. 3. Hedrich, C. M., and G. C. Tsokos. 2011. Epigenetic mechanisms in systemic lupus erythematosus and other autoimmune diseases. Trends Mol Med. 17: 714–724. 4. Hedrich, C. M., and J. H. Bream. 2010. Cell type-specific regulation of IL-10 expression in inflammation and disease. Immunol Res. 47: 185–206. 5. Brenner, C., and F. Fuks. 2006. DNA methyltransferases: facts, clues, mysteries. Curr Top Microbiol Immunol. 301: 45–66. 6. Brenner, C., and F. Fuks. 2007. A methylation rendezvous: reader meets writers. Dev Cell. 12: 843–844. 7. Deplus, R., C. Brenner, W. A. Burgers, P. Putmans, T. Kouzarides, de, L. Y., and Fuks, F. 2002. Dnmt3L is a transcriptional repressor that recruits histone deacetylase. Nucl Acids Res. 30: 3831–3838. 8. Esteve, P. O., H. G. Chin, A. Smallwood, G. R. Feehery, O. Gangisetty, A. R. Karpf, M. F. Carey, and S. Pradhan. 2006. Direct interaction between DNMT1 and G9a coordinates DNA and histone methylation during replication. Genes Dev. 20: 3089–3103. 9. Smallwood, A., P. O. Esteve, S. Pradhan, and M. Carey. 2007. Functional cooperation between HP1 and DNMT1 mediates gene silencing. Genes Dev. 21: 1169–1178. 10. Brooks, W. H., D. C. Le, J. O. Pers, P. Youinou, and Y. Renaudineau. 2010. Epigenetics and autoimmunity. J Autoimmun. 34: J207–J219. 11. Grolleau-Julius, A., D. Ray, and R. L. Yung. 2010. The role of epigenetics in aging and autoimmunity. Clin Rev Allergy Immunol. 39: 42–50. 12. Renaudineau, Y. 2010. The revolution of epigenetics in the field of autoimmunity. Clin Rev Allergy Immunol. 39: 1–2. 13. Renaudineau, Y., and P. Youinou. 2011. Epigenetics and autoimmunity, with special emphasis on methylation. Keio J Med. 60: 10–16. 14. Wilson, C. B., K. W. Makar, M. Shnyreva, and D. R. Fitzpatrick. 2005. DNA methylation and the expanding epigenetics of T cell lineage commitment. Semin Immunol. 17: 105–119. 15. Richardson, B. C. 2008. Epigenetics and autoimmunity. Overview Autoimmunity. 41: 243–244. 16. Strickland, F. M., and B. C. Richardson. 2008. Epigenetics in human autoimmunity. Epigenetics in autoimmunity – DNA methylation in systemic lupus erythematosus and beyond. Autoimmunity. 41: 278–286. 17. Schoenborn, J. R., and C. B. Wilson. 2007. Regulation of interferon-gamma during innate and adaptive immune responses. Adv Immunol. 96: 41–101. 18. Hofmann, S. R., A. Rosen-Wolff, G. C. Tsokos, and C. M. Hedrich. 2012. Biological properties and regulation of IL-10 related cytokines and their contribution to autoimmune disease and tissue injury. Clin Immunol. 143: 116–127.

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