www.nature.com/cmi REVIEW ARTICLE OPEN Epigenetic regulation of B cells and its role in autoimmune pathogenesis ✉ Fan Xiao 1, Ke Rui2, Xiaofei Shi3, Haijing Wu4, Xiaoyan Cai5, Kathy O. Lui 6 , Qianjin Lu4, Esteban Ballestar 7,8 , Jie Tian2 , ✉ ✉ Hejian Zou9 and Liwei Lu 1,10 © The Author(s) 2022 B cells play a pivotal role in the pathogenesis of autoimmune diseases. Although previous studies have shown many genetic polymorphisms associated with B-cell activation in patients with various autoimmune disorders, progress in epigenetic research has revealed new mechanisms leading to B-cell hyperactivation. Epigenetic mechanisms, including those involving histone modifications, DNA methylation, and noncoding RNAs, regulate B-cell responses, and their dysregulation can contribute to the pathogenesis of autoimmune diseases. Patients with autoimmune diseases show epigenetic alterations that lead to the initiation and perpetuation of autoimmune inflammation. Moreover, many clinical and animal model studies have shown the promising potential of epigenetic therapies for patients. In this review, we present an up-to-date overview of epigenetic mechanisms with a focus on their roles in regulating functional B-cell subsets. Furthermore, we discuss epigenetic dysregulation in B cells and highlight 1234567890();,: its contribution to the development of autoimmune diseases. Based on clinical and preclinical evidence, we discuss novel epigenetic biomarkers and therapies for patients with autoimmune disorders. Keywords: B cells; Epigenetic regulation; Autoimmune disease; Biomarker; Therapy Cellular & Molecular Immunology (2022) 19:1215–1234; https://doi.org/10.1038/s41423-022-00933-7 INTRODUCTION including B cells [6, 7]. These autoimmunity-associated risk Immune dysregulation contributes to the breakdown of immune variants have functions that are highly enriched for cellular tolerance, leading to autoimmune pathogenesis. In recent processes that regulate B-cell proliferation, differentiation, and decades, numerous studies have revealed the significant roles of activation [8]. However, the low concordance rates of many both innate and adaptive immune cells, especially B cells, in the autoimmune diseases in monozygotic twins suggest the roles of development of autoimmune diseases [1–3]. The presence of environmental factors and epigenetic mechanisms in disease autoantibodies and hyperactivation of B cells have been development [9]. Increasing evidence shows that epigenetic characterized as salient features in patients with various auto- modifications, which regulate gene expression without affecting immune diseases, such as systemic lupus erythematosus (SLE), DNA sequence, are critically involved in the breakdown of B-cell primary Sjögren’s syndrome (pSS), and rheumatoid arthritis (RA), tolerance, contributing to autoimmune inflammation and disease which highlights a pivotal role of B cells in the pathogenesis of progression in patients [9–11]. Moreover, the differentiation and autoimmune diseases [4, 5]. It has been recognized that B-cell function of B-cell subsets are regulated by diverse epigenetic dysregulation is critically involved in the initiation and perpetua- events, including changes in histone modifications, DNA methyla- tion of autoimmunity. However, the mechanisms and conse- tion profiles, and noncoding RNAs. Epigenetic dysregulation may quences of B-cell tolerance breakdown in the pathogenesis of lead to aberrant expansion of pathogenic B-cell subsets in the autoimmune diseases are still not fully understood. development of autoimmune diseases [12, 13]. Genome-wide Genome-wide association studies (GWAS) have identified epigenomic analysis has identified significant alterations in hundreds of genetic polymorphisms that are associated with the epigenetic profiles of B cells from patients with autoimmune increased risks of developing autoimmune diseases, among which diseases [14]. Recent studies have revealed that epigenetic many affect genes that modulate the functions of immune cells, signatures, including DNA methylation profiles, may serve as 1 Department of Pathology, Shenzhen Institute of Research and Innovation and Shenzhen Hospital, The University of Hong Kong, Hong Kong; Chongqing International Institute for Immunology, Chongqing, China. 2Institute of Medical Immunology, Affiliated Hospital of Jiangsu University, Zhenjiang, China. 3Department of Rheumatology and Immunology, The First Affiliated Hospital and School of Medicine, Henan University of Science and Technology, Luoyang, China. 4Department of Dermatology, Second Xiangya Hospital, Central South University, Hunan Key Laboratory of Medical Epigenomics, Changsha, Hunan, China. 5Department of Rheumatology, Guangzhou First People’s Hospital, School of Medicine, South China University of Technology, Guangzhou, China. 6Department of Chemical Pathology, Faculty of Medicine, Prince of Wales Hospital, The Chinese University of Hong Kong, Hong Kong, China. 7Epigenetics and Immune Disease Group, Josep Carreras Research Institute, Badalona, 08916 Barcelona, Spain. 8Epigenetics in Inflammatory and Metabolic Diseases Laboratory, Health Science Center, East China Normal University, Shanghai, China. 9Department of Rheumatology, Huashan Hospital, Fudan University, Shanghai, China. 10Centre for Oncology and Immunology, Hong Kong Science Park, Hong Kong, China. ✉email: tianjie@ujs.edu.cn; hjzou@fudan.edu.cn; liweilu@hku.hk Received: 12 July 2022 Accepted: 19 September 2022 Published online: 12 October 2022 F. Xiao et al. 1216 Fig. 1 Major epigenetic mechanisms. Histone modifications and DNA methylation affect transcriptional accessibility. MiRNAs bind to target mRNAs and lead to the cleavage and degradation of the mRNAs useful biomarkers that can reflect disease prognosis, severity, and histone modifications have been reported, and histone acetylation responses to therapies [15, 16]. Moreover, clinical investigations and methylation are most commonly described in the regulation and animal studies suggest that targeting epigenetic modifiers of B-cell functions and autoimmune pathogenesis. Histone could be a novel effective therapeutic strategy [10]. Therefore, the acetylation is usually associated with an open chromatin structure characterization of global epigenetic profiles by high-throughput and easy binding of transcriptional machinery to transcription technologies may facilitate the development of personalized sites with increased gene expression. Histone methylation can medicine for treating patients. In this review, we discuss either promote or suppress gene transcription. Trimethylation at epigenetic modifications in different functional B-cell subsets the Lys4 residue of histone H3 (H3K4me3) activates gene and their roles in disease pathogenesis and clinical therapies for transcription and is usually enriched in active promoters around patients with autoimmune diseases. transcription start sites (TSSs) [18, 19]. However, trimethylation at Lys9 and Lys27 of histone H3 (H3K9me3 and H3K27me3, respectively) can function as a silencer to repress gene expression EPIGENETIC MODIFICATIONS REGULATE B-CELL by modifying chromatin architecture and interactions [20, 21]. DIFFERENTIATION Other histone modifications, including ubiquitination, phosphor- Epigenetic mechanisms ylation, and sumoylation, also directly influence interactions In the nucleus, DNA is packaged into chromatin through among histones, DNA, and transcription complexes [22, 23]. interactions with histones and many other proteins. Epigenetic Histone acetylation is catalyzed by histone acetyltransferases changes, such as cytosine methylation in DNA and posttransla- (HATs) and histone deacetylases (HDACs) that can add or remove tional modification of histone amino acid residues, strongly affect acetyl groups, respectively. HAT family members have conserved chromatin relaxation and condensation, which are closely protein–protein interaction and substrate-specific binding associated with transcriptional activity. Moreover, epigenetic domains that can recognize particular genomic sites [24]. Both modifications can directly influence the binding of transcription HATs and HDACs are important regulators during B-cell develop- factors to gene promoters and enhancers. Noncoding RNAs, ment and autoimmune diseases. Although HDACs and HATs show including long noncoding RNAs (lncRNAs) and microRNAs contrary functions in histone modification, they are counter- (miRNAs), are actively involved in the regulation of chromatin balanced during B-cell development and show complex roles in structure, gene silencing, and posttranscriptional events. Epige- B-cell homeostasis. netic control of RNAs via factors such as N6-methyladenosine (m6A) has been shown to play important roles in regulating RNA DNA methylation. Methylation of the 5’ position of cytosines splicing, translation, and stability. m6A is initially installed by (5mC), in the context of CpG dinucleotides, is the main form of various methyltransferase complexes (“writers”), including DNA methylation in mammals and is usually associated with gene methyltransferase-like protein 3 (METTL3), and removed by m6A suppression. Recently, DNA N6-methyldeoxyadenine (6mA) has demethylases (“erasers”), such as fat-mass and obesity-associated also been identified in the human genome [25, 26]. These protein and alkylation repair homolog protein 5. The effects of epigenetic modifications in DNA are usually stable and heritable. m6A are largely dependent on m6A-binding proteins (“readers”), In the mammalian genome, 5mC can be found in isolated CpG such as YT521-B homology (YTH) domain family members [17]. sites or in the context of CpG islands, and its addition is catalyzed These epigenetic modifications are important regulators during by various DNA methyltransferases (DNMTs) with distinct capa- B-cell differentiation and activation (Fig. 1). cities. DNMT1 plays a dominant role in the maintenance of the DNA methylation profiles during cell division, while DNMT3A and Histone posttranslational modifications. As the basic structural DNMT3B are responsible for adding new methyl groups to unit of the chromosome, the nucleosome is composed of a 147 bp unmethylated DNA [27]. Apart from their roles in establishing segment of DNA that is wrapped around an octamer of core and maintaining DNA methylation patterns, DNMTs have been histone proteins consisting of two copies of each of the histones reported to serve as versatile tools for epigenetic regulation H2A, H2B, H3, and H4. Histones can be modified at N-terminal [27, 28]. Recent evidence has indicated diverse functions of residues through various histone-modifying enzymes. These DNMTs, including transcriptional silencing, transcriptional activa- modifications regulate gene expression by changing chromatin tion, and posttranscriptional regulation [27]. Active DNA demethy- structure and accessibility to transcription complexes. Many lation is a multistep process that starts with the participation of Cellular & Molecular Immunology (2022) 19:1215 – 1234 F. Xiao et al. 1217 enzymes of the ten-eleven translocation (TET) family. TET enzymes oxidize methylcytosine to hydroxymethylcytosine, formylcytosine, and carboxycytosine, which are then excised through the activity of thymine DNA glycosylase, followed by base excision repair. TETs and DNMTs cooperate to sustain homeostasis of gene transcrip- tion with site-specific dependency [29]. In prokaryotes and protists, DNA 6mA modification is frequently detected. Recent developments in 6mA detection techniques have revealed the presence of 6mA in the genomic DNA of eukaryotes, including mammals [25, 26]. Unlike 5mC, which increases DNA helix stability, 6mA destabilizes the helix and induces DNA unwinding [30]. 6mA modification is a reversible process mediated by 6mA-specific methyltransferases and demethylases. Many studies have shown diverse biological and pathological roles of 6mA in regulating gene transcription, chromatin, and tumor progression [25, 26]. Available data indicate that 6mA may have a conserved role in recognizing and eliminating foreign DNA and thus participate in immune modulation [25]. Alkylation repair homolog 1 (ALKBH1) is a demethylase that regulates 6mA turnover in unpaired regions associated with dynamic chromosome regulation [31]. A recent study showed that ALKBH1 regulates the microenvironment in the bone marrow, where B cells are generated [32], suggesting that ALKBH1 may indirectly regulate B-cell homeostasis. Current evidence on the roles of 6mA in regulating B-cell responses is still limited, and further investigations are needed. Noncoding RNAs. The pervasive transcription of DNA yields the production of numerous noncoding RNAs, including lncRNAs, miRNAs, and circular RNAs. These noncoding RNAs do not encode functional proteins, but they exert diverse functions in regulating gene expression. LncRNAs are generally over 200 nt in length, while miRNAs are much smaller, with 21–25 nucleotides. LncRNAs act as important regulators of chromatin remodeling, gene transcription, and posttranscriptional modification [33]. LncRNAs interact with a variety of targets, including proteins and RNAs, via Fig. 2 Key epigenetic events in B-cell subsets. A The histone allosteric effects. Moreover, they can serve as a molecular scaffold demethylase LSD1 controls marginal zone B-cell and follicular B-cell to recruit target chromatin-modifying proteins [34]. Unlike fate. B Naive B cells express high levels of Sirt1, which suppresses lncRNAs, miRNAs generally target mRNAs in the 3′ untranslated AICDA expression. C In germinal center (GC) B cells, TET proteins region (3′UTR), which leads to the cleavage and degradation of promote AICDA gene transcription through DNA demethylation. mRNAs and inhibits protein translation. The biogenesis of miRNAs AICDA mRNA is targeted by various miRNAs. D In plasma cells, PRDM1 mRNA is targeted by miRNAs. Blimp1 suppresses several is directly regulated by DNA methylation [35]. MiRNAs also target transcription factors that support other B-cell subsets by interacting various DNMTs and thus regulate DNA methylation [36], indicating with epigenetic modifiers. MiR-148a is regulated by Blimp1 and crosstalk between different epigenetic events. serves as a key link between Blimp1 and metabolic regulation in Emerging evidence suggests that noncoding RNAs play an plasma cells. DNMT3A/B and TET2/3 sustain the DNA methylation important role in immune homeostasis and the etiology of human balance and regulate the transcription of genes associated with diseases, including autoimmune diseases [37]. It has been shown plasma cell differentiation. E Summary of key epigenetic regulatory that various noncoding RNAs sustain B-cell lineage-specific gene events in B-cell subsets expression profiles and are involved in B-cell-related diseases [38]. The aberrant expression profiles of several noncoding RNAs, Epigenetic modifications in functional B-cell subsets including miR-150, miR-155, and small nucleolar RNA host gene During ontogeny, B cells differentiate from hematopoietic stem 14, are associated with B-cell malignancies and autoimmune cells and undergo an ordered maturation and selection process in diseases [39, 40]. the bone marrow. Many studies have demonstrated that epigenetic modifications, including those related to DNA methyla- N6-methyladenosine RNA methylation. m6A is a dynamic and tion, histone modifications, noncoding RNAs, and m6A mRNA reversible posttranscriptional epigenetic modification that is the methylation, play an important role during the B-cell develop- most prevalent type of mRNA methylation in eukaryotes. METTL3 mental process in the bone marrow [38]. Newly formed immature and METTL14 are two important methyltransferases responsible B cells migrate from the bone marrow and differentiate into for m6A. Deficiency of METTL14 reduces mRNA m6A methylation various mature functional B-cell subsets, including marginal zone in B cells and results in severely inhibited proliferation of pro-B (MZ) B cells, germinal center (GC) B cells, plasma cells, memory B cells with severely abnormal gene expression profiles associated cells, and regulatory B (Breg) cells, in the peripheral lymphoid with B-cell development, suggesting that m6A mRNA methylation organs. These B-cell subsets exhibit diverse functions, including plays an essential role in B-cell development in bone marrow [41]. antigen presentation, antibody secretion, and cytokine produc- Consistently, the cytoplasmic m6A reader YT521-B homology tion, under different conditions. Maturation and activation of domain family member 2 sustains IL-7-induced pro-B-cell pro- peripheral B cells are mainly dependent on environmental factors liferation by suppressing a group of transcripts [41]. METTL3- such as B-cell activating factor (BAFF), ligands of Toll-like receptors dependent m6A mRNA methylation controls early B-cell differ- (TLRs), and IL-21. Recent studies have highlighted the essential entiation from hematopoietic stem cells in bone marrow [42]. roles of epigenetic regulation in different B-cell subsets (Fig. 2), Cellular & Molecular Immunology (2022) 19:1215 – 1234 F. Xiao et al. 1218 and this epigenetic regulation contributes to the pathogenesis of identified by the high expression levels of several key transcription autoimmune diseases, including SLE, pSS, and RA [5, 8, 43, 44]. factors. B-cell lymphoma 6 (BCL6) is a key transcription factor for GC B cells. The epigenetic regulation of BCL6 plays a critical role Marginal zone B cells. Immature B cells migrate from the bone during the GC reaction. Deletion of a GC-specific region upstream marrow and enter the spleen as transitional B cells, where they of Bcl6 that shows frequent intrachromosomal interactions results encounter survival signals, including BAFF, in the tissue micro- in diminished GC formation without affecting other develop- environment and complete their fate decision as either MZ B cells mental stages [53]. Furthermore, dysregulated epigenetic regula- or follicular B cells. MZ B cells are located at the borders of red tion of BCL6 in B cells is associated with GC-derived lymphoma pulp and white pulp. Early studies showed that MZ B cells are [56]. T follicular helper (Tfh) cells, characterized by high expression innate-like lymphocytes and are important for housekeeping of BCL6, play an essential role in the GC reaction and autoimmune functions such as the clearance of apoptotic cell debris. MZ B cells pathogenesis [3]. Ubiquitin-like with PHD and RING finger express B-cell receptors (BCRs) with polyreactivity and respond to domains 1 (UHRF1) suppresses DNA methylation and decreases various TLR stimulations, which drives the rapid production of low- the level of H3K27m3 within BCL6 promoter regions and thus affinity antibodies against both T-cell-independent and T-cell- controls Tfh cell differentiation and the GC reaction. Decreased dependent antigens [45, 46]. MZ B cells are characterized by low expression of UHRF1 results in abnormal Tfh expansion and GC expression levels of surface IgD and high expression levels of IgM responses, which promotes the development of SLE [57]. and CD21/CD35. GC B cells express high levels of AID. As an essential step in SHM Expanded MZ B-cell populations that produce massive levels of and CSR of immunoglobulin genes, AID induces the conversion of autoantibodies are detected in some patients with autoimmune deoxycytidine to deoxyuracil and drives base pair mismatches in diseases [45]. However, autoimmune vasculitis patients show DNA. The recruitment of AID at IgH switch regions is regulated by reduced frequencies and numbers of circulating MZ-like B cells, the histone methyltransferase multiple myeloma SET domain, the suggesting complex roles of MZ B cells in autoimmune disorders RNA exosome cofactor MPP6 and the nuclear m6A-modified RNA [47]. Although MZ B cells undergo clonal expansion with aberrant reader YTH domain containing 1, suggesting epigenetic regulation autoantibody production and can enter lymphoid follicles to of AID-mediated DNA breaks during CSR [58, 59]. Although AID interact with CD4 T cells in lupus-prone mice [48, 49], a recent mainly binds to the Ig gene locus, chromatin immunoprecipitation study showed defective MZ B-cell differentiation in SLE patients analysis shows a genome-wide presence of AID in activated B cells [50]. Reduced MZ B-cell frequency is observed in SLE patients, and [60], suggesting diverse functions of AID. In addition to inducing the reduction is even more prominent in those with lupus base pair mismatch, AID also shows epigenetic regulatory nephritis, which might be associated with the reduction of their functions during the GC reaction. Although GC B cells show putative transitional B-cell precursors [50]. Recently, it has been marked locus-specific loss of DNA methylation, deficiency of AID reported that MZ B-cell differentiation is regulated by lysine- results in abrogated CpG hypomethylation during the GC reaction specific demethylase 1 (LSD1), a histone demethylase that targets [61]. Moreover, AID also contributes to the epigenetic diversity of H3K4me1, H3K4me2, H3K9me1, and H3K9me2 through flavin GC B cells [61, 62]. Overexpression of AID increases cytosine adenine dinucleotide-dependent amine oxidation [51, 52]. Condi- methylation heterogeneity, whereas AICDA depletion leads to tional deletion of LSD1 in B cells results in a dramatic reduction in reduced heterogeneity characteristic of normal GC B cells [62]. MZ B cells, while follicular B cells are not affected. Moreover, LSD1 These epigenetic effects of AID are dependent on TET2, as AID- regulates chromatin accessibility at the motifs of several key mediated demethylation is markedly impaired in Tet2-deficient GC transcription factors for B-cell development and thus sustains the B cells [63]. Interestingly, AID might be involved in the establish- transcriptional identity of MZ B cells [51]. Mechanistically, LSD1 ment of DNA methylation patterns even before the GC reaction acts as an epigenetic regulator and cooperates with p52, a subunit [64]. AID induces DNA breaks and drives CSR in GC B cells. of NF-κB with DNA binding abilities, to drive the fate determina- AID expression can hardly be detected in resting B cells but is tion of MZ and follicular B cells (Fig. 2A) [51]. strongly induced by T-cell-dependent and T-cell-independent antigenic stimuli and effector molecules, including CD40L, TLR Germinal center B cells. Upon proper antigen stimulation, naive B ligands, and cytokines. The induction of Aicda, which encodes AID, cells undergo rapid proliferation with the help of cognate CD4 is regulated by epigenetic modifications, including histone T cells within lymphoid follicles and form GCs, a specialized acetylation and DNA demethylation around the gene locus microanatomical structure that supports cell division, somatic [65, 66]. B-cell-specific deficiency of HDAC3 results in an impaired hypermutation (SHM), and class switch recombination (CSR) of GC reaction with a reduction in dark zone centroblasts and activated B cells. GC B cells are characterized by surface accumulation of light zone centrocytes [67]. The NAD+-dependent phenotypic markers of peanut agglutinin (PNA)+, Fas+, and IgDlo. class III HDAC sirtuin 1 (Sirt1), a nonclassical class III HDAC, is highly In lymphoid tissues, GC structures can be divided into two major expressed in resting B cells but downregulated upon B-cell areas: the light zone and the dark zone. GC B cells undergo activation (Fig. 2B). Deficiency of Sirt1 results in increased Aicda intensive proliferation in the dark zone, where activation-induced expression through acetylation of Aicda promoter histone and cytidine deaminase (AID) critically regulates SHM at the variable nonhistone proteins, suggesting that Sirt1 acts as a key regions of immunoglobulin genes to increase BCR affinities. In the suppressive epigenetic regulator of Aicda during B-cell activation light zone, B cells expressing BCRs with high affinity for the [66]. DNA methylation and demethylation markedly affect the GC antigens are selected. The bidirectional movement of GC B cells in B-cell reaction (Fig. 2C). Deficiency of Tet2 and Tet3 promotes GC the light zone and dark zone results in iterative rounds of SHM B-cell responses, while deletion of Dnmt1 abrogates this effect and selection, which ultimately generates B cells with high affinity [68]. Moreover, combined Tet2 and Tet3 loss-of-function in GC B for the antigen. cells increases C-to-T and G-to-A transition mutagenesis, suggest- Compared with naive B cells, GC B cells are predominantly ing that TET enzymes may affect SHM [69]. Indeed, TET enzymes hypomethylated and show dramatic reorganization of the promote DNA demethylation and maintain chromatin accessibility genomic architecture with massive unpacking of chromosomes at the superenhancer region of the Aicda locus [65]. Furthermore, [53, 54]. Mettl3-deficient GC B cells exhibit reduced cell cycle basic leucine zipper transcription factor ATF-like (BATF) plays a key progression and decreased expression of proliferation- and role in TET-mediated Aicda expression [65]. The short-chain fatty oxidative phosphorylation-related genes, suggesting that m6A acids (SCFAs) butyrate and propionate are common metabolites modifications by METTL3 are required for GC maintenance [55]. with diverse immunomodulatory functions. It was recently found GC B cells exhibit a unique transcriptional network. GC B cells are that SCFAs inhibit Aicda expression in a dose-dependent manner Cellular & Molecular Immunology (2022) 19:1215 – 1234 F. Xiao et al. 1219 by increasing the levels of certain miRNAs that target the Aicda 3′- demethylation is dispensable for initial IRF4 expression but is UTR through inhibition of HDACs targeting miRNA host genes [70]. essential for sustaining high IRF4 expression, which is required for The transcription of Aicda is also regulated by noncoding RNAs plasma cell differentiation [82]. Interestingly, ascorbic acid, an such as miR-29b and miR146a (Fig. 2C) [71, 72]. Moreover, m6A essential vitamin for humans, promotes plasma cell differentiation modifications by METTL3 are essential for maintaining GC and humoral responses by increasing TET2/3-mediated DNA responses in peripheral lymphoid organs [55]. METTL3 enzyme- demethylation [83], supporting an important role of DNA catalyzed m6A in IgH locus-associated G-rich long noncoding RNA demethylation during plasma B-cell differentiation. (SμGLT) drives recognition and 3’-end processing by RNA Many miRNAs regulate the differentiation of plasma cells exosomes, which suppresses chromosomal translocation and through diverse effector mechanisms. MiR-148a is abundantly CSR [59]. METTL3 also suppresses IgH-associated abnormal DNA expressed in plasma cells. B-cell-specific deficiency of miR-148a breaks and improves genomic stability [59]. The RNA exosome results in reduced numbers of antibody-secreting plasma cells. complex is a critical regulator of noncoding RNAs. DIS3, an Moreover, miR-148a promotes plasmablast differentiation from GC important RNase subunit within RNA exosomes, was recently B cells in mice. Transcriptome and metabolic analyses suggest that found to regulate chromosomal architecture and SHM in B cells miR-148a favors plasma cells by fine tuning glucose uptake and [73]. Ablation of DIS3 results in insufficient noncoding RNA oxidative phosphorylation [84]. Longevity is a hallmark of long- turnover and accumulation of DNA‒RNA hybrids, which affects lived plasma cells derived from GC B cells. Many miRNAs, including somatic mutation at the Igh locus [73]. Overexpression of miR-29b miR-155, are important for sustaining plasma cell survival [85]. in human B cells decreases AID expression and impairs CSR to IgE Recently, it was found that miR-29 controls apoptosis of mature B [71]. TGF-β imitates CSR to IgA by activating Smad2, Smad3, and cells via the phosphatase and tensin homolog deleted on Smad4. miR146a is abundantly expressed in resting B cells, chromosome 10 (PTEN)-phosphoinositide 3-kinase (PI3K) axis whereas activated B cells undergoing CSR show diminished signaling pathway, suggesting that miR-29 may play a role in miR146a levels [74]. Deletion of miR146a leads to increased levels sustaining plasma cell survival [86]. of Smad2, Smad3, and Smad4 and promotes CSR to IgA, One of the most prominent biological features of plasma cells is suggesting that miR146a may regulate CSR by targeting TGF-β their unique metabolic pattern that sustains the massive signaling [74]. production of antibodies. Plasma cells primarily rely on oxidative phosphorylation to support the energy demand for producing Plasma cells. Plasma cells are terminally differentiated B cells antibodies, whereas GC B cells utilize fatty acid oxidation more with potent antibody-secreting abilities that are generated either than glycolysis and oxidative phosphorylation [87, 88]. The from follicular GC reactions or from activated extrafollicular B cells. longevity of plasma cells is also dictated by metabolic pathways GC B-cell-derived plasma cells produce class-switched antibodies [89, 90]. The import of pyruvate into mitochondria is important, as with high affinity and have a long lifespan, whereas extrafollicular the loss of mitochondrial pyruvate carriers leads to a progressive B-cell-derived plasma cells are short-lived cells that mainly decrease in long-lived plasma cells in mice [91]. Plasma cells also produce IgM with low affinity. Numerous studies have shown require a high rate of glucose uptake for glycolysis and antibody the diverse functions of plasma cells in infections, cancers, and glycosylation [92]. The metabolic signature is largely dependent autoimmune diseases [43, 75]. Plasma cells exhibit unique on Blimp1, the key transcription factor of plasma cells [76]. phenotypic, transcriptional, metabolic, and functional character- Interestingly, miR-148a is regulated by Blimp1 and serves as a key istics. The differentiation of antibody-secreting plasma cells is a link between Blimp1 and metabolic regulation in plasma cells coordinated process with both genetic and epigenetic regulation (Fig. 2D) [84]. Blimp1-induced miR-148a controls energy metabo- [76]. lism in plasma cells by regulating Glut-1-mediated glucose uptake Plasma cells show high expression levels of B-lymphocyte- and mitochondrial respiration [84]. Enhancer of zest 2 (EZH2) is the induced maturation protein 1 (Blimp1), which plays a central role catalytic subunit of polycomb repressive complex 2 (PRC2), which during plasma cell differentiation. The mRNA of the Prdm1 gene mediates H3K27 trimethylation. EZH2 directly interacts with Bcl6 that encodes Blimp1 has a long 3’-UTR and is targeted by many and controls GC formation in mice [93]. Moreover, EZH2 is miRNAs, such as miR30 family members (Fig. 2D) [77, 78]. These required for the metabolic programming of plasma cells. miRNAs directly target Prdm1 mRNA, leading to suppressed Deficiency of Ezh2 results in impaired plasma cell differentiation Blimp1 expression and inhibited plasma cell differentiation [78]. accompanied by reduced glycolysis and oxidative phosphoryla- Blimp1 suppresses several transcription factors that support other tion [94]. B-cell subsets, such as BCL6, through diverse epigenetic modifica- tions (Fig. 2D). Blimp1 interacts with HDACs, regulates histone Memory B cells. Memory B cells are important for rapid immune acetylation and thus suppresses the expression of target genes responses against repeated infection. The effectiveness of such as Bcl6 and cMyc [77, 79]. Moreover, Blimp1 recruits vaccines against infectious pathogens is largely dependent on methyltransferases and promotes H3K9me3 deposition in the the formation of memory B cells [95, 96]. Both memory B cells and promoter regions of Pax5 and Spib [77, 80]. plasma cells can be derived from GC-dependent and GC- DNA methylation not only is important for GC B-cell differentia- independent pathways, but these two B-cell populations show tion but also plays a key role in sustaining plasma cell identity distinct transcriptional, phenotypic, and functional features [97]. (Fig. 2D). Genetic deletion of DNMT3 promotes the expansion of Unlike plasma cells, memory B cells are normally in a resting state GC B cells and plasma cells. Gene expression is mostly normal in and can mount a strong and rapid response to a second challenge naive and GC B cells but is prominently dysregulated in Dnmt3- [98]. The terminal differentiation of GC B cells into plasma cells deficient plasma cells [81]. In particular, dysregulated DNMT3- versus memory B cells is largely regulated by the extent of signals dependent DNA methylation coincides with E2A and PU.1- received from BCR engagement and T-cell help. BTB domain and interferon regulatory factor (IRF) composite-binding motifs in CNC homology 2 (BACH2) is a transcription factor that represses plasma cells, suggesting that de novo DNA methylation by DNMT3 the expression of plasma cell-defining factors, including Blimp1. inhibits plasma cell differentiation by repressing the gene GC B cells with weak T-cell help do not transduce sufficient NF-κB expression program of key B-cell fate and activation genes [81]. signals to repress BACH2 and have a strong potential to In contrast, DNA demethylation mediated by TET2 and TET3 is differentiate into memory B cells [97]. In contrast, epigenetic essential for plasma cell differentiation since conditional Tet2/3 suppression of BACH2 by EZH2 promotes plasma cell but not double-KO B cells fail to differentiate into plasma cells upon memory B-cell differentiation [99]. Integrative analysis of tran- antigen immunization [82]. TET2- and TET3-dependent scriptome factors, including mRNAs, miRNAs, lncRNAs, chromatin Cellular & Molecular Immunology (2022) 19:1215 – 1234 F. Xiao et al. 1220 accessibility, and cis-regulatory elements, revealed a core mRNA- agents for treating autoimmune diseases and transplant rejection. noncoding RNA transcriptional signature of human memory B SCFAs from the environment not only epigenetically regulate the cells [100]. Memory B cells have an accessible chromatin generation of GC B cells and plasma cells but also promote IL-10 architecture around several plasma cell-specific genes, including expression dependent on their HDAC inhibitory activity [70, 122]. Xbp1, Prdm1, and Irf4, suggesting the reactivation potential of memory B cells [101]. B1 cells. B cells are divided into two major lineages: B1 cells and Memory B cells are heterogeneous and have various subsets. B2 cells. B2 cells are conventional B cells that can be activated and Previous studies identified different memory B-cell subsets in mice produce antigen-specific antibodies. B1 cells are long-lived self- based on the expression of CD80, CD86, PDL2, and BCR isotype renewing cells that spontaneously produce natural antibodies. [97, 98]. Recently, atypical memory B cells were identified to be B1 cells are mostly located in peritoneal and pleural cavities and characterized by high expression of CD11c and T-bet and low exhibit innate-like features. B1 cells are characterized by surface expression of CD27 and CD21 [5, 98, 102]. Increasing evidence expression of CD45RA, CD11b, and CD43. Based on the expression shows the critical involvement of atypical memory B cells during levels of CD5, B1 cells are further divided into B1a (CD5+) and B1b infections and autoimmune diseases [103, 104]. Expansion of (CD5-) subsets [123, 124]. B1 and B2 cells also show different CD11c+ atypical memory B cells is observed in the peripheral transcriptomes, metabolic statuses, and epigenetic modifications blood of SLE patients. Moreover, CD11c+ B cells from SLE patients [123, 125]. can differentiate into autoantibody-producing plasma cells upon Genome-wide CpG methylation analysis has revealed that B1a IL-21 stimulation in culture [104, 105]. It has been well recognized cell development is characterized by programmed demethylation that the lncRNA Xist can regulate X-chromosome inactivation at enhancers that are methylated in B2 cells, which is associated (XCI). Epigenetic profiling shows that Xist is lost from the with B1 lineage-specific gene expression [125]. Interestingly, B- inactivated chromosome at the pro-B-cell stage but is restored cell-specific DNMT3a deficiency results in the selective expansion upon B-cell activation, suggesting that Xist RNA localization is of B1a cells, suggesting that DNMT3a-dependent CpG methylation important for regulating XCI during B-cell development and may control B-cell lineage-specific gene expression [125]. Inhibi- activation in females [106]. Conventional memory B cells show tion of HDAC activity promotes the migration and function of dispersed Xist RNA signals across the genome, while atypical B1 cells, suggesting an important role of histone acetylation in memory B cells exhibit distinct Xist RNA localization patterns [107]. regulating B1-cell functions [126]. Since B1 cells require Blimp1 for The loss of Xist promotes the differentiation of CD11c+ B cells, antibody secretion in early protection against pathogens suggesting that Xist-mediated XCI maintenance may contribute to [127, 128], the epigenetic modification of Prdm1 may be important the generation of CD11c+ atypical memory B cells during SLE for sustaining the normal functions of B1 cells. CD5 is an important development [13]. marker of B1a cells. It has been reported that CD5 expression in B cells from SLE patients can be regulated by IL-6-induced DNA Regulatory B cells. Breg cells represent a special B-cell subpopu- methylation, indicating a potential role of cytokines in regulating lation with potent immunosuppressive functions. Bregs maintain B-cell phenotypes [129]. immune tolerance by suppressing various inflammatory popula- tions, including Th17 cells, Th1 cells, CD8 T cells, monocytes, and dendritic cells (DCs) [108]. Bregs produce many suppressive EPIGENETIC DYSREGULATION OF B CELLS IN AUTOIMMUNE cytokines, such as IL-10, TGF-β, and IL-35 [108]. Bregs are DISEASES heterogeneous and have various phenotypes, including During the past decades, extensive evidence from clinical CD5+CD1dhi B cells, Tim-1+ B cells, and LAG-3+CD138hi plasma investigations and animal studies has demonstrated the signifi- cells [108, 109]. Currently, the developmental origin of Bregs is cance of B-cell hyperactivation during the development of unclear. Immature B cells, MZ B cells, and plasma cells all have the autoimmune diseases. B-cell activation and differentiation are potential to differentiate into IL-10-producing B cells in the critically regulated by epigenetic modifications (Fig. 2E). Impor- presence of BAFF, lipopolysaccharide (LPS), CD40L, IL-21 or other tantly, successful applications of B-cell-targeted therapies in some stimuli [108, 110, 111], suggesting that environmental factors and autoimmune diseases have highlighted the pathological signifi- not lineage specificity determine the generation of Bregs. cance of B cells in autoimmune pathogenesis. Despite recent Numerous studies have demonstrated an important role of Bregs studies revealing a pivotal role of B cells in autoimmunity, the in infection, inflammation, cancer, and autoimmunity triggers and subsequent consequences of B-cell tolerance break- [44, 110, 112–115]. Adoptive transfer of Bregs suppresses T-cell down have not been fully elucidated. responses and ameliorates disease development in mice with experimental Sjögren’s syndrome, collagen-induced arthritis (CIA), Dysregulated B-cell signaling in autoimmune diseases and SLE, suggesting therapeutic potential of Bregs in autoimmune Hyperactivation of B cells with massive production of autoanti- diseases [44, 116, 117]. bodies and cytokines are hallmark features of many autoimmune Although it is unclear whether Breg identity is determined by a diseases, including SLE, pSS, and RA. Accumulating evidence specific transcription factor, increasing evidence shows a pivotal suggests that dysregulated B-cell signaling drives autoimmune role of epigenetic regulation in Bregs. IL-10 is a key effector development by promoting the activation and differentiation of cytokine for the suppressive functions of Bregs. DNA methylation autoreactive B-cell clones [8]. It has been well recognized that the profiling has shown that IL-10-producing B cells are characterized microenvironment is important for B-cell differentiation and by a specific methylation signature around the IL10 TSS, activation in the bone marrow and peripheral lymphoid organs. suggesting an important role of DNA methylation in regulating Intrinsic BCR signals, cytokines, and TLR ligands shape B-cell IL-10 production by B cells [118]. Moreover, histone modifications phenotypes and drive autoimmune GC reactions. Consequently, are also involved in the induction of IL-10-producing B cells. the breakdown of immune tolerance leads to the development of Inhibition of HDAC11 increases IL-10 production by B cells in systemic autoimmunity [8]. patients with allergic rhinitis [119]. Entinostat, an HDAC inhibitor, inhibits the binding of HDAC1 to the promoter region of Il10 and BCR signaling. During B-cell development, BCR signaling controls promotes the induction of IL-10-producing B cells by LPS [120]. the negative and positive selection of immature B cells in the Another HDAC inhibitor, trichostatin A, increases the frequency of bone marrow, which largely shape the BCR repertoire. A sustained IL‑10- and TGF‑β‑producing CD5+CD1dhigh B cells in vitro and BCR signal is essential for the survival of both mature and in vivo [121], suggesting that HDAC inhibitors might be potential immature B cells, whereas strong BCR signaling may also promote Cellular & Molecular Immunology (2022) 19:1215 – 1234 F. Xiao et al. 1221 B-cell apoptosis. Thus, an intermediate BCR intensity is optimal for development of personalized medicine for the treatment of sustaining B-cell survival. In general, B cells with BCRs that autoimmune diseases. recognize self-antigens are removed during negative selection. However, genetic polymorphisms associated with BCR signaling CD40. During an adaptive immune response, cognate T cells may affect the generation of autoreactive B cells. A single- help sustain B-cell survival and promote B-cell differentiation. nucleotide polymorphism (SNP) in protein tyrosine phosphatase CD40 is a TNF receptor superfamily member widely expressed in nonreceptor type 22 (PTPN22) (W620) that negatively regulates various immune and nonimmune cell populations. The interaction BCR downstream signaling is associated with increased risks of between CD40 expressed on B cells and its binding ligand CD40L several autoimmune diseases, including SLE, RA, and type 1 (CD154) on CD4 T cells plays an important role during the GC diabetes (T1D), suggesting a fundamental role for BCR signaling in reaction and B-cell differentiation into plasma cells. Many studies the development of autoimmunity [130–132]. PTPN22 has broad have reported aberrant CD40 signaling in patients with auto- effects on both T-cell and B-cell selection and function. Although it immune diseases, which supports the notion that the CD40-CD40L is not clear whether the SNP is a gain-of-function or loss-of- interaction contributes to B-cell hyperactivation and the main- function variant, studies of animal models suggest a gain-of- tenance of autoimmunity [142]. Blockade of CD40L has been function for the PTPN22 variant in the development of auto- shown to inhibit autoantibody production and tissue inflamma- immune diseases [8, 133]. Nonobese diabetic (NOD) mice with tion in mice with autoimmune thyroid diseases and NOD mice Ptpn22 deficiency show reduced numbers of plasma cells, and B- with SS-like phenotypes, suggesting a critical role of the CD40- cell-specific ablation of Ptpn22 decreases the incidence of diabetes CD40L interaction in these diseases [143]. Many biological agents in NOD mice, suggesting that Ptpn22 variation may contribute targeting CD40 signaling have been evaluated in clinical trials for to T1D by modifying B-cell maturation [8, 133]. Moreover, PTPN22 their therapeutic efficacy in patients with autoimmune diseases (W620) affects the expression of various surface receptors in B and have achieved partially satisfactory outcomes [142]. cells and may modulate B-cell tolerance via diverse mechanisms [8, 132]. The PTPN22 (W620) risk allele shows a dominant effect in TLRs. Apart from T-cell-dependent stimulation, B cells can also be regulating autoreactive B cells even before the onset of activated independent of T cells in response to pathogen- autoimmunity, which might be associated with altered BCR associated molecular patterns (PAMPs) and damage-associated signaling and upregulation of genes that promote B-cell molecular patterns (DAMPs) by TLRs. Bacterial and viral-derived responses such as CD40, TNF receptor-associated factor 1 (TRAF1), PAMPs such as LPS, RNA, and DNA can be recognized by surface and IRF5 [134]. and cytosolic TLRs, which directly activate B cells even without Bruton’s tyrosine kinase (BTK) is a well-known downstream BCR engagement. Moreover, B cells can also be activated by self- molecule of BCR signaling. BTK is essential for B-cell survival, derived DAMPs, including double-stranded DNA (dsDNA), which proliferation, and function. Spontaneous mutations that lead to may drive the development of autoimmunity [144]. During B-cell insufficient BTK function usually cause a dramatic loss of mature B development, combined BCR and TLR signals orchestrate the cells and reduced levels of serum antibodies, resulting in X-linked selection of self-reactive B cells in peripheral organs [8]. Myeloid agammaglobulinemia [135]. Many studies have shown that BTK differentiation factor 88 (MyD88) and interleukin-1 receptor- inhibitors effectively ameliorate disease pathology in various associated kinase 4 (IRAK-4) are important adaptors of most TLR animal models of autoimmune diseases [136, 137]. Administration signaling pathways. Patients with a deficiency of MyD88 or IRAK-4 of evobrutinib, a novel BTK inhibitor, shows robust efficacy in exhibit defective central and peripheral B-cell tolerance check- mouse models of RA and SLE, as reflected by significant points and accumulation of autoreactive naive B cells in blood reductions in disease severity and histological damage [137]. [145]. In patients with Wiskott–Aldrich syndrome, an X-linked The activated B-cell subtype of diffuse large B-cell lymphoma immunodeficiency disease frequently associated with systemic requires chronic active BCR signaling for survival and is resistant to autoimmunity, modest alterations in BCR and TLR signaling can the BTK inhibitor ibrutinib [138]. This resistance is due to disrupt B-cell tolerance by regulating the positive selection of self- epigenetic rather than genetic changes that circumvent BTK reactive B cells [146]. blockade, suggesting the potential roles of epigenetic regulation Although human B cells express low levels of TLR4, our previous in BCR-driven B-cell hyperactivation [138]. Several clinical trials study identified a novel TLR4+CXCR4+ plasma cell subset that have shown promising results of BTK inhibitors for treating played a pathogenic role in the pathogenesis of SLE [147]. patients with various autoimmune diseases, such as RA and Blockade of TLR4 significantly decreased serum autoantibody multiple sclerosis (MS) [136]. levels and attenuated renal damage in lupus mice, suggesting that The diversity of the BCR repertoire is an important factor that TLR4 might be a potential target for prohibiting plasma cell modulates the development of autoimmune diseases. Single-cell responses and treating SLE [147]. In addition to classic RNA sequencing (scRNA-seq) and high-throughput sequencing Myd88 signaling, TLR4 signals can also be transduced by BCR technologies have made significant contributions to the systemic and its key adaptor spleen tyrosine kinase in B cells [148]. Genetic analysis of the BCR repertoire. Compared with those from healthy association studies have identified TLR polymorphisms as donors, B cells from SLE patients show increased BCR clonotypes important risk factors for autoimmune diseases, including SLE. In and biased usage of BCR V(D)J genes [139]. In particular, B cells particular, increased expression of TLR7, which recognizes single- from SLE patients who are sensitive to immunosuppressive drugs stranded RNA, is suggested to be a risk factor for SLE [149]. show significantly decreased BCR expression and clonal diversi- Interestingly, TLR7 usually escapes interaction with XCI, which may fication, while these changes are undetectable in nonsensitive lead to gender bias in SLE patients [150]. Biallelic B cells with lupus patients, indicating that alterations of the BCR repertoire are higher TLR7 expression exhibit a significantly higher propensity to associated with sensitivity to immunosuppressive therapy [140]. It switch to the IgG class than monoallelic B cells during plasma cell has also been reported that the nonresponse rates to rituximab differentiation due to the elevated responsiveness to TLR7 ligands among RA patients are closely associated with marked disruption [150]. Evidence from mice with overexpression or deficiency of of the BCR repertoire, suggesting that BCR clonality may serve as a TLR7 further demonstrates a critical involvement of TLR7 signaling predictor of the responses of RA patients to B-cell-depletion in autoimmunity [8, 149, 151]. Lupus-prone mice with B-cell- therapy [141]. Although the mechanisms underlying the establish- specific Tlr7 deletion show inhibited B-cell responses, decreased ment and changes of the BCR repertoire in autoimmunity are still levels of class-switched autoantibodies against RNA-associated not fully understood, further studies will shed light on the autoantigens, and diminished systemic autoimmunity [152]. generation of autoreactive B cells, which will facilitate the TLR7 signaling in B cells is most prominently associated with GC Cellular & Molecular Immunology (2022) 19:1215 – 1234 F. Xiao et al. 1222 reactions, leading to systemic autoimmunity [146]. In contrast, produced by myeloid cells such as neutrophils, DCs, and TLR9, which interacts with DNA containing unmethylated monocytes. Moreover, Tfh cells within GC regions might be an cytosine-phosphate-guanosine motifs, plays a protective role in important source of local BAFF that promotes the selection of many autoimmune diseases. SLE patients show impaired TLR9 but high-affinity B-cell clones, which may contribute to autoantibody intact TLR7 responses in B cells [153]. B-cell-intrinsic Tlr9 deficiency production and systemic autoimmunity [8, 164]. Many biological results in increased systemic inflammation and immune complex- agents that target BAFF have been developed, and clinical trials related glomerulonephritis but decreased anti-nucleosome anti- suggest promising therapeutic effects in patients with autoim- body levels in lupus mice, suggesting that TLR9 regulates lupus mune diseases such as SLE [156]. Further investigations of development independent of autoantibodies [152, 154]. Defi- strategies targeting different forms of BAFF might be important ciency of Tlr9 in other immune populations, including DCs, for the personalized treatment of autoimmune patients. plasmacytoid DCs, and neutrophils, has undetectable effects on disease manifestations [154]. Overexpression of TLR9 in B cells Epigenetic regulation of B-cell hyperactivation. Recent studies ameliorates nephritis in lupus mice, further indicating a protective have revealed the significant roles of epigenetic regulation during role of B-cell-intrinsic TLR9 signaling in autoimmunity [154]. B-cell hyperactivation, which may contribute to immune tolerance However, B-cell-specific deletion of TLR9 also protects mice from breakdown and the development of autoimmunity. B-cell T1D development. The protective effects might be associated with hyperactivation is closely associated with multiple signaling increased production of IL-10 by B cells [155]. The functional molecules, including BCR, CD40, and TLRs. During GC reactions interaction between TLR7 and TLR9 within B cells is important for and plasma cell differentiation, SHM and CSR largely shape the B-cell dysregulation in autoimmunity. Unlike TLR4, which is BCR repertoire, which is tightly regulated by many epigenetic located on the plasma membrane, TLR7 and TLR9 can only events. AID, an important regulator of SHM, increases BCR recognize internalized ligands in endosomes. Similar pathways affinities by diversifying the variable regions of immunoglobulin participate in the internalization of ligands for TLR7 and TLR9, as genes. The transcription and expression of AICDA are regulated by both processes require BCR-mediated endocytosis and the histone modifications, DNA methylation, and various miRNAs formation of endosomes. The antagonistic interaction between (Fig. 2) [63, 65, 66, 71, 72], which directly modulate BCR clonotypes TLR7 and TLR9 in B cells might be partially explained by their during autoimmune disease progression. Epigenetic mechanisms trafficking to endosomes [149]. Further investigations are needed are involved in regulating CD40 expression, as revealed by the to elucidate the differential roles of TLRs in B-cell dysregulation finding that HDAC inhibitors can alter the acetylation of histones and the development of autoimmunity. in chromatin and enhance CD40 expression [165]. Moreover, miRNAs such as miR146a target multiple genes associated with BAFF. As an important survival factor for B cells, BAFF is the CD40 pathway and regulate B-cell activation and GC reactions. considered an important player in systemic autoimmune diseases Although miR146a does not directly target the CD40 3’UTR, it can [156]. BAFF binds to three distinct receptors, namely, BAFFR, bind to mRNAs of IKKA, REL, and TRAF6, all of which are important transmembrane activator and CAML interactor, and B-cell components of the CD40 signaling pathway [72]. MiR146a also maturation antigen. Soluble BAFF exists as a 3-mer or in multimers indirectly controls CD40 expression by targeting STAT1, which is of up to 60, and its different forms may have different binding indispensable for interferon γ-driven CD40 induction [72]. abilities to the three receptors [156]. BAFF signaling together with Epigenetic regulation of TLRs is observed in diverse conditions. BCR pathways orchestrates B-cell development. The interplay The lncRNA Xist maintains XCI in normal B cells, whereas Xist between BAFF receptors and BCR promotes B-cell survival and dysregulation leads to the escape of X-linked genes such as TLR7 modulates B-cell selection [8]. BAFFR is widely expressed in [150]. Earlier studies suggested that DNA methylation in the various B-cell subsets. Although BAFF is essential for maintaining human TLR2 promoter region was associated with suppressed naive B-cell survival, B-cell-intrinsic BAFFR is dispensable for the TLR2 expression in monocytes [166]. Gene transcription of TLR4 is survival and function of GC B cells but controls GC-independent also downregulated by histone deacetylation and DNA methyla- memory B-cell responses in early immune defense [157]. Memory tion [166]. However, the exact epigenetic regulation mechanisms B cells express BAFFR and require both BCR and BAFFR signaling affecting TLR signaling pathways during B-cell hyperactivation for their long-term survival [158]. Although BAFF is mostly known need further investigation. As an important epigenetic regulator, as a key B-cell survival factor, it exerts diverse functions in the miRNAs can regulate TLR signaling pathways by targeting TLRs B-cell response. BAFF promotes IL-10 and IL-35 production by MZ and associated adaptor proteins. Many miRNAs, such as miR-155, B cells with increased suppressive abilities [156, 159]. BAFF miR-146, and miR-21, are induced by TLR activation and in turn signaling also increases metabolic capacity and regulates redox regulate the expression of TLR pathway components and TLR- balance in B cells. BAFF promotes glycolysis and mitochondrial induced cytokines [167]. LPS stimulation activates TLR4 signaling oxidative phosphorylation through the PI3K/Akt pathway, which and induces the expression of miR-155 in B cells, which is crucial provides necessary molecular building blocks and energy that for GC responses and the production of cytokines by B cells support cell mass generation [160]. [167–169]. The activation and differentiation of B cells also A variant of TNFSF13B, which encodes BAFF, is associated with require negative regulators. A recent study observed high MS and SLE because it increases soluble BAFF levels and enhances expression of Jmjd1c, a member of the JmjC domain-containing humoral immunity [161]. Indeed, increased levels of BAFF are histone demethylase family, in B cells but not in other observed in many patients with autoimmune diseases, including immune cells, which protected mice from autoimmune arthritis SLE, pSS, and RA [156]. Although it is still controversial, several development [170]. MiR146a also serves as a negative feedback cross-sectional studies suggest that BAFF serum levels are regulator for B-cell hyperactivation [72]. Collectively, the available positively correlated with disease severity in SLE patients [162]. evidence shows critical epigenetic regulation during B-cell BAFF transgenic mice show several hallmark features of SLE and hyperactivation. SS, including the presence of autoantibodies, immune complex- mediated glomerulonephritis, and salivary gland dysfunction and Epigenetic dysregulation of B cells contributes to autoimmune inflammation [8, 163]. The mechanisms by which BAFF promotes disease development autoimmunity are not yet fully understood. Current evidence Altered B-cell signaling and hyperactivated humoral responses are suggests that excess BAFF levels contribute to the selection of key drivers of human autoimmunity. Emerging evidence has autoreactive B cells and promote the long-term survival of revealed that epigenetic regulation plays significant roles in B-cell autoantibody-secreting plasma cells [8, 156]. BAFF is mainly responses, contributing to the initiation and perpetuation of Cellular & Molecular Immunology (2022) 19:1215 – 1234 F. Xiao et al. 1223 Fig. 3 Epigenetic dysregulation of atypical memory B cells contributes to autoimmune diseases. ① Atypical memory B cells from patients with autoimmune diseases, including SLE, show high transcriptional accessibility to genes associated with B-cell differentiation and activation, such as TBX21, IRF9, JUN, ATF3, and EGR family genes. ② The lncRNA Xist maintains X-chromosome inactivation (XCI) through continuous deacetylation of H3K27ac at promoters of target genes in the X-chromosome. During autoimmune disease progression, the loss and mislocalization of Xist lead to H3K27 acetylation and escape of X-linked genes such as TLR7 from XCI in atypical memory B cells autoimmune diseases. Epigenetic modifications in B cells regulate characterized by the abundant expression of CD11c and T-bet. the selection, activation, and differentiation of autoreactive B cells. Epigenetic dysregulation of these cells promotes the development of various autoimmune diseases, including SLE (Fig. 3). Atypical memory Systemic lupus erythematosus. SLE is a systemic autoimmune B cells show more accessible motifs for the T-bet, ISGF3, AP-1, and disease characterized by immune dysregulation, including B-cell early growth response (EGR) transcription factor families than other hyperactivation and prominent autoantibody production. Recent B-cell subsets [12]. Interestingly, atypical memory B cells also show studies have identified promoter and enhancer site-specific enriched accessible chromatin at T-bet and AP-1 motifs in SWEF- hypomethylation of the CD40L gene in CD4+ T cells with disease family-deficient mice that develop systemic autoimmunity [176]. SLE activity in SLE patients [171, 172]. Evidence has also shown DN2 B cells show activation of activating transcription factor 3 (ATF3) epigenetic dysregulation of B cells in patients with SLE and animal response pathways that are induced by BCR and TLR stimulation. The models with SLE-like phenotypes. Further studies have revealed ATF3 and EGR families may act in synergy with T-bet to shape the the molecular mechanisms and functional significance of B-cell epigenome of expanded SLE DN2 B cells [12]. epigenetic regulation during SLE pathogenesis. The lncRNA Xist sustains XCI during the early development of Genome-wide DNA methylation analysis of SLE twin cohorts has female cells. It was recently found that B cells from SLE patients shown hypermethylated CpG islands in B cells and identified TNF showed abnormal XCI with escape of many X-linked genes and Xist and EP300 as the most important upstream regulators [173]. RNA interactome genes [13, 107]. Xist is required to suppress the Moreover, B cells and other immune populations show marked expression of various X-linked immune genes, such as TLR7, that play hypomethylation of interferon-regulated genes, including IFI44L, a significant role in human SLE [13, 177]. The escape of TLR7 from XCI PARP9, and IFITM1, which might be associated with disease flares increases TLR7 gene products and enhances the B-cell response to [173]. Scharer et al. studied the transcriptomic and epigenetic TLR7 engagement, which promotes autoantibody production and programs of circulating B-cell subsets in an African–American may drive SLE progression [150]. In particular, scRNA-seq analysis cohort with high disease activity. Although the core features of showed Xist dysregulation in CD11c+ atypical memory B cells from B-cell development are similar, the resting naive B cells from SLE female SLE patients [13]. Moreover, TLR7 activation and XIST patients are epigenetically distinct from those of healthy controls inactivation promote the generation of isotype-switched CD11c+ [12]. Moreover, naive SLE B cells exhibit a unique chromatin atypical memory B cells [13]. Mechanistically, TRIM28 was identified as architecture. The SLE-specific chromatin accessibility signatures a B-cell-specific Xist cofactor [13]. Xist-dependent type I interferon include patterns related to areas surrounding genes that encode and extrafollicular B-cell pathways are also defective in some SLE transcription factors involved in B-cell activation and differentia- patients [178]. Mislocalized Xist RNA predisposes SLE B cells to tion, such as NF-κB, activator protein-1 (AP-1), BATF, IRF4, and aberrant X-linked gene expression from the inactivated chromosome Blimp1 [174]. Epigenetic alterations were observed in immature B and likely contributes to the female bias in SLE [107]. cells emerging from bone marrow in African–American SLE TET enzymes, which are involved in active DNA demethylation, patients, whereas defects developed at a late stage of B-cell have pleiotropic roles in B-cell homeostasis [68, 69]. A recent study development in European American patients, suggesting that SLE- suggests that TET-mediated chromatin modification regulates auto- specific DNA methylation signatures might be ethnicity depen- reactive B cells and prevents autoimmunity. Deficiency of Tet2 and dent [175]. Collectively, these data show global epigenetic Tet3 within B cells causes spontaneous B-cell activation and SLE-like changes in SLE B cells, which may contribute to B-cell disease characteristics, including increased levels of autoantibodies, hyperactivation and SLE pathogenesis. enhanced proteinuria, and kidney tissue damage [179]. Moreover, Epigenetic changes in functional B-cell subsets are recognized as Tet2 and Tet3 suppress Cd86 gene activity dependent on HDAC important factors in SLE development. Although many B-cell subsets binding and DNA demethylation at the intron region of Cd86 [179]. show epigenetic alterations, the CpG methylation status identifies The available evidence suggests that TET proteins participate in the the differentiation hierarchies. DN2 (CD27–CD11c+T-BET+CXCR5–) breakdown of B-cell tolerance. Indeed, TET3 was determined to be a B cells and atypical memory B cells show many similarities and are potential SLE susceptibility risk gene by GWAS [6]. Cellular & Molecular Immunology (2022) 19:1215 – 1234 F. Xiao et al. 1224 Histone modifications participate in regulating B-cell activation and expression levels of miRNA-155 are increased in B cells from RA differentiation. Inhibition of HDACs shows therapeutic benefits patients compared with the levels in B cells from healthy associated with suppressed B-cell responses in various lupus models. individuals. In particular, miRNA-155 is highly expressed in Selective inhibition of HDAC6 by the small molecule ACY-738 IgD-CD27- memory B cells from ACPA-positive RA patients. In decreased the occurrence of glomerulonephritis and the number of synovial tissues, miRNA-155 expression is negatively associated plasma cells in New Zealand black × New Zealand white (NZB/W) F1 with PU.1 expression in B cells. Inhibition of endogenous miRNA- mice [180]. The HDAC inhibitor panobinostat significantly reduced 155 in B cells restores PU.1 expression and decreases antibody the number of autoreactive plasma cells and ameliorated nephritis in production, suggesting that miRNA-155 modulates B-cell func- MRL/lpr mice, whereas other immune populations were largely tions by suppressing PU.1 in RA patients [189]. unaffected [181]. SCFAs exhibit HDAC inhibitor activities. Oral feeding of SCFAs inhibited plasma cell differentiation, suppressed B-cell class Primary Sjögren’s syndrome. As a chronic autoimmune disorder, switching, and alleviated lupus skin lesions and kidney pathology in pSS is characterized by inflammation and tissue destruction in both MRL/lpr and NZB/W F1 mice [70]. In contrast, the nonclassical salivary glands and lacrimal glands, which cause dry mouth and class III HDAC Sirt1 acts as a suppressive epigenetic regulator of Aicda dry eyes. Although many immune cell populations and cytokines and thus regulates SHM during B-cell activation [66]. Sirt1 plays an are involved in pSS development, clinical observations support a intrinsic role in modulating autoantibody production during SLE critical role of B cells in pSS pathogenesis since patients have development. The expression of Aicda is negatively correlated with diverse autoantibodies and increased risks of developing B-cell Sirt1 levels in B cells isolated from SLE patients and lupus-prone MRL/ lymphoma [7, 190–192]. Autoantibodies specific for pSS may even lpr mice. Sirt1 deletion in activated B cells leads to massive production develop long before symptoms emerge, suggesting a key role of B of class-switched autoantibodies, including anti-dsDNA, anti-histone, cells in disease initiation and perpetuation. GWASs have revealed and anti-ribonucleoprotein antibodies, in normal female C57BL/6 risk genes associated with B-cell activation, such as BLK, CXCR5, mice [66]. Tightly regulated B-cell activation by HDACs sustains B-cell and PRDM1 [7]. An epigenetic analysis showed that DNA homeostasis. Breakdown of epigenetic balance may lead to methylation changes are mainly present in B cells but not in dysregulated B-cell responses and contribute to autoimmunity in T cells. Moreover, genes with differentially methylated probes SLE pathogenesis. overlapped with the risk genes identified by GWAS, including BLK and CXCR5, suggesting genetic and epigenetic dysregulation of Rheumatoid arthritis. RA is a common chronic autoimmune these key genes during pSS development [193]. Interferon- disease characterized by immune dysregulation and progressive regulated genes are hypomethylated in B cells and are associated joint damage. B cells are one of the most important players in RA with increased B-cell numbers [193, 194]. Methylation changes in development. Massive infiltration of B cells and plasma cells is these genes in B cells are positively correlated with disease observed in the synovial tissues of RA patients. Importantly, more activity, further emphasizing the central role of B cells and the than 80% of RA patients are seropositive for autoantibodies, relevance of DNA methylation changes in pSS pathogenesis including rheumatoid factor and anti-citrullinated protein anti- [193, 194]. bodies (ACPAs), which further highlights the significant roles of B A genome-wide case‒control study revealed many differentially cells in RA [182]. methylated areas in inflamed minor salivary gland biopsies from Epigenetic changes in RA patients have been studied in various pSS patients. Although this study used a mixture of epithelial cells immune and nonimmune populations. Aberrant DNA methylation and infiltrating immune cells, the enrichment analysis showed and histone modification patterns and miRNA expression levels dysregulated epigenetic control of genes associated with B-cell are considered important contributors to RA pathogenesis [183]. survival and functions, such as CXCR5 and TNFRSF13B [195]. An epigenome-wide association study (EWAS) of RA patients with B cells from pSS patients and healthy controls show major three different replication cohorts identified several important differences in miRNA expression patterns. Several differentially disease-specific alterations of DNA methylation in B cells [184]. expressed miRNAs, including hsa-mir-30b-5p, have been identified DNA hypermethylation at CD1C and hypomethylation at TNFSF10 in B cells. Inhibiting hsa-mir-30b-5p increases BAFF expression, genes are associated with elevated RA risk. Casitas B-lineage suggesting a functional role of the miRNA in B-cell responses lymphoma (CBL) proteins interact with BCR downstream signaling [196]. A transcriptomic analysis showed increased expression of factors and control the B-cell-intrinsic checkpoint of immune the lncRNA LINC00487 in various B-cell subsets from pSS patients, tolerance [185]. B cells from RA patients show differential which was correlated with disease activity scores [197]. These methylation at the genes involved in CBL pathways, which may studies show dysregulated epigenetic control of B cells in pSS have functional significance in RA pathogenesis. These epigenetic patients, highlighting an important role of B cells during pSS alterations are also detected in SLE patients, suggesting that development. patients with these two conditions have shared methylation alterations [184]. There is a potential impact of therapeutic treatments on epigenetic factors, but genome-wide profiling Multiple sclerosis. MS is an autoinflammatory and neurogenera- revealed a novel methylation signature related to 113 sites within tive disease that leads to demyelination of neurons and axonal B cells in treatment-naive early RA patients [186]. Both early and loss in the central nervous system (CNS). Although MS has been established RA patients show similar DNA methylation patterns in traditionally considered a T-cell-dominated autoimmune disease, B cells [186]. recent studies in the past decade have revealed the critical Small RNA sequencing of B cells revealed 27 miRNAs that are involvement of B cells during MS pathogenesis [198]. Abnormal differentially expressed in MTX-treated RA patients compared with cytokine profiles in B cells have been observed in MS patients. healthy controls. However, no significant differences were These B cells produce an excessive amount of proinflammatory observed between newly diagnosed RA patients and healthy cytokines, including TNF, IL-6, and GM-CSF [198]. B cells may donors [187]. The differentially expressed miRNA target genes directly target oligodendrocytes and neurons by producing involved in B-cell activation, differentiation, and BCR signaling cytotoxic mediators. Furthermore, the interplay between B and included STAT3, PRDM1, and PTEN. miRNA-155 is a critical T cells may have a pivotal role since it has been shown that B cells regulator of GC and plasma cell responses [85]. Mice with miR- drive the proliferation and migration of brain-homing, autoreac- 155 deficiency are resistant to CIA, with significantly suppressed tive CD4 T cells in MS [199]. Clinical trials show the therapeutic antigen-specific Th17-cell and autoantibody responses and benefits of B-cell depletion therapies in many MS patients, markedly reduced joint inflammation [188]. Moreover, the supporting the role of B cells in MS pathogenesis [200]. Cellular & Molecular Immunology (2022) 19:1215 – 1234 F. Xiao et al. 1225 Compared with monocytes and T cells, B cells display the most presentation by B cells [211]. In a larger EWAS, differentially significant differences in methylated areas, showing a global DNA variable CpG positions were detected across 406,365 CpGs in CD4 hypomethylation signature [201, 202]. In a cohort involving 24 T cells, B cells, and monocytes in 52 monozygotic twin pairs patients with relapsing-remitting MS and 24 healthy controls, EWAS discordant for T1D [212]. Twins with T1D showed substantial analysis of DNA methylation also revealed extensively altered enrichment of differentially variable CpG positions compared with methylation patterns in B cells of MS patients [203]. The genes with their healthy twin and unrelated healthy individuals [212]. differentially methylated areas were found to be involved in innate Evidence from studies of cord blood of newborns who developed immunity and BCR signaling. Notably, hypermethylated areas were T1D suggests that these CpG modifications are likely to emerge identified around the transcriptional start site of the LTA gene, after birth [212]. An analysis of cell type-specific gene regulatory which encodes the proinflammatory cytokine lymphotoxin alpha circuits highlighted immunometabolism and mTOR signaling as secreted by B cells and other immune cells [203]. These epigenetic enriched pathways in B cells [212]. T1D-associated genetic and features highlight specific epigenetic programs involved in B-cell epigenetic variants seem to act independently since the differentiation, activation, and function. differentially variable CpG positions are not enriched in genetic susceptibility loci [212]. Systemic sclerosis. Systemic sclerosis (SSc) is a severe autoim- mune disease characterized by vascular damage and immune dysregulation that leads to fibrosis of the skin and internal organs. EPIGENETIC BIOMARKERS AND THERAPIES FOR AUTOIMMUNE Although genetic susceptibility is important in SSc, epigenetic DISEASES modifications constitute the driving force for disease initiation Epigenetic biomarkers for autoimmune diseases [204]. Environmental factors such as exposure to silica or viruses Current diagnostic criteria for autoimmune diseases have been can trigger epigenetic modifications and are linked to genetic developed largely based on clinical manifestations and laboratory susceptibility [204]. Genetic and epigenetic factors ultimately tests, and there is a lack of biomarkers with high sensitivity and cause a series of cellular processes that lead to tissue fibrosis. specificity [213]. The shared clinical parameters among different Altered DNA methylation and histone modification profiles and autoimmune diseases and heterogeneity of the patients highlight dysregulated levels of noncoding RNAs together contribute to the an essential need for novel biomarkers that can reflect disease development of SSc. Although it has been clear that genetic characteristics and predict therapy responses. Epigenetic changes predisposition can only partially explain SSc occurrence, cell- are considered one of the earliest factors associated with disease specific epigenetic regulation during SSc is not fully understood. initiation before clinical manifestations. There has been rapid B cells, T cells, monocytes, and fibroblasts show altered histone development of molecular biology techniques, and increasing modification signatures in SSc patients compared with healthy evidence derived from such techniques shows that abnormal controls, suggesting a role of epigenetic events in immune epigenetic events may serve as promising biomarkers for dysregulation and fibrogenesis. Activated B cells can affect tissue autoimmune diseases. fibrosis through diverse effector mechanisms in SSc [205]. Notably, Genome-wide DNA methylation studies have already revealed B-cell depletion inhibits tissue fibrosis by suppressing profibrotic differentially methylated sites in patients with autoimmune macrophage differentiation in a mouse model of SSc, further diseases, including SLE, RA, and SSc [14]. Integrative analysis of supporting an important role of B cells in the pathogenesis of SSc a multiple autoimmune disease methylation dataset revealed that [206]. In SSc patients, global histone H4 hyperacetylation and hypomethylation of IFN-related genes is a common feature of RA, histone H3K9 hypomethylation are detected in B cells. Moreover, SLE, and SSc, suggesting that the DNA methylation profile of IFN- H4 acetylation levels are negatively correlated with HDAC2 related genes could be a biomarker for the diagnosis of expression but positively correlated with disease activity, suggest- autoimmune diseases [14]. In SLE patients, altered epigenetic ing a direct association between B-cell epigenetic alterations and modifications support the dysfunction of various B-cell subsets, clinical disease severity in SSc patients [205]. In addition to B cells, such as DN2 B cells [12]. Importantly, DNA methylation profiling monocytes have altered histone modifications correlated with an studies have identified distinct molecular signatures of differen- enhanced IFN signature in SSc patients [207]. Interestingly, a tially methylated loci that could stratify healthy controls and SLE genomic methylation study of whole blood from SSc discordant patients [12]. These signature loci include hypomethylated sites twin pairs showed shared methylated sites between SSc and SLE near interferon-induced genes such as IFI44 and IFITM1 and patients across various immune populations, suggesting that hypermethylated CpGs surrounding SOX12, ARFGAP3, CCDC81, these epigenetic features may be common biomarkers for and MEG3. The DNA methylation patterns in B cells from healthy autoimmune diseases [208]. controls and SLE patients show clear differences, suggesting that the identified differentially methylated loci could be used as SLE Type 1 diabetes. T1D is one of the most common autoimmune biomarkers [12]. Since altered DNA methylation modifications are diseases and is characterized by the destruction of insulin- detected in diverse B-cell subsets, the associations between producing β cells in the pancreas. Although previous studies epigenetic modifications and disease status deserve further showed a dominant role of T cells in pancreatic tissue destruction, investigation. Synovial and blood monocytes of undifferentiated accumulated evidence suggests that T1D is associated with the arthritis patients show marked alterations in methylation profiles tolerance breakdown of B cells [209]. These autoreactive B cells compared with those from healthy donors [15]. DNA methylation- contribute to T1D development by producing autoantibodies and based biomarkers are closely associated with prognosis, disease presenting autoantigens to T cells. Importantly, recent studies activity, and treatment efficacy in patients with early inflammatory suggest that the generation of islet-reactive B cells is associated arthritis, suggesting that the DNA methylation signatures of many with certain genetic polymorphisms and aberrant epigenetic immune subsets could serve as important biomarkers for events [209, 210]. personalized clinical management of autoimmune disease Epigenetic studies of monozygotic twins discordant for T1D patients [15, 16]. have been performed to investigate the effects of nongenetic It has been well characterized that type I IFN pathways are factors during disease development. A genome-wide DNA activated and involved in many autoimmune diseases, including methylation study identified 88 CpG sites displaying significant SLE. Increased type I IFN levels are observed in and associated methylation changes in B cells from 3 pairs of T1D-discordant with nephritis, mucocutaneous manifestations, and the presence monozygotic twins [211]. Functional annotation suggested that of autoantibodies in SLE patients [214]. The production of type I these epigenetic changes may be associated with antigen IFNs is triggered by the activation of nucleic acid-binding pattern Cellular & Molecular Immunology (2022) 19:1215 – 1234 F. Xiao et al. 1226 recognition receptors, including TLR7, cyclic GMP-AMP synthase, expression is associated with disease activity, clinical manifesta- and RIG-I-like receptors. Several genetic and epigenetic studies tions, and laboratory parameters of SLE, suggesting that Xist have shown a clinical association of aberrant IFN-stimulated gene might be a biomarker for SLE [219–221]. expression with SLE development [214]. Notably, SLE patients show significant hypomethylation of two CpG sites within the Targets for epigenetic therapy promoter region of IFI44L, a typical type I interferon-stimulated EZH2. EZH2 is a histone methyltransferase subunit of the gene, when compared with healthy controls, RA patients, and pSS polycomb repressive complex that mediates trimethylation of patients [215]. In a paired analysis of twins discordant for SLE, B Lys27 in histone H3. EZH2 plays an important role in regulating cells, T cells, and monocytes in the diseased twin all exhibited B-cell differentiation and metabolism during the GC reaction and significant hypomethylation of interferon-stimulated genes, plasma cell formation [93, 94]. EZH2 also controls T-cell functions including IFI44L, which was even more pronounced in twins in cooperation with transcription factors and has been proposed who experienced a disease flare within the past 2 years [173]. In as a novel therapeutic target for treating SLE patients [222]. addition, the methylation levels around the IFI44L promoter are In SLE patients, the expression of EZH2 is elevated and negatively associated with renal damage. The DNA methylation positively correlated with the overexpression of IFN-stimulated status at the two IFI44L promoter sites has higher sensitivity and genes in PBMCs and renal tissues of SLE patients [223]. specificity than most available tests, suggesting that the methyla- Notably, increased EZH2 expression is detected in whole blood, tion level of IFI44L is a highly sensitive and specific diagnostic neutrophils, monocytes, B cells, and CD4+ T cells [224]. Treatment marker for SLE [215]. Hypomethylation at other IFN-stimulated with the EZH2 inhibitor GSK126 prolonged survival time, reduced genes, such as IFI44 and IFITM1, in B cells also discriminates SLE the levels of anti-dsDNA autoantibodies, and improved lupus patients from healthy controls, providing further evidence that nephritis in NZB/W F1 mice [223]. Moreover, an EZH2 inhibitor DNA methylation around IFN-stimulated genes is a promising decreased the expression of IFN-stimulated genes in the kidneys of biomarker for SLE [12]. Interestingly, bioinformatic analysis and these mice, suggesting that EZH2 inhibition interferes with the machine learning have identified IFI44 as an optimal diagnostic activation of type I IFN signaling pathways [223]. In lupus-prone marker of SLE, which was verified by quantitative real-time PCR in MRL/lpr mice, intraperitoneal administration of 3-deazaneplanocin an independent cohort [216]. Genome-wide DNA methylation A (DZNep), an EZH2 inhibitor, improved survival and significantly profiling also revealed prominent hypomethylation of interferon- reduced anti-dsDNA antibody levels. DZNep-treated mice exhibited stimulated genes such as MX1, IFI44L, and PARP9 in B cells from a significant reduction in renal damage, splenomegaly, and pSS patients, suggesting that methylation levels at different IFN- lymphadenopathy with a decrease in the levels of many cytokines stimulated gene loci may be biomarkers for different autoimmune and chemokines [224]. In an allogeneic T-cell-induced lupus model, diseases [194]. deficiency or pharmacological inhibition of EZH2 suppresses GC Regarding histone modifications, early studies showed signifi- formation and autoantibody production [225]. Increased expression cant alterations of H3K4me3 in peripheral blood mononuclear of EZH2 seems to be associated with metabolic disorders since cells (PBMCs) of SLE patients and H3K9 hypomethylation in SSc mTORC1-mediated glycolysis controls EZH2 expression in SLE patients [205, 207, 217]. These histone methylation changes are T cells, suggesting that glycolytic pathways might be indirect associated with disease activity and may be clinical and biological targets for suppressing EZH2 expression [226]. In addition to SLE, markers. EZH2 is involved in other autoimmune diseases, including Increasing evidence suggests the strong involvement of autoimmune hepatitis. High expression of EZH2 promotes immune noncoding RNAs in B-cell responses and autoimmunity. Many activation and liver fibrosis through H3K27me3 whereas DZNep studies have shown a particularly important role of aberrant treatment attenuates hepatic inflammation and liver fibrosis in mice noncoding RNAs, including miRNAs, in autoimmune disease with autoimmune hepatitis [227]. Currently, supportive evidence pathogenesis, which suggests that noncoding RNAs could serve from clinical trials on the efficacy of EZH2 inhibitors in autoimmune as promising biomarkers for various autoimmune diseases [218]. patients is still lacking. However, active clinical investigations of The expression levels of miRNAs are relatively stable in serum and EZH2 inhibitors in patients with cancer will shed new light on the thus could be assessed in a reproducible and consistent manner. future development of EZH2-targeted therapies for patients with miRNAs can be detected in various tissues in a rapid and precise autoimmune diseases. way, showing promising clinical application potential. Genome-wide miRNA expression profiling has revealed sig- HDACs. Histone acetylation balance has shown major effects on nificantly altered miRNA signatures in naive and memory B cells chromatin remodeling and gene transcription. Histone acetylation from renal and nonrenal severe SLE patients of Latin American and deacetylation are catalyzed by HATs and HDACs, respectively. background. B-cell subset-specific miRNAs were characterized in HDACs are important for B-cell development and activation. In healthy donors and SLE patients. Specific miRNA signature profiles recent decades, many HDAC inhibitors have been developed and could be applied to discriminate naive and memory B cells of SLE have shown therapeutic potential for treating autoimmune patients from those of healthy controls. Moreover, six miRNAs diseases. were found to be associated with specific pathologic features In MRL/lpr mice with SLE-like phenotypes, HDAC6 is highly representing renal outcomes in SLE patients, suggesting that they expressed in B cells. A selective HDAC6 inhibitor, ACY-738, inhibits are promising biomarkers for molecular diagnosis. Many miRNAs pre-B-cell proliferation by regulating Bax-mediated pathways have been shown to have an abnormal expression in serum, [228]. Treatment with ACY-738 also significantly decreased PBMCs, T cells, and tissue cells from patients with SLE as well as proteinuria scores and prevented lupus nephritis in NZB/W F1 other autoimmune diseases [213], suggesting that combined mice. Moreover, ACY-738 administration dramatically reduces the miRNA signatures in various organs and cell populations may numbers of GC B cells and plasma cells, leading to a reduction in provide precise clues for the diagnosis and clinical management serum autoantibody levels and attenuated IgG deposition in the of autoimmune diseases. SLE B cells show aberrant expression and glomerulus [229]. A novel HDAC6-selective inhibitor, CKD-506, localization of the lncRNA Xist. In particular, Xist-mediated escape exerts prophylactic and therapeutic effects in experimental of many X-linked genes, including TLR7, contributes to dysregu- autoimmune encephalomyelitis (EAE) mice by regulating periph- lated B-cell responses and SLE development [13, 107]. SLE T cells eral immune responses and maintaining blood‒brain barrier also show altered expression of XIST RNA interactome genes, integrity [230]. Another HDAC inhibitor, panobinostat, which showing dysregulated Xist-associated XCI expression in diverse targets class I, II, and IV HDACs, ameliorates renal damage and immune cells [219, 220]. Xist is aberrantly expressed, and Xist decreases disease severity while significantly reducing plasma cell Cellular & Molecular Immunology (2022) 19:1215 – 1234 F. Xiao et al. 1227 responses and autoantibody levels in MRL/lpr mice [181]. different cell subsets show distinct DNA methylation features. However, other immune parameters are largely unaffected [181]. Inhibiting DNMTs in CD4 and CD8 T cells with 5-azaC and a Interestingly, panobinostat has shown promising therapeutic nanolipogel delivery system dramatically ameliorates lupus- efficacy for treating multiple myeloma, a B-cell malignancy [231]. related pathology through distinct mechanisms. Targeted inhibi- Dysregulated expression of HDACs is detected in synovial tion of DNMTs in CD4 T cells promotes Treg expansion, whereas tissues from RA patients and linked to disease pathology [232]. inhibiting DNMTs in CD8 T cells restrains pathogenic CD4-CD8- Although the functions of HDACs in immune tolerance breakdown double-negative T cells in MRL/lpr mice, indicating the signifi- and joint damage are not fully understood, preclinical studies cance of cell-specific inhibition of DNA methylation [237]. Since have shown that HDAC inhibitors exert both prophylactic and hypomethylation of IFN-related genes is common in B cells and therapeutic benefits [232]. For example, MPT0G009 shows potent other immune cells from patients with SLE or SSc [14], inhibiting inhibitory effects on various HDAC isoforms and inhibits the DNA methylation may not be an optimal therapeutic approach. In development of arthritis in an adjuvant-induced arthritis model diseases with global hypomethylation, future investigations to [233]. A well-studied HDAC inhibitor, givinostat, exhibits signifi- explore novel agents that can specifically increase DNA methyla- cant therapeutic benefit and good safety profiles in patients tion in B cells and other populations without triggering severe side with systemic-onset juvenile idiopathic arthritis [234]. The avail- effects are important. able studies suggest that HDAC inhibitors exhibit major effects on synoviocytes and T cells in arthritic mice [232]. Further studies are miRNAs. Various miRNAs have been identified as important needed to investigate the effects of HDAC inhibitors on B-cell- regulators during B-cell activation and the development of various mediated arthritis development in mice and patients with RA. autoimmune diseases [38]. Dysregulated miRNA profiles are Many HDAC inhibitors have been developed, such as panobinostat, involved in the pathogenesis of autoimmune diseases via diverse entinostat, and ricolinostat. Some of these agents have been tested in effector mechanisms, including regulating immune cell activation, clinical trials for patients with cancers. Apart from small molecules that cytokine release, and autoantibody production [218]. Moreover, target HDACs, recent studies suggest that natural metabolites may miRNA signature profiles may serve as useful clinical biomarkers. also serve as HDAC inhibitors. SCFAs directly modulate plasma cell Preclinical studies have shown that miRNAs may also be therapeutic differentiation and antibody production. By acting as HDAC inhibitors, targets. B cells from MRL/lpr mice show high levels of miR-7, which SCFAs significantly decrease AID and Blimp1 expression in B cells, directly targets Pten mRNA. Treatment with a miR-7 antagomir reduce serum levels of class-switched autoantibodies, and abolish significantly reduces hypertrophy of the spleen, decreases protei- lupus-like skin lesions and kidney pathology in both MRL/lpr and NZB/ nuria, and inhibits immune complex deposition in glomeruli in MRL/ W F1 mice, suggesting that dietary fibers and related catabolites lpr mice. Notably, a miR-7 antagonist normalized B-cell hyperre- produced by intestinal microbiota may exert beneficial effects for sponsiveness, suggesting that inhibition of miR-7 alleviates lupus ameliorating autoimmunity [70]. nephritis by modulating B-cell responses [238]. MiR-155 is important Hundreds of clinical trials associated with HDAC inhibitors have in GC and plasma cell responses and is associated with RA been registered, most of which are designed to evaluate safety and development [85, 188, 189]. Moreover, administration of a miR-155 efficacy in patients with different cancers, inflammatory diseases, and antagomir attenuates pristane-induced lupus alveolar hemorrhage neurodegenerative disorders. Although available clinical evidence of in mice [239]. Treatment with an antisense oligonucleotide that HDAC inhibitors in autoimmune disease patients is limited, studies on targets miR155 reduces CNS inflammation and decreases the immune system disorders will provide more clues and evidence for disease severity of EAE mice before and after the appearance of future investigations. clinical symptoms [240], suggesting that miR-155 may represent an attractive therapeutic target. DNMTs. DNMTs are a conserved family of cytosine methyltrans- Recently, many miRNA-based therapies have been developed. ferases. DNMTs and TETs sustain a homeostatic balance of DNA Compared with other biological agents, such as monoclonal methylation. It has been reported that both DNMT3A and antibodies, miRNAs show unique advantages in terms of drug DNMT3B suppress the expansion of GC B cells and plasma cells development. miRNAs are often conserved among species with [81] and may control B-cell lineage-specific gene expression [125]. known sequences. Therefore, it is possible to deliver synthetic As discussed, DNA methylation signatures may serve as promising oligonucleotides or small molecules that can either supplement or biomarkers for the diagnosis and clinical management of SLE and block the effects of target miRNAs. The available platforms for other autoimmune diseases. Some currently used immunosup- delivering therapeutic oligonucleotides provide a solid foundation pressive drugs for patients with autoimmune diseases can induce for miRNA-based therapies. However, since miRNAs can regulate epigenetic changes, which could partially account for their various targets during different cellular processes, the incomplete therapeutic effectiveness [11]. In mice with EAE, low-dose understanding of miRNA biology may restrain translational studies treatment with 5-aza-2’-deoxycytidine (5-azadC), an inhibitor of of miRNA therapies in the clinic due to potential off-target and side DNMTs, increases the immunosuppressive function of regulatory effects [241]. Currently, the clinical application of miRNA-based T cells, suppresses CNS inflammation, and inhibits EAE develop- therapies is at an early stage. Although available evidence shows ment [235]. Similarly, low-dose treatment with 5-azacytidine (5- promising therapeutic potential of epigenetic therapies (Table 1), azaC), another DNMT inhibitor, reduces disease severity and halts further clinical trials will be needed for the validation of novel joint inflammation in mice with proteoglycan-induced arthritis therapeutic strategies in patients with autoimmune diseases. [236]. Moreover, 5-azaC treatment compromises GC formation, leading to suppressed SHM, CSR, and IgG1 antibody production [236]. These findings suggest that inhibition of DNMTs by CHALLENGES AND FUTURE PERSPECTIVES pharmacological small molecules can modulate B-cell and T-cell The epigenome in B-cell subsets and its roles in autoimmunity responses and provide therapeutic benefits in mouse models of Mounting evidence has shown the important roles of epigenetic autoimmune diseases. 5′-Azacytidine has been approved for the alterations in many B-cell subsets and B-cell-mediated autoimmu- treatment of several blood cell malignancies, suggesting the nity. During B-cell activation and differentiation, there is a therapeutic potential of DNMT inhibitors in autoimmune diseases. dramatic change in the epigenome that involves diverse effector However, it is important to note that DNMT inhibition by 5-azadC mechanisms, including those related to DNA methylation, histone may also increase antibody production by B cells, indicating modifications, and noncoding RNAs. With the rapid development complex effects on disease development under different condi- of genetic approaches, the roles of epigenetic processes can be tions [11]. Targeting DNMTs in specific cell types is important since studied using mice with B-cell-conditional deficiency of key Cellular & Molecular Immunology (2022) 19:1215 – 1234 F. Xiao et al. 1228 Table 1. Epigenetic targets for autoimmune diseases Targets Agents Diseases/models Therapeutic results EZH2 GSK126 NZB/W mice Increased survival time, reduced levels of anti-dsDNA autoantibodies, and improved lupus nephritis [223] DZNep MRL/lpr mice Improved survival rates and reduced anti-dsDNA antibody levels [224] GSK503 Murine bm12 model of lupus-like Suppressed germinal center formation and autoantibody chronic graft versus host disease production [225] DZNep Induced autoimmune hepatitis mice Attenuated hepatic inflammation and liver fibrosis [227] HADCs ACY-738 NZB/W mice Decreased proteinuria scores and serum autoantibodies levels, attenuated IgG deposition in glomerulus, and ameliorated lupus nephritis [229] CKD-506 EAE mice Decreased clinical scores and central nervous system inflammation [230] Panobinostat MRL/lpr mice Ameliorated renal damage, reduced autoantibody levels, and decreased disease severities [181] MPT0G009 Adjuvant-induced arthritis mice Ameliorated joint inflammation and cartilage damage [233] Givinostat Systemic-onset juvenile idiopathic Significant therapeutic benefit and tolerated safety profiles in the arthritis patients patients [234] SCFAs MRL/lpr mice and NZB/W mice Reduced serum levels of class-switched autoantibodies, abolished lupus-like skin lesions and kidney pathology [70]. DNMTs 5-azadC EAE mice Suppressed central nervous system inflammation [235] 5-azaC Proteoglycan-induced arthritis mice Reduced disease severity and halted joint inflammation [236] 5-azaC MRL/lpr mice Ameliorated lupus-related pathology [237] miRNAs miR-7 antagomir MRL/lpr mice Reduced spleen hypertrophy, decreased proteinuria, and inhibited immune complex deposition in glomeruli [238] miR-155 antagomir CIA mice Reduced disease severity [245] miR-155 antagomir Pristane-induced lupus mice Attenuated pristane-induced lupus alveolar hemorrhage [239] miR-155 antagomir EAE mice Reduced central nervous system inflammation and decreased disease severities [240] DZNep 3-Deazaneplanocin A, EAE experimental autoimmune encephalomyelitis, CIA collagen-induced arthritis, SCFAs short-chain fatty acids enzymes. However, the roles of epigenetic events in B-cell subsets be novel biomarkers for clinical diagnosis and may provide new can hardly be determined using these genetic methods. Since B epigenetic targets for therapies [10]. Targeting epigenetic cells may exert different functions under different disease modifications may have side effects since epigenetic events conditions due to cellular heterogeneity, it is important to usually have tissue-specific and cell-specific functions. Although investigate the epigenomes of functional B-cell subsets. many clinical trials have been performed on cancer patients, Plasma cells are terminally differentiated B cells that exert limited clinical evidence has been obtained regarding the safety diverse functions by producing antibodies as well as cytokines and effectiveness of epigenetic therapies in patients with such as IL-10 [5]. It has been well recognized that plasma cells play autoimmune diseases. The insufficient understanding of epige- crucial roles in the development of many autoimmune diseases. netic mechanisms in disease pathology and the side effects of The differentiation of plasma cells is tightly regulated by intrinsic current epigenetic therapies are major challenges for clinical transcription factors and the surrounding microenvironment [242]. applications. Although recent studies suggest that HDACs, DNMTs, and TETs The personalized treatment of cancer patients by integrating contribute to plasma cell generation, it remains largely unclear genomic information and other data, including clinical profiles how epigenetic events sustain plasma cell identity and regulate and epigenetic results, has been proposed [243]. Personalized plasma cell functions. Atypical memory B cells serve as a key medicine has been extensively discussed in the context of cancer pathogenic player in autoimmune diseases. Dysregulated epige- treatment and is receiving increasing attention in the field of netic events, including DNA hypomethylation and mislocated Xist, autoimmune diseases. Patients usually show variable responses to have been identified in SLE patients [12, 13]. Although the the same therapy, suggesting heterogeneity of patients and the available evidence suggests a role for epigenetic alterations in need for more precise therapies. Available evidence suggests that the formation of atypical memory B cells, further investigations on epigenomic profiles are not only important for understanding the detailed mechanisms and functions of other epigenetic events autoimmune disease pathogenesis but also valuable for evaluat- in these cells are warranted. Future studies on epigenetic ing disease progression and may provide clues for predicting regulation in functional B-cell subsets will further enhance the responses to specific therapies [16, 213, 244]. understanding of autoimmune pathogenesis and may provide Many studies suggest that epigenetic signatures may serve as novel therapeutic targets for treating patients. useful biomarkers for the diagnosis and clinical management of patients [213]. As previously discussed, several studies have Epigenetics for clinical therapy and personalized medicine revealed significant epigenetic changes around genes associated Over the past years, there has been enormous progress in with type I IFN and identified potential diagnostic markers for SLE genome-wide epigenetic studies with fruitful achievements. The [14, 213, 215, 216]. 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Cell Mol Immunol. 2022;19:863–5. and 17103821), RGC Theme-based Research Scheme (TRS) (T12-703/19-R), and the 223. Wu L, Jiang X, Qi C, Zhang C, Qu B, Shen N. EZH2 inhibition interferes with the Centre for Oncology and Immunology under the Health@InnoHK Initiative funded by activation of type I interferon signaling pathway and ameliorates lupus nephritis the Innovation and Technology Commission, Hong Kong, China. The figures were in NZB/NZW F1 mice. Front Immunol. 2021;12:653989. created with BioRender.com. Cellular & Molecular Immunology (2022) 19:1215 – 1234 F. Xiao et al. 1234 AUTHOR CONTRIBUTIONS Open Access This article is licensed under a Creative Commons LL, JT, and HZ conceptualized the study. FX wrote the manuscript. 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