The Role of Oxidative Stress in Epigenetic Changes Underlying Autoimmunity

Abstract

Significance:

Epigenetic dysregulation plays an important role in the pathogenesis and development of autoimmune diseases. Oxidative stress is associated with autoimmunity and is also known to alter epigenetic mechanisms. Understanding the interplay between oxidative stress and epigenetics will provide insights into the role of environmental triggers in the development of autoimmunity in genetically susceptible individuals.

Recent Advances:

Abnormal DNA and histone methylation patterns in genes and pathways involved in interferon and tumor necrosis factor signaling, cellular survival, proliferation, metabolism, organ development, and autoantibody production have been described in autoimmunity. Inhibitors of DNA and histone methyltransferases showed potential therapeutic effects in animal models of autoimmune diseases. Oxidative stress can regulate epigenetic mechanisms via effects on DNA damage repair mechanisms, cellular metabolism and the local redox environment, and redox-sensitive transcription factors and pathways.

Critical Issues:

Studies looking into oxidative stress and epigenetics in autoimmunity are relatively limited. The number of available longitudinal studies to explore the role of DNA methylation in the development of autoimmune diseases is small.

Future Directions:

Exploring the relationship between oxidative stress and epigenetics in autoimmunity will provide clues for potential preventative measures and treatment strategies. Inception cohorts with longitudinal follow-up would help to evaluate epigenetic marks as potential biomarkers for disease development, progression, and treatment response in autoimmunity. Antioxid. Redox Signal. 36, 423–440.

Introduction

A healthy immune system can distinguish self- from non-self-components under delicate and complex regulation. If self-tolerance fails, autoantibodies and autoreactive T cells will target self-tissues and cells to cause damage, which leads to autoimmune diseases (ADs). The ADs include but are not limited to systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), inflammatory bowel disease (IBD), and multiple sclerosis (MS). Autoimmunity is caused by interactions of genetic, epigenetic, and environmental factors. Epigenetics has been proposed to mediate the interaction between environmental triggers and genetic susceptibility factors, leading to the development of ADs (92, 121). An epigenetic trait is defined as “a stably heritable phenotype resulting from changes in a chromosome without alterations in the DNA sequence” (10). Epigenetic mechanisms include DNA methylation, histone modifications (acetylation, methylation, citrullination, ubiquitination, phosphorylation, and sumoylation), and noncoding RNAs (116). Environmental factors involved in the pathogenesis of ADs include dietary components, hormones, sunlight, air pollution, drugs, and infection (87, 113). Oxidative stress is one mechanism by which environmental triggers can induce ADs (83, 106, 135). To illustrate the relationship between oxidative stress and epigenetic dysregulation in the context of autoimmunity, we focus on research progress related to DNA methylation and histone modification changes in ADs, and the contribution of oxidative stress to epigenetic aberrancies in autoimmunity.

DNA Methylation and Histone Modifications

DNA methylation

DNA methylation is a covalent addition of a methyl group at the 5-carbon of the cytosine ring, which usually occurs on CG dinucleotides within CG-rich regions, including CpG islands. The methyl donor in cells is S-adenosyl-methionine (SAM). The addition of methyl group is mediated by DNA methyltransferases (DNMTs) (“writers”). The DNMT family includes DNMT1, which catalyzes methylation of newly replicated DNA, and DNMT3A and DNMT3B, which have a role in de novo DNA methylation (64). The DNA methylation changes are interpreted by methyl-CpG-binding domain (MBD) family proteins (“readers”), which include methyl-CpG-binding protein 2 (MeCP2), MBD1-6, SETDB1, SETDB2, BAZ2A, and BAZ2B (33). To remove DNA methylation (“erasers”), ten-eleven translocation (TET) proteins oxidize 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), 5-formalcytosine (5fC), and 5-carboxylcytosine (5acC). This is followed by base excision repair by glycosylases TDG/SUMG1 to generate an abasic site, which is then replaced by an unmodified cytosine in mammalian cells (11, 69). Activation-induced cytidine deaminase/apolipoprotein B mRNA-editing catalytic polypeptides (AID/APOBEC) family members are also involved in DNA demethylation by 5mC or 5hmC deamination (11). DNA methylation in promoters and enhancer regions is associated with repressed transcription. The genomic CpG methylation landscape could be regulated by histone lysine methylation, acetylation, and citrullination through mediation of DNMTs stability (28, 29).

Histone modifications

Among various histone modifications, we will briefly introduce histone methylation, acetylation, and citrullination. Histone methylation changes in autoimmunity will be discussed in detail along with DNA methylation. Key modulators in histone acetylation and citrullination will be emphasized to highlight how oxidative stress influences epigenetics.

Histone methylation happens at two residues: lysine and arginine. Three classes of histone methyltransferases (HMTs) have been found: SET domain lysine methyltransferase, non-SET domain lysine methyltransferase, and arginine methyltransferase (105). Histone lysine demethylases (HKDMs) are classified into two classes: LSD1 and JmjC, based on their demethylase domain (105). Histone methylation regulates transcriptional repression or activation depending on the position, methylation state, and interaction with other histone modifications. Generally, H3K9, H3K27, and H4K20 methylations are markers of compacted and silenced chromatin, whereas methylation in H3K4, H3K36, and H3K79 are associated with active transcription (58).

The protein arginine methyltransferases (PRMTs) family includes nine members divided into four types. Type I PRMTs (PRMT1-4, PRMT6, and PRMT8) catalyze asymmetric dimethylarginine (aDMA), Type II PRMTs (PRMT5, PRMT7, and PRMT9) catalyze symmetric dimethylarginine (sDMA), Type III PRMTs (PRMT7) catalyze monomethylarginine (MMA) in mammals, and Type IV is only found in fungi (67). Arginine demethylases include JMJD6, PAD4, and HKDMs such as JMJD1B (136). Histone arginine methylation also regulates gene expression. H3R2me2s, H3R17me2a, H3R26me2a, and H4R3me2a are active transcription markers, whereas H3R2me2a, H3R8me2a, H3R8me2s, and H4R3me2s repress gene expression (12). Non-histone arginine methylation is involved in DNA damaging signaling, pre-mRNA splicing, mRNA translation, and growth factor-mediated signal transduction (49).

Histone acetylation is catalyzed by histone acetyltransferases (HATs) to transfer acetyl-CoA to lysine residues. There are three HAT families, GNAT, MYST, and p300/CBP. Histone deacetylases (HDACs) remove acetyl groups and are divided into four classes (105). Class I (HDAC1-3 and HDAC8), class II (HDAC4-7 and HDAC9-10), and class IV (HDAC11) have an active-site metal dependent catalytic mechanism. Class III (Sirt1-7) has an NAD^+-dependent catalytic mechanism. Histone lysine acetylation promotes gene activation, and deacetylation promotes gene silencing (118, 127).

Citrullination is a Ca²⁺-dependent process by which peptidylarginine is converted to citrulline by peptidylarginine deiminase (PAD). The PAD family has five isozymes, PAD1-4 and PAD6. PAD4 can enter into the nucleus and citrullinate H3 on arginine 2, 8, 17 and 26, and also H2A and H4 on arginine 3 (102). In addition to its role as histone arginine demethylase (105), PAD4-mediated histone citrullination plays critical roles in chromatin decondensation in granulocytes (122).

DNA Methylation and Histone Methylation Changes in Autoimmunity

DNA methylation and histone methylation differences are widely seen in patients with ADs compared with healthy controls (HC). Aberrant biological pathways in the immune system and organ-specific tissues regulated by DNA methylation and histone methylation are involved in autoimmunity, including overactive inflammation induced by interferon (IFN), tumor necrosis factor (TNF), and IL-6 related pathways, dysregulated cellular responses to stress and organ development, and abnormal extracellular matrix synthesis/degradation (Fig. 1). The methylation changes in four ADs, including RA, SLE, IBD, and MS, will be briefly discussed later.

FIG. 1.

The major targets and pathways altered in DNA methylation and histone methylation in autoimmune diseases, including RA, SLE, IBD, and MS. Inflammatory pathways including IFN and TNF pathways, the signature TF of regulatory T cells FOXP3, and histone methyltransferase EZH2 are common targets across multiple autoimmune diseases. ECM, extracellular matrix; HKMT, histone lysine methyltransferase; IFN, interferon; PRMT, protein arginine methyltransferase; PRC, polycomb repressive complex; HKDM, histone lysine demethylase; IBD, inflammatory bowel diseases; MS, multiple sclerosis; RA, rheumatoid arthritis; SLE, systemic lupus erythematosus; TF, transcription factor; TNF, tumor necrosis factor. Color images are available online.

Methylation changes in RA

RA is the most prevalent systemic AD affecting around 1% of the population worldwide (24). Aberrantly active immune cells and synovial fibroblasts (SF) attack and destroy cartilage and bone. Pain and swelling in joints, and damage to organs throughout the body can be seen in patients with RA. Besides symptoms, blood tests including rheumatoid factor and RA specific anti-citrullinated peptide antibodies (ACPA) are helpful for RA diagnosis.

DNA methylation changes in RA

Fibroblast-like synoviocytes (FLS), also known as SF, are the major non-immune cells in synovial tissues and contribute to cartilage destruction in RA through interaction with immune cells and by acting as an effector in synovial hyperplasia, angiogenesis, and tissue invasion (132). Genes involved in inflammation and skeletal development are differentially methylated in FLS from patients with RA. Genome-wide DNA methylation studies revealed DNA methylation profiles that can distinguish between RA FLS and osteoarthritis (OA) FLS, and samples derived from the hip versus the knee joints (3). The IL-6/JAK-STAT pathway and multiple HOX genes are differentially methylated between knee and hip FLS, with significantly lower IL6 mRNA levels in the knee compared with the hip in RA.

Peripheral blood mononuclear cells (PBMC) showed cell type-specific methylation changes correlated with RA in genome-wide DNA methylation studies, and naïve CD4⁺ T cells have more differentially methylated CpG sites compared with other cell types (42, 93, 95). Naive CD4⁺ T cells have been shown to share hypermethylation profiles with FLS in RA (95). Differential DNA methylation involves pathways such as IL-6/JAK1/STAT3, TNF signaling, IFN signaling, and Th17 differentiation in functional analysis of naive CD4⁺ T cells from treatment-naive RA compared with HC (93). Hypermethylation of CD1C, which encodes a glycoprotein involved in lipid antigen presentation to T cells, and hypomethylation of TNFSF10, which encodes TRAIL that regulates apoptosis in B cells, have been found in three European RA cohorts (63). Six DNA methylation-mRNA-RA pathways were found in RA PBMC compared with HC, and an IFN-based network was formed combing differentially methylated positions (DMP) and differentially expressed genes. Functional experiments demonstrated that cg00959259-PARP9, which is involved in the IFN pathway, modulated Jurkat T cell proliferation, cell activation, and IL-2 expression (144).

ACPA can exist years before RA diagnosis. Ekstrom and colleagues showed differentially methylated regions (DMR) in whole blood of 5 pairs of ACPA-positive versus ACPA-negative healthy monozygotic (MZ) twins, also in 7 pairs of ACPA-positive RA patients versus ACPA-negative healthy MZ twins (43). Importantly, DMR of protocadherin gene PCDHB14, which functions in self-recognition, may temporally connect ACPA-positivity to clinical RA (43). In another 79 MZ twin pairs discordant for RA, methylation changes suggested important roles of stress response pathways, including K63 ubiquitination, which regulates oxidative stress, and potential methylation-influenced disruption of RUNX3 transcription factor (TF) binding sites, in the etiology and development of RA (125).

Treatment with the DNMT inhibitor 5′-azacytidine (5′-azaC) alleviated arthritis and showed an attenuation of IgG1 production in a murine model of RA. 5′-azaC demethylated the aryl hydrocarbon receptor gene (Ahr) in B cells, resulting in Ahr-mediated downregulation of Aicda gene expression and suppression of high-affinity antibody maturation and germinal center formation (114).

Histone methylation changes in RA

Various studies have shown that the expression of several histone lysine methyltransferases (HKMTs) and HKDMs is dysregulated in RA synovial fibroblasts (RASF) as well as in peripheral blood.

It has been shown that after TNFα stimulation, mRNA levels of 12 HKMTs were significantly higher in RASF compared with OA synovial fibroblasts (OASF), including MLL1, MLL3, SUV39H1, SUV39H2, PRDM2, EZH2, SETD2, NSD2, NSD3, SMYD4, DOT1/DOT1L, and PR-set7 (8). Besides, mRNA of 4 HKDMs were also higher in RASF compared with OASF: FBXL10, NO66, JMJD2D, and FBXL11. The mRNA expression levels of DOT1/DOT1L, which mediates methylation of H3K79, a transcriptionally permissive histone mark, were also increased in synovial biopsies from patients with RA compared with OA or HC. However, protein levels of DOT1L and global H3K79me2 were not significantly different between RA and OA (51). DOT1/DOT1L binding to the proximal promoters of mouse Il6 and Ifnb1 promoted IL-6 and IFN-β production in macrophages (17). Karouzakis et al. found that T-box transcription factor 5 (TBX5) was significantly hypomethylated and was enriched in H3K4me3 and H3 acetylation in RASF, which led to TBX5 overexpression in RASF compared with OASF, as well as overexpression of TBX5 targets, including IL-8, CXCL12, and CCL20 (66).

RASF produce proteolytic enzymes to degrade articular cartilage, resulting in joint destruction. Matrix metalloproteinases (MMPs) degrade extracellular matrix (ECM), the main component of articular cartilage. H3K4me3 was found to be significantly enriched, whereas H3K27me3 was significantly depleted in MMPs promoter regions in RASF compared with OASF, resulting in enhanced transcription of MMPs in RASF (9). The MMPs expression could also be regulated by the IL-6/STAT3 pathway.

An integrated epigenomic assessment in RASF, which included characterization of histone modifications, chromatin accessibility, DNA methylation, and RNA expression, revealed a role for Huntington-interacting protein-1 (HIP1) in RA. Knockdown of HIP1 in RASF provided evidence for its involvement in SF invasion (4).

EZH2, which is the catalytic component of polycomb repressive complex 2 (PRC2), is an H3K27me3 methyltransferase and it plays important roles in both RASF and CD4⁺ T cells (Fig. 2A). EZH2 was found to be overexpressed in RASF compared with OASF and could be induced by the TNFα/NFκB/JNK pathway (115). Global H3K27me3 was not significantly different between RASF and OASF with and without TNFα stimulation, whereas low expression of the EZH2 target gene SFRP1 (secreted frizzled-related proteins 1), a Wnt signaling inhibitor, was correlated with increased H3K27me3 and decreased H3K4me3 at the promoter region, resulting in upregulation of Wnt signaling and RASF activation. Recently, Zhang and colleagues found that EZH2 was significantly lower in PBMC and CD4⁺ T cells from patients with RA compared with HC (129). EZH2 inhibition with GSK126 or EZH2 siRNA suppressed FOXP3 expression by downregulating RUNX1 and upregulating SMAD7 in CD4⁺ T cells, thus inhibiting Treg differentiation. The role of EZH2 in regulating FOXP3/Tregs has also been shown in MS and IBD mouse models (34, 100).

FIG. 2.

The role of histone lysine methyltransferase EZH2 in autoimmunity. (A) EZH2 is upregulated by TNFα via JNK and NF-κB pathways (IKKβ is an active subunit of NF-κB) in RASF. A Wnt signaling inhibitor sFRP1 (secreted frizzled-related proteins 1) is inhibited with increased H3K27me3 enrichment and decreased H3K4me3 enrichment at the promoter region (upper panel). EZH2 is repressed in CD4⁺ T cells from RA patients, resulting in RUNX1 suppression and SMAD7 activation, and then FOXP3 suppression and inhibited regulatory T cell (Treg) differentiation (lower panel). (B) EZH2 in CD4⁺ T cells promotes SLE in at least two ways: (1) EZH2 induces hypomethylation in junctional adhesion molecule A (JAM-A) and increases JAM-A expression to promote T cell adhesion and migration; (2) EZH2 promotes T cell proliferation and B cells hyperstimulation possibly through the TCR (T cell receptor) mediated-MEK-ERK-IL2/IL2RA pathway. (C) EZH2 promotes NF-κB dependent cytokine production in macrophages/microglia, and cellular extravasation and migration of neutrophils and dendritic cells in the mouse model of MS and IBD. EZH2 conditional knockout increases expression of SOCS3 (suppressor of cytokine signaling 3), which increases Lys48-linked ubiquitination and degradation of TRAF6 (TNF receptor-associated factor 6) to inhibit TLR-induced MyD88-dependent NF-κB activation. Cytosolic EZH2 methylates K2454 in talin, a key regulator of cell migration, disrupting talin binding to F-actin. This effect is dependent on the interaction between EZH2 and cytoskeletal-reorganization effector Vav1. EZH2 knockout increases cell adhesion and stops cell migration and extravasation. RASF, RA synovial fibroblast. Color images are available online.

Methylation changes in SLE

The SLE is an AD that affects multiple organ systems. The disease is characterized by the production of antinuclear antibodies and a type I interferon signature. Strong evidence suggests a role for genetic predisposition, environmental triggers, and epigenetic dysregulation in the pathogenesis of this remitting-relapsing disease.

DNA methylation changes in SLE

DNA methylation changes in SLE reflect the interaction with genetic variation and interaction with environmental exposures that can trigger the disease. Differentially methylated CpG sites are predominantly hypomethylated in most blood cell types from patients with SLE compared with controls. Significant hypomethylation in type I interferon (IFN) and IFN-related genes is observed. Hypomethylation in the interferon induced protein 44 like (IFI44L) promoter region has been demonstrated to be a potential diagnostic biomarker for SLE (140).

Genome-wide DNA methylation studies showed hypomethylation in CD4⁺ T cells (2, 19, 20, 61, 120), B cells (2, 14), monocytes (2, 120), granulocytes (23, 120), whole blood (59), and PBMC (62) in SLE compared with HC, especially in genes related to type I IFN (2, 14, 20, 23, 59, 62, 120, 130). Other reports suggested predominantly more hypermethylation in SLE B cells (120). The differences in DNA methylation pattern between SLE B cells studies may be due to differences in B cell subpopulations (120). Hypomethylation can be expected in a high proportion of memory and plasma B cells, which was observed in SLE (99).

Contrary to the earlier cited cell types, more loci were found to be hypermethylated than hypomethylated in adult SLE CD8⁺ T cells (79). Elegant work has demonstrated that DNMT3 and H3K9 methyltransferase G9a are recruited to regulatory regions of the CD8 gene cluster by cAMP responsive element modulator α (CREMα), which increases DNA methylation and H3K9me3, silencing CD8A and CD8B, and leading to double negative T cells expansion in the PBMC of patients with SLE (53). The most hypomethylated sites in SLE CD8⁺ T cells were within major histocompatibility complex class II gene HLA-DRB1, which was found to be regulated by the IFNα/STAT1/CIITA pathway. The SLE CD8⁺ T cells could activate autologous naive CD4⁺ T cells dependent on type I IFN induction and HLA-DRB1 expression (79).

Recent studies also examined ancestral differences in the epigenetic profiles of patients with SLE. African Americans have a higher genetic risk for SLE and show hypomethylation in proapoptotic and proinflammatory genes compared with European Americans (21, 22, 40).

In a longitudinal study with ∼4 years of follow-up, DNA methylation patterns in SLE neutrophils across ancestries were found to be, in part, determined by genetic variants and remained largely stable over time (22). Demethylation of polypeptide N-acetylgalactosaminyltransferase 18 (GALNT18) was found to be associated with the development of active lupus nephritis during follow-up.

Histone methylation changes in SLE

Histone methylation regulates immune cells activation, proliferation, and differentiation, as well as autoantibody production in SLE. H3K4me2 and H3 acetylation levels (both activating epigenetic marks) within the promoter of TNFSF7, which encodes CD70, in CD4⁺ T cells were positively correlated with SLE disease activity (143). CREMα levels in T cells positively correlated with SLE disease activity (52). Set1 enrichment at the CREMα promoter upregulates H3K4me3, consequently leading to CREMα overexpression and IL-17 production (138).

EZH2 is a transcriptional repressor that tri-methylates H3K27 and has a potential pathogenic role in both CD4⁺ T cells and CD19⁺ B cells in SLE (Fig. 2B). EZH2 mRNA and protein levels, as well as H3K27me3, were upregulated in lupus naive CD4⁺ T cells compared with HC (119). Transfection of an EZH2 plasmid into CD4⁺ T cells isolated from HC led to upregulation of junctional adhesion molecule A (JAM-A) and increased T cell-endothelial cell adhesion, similar to CD4⁺ T cells isolated from patients with SLE (119). Inhibition of EZH2 significantly prolonged survival and improved lupus-like features in MRL/lpr lupus-prone mice (96). Ezh2-deficient CD4⁺ T cells and EZH2-specific inhibitor GSK503 induced fewer geminal centers and decreased autoantibody production because of fewer T follicular helper cells (Tfh) cells to activate B cells in a model of allogeneic T cell-induced lupus disease (141). Dobenecker et al. showed that EZH2 regulates TCR-mediated MEK-ERK1/2-IL2 pathway to promote T cells proliferation. GSK503 significantly prevented or even cured Treg-depletion induced autoimmunity in mouse models (32). In addition, EZH2 is recruited via BCL-6 to the promoter region of miR-142-3p/5p to decrease miR-142-3p/5p expression in SLE CD4⁺ T cells, which correlates with T cell overactivation and B cell hyperstimulation (30, 101).

Methylation in IBD

IBD is chronic and can affect the entire digestive tract (Crohn's disease, CD) or only the colon (ulcerative colitis, UC). The IBD symptoms predominantly reflect the gastrointestinal involvement; however, systemic inflammatory complications affecting skin, eyes, and other organs can occur. The IBD development during childhood usually carries a more aggressive and severe disease course (97).

DNA methylation in IBD

DNA methylation analysis has been done in colon biopsy and blood samples of patients with IBD. DNA methylation profiles of intestinal cells provide insights into disease pathogenesis, complications, and prognosis of IBD, whereas DNA methylome analysis in peripheral blood cells needs more investigation.

In colonoscopy-collected biopsy samples from patients with IBD compared with HC, differentially methylated profiles reflected changes in immune functions, gastrointestinal development, and metabolism. In pediatric patients, comparing CD versus UC and CD versus HC in terminal ileum epithelium, and CD versus HC and UC versus HC in sigmoid colon epithelium, DMPs were mainly hypomethylated, which were partly independent of inflammation. In addition, these methylation differences were stable over time and can predict disease status and correlate with clinical outcomes (55). In another study, genes involved in innate immune defense, including PIGR, MUC2, TLR3, and IL6R, were hypomethylated, and DNA demethylase TET1 was hypermethylated in intestinal epithelial cells from newly diagnosed pediatric IBD (70). The DMPs in pediatric IBD overlapped with CpG methylation changes during gastrointestinal development by comparing fetal and pediatric epithelium (70). Fibrosis is a common complication in CD. The genes WNT2B and PTGIS were hypermethylated, and PTGDS was hypomethylated in intestinal fibroblasts isolated from patients with fibrostenotic CD (98). WNT2B is involved in development, whereas PTGIS and PTGDS are linked to extracellular matrix synthesis.

UC is correlated with an increased risk for colon cancer in the long term. Genes involved in biosynthetic processes, regulation of metabolism, and nitrogen compound metabolic processes were hypermethylated and associated with increased UC duration and severity, which are related to tumorigenesis in this disease (110). A tumor suppresser gene, fragile histidine triad (FHIT) was also found to be hypermethylated in CD (68). In the murine IBD model induced by dextran sodium sulfate (DSS), hypermethylation in intestinal epithelial cells and hypomethylation in sperm cells could be transmitted to offspring and increased the susceptibility to DSS-induced colitis (117).

DNA methylation in blood samples showed changes between IBD and control populations. For example, hypomethylation of the promoter region of TRIM39-RPP21, which regulates the type I IFN pathway, was observed in PBMCs from patients with CD and UC compared with HC. Hypermethylation and decreased mRNA levels of TRAF6, a key component in TNF and NF-κB signaling, were found in both CD and UC patients as well (78). Somineni et al. also showed DNA hypermethylation in blood samples from pediatric CD patients at diagnosis; annotated genes included TNF, JAK3, RPS6KA2, ITGB2, TMEM49, and LOC100996291 (107). However, the DNA methylation signatures were strongly correlated with plasma C-reactive protein levels (an inflammation marker), and most CpG methylation profiles at diagnosis disappeared with the exception of only 10 CpG sites that retained the same methylation level trends after treatment despite disease progression in 1–3 years of follow-up. Thus, the authors suggested blood methylation as a marker of inflammation, not of CD development or progression. In another study, DNA methylation profiles in CD8⁺ T cells from pediatric IBD patients correlated with age and gender, but not disease outcome in three cohorts with at least 18 months of follow-up (39).

Histone methylation in IBD

Research in both histone repression and activation marks has identified potential treatment targets in IBD. H3K27me2/3 demethylase Kdm6a was shown to promote IL-6 and IFN-β production through demethylase-dependent and -independent mechanisms in macrophages, respectively (72). Another H3K27me2/3 demethylase, JMJD3, has been shown to promote nuclear factor-erythroid 2-related factor 2 (Nrf2) expression by decreasing repressive H3K27me3 in the promoter region (56). Nrf2 promoted NLRP3 inflammasome activation, which has a fundamental role in the pathogenesis of IBD (133). Indeed, a JMJD3 inhibitor reduced colitis disease activity index, and it rescued body weight loss and colonic shortening in the DSS-induced IBD mice model (56). Another repressive histone mark studied in IBD is H3K9me3. Activating transcription factor 7 interacting protein (ATF7ip) inhibited IL-2 via mediating H3K9me3 in the Il2-Il21 intergenic region. T cell specific deletion of ATF7ip increased IL-2 production, and it subsequently decreased Th17 differentiation and induced resistance to colitis (103).

H3K4me promotes active transcription and plays a role in IBD. H3K4 methyltransferase Ash1l demonstrated a protective effect against T cell-mediated colitis in mice by promoting Foxp3 expression through enhancing the TGF-β-Smad2/3 pathway by recruiting Ash1l to the promoter region of Smad2/3 on TGF-β stimulation (128).

Methylation changes in MS

MS affects the central nervous system (CNS), because a dysregulated immune system attacks myelin (the protective covering of neural fibers) and neural cells, leading to neurological complications. Patients with MS may experience numbness, stiffness, walking difficulty, and cognitive and mood changes.

DNA methylation changes in MS

In brain tissue and blood, DNA methylation is shown to causally correlate with MS. Dysregulated pathways involve cellular survival, proliferation, metabolism, and inflammation.

In pathology-free regions from patients with MS and control brains, hypermethylation occurred in genes (BCL2L2 and NDRG1) regulating the survival of oligodendrocytes whose main function is to myelinate CNS axons, whereas hypomethylation was found in genes related to proteolytic processing (LGMN and CTSZ) (57). In experimental autoimmune encephalomyelitis (EAE; a MS mouse model), and in patients with MS, a subset of astrocytes characterized by a defect in the methionine adenosyltransferase IIα (MAT2α)-dependent DNA methylation pathway, with potential proinflammatory consequences, has been recently identified (126).

Integrating data from large-scale genomic studies in MS, 178 methylation sites were suggested to causally associate with MS by using summary data-based Mendelian randomization (80). For example, 13 methylation sites were found in the discoidin domain receptor tyrosine kinase 1 (DDR1), which regulates cell growth, differentiation, and metabolism. In a large MS genome-wide association study, the gene SHMT1, which encodes a serine hydroxymethyl-transferase required for generating the methylation substrate SAM, was identified as a novel MS susceptibility locus, suggesting that methylation homeostasis is important in MS pathogenesis (7). An indication of hyper-methylation was observed in CD8⁺ T cells but not CD4⁺ T cells or whole blood in treatment-naive MS patients, without significant genome-wide DMP (13). Distinctive DNA methylation profiles in CD8⁺ T cells compared with CD4⁺ T cells in the same relapsing-remitting MS population were observed (75). Hypermethylation was found in CD19⁺ B cells in relapsing-remitting MS patients compared with HC, especially in lymphotoxin alpha locus (LT-α, also known as TNF-β) with 19 DMPs spanning 860 bp (76). Other hypermethylated DMRs included CARD11, CXCR5, CD19, and IL21R, which are involved in apoptosis and inflammation.

Environmental effects on MS pathogenesis can be mediated through DNA methylation. High BMI has been shown to increase the risk for MS. Genes regulating anti-proliferation (NRXN1, FZD7, and TP63) were found to be hypermethylated in monocytes, which had higher cell counts in MS patients with high BMI compared with normal BMI. Hypermethylation can be induced by high ceramide in cell culture, which may come from de novo synthesis from diet and recycling of damaged lipids. Higher plasma ceramide levels were also detected in MS patients with high BMI (15). In 2-years follow-up, high numbers of monocytes were correlated with worsened clinical disability in high-BMI MS patients.

Another example of the effect of diet on epigenetic changes in MS is vitamin D. Vitamin D supplementation protected mice from EAE by decreasing CD4⁺ T cells proliferation and Th17 cells. Vitamin D downregulated JAK/STAT, ERK/MAPK, and PI3K/Akt/mTOR pathways; it induced genome-wide DNA hypomethylation and upregulation of microRNAs (134). Smoking as an established environmental risk factor can affect blood DNA methylation of MS patients, especially in populations with an increased genetic risk for MS. Aryl-hydrocarbon receptor repressor (AHRR), which is involved in regulation of xenobiotic metabolism, was hypomethylated and overexpressed by smoking in PBMC from MS patients (77).

Histone methylation changes in MS

Histone lysine and arginine methylation studies in MS have identified H3K9me2/3 as a potential treatment response biomarker, and EZH2 and PRMT5 as potential therapeutic targets.

H3K9me2 catalyzed by methyltransferase G9a was inversely correlated with IFNβ and IFN-stimulated gene (ISG) expression (35). SIRT1, H3K9me2, phosphorylated SIRT1 (p-SIRT1), and H3K9me3 were significantly lower in the PBMCs from relapsing compared with stable MS patients. Further, SIRT1, H3K9me2, and H3K9me3 can potentially predict responses to glatiramer acetate treatment (18, 54). Jarid2, a member of PRC2, inhibited H3K9me3 recruitment to the Zbtb16 locus, which encodes promyelocytic leukemia zinc finger (PLZF), a signature TF for invariant natural killer (iNK) T cells. Mice with Jarid2-deficient T cells had increased PLZF expression and high frequency of iNK T cells, perturbed Th17 differentiation, and reduced Th17-driven EAE (89).

The histone lysine methyltransferase EZH2 plays a role in MS by mediating the NF-κB pathway and cell adhesion and migration (Fig. 2C). Besides its role in regulating FOXP3 in Treg cells (34, 100), Zhang and colleagues showed that EZH2 in macrophages/microglia had a pathogenetic role in EAE and DSS-induced colitis models through repressing the suppressor of cytokine signaling 3 (SOCS3) (139). Microglia/myeloid cell-conditional EZH2 knockout promoted H3K4me3 in the Socs3 enhancer region and increased SOCS3 expression, which increased Lys48-linked ubiquitination and degradation of TNF receptor-associated factor 6 (TRAF6), and it inhibited the TLR-induced MyD88-dependent NF-κB pathway and proinflammatory cytokines production (139). In addition, cytosolic EZH2 can regulate integrin-dependent cell adhesion and migration (50). EZH2 conditional knockout in innate leukocytes decreased extravasation and cell migration and attenuated EAE.

Histone arginine methyltransferase PRMT5 plays an important role in MS pathogenesis by regulating T cells homeostasis (Fig. 3). Inhibiting PRMT5 significantly reduced memory T helper (Th) cell expansion, thus reducing inflammation and disease in EAE in both prophylactic and therapeutic models (123). PRMT5 was required for thymic and peripheral homeostasis of CD4⁺, CD8⁺, and iNK T cells and T cells activation (60, 112, 124). Inducible PRMT5 knockout in CD4⁺ T cells suppressed Th17 differentiation and ameliorated EAE. PRMT5 knockout inhibited symmetrical dimethylation of cholesterol biosynthesis regulator SREBP1 at R321 and promoted SREBP1 degradation. Retinoid-related orphan receptor (RORγt) activity, which is the lineage-specific TF of Th17 cells, was subsequently inhibited with decreased RORγt agonist desmosterol produced during cholesterol biosynthesis (124) (Fig. 3A). PRMT5 is also necessary for T cells to express the common γ chain (γc) shared by IL-2 and IL-7 receptors to survive and proliferate with TCR activation (60, 112). PRMT5 symmetrically dimethylates pre-mRNA splicesome SmD3 to promote splicing of Il2rg (encodes γc, a subunit for IL2R) and regulate γc family cytokine receptor expression (60) (Fig. 3B). However, conditional knockout of PRMT5 in Tregs results in scurphy-like autoimmunity symptoms, because PRMT5 binding to FOXP3 symmetrically dimethylates R27, R51, and R146 to maintain Treg functions (82) (Fig. 3C).

FIG. 3.

Protein arginine methyltransferase PRMT5 plays important roles in regulating T cell homeostasis and autoimmunity. (A) Conditional knockout of PRMT5 in CD4⁺ T cells inhibits symmetrical dimethylation of cholesterol biosynthesis regulator SREBP1 and promotes SREBP1 phosphorylation and degradation. Th17 lineage-specific TF retinoid-related orphan receptor (RORγt) activity is suppressed with decreased desmosterol levels. Th17 differentiation is repressed, and mice are protected from EAE. (B) PRMT5 dimethylates SmD3, a component of pre-mRNA splicesome. Conditional knockout of PRMT5 inhibits the IL2-JAK3-STAT5 pathway by preventing Il2rg (encodes γc, a subunit for IL2R) and Jak3 splicing by SmD3. Common γc family cytokine receptors (such as CD25) are downregulated and populations of iNKT cells (invariant natural killer T cells), CD4⁺ and CD8⁺ T cells are suppressed by PRMT5 knockout. (C) PMRT5 dimethylates FOXP3 at R27, R51, and R146 to maintain regulatory T cells (Treg) function. Conditional knockout of PRMT5 in Tregs inhibits FOXP3-regulated gene expression (such as Nrp-1, CTLA4, ICOS, and Ki67) and promotes autoimmunity. EAE, experimental autoimmune encephalomyelitis. Color images are available online.

Oxidative Stress and Its Role in Autoimmunity

Oxidative stress occurs when the balance of production and removal of reactive oxygen species or reactive nitrogen species (ROS, RNS) is lost (104). Mitochondrial electron transport chain, NADPH oxidases (NOX), nitric oxide synthases, and nitrite reductases are producers of ROS and RNS. Scavengers include antioxidant molecules, such as glutathione and vitamin C, and antioxidant enzymes. The glutathione and thioredoxin systems are the most important antioxidant enzyme systems. Oxidative stress can be triggered by environmental exposure, such as infection, UV light, and drug use, and has been shown to be involved in ADs (74, 104). Elevated oxidative stress increases mammalian target of rapamycin (mTOR) activity (36), and it affects redox-sensitive TFs (Nrf2 (31), AP-1 (1), and NFκB (81)) and cytokine production (IL-2, IL-10, and TNF) in cells (41, 90, 91). Here, we will discuss how oxidative stress alters the epigenetic landscape in autoimmunity.

ROS and epigenetics in autoimmunity

ROS are highly reactive molecules, including superoxide (O₂⁻), hydrogen peroxide (H₂O₂), and hydroxyl radical (OH). Evidence suggests that ROS can interact with epigenetic regulation in autoimmunity via three different mechanisms: A) DNA damage repair; B) cellular metabolism and local redox environment; and C) key redox-sensitive TFs (Fig. 4).

FIG. 4.

Mechanisms of epigenetic regulation by ROS. (A) ROS regulate epigenetics through the DNA damage repair mechanism. To repair DNA damage caused by ROS, a complex containing DNMT1, SIRT1, DNMT3B, EZH2, SUZ12, and EED2 would form and translocate to actively transcribed genes. The complex decreases H3K4me3 and H4K16ac, increases H3K27me3 and DNA methylation to decrease transcription. (B) ROS activate mTORC1, leading to increased glycolysis and EZH2 overexpression in SLE T cells. The ratio of reduced GSH to glutathione disulfide (GSSG) is decreased by ROS, which inhibits MAT that synthesizes SAM, DNMT1, histone demethylases, DNA demethylases (TET), and PAD4, and activates class I/II HDAC. (C) Key redox-sensitive TFs, such as Nrf2, can regulate epigenetics. In astrocytes from EAE, Nrf2 is downregulated; therefore, antioxidant and anti-inflammatory genes mediated by Nrf2 are inhibited. Granulocyte-macrophage colony-stimulating factor (GM-CSF)-mediated overexpressed MAFG cooperates with MAT2α to promote DNMTs activity and increase methylation at promoters of anti-inflammatory genes, which promote proinflammation and EAE development. DNMT1, DNA methyltransferase 1; GSH, glutathione; HDAC, histone deacetylases; MAT, methionine adenosyltransferase; PAD4, peptidylarginine deiminase 4; ROS, reactive oxygen species; SAM, S-adenosyl-methionine. Color images are available online.

Oxidative stress causes DNA damage, and base excision repair involves histone acetylase and deacetylase reactions (111). Large complexes, including DNMT1, SIRT1, DNMT3B, EZH2, SUZ12, and EED2, are formed to repair DNA damage induced by ROS, and DNMT1 in this complex acts as an upstream regulator to recruit additional molecules (86). This complex can translocate to promoters of genes that are highly expressed and contain CpG islands. The translocation decreases H3K4me3 and H4K16ac, increases H3K27me3 and CpG methylation, leading to repressed transcription (Fig. 4A).

Epigenetics can be regulated by oxidative stress through cellular metabolism (Fig. 4B). For example, H₂O₂ treatment increased EZH2 expression in naive CD4⁺ T cells (142). mTORC1 can be activated under oxidative stress and increases glycolysis in CD4⁺ T cells. EZH2 can be downregulated in CD4⁺ T cells by inhibiting mTORC1 or inhibiting glycolysis (142). Further, EZH2 levels in lupus B cells are upregulated by mTORC1 and Syk in the presence of methionine (137).

Redox balance affects methyl donor SAM production and activity of key epigenetic enzymes (Fig. 4B). Methionine adenosyltransferase (MAT), which synthesizes SAM, and DNMT1 contain redox-sensitive cysteine residues in catalytic sites (48). ROS inhibit MAT and DNMT1 activity, and ROS are closely regulated by glutathione (GSH) (38). GSH as the major intracellular antioxidant can be oxidized to glutathione disulfide (GSSG) under oxidative stress. In addition, NAD+/NADH regulates epigenetics through disruption of DNA demethylases (TET) and HDACs, including sirtuin (25). Global DNA methylation was shown to be increased by H₂O₂ treatment (85), which might suggest that TET activity was inhibited more than MAT and DNMT1 by H₂O₂.

Histone modifications can also be regulated by ROS. Histone demethylases containing JmjC domain require iron Fe (II) and α-ketoglutarate to function. Exposure to H₂O₂ increases H3K4me3 and H3K27me3 as Fe (II) is oxidized to Fe (III), and decreases H3K9ac and H4K8ac due to increased HDAC activity (85).

Histone citrullination can also be regulated by GSH/GSSG metabolism and the local redox environment. H₂O₂>40 μM inhibited extracellular PAD2- and PAD4-induced citrullination (26). However, H₂O₂ treatment (1–1000 μM, 2 h or 3 h) promoted PAD4-mediated intracellular histone citrullination in neutrophils from humans and mice (71, 84). The catalytic function of PAD requires reduced GSH in vivo and the reducing agent dithiothreitol in vitro (27). In synovial fluid from RA patients, the activity of glutathione reductase (GR) (65), which converts GSSG to GSH, and antioxidant thioredoxin (131) was significantly higher compared with OA patients. Further, ROS promote calcium transport to the nucleus (26, 47). Therefore, intracellular PAD4 activity can be enhanced under oxidative stress with increased GR and thioredoxin to mitigate the inhibitory effect of high H₂O₂ concentration. Tsung and colleagues showed that superoxide induces histone H3 citrullination and the formation of neutrophil extracellular traps (5).

Lastly, epigenetics can be regulated by key redox-sensitive enzymes in autoimmunity. Nrf2, which is a master regulator of oxidative stress, suppresses EAE as a negative regulator of inflammation in astrocytes (126) (Fig. 4C). Reduced Nrf2 activity in astrocytes was associated with enhanced SAM biosynthesis and DNMT activity. MAFG acts as a cofactor with Nrf2 to bind to antioxidant response element sequences and induces transcription. When Nrf2 expression is reduced, MAFG cooperates with MAT2α, which generates SAM to increase DNA methylation in astrocytes and promote inflammation as seen in MS patients (126). Nrf2-mediated epigenetic changes were also demonstrated in the streptozotocin-induced type 1 diabetic model (37).

RNS and epigenetics in autoimmunity

Intracellular superoxide (O₂⁻) combined with nitric oxide (NO) would form peroxynitrite (ONOO⁻) (a molecule in the RNS family) to nitrate protein tyrosine. Serum 3-nitrotyrosine levels were upregulated and correlated with disease activity in patients with SLE and Sjogren's syndrome (109). Defective PKCδ phosphorylation in CD4⁺ T cells is associated with active disease in SLE patients (44). Dr. Richardson and colleagues showed that nitrated PKCδ prevented T505 PKCδ phosphorylation, resulting in impaired ERK signaling, decreased DNMT1, and demethylation in lupus T cells (46). By treating CD4⁺ T cells cultured in low methionine medium with peroxynitrite, the expression of methylation-sensitive genes (including CD70, KIR genes, and perforin) was significantly increased (94). H₂O₂ and ONOO⁻ exposure in CD4⁺ T cells from HC decreased PKCδ/ERK/DNMT1, resulting in demethylation and overexpression of the same methylation sensitive genes overexpressed in T cells from patients with SLE (73, 108). Adoptive transfer of H₂O₂- and nitric oxide-treated CD4⁺ T cells in syngeneic mice induced anti-dsDNA antibody and glomerulonephritis (108). In addition, a transgenic mouse with an inducible dominant negative PKCδ (dnPKCδ) in T cells provided further evidence for a key regulatory role of PKCδ on T cell DNA methylation and the pathogenesis of lupus (45). The dnPKCδ transgenic mice showed decreased ERK signaling and DNMT1 expression, upregulation of CD70 and CD11a, dsDNA autoantibody production, and glomerulonephritis. A follow-up study revealed that protein phosphatase 5 (PP5) may also mediate the effects of oxidative stress on DNMT1 and epigenetically regulated genes (88) (Fig. 5).

FIG. 5.

RNS mediate DNA demethylation through the PKCδ/ERK/DNMT1 pathway. Peroxynitrite (ONOO⁻) nitrates PKCδ, which prevents T505 PKCδ phosphorylation and ERK activation, resulting in reduced DNMT1 expression and activity. ONOO⁻ may inhibit DNMT1 activity also by increasing protein phosphatase 5 (PP5) expression. DNA methylation-sensitive genes, including CD40LG, CD70, KIR, and perforin, are hypomethylated and overexpressed. RNS, reactive nitrogen species. Color images are available online.

Conclusions

DNA methylation and histone modification are involved in the pathogenesis of ADs. Epigenetically dysregulated pathways in autoimmunity result in overactive inflammatory changes (IFN, TNF, and IL-6), increased extracellular matrix degradation (MMPs and HIP1) or synthesis (PTGDS and PTGIS), and dysregulated stress response (Nrf2 and K63 ubiquitination) and key TFs (FOXP3 and Nrf2) (Fig. 1). Epigenetic changes involving IFN and TNF pathways, FOXP3, and EZH2 appear to be common to several ADs. Further, some CpG sites with altered methylation levels in RA B cells were replicated in SLE B cells (63). Overlaps in DNA methylation patterns of MZ twins discordant for RA and type 1 diabetes were more than expected by chance (125). Hypomethylation of type I IFN genes in CD4⁺ T cells has been found in SLE, Sjogren's syndrome, RA, Graves' disease, and systemic sclerosis (6, 16).

Epigenetics can act as a bridge to connect genetics and the environment in the context of AD etiology. This is mediated, in part, through the interaction between environmental triggers inducing oxidative stress and epigenetic mechanisms sensitive to ROS or RNS in a genetically susceptible host (Fig. 6). Oxidative stress affects DNA methylation and histone modifications through cellular metabolism, DNA repair mechanisms, redox-sensitive TFs, and the PKCδ/ERK/DNMT1 pathway. Increased levels of oxidative stress markers correlate with disease flare in several ADs. Targeting oxidative stress could potentially help in treating ADs through epigenetic modulation. Additional work to more comprehensively understand the interplay between metabolic regulation, redox environment, and epigenetic modulation in autoimmunity is warranted.

FIG. 6.

Proposed model of the interactive relationships between the environment, genetic predisposition, epigenetics, oxidative stress, and inflammation. Color images are available online.

Abbreviations Used

ACPA

anti-citrullinated peptide antibodies

AD

autoimmune disease

CD

Crohn's disease

CNS

central nervous system

CREMα

cAMP responsive element modulator α

DMP

differentially methylated positions

DMR

differentially methylated regions

DNMTs

DNA methyltransferases

DSS

dextran sodium sulfate

EAE

experimental autoimmune encephalomyelitis

ECM

extracellular matrix

FLS

fibroblast-like synoviocytes

GR

glutathione reductase

GSH

glutathione

GSSG

glutathione disulfide

HATs

histone acetyltransferases

HC

healthy controls

HDACs

histone deacetylases

HKDMs

histone lysine demethylases

HKMTs

histone lysine methyltransferases

HMTs

histone methyltransferases

IBD

inflammatory bowel disease

IFN

interferon

iNK

invariant natural killer

MS

multiple sclerosis

mTOR

mammalian target of rapamycin

MZ

monozygotic

Nrf2

nuclear factor erythroid-2 related factor 2

OA

osteoarthritis

PAD

peptidylarginine deiminase

PBMC

peripheral blood mononuclear cells

PLZF

promyelocytic leukemia zinc finger

PRMTs

protein arginine methyltransferases

RA

rheumatoid arthritis

RNS

reactive nitrogen species

ROS

reactive oxygen species

SAM

S-adenosyl-methionine

SF

synovial fibroblasts

SLE

systemic lupus erythematosus

TET

ten-eleven translocation

TNF

tumor necrosis factor

UC

ulcerative colitis

Authors' Contribution

Both authors contributed to writing and revising this article.

Author Disclosure Statement

The authors declare no financial conflict of interest.

Funding Information

This work was supported by the National Institute of Allergy and Infectious Diseases (NIAID) and the National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS) of the National Institutes of Health (NIH) grants number R01AI097134 and R01AR070148, and the Lupus Research Alliance.