Epigenetics and Allergy: from Basic Mechanisms to Clinical Applications

Figures & data

Figure 1. Allergic (type 2) inflammation: basic mechanisms and pathophysiology, and selected clinical consequences.

Th2 cytokines can be synthesized also by innate lymphoid cells type 2 (ILC2), which is not shown. Contribution of Th9 cells, major IL-9 producers, is also not presented. MHC-II denotes MHC class II molecules; TCR, T cell receptor. For remaining abbreviations, detailed description of the figure content and additional information, please, refer to the main text. This figure was inspired by several previously published images, especially by the one by Gandhi et al. [23].

Figure 2. Major types of Th cells, their differentiation and its epigenetic regulation, and their crucial allergy-related functions.

DNA methylation (CpG) status of the loci pivotal for Th lineages is shown. Levels of epigenetic histone marks at loci specific for Th subpopulations is depicted (selection). For more details and abbreviations, please, refer to the main text. This figure was inspired by several previously published images, especially by the one by Suarez-Alvarez et al. [83].

Table 1. Overview of epigenome-wide association studies performed in allergic diseases.

Study	Allergy-related trait	Asthma definition	Technology used	Sample size of primary screen (cases/controls)	Tissue used/correction for cellular heterogeneity	Main findings	Validation	Comments	Ref.
Liang et al.	Serum IgE levels	Asthma with high and low IgE levels; physician-diagnosed asthma for replication	27K/450K	355 adults and children including 113 asthmatic children	PBMCs and eosinophils/yes	36 loci validated in all three cohorts with high confidence; a number of CpGs with correlation between methylation and IgE levels including IL4, IL5RA, LPCAT2, ZNF22, SLC25A33	Two cohorts: n = 149 (extreme IgE levels) and n = 160 asthmatic families)	Study shows a tenfold greater capacity of DNA methylation to explain variability in serum IgE levels compared with all so far performed GWAS	[210]
Hong et al.	CMA	–	450K	106/76 children	Whole blood/yes	575 DMPs (568 hypo/7 hyper) including CpGs in or close to NDFIP2, EVL, TRAPPC9, RPS6K2 (all> 5%), ILIRL1, IL5RA, STAT4, IL4, CCL18	Yes; 10 loci/2 cohorts: 5 CMA cases/20 controls whole blood, white children and 8 CMA cases/132 controls CB black children	Only 5 CpGs with diff. methylation >5%; reasonable replication rate in blood, not in CB; potential functional importance as in gene regulatory regions such as CTCF sites	[303]
Petrus et al.	CMA	–	450K	20/23 children and including 10 boys that outgrew CMA	Whole blood/no	Hypermethylation in CMA	Amplicon bisulfite sequencing of four genes (n = not specified)	No correction for cellular heterogeneity, small sample sizes	[207]
Martino et al.	Various IgE-mediated FA (mainly hen’s egg)	–	450K	12 children with FA/12 controls; 2 time points (birth and 1 year)	CD4^+ T cells from CBMCs and PBMCs/NA	179 CpGs associated with FA at 1 year, 136 CpGs at birth, 92 common to both time points	No	Longitudinal study with sample at birth and 1 year showing presence of epigenetic alterations predicting later food allergy; measurement of gene expression	[206]
Martino et al.	Multiple FA	–	450K	58 food-sensitized children (50% clinically reactive)/13 controls	PBMCs/yes	DNA methylation signature comprised of 96 CpGs predicted clinical outcome	Yes; 48 samples from an independent FA cohort (79% accuracy of the methylation signature)	DNA methylation signature outperformed IgE and skin prick testing for oral food challenge outcome → potential for novel diagnostic assays	[221]
Nestor et al.	SAR	–	450K	8 cases/8 controls; sampled in and outside the pollen season	CD4^+ T cells/NA	DNA methylation signatures separate patients and controls even outside the pollen (357 genes diff. methylated, including mainly CpGs in gene bodies of HSPD1, TGFBR2, CD134 (TNFRSF4, OX40), CD45 (PTPRC), SPN (CD43), IL2RA, IL13RA1)	Technical validation of 3 CpGs in 4 SAR patients and 4 controls; similar affects seen by in vitro stimulation of PBMCs	Demonstrates the potential of DNA methylation based signatures; but very small sample size, and small differences per CpG site; expression profiles do not separate patients and controls; no single CpG differentiates patients and controls	[166]
Lockett et al.	Impact of season of birth on allergy development	–	450K	367 individuals (age 18)	Whole blood/yes	92 CpGs associated with season of birth, 20 CpGs nominally significant with allergy	207 children (age 8 years) replicates four of the CpGs	Methylation in newborns is not associated with season, suggesting a postnatal occurrence; longitudinal data available	[304]
Stefanowicz et al.	Atopy with/or without asthma	IgE skin prick test, but combined analysis of asthmatics (independent of atopy status)	GoldenGate	25 children	PBMCs and AECs/No	No diff. methylated CpGs in PBMCs, eight in AECs (atopy vs asthma) including STAT5A	No	Identification of diff. methylated CpGs between PBMCs and AECs in each subject category (∼10% of analyzed CpGs); expression analysis, but in a different data set	[209]
Morales et al.	Wheezing before age of 6 versus controls	–	GoldenGate	122 children	Whole blood/partly (eosinophil counts)	Hypomethylation of ALOX12 associated with persistent wheezing, partly related to DDE exposure	Independent cohort of 236 children	Epigenetic variation determined to a large part by genetic polymorphisms	[199]
Breton et al.	Asthmatics/prenatal tobacco smoke exposure	Symptoms of asthma or use of asthma medication	27K	527 children (age 5–12 years)	Whole blood/yes	19 CpGs altered in function of prenatal tobacco smoke exposure	2 CpGs replicated in independent cohorts (CB and adult whole blood)	Analyses show some loci to be replicated in different tissues; promoter-focused design limits the regions that can be analyzed	[183]
Joubert et al.	Prenatal tobacco smoke exposure	–	450K	1062 children	CB/yes	26 CpGs in 10 genes altered in function of prenatal tobacco smoke exposure	21/26 CpGs validated in a small cohort (n = 36)	Smoking assessed through maternal cotinine levels	[305]
Markunas et al.	Prenatal tobacco smoke exposure	–	450K	889 children	Whole blood shortly after delivery/yes	185 CpGs in 100 gene regions	In silico validation of ten gene regions using data from [305]	Smoking assessed through questionnaire; some overlap with previous EWAS	[306]
Richmond et al.	Prenatal tobacco smoke exposure	–	450K	800 children: three time points per child (birth, age 7 and 17 years)	CB/yes	15 CpGs sites associated with maternal smoking; dose – response relationship in function of smoking intensity and duration for some CpGs	No	Longitudinal design allows to identify CpGs that reverse methylation and where methylation persists	[307]
Joubert et al.	Prenatal tobacco smoke exposure	–	450K	6685 children (13 cohorts)	Various/yes	>6000 CpGs differentially methylated, including 2000 new ones	Replication in older children (n = 3187)	Large-scale EWAS identifying a huge number of new loci associated with maternal smoking supporting the need for large-scale analyses	[192]
Kim et al.	Atopic asthma	Differentiation of IgE-associated and non IgE-mediated asthma	27K	Atopic asthma (n = 7) versus nonatopic asthma (n = 7) versus controls (n = 10)	Bronchial mucosal tissue/not precised	Differential methylation in 52 genes in atopic asthma versus asthma, most of them hypomethylated	No	Atopy to dust mite	[308]
Rossnerova et al.	Asthma/pollution	Not precised	27K	200 children (age 7–15 years)	Whole blood/no	No methylation differences for asthma; 10K CpG sites diff. methylated between highly polluted and control regions	No	Very large number of diff. methylated CpGs might point to unknown confounders (e.g., blood cell composition)	[278]
Jiang et al.	Asthma/diesel exhaust exposure	Physician-diagnosed asthma	450K	16 asthmatics randomized to filtered air or diesel exhaust	PBMCs/yes	2827 CpG sites, mainly hypomethylated, enriched in protein kinase and NF-kB signaling	No	In addition, methylation changes in repetitive elements were observed; however, small sample size, no validation, replication, no inclusion of healthy controls	[309]
Somineni et al.	Asthma/pollution	Physician-diagnosed	450K	12/12 African American siblings	Nasal epithelial cells/no	Demethylation at a single CpG site in the TET1 promoter associated with asthma	35 additional siblings pairs with saliva, PBMCs, lung bronchial epithelial cells available; independent cohort n = 186	TET1 hypomethylation correlated with higher TET1 expression and higher global 5 hmC levels; increased methylation in function of traffic pollution; EWAS with robust multitissue validation	[169]
Clifford et al.	Allergen/diesel exhaust exposure	IgE-related diseases including asthma	450K	17 adult patients (47% asthmatics)	Bronchial epithelial cells/not precised	Altered methylation at 7 CpG sites 48 hours after exposure; if allergen and diesel exhaust were separated by 1 month, 500 diff. methylated (mainly hypomethylated) CpGs were identified suggesting a priming mechanism	1 CpG by pyrosequencing in the same samples	Exposure to filtered air or diesel exhaust followed by bronchoscopy for allergen delivery; CpGs differ in function of the order of allergen/diesel exposure; small cohort size	[310]
Yang et al.	Asthma	IgE-related asthma	450K	97 patients versus 97 controls	PBMCs/yes	81 DMRs between asthmatics and controls with hypomethylation of several immune related genes (IL13, RUNX3, TIGIT)	No	Few, but some DMRs related to IgE levels and percentage predicted FEV1 levels; integration of gene expression and DNA methylation data	[279]
Rastogi et al.	Asthma and obesity	Not precised	1.8–2 M CpG sites (sequencing based)	32 preadolescent children (8 per group asthmatics and controls with obesity or not)	PBMCs/no	Diff. methylated CpGs in immune related genes including CCL5, IL2RA, TBX21, FCER2, TGFB1	No	T cell signaling and macrophage activation pathways selectively hypomethylated in obese asthmatics	[311]
Amarasekara et al.	Allergy protective effects of fish oil supplementation during pregnancy	–	450K	70 mother–infant pairs; fish oil supplementation (n = 36), control group (n = 34)	CD4^+ T cells from CBMCs	No statistically significant CpGs at the genome-wide level in relation to treatment or PUFA levels	No	Some CpGs showed nonetheless a correlation to PUFA levels	[257]
Rodriguez et al.	Atopic dermatitis	–	27K	28 patients with atopic dermatitis, 29 controls	Whole, blood, T cells, B cells, skin epidermis	A large number of methylation differences between patients and controls are only found in the skin	No	Demonstrates the need for tissue-specific DNA methylation analyses; complementary expression analyses which show some overlap predominantly in genes implicated in epidermal differentiation and innate immune response	[208]

Table 1 summarizes a selection of EWAS analyzing at least 500 genes simultaneously in allergic diseases in humans. Studies in animal models are not listed.

450K: Infinium Human Methylation450 Bead Array (485,000 CpGs covering all genes); 27K: Infinium HumanMethylation27 BeadChip (27,578 CpG covering >14,000 genes); 5 hmC: 5-Hydroxymethylcytosine; AEC: Airway epithelial cell; CMA: Cow’s milk allergy; CB: Cord blood; CBMC: CB mononuclear cell; DDE: Dichlorodiphenyldichloroethylene; Diff.: Differentially; DMP: Differentially methylated position; FA: Food allergy; FEV1: Forced expiratory volume in one second; GoldenGate: Illumina GoldenGate BeadArray (1505 CpG sites – 807 genes); PBMCs: Peripheral blood mononuclear cells; PUFA: Polyunsaturated fatty acid; SAR: Seasonal allergic rhinitis.

Table

Abbreviation
A. lwoffii	Acinetobacter lwoffii
AD	Atopic dermatitis
AECs	Airway epithelial cells
AHR	Airway hyper-responsiveness
APCs	Antigen-presenting cells
AR	Allergic rhinitis
CB	Cord blood
CBMCs	CB mononuclear cells
ccfDNA	Circulating cell-free DNA
CMA	Cow’s milk allergy
CNS	Conserved non-coding sequence (e.g., CNS2)
CpG (site)	DNA sequence where a cytosine nucleotide (C) is directly followed by a guanine nucleotide (G)
DDE	Dichlorodiphenyldichloroethylene
DEPs	Diesel exhaust particles
Diff.	Differentially
DMPs	Diff. methylated positions
DNMTs	DNA methyltransferases (e.g. DNMT1, DNMT3A)
EPIT	Epicutaneous immunotherapy
EWAS	Epigenome-wide association study
FA	Food allergy
FACS	Fluorescent activated cell sorting
Fc RI	High-affinity IgE receptor
Fc RII (CD23)	Low-affinity IgE receptor
FOXP3	Forkhead box protein 3
GATA3	GATA binding protein 3
GEM (software)	Gene, Environment and Methylation (software)
GoldenGate	Illumina GoldenGate BeadArray (1,505 CpG sites – 807 genes)
GWAS	Genome-wide association study
H3/H4ac	Histone H3 or H4 acetylation (e.g. H3K9ac, H3K18ac)
H3K(X)me(x)	Histone H3K (X = e.g. K4, K27) di- (x = 2) or tri- (x = 3) methylation (e.g. H3K4me2, H3K27me3)
HATs	Histone acetyltransferases
HDACs	Histone deacetylases (e.g. HDAC9)
HDACi	HDAC inhibitors
HMTs	Histone methyltransferases
HRVs	Human rhinoviruses
IFN-	Interferon-
IgE	Immunoglobulin E
IL	Interleukin (e.g., IL-4, IL-5)
ILCs	Innate lymphoid cells (e.g., ILC2)
IRF4	Interferon regulatory factor 4
JNK2	c-Jun N-terminal kinase 2
LCR	Locus control region
MAPK9	Mitogen-activated protein kinase 9
MEDALL (consortium)	Mechanisms of the Development of Allergy (consortium)
MHC-II	Major histocompatibility complex class II
miRNA	MicroRNA
NOS	Nitric oxide synthase (e.g. NOS1, NOS2)
OIT	Oral immunotherapy
PACE (consortium)	Pregnancy And Childhood Epigenetics (consortium)
PAHs	Polycyclic aromatic hydrocarbons
PBMCs	Peripheral blood mononuclear cells
PUFAs	Polyunsaturated fatty acids
RISC	RNA-induced silencing complex
RORC2	RAR related orphan receptor C isoform 2 (traditionally called also ROR T)
RSV	Respiratory syncytial virus
SAR	Seasonal allergic rhinitis
SNPs	Single-nucleotide polymorphisms
SLIT	Sublingual immunotherapy
TBX21	T-box 21 (traditionally called also T-bet)
TCR	T-cell receptor
TET1	Tet methylcytosine dioxygenase 1
TFs	Transcription factors
TGF-	Transforming growth factor-
Th (cells)	T-helper (cells; e.g. Th2, Th9)
Treg cells	Regulatory T-cells
TSDR	Treg-specific demethylated region
TSLP	Thymic stromal lymphopoietin
27K	Infinium HumanMethylation27 BeadChip (27,578 CpG covering >14,000 genes)
450K	Infinium HumanMethylation450 BeadArray (485,000 CpGs covering all genes)
5hmC	5-hydroxymethylcytosine

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