epigenetics in human disease || epigenetic aberrations in human allergic diseases

CHAPTER 18

Epigenetic Aberrations inHuman Allergic Diseases
Manori Amarasekera1, David Martino2, Meri K. Tulic1, Richard Saffery2, Susan Prescott11University of Western Australia, Subiaco, WA, Australia2Murdoch Children’s Research Institute, Parkville, VIC, Australia
T.

C

CHAPTER OUTLINE

369

18.1 Introduction and Context: TheRising Prevalence of AllergicDiseases 369

18.2 Mechanisms of AllergicResponse 370

18.3 Fetal life: The Critical Period ofImmune Development 371

18.4 Developmental Differences inGene Expression in AllergicDisease 371

18.5 Epigenetic Regulation ofImmune Development 372

18.6 Factors that Modulate AllergicDisease Risk ThroughEpigenetic Mechanisms 37418.6.1 Epigenetic Effects of

Bacterial Exposure on

Immune

Development 374

Tollefsbol (Ed): Epigenetics in Human Disease. DOI: 10.1016/B978-0-12-3

opyright � 2012 Elsevier Inc. All rights reserved.

18.6.2 Epigenetic Effects of

Maternal Diet on Immune

Function 376

18.6.3 Epigenetic Effects of

Tobacco Smoke 377

18.6.4 Effects of Air Pollutants

and Other Outdoor

Pollutants on

Epigenetics 378

18.6.5 Other Maternal Factors

that May Modulate

Allergic Propensity in the

Newborn Through

Epigenetic

Mechanisms 379

18.7 Conclusions 379References 380

18.1 INTRODUCTION AND CONTEXT: THE RISING PREVALENCEOF ALLERGIC DISEASESThe prevalence of allergic diseases such as asthma, allergic rhinitis, and eczema has risen at analarming rate over the past 4e5 decades [1,2]. This has been clearly associated with the marked

environmental changes associated with transition to more modern lifestyles. Moreover, the

parallel rise in a wide range of other immune diseases during this short period providesadditional strong evidence that the immune system is highly susceptible to these environ-

mental changes [3]. Furthermore, there is mounting evidence that the effects of environmental

change are potentially greatest during critical periods of life, when epigenetic modifications inimmune gene expression can alter subsequent disease susceptibility.

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Epigenetics in Human Disease

The “allergy epidemic” was first evident in industrially developed countries initially as a surgeof respiratory diseases such as asthma, rising at approximately 5% per year towards the new

millennium [1]. The burden of these disorders is enormous, with more than 40% of the

population in developed countries experiencing allergic symptoms [4,5]. While the prevalenceof asthma and rhinitis may have reached a plateau, or may even have begun to decline in some

regions [6e8], the global burden of these diseases continues to rise as the prevalence ofrespiratory allergies increases in developing countries as they undergo economic and lifestyle

transition [6]. Of further concern, is an apparent “second wave” of allergic disease, manifest by

a much more recent rise in food allergy, now looming as an epidemic in developed countries[9]. Food allergy was still uncommon at the time of the “first wave” of respiratory allergic

disease, only emerging as a significant problem in the last 10e15 years. The reason for this

earlier and more dramatic presentation of the allergic phenotype is not clear, but as thisappears linked with ongoing environmental change, the same trends can also be anticipated in

developing regions. This rise in disease burden is most evident in infants and children under

3 years of age, further highlighting the likely role of early environmental exposures. Of yetfurther concern, there is also evidence of increasing disease persistence. Food allergies (such as

egg and milk allergy) which were previously transient in nature, are now more likely to persist

into late childhood and adolescence [10]. Less common presentations of food allergy, forexample eosinophilic eosophagitis, have also increased in recently [11]. Collectively, these

changes in disease patterns are placing growing demands on healthcare systems globally.

While environmental change may be driving the recent rise in disease, differences in suscep-tibility and familial aggregation of allergic diseases also implies a genetic contribution to the

risk of these diseases. Variations in genetic susceptibility cannot explain the rise in disease, but

there was hope that identification of specific atopy/asthma genes could provide valuableinsight into the causal pathways and disease pathogenesis. Although a large number of

candidate genes have been associated with the asthma/allergy phenotype, the findings have

been highly variable with poor reproducibility between populations [12,13]. A study carriedout in early 1990s reported that monozygotic twins showed a greater concordance than

dizygotic twins, whether reared apart or together, for asthma and rhinitis, indicating herit-

ability as the major factor on expression of these disorders [14]. However, subsequent twinstudies, with higher levels of within-pair discordance, revealed that environmental factors are

equally or even more important in the development of disease [15,16]. This reflects thesignificant heterogeneity of these conditions that arise as a result of multiple and variable

genetic and environmental influences. It is important to elucidate how environmental

modifiers confer changes to gene expression to fully understand the geneeenvironmentinteractions.

18.2 MECHANISMS OF ALLERGIC RESPONSEDifferentiation of naive CD4þ T cells into type 2 helper (Th2) cells to an “innocuous”

environmental antigen (allergen) is a hallmark of the allergic response, and produces the

propensity for IgE production (atopy) to that specific allergen. Once this pattern of response isestablished, subsequent exposure to the allergen leads to crosslinking of IgE on mast cells and

an inflammatory cascade that culminates in the release of histamine and other mediators

which produce the many signs and symptoms of allergic disease. The clinical pattern andseverity vary according to the route of the allergen exposure, dose of the allergen, the pattern of

tissues affected, and other individual factors. Although the culminating events in the IgE

cascade and the underlying characteristics of the Th2 cellular response are well characterised[17]17, the factors initiating and driving this process are less clear. Naive CD4þ T cells have the

capacity to differentiate into a range of effector cells or regulatory cells depending on the local

milieu at the time of allergen/antigen encounter (as discussed in more detail below). Forexample, the presence of interleukin 12 (IL-12) secreted from antigen-presenting cells (APC)

CHAPTER 18Epigenetic Aberrations in Human Allergic Diseases

promotes differentiation into Th1 subset. On the other hand, relative absence of IL-12 and thepresence of IL-4 in the local microenvironment promote Th2 differentiation over the Th1

pathway. While the environmental changes favoring Th2 differentiation are not fully under-

stood, declining microbial exposure (a potent stimulant for APC induced IL-12 production)has been a leading candidate. The range of other environmental factors which can also modify

IL-12 production or T-cell differentiation, includes smoking [19], vitamin D [19], and anti-oxidants [20], which have all been implicated in the rise in allergic disease. Therefore, the

processes regulating T-cell gene expression during differentiation and maturation are of central

interest, particularly with emerging evidence that some of the environmental factors regulatingthe differentiation process have epigenetic effects which could influence CD4þ T-cell lineage

commitment and subsequent allergic propensity, as further discussed below.

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18.3 FETAL LIFE: THE CRITICAL PERIOD OF IMMUNEDEVELOPMENTAs allergic disease is often first manifest in early childhood, it is clear that preceding events in

development are important. Early life therefore represents a critical period when geneeenvironmental interactions play a determining role in specifying immune tolerance. Clear

differences in the neonatal immune function of children who subsequently develop allergy

strongly suggests that these disorders have their origins in fetal life [21e26]. The DevelopmentalOrigins of Health and Disease (DOHaD) hypothesis proposes that prenatal exposures have the

potential to modify a range of developmental processes in the fetus, and these exposures may

program susceptibility to many chronic diseases in later life [27,28]. There is some evidence thatthese changes in disease predisposition are the result of altered fetal gene expression induced

through epigenetic changes. Although this has been best studied in the context of cardiovascular

and metabolic diseases, epigenetic effects of environmental changes are also now being inves-tigated as a mechanism of the dramatic rise in allergic diseases [29e31].

Complex immunological mechanisms have evolved to allow fetal and maternal immune

systems to coexist during pregnancy. The maternal immune system adapts in a subtle way toa “Th2-state” in order to down-regulate Th1-mediated IFN-g responses to fetal antigens which

can adversely affect the developing fetus [32e34]. Reflecting this, neonatal cytokine produc-

tion is dominated by Th2 cytokines, with relative suppression of IFN-g production [35]. Thisdown-regulation of Th1 response in the fetal environment is generally attributed to the

production of Th1-antagonistic mediators produced by the placenta; however there is also

evidence of direct epigenetic control of gene transcription (further discussed below). Regula-tory T cells (Tregs) expand during pregnancy and are recruited to the fetoematernal interface

where they orchestrate immune tolerance towards the fetus [36] which may also be under

epigenetic control. Together, these observations suggest a role for epigenetic regulation in theestablishment and maintenance of the fetal environment.

While the transition in early gene expression patterns from fetal to postnatal patterns isdevelopmentally regulated, environmental forces, such as microbial exposure (which is

known to promote Th1 and Treg differentiation), also appear to play a critical role in the

success of this process [37]. A better understanding of these effects is important for developingstrategies to prevent or suppress the allergic phenotype.

18.4 DEVELOPMENTAL DIFFERENCES IN GENE EXPRESSION INALLERGIC DISEASEMany presymptomatic differences of immune maturation pathways have been observed

between allergic and non-allergic children. Of these, relative immaturity of neonatal Th1

immune function has been one of the prominent antecedents of allergic disease [21,38].Although Th1 responses are generally suppressed at birth, this appears to be more marked in


372

individuals who develop subsequent allergic disease [21,38]. This is also coupled witha delayed postnatal maturation of Th1 immunity in high-risk children [22,26]. Differences in

innate immunity [26] and Treg function [39,40] are also observed at birth between allergic

and non-allergic children. These differences could reflect both genetic predisposition andenvironmental exposures in pregnancy at a time when the fetal immune system is potentially

more vulnerable to epigenetic changes in gene expression, as discussed further below.

18.5 EPIGENETIC REGULATION OF IMMUNE DEVELOPMENTDevelopment of the immune system, like all other systems/organs, is under epigenetic control.

Changes in epigenetic profile have been observed in developmental maturation of T cells withage [41]. In the fetus, there is low-level methylation of CpG sites within the promoter regions

of the IFNG and Th2 cytokine loci (gene silencing) in naive CD4þ T cells [42]. With age there is

progressive demethylation of IFNG which is accompanied by an increased capacity of IFNg

production by adult naive CD4þ T cells.

The best evidence of epigenetic regulation of immune pathways has been observed for T-cell

differentiation [43e45]. Polarization of naive CD4þ T cells by the cytokine milieu ismediated through activation of signaling pathways and transcription factors that are distinct

for Th1 and Th2 subsets. IL-4 activation of STAT-6 signaling and the expression of the

transcription factor GATA-3 promotes Th2 differentiation. On the other hand, IL-12-induced differentiation into IFN-g producing Th1 cells is mediated through the activation of

STAT-4 signaling and the transcription factor T-bet. Regulation of these Th cell lineages is

governed by reciprocal inhibition; i.e. IL-4 activated GATA-3 inhibits Th1 cytokine expres-sion, while inhibition of Th2 cytokine expression by T-bet ensures that once committed

naive T helper cells generally differentiate in one dominant direction [46,47]. More recently

another transcription factor, Runt-related transcriptional factor 3 (RUNX3), was found to beinduced by T-bet, which together enhances expression of IFNG while repressing IL4

expression [48].

In naive CD4þ T cells, both IFNG and IL4 are methylated, resulting in chromatin remodeling

that is transcriptionally non-permissive [43,45]. During Th1 lineage commitment the

promoter region of the IFNG gene undergoes progressive demethylation (activation), whilethe IL4 is hypermethylated (silenced), thus inducing chromatin remodeling compatible with

IFNG but not IL4 expression. On the other hand, differentiation of naive CD4þ cells to Th2

cells involves hypomethylation of the IL4 and concomitant silencing (through methylation) ofIFNG [42e44,46,49]. GATA-3-mediated chromatin remodeling at the Th2 cytokine loci (IL4/

IL5/IL13/RAD50) is also essential for the Th2 lineage commitment and maintenance of Th2

phenotype through cell division [50]. Changes in histone modifications following T-cellreceptor (TCR) signaling appear to be involved in this process. These modifications are

orchestrated by site-specific enzymes including histone acetyltranferases (HATs) and histone

deacetylases (HDACs) [46]. Acetylation of histone by HAT is generally associated with geneexpression, whereas removal of acetyl group by HDAC is associated with more closed chro-

matin structure that makes the gene transcriptionally non-permissive. The importance of

endogenous HDAC activity on Th cell differentiation was demonstrated by a shift in recall(memory) responses toward a Th2 phenotype when HDAC activity was inhibited by

trichostatin-A (TSA). Bronchial biopsies from untreated asthmatics revealed a higher level of

HAT and lower level of HDAC activity [51]. Of note, treatment with inhaled steroids wasassociated with a reverse in HAT/HDAC levels to that of controls, suggesting therapeutic

pathways also involve epigenetic modulation.

IL-2, another important cytokine in maintaining survival and proliferation of activated T cells,

provides another example of the epigenetic regulation of immune pathways. In naive T cells,


373

the IL2 promoter region is also methylated, undergoing demethylation following activation ofthe T cell with enhancing IL2 expression [52,53].

Regulatory T-cell differentiation is also under epigenetic control [54,55]. Expression of theforkhead transcription factor 3 (FOXP3) is known to be critical for the development and

regulatory functions of Tregs. The regulation of FOXP3 expression is not completely under-

stood, however, recent findings indicate a pivotal role for DNA methylation at both promoterand enhancer sequences [55]. In undifferentiated naive T cells, CpG sites within a CpG island

associated with the FOXP3 promoter are highly methylated and upon stimulation by TGF-b,

these methylationmarks are removed, ensuring stable expression of FOXP3 and differentiationinto Treg cell lineage.

A more recently recognized distinct helper-T cell subset, the Th17 lineage associated witha number of autoimmune diseases as well as with severe forms of allergic diseases [56e58]

also appears to be regulated through changes in histone acetylation [59]. Although the

developmental role of this IL-17-producing lineage is not clear, a recent study revealed thatTh17 cells can differentiate from Treg cells, and that HDAC inhibitor TSA had a profound

negative effect on the emergence of these IL-17-producing cells [60].

These observations have led to speculation that factors that increase methylation (i.e. of IFNGand FOXP3) may increase the risk of disease by silencing the Th1 and Treg pathways. While

this notion may appear simplistic, there are now a number of studies pursuing this general

concept that environmental changes can alter patterns of gene methylation.

While there is solid evidence that epigenetic machinery regulates genes/pathways directly

linked to allergic response, epigenetic regulation of “master” transcription factors (such asNFkB) indirectly controls a wider range of downstream immune and inflammatory responses

[61,62]. For example, glucocorticoid receptor (GR) function is regulated by HDAC2 which is

sensitive to oxidative stress [63]. Reduced GR function up-regulates the NFkB activity [64]which in turn enhances the expression of inflammatory genes such as IL8 [63]. This highlights

how disease risk may be modulated at multiple levels.

In addition to histone modifications and DNA methylation, other important gene regulatorynetworks contribute to the control of gene expression, including microRNAs (miRNAs), small

interfering RNAs (siRNAs), and long non-coding RNAs (ncRNA). There is accumulatingevidence that miRNAs are important for T-cell differentiation [65,66] and may be linked to

development of inflammatory disease [67,68]. Since miRNAs often target hundreds of genes

[69], they may be more important epigenetic regulators that mediate environmental assaultsin specific inflammatory pathways.

Although early models of “lineage commitment” proposed distinct terminally differentiated

Th subsets, more recent evidence argues against this more static view [70]. While CD4þ cellsdifferentiate according to their local cytokine milieu during stimulation, they retain a degree of

cellular plasticity [70e72]. Epigenetic modifications are undoubtedly involved in regulating

the switch that controls signature-cytokine genes determining naive CD4þ cell fates, but maybe equally important in contributing to the potential cellular flexibility.

Now there is a growing body of data in support of epigenetic regulation of cellular plasticity ofTh cells [70]. While Foxp3 is essential for the maintenance of suppressive function in Treg cells,

it has been shown that depletion of Foxp3 expression results in acquisition of “effector”

functions by these cells with concomitant loss of regulatory properties [73,74]. These obser-vations have led to the speculation that “Th-effector” functions in Treg cells are continuously

maintained in a dormant state by an active Foxp3-mediated mechanism. Recently, Beyer et al.

reported that repression of “Th-effector” functions of Treg cells are mostly mediated by SATB1,a chromatin organizer and a transcription factor, and appear to be under direct transcriptional

control of Foxp3 (at SATB1 locus) as well as indirectly by Foxp3-dependent miRNA [75].

FIGURE 18.1Environmental influences on de

This illustrates the environment

shown to modify epigenetic pro

during early development. This

the color plate section.


374

Furthermore, release of SATB1 from Foxp3 control was able to reprogram the Treg to gain“effector” function while losing its suppressive function. This shows that Treg population

represents a differentiated cell lineage committed to a specific function but retains develop-

mental plasticity that may be mediated through epigenetic mechanisms.

Challenging traditional models of epigenetic control of T-cell lineage commitment, Wei et al.

mapped active and repressive histone marks genome-wide across a spectrum of Th1, Th2,Th17, natural and induced Treg phenotypes [76]. Modifications at signature cytokine genes

conformed to previous models of T-cell commitment, however, master transcription factors,

such as the gene encoding T-bet (TBX21) and GATA3, were found to exhibit a mixture of activeand repressive (bivalent) chromatin states across these phenotypes. It is speculated that

bivalent epigenetic marks in master regulators of the Th differentiation maintain these tran-

scription factors at a “poised” state for expression in non-expressing cell lineages and underappropriate conditions they can be induced leading to an alternate cell fate [76,77]. This shows

that epigenetic mechanisms play a dual role in Th differentiation: ensure a “committed” state

of Th-cell response upon activation while conferring cellular plasticity.

18.6 FACTORS THAT MODULATE ALLERGIC DISEASE RISKTHROUGH EPIGENETIC MECHANISMSThere is intense interest in theprenatal factors thatmaymodifyoptimal patterns of immune gene

activation or silencing. A range of exposures already implicated in the rise in allergic disease have

been shown to have potential epigenetic effects on fetal immune function, including microbialexposure, maternal diet, and pollutants [37,38] (Figure 18.1). Table 18.1 summarizes the envi-

ronmental factors that modulate disease risk possibly through epigenetic modifications.

18.6.1 Epigenetic Effects of Bacterial Exposure on ImmuneDevelopment

Declining microbial exposure has long been implicated in the rise in allergic diseases [3],

although the original “hygiene hypothesis” was focused more on the allergy-protective effectsof infectious exposures rather than more general microbial burden [79]. The initial explan-

ations for this protective effect centered around the ability of microbial components to up-

regulate IFN-g production and inhibit pro-allergic Th2 response [3]. This notion is supported

veloping immune system:

al factors that have been

file and gene expression

figure is reproduced in

TABLE 18.1 Environmental Factors Known to be Associated with Epigenetic Modifications

EnvironmentalFactor Epigenetic Modification

Effect of Epigenetic Changes onAllergy Reference

Prenatal exposure tomicrobial products

H4 acetylation of IFNG promoter inmice

Increase in IFNG expression [80]

Demethylation of Treg-specificdemethylated region (TSDR) inneonates

Induce FOXP3 expression [87]

Maternal high-folatediet

Methylation changes in 82 gene lociincluding RUNX3 in mice

Increase in airwayhyperresponsiveness, airwayeosinophilia, and production ofinflammatory cytokines

[94]

Exposure to tobaccosmoke

Suppress HDAC activity and overallHDAC activity in alveolarmacrophages and bronchial biopsiesin healthy smokers

Increase in expression ofinflammatory mediators GM-CSF,IL-8, and TNF-aReduced response to corticosteroids

[63]

Hypomethyalation of MAOBpromorter in circulating platelets andPBMCs in smokers

Significance to allergy is not yetknown

[111]

Prenatal exposure totobacco smoke

Global DNA hypomethylation,hypermethylation of AXL andPTPRO, varied pattern of LINE1methylation pattern by child’sGSPT1haplotype

Relevance to pathogenesis of allergicdisease is not yet known

[116]

Exposure to blackcarbon particles

Hypomethylation of LINE1 ofleukocyte DNA of elderly people

Relevance to allergy has not yetidentified

[121]

Prenatal exposure toPAH

Hypermethylation of ACSL3 in cordblood mononuclear cells

Direct relevance to allergic diseasepathogenesis has not been defined

[126]

Exposure to dieselexhaust particles

Hypermethylation of IFNG promoterand hypomethylation of IL4promorter in splenic CD4þ cells inmice

Increase in the production of IgEupon intranasal administration ofAspergillus fumigates

[133]

Prenatal exposure tolead

Global DNA hypomethylation inneonates

Outcome on allergic disease is notyet known

[146]


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by the impaired postnatal maturation of IFN-g pathways in allergic disease [22,26]. Further-more, an array of microbial exposures (such as enteric flora, farm animals, and house dust

endotoxin) has been shown to both increase early IFN-g production (Th1 response) and

decrease the risk of allergic disease. Given the fact that ultimate regulation of IFN-g expressionis linked to methylation/demethylation events in the IFNG promoter in CD4þ T cells, it is

tempting to speculate that microbial exposures induce demethylation/acetylation of IFNG in

CD4þ T cells [31]. Preliminary results from an animal model provide some evidence that this ispossible. Non-pathogenic microbial strains (Acinetobacter lwoffi) isolated from farming

environments can induce epigenetic effects when administered to pregnant animals and

protect the offspring from experimental postnatal asthma [80]. This effect depends onincreased expression of IFN-g mediated by an increase in H4 acetylation of the IFNG

promoter. Notably, these effects were abolished by inhibition of histone acetylation following

garcinol treatment. Even though there is no direct evidence for such association in humans,bacterial, viral, and parasitic agents have been shown to hold the capacity of inducing

methylation events in host DNA [81e84]. This is an area for future research with clearimplications on therapeutic as well as preventive strategies for allergic diseases.

While the postnatal microbial exposure has the most obvious implications for the developing

immune system, there is emerging evidence that effects of microbial exposure may begin muchearlier with maternal microbial exposure showing potential to modulate fetal immune


376

function. Both human and animal studies clearly demonstrate that in utero exposure to bothpathogenic and non-pathogenic microbial products can prevent allergic outcomes in the

offspring, independent of postnatal exposure [85e88]. This protective effect has been asso-

ciated with enhanced neonatal Treg numbers and function [87] along with increased IFNGexpression [80]. Interestingly, exposure to microbial products in a farming environment

appeared to stimulate FOXP3 expression in neonates by demethylating an evolutionaryconserved element within the FOXP3 locus, Treg-specific demethylated region (TSDR) [87].

These findings together, are highly suggestive of microbial exposure during pregnancy can

modify fetal immune responses through epigenetic mechanisms [81e83].

18.6.2 Epigenetic Effects of Maternal Diet on Immune Function

Modern diets differ in many aspects from more traditional diets with more processed and

synthetic foods and less fresh fish, fruits, and vegetables. Of immediate relevance here, thisdietary pattern in pregnancy appears to provide less tolerogenic conditions during early

immune development promoting allergic outcomes in the offspring [89]. The specific nutri-

tional changes implicated in the rising prevalence of asthma and other allergic diseases includesa decline in consumption of polyunsaturated fatty acids (PUFA) [90], soluble fiber [91], anti-

oxidants, and other vitamins [92,93]. Indeed, diet and nutrition in pregnancy have been

a dominant basis for notions of the “developmental origins” of many diseases [28]. The firstevidence that maternal dietary changes in pregnancy can alter immune function and allergic

outcomes through epigenetic modifications came from animal studies. A diet rich in methyl

donors (folate) fed to pregnant mice induced allergic airway disease and a Th2 phenotype to inthe offspring (F1 generation) [94]. This folate-rich maternal diet induced methylation changes

in 82-gene loci in the offspring, resulting in increased airway hyperresponsiveness, airway

eosinophilia, and production of inflammatory cytokines. This trait was then inherited to thesubsequent F2 generation, demonstrating the transgenerational effects of environmental

modification. Among these genes, RUNX3, a gene known to regulate T-lymphocyte develop-

ment and to suppress airways inflammation [95] was hypomethylated with concordant tran-scriptional silencing of this gene in the progeny [94]. While some human studies reported that

folic acid supplementation during pregnancy is associated with an increased risk of asthma and

respiratory infections in infants [96], a recent Dutch study revealed no association betweenmaternal folic acid supplementation and allergic outcomes inneonates [97].However, until this

is fully explored in human studies and the mechanistic pathways are clearly delineated, it is not

appropriate to change the current practice of folate fortification to prevent neural tube defects.

The role of vitamin D as an immune-modulatory substance is currently under much debate.

Epidemiological associations between vitamin D levels and allergic diseases remain incon-clusive. Vitamin D intake during pregnancy has been associated with either increased risk

[98] or decreased risk [99,100] of allergic disease in infants. At a cellular level, 1a,25-

dihydroxyvitamin D3 (active metabolite of vitamin D) also appears to have diverse actions onnuclear factor kappa B (NF-kB)-driven transcription of inflammatory genes [101,102]. However,

it appears that HDAC activity is required for vitamin D-mediated NF-kB modulation [101].

Supplementation of fish oil (n-3 PUFA) is associated with effects on immune function of theoffspring [90]. However, at this stage it is not clear whether it is related to epigenetic modu-

lation. Similarly, antioxidants have the capacity to induce T-cell tolerance [103] and enhance

the production of IL-12 by dendritic cells [104]. It can be postulated that through these effectsantioxidants can favor the development of Th1 cells while suppressing the Th2 development.

The effect of dietary antioxidants during pregnancy on fetal immune development is limited

[105]. Evidence that oxidative stressors can modify the disease risk through epigeneticmechanisms suggests a role for these pathways [106].

Purified compounds isolated from garlic and broccoli have been reported to have epigeneticeffects [107]. These bioactive compounds are found to be associated with HDAC inhibitory


activity in animal models and may be related to increased cancer risk. Based on the immunemodulatory property of these extracts, these common dietary components may be an addi-

tional source of epigenome modifiers in allergy risk and warrant further study.

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18.6.3 Epigenetic Effects of Tobacco Smoke

Exposure to tobacco smoke represents a major risk factor for an array of diseases including

asthma [108], chronic obstructive pulmonary disease (COPD) [109], and lung cancers [110].There is mounting evidence that epigenetic modifications induced by tobacco smoke are

associated with the development of these chronic diseases [78]. One possible epigenetic effect

of tobacco smoke in the pathogenesis of respiratory inflammatory diseases is throughperturbing the balance between the HAT/HDAC homeostasis of the airway immune cells.

Bronchial biopsies and alveolar macrophages taken from healthy smokers and age-matched

healthy non-smokers reveal that tobacco smoke suppresses the HDAC2 expression and overallHDAC activity and enhanced the expression of inflammatory mediators GM-CSF, IL-8, and

TNF-a [63]. Of note, cigarette smoking reduces the response to corticosteroids by decreasing

HDAC activity in key inflammatory cells such as alveolar macrophages, explaining theattenuated response to steroid therapy in patients with COPD (which also has a strong link

with chronic exposure to tobacco smoke). The HDAC inhibitor TSA has been shown to reverse

the proinflammatory changes and glucocorticoid responsiveness in the macrophages [63],implying their usefulness as an adjuvant drug for the treatment of asthma.

In addition to altering the HAT/HDAC balance, tobacco smoke can modulate the DNAmethylation status of regulatory regions in a number of genes. Direct evidence for

modified DNA methylation of epigenetic tags comes from a report that smoking leads to

hypomethylation of monoamine oxidase (MAO) type B promoter in circulating platelets[111]. Furthermore, the authors describe similar results for PBMC of smokers and imply

smoking-induced MAO gene deregulation could have a more general impact than vascular

effects.

Exposure to cigarette smoke in pregnancy has many adverse effects on the fetus, including

effects on lung function and asthma risk [112,113]. Smoking in the last trimester has beenassociated with early onset of airway hyperreactivity (likely asthma) by the age of 1 year [114].

Moreover, both maternal and grandmaternal smoking during pregnancy are associated with

increased risk of childhood asthma, suggesting a persistent heritable effect [115]. Comparisonof DNAmethylation pattern of buccal cells from children born according to maternal smoking

habits during pregnancy, revealed that exposure to tobacco smoke was associated with global

DNA hypomethylation. Exposed children had significantly lower methylation of short inter-spersed nucleotide element AluYb8, a surrogate marker of global DNA methylation [116]. In

addition, the study revealed that smoking affects epigenetic marks in gene specific manner.

Using a CpG loci screen, eight genes were found differentially methylated in exposed childrenas opposed to unexposed children. Two genes, AXL and PTPRO, were validated by pyrose-

quencing and showed significant hypermethylation in exposed children but their significance

in relation to asthma pathogenesis remains unclear. Moreover, methylation status of the DNArepetitive element LINE1 was observed only in children with common GSTM1-null genotype,

while methylation pattern in exposed children varied with a common GSPT1 haplotype

implying the genetic influence over the epigeneticeenvironmental interactions. It is interestingthat maternal smoking had dual effects on fetal methylation profile; global DNA hypo-

methylation and hypermethylation of some specific genes [116]. Global hypomethylation

could result from DNA damage caused by smoke-induced oxidative stress to DNA thatinterferes with the binding of DNA methyltransferase (DNMT) preventing DNA methylation

[117]. The effect of tobacco smoke exposure on methylation of specific genes could possibly be

due to de novo methylation in specific gene promoters, perhaps by incomplete erasure duringmethylation reprogramming that occurs in the embryo after fertilization [118].


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18.6.4 Effects of Air Pollutants and Other Outdoor Pollutants onEpigenetics

Airborne pollutants such as particulate matter (PM) and noxious gases including benzene havebeen shown to be associated with asthma and other respiratory diseases [119,120]. These

agents cause exacerbation of asthma symptoms in affected individuals but a causative link to

asthma has not been well defined. Ambient level of black carbon particles, a marker of trafficpollution, has been consistently associated with a variety of adverse health outcomes and

exposure even for a short duration was associated with hypomethylation of LINE1 but not Alu

in blood DNA samples taken from a cohort of elderly people [121]. Polycyclic aromatichydrocarbons (PAH) are one of the most widespread organic pollutants and also a major

component of PM of urban aerosol. In addition to their presence in oil, coal, and tar deposits

they are also formed by the incomplete combustion of carbon-containing fuels such as wood,coal, diesel, fat, tobacco, and incense. Grilled, smoked, or barbecued meats also appear to

contain high levels of PAH [122,123]. In addition to its carcinogenic properties [124], it has

been found not only to impair functions of airway cells and smooth muscle cells but alsodiminish responsiveness to standard therapy given to asthmatics [125]. Recently, a novel

epigenetic marker for PAH-associated asthma has been identified and cord blood mono-

nuclear cells (CBMC) of children born to mothers who had been exposed to considerable levelof PAH showed hypermethylation of the acyl-CoA synthetase long-chian family member 3

(ACSL3) promoter [126]. Furthermore, the exposure level was highly correlated with increasedrisk of asthma symptoms in the offspring before age 5 years. ACSL3 genes are expressed in

lungs and thymus tissue and encode key enzymes in fatty acid metabolism [127,128]. Given

the fact that ACSL3 is located in 2q36.1 which is associated with regions of the asthmasusceptibility loci in specific populations [129,130], it is tempting to speculate that hyper-

methylation of this gene in lung tissues can potentially influence the fatty acid metabolism and

phospholipid composition of the membranes and lung function. The direct relevance of thisfinding to asthma pathogenesis has not been defined but epidemiological studies have

revealed the alterations in fatty acid composition in the diet [131] and cord blood [132] are

associated with the increased risk of asthma.

Diesel exhaust particles (DEP), in addition to a source of PAH, give rise to an array of chemicals

dispersed in the air as ultrafine particles. Exposure of mice to inhaled DEP for 3 weeks can

augment the production of IgE upon intranasal administration of Aspergillus fumigates [133].Hypersensitization occurred through hypermethylation of IFNG promoter with concomitant

hypomethylation of IL4 promoter in DNA from splenic CD4þ cells. The effects of PM could be

mediated in the airways through induction of oxidative stress. Treatment of A549 cells(adenocarcinomic human alveolar basal epithelial cells) with either PM-10 or H2O2 increased

the expression and release of IL-8, which increased the HAT activity, hence remodeled the IL8

promoter region [134] suggesting PM exert their effects through chromatin remodeling of thesusceptible genes.

Benzene, toluene, xylene, and other volatile organic compounds like PM are associated withadverse health effects including asthma. Children exposed to benzene have an increased risk of

asthma symptoms [135] and this could possibly be mediated through changing the DNA

methylation profile since exposure to benzene induced changes in DNA methylation ina global and gene-specific manner [136].

Although less is known about the immune effects of other pollutants released to the envir-onment by agricultural and modern industrial processes, it has been shown that highly lipid

soluble substances such as polychlorinated biphenyl compounds, organochlorine pesticide,

dioxins, and phthalates accumulate in human tissue with age [137] possibly throughcontaminated water, food, and clothing. Of most concern, some of these products have been

measured in breast milk, cord blood, and placental tissue [138e140] emphasizing the possible

adverse outcome in early development and subsequent disease pathogenesis in offspring.


Higher levels of organic pollutants were associated with higher levels of cord blood IgEantibodies [141]. At higher levels these products can have immunosuppressive effects in

humans [142], whereas at low levels some appear to selectively inhibit type 1 immune

responses [143], leading to speculation that this could possibly favor allergic (type 2) immuneresponses. Many of these organic pollutants now have been associated with modified epige-

netic tags in humans, as evidenced by variations in global DNA hypomethylation patterns withpersistent low-dose exposure [144]. More evidence for epigenetic alterations induced by

exposure to organic pollutants comes from rodent studies [145]. Prenatal exposure to lead was

associated with global DNA hypomethylation in a human study suggesting epigenome of thedeveloping fetus can be influenced by maternal cumulative lead burden [146]. This may

influence long-term epigenetic programming and disease susceptibility throughout the life

course. The levels of many pollutants are declining in some regions as a result of restrictionsimposed on the use of pesticides and other toxic chemicals, and this is reflected in declining

levels measured in adipose tissue [147]. Nevertheless, the effects of these factors should not be

ignored, as epigenetic effects may potentially reflect exposure of subsequent several genera-tions and this relationship may be obscured in cross-sectional epidemiological studies.

379

18.6.5 Other Maternal Factors that May Modulate Allergic Propensity inthe Newborn Through Epigenetic Mechanisms

In the context of asthma and allergic diseases, the maternal phenotype appears to be a major

factor determining the subsequent outcomes in the offspring. Maternal asthma and allergicstatus has a stronger effect than paternal allergy on both allergic diseases and Th1-IFNg

production in the neonate [148]. Lower IFNg responses to HLA-DR-mismatched fetal antigens

have also been observed in allergic mothers compared to non-allergic mothers [149]. This mayaffect the cytokine milieu at the fetoematernal interface and could be a mechanistic link of

attenuated Th1 responses commonly observed in infants born to atopic mothers [38]. Rising

rates of maternal allergy mean that the endogenous effects of the maternal allergic phenotypehave the potential to amplify the effects of a proallergic exogenous environment. The underlying

mechanisms, yet unclear, may involve epigenetic modifications of the specific immune genes.

The hypothalamicepituitaryeadrenal (HPA) axis is a major component of the neuroendocrine

system. This axis controls many body processes and plays a major role in controlling stress

responses. The immune system and the HPA axis are closely linked, particularly through theeffects of glucocorticoids which connect these two critical systems. In pregnancy, the placental

immune system is, at least in part, regulated by glucocorticoids. Under adverse (stressful)

conditions HPA axis activation can induce up-regulation of placental Th1 cytokine production,resulting in poor fetal outcomes. In animal models, it has been shown that maternal stress can

alter placental gene expression, in particular, genes involved in DNA methylation and histone

modification and cell cycle regulation [150] and strongly suggest that maternal stress can induceepigenetic effects on fetal immune function with implications for the subsequent risk of disease

in childhood. This is still poorly understood and an important area for ongoing research.

18.7 CONCLUSIONSExposure to a plethora of environmental factors (microbial exposure, maternal diet and

smoking) during critical periods of early immune development, has the potential to modify

the fetal immune development and the risk of subsequent disease. Notions of plasticity in geneexpression that may be epigenetically modified by the early environment provide a newmodel

to understand the geneeenvironmental interactions that contribute to the rising prevalence of

asthma, allergy, and other immune diseases. Of greatest significance, this epigenetic plasticitymay pave the way to develop novel early interventions to curb the epidemic of immune

disease, ideally through primary prevention in early life.


380

The discovery of epigenetics as a key mechanism modulating immune machinery hasprofoundly changed perspectives and research approaches to allergy disease. However, many

unanswered questions need to be addressed before these findings will be of any therapeutic

value, including: can epigenetic profiles be used to accurately predict disease risk andsusceptibility to treatment at the individual and population levels? How long will epigenetic

memory last and can we reverse any events that occurred in early life at a later stage? Can weerase the epigenetic marks passed through generations by modulating the environment of the

next generations or with therapeutic interventions?

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epigenetics in human disease || epigenetic aberrations in human allergic diseases

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