Immune development is under epigenetic regulation

Epigenetic regulation is fundamental to cellular differentiation and all aspects of normal development. Specifically, changes in the methylation of DNA and histones, and histone acetylation regulate gene expression by altering the DNA compaction and accessibility for gene transcription⁽Reference Song, Mahmood and Ghosh¹²⁾. There is clear evidence that T-cell differentiation is under epigenetic control⁽Reference Janson, Winerdal and Winqvist¹³⁾, including Th1 and Th2 differentiation⁽Reference Fields, Kim and Flavell^14–Reference Santangelo, Cousins and Winkelmann¹⁸⁾, FoxP3 expression and Treg differentiation⁽Reference Janson, Winerdal and Marits^19, Reference Polansky, Kretschmer and Freyer²⁰⁾ and Th17 differentiation⁽Reference Koenen, Smeets and Vink²¹⁾.

The main epigenetic mechanism controlling Th1 expression is methylation of the interferon-γ gene promoter. This is hypermethylated (i.e. underexpressed) in neonatal CD4+ T-cells and shows progressive demethylation by adulthood⁽Reference White, Watt and Holt²²⁾. Changes in methylation (demethylation) are also prerequisite for FOXP3 expression and Treg differentiation⁽Reference Janson, Winerdal and Marits^19, Reference Polansky, Kretschmer and Freyer²⁰⁾. Another major mechanism of epigenetic regulation is histone acetylation. Removal of acetyl groups by histone deacetylase generally leads to gene silencing, whereas acetylation by histone acetyl transferase opens chromatin structure for enhanced gene transcription⁽Reference Bhavsar, Ahmad and Adcock²³⁾. Exposures that inhibit histone deacetylase such as oxidative stress up-regulate Th2 cytokine (IL-13 and IL-5) and GATA3-mediated T-cell responses⁽Reference Bhavsar, Ahmad and Adcock^23, Reference Su, Becker and Kozyrskyj²⁴⁾. The Th17 lineage also appears to be regulated through similar epigenetic mechanisms⁽Reference Janson, Winerdal and Winqvist^13, Reference Koenen, Smeets and Vink²¹⁾.

These insights have logically led to interest in factors which may promote allergic propensity by increased histone acetylation (Th2 promotion) and/or increased gene methylation (Th1 and Treg silencing)⁽Reference Bousquet, Jacot and Yssel^25, Reference Miller and Ho²⁶⁾. As discussed further later, the first evidence of this comes from an animal model in which maternal folate supplementation (a dietary methyl donor) resulted in hypermethylation (suppression) of regulatory genes and the development of allergic disease in the offspring⁽Reference Hollingsworth, Maruoka and Boon³⁾. At this stage, the implications in human subjects are not clear, but this provides a platform for investigating epigenetic pathways as a mechanism for gene–environmental interactions in allergic disease.

Altered patterns of immune response begin to emerge in fetal life

There have been numerous studies showing pre-symptomatic differences in the immune responses of newborns who later develop allergic disease (reviewed in Prescott and Clifton⁽Reference Prescott and Clifton²⁷⁾ and Prescott⁽Reference Prescott²⁸⁾). This was initially thought to largely reflect inherited genetic risk. However, the epidemic rise in allergic disease has raised the alternative hypothesis that this may be due to more complex alteration in immune gene expression conferred by gene–environmental interactions in utero. Thus, at least some of the environmental effects driving the rise in allergic disease may begin in utero, and the differences in neonatal immune function may be the first signs of this increasing allergic predisposition.

A relative immaturity of neonatal Th1 immune function has been one of the clearest and most replicated neonatal associations with allergic disease⁽Reference Rinas, Horneff and Wahn^29–Reference Warner, Miles and Jones³¹⁾. Although Th1 responses are generally suppressed at birth under the Th2-dominant influence of pregnancy, this appears to be more marked in neonates with allergic predisposition or subsequent allergic disease⁽Reference Rinas, Horneff and Wahn^29–Reference Warner, Miles and Jones³¹⁾. Other aspects of neonatal effector T-cell function may be impaired in this population⁽Reference Prescott, Macaubas and Holt¹⁰⁾. More recently, there has been emerging evidence that allergic disease is also associated with attenuated neonatal Treg function⁽Reference Smith, Tourigney and Noakes^32, Reference Schaub, Liu and Hoppler³³⁾ and differences in innate immunity⁽Reference Prescott, Noakes and Chow^34–Reference Amoudruz, Holmlund and Malmstrom³⁷⁾. A number of other neonatal markers have been identified in relation to allergic disease (reviewed in Prescott and Clifton⁽Reference Prescott and Clifton²⁷⁾), though none of these has so far been shown to be of accurate predictive value. Further research is needed to understand the functional significance and the possible contribution to the disease pathogenesis. It is possible that that impaired Th1 and Treg function may contribute to a reduced capacity to suppress Th2 responses in the early postnatal period; however, this is likely to be oversimplistic. While there has been long-standing speculation that dysfunction of antigen presenting cells and innate immunity may contribute to the apparent immaturity of Th1 activity, there is still only indirect evidence to support this⁽Reference Upham, Holt and Taylor^38, Reference Gabrielsson, Soderlund and Nilsson³⁹⁾. Furthermore, while some groups have shown that markers of innate activity (such as Toll-like receptor function or expression) are lower in neonates at risk of allergic disease⁽Reference Schaub, Campo and He^35, Reference Amoudruz, Holmlund and Malmstrom³⁷⁾, we have shown the opposite⁽Reference Prescott, Noakes and Chow³⁴⁾. While this needs to be examined further, collectively these observations do suggest that differences in neonatal immune function confer increased susceptibility to subsequent postnatal environmental influences and contribute to an evolving allergic phenotype.

Maternal dietary influences on immune development

Maternal nutrition is critically important for most aspects of fetal development, including the immune system. Complex dietary changes with progressive industrialisation have been implicated with the rise of allergic disease. As with other exposures, nutritional changes are likely to have more profound effects on pregnancy when the organ systems and physiological responses are developing. Many dietary nutrients have recognised immunomodulatory properties and plausible biological mechanisms of influence⁽Reference Shaheen⁴⁵⁾. This includes PUFA⁽Reference Calder⁴⁶⁾, antioxidants and other vitamins⁽Reference Devereux⁴⁷⁾. Of these, PUFA are among the most extensively studied in this context. A declining intake of anti-inflammatory n-3 PUFA (found in oily fish) has been implicated in the rise in allergic disease⁽Reference Black and Sharpe⁴⁸⁾, and a series of studies have shown a protective relationship between maternal n-3 PUFA consumption in pregnancy and subsequent infant allergic disease⁽Reference Salam, Li and Langholz^49–Reference Sausenthaler, Koletzko and Schaaf⁵⁴⁾, though not all were significant after allowing multiple comparisons⁽Reference Newson, Shaheen and Henderson⁵⁵⁾. Several intervention studies using fish-oil supplementation in pregnancy have also suggested protective effects against allergic disease in early childhood⁽Reference Dunstan, Mori and Barden^40, Reference Furuhjelm, Warstedt and Larsson⁵⁶⁾ and a long-term (16-year) follow-up study showed a reduction in subsequent asthma⁽Reference Olsen, Osterdal and Salvig⁵⁷⁾. A number of other studies are currently in progress to hopefully assess this more definitively.

In the last 12 months, dietary folate has become one of the most topical dietary nutrients in this area. As a dietary methyl donor, there has been established interest in the broader context of epigenetics; however, the recent pro-allergic effects demonstrated in an animal model have extended this interest into the field of allergic disease, and for the first time provided an epigenetic model for these immune disorders. As indicated earlier, supplementation with folate in pregnancy induced hypermethylation (silencing) of regulatory genes in lung tissue, and was associated with the development of allergic airway disease and systemic allergic responses⁽Reference Hollingsworth, Maruoka and Boon³⁾. This effect was also transmitted epigenetically to subsequent generations. This has been followed by reports in human subjects⁽Reference Haberg, London and Stigum^58, Reference Whitrow, Moore and Rumbold⁵⁹⁾ linking folic acid supplementation during pregnancy with increased risk of asthma and respiratory disease in infants. There have been links between folate status in the postnatal period and allergic disease, but if anything, folate was protective⁽Reference Matsui and Matsui⁶⁰⁾. At this stage, the significance of these findings is not yet clear. At this stage, it is premature to change current practice based on animal studies and preliminary epidemiological reports, but there is now an urgent call for further studies to address this now pressing question⁽Reference Miller⁶¹⁾.

Most other nutrients linked with allergic disease in epidemiological studies have not been investigated in intervention studies. Maternal antioxidant vitamins have been associated with differences in neonatal immune function⁽Reference Devereux, Barker and Seaton⁴¹⁾, as well as reduced risk of possible allergic outcomes including recurrent wheezing (vitamin C⁽Reference Farchi, Forastiere and Agabiti^62, Reference Forastiere, Pistelli and Sestini⁶³⁾, vitamin D⁽Reference Devereux, Litonjua and Turner⁶⁴⁾, vitamin E⁽Reference Devereux, Turner and Craig^65, Reference Litonjua, Rifas-Shiman and Ly⁶⁶⁾ and Zn⁽Reference Litonjua, Rifas-Shiman and Ly⁶⁶⁾), and eczema (vitamin E⁽Reference Martindale, McNeill and Devereux⁶⁷⁾). However, these correlative findings are not consistent between studies and concerns have been raised about the potential for paradoxical or adverse effects of antioxidants on allergic disease⁽Reference Murr, Schroecksnadel and Winkler⁶⁸⁾. This is based on speculation that antioxidant supplementation could promote Th2 differentiation by inhibiting oxidative stress⁽Reference Murr, Schroecksnadel and Winkler⁶⁸⁾. At this stage, there is no place for specific dietary supplementation in pregnancy for allergy prevention, although this may be included in future strategies once these relationships are more fully understood.

Maternal microbial exposure

The decline in the level and diversity of microbial exposure is a leading candidate in the allergy epidemic. While focus has been on the role of postnatal microbial exposure, animal studies clearly demonstrate that in utero (maternal) exposure to both pathogenic⁽Reference Blumer, Herz and Wegmann⁶⁹⁾ and non-pathogenic microbial products⁽Reference Blumer, Sel and Virna⁷⁰⁾ can inhibit the development of allergic phenomena in the offspring. In human subjects, maternal exposure to high microbial burden in German farming environments has been associated with altered expression of innate immune genes and reduced risk of allergic disease in the children⁽Reference Ege, Bieli and Frei³⁶⁾. Similar protective effects of farming environments have also been observed in New Zealand⁽Reference Douwes, Cheng and Travier⁷¹⁾. This effect was independent of postnatal exposure in both studies⁽Reference Ege, Bieli and Frei^36, Reference Douwes, Cheng and Travier⁷¹⁾. In what may be another example of epigenetic regulation, there is preliminary evidence that an apathogenic microbial strain isolated from the German farming environment can mediate allergy-protective effects by epigenetic changes⁽Reference Teich, Conrad and Ferstl⁷²⁾. Intranasal administration of this strain (Acinetobacter lwoffii) to pregnant mice was associated with significant effects on the ontogeny of splenic CD4+ Th1 interferon-γ production in the offspring of exposed mothers. These differences were directly related to epigenetic changes in the interferon-γ promoter⁽Reference Teich, Conrad and Ferstl⁷²⁾. This supports notions that microbial exposure may modify foetal gene expression and provides a potential epigenetic mechanism. Intervention studies using microbial products in human pregnancy are mainly limited to probiotics⁽Reference Johannsen and Prescott⁷³⁾. Although there is some evidence that these products may reduce the risk of eczema⁽Reference Osborn and Sinn⁷⁴⁾, there is wide heterogeneity in study protocols and findings between studies. This appears to be species-dependent⁽Reference Wickens, Black and Stanley⁷⁵⁾ and although there has been speculation that antenatal supplementation may explain the beneficial effects in some studies⁽Reference Abrahamsson, Jakobsson and Bottcher⁷⁶⁾, this is also not consistent⁽Reference Johannsen and Prescott⁷³⁾. Furthermore, while one study suggested that probiotic bacteria during the final weeks of pregnancy was associated with an increase in cytokine (interferon-γ) detection in cord blood⁽Reference Prescott, Wickens and Westcott⁷⁷⁾, another more comprehensive investigation found no effects on any aspect of neonatal immune function⁽Reference Boyle, Mah and Chen⁷⁸⁾. At this stage, the role of probiotics in the prevention of allergic disease is still unclear and no specific recommendation can be made.

Maternal allergen exposure

Although early allergy prevention strategies focused on allergen avoidance, there is little clear evidence that changes in food or inhalant allergen exposure in pregnancy are responsible for the rise in allergic disease. Moreover, there is no clear evidence that restrictive dietary recommendations actually prevent allergic disease. In contrast, there are a growing number of reports that an attempt to avoid or delay allergen exposure may actually increase the risk of allergic sensitisation⁽Reference Woodcock, Lowe and Murray^79, Reference Snijders, Thijs and van Ree⁸⁰⁾. Many international expert bodies have independently concluded that there is insufficient evidence to justify the continued use of these allergen-restrictive diets in either pregnancy or early infancy⁽Reference Greer, Sicherer and Burks^81–Reference Agostoni, Decsi and Fewtrell⁸³⁾.

Maternal smoking

Maternal cigarette smoking in pregnancy has many adverse effects on the fetus, including effects on lung function and asthma risk. While there are documented effects on neonatal immune function⁽Reference Devereux, Barker and Seaton^41–Reference Noakes, Hale and Thomas⁴³⁾, the relationship with other allergic sensitisation has been less clear. Regardless of this, the avoidance of cigarette smoke is an unequivocal recommendation in view of the many toxic effects on the fetus.

Other maternal exposures

A range of other maternal exposures could potentially influence fetal immune development. Firstly, the use of a number of medications in pregnancy has been associated with an increased risk of childhood asthma. The most consistent relationship has been seen with paracetamol, with a series of independent studies⁽Reference Rebordosa, Kogevinas and Sorensen^84–Reference Persky, Piorkowski and Hernandez⁸⁷⁾ supporting the initial reports⁽Reference Shaheen, Newson and Sherriff⁸⁸⁾. Documented depletion of antioxidant glutathione has been proposed as a mechanism of effect on immune function and lung development. Another notable relationship has been a recent large-scale study showing that acid-suppressive medications in pregnancy are associated with an increased risk of developing childhood allergy⁽Reference Dehlink, Yen and Leichtner⁸⁹⁾. Although the mechanisms are unclear, it does follow animal studies showing similar effects.

Secondly, modern environmental changes have been associated with the appearance of many new chemicals and persistent organic pollutants. A number of these products of industry and agriculture have been linked with immune disorders⁽Reference Hertz-Picciotto, Park and Dostal⁹⁰⁾ because of oestrogenic (proTh2) properties, which have earned them the title of ‘hormone imposters’. Although environmental measures have helped reduce the level of exposure, we have detected pesticides in 94% of maternal abdominal fat samples (collected at caesarean section) and 62% breast milk samples, albeit at very low levels⁽Reference Noakes, Taylor and Wilkinson⁹¹⁾. The effects of these and other modern environmental exposure on immune development is still unclear and difficult to investigate, but should not be ignored as potential contributors to the rise in modern diseases.