The Genetics and Epigenetics of Atopic Dermatitis Filaggrin and Other Polymorphisms

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1 DOI /s The Genetics and Epigenetics of Atopic Dermatitis Filaggrin and Other Polymorphisms Yunsheng Liang 1 & Christopher Chang 2 & Qianjin Lu 1 # Springer Science+Business Media New York 2015 Abstract Atopic dermatitis (AD) is a chronic inflammatory skin disease caused by a combination of genetic and environmental factors. Genetic evidences depict a complex network comprising by epidermal barrier dysfunctions and dysregulation of innate and adaptive immunity in the pathogenesis of AD. Mutations in the human filaggrin gene (FLG) are the most significant and well-replicated genetic mutation associated with AD, and other mutations associated with epidermal barriers such as SPINK5, FLG-2, SPRR3, and CLDN1 have all been linked to AD. Gene variants may also contribute to the abnormal innate and adaptive responses found in AD, including mutations in PRRs and AMPs, TSLP and TSLPR, IL-1 family cytokines and receptors genes, vitamin D pathway genes, FCER1A, and Th2 and other cytokines genes. GWAS and Immunochip analysis have identified a total of 19 susceptibility loci for AD. Candidate genes at these susceptibility loci identified by GWAS and Immunochip analysis also suggest roles for epidermal barrier functions, innate and adaptive immunity, interleukin-1 family signaling, regulatory T cells, the vitamin D pathway, and the nerve growth factor pathway in the pathogenesis of AD. Increasing evidences show the modern lifestyle (i.e., the hygiene hypothesis, Western diet) * Qianjin Lu epigenetics2010@126.com 1 2 Christopher Chang c3chang@yahoo.com Hunan Key Laboratory of Medical Epigenomics & Department of Dermatology, The Second Xiangya Hospital, Central South University, 139 Renmin Middle Rd, Changsha, Hunan , People s Republic of China Division of Rheumatology, Allergy and Clinical Immunology, University of California at Davis, Davis, CA 95616, USA and other environmental factors such as pollution and environmental tobacco smoke (ETS) lead to the increasing prevalence of AD with the development of industrialization. Epigenetic alterations in response to these environmental factors, including DNA methylation and microrna related to immune system and skin barriers, have been found to contribute to the pathogenesis of AD. Genetic variants and epigenetic alteration might be the key tools for the molecular taxonomy of AD and provide the background for the personalized management. Keywords Atopic dermatitis. Genetics. Epigenetics. Epidermal barrier. Filaggrin. Polymorphism Introduction Atopic dermatitis is a chronic inflammatory skin disease, which results from a mixture of genetic and environmental factors [1]. The growing repertoire of genetic studies depict a complex network of epidermal barrier dysfunctions and dysregulation of innate and adaptive immunity, interleukin-1 family signaling, regulatory T cells, the vitamin D pathway, and nerve growth factor pathway in the pathogenesis of AD [2 7]. Increasing evidence shows that the modern lifestyle (i.e., the hygiene hypothesis, Western diet) and other environmental factors such as pollution and environmental tobacco smoke (ETS) lead to an increasing prevalence of AD, also correlating with the development of industrialization [8 11]. However, our understanding of the underlying mechanisms through which these environmental factors increase the prevalence of AD remains incomplete. Epigenetics provide a new explanation of gene-environment interactions [12, 13]. Abnormal epigenetic modifications mediated by environmental exposures have been suggested to contribute to the pathogenesis of AD

2 [11, 14 18]. In this article, we review recent insights into the roles of genetics and epigenetic factors in the pathogenesis of AD. Genetics and AD Polymorphisms in Genes of Epidermal Barriers The epidermal barrier, particularly the stratum corneum (SC) layer, is the first line of defense between the host organism and its environment. The SC layer is the culminated product of a complex keratinocyte differentiation process. The SC matrix, corneodesmosomes and tight junctions form a strong but resilient physical barrier which minimizes water loss and protects the body from allergen or microbial penetration [19]. The critical role of skin barrier dysfunction as a causative factor in AD is supported by reports demonstrating that loss-offunction mutations in the filaggrin gene (FLG) are the most significant and well-replicated risk factor for development of AD [20 22]. Recent studies indicate that defects in terminal keratinocyte differentiation leading to reduced levels of ceramides, filaggrin, and antimicrobial peptides contributing greatly to the triggering and perpetuation of skin inflammation in AD [23]. FLG Mutations Filaggrin is a major structural protein in the SC. Filaggrin is generated from the precursor proprotein profilaggrin which is expressed in terminally differentiating keratinocytes and is the major constituent of keratohyalin granules in the stratum granulosum. During terminal differentiation at the granular to cornified layer transition, profilaggrin is rapidly dephosphorylated and cleaved by several endoproteases to generate functional filaggrin monomers. Filaggrin aggregates the keratin cytoskeleton to facilitate the flattening of keratinocytes in the outermost skin layer [19, 20, 22]. In the upper layers of the SC, filaggrin is proteolyses into trans-urocanic acid (trans- UA) and pyrrolidine carboxylic acid (PCA) which contribute to the composition of natural moisturizing factor (NMF) [20]. Additionally, these organic acid breakdown products help maintain the acidic ph gradient of the epidermis. The acidic ph is key for many functions of the SC and affects or modulates antimicrobial activity, the activity of enzymes involved in ceramide metabolism and the serine protease cascade required for epidermal differentiation and cornified cell envelope formation [21]. FLG is located in the epidermal differentiation complex on chromosome 1q21. Exon 3 of FLG encodes almost the entire profilaggrin protein. Forty-nine truncating mutations, including the two initially reported mutations (R501X and 2282del4), throughout the length of the profilaggrin molecule have been described and the incidence of these mutations varies considerably among different ethnic groups and populations. Loss-of-function mutations within exon 3 result in a truncated profilaggrin molecule lacking the C-terminus and hence an absence of filaggrin [22]. FLG mutations were initially shown to be strongly associated with AD in an Irish population and with atopic dermatitis plus asthma in a Scottish population in 2006 [24]. These findings have been widely replicated in more than 30 independent studies. Meta-analyses of studies involving many thousands of patients have confirmed these associations with an overall odds ratio for atopic dermatitis ranging from 3.12 to 4.78 [25, 26]. Population cohort studies have shown moderate severe AD cases has a higher odds ratio for filaggrin mutations than mild moderate AD cases. Approximately 50 % of moderateto-severe AD cases can be attributed, at least in part, to FLGnull mutations, whereas up to 15 % of mild-to-moderate AD cases can be explained by FLG [27]. Patients with AD who carry FLG mutations (AD FLG )havemoreearly-onset,severe, and persistent disease [28 30], a higher incidence of eczema herpeticum [31], and a greater risk of multiple allergies and asthma than patients with atopic dermatitis without FLG mutations (AD NON-FLG )[32 34]. FLG loss-of-function mutations confer an overall risk of 1.48 to 1.79 for asthma, but this risk is limited to those who have AD or a history of the disease [32, 35]. Patients with AD FLG have a much greater risk of asthma than patients with AD NON-FLG. Asthmatic patients with FLG mutations have a more difficult disease course and more frequent exacerbations [33]. FLG mutations also confer a significant risk for peanut allergy with overall OR of 5.3 and a residual OR of 3.8 when corrected for AD [34]. FLG mutations are associated with the filaggrin breakdown products trans-ua and PCA which are major components of NMF [20]. These products have been shown to be accurate proxy markers of FLG genotype in patients with moderate tosevere AD [36]. FLG mutations are associated with SC IL-1 cytokines in patients with AD FLG [37]. IL-1 cytokine overexpression produced by keratinocytes leads to cutaneous inflammation [38]. Multiple proteases are necessary for epidermal homeostasis and cleavage of IL-1 cytokines at optimal acidic ph values. SC IL-1α, IL-1β IL-18, and IL-1 receptor antagonist levels were recently shown to be increased in the uninvolved skin of patients with moderate-to-severe AD FLG compared with that seen in patients with AD NON-FLG [37]. Thus, it is possible that a reduction in filaggrin and its acidic breakdown products increases ph and serine protease activity, contributing to the generation of the active IL-1 cytokines, which might be the first stage of the cytokine cascade that contributes to AD inflammation. This work suggests that there might be a preexisting or enhanced proinflammatory status in the skin of patients with AD FLG.

3 AD risk is also related to filaggrin CNV in a dosedependent fashion. At least three copy number variants (CNV), 10, 11, or 12 repeat, in filaggrin alleles are recognized in normal populations. The lowest CNV genotype (10-10 filaggrin repeats) carried by 11.5 % of the Irish population had an eczema odds ratio (OR) of 1.67 independently of FLG loss-of-functions mutations. The addition of each additional filaggrin repeat decreased the OR for AD by 0.88 [39]. These studies suggest that patients with AD FLG might have a distinct AD endophenotype and profile compared with patients with AD NON-FLG, and highlight the importance of skin barrier function in the pathogenesis of AD. This has driven a surge in research characterizing the filaggrin-deficient skin barrier and its consequences [20 22]. SPINK5 Mutations The serine protease inhibitor Kazal-type 5 (SPINK5)is located in the cluster of serine peptidase inhibitors on chromosome 5q31 and encodes the serine protease inhibitor lymphoepithelial Kazal-type-related inhibitor (LEKTI) expressed in the epithelium and mucous membranes [40, 41]. LEKTI contributes to the regulation of proteolysis in keratinocyte differentiation and the generation of normal epithelium through inhibiting the activity of kallikreins (KLKs), the serine protease in epidermis [41]. SPINK5 mutations are associated with AD in certain populations, especially eastern Asian populations [42 46]. Loss-of-function mutations in SPINK5 might result in excessive KLKs activity which degrades lipid-processing enzymes and corneodesmosome constituent proteins, and then provokes permeability barrier defects [41, 47]. Interestingly, the LEKTI-deficient mouse model provides unique insights into the pathogenesis of AD that kallikrein 5 binds to the protease activated type 2 receptor (PAR2), stimulating NFκB dependent production of thymic stromal lymphopoietin (TSLP) [48]. A recent study suggests that the E420K LEKTI variant is a risk factor for AD through an increase in the expression of TSLP [47]. Other Mutations in Genes Related to Epidermal Barrier Recent studies have found many other mutations involved in corneocyte envelope (CE) formation are associated with AD. Defects of filaggrin-like proteins have been found in both lesional and nonlesional atopic skin [49, 50]. Filaggrin-2 (FLG-2) is a filaggrin-like protein as differentiation-specific components of the corneocyte envelope [51]. FLG-2 mutations are linked to more persistent AD in African American populations [52]. Mutations in the cornified envelope precursor (SPRR3) which encodes a CE precursor protein, including an extra 24-bp defect in the central domain, as well as additional in-frame deletions and insertions, have been associated with AD [53, 54]. These mutations result in overexpression of SPRR3 in patients with AD which likely impair the supramolecular organization of lamellar body-derived lipids into normal bilayer structures through production of a CE scaffold. Very recent studies demonstrate that mutations which impair the delivery of lamellar body contents are associated with AD. Nonsense and missense mutations in the gene TMEM79, which encodes the protein mattrin, have been identified in some Irish patients with AD who lack FLG mutations [55]. Mattrin localizes to the cytosol and more specifically within the trans-golgi network in the outermost cells of the granular layer. Reductions in mattrin levels block the secretion of lamellar body contents, including desquamatory proteases, antiproteases, and lamellar-derived lipids [56]. Defects in tight junctions have been revealed in patients with AD. A strikingly reduced expression of the tight junction proteins, claudin-1 and claudin-23, and a remarkable impairment of the bioelectric barrier function has been found in the epidermis of AD patients [57]. CLDN1 haplotype-tagging single nucleotide polymorphisms reveal associations with AD in two North American populations [57]. Genetic variants in CLDN1 are also associated with risk of eczema herpeticum in AD subjects [57]. Polymorphisms in Inflammation and Immune-Related Genes AD is a complex chronic inflammatory disease and is characterized by an epidermal barrier abnormality, cutaneous inflammation, immune dysregulation with a systemic Ballergic^ Th2 skewing, and a Th17/Th22 cell response. Gene variants related to these immunological events contribute to abnormal inflammation and immune responses in AD [58, 59]. Mutations in PRRs and AMPs Pattern-recognition receptors (PRRs) protect organisms from microbial pathogens through recognition of pathogenassociated molecular patterns (PAMPs). In addition, antimicrobial peptides (AMPs) play a key role in the clearance of pathogens in skin barriers [60]. Mutations in PRRs such as toll-like receptors (TLRs) and nucleotide-binding oligomerization domain-like receptors (NLRs) are associated with AD. The TLR2 polymorphisms A-16934T and R753Q were found in severe AD patients [61, 62]. The TLR2 R753Q mutation impairs TLR2 expression and modifies many cytokine production (IL-2, IL-6, IL-8, and IL-12) in AD patients [63 65]. The TLR4 A-896G mutation determines severe and complicated course of in children AD [66]. Two TLR6 polymorphisms had been found in AD children [67, 68]. The TLR9 promoter polymorphism C-1237T had been reported in patients with intrinsic AD [69, 70]. Several NLR gene polymorphisms in CARD4, CARD12, CARD15, NALP1, NALP12, and NOD1 had also been reported in AD patients [70 72].

4 Several SNPs of human β-defensin 1 (DEFB1) gene had been found to be associated with AD, and some of these SNPs had correlate with disease severity, hypereosinophilia, and specific IgE [73 76]. Mutations in IL-1 Family Cytokines and Receptors Genes IL-1 family cytokines play key roles in innate immunity and contribute to the pathogenesis of AD, including IL-1α,IL-1β, IL-18, and IL-33. SC IL-1α, IL-1β IL-18, and IL-1 receptor antagonist levels were recently shown to be increased in the uninvolved skin of patients with moderate-to-severe AD. IL-1 cytokines might represent the first stage of the cytokine cascade that contributes to AD inflammation [37]. Serum IL- 18R1/IL-18RAP may be a biomarker of AD severity [77]. IL-33 and its receptors, ST2 and IL-1RAcP, are increased in lesional skin of patients with AD. The IL-33/IL-1RL1 axis triggers the release of several proinflammatory mediators and induces systemic Th2-type inflammation [78]. The 2q12 contains the receptors of the IL-1 family cytokines (IL1RL1, IL18R1, and IL18RAP) and is a susceptibility loci for AD identified by GWAS [5]. Two studies have reported variants in the IL-18 gene that are associated with AD [79, 80]. Mutations in TSLP and TSLPR Thymic stromal lymphopoietin (TSLP) is mainly produced by keratinocytes and other epithelial cells and overexpressed in the epidermis in lesions of AD. TSLP exerts its biological functions by binding to a heterodimeric receptor consisting of the IL-7 receptor alpha chain (IL-7Rα) and the TSLP receptor chain (TSLPR) [81]. TSLP plays a crucial role in DCmediated Th2 inflammatory responses [82]. TSLP and IL7RA variations are associated with AD [83, 84], and a recent study found that TSLP variation is associated with less persistent AD [85]. Mutations in Vitamin D Pathway Genes Vitamin D plays crucial roles in cutaneous immunity, and vitamin D treatment improved eczema control in small clinical trials [86]. A case-control study investigating genetic association between childhood eczema and vitamin D pathway genes found rs in CYP27A1 to be associated with AD in southern Chinese. Peripheral blood eosinophil percentage was modulated by rs and rs of CYP2R1 as well as their haplotypes. CYP2R1 and VDR haplotypes altered eczema susceptibility, and GC rs7041 and CYP2R1 rs interacted to modulate total IgE among eczema patients [87]. Mutations in Th2 and Other Cytokines Gene The adaptive immune response in AD is associated with increased expression of the Th2 cytokines (IL-4, IL-5, IL-13, and IL-31) and the Th22 cytokine, IL-22 during the acute phase of AD [58, 59]. Several distinct polymorphisms of IL- 4, IL-5, IL-13, IL-4 receptor alpha (IL-4RA), IL-5 receptor alpha (IL-5RA), and IL-13 receptor alpha (IL-13RA) have been found to influence the susceptibility to AD in different populations [88 97]. Variants in signal transducer and activator of transcription 6 (STAT6), a key downstream transcription factor in IL-4- and IL-13-mediated responses, have been found to be associated with allergic diseases [98, 99]. A common haplotype encoding IL-31, a Th2 cytokine which induces severe pruritus, has been reported to be associated with the intrinsic non-ige-associated form of AD [100, 101]. Besides a strong intrinsic Th2 deviation, Th1 immune responses are attenuated in AD. Genetic variants in IL-12 and IL-12R [102, 103], IFNG and IFNGR1, as well as interferon regulatory factor (IRF)-2 were significantly associated with AD and eczema herpeticum (EH) [104, 105]. Other cytokine and receptor variants were also identified in AD, including IL-2, IL- 6, IL-9, and IL-10 [83, ]. Mutations in FCERIA Genetic polymorphisms of the alpha-chain of the high-affinity receptor for IgE (FCERIA) have been correlated with AD. SNPs in the promoter region of FCERIA have been associated with AD and/or high total IgE serum levels [ ]. The FCERIA gene plays a major role in the IgE response and allergic sensitization is its role in AD is supported by a large and replicated GWAS [115]. GWAS and Immunochip Analysis of AD GWAS is a comprehensive and unbiased approach to identify the genetic components of complex human diseases [116]. Immunochip analysis was recently developed to conduct deep replication of major autoimmune and inflammatory diseases, and fine mapping of established loci identified by GWAS [117]. GWAS and Immunochip analysis for AD have identified a total of 19 associated loci with a genome-wide level of significance (P< )[2 6]. The first GWAS of AD was reported in 2009 [6]. A susceptibility region on chromosome 11q13 located 38 kb downstream of C11orf30 was identified. The peak association was observed 68 kb upstream of leucine-rich repeat containing 32 (LRRC32). LRRC32 was previously reported to have specific expression in activated human naturally occurring regulatory Tcells[118]. Another association was observed at rs in the epidermal differentiation complex (EDC) on chromosome 1q21. Rs is in high LD with the FLG mutations,

5 and exclusion of FLG mutation carriers abolished the observed association. In 2011, a GWAS for AD in the Chinese Han population reported two new susceptibility loci, 5q22.1 and 20q13.33 [3]. The 5q22.1 region contains transmembrane protein 232 (TMEM232) and solute carrier family 25, member 46 (SLC25A46), and the TSLP gene is located adjacent ( 300 kb downstream) to the associated region. The 20q13.33 region contains the tumor necrosis factor receptor superfamily, member 6b (TNFRSF6B) gene that encodes decoy receptor 3 (DcR3). DcR3 is a secreted protein belonging to the tumor necrosis factor receptor family (TNFRSF) and it binds to LIGHT (TNFSF14). LIGHT is a target for asthma airway remodeling [119]. In 2011, a meta-analysis of GWAS for AD identified a total of three novel risk loci: 5q31.1 (KIF3A/IL4/IL13), 11q13.1 (OVOL1), and 19p13.2 (ACTL9) [4]. The 5q31.1 region contains a clustered family of Th2 cytokine genes, including IL13 and IL4. The 11q13.1 locus contains OVO homolog-like 1 (OVOL1), Ovol1 regulates the growth arrest of embryonic epidermal progenitor cells and represses c-myc transcription [120]. The 19p13.2 region contains the Ba disintegrin and metalloproteinase with thrombospondin motifs^ 10 (ADAMTS10) and actin-like 9 (ACTL9) genes. ADAMTS proteins are secreted zinc-dependent metalloproteinases and play a role in connective tissue remodeling and extracellular matrix turnover [121]. In 2012, GWAS for AD in the Japanese population identified eight novel regions of interest: 2q12 (IL1RL1/IL18R1/ IL18RAP), 3p21.33 (GLB1), 3q13.2 (CCDC80), 6p21.3 (the MHC region), 7p22 (CARD11), 10q21.2 (ZNF365/EGR2), 11p15.4 (OR10A3/NLRP10), and 20q13 (CYP24A1/ PFDN4) [5]. The 2q12 contains the receptors of the IL-1 family cytokines (IL1RL1, IL18R1, and IL18RAP). IL-1 family cytokines, including IL-1α, IL-1β, IL-18, and IL-33, play key roles in innate immunity and contribute to the pathogenesis of AD [38, 78]. The 3p21.33 region is adjacent to the CCR4 gene, which encodes a Th2-associated chemokine receptor for CCL22 and CCL17 (TARC). Serum TARC levels are useful for evaluation of the disease activity of AD [122, 123]. CCR4 is a skin-homing receptor of Th22 which is important in chronic AD [124]. The region at 3q13.2 contains CCDC80. CCDC80 is involved in induction of C/EBP α and peroxisome proliferator-activated receptor (PPAR) [125]. The region at 7p22 contains CARD11, which encodes CARMA1. CARMA1 has a critical role in the regulation of JunB and GATA3 and subsequent production of Th2 cytokines. The region at 10q21.2 contains early growth response protein 2 (EGR2), a T cell anergy-associated transcription factor involved in the negative regulation of T cell proliferation and inflammation [126]. The region at 11p15.4 contains NLRP10. NLRP10 is essential to initiate adaptive immunity and plays a role in the control of fungal infection through adaptive immunity [127]. The region at 20q13 includes CYP24A1, which encodes a mitochondrial cytochrome p450 superfamily enzyme. The protein acts as a degradation enzyme of 1,5- dihydroxyvitamin D3 [128]. A recent immunochip analysis for AD revealed four new susceptibility loci: 4q27 (IL-2/IL21), 11p13 (PRR5L), 16p13.13 (CLEC16A/DEXI), and 17q21.32 (ZNF652) [6]. The 4q27 region contains the IL2 and IL21 genes. The TNF receptor associated factors (TRAF6) (11p13), recombinationactivating gene 1 (RAG1) (11p13), RAG2 (11p13), suppressor of cytokine signaling 1 (SOCS1) (16p13.13), and nerve growth factor receptor (NGFR) (17q21.32) genes are located adjacent to those associated regions. The candidate genes identified by GWAS and Immunochip analysis suggest roles for epidermal barrier functions, innateadaptive immunity, interleukin-1 family signaling, regulatory T cells, the vitamin D pathway, andthe nervegrowthfactorpathway in the pathogenesis of atopic dermatitis [2 6](Table1). Epigenetics, Environment, and AD The prevalence of pediatric AD has increased throughout the world, and nor ranges from 10 to 20 % in developed countries. AD remains a major public health problem in many countries, particularly in the developing world, where the disease is still very much on the rise [1, 9]. Genetics alone is unable to explain the increasing prevalence of AD. With the development of industrialization, several factors have been proposed to account for this increase, including the modern lifestyle (i.e., the hygiene hypothesis, Western diet) and other environmental factors such as environmental tobacco smoke (ETS) and pollution [11, 18, ]. The introduction of epigenetics in the context of allergic diseases has opened a novel era to study the role of environmental factors in the pathogenesis of AD. Epigenetics is the study of heritable changes in gene expression that occur without directly altering the DNA sequence. Three main classes of epigenetic marks are DNA methylation, modifications of histone tails, and noncoding RNAs [132]. Recent evidence suggests that the epigenome is dynamic and changes in response to the environment, diet, and aging [12]. Abnormal epigenetic regulation of the immune system and skin barriers has been found to contribute to the pathogenesis of AD (Table 2). Abnormal Epigenetic Related to Immune System During immune cell development, epigenetic mechanisms mediate age-dependent and individual-specific changes in immune responses. The perinatal period surrounding birth is a Bprime-time^ for the induction of immunological tolerance and late-stage maturation of the immune system [133]. Mounting evidence suggests that disruption of the epigenetic

6 Table 1 Theroleofgeneticsinatopicdermatitis Epidermal barriers Innate immunity Gene and loci Rationale Reference FLG FLG mutations with a strong association with AD have been widely replicated [24 26] in more than 30 studies; FLG mutations result in an absence of filaggrin, which then leads to a functional epidermal barrier defect and allergic sensitization. SPINK5 SPINK5 mutations may result in excessive KLKs activity which degrades [41 47] lipid-processing enzymes and corneodesmosome constituent proteins, which then provokes permeability barrier defects. FLG-2 Filaggrin-2 is a filaggrin-like protein and is a component of the corneocyte [52] envelope. SPRR3 SPRR3 mutations result in overexpression of SPRR3, which likely impairs the [53, 54] supramolecular organization of lamellar body derived lipids into normal bilayer structures. TMEM79 TMEM79 encodes the protein mattrin, which regulates the secretion of [55, 56] lamellar body contents. CLDN1 CLDN1 encodes the tight junction protein claudin-1. A strikingly reduced [57] expression of the tight junction proteins, claudin-1 and claudin-23, has been found in the epidermis of AD patients. 1q21.3 rs in the epidermal differentiation complex (EDC), which is in high [6] LD with the FLG mutations. 11q13.1 The 11q13.1 locus contains OVO homolog-like 1 (OVOL1), Ovol1 regulates the growth arrest of embryonic epidermal progenitor cells and represses c-myc transcription. [4] TLRs The TLR2 polymorphisms A-16934T and R753Q have been found in severe PRRs AD patients. The TLR4 A-896G mutation is associated with a severe and complicated course of AD in children. TLR6 polymorphisms have been found in AD children. The TLR9 promoter polymorphism C-1237T has been reported in patients with intrinsic AD. NLRs Several NLR gene polymorphisms in CARD4, CARD12, CARD15, NALP1, NALP12, and NOD1 have also been reported in AD patients. AMPs DEFB1 Several SNPs of human β-defensin 1 (DEFB1) gene have been found to be associated with AD, and some of these SNPs have correlated with disease severity, hypereosinophilia, and specific IgE. IL-1 family cytokines and receptors 2q12, IL-18 IL-1 family cytokines play key roles in innate immunity and contribute to the pathogenesis of AD, including IL-1α, IL-1β, IL-18, and IL-33. The 2q12 contains the receptors of the IL-1 family cytokines(il1rl1, IL18R1, and IL18RAP), and variants in the IL-18 gene are associated with AD TSLP and TSLPR TSLP, IL7RA TSLP plays a crucial role in DC-mediated Th2 inflammatory responses. TSLP and IL7RA variations are associated with AD, and a recent study found that TSLP variation is associated with less persistent AD. Vitamin D pathway Adaptive immunity Th2 immunity Th1 immunity CYP27A1, CYP2R1, VDR, 20q13 IL-4, IL-5, IL-13, IL-4RA, IL-5RA, IL-13RA STAT6 IL-31 FCERIA IL-12, IL-12R, IFNG, IFNGR1, IRF-2 Vitamin D plays crucial roles in cutaneous immunity, and vitamin D treatment improved eczema control in small clinical trials. Variants in CYP27A1, CYP2R1, VDR, and 20q13 including CYP24A1 are associated with AD. Several distinct polymorphisms of IL-4, IL-5, IL-13, IL-4 receptor alpha (IL-4RA), IL-5 receptor alpha (IL-5RA) and IL-13 receptor alpha (IL-13RA) have been found to influence the susceptibility to AD in different populations. STAT6, a key down-stream transcription factor in IL-4 and IL-13 mediated responses, has been found to be associated with allergic diseases. A common haplotype encoding IL-31, Th2 cytokine which induces severe pruritus, has been reported to be associated with the intrinsic non-igeassociated form of AD. SNPs in FCERIA have been associated with AD and/or high total IgE serum levels. Genetic variants in IL-12 and IL-12R, IFNG and IFNGR1, as well as interferon regulatory factor (IRF)-2 were significantly associated with AD and eczema herpeticum (EH). [61 70] [70 72] [73 76] [5, 37, 77 80] [82 85] [5, 87] [88 97] [98, 99] [100, 101] [ ] [ ]

7 Table 1 (continued) Gene and loci Rationale Reference Treg LRRC32 at 11q13 LRRC32 is specific expressed in Treg cells. [6] Other cytokines IL-2, IL-2RA, IL-6, IL-6R, IL-9, IL-9R, IL-10 Other cytokine and receptor variants were also identified in AD, including IL-2, IL-6, IL-9, and IL-10. [6, 83, ] The susceptibility loci 4q27 region contains the IL2 and IL21 genes GAWS 5q11.1 TMEM232/SLC25A46 [3] 20q13.3 TNFRSF6B/ZGPAT [3] 19p13.2 ACTL9 [4] 3q13.2 CCDC80 [5] 6p21.3 The MHC region [5] 7p22 CARD11 [5] 10q21.2 ZNF365/EGR2 [5] 11p15.4 OR10A3/NLRP10 [5] Immunochip 11p13 PRR5L [6] 16p13.13 CLEC16A/DEXI [6] 17q21.32 ZNF652 [6] program mediated by environment factors, including environmental tobacco smoke (ETS), pollutants, microbial contact, and nutrition, during early life might have lasting adverse consequences for immune development and allergic disease [133, 134]. Regulatory T cells (Tregs) play an essential role in early immune programming and shaping the immune response toward a proallergic or tolerant state. Tregs are best characterized by the expression of forkhead box transcription factor 3 (Foxp3), which is essential for Treg induction and stability [135]. Foxp3 is regulated by DNA methylation of its transcriptional regulatory regions. Demethylation of the Tregspecific demethylated region (TSDR) corresponds with the stability of FOXP3 expression in Tregs [136, 137]. Recent studies suggest that prenatal environmental factors (maternal allergy, maternal cytokine production, exposure to tobacco smoke) might modify DNA methylation of the FOXP3 locus in cord blood. Children with low Treg numbers, detected by means of demethylation of TSDR, at birth might be at a higher risk for developing AD or sensitization to food allergens in the first 3 years of life [14, 15, ]. Overexpression of the high-affinity receptor for immunoglobulin E (FcεRI) on monocytes and dendritic cells contributes to the pathogenesis of AD. Our study found that monocytes from AD patients show that a global DNA hypomethylation and demethylation of specific regulatory elements within the FCER1G locus contributes to FcεRI overexpression on monocytes from patients with AD [141]. Further study has found that TSLP-activated pstat5 pathway contributes to FCER1G demethylation and FcεRI overexpression on monocytes by recruiting DNA demethylase TET2 (Liang Y, unpublished data). In the neonatal immune system, epigenetic regulation might be biased against robust Th1-mediated immunity to prevent harmful cell-immune responses toward the developing fetus. The IFN-gamma (IFNG) gene, the prototypical Th1 cytokine gene, is hypermethylated in resting neonatal CD4+ cells compared with adult CD4+ cells [142]. Similarly, chromatin accessibility at the TBX21 locus, the master regulator of Th1 lineage commitment, is attenuated in neonatal CD4+ cells relative to adult cells [143]. After birth, exposure to a diverse range of microorganisms and establishment of the microbiota promotes necessary upregulation of Th1 immune responses via epigenetic modification. In mice, it has been shown that prenatal administration of Gram-negative bacteria results in histone H4 acetylation at the IFNG gene and associated upregulation of IFN-γ production in the offspring [144]. Epidemiologic studies have identified that alterations in the composition of commensal bacterial communities are associated with allergic disease [145]. For example, infants who develop allergies have altered commensal populations early in life [146, 147], and children who have undergone early treatment with antibiotics are at an increased risk of developing allergic diseases [148]. Studies in animal model systems have further implicated commensal-derived signals in influencing the development of Th2 cytokine-mediated allergic inflammation [149, 150]. These are some good examples of proof of the principle of hygiene hypothesis as a risk factor in allergic disease. However, the epigenetic mechanisms that contribute to the pathogenesis of allergic diseases remain poorly characterized. Exposure to environmental pollutants has been identified as a factor that can alter epigenetic profiles in the developing offspring and associate with allergic diseases. Altered methylation has been reported at the ACSL3 gene promoter (measured in cord blood) in association with maternal airborne polycyclic aromatic hydrocarbon (PAH) exposure and

8 Table 2 The role of epigenetics in atopic dermatitis Cell /tissue types Epigenetic alteration Outcome Reference Cord blood Methylation of TSDR in FOXP3 Maternal allergy, maternal cytokine production, and exposure to tobacco smoke may modify DNA methylation of the FOXP3 locus in cord blood. Children with low Treg numbers, detected by means of demethylation of TSDR, at birth might be at a higher risk of developing AD or food allergy in the first 3 years of life. Cord blood mir-223 Maternal tobacco exposure during pregnancy correlated with high mirna-223 and low Treg cell numbers, and the children with low Treg cell numbers at birth had a predisposition to AD during the first 3 years of life. Monocytes FCER1G metylation Monocytes from AD patients show that global DNA hypomethylation and demethylation of specific regulatory elements within the FCER1G locus contributes to FcεRI overexpression on monocytes from patients with AD. Epidermis DNA methylation Striking DNA methylation differences and correlation with altered transcript levels of genes were observed between lesional epidermis from patients with AD and healthy control epidermis. These genes are predominantly relevant for epidermal differentiation and innate immune response, including S100A genes located within the epidermal differentiation complex, KRT6A and KRT6B genes encoding keratins located within the keratin clusters, as well as OAS1, OAS2, and OAS3, which belong to a family of proteins regulated by IFN and are involved in the innate immune response. Keratinocyte TSLP methylation DNA demethylation of a specific regulatory region of the TSLP gene may contribute to TSLP overexpression in skin lesions in patients with AD. Lesions mirna arrays in AD lesions Upregulation: mir-155, MiR-21, mir146a, mir-17-5p, mir223, mir142-3p/5p, etc. Downregulation: mir-122a, mir-326, mir-133b, mir-375, mir-125b, etc. Lesions Upregulated mir-155 in AD lesions Topical exposure of nonlesional skin of patients with atopic dermatitis to relevant allergens has been found to induce mir-155 expression. Upregulation of mir-155 has been shown to suppress CTLA-4, a negative regulator of T cell function. This CTLA-4 suppression in turn enhances the T cell proliferative response, which then leads to maintenance of a chronic inflammatory state. [14, 15] [14] [141] [16] [167] [172, 173] [173] parent-reported asthma in children prior to age 5 [151]. A follow-up study reported that maternal PAH exposure was associated with increased promoter methylation of IFNG in cord blood DNA, and in vitro exposure of Jurkat cell lines to noncytotoxic doses of PAH confirmed increased IFNG methylation and reduced expression of IFNG [152]. Epidemiologic data suggest that changes in dietary patterns (Western diet) may also partially explain the increasing prevalence of allergic diseases in industrialized countries [18, 153, 154]. Potential mechanisms through which diet is suspected to effect allergic diseases have been purported to be epigenetic changes, including DNA methylation. Dietary methyl donors, including folate and choline, are necessary for the one-carbon metabolic pathway that produces S-adenosyl-methionine (SAM), which is the universal methyl donor that is essential for the DNA methylation process to occur. Vitamin B12 and betaine, which are additional agents derived from the diet, are also necessary for methionine synthesis, which is another important step in DNA methylation. Therefore, differential intake of these nutrients might lead to differences in DNA methylation and ultimately alter gene expression [18]. It is been shown that the methylation changes induced by a diet high in methyl donors might result in increased atopic diseases in a mouse model. In utero supplementation of a diet high in methyl donors (including folic acid, choline, and vitamin B12) resulted in increased atopy and airway responsiveness in the exposed progeny, which is associated with increased methylation of the Runt-related transcription factor (Runx3) gene and decreased expression of Runx3. Runx3 is known to negatively regulate allergic airway disease [155]. However, the results of the studies investigating prenatal and postnatal dietary methyl donor intake or status that have been published to date have been mixed [ ], thereby precluding any definitive conclusions on the role of dietary methyl donors with regard to its association with allergic diseases. Abnormal Epigenetic Alterations to Skin Barriers In a recent epigenome-wide association study, striking DNA methylation differences and correlation with altered transcript levels of genes were observed between lesional epidermis from patients with AD and healthy control epidermis [16]. These genes are predominantly relevant for epidermal differentiation and the innate immune response, including S100A genes located within the epidermal differentiation complex, KRT6A and KRT6B genes encoding keratins located within the keratin clusters [163, 164], and OAS1, OAS2, and OAS3, which belong to a family of proteins regulated by IFN and which are involved in the innate immune response [165]. Together, these findings indicate altered methylation and expression of genes important for keratinocyte differentiation,

9 proliferation, and innate immune response in the lesional epidermis of AD patients. TSLP plays a crucial role in the development of allergic symptoms, especially in asthma and atopic dermatitis (AD). Human TSLP is overexpressed in keratinocytes of patients with acute and chronic AD [166]. Our study found that DNA demethylation of a specific regulatory region of the TSLP gene may contribute to TSLP overexpression in skin lesions in patients with AD [167]. Interestingly, a recent study found that prenatal tobacco smoke exposure may lead to TSLP DNA demethylation in human cord blood, which is positively associated with AD [11]. MicroRNAs in AD MicroRNAs (mirnas) are short, single-stranded RNA molecules that function together with their partner proteins and cause degradation of target mrnas or inhibit their translation [168]. mirnas play essential roles in a wide variety of biological processes, including proliferation, differentiation, cell fate determination, apoptosis, signal transduction, and organ development [169]. Furthermore, mirna-mediated control of gene expression has emerged as an important regulatory mechanism in immune homeostasis and the development of the lymphoid lineages [170]. Inhibition or overexpression of deregulated mirnas have been shown to have therapeutic consequences in human diseases [171], and thus, mirnas represent important potential targets for therapeutics and diagnostics in human diseases. mirnas upregulated in patients with other allergic disorders, including mir-21, mir-146, and mir-223, are also upregulated in the skin of patients with atopic dermatitis [172, 173]. A recent study showed that maternal tobacco exposure during pregnancy correlated with high mirna-223 and low Treg cell numbers, and the children with low Treg cell numbers at birth had a predisposition to AD during the first 3 years of life [14]. Notably, Sonkoly et al. found that mir-155 was one of the most upregulated mirnas in skin lesions from patients with AD compared with healthy control subjects. Topical exposure of nonlesional skin of patients with atopic dermatitis to relevant allergens was found to induce mir-155 expression. Functionally, mir-155 has been shown to suppress cytotoxic T lymphocyte associated protein 4 (CTLA- 4), a negative regulator of T cell function. This CTLA-4 suppression in turn enhances the T cell proliferative response, which may then lead to maintenance of a chronic inflammatory state [173]. Summary AD is common complex inflammatory skin disease with a strong genetic background. However, the 2-fold to 3-fold increase in the prevalence of AD in developed countries that coincided with the development of industrialization suggests that environmental factors are critical in determining disease expression [1, 9]. Several factors have been proposed to account for this increase, including the modern lifestyle (i.e., the hygiene hypothesis, Western diet) and environmental factors such as environmental tobacco smoke (ETS) and pollution [11, 18, ]. Interactions between susceptibility genes, environment, and immunologic factors contribute to the pathogenesis of AD (Fig. 1). Genetic evidence indicates that AD is a genetically complex disorder with epidermal barrier dysfunction and dysregulation of innate and adaptive immunity. Genetic variants in epidermal barrier, innate and adaptive immunity, interleukin-1 family signaling, regulatory T cells, the vitamin D pathway and the nerve growth factor pathway might contribute to the pathogenesis of AD. Epigenetics has opened a novel opportunity to study the role of gene-environment interactions in the pathogenesis of AD. Abnormal epigenetic regulation in immune system and skin barriers which may be mediated by environmental factors has been found to be associated with AD. For example, exposure to tobacco smoke may modify DNA methylation of the FOXP3 locus and mirna-223 expression in cord blood, which is associated with low Treg numbers and an increased risk for AD [14]. Abnormal DNA methylation may regulate expression of genes important for keratinocyte differentiation, Fig. 1 The possible genetic and epigenetic mechanisms involved in the pathogenesis of AD. Inherited genetic mutations and abnormal epigenetic modifications (i.e., DNA methylation, histone modification, and micrornas) mediated by the modern lifestyle (i.e., the hygiene hypothesis, Western diet) and environmental exposures such as pollution and environmental tobacco smoke (ETS) may lead to epidermal barrier dysfunctions as well as dysregulation of innate and adaptive immunity. The interactions between epidermal barrier dysfunctions and dysregulation of innate and adaptive immunity contribute to the pathogenesis of AD

10 proliferation, and innate immune response in the lesional epidermis of AD patients [16]. However, epigenetic evidence relevant to AD is still currently very limited, and further research must be done to clarify the underlying epigenetic mechanisms mediated by environmental factors in the pathogenesis of AD. Some of the areas of critical importance include epigenome alteration (i.e., DNA methylation, histone modification, and microrna) and epigenetic modifying zymogram of different cell types of the skin barriers and immune system of AD patients, the effects of potential environmental factors (i.e., ETS, dietary methyl donors, microbiota in skin and gut, pollution) on these epigenome alterations, and genetic and epigenetic interaction in AD-related genes. Accomplishing these monumental tasks can be partially fulfilled with the development of big-data biotechnology, which may bring novel epigenetic interventions and treatment in AD. AD has heterogeneous clinical phenotypes such as AD with early-onset, childhood AD as compared to adulthood AD, or AD with IgE-mediated allergic reactions, which may be distinguished by distinct genetic and epigenetic dispositions [174]. As an example, patients who carry FLG mutations (AD FLG ) would be predicted to have an early onset, severe, and persistent disease associated with a disturbed epidermal barrier function and a significant risk for subsequent IgE sensitization and the Batopic march^ [21]. Genetic variants and epigenetic alteration may be the key tools for the molecular taxonomy of AD and provide the background for personalized management strategies. Acknowledgments This study was supported by the National Natural Science Foundation of China (Nos , , and ). References 1. Bieber T (2008) Atopic dermatitis. N Engl J Med 358: Esparza-Gordillo J, Weidinger S, Folster-Holst R et al (2009) A common variant on chromosome 11q13 is associated with atopic dermatitis. 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