The hygiene hypothesis was proposed more than 20 years ago by Strachan to explain the dramatic increase in the prevalence of allergic diseases over the last few decades. In its original form, this hypothesis claimed that limiting early-life infections impedes the development of the natural immune system and thereby predisposes individuals to allergic disease.29 The modified "microflora hypothesis" proposes that the overlying hygienic Western lifestyle limits not only infections, but also general microbial exposure and it alters the colonization of the human gut, which in turn disrupts the development of immune systems and ultimately leads to allergic diseases.18,30,31 Infancy and early childhood have been identified as important and vulnerable periods in the development of the gut microbiota, which shapes the disposition to allergic diseases.

The importance of microbial exposure during the perinatal period has been studied in animal models. It is difficult to achieve oral tolerance in germ-free mice because they lack Th1 responses and have stronger Th2 responses, as demonstrated by the increased production of IL-4.32 Administration of lipopolysaccharides together with food antigens in mice increased the tolerance of foods.33 In addition, the proper development of oral tolerance seems to require a complex intestinal microbiota rather than colonization with a single microorganism.34 These results suggested that the gut microbiota is a key factor that shapes the development and regulation of the immune system early in life.

After birth, the diversity of the gut microbiota increases with age.35 Since healthy human fetuses are thought to develop within a bacteria-free environment, the nature of the first contact of the neonate with the germ-containing environment may markedly shape the early microbiota composition. Indeed, the mode of delivery has been shown to influence the early colonization pattern in neonates: vaginally delivered infants acquire bacterial communities that resemble the microbiota of their mother's birth canal, which are dominated by Lactobacillus, Prevotella, or Sneathia spp., whereas, infants born by Cesarean section harbor bacterial communities that are similar to those found on the skin surface and are dominated by Staphylococcus, Corynebacterium, and Propionibacterium spp.18,36 A meta-analysis also showed that Cesarean delivery can be a risk factor for allergic rhinitis, asthma, hospitalization for asthma, and perhaps food allergy/food sensitization, but not for inhalant sensitization or atopic dermatitis.37 Moreover, the gut flora composition of atopic infants is similar to that of infants born by Cesarean section.38 Other factors have also been associated with allergy, such as probiotics use and antibiotic use in neonates and infants that alter the gut micriobiota. A Korean birth cohort study (The COhort for Childhood Origin of Asthma and allergic disease, COCOA) showed that Cesarean delivery and prenatal antibiotic exposure may affect the gut microbiota, which may in turn influence the risk of atopic dermatitis in infants.18

To confirm the pathogenesis of allergic diseases shaped by microbiota composition, it is necessary to perform prospective studies which show the different population of microbiota, and function of immune system in allergic and non-allergic infants. Moreover, only a few prospective studies have used metagenomic analysis to examine the association between gut microbiome and allergic diseases. Two of these showed that low microbial diversity early in life is associated with an increased risk of allergic diseases.39,40 One study showed that reduced gut bacterial diversity in infants associates with an increased risk of allergic sensitization, allergic rhinitis, and peripheral blood eosinophilia in the first 6 years of life.39 These results support the general hypothesis that imbalances of gut microbiota influence the development of allergic diseases.

The hygiene hypothesis also suggests that microbial biodiversity in the environment may influence our immune system and health later in life. Supporting this is the fact that microbial-rich environments confer protection against allergic diseases41 and poor biodiversity is associated with human immune dysfunction. For example, the homes of atopic individuals have significantly lower environmental biodiversity than those of healthy adolescents.42 The atopic adolescents also have significantly less diverse gram-negative gammaproteobacteria on their skin. Furthermore, the abundance of the genus Acinetobacteria on the skin correlates positively with the expression of IL-10 in peripheral mononuclear cell. These observations suggest that the Western lifestyle and living in urban areas hampers the exposure to microbes that is needed to develop a healthy microbiota, which in turn is essential for the proper development of immune tolerance and homeostasis.

However, the relationship between environmental and human microbiota remains unclear. Repeated skin and fecal measurements in 2 subjects showed that only a small fraction of all taxa found within a single body site are present across all time points.43 This suggests that the human core microbiota is small and that many taxa are present on some but not all occasions. While it seems plausible that the composition of the human microbiota can be temporarily changed by external influences, such as inhalation or ingestion of microbiota and that farm- or animal-associated microbiota may compete with the microbiota that promote asthma, it remains unclear whether such exposure can permanently change the colonization patterns.

The above studies do not exclude the possibility that the microbiota composition is influenced by the immune system rather than the other way around. However, there are several lines of evidence that changes in the human microbiota can lead to different stable immune states.44,45 Commensal microbiota functions in the immunoregulatory network and the Toll-like receptor (TLR) system. The development and maintenance of epithelial-cell integrity, tolerance, and tissue repair requires the interaction with microbes and immune cells via their specific receptors. When these microbial stimuli are absent, it cannot adequately induce the immunoregulatory circuits including regulatory dendritic cells, regulatory T (Treg) cells and regulatory cytokines such as IL-10, transforming growth factor-β (TGF-β).46 Interestingly, it has also been shown that an inflammatory milieu enhances the conversion of Treg cells to inflammatory Th17 cells and enriches the bacteria that tolerate to the inflammatory mediators in microbiota, thereby creating a self-perpetuating system. These molecular findings further support the hygiene hypothesis, which states that a sedentary lifestyle in affluent urban environments does not provide adequate microbial exposure for the development of a healthy microbiota.

Epigenetic mechanisms have also received considerable attention as a possible explanation as to how environmental exposure modulates the immune system. In general, microbe-rich environments induce both pro-inflammatory and immunoregulatory circuits early in life, which indicates an early activation of the relevant genes.47

The importance of microbial contact during the crucial periods of fetal life, delivery, and infancy for the healthy immune system and metabolic programming is only now being understood. This understanding creates new opportunities for improving infant's health and reducing the risk of disease in later life (Fig. 1). However, the underlying mechanisms, the cause-effect relationship, and particularly the crucial individual microbes and microbial compounds still have to be elucidated.

Air pollution has been markedly increased by the rapid growth of the world's motor vehicle fleet along with recent urbanization. Motor vehicles emit large quantities of noxious pollutant including CO₂, carbon monoxide (CO), hydrocarbons (HC), nitrogen oxides (NOx), and PM. Traffic-related air pollution (TRAP), including these primary air pollutants and secondary by-product such O₃, can have adverse effects on health and the environment. Among these pollutants, particle pollution and ground-level ozone are the most widespread threats to human health.

PMs are major pollutants along with gaseous pollutants such as NOx and O₃. They consist of variously sized particles but the smaller particles have greater adverse health effects because they can penetrate the lung as well as the blood stream more easily. Diesel exhaust particles (DEPs) emitted from motor vehicles are major sources of airborne PM and account for up to 90% of the airborne PM in urban areas. DEPs are known to generating reactive oxidative stress (ROS).

Ozone is a secondary air pollutant that is produced by photochemical reaction of primary pollutants such as NOx and volatile organic compounds (VOCs) under ultraviolet light. NO₂ is generated by most combustion processes that use air as the oxidant. Since the most prominent sources of NO₂ are the internal combustion engines of automobiles, NO₂ levels may be an indicator of TRAP. In addition, NO₂ and other nitrogen oxides also contribute to the generation of ozone and other oxidant pollutants. It is difficult to determine the independent effect of NO₂ on human health in epidemiological studies because NO₂ is often seen together with other pollutants derived from combustion exhaust and it acts as a precursor for secondary pollutants. Nevertheless, several studies have shown that long-term exposure to NO₂ can decrease lung function and increase respiratory symptoms and it is also associated with immunological impairments.

Air pollution combined with exercise exacerbates allergic asthma and decreases the lung function in asthmatics.48 Air pollution also causes asthma and increases airway hyperresponsiveness.49 Interestingly, it may act synergistically with host susceptibility (as indicated by past episodes of bronchiolitis) to promote the development of asthma.50 Allergic rhinitis is also closely associated with air pollution. The prevalence of allergic rhinitis is higher in populations that reside in more polluted areas or near roads with heavier traffic than in populations with less pollutant exposure.16,51 While air pollution is considered to be a respiratory environmental factor because it is primarily inhaled into respiratory systems, it is also associated with eczema and skin disease.52 Indeed, a recent study has shown that the amount of air pollutants such as NO, toluene, and VOCs correlate with eczema symptoms.53 Although there is a great deal of consistent evidence that shows positive associations between air pollution and the development of allergic symptoms, it is still controversial whether air pollution can cause allergic sensitization.54 Recent human and animal studies showed that air pollutants such as ozone and DEPs increase the prevalence of allergic sensitization and stimulate the production of allergen-specific IgE antibodies, respectively.16,55

Lastly, in East Asia, Asian sand dust that typically occur in early spring have been receiving increased attention due to their significant ecological and health effects, contribution to the aggravation of pre-existing allergic diseases, and augmentation of harmful effects by air pollution.

Future studies using relevant biomarkers reflecting exposure to air pollutants and the consequent effects on target organs will give more information about how to cope with air pollution. Moreover, while the independent effect of each air pollutant should be determined, it is also important to assess their effects when they are combined with other environmental or host factors.

The effect of indoor air pollution should also be assessed closely. Indoor air pollutant concentrations can be 2- to 5-fold higher than outdoor levels and most people spend as much as 90% of their time indoors.56 Such chronic exposure to indoor pollutants at home or in the work-place warrants careful assessment of their health impacts. The sources of indoor air pollution include the combustion of fuel for heating the home and cooking, tobacco smoke, chemical products used for cleaning, building and decorating, and dust from outside.

The indoor air pollution in residences, day-care centers, retirement homes and other special environments affects specific population groups that are particularly vulnerable due to their health status or age. Moreover, dampness is more likely to occur in modern buildings, which are crowded and lack appropriate ventilation, may have higher levels of microbial pollution that involve hundreds of species of bacteria and fungi that grow indoors easily. Exposure to such microbial contaminants is associated clinically with respiratory symptoms, allergies, asthma and immunological reactions.57

Environmental tobacco smoke (ETS) has been identified as a major source of indoor air pollution.58 Several studies showed that especially fetal and infantile exposure to tobacco smoke increased the risk of asthma symptoms and lower respiratory tract infection in early childhood.59,60 ETS exposure also increases the vulnerability of the lungs to other air pollutants such as PM.61 Additionally, a recent study showed that prenatal exposure to cockroach allergen increased the risk of allergic sensitization in children at the age of 5-7 years and that exposure to nonvolatile polyaromatic hydrocarbons (PAHs) augmented the risk.62 Moreover, VOCs are emitted from interior products such as synthetically coated furniture, carpets, and polyvinylchloride flooring, which means that newly built or renovated houses have higher levels of these compounds. Children have been shown to have increased levels of pro-inflammatory cytokines such as TNF-α and IL-6 after indoor activities63 and exposure to VOCs is associated with respiratory symptoms and allergic manifestations through increased inflammation as a result of oxidative stress.64

Modern people are often exposed to a variety of chemical pollutants as well as increased levels of air pollution at home or in the workplace during their daily life. Heavy metals such as arsenic and nickel are widespread environmental chemicals that are associated with various adverse health effects, including cancer, cardiovascular diseases, neurological disease, and autoimmune disease. They exert their deleterious effects by inducing ROS, which in turn cause the oxidative deterioration of biomolecules or epigenetic changes.65 Pesticides and various chlorination byproducts in drinking water have also been shown to induce epigenetic alterations that are associated with an increased risk of cancer.66,67 However, the effect of these environmental chemicals on allergic disease has been poorly studied to date.

Oxidative stress plays a central role in the mechanisms by which air pollutants damage human health. Many air pollutants exert their deleterious effects by inducing oxidative stress in the cells and tissues with which they have contact. In particular, TRAPs are known to form ROS such as superoxide anions, hydrogen peroxide, and hydroxyl radicals. Oxidative stress arises when ROS overwhelms antioxidant defenses. After this imbalance is reached, ROS react readily with proteins, lipids, and DNA, resulting in a number of pathological consequences (Fig. 2). A primary consequence of oxidative stress is the lipid peroxidation or the oxidative degeneration of lipids. If this process is not stopped, this reaction can lead to cell death through damaging cell membranes. Activation of signaling pathways is another way by which oxidant pollutants may induce pathological responses in the lung. Given these observations, it is likely that individual susceptibility to air pollution may depend on genetic variation in the antioxidant system. Indeed, common genetic variants of the glutathione-S-transferases, which play a role in cellular protection against oxidative stress,68 are associated with the allergic diseases in a general population with exposure to air pollution. As for air pollution, NO₂, PM_2.5, PM₁₀, and ozone were often estimated.56,58 Genetic polymorphisms of the antioxidant system and their interactions with environmental factors will be discussed later.

It has been the subject of considerable research that environmental factors, especially air pollution, are able to induce epigenetic changes that promote allergic diseases. These studies are summarized in Table 1. The studies in humans showed that PM decreases the degree of methylation of the iNOS promoter, which is involved in defense to oxidative stress.69,70 An animal study also showed that DEP challenge induces the hypermethylation of IFN-γ and the hypomethylation of IL-4, thereby skewing the immune system towards Th2 responses.71

The epigenetic effect of smoking also has been investigated in children. Cigarette smoking can affect the development of asthma by inducing global and gene-specific methylation or by modifying histone protein.72,73,74 Interestingly, regardless of maternal smoking, the smoking of grandmothers associates significantly with asthma in their grandchildren at the age of 5.75 This suggests that there is transgenerational inheritance of epigenetic changes. In addition, in utero exposure to traffic-related PAH induces a specific methylation mark that serves as a potential surrogate marker for the subsequent development of asthma in offspring.76 Besides the epigenetic effects by air pollution, an animal study that assessed the protective effect of farm exposure on asthma revealed that maternal exposure to farm-derived gram-negative bacteria protected the offspring from asthma by preventing the reduction of histone 4 acetylation within the IFN-γ promoter.77 In general, low early-life microbial exposure is associated with hypermethylation of the IFN-γ gene of naïve T cells, which is in turn associated with an increased risk of allergy (Fig. 3).78 These observations call for more research on the role of microbial stimuli in the epigenetic modulation of T cells, particularly Treg cell function.

Not only environmental factor but also genetic variation can affect or interact with the epigenetic pattern. DNA methylation of the filaggrin gene worsens the effect of heterozygosity of loss-of-function variants on eczema.79 A recent remarkable study also demonstrated three-way interactions among DNA genetic variations, epigenetic variations, and air pollution in terms of phenotype expression (Fig. 4).69 These findings suggest that the modification of epigenetic pattern by genetic variation may further interact with environmental factors, thus resulting in phenotypic alterations.

In terms of methodology, epigenetic changes are dependent on specific tissues or cell types and they may be generated in a time-dependent manner. For these reasons, it is recommended that studies on asthma-related epigenetics involve several measurements over time and that the most appropriate target cells are selected. Of particular use in such research will be birth cohorts, where pregnant women are enrolled and followed up. This will allow the epigenetic modifications that occur during the critical period of life to be identified as well as the life-long effects of these modifications. Since epigenetic changes can pass into the next generation, such epigenetics research may shed more light on the most effective methods of preventing allergic disease.

The concept of gene-environmental (GxE) interactions has evolved in the last century and has now become a central theme in studies to assess the causes of allergic disease.80 Although many studies have been performed to search for genes associated with allergic disease, genes alone cannot explain the recent increase of allergic disease. Another explanation is that genes act in association with environmental change to develop the allergic diseases. Technological advances have greatly influenced the recent burst of research into GxE interactions in allergic diseases.81 These studies have led to the identification of novel associations of genetic variants with disease, the improved measurement of environmental factors by developing exposure biology tools, and the development of new methods for identifying GxE interactions (Table 2).82,83,84,85,86,87,88,89,90,91,92,93

With regard to asthma, the most well-known and extensively studied GxE interactions are those between (1) the CD14 gene and endotoxin exposure,94 (2) HLA genes and allergens,95 and (3) TLR genes and infectious agents.96 Recent genome-wide environment interaction studies (GEWIS) on asthma also discovered interactions between genome-wide single nucleotide polymorphisms and farm-related exposure in Central Europe82 and smoking exposure.97

Given the increasing evidence showing that air pollution promotes allergic diseases, many studies have investigated the interplay between genetic susceptibility and air pollution on allergic diseases. Since air pollution induces oxidative stress and thereby promotes inflammation, most of these studies focused on genes in the oxidative stress and inflammatory pathways.

The antioxidant system is a complex network that is composed of many enzymes, of which glutathione S-transferase (GST) enzymes play a particularly important role. They are essential for glutathione homeostasis, which is a determinant of the cellular response to oxidative stress and cytoprotection from the byproducts of oxidative stress. The GSTM1 and GSTP1, sub-classes of GST, have been studied most frequently. These studies assessed how the polymorphisms in these enzymes modify individual susceptibility to air pollution and promote its deleterious effects. However, there is some debate about whether children who inherit a Val (105) variant allele in GSTM1 are protected from a higher risk of asthma with exercise, especially in high ozone levels.98 The inconsistency of the results suggests that gene to gene interactions (i.e., interactions between GSTP1 and other antioxidant genes) and epigenetic mechanisms should be considered along with GxE interactions.99

A previously described study showed that PAH augmented allergic sensitization in offspring with prenatal exposure to cockroach allergen, especially in children with GSTM1 null mutations.62 These findings suggest that in utero exposure may predetermine the physiological and metabolic adaptations that are made in later life, especially in genetically susceptible individual.

Several recent studies that detected GxE interactions examined diverse environmental determinants, particularly smoking, farming, indoor and occupational environments, air pollution, and diet or nutrition.82,83,84,85,86,87,88,89 To improve our understanding of the GxE influences on allergic diseases, newly identified genetic and environmental factors should be studied. The latter factors include microbiota and stress, especially during the prenatal or early postnatal period. Future studies should also clearly define the outcomes (phenotypes) of allergic diseases. In addition, such studies should consider various times of the GxE interaction because so many studies show that timing of exposure has crucial effects on susceptibility to disease due to lung growth, immunological maturation, and epigenetic alterations.80 Longitudinal studies with multidisplinary research teams are also needed to assess the effect of the environment on allergic diseases properly.