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REVIEW

Epigenetic modifications of the immune system in health and disease Yuuki Obata1,2, Yukihiro Furusawa1,3 and Koji Hase1,3 Vertebrate animals have developed sophisticated host defense mechanisms against potentially hostile antigens. These mechanisms mainly involve the immune system and the epithelial cells that cover the body surface. Accumulating studies have revealed that epigenetic mechanisms in collaboration with signal transduction networks regulate gene expression over the course of differentiation, proliferation and function of immune and epithelial cells. The epigenetic status of these cells is fine-tuned under physiological conditions; however, its disturbance often results in the development of immunological disorders, namely inflammation. Certain environmental factors influence the differentiation and function of immune cells through epigenetic alterations. For example, commensal microbiota-derived metabolites inhibit histone deacetylases to induce regulatory T cells, whereas some infectious agents induce DNA methylation, resulting in the development of cancer. These data imply that epigenetic regulation of host defense cells, which are usually the first to encounter external antigens, is implicated in disease development. Here, we highlight recent advances in our understanding of the molecular mechanisms by which the epigenetic status of immune and epithelial cells is controlled. Immunology and Cell Biology advance online publication, 10 February 2015; doi:10.1038/icb.2014.114 Epigenetic regulation including DNA methylation and histone modifications is a heritable modification that influences gene expression.1 The chemical modifications on the amino-terminal tails of histones, namely, acetylation, methylation, phosphorylation and ubiquitylation, are associated with conformational changes in chromatin structures.2,3 Among them, acetylation of histone tails is known to generate an open chromatin structure that contributes to incite active transcription.4 Compelling evidence has suggested that epigenetic machineries play an important role in immune response by regulating the cell-fate decision and maintenance of immune cell lineages.5–7 This is exemplified by the observations that specific epigenetic signatures along with the expression of master transcription factors dictate phenotypes and stability of differentiated CD4+ T cells. Loss-of-function studies on epigenetic-modifying enzymes (for example, DNA methyltransferases (DNMTs) and histone deacetylases (HDACs)) support the biological significance of epigenetic regulation in immune cells. Importantly, the epigenetic status of immune cells seems to be controlled by both cellautonomous programs and bioenvironmental factors such as cytokines and microbial factors. Here, we discuss recent advances in our understanding of the epigenetic regulation of the immune system and its implication in health and disease. EPIGENETIC REGULATION OF THE IMMUNE SYSTEM Epigenetic regulation of macrophage homeostasis and disease The functions of macrophages are regulated by histone modifications. In particular, activation of Toll-like receptor (TLR) signaling in 1

macrophages induces histone lysine acetylation of inflammatory cytokine genes.8–11 Acetylated lysine residues in the histone tail are recognized by the bromodomain and extraterminal (BET) proteins that are composed of Brd2, Brd3, Brd4 and Brtd.12 Among them, Brd4 is well known to contribute to the transcription of inflammatory cytokine genes by forming a complex with positive transcription elongation factor-b and RNA polymerase II at the transcription start site.8,13,14 A synthetic compound, I-BET, suppresses the expression of inflammatory cytokine genes in lipopolysaccharide (LPS)-stimulated macrophages by interfering with binding of BET proteins to acetylated histones.11 Administration of I-BET protected mice from endotoxin shock induced by LPS treatment or heat-killed Salmonella typhimurium treatment.11 This finding demonstrated the therapeutic potential of drugs that interfere with the binding of BET to acetylated histones in the prevention of inflammatory disorders. Another study has shown that HDAC11 (class IV HDAC) serves as an epigenetic silencer of interleukin (IL)-10 expression in macrophages.15 HDAC11 interacts with the distal promoter region of Il10, leading to the development of a hypoacetylated and condensed chromatin structure. Such structural changes inhibit the binding of STAT3 (signal transducer and activator of transcription 3) on the distal promoter of the Il10 gene, resulting in the silencing of Il10 expression. Histone lysine methylation also plays an important role in the maintenance of macrophage homeostasis. Ash1l, a mammalian homolog of the Drosophila Ash1 gene, functions as an H3K4 methyltransferase.16–18 Ash1l promotes A20 expression in

Division of Mucosal Barriology, International Research and Development Center for Mucosal Vaccines, Institute of Medical Science, University of Tokyo, Tokyo, Japan; Department of Immune Regulation, Graduate School of Medical and Pharmaceutical Sciences, Chiba University, Chiba, Japan and 3Department of Biochemistry, Faculty of Pharmacy, Keio University, Tokyo, Japan Correspondence: Dr K Hase, Department of Biochemistry, Faculty of Pharmacy, Keio University, 1-5-30 Shibakoen, Minato-ku, Tokyo 105-8512, Japan. E-mail: [email protected] Received 7 November 2014; accepted 6 December 2014 2

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macrophages by enhancing H3K4 methylation at the promoter of Tnfaip3 (which encodes A20) that in turn facilitates A20-mediated deubiquitination of nuclear factor (NF)-κB essential modulator (NEMO) and TRAF6. The end result is the suppression of NF-κB and mitogen-activated protein kinase signaling pathways, and the subsequent production of IL-6.19 Therefore, knockdown of Ash1l increases LPS-induced IL-6 production by macrophages in vitro. Ash1l-deficient mice were more susceptible to Escherichia coli-induced sepsis and collagen-induced arthritis, and more prone to spontaneous systemic autoimmune symptoms compared with control mice.19 These results suggest that Ash1l-dependent epigenetic regulation is critical to protect mice from autoimmune diseases. Another H3K4 methyltransferase, Wpp7,20 is required for the function and antimicrobial response of macrophages.21 Wbp7-deficient macrophages exhibit a remarkable downregulation of glycosylphosphatidylinositol (GPI)-anchored proteins on the cell surface membrane because of epigenetic silencing of Pigp,21 a component of the GPI-GlcNAc transferase22 responsible for GPI anchor synthesis.23 Wbp7-deficient macrophages show impaired responses to LPS because of a lack of surface CD14, a GPI-anchored protein that functions as a co-receptor for LPS.21 Thus, epigenetic regulation by Wbp7 secures macrophage responsiveness to microbial stimuli through the control of GPI anchor synthesis. Macrophages are classified into two subsets, M1 and M2 macrophages, based on their function.24 M1 macrophages are characterized by the production of high amounts of proinflammatory cytokines and nitric oxide upon recognition of pathogens through the patternrecognition receptor. Based on these functions, they play a central role in the clearance of bacteria, virus and fungi. On the other hand, M2 macrophages are characterized by high expression of arginase-1 (Arg1), found in inflammatory zone-1 (Fizz1), and chitinase 3-like 3 (Chi3l3), and play an essential role in tissue remodeling, immune response to parasite infection and angiogenesis.25 Polarization of macrophages into M1 or M2 cells is also regulated by epigenetic mechanisms. Satoh et al.28 have demonstrated that Jumonji domaincontaining 3 (Jmjd3), an H3K27 demethylase that catalyzes the conversion of trimethylated H3K27 (H3K27me3) to monomethylated H3K27 (H3K27me1),26,27 is essential for the polarization of M2 macrophages.28 Jmjd3 is known to be induced in macrophages by TLR stimulation in an NF-κB-dependent manner.27 Deficiency of Jmjd3 affects the expression of M2 markers in bone marrow-derived macrophages under M2-polarizing conditions in vitro. The trimethylation levels of H3K27 at M2 marker genes are not changed in the absence of Jmjd3, whereas those at the IRF4 transcription start site are higher in Jmjd3-deficient macrophages than in Jmjd3-sufficient macrophages. In agreement with the epigenetic status, Irf4 expression is diminished in Jmjd3-deficient macrophages. Furthermore, the M2 macrophage polarization is defective in Irf4-deficient mice. These results suggest that Jmjd3-dependent upregulation of Irf4 is responsible for M2 macrophage polarization.28 Given that Jmjd3-deficient chimeric mice show impaired immune response to Nippostrongylus brasiliensis infection, a potent inducer of type 2 immunity in the lung,29 the Jmjd3–IRF4 axis in macrophages plays a significant role in the host defense against helminth infection. Another study has reported that Jmjd3 is essential for the polarization of M2-type microglia cells, and this regulation may be implicated in the pathogenesis of Parkinson’s diseases.30 Knockdown of Jmjd3 in microglia cells compromises M2 polarization and reciprocally increases M1 polarization of microglial cells that evokes inflammatory responses and leads to neuron death. Similarly, in vivo knockdown of Jmjd3 in the substantia nigra induces death of dopaminergic neurons Immunology and Cell Biology

in the mouse model of Parkinson’s diseases because of aberrant activation of microglia cells. These observations raise the possibility that Jmjd3 could be a therapeutic target for inflammatory diseases. Indeed, a selective jumonji H3K27 demethylase inhibitor, GSK-J4, was recently reported to inhibit LPS-induced production of inflammatory cytokines, including tumor necrosis factor-α.31 Epigenetic regulation of Treg cells and disease Aberrant immune response to trillions of commensal bacteria causes intestinal inflammation. To avoid this risk, the intestinal mucosa possesses unique immune regulatory systems represented by accumulation of regulatory T (Treg) cells. Treg cells are currently classified into thymus-derived Treg and peripherally induced Treg cells, according to their origin.32 Several recent studies have identified bacterial strains responsible for the induction of functional Treg cells in the colon. For example, TLR2 stimulation of CD4+ T cells with polysaccharide A of Bacteroides fragilis induces development of IL-10producing Treg cells.33 Treatment of a cocktail of intestinal bacteria belonging to Clostridiales clusters IV, XIVa and XVIII from mouse or human feces are sufficient to promote the development of Helios− peripherally induced Treg cells in the colon of germ-free (GF) mice.34,35 Furthermore, inoculation of altered Schaedler flora composed of eight different commensal bacteria in GF mice induces de novo generation of colonic Treg cells that in turn suppresses T helper type 1 cell (Th1) and Th17 responses to maintain gut immune homeostasis.36 The bacterial community harbored by Clostridialescolonized mice is actively involved in digestion of dietary fibers to produce small-molecule metabolites, leading to production of shortchain fatty acids.37 These short-chain fatty acids promote the migration and differentiation of colonic Treg cells.37–39 These observations indicate that interplay between commensals and host cells may play a critical role in the maintenance of gut immune homeostasis. Butyrate, one of the short-chain fatty acids produced by commensal microbiota, is well documented to inhibit class I and IIa HDACs among the four classes of HDACs.40,41 Treatment of naive CD4+ T cells with butyrate upregulates Foxp3 expression by enhancing the acetylation status of histone H3 in the promoter, CNS1 and CNS3 enhancer regions of the Foxp3 gene locus, thus augmenting Treg induction (Figure 1).37 Consistent with the in vitro observations, intake of butyrylated starch42 strongly induces IL-10-producing functional Treg cells, thereby ameliorating experimental colitis induced by adoptive transfer of CD4+CD45RBhi T cells into Rag1− / − mice.37 Other studies have shown that butyrate suppresses proinflammatory cytokine expression in macrophages and dendritic cells, most likely through HDAC inhibition.38,43 In addition, the activation of Gpr109a, a receptor for butyrate, induces Raldh1 expression on dendritic cells and promotes the induction of Treg differentiation (Figure 1).44 These results suggest that commensal-derived butyrate is important for the prevention of inflammatory disorders such as inflammatory bowel disease in mice. In support of this notion, the frequency of butyrate-producing bacteria is found to be decreased in the fecal samples from patients with inflammatory bowel disease.45 Further investigation is necessary to clarify the mechanism by which butyrate selectively affects the epigenetic status of the Foxp3 gene. The DNA methylation machinery is critical for Treg homeostasis in the colon, as demonstrated by the reduction of proliferating Treg cells in mice with a T cell-specific deletion of Uhrf1 (Ubiquitin-like, with PHD and RING finger domains 1; Uhrf1 cKO mice).46 Uhrf1 plays a nonredundant role in the maintenance of DNA methylation by recognizing and recruiting Dnmt1 to hemi-methylated DNA in proliferating cells.47 Colonization of GF mice with gut microbiota

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Figure 1 Commensal microbiota-dependent regulation of intestinal immune system. Commensal bacteria actively consume indigestible materials and produce a diversity of metabolites, including short-chain fatty acids (SCFAs). Among them, butyrate upregulates histone H3 acetylation at the Foxp3 gene locus. Butyrate also binds to GPR109a on dendritic cells (DCs) to induce production of retinoic acids (RAs) and IL-10. Butyrate together with acetate and propionate induce transforming growth factor-β (TGF-β) secretion by IECs. Collectively, these biological effects of SCFAs facilitate differentiation of naive CD4+ T cells into Treg cells. Dnmt1-dependent DNA methylation contributes to repression of ISC-associated genes in IECs, critical for differentiation and proliferation of IECs. In addition, IEC-intrinsic HDACs regulate epithelial composition and barrier functions. Commensal bacteria epigenetically downregulate the expression of epithelial TLR4 to maintain hyporesponsiveness to commensal bacteria. A full color version of this figure is available online at the Immunology and Cell Biology website.

promotes Uhrf1-dependent proliferation of colonic Treg cells.46 Notably, ablation of Uhrf1 affects the development of functional Treg cells in the colon. As a consequence, Uhrf1 cKO mice spontaneously develop severe colitis. A similar phenotype is also observed in mice with T cell-specific deletion of Dnmt1 (Y Obata et al., unpublished observation), suggesting that DNA methylation machinery via the Uhrf1–DNMT1 axis is essential for the expansion and functional maturation of colonic Treg cells to establish microbe–T-cell mutualism. Uhrf1 epigenetically silences cyclin-dependent kinase inhibitor 1a (Cdkn1a), a negative regulator of G1-phase progression, via CpG methylation at the distal promoter region.48 The epigenetic regulator Trim28 (tripartite motif protein 28), also known as KAP1 and TIF1, contributes to gene repression by interacting with heterochromatin proteins and recruiting histone methyltransferases, SETDB1 and HDACs.49,50 Trim28 plays an essential role in the maintenance of T-cell homeostasis in vivo51 because deficiency of Trim28 in T cells impairs cell-cycle progression, IL-2 production and IL-2 response. In addition, trimethylation level of H3K9 (repression mark) in exon 1 of the Tgfb3 gene of Trim28deficient T cells is remarkably decreased, whereas acetylation level of H3K9 (activation mark) is enhanced. Because these epigenetic alternations abnormally upregulate transforming growth factor-β3 and induce autoreactive Th17 cells, Trim28-deficient mice display

exacerbation of experimental autoimmune encephalomyelitis. This finding indicates the importance of epigenetic regulation in preventing autoimmune reactions. EPIGENETIC MODIFICATION OF IECS AND COLORECTAL CANCER Intestinal epithelial cells (IECs) covering the gastrointestinal mucosa serve as a barrier to protect the host from invasion by potential pathogens and also as sentinels to warn of their presence.52 Intestinal stem cells (ISCs) located at the crypt bottom divide to produce transitamplifying cells that differentiate into functional epithelial cell lineages: absorptive enterocytes and three types of secretory epithelial cells, namely, mucus-secreting goblet cells,53 gastrointestinal hormoneproducing enteroendocrine cells54 and antimicrobial peptideproducing Paneth cells.55 DNA methylation is important for the regulation of gene expression during the development of self-renewing tissues, including IECs. DNA methylation patterns are established and maintained by the de novo methyltransferases (DNMT3a and DNMT3b) and the maintenance methyltransferase (DNMT1).56 Previous study has shown that DNMT1 is highly expressed in the ISCs and proliferative progenitor cells.57 Mice with IEC-specific deletion of DNMT1 (DNMT1F/F; Villin-Cre-ERT2) showed an increase in the number of proliferative progenitor cells and a reciprocal decrease in Immunology and Cell Biology

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the number of differentiated enterocytes (Figure 1).58 This defect is at least partly attributed to hypomethylation and increased expression of Olfm4 and Hes1 that contribute to the maintenance of ISCs. Genomewide DNA methylomics analysis of ISCs and differentiated IECs demonstrated that genes highly expressed in ISCs, such as Olfm4 and Hes1, are methylated to repress their expression during differentiation, whereas genes associated with differentiation, such as Alpi and Lct, are demethylated to ensure epithelial expression.58 Thus, DNA methylation by DNMT1 is essential for the repression of ISC-associated genes during differentiation and for the maintenance of balance between proliferation and differentiation of IECs. Considering that balanced differentiation and proliferation of IECs is critical for the establishment of mucosal barrier functions against commensal bacteria,59 DNA methylation-dependent control of IEC homeostasis may be important for the avoidance of intestinal disease, such as inflammatory bowel disease. However, the molecular mechanism controlling the expression of DNMT1 in IECs is largely unknown. In addition to DNA methylation, histone modifications also contribute to the maintenance of intestinal epithelial homeostasis. HDACs are evolutionally conserved enzymes that remove acetyl groups from histones and play a critical role in epigenetic gene silencing. A recent study has demonstrated that IEC-specific deletion of HDAC3 (HDAC3IEC-KO) causes an impairment in epithelial homeostasis characterized by a loss of Paneth cells, increased intestinal permeability, bacterial translocation, spontaneous inflammation and alterations in the gut microbiota.60 HDAC3IEC-KO mice also showed increased susceptibility to oral Listeria monocytogenes infection as well as development of dextran sodium sulfate-induced colitis. Because depletion of the commensal bacteria by rederivation of these mice into GF conditions partly canceled epithelial dysfunction, IEC-intrinsic HDAC3 seems to regulate epithelial homeostasis in the presence of commensal bacteria (Figure 1). Furthermore, other class I HDACs, including HDAC1 and HDAC2, also play a critical role in the regulation of epithelial homeostasis. The mice with IEC-specific deletion of both HDAC1 and HDAC2 showed remarkable reduction of secretory epithelial cells, such as goblet cells and Paneth cells, and increase in epithelial proliferation and migration.61 Although further analysis will be required to define the molecular mechanisms underlying the regulation of HDACs in IEC homeostasis, these findings provide new insight into the epigenetic regulation of barrier function in the gut. Epigenetic regulation plays a key role in tumor initiation and progression in colorectal cancer (CRC). Prostaglandin E2 was shown to upregulate the expression levels of DNMT1 and DNMT3B in human CRC cell lines.62 Therefore, prostaglandin E2 epigenetically silences tumor-suppressor genes and DNA-repair genes by inducing DNA methylation in their promoter region in human CRC cell lines and primary colonic tumor cells from ApcMin/+ mice.62 A recent study has shown that intestinal immune system also has an impact on the development of CRC through epigenetic modifications. For example, intestinal IL-22+CCR6+CD4+ T cells infiltrate into the human colon cancer region, where CCL20, a ligand for CCR6, is expressed. IL-22 potentiates the stemness of CRC through the activation of the transcription factor STAT3 that directly binds to the promoter of stem cell-associated genes, including Sox2.63 In addition, activated STAT3 also directly binds to the promoter region of DOT1L, an H3K79 methytransferase.64 DOT1L then enhances H3K79 methylation at genes involved in cancer stemness, such as Nanog, Sox2 and Pou5F1.63 Furthermore, IL-22-producing colonic innate lymphoid cells have also been demonstrated to promote colonic cancer during inflammation induced by Helicobacter hepaticus infection.65 These Immunology and Cell Biology

studies emphasize the pathophysiological significance of IL-22 in the development of colon cancer. In support of this notion, mice lacking soluble IL-22-binding protein (IL-22BP), which prevents the binding of IL-22 to membrane-bound IL-22Rα1, showed increased tumorigenesis in a colitis-associated colon cancer model.66 Furthermore, previous single-nucleotide polymorphism analysis has shown that IL22 polymorphisms are associated with an increased risk of colon cancer.67 Considering that IL-22 signaling plays a central role in the regulation of inflammatory response, epithelial repair and host defense against intestinal pathogens,68–70 these findings may provide mechanistic insights into the development of inflammation-associated cancer. MICROBIAL MODIFICATION OF HOST EPIGENETIC STATUS Many studies have shown that certain infectious agents and commensal microbes can change the epigenetic status of mammalian host cells during infection and symbiosis. Gastric infection with Helicobacter pylori is a predisposing factor for chronic gastritis, peptic ulcers and gastric cancer.71 Several studies have demonstrated that H. pylori infection induces aberrant DNA methylation in a number of gene promoters in gastric cells.72,73 Among the genes hypermethylated during H. pylori infection is a tumor-suppressor gene, Foxd3.74 In addition, DNA methylation levels in miR-124a-1–3, which are known as tumor-suppressor miRs, were significantly higher in the gastric mucosae of H. pylori-infected healthy volunteers than in those of uninfected subjects, suggesting that H. pylori infection may silence these micro RNAs.75 A more recent study has shown that chronic H. pylori infection downregulates miR-210 expression in gastric mucosa because of increased levels of DNA methylation and upregulates the miR-210-target genes STMN1 and DIMT1, both of which enhance epithelial proliferation.76 These observations provide a mechanistic link between chronic infection, epigenetic modifications and carcinogenesis. Moreover, uropathogenic E. coli infection significantly increases the expression of Dnmt1 in uroepithelial cell lines, resulting in a decrease in Cdkn2a because of enhanced DNA methylation at its promoter region.77 Given that Cdkn2a functions as a tumor suppressor,78 this observation implies that uropathogenic E. coli infection may increase the risk of bladder cancer.79,80 Translocation of the periopathogenic bacterium Campylobacter rectus into the placenta during pregnancy is implicated in preterm birth. Maternal infection with C. rectus results in downregulation of Igf2 in the placental tissue due to hypermethylation in the promoter region of Igf2 (insulin-like growth factor 2), an imprinted gene important for fetal growth and placental development.81,82 Remarkably, placental tissue, which was considered to be sterile, actually harbors various commensal bacteria such as Firmicutes, Tenericutes, Proteobacteria, Bacteroidetes and Fusobacteria.83 Variation in the placental microbiome is also associated with preterm birth.83 Therefore, the placental microbial community and infection during pregnancy may regulate genes critical for fetal development. Interestingly, in mice, maternal exposure to Acinetobacter lwoffii F78 prevents the offspring from developing asthma. T cells from the offspring of A. lwoffii F78-exposed mother display reduced capacity to produce Th2 cytokines IL-4, IL-5 and IL-13, a reciprocal increase in interferon-γ production via enhancement of histone H4 acetylation at the interferon-γ promoter and the reduction of acetylation at the IL-4 promoter.84 Certain oral bacteria such as Porphyromonas gingivalis and Fusobacterium nucleatum can also influence the expression levels of chromatin-modifying enzymes, including DNMTs and HDACs, in the gingival epithelial cells.85 This epigenetic response seems to

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regulate the expression of antimicrobial proteins such as human βdefensin 2 (hBD2) and CC chemokine ligand 20 (CCL20). In addition, Chlamydophila pneumoniae is an intracellular pathogen that causes acute respiratory diseases. The C. pneumoniae genome encodes a SET domain-containing protein that controls chromatin remodeling through histone methyltransferase activity. The chlamydial SET domain protein directly interacts with chlamydial histone H1-like proteins such as Hc1 and Hc2 and murine histone H3 in vitro.86 However, the pathological role of chlamydial SET domain-dependent epigenetic regulation in infection remains to be elucidated. Considering that many types of bacteria have SET domain genes,86 histone methyltransferase-dependent epigenetic modifications may contribute to the establishment of host–microbe symbiosis as well as infection. Epigenetic modifications are thought to be one of the mechanisms by which intracellular pathogens survive within host cells. For example, Anaplasma phagocytophilum, a causative agent of human granulocytic anaplasmosis, downregulates the expression of host defense genes such as defensins in the infected cells.87 This downregulation is attributed to the upregulation of HDAC1 that binds to the promoter region of defense genes and epigenetically silences their expression. Listeria monocytogenes also epigenetically regulates host gene expression by inducing deacetylation of H3K18. This epigenetic modification is attribute to the translocation of cytoplasmic sirtuin 2 (SIRT2) into the nucleus of host cells during infection. SIRT2deficient mice show increased resistance to L. monocytogenes infection, suggesting the significant role of SIRT2-dependent epigenetic regulation in the establishment of in vivo infection.88 Therefore, the elucidation of the mechanism by which pathogens exert epigenetic modifications may lead to the development of novel therapeutics for infectious disease. In addition to bacterial pathogens, commensal bacteria also epigenetically control the expression of host genes like TLR4 that recognizes bacteria-derived LPS.89,90 The frequency of DNA methylation in the 5′ region of the TLR4 gene was significantly lower in colonic epithelial cells from GF mice lacking commensal bacteria than those from conventional mice.90 This epigenetic modification represses the expression of TLR4. This mechanism is important for the maintenance of IEC hyporesponsiveness to LPS. Given that the recognition of luminal microbes by epithelial TLRs would initiate mucosal immune responses, epigenetic silencing of TLR4 may contribute to establishment of symbiotic relationships between IECs and commensal bacteria. CONCLUSION Recent loss-of-function studies on epigenetic modifiers have indicated that DNA methylation and histone modifications significantly contribute to cell-fate decisions, cell lineage stability and functions of various immune cell subsets. Some genome-wide epigenetic analyses have uncovered immune cell type-specific epigenetic signatures.91,92 These investigations also suggest that appropriate control of epigenetic status in those cells is critical to maintain immune homeostasis. It is, therefore, becoming more important to elucidate the molecular machineries involved in the epigenetic regulation of the immune system, as well as the bioenvironmental factors (for example, cytokines, hormones, diets, stress and microbial products) that potentially influence the epigenetic status of immune cells. Because alterations of epigenetic status are frequently heritable to daughter cells, accumulation of epigenetic alterations could provoke life-long inflammatory response, autoimmunity and allergic diseases. This concept is exemplified by the hygiene hypothesis that proposes that microbial exposure early in life is essential to suppress Th2-dependent

IgE responses in adulthood. Collectively, further studies on epigenetic regulation in immune cells are important for understanding the mechanism by which the immune system is established and to open up new avenues for novel epigenetic therapies in diverse immunological disorders.

CONFLICT OF INTEREST The authors declare no conflict of interest.

1 Cedar H, Bergman Y. Linking DNA methylation and histone modification: patterns and paradigms. Nat Rev Genet 2009; 10: 295–304. 2 Jenuwein T. Translating the histone code. Science 2001; 293: 1074–1080. 3 Zhang Y. Transcriptional regulation by histone ubiquitination and deubiquitination. Genes Dev 2003; 17: 2733–2740. 4 Lee DY, Hayes JJ, Pruss D, Wolffe AP. A positive role for histone acetylation in transcription factor access to nucleosomal DNA. Cell 1993; 72: 73–84. 5 Weng N-P, Araki Y, Subedi K. The molecular basis of the memory T cell response: differential gene expression and its epigenetic regulation. Nat Rev Immunol 2012; 12: 306–315. 6 Zediak VP, Wherry EJ, Berger SL. The contribution of epigenetic memory to immunologic memory. Curr Opin Genet Dev 2011; 21: 154–159. 7 Turner SJ. T cell immunity as a tool for studying epigenetic regulation of cellular differentiation. 2013; 4: 218. 8 Takeuch O, Akira S. Epigenetic control of macrophage polarization. Eur J Immunol 2011; 41: 2490–2493. 9 Leng J, Butcher BA, Egan CE, Abi Abdallah DS, Denkers EY. Toxoplasma gondii prevents chromatin remodeling initiated by TLR-triggered macrophage activation. J Immunol 2009; 182: 489–497. 10 Aung HT, Schroder K, Himes SR, Brion K, van Zuylen W, Trieu A. LPS regulates proinflammatory gene expression in macrophages by altering histone deacetylase expression. FASEB J 2006; 20: 1315–1327. 11 Nicodeme E, Jeffrey KL, Schaefer U, Beinke S, Dewell S, Chung C-W. Suppression of inflammation by a synthetic histone mimic. Nature 2010; 468: 1119–1123. 12 Belkina AC, Denis GV. BET domain co-regulators in obesity, inflammation and cancer. Nat Rev Cancer 2012; 12: 465–477. 13 Belkina AC, Nikolajczyk BS, Denis GV. BET protein function is required for inflammation: Brd2 genetic disruption and BET inhibitor JQ1 impair mouse macrophage inflammatory responses. J Immunol 2013; 190: 3670–3678. 14 Khan YM, Kirkham P, Barnes PJ, Adcock IM. Brd4 is essential for IL-1β-induced inflammation in human airway epithelial cells. PLoS ONE 2014; 9: e95051. 15 Villagra A, Cheng F, Wang H-W, Suarez I, Glozak M, Maurin M. The histone deacetylase HDAC11 regulates the expression of interleukin 10 and immune tolerance. Nat Immunol 2009; 10: 92–100. 16 Gregory GD, Vakoc CR, Rozovskaia T, Zheng X, Patel S, Nakamura T. Mammalian ASH1L is a histone methyltransferase that occupies the transcribed region of active genes. Mol Cell Biol 2007; 27: 8466–8479. 17 Byrd KN, Shearn A. ASH1, a Drosophila trithorax group protein, is required for methylation of lysine 4 residues on histone H3. Proc Natl Acad Sci USA 2003; 100: 11535–11540. 18 Klymenko T, Müller J. The histone methyltransferases Trithorax and Ash1 prevent transcriptional silencing by Polycomb group proteins. EMBO Rep 2004; 5: 373–377. 19 Xia M, Liu J, Wu X, Liu S, Li G, Han C. Histone methyltransferase Ash1l suppresses interleukin-6 production and inflammatory autoimmune diseases by inducing the ubiquitin-editing enzyme A20. Immunity 2013; 39: 470–481. 20 Ernst P, Vakoc CR. WRAD: enabler of the SET1-family of H3K4 methyltransferases. Brief Funct Genomics 2012; 11: 217–226. 21 Austenaa L, Barozzi I, Chronowska A, Termanini A, Ostuni R, Prosperini E. The histone methyltransferase Wbp7 controls macrophage function through GPI glycolipid anchor synthesis. Immunity 2012; 36: 572–585. 22 Watanabe R, Murakami Y, Marmor MD, Inoue N, Maeda Y, Hino J. Initial enzyme for glycosylphosphatidylinositol biosynthesis requires PIG-P and is regulated by DPM2. EMBO J 2000; 19: 4402–4411. 23 Kinoshita T, Fujita M, Maeda Y. Biosynthesis, remodelling and functions of mammalian GPI-anchored proteins: recent progress. J Biochem 2008; 144: 287–294. 24 Murray PJ, Wynn TA. Protective and pathogenic functions of macrophage subsets. Nat Rev Immunol 2011; 11: 723–737. 25 Mantovani A, Sozzani S, Locati M, Allavena P, Sica A. Macrophage polarization: tumor-associated macrophages as a paradigm for polarized M2 mononuclear phagocytes. Trends Immunol 2002; 23: 549–555. 26 Hong S, Cho Y-W, Yu L-R, Yu H, Veenstra TD, Ge K. Identification of JmjC domaincontaining UTX and JMJD3 as histone H3 lysine 27 demethylases. Proc Natl Acad Sci USA 2007; 104: 18439–18444. 27 De Santa F, Totaro MG, Prosperini E, Notarbartolo S, Testa G, Natoli G. The histone H3 lysine-27 demethylase Jmjd3 links inflammation to inhibition of polycomb-mediated gene silencing. Cell 2007; 130: 1083–1094.

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Epigenetic regulation of the immune system Y Obata et al 6 28 Satoh T, Takeuchi O, Vandenbon A, Yasuda K, Tanaka Y, Kumagai Y. The Jmjd3-Irf4 axis regulates M2 macrophage polarization and host responses against helminth infection. Nat Immunol 2010; 11: 936–944. 29 Shinkai K, Mohrs M, Locksley RM. Helper T cells regulate type-2 innate immunity in vivo. Nature 2002; 420: 825–829. 30 Tang Y, Li T, Li J, Yang J, Liu H, Zhang XJ. Jmjd3 is essential for the epigenetic modulation of microglia phenotypes in the immune pathogenesis of Parkinson's disease. Cell Death Differ 2014; 21: 369–380. 31 Kruidenier L, Chung C-W, Cheng Z, Liddle J, Che K, Joberty G. A selective jumonji H3K27 demethylase inhibitor modulates the proinflammatory macrophage response. Nature 2012; 488: 404–408. 32 Abbas AK, Benoist C, Bluestone JA, Campbell DJ, Ghosh S, Hori S. Regulatory T cells: recommendations to simplify the nomenclature. Nat Immunol 2013; 14: 307–308. 33 Round JL, Mazmanian SK. Inducible Foxp3+ regulatory T-cell development by a commensal bacterium of the intestinal microbiota. Proc Natl Acad Sci USA 2010; 107: 12204–12209. 34 Atarashi K, Tanoue T, Shima T, Imaoka A, Kuwahara T, Momose Y et al. Induction of colonic regulatory T cells by indigenous Clostridium species. Science 2011; 331: 337–341. 35 Atarashi K, Tanoue T, Oshima K, Suda W, Nagano Y, Nishikawa H. Treg induction by a rationally selected mixture of Clostridia strains from the human microbiota. Nature 2013; 500: 232–236. 36 Geuking MB, Cahenzli J, Lawson MAE, Ng DCK, Slack E, Hapfelmeier S. Intestinal bacterial colonization induces mutualistic regulatory T cell responses. Immunity 2011; 34: 794–806. 37 Furusawa Y, Obata Y, Fukuda S, Endo TA, Nakato G, Takahashi D. Commensal microbe-derived butyrate induces the differentiation of colonic regulatory T cells. Nature 2013; 504: 446–450. 38 Arpaia N, Campbell C, Fan X, Dikiy S, van der Veeken J, deRoos P. Metabolites produced by commensal bacteria promote peripheral regulatory T-cell generation. Nature 2013; 504: 451–455. 39 Smith PM, Howitt MR, Panikov N, Michaud M, Gallini CA, Bohlooly-Y M. The microbial metabolites, short-chain fatty acids, regulate colonic Treg cell homeostasis. Science 2013; 341: 569–573. 40 Candido EP, Reeves R, Davie JR. Sodium butyrate inhibits histone deacetylation in cultured cells. Cell 1978; 14: 105–113. 41 Licciardi PV, Wong S-S, Tang ML, Karagiannis TC. Epigenome targeting by probiotic metabolites. Gut Pathogens 2010; 2: 24. 42 Annison G, Illman RJ, Topping DL. Acetylated, propionylated or butyrylated starches raise large bowel short-chain fatty acids preferentially when fed to rats. J Nutr 2003; 133: 3523–3528. 43 Chang PV, Hao L, Offermanns S, Medzhitov R. The microbial metabolite butyrate regulates intestinal macrophage function via histone deacetylase inhibition. Proc Natl Acad Sci USA 2014; 111: 2247–2252. 44 Singh N, Gurav A, Sivaprakasam S, Brady E, Padia R, Shi H. Activation of gpr109a, receptor for niacin and the commensal metabolite butyrate, suppresses colonic inflammation and carcinogenesis. Immunity 2014; 40: 128–139. 45 Frank DN, Amand AL St, Feldman RA, Boedeker EC, Harpaz N, Pace NR. Molecular-phylogenetic characterization of microbial community imbalances in human inflammatory bowel diseases. Proc Natl Acad Sci USA 2007; 104: 13780–13785. 46 Obata Y, Furusawa Y, Endo TA, Sharif J, Takahashi D, Atarashi K. The epigenetic regulator Uhrf1 facilitates the proliferation and maturation of colonic regulatory T cells. Nat Immunol 2014; 15: 571–579. 47 Sharif J, Muto M, Takebayashi S-I, Suetake I, Iwamatsu A, Endo TA. The SRA protein Np95 mediates epigenetic inheritance by recruiting Dnmt1 to methylated DNA. Nature 2007; 450: 908–912. 48 Yang W, Bancroft L, Augenlicht LH. Methylation in the p21WAF1/cip1 promoter of Apc+/-, p21+/- mice and lack of response to sulindac. Oncogene 2005; 24: 2104–2109. 49 Nielsen AL, Ortiz JA, You J, Oulad-Abdelghani M, Khechumian R, Gansmuller A. Interaction with members of the heterochromatin protein 1 (HP1) family and histone deacetylation are differentially involved in transcriptional silencing by members of the TIF1 family. EMBO J 1999; 18: 6385–6395. 50 Schultz DC, Ayyanathan K, Negorev D, Maul GG, Rauscher FJ. SETDB1: a novel KAP-1-associated histone H3, lysine 9-specific methyltransferase that contributes to HP1-mediated silencing of euchromatic genes by KRAB zinc-finger proteins. Genes Dev 2002; 16: 919–932. 51 Chikuma S, Suita N, Okazaki I-M, Shibayama S, Honjo T. TRIM28 prevents autoinflammatory T cell development in vivo. Nat Immunol 2012; 13: 596–603. 52 Peterson LW, Artis D. Intestinal epithelial cells: regulators of barrier function and immune homeostasis. Nat Rev Immunol 2014; 14: 141–153. 53 Kim YS, Ho SB. Intestinal goblet cells and mucins in health and disease: recent insights and progress. Curr Gastroenterol Rep 2010; 12: 319–330. 54 Furness JB, Rivera LR, Cho H-J, Bravo DM, Callaghan B. The gut as a sensory organ. Nat Rev Gastroenterol Hepatol 2013; 10: 729–740. 55 Clevers HC, Bevins CL. Paneth cells: maestros of the small intestinal crypts. Annu Rev Physiol 2013; 75: 289–311. 56 Robertson KD. DNA methylation and human disease. Nat Rev Genet 2005; 6: 597–610. 57 Suetake L, Shi L, Watanabe D, Nakamura M, Tajima S. Proliferation stage-dependent expression of DNA methyltransferase (Dnmt1) in mouse small intestine. Cell Struct Funct 2001; 26: 79–86.

Immunology and Cell Biology

58 Sheaffer KL, Kim R, Aoki R, Elliott EN, Schug J, Burger L. DNA methylation is required for the control of stem cell differentiation in the small intestine. Genes Dev 2014; 28: 652–664. 59 Obata Y, Takahashi D, Ebisawa M, Kakiguchi K, Yonemura S, Jinnohara T. Epithelial cell-intrinsic Notch signaling plays an essential role in the maintenance of gut immune homeostasis. J Immunol 2012; 188: 2427–2436. 60 Alenghat T, Osborne LC, Saenz SA, Kobuley D, Ziegler CGK, Mullican SE. Histone deacetylase 3 coordinates commensal-bacteria-dependent intestinal homeostasis. Nature 2013; 504: 153–157. 61 Turgeon N, Blais M, Gagné J-M, Tardif V, Boudreau F, Perreault N. HDAC1 and HDAC2 restrain the intestinal inflammatory response by regulating intestinal epithelial cell differentiation. PLoS ONE 2013; 8: e73785. 62 Xia D, Wang D, Kim S-H, Katoh H, DuBois RN. Prostaglandin E2 promotes intestinal tumor growth via DNA methylation. Nat Med 2012; 18: 224–226. 63 Kryczek I, Lin Y, Nagarsheth N, Peng D, Zhao L, Zhao E. IL-22(+)CD4(+) T cells promote colorectal cancer stemness via STAT3 transcription factor activation and induction of the methyltransferase DOT1L. Immunity 2014; 40: 772–784. 64 Feng Q, Wang H, Ng HH, Erdjument-Bromage H, Tempst P, Struhl K. Methylation of H3-lysine 79 is mediated by a new family of HMTases without a SET domain. Curr Biol 2002; 12: 1052–1058. 65 Kirchberger S, Royston DJ, Boulard O, Thornton E, Franchini F, Szabady RL. Innate lymphoid cells sustain colon cancer through production of interleukin-22 in a mouse model. J Exp Med 2013; 210: 917–931. 66 Huber S, Gagliani N, Zenewicz LA, Huber FJ, Bosurgi L, Hu B. IL-22BP is regulated by the inflammasome and modulates tumorigenesis in the intestine. Nature 2012; 491: 259–263. 67 Thompson CL, Plummer SJ, Tucker TC, Casey G, Li L. Interleukin-22 genetic polymorphisms and risk of colon cancer. Cancer Causes Control 2010; 21: 1165–1170. 68 Zheng Y, Valdez PA, Danilenko DM, Hu Y, Sa SM, Gong Q. Interleukin-22 mediates early host defense against attaching and effacing bacterial pathogens. Nat Med 2008; 14: 282–289. 69 Sabat R, Ouyang W, Wolk K. Therapeutic opportunities of the IL-22-IL-22R1 system. Nat Rev Drug Discov 2014; 13: 21–38. 70 Behnsen J, Jellbauer S, Wong CP, Edwards RA, George MD, Ouyang W. The cytokine IL-22 promotes pathogen colonization by suppressing related commensal bacteria. Immunity 2014; 40: 262–273. 71 Polk DB, Peek RM. Helicobacter pylori: gastric cancer and beyond. Nat Rev Cancer 2010; 10: 403–414. 72 Nakajima T, Maekita T, Oda I, Gotoda T, Yamamoto S, Umemura S. Higher methylation levels in gastric mucosae significantly correlate with higher risk of gastric cancers. Cancer Epidemiol Biomarkers Prev 2006; 15: 2317–2321. 73 Maekita T, Nakazawa K, Mihara M, Nakajima T, Yanaoka K, Iguchi M. High levels of aberrant DNA methylation in Helicobacter pylori-infected gastric mucosae and its possible association with gastric cancer risk. Clin Cancer Res 2006; 12: 989–995. 74 Cheng ASL, Li MS, Kang W, Cheng VY, Chou J-L, Lau SS. Helicobacter pylori causes epigenetic dysregulation of FOXD3 to promote gastric carcinogenesis. Gastroenterology 2013; 144: 122–133 e9. 75 Ando T, Yoshida T, Enomoto S, Asada K, Tatematsu M, Ichinose M. DNA methylation of microRNA genes in gastric mucosae of gastric cancer patients: its possible involvement in the formation of epigenetic field defect. Int J Cancer 2009; 124: 2367–2374. 76 Kiga K, Mimuro H, Suzuki M, Shinozaki-Ushiku A, Kobayashi T, Sanada T. Epigenetic silencing of miR-210 increases the proliferation of gastric epithelium during chronic Helicobacter pylori infection. Nat Commun 2014; 5: 4497. 77 Tolg C, Sabha N, Cortese R, Panchal T, Ahsan A, Soliman A. Uropathogenic E. coli infection provokes epigenetic downregulation of CDKN2A (p16INK4A) in uroepithelial cells. Lab Invest 2011; 91: 825–836. 78 Rocco JW, Sidransky D. p16(MTS-1/CDKN2/INK4a) in cancer progression. Exp Cell Res 2001; 264: 42–55. 79 Kantor AF, Hartge P, Hoover RN, Narayana AS, Sullivan JW, Fraumeni JF. Urinary tract infection and risk of bladder cancer. Am J Epidemiol 1984; 119: 510–515. 80 Jiang X, Castelao JE, Groshen S, Cortessis VK, Shibata D, Conti DV. Urinary tract infections and reduced risk of bladder cancer in Los Angeles. Br J Cancer 2009; 100: 834–839. 81 Constância M, Hemberger M, Hughes J, Dean W, Ferguson-Smith A, Fundele R. Placental-specific IGF-II is a major modulator of placental and fetal growth. Nature 2002; 417: 945–948. 82 Bobetsis YA, Barros SP, Lin DM, Weidman JR, Dolinoy DC, Jirtle RL. Bacterial infection promotes DNA hypermethylation. J Dent Res 2007; 86: 169–174. 83 Aagaard K, Ma J, Antony KM, Ganu R, Petrosino J, Versalovic J. The placenta harbors a unique microbiome. Sci Transl Med 2014; 6: 237ra65. 84 Brand S, Teich R, Dicke T, Harb H, Yildirim AÖ, Tost J. Epigenetic regulation in murine offspring as a novel mechanism for transmaternal asthma protection induced by microbes. J Allergy Clin Immunol 2011; 128: 618–625 e1–7. 85 Yin L, Chung WO. Epigenetic regulation of human β-defensin 2 and CC chemokine ligand 20 expression in gingival epithelial cells in response to oral bacteria. Mucosal Immunol 2011; 4: 409–419. 86 Murata M, Azuma Y, Miura K, Rahman MA, Matsutani M, Aoyama M. Chlamydial SET domain protein functions as a histone methyltransferase. Microbiology 2007; 153: 585–592.

Epigenetic regulation of the immune system Y Obata et al 7 87 Garcia-Garcia JC, Barat NC, Trembley SJ, Dumler JS. Epigenetic silencing of host cell defense genes enhances intracellular survival of the rickettsial pathogen Anaplasma phagocytophilum. PLoS Pathog 2009; 5: e1000488. 88 Eskandarian HA, Impens F, Nahori M-A, Soubigou G, Coppée J-Y, Cossart P. A role for SIRT2-dependent histone H3K18 deacetylation in bacterial infection. Science 2013; 341: 1238858. 89 Takahashi K, Sugi Y, Hosono A, Kaminogawa S. Epigenetic regulation of TLR4 gene expression in intestinal epithelial cells for the maintenance of intestinal homeostasis. J Immunol 2009; 183: 6522–6529.

90 Takahashi K, Sugi Y, Nakano K, Tsuda M, Kurihara K, Hosono A. Epigenetic control of the host gene by commensal bacteria in large intestinal epithelial cells. J Biol Chem 2011; 286: 35755–35762. 91 Ohkura N, Hamaguchi M, Morikawa H, Sugimura K, Tanaka A, Ito Y. T cell receptor stimulation-induced epigenetic changes and Foxp3 expression are independent and complementary events required for Treg cell development. Immunity 2012; 37: 785–799. 92 Ciofani M, Madar A, Galan C, Sellars M, Mace K, Pauli F. A validated regulatory network for Th17 cell specification. Cell 2012; 151: 289–303.

Immunology and Cell Biology