Gut Microbiome and the Development of Food Allergy and Allergic Disease

Abstract

The prevalence of food allergy and other allergic diseases continues to rise within the industrialized world, yet the cause of this epidemic remains elusive. Environmental factors such as microbial exposures have more recently been implicated as one possible driving factor behind the increasing burden of allergic disease. The impact of gut microbiome on human development, nutritional needs, and disease has become evident with advances in our ability to study these complex communities of microorganisms, and there is a growing appreciation for the role of the microbiome in immune regulation. Several studies have examined associations between changes in the commensal microbiota and the development of asthma, allergic rhinitis, and asthma, but far less have evaluated the impact of the microbiome on the development of food allergy. In this article we review the human gastrointestinal microbiome, focusing on the theory and evidence for its role in the development of IgE-mediated food allergy and other allergic diseases.

Introduction

Food allergy, defined as an adverse, immune-mediated reaction to a food that is reproducible upon a subsequent exposure,¹ affects nearly 5% of all adults² and up to 8% of children in the United States.³ Recent data from the US Centers for Disease Control and Prevention (CDC) have found that the prevalence among children 0 to 17 years increased by 50% from 1999 to 2011.⁴ Even prior to this increase in prevalence, food allergies were the leading cause of anaphylaxis in patients presenting to the emergency department in the US.⁵ Studies have also shown that a diagnosis of food allergy results in a significantly lower quality of life.^6–8 Despite the increase in prevalence, the life-threatening potential, and the disease burden of food allergies, the cause of this epidemic remains elusive.

One of the leading theories to explain this modern day allergy epidemic was introduced by Strachan in 1989 as the hygiene hypothesis. In his hypothesis, Strachan proposed that a larger family size was protective against allergic disease because of early life exposure to sibling infections.⁹ However, since its introduction, others have revisited this idea, suggesting that that changes in early life viral and bacterial exposures and intestinal colonization patterns in western countries have contributed to the failure to induce and maintain tolerance, a state of unresponsiveness to harmless antigens.^10,11

The Human Microbiome

It has been estimated that the human gut is populated with up to 100 trillion microbes.¹² Rough estimates are that the microbiota (previously termed flora or microflora) contain on the order of 150-fold more genes than are encoded in the human genome.¹³ The ancient symbiotic relationship between multicellular animals and resident microbes has shaped the evolution of our immune system into its present state.¹⁴ Although the composition of the microbiota changes substantially from infancy to adulthood, the majority of organisms come from the four phyla Actinobacteria, Bacteroidetes, Firmicutes, and Proteobacteria.¹⁵

The advent of high-throughput DNA deep sequencing technologies has revolutionized the ability to characterize microbial diversity and compare this diversity across organs and individuals. Sequencing of diagnostic regions of the 16S rRNA gene sequence provides a robust method to identify the bacteria present in a sample. Because clinical samples can be sequenced directly, organisms are identified even if they cannot yet be cultured, and the resulting 16S rRNA sequence provides a reference for known bacterial taxa (i.e., species) and for novel ones.^16,17 This bacterial census provides information on specific taxa that are present; loss of specific taxa and alterations in the community structure are associated with disease progression (e.g., infection by Clostridium difficile).¹⁸ Beyond 16S data, genome sequencing from microbial communities (i.e., metagenomics) can enable functional studies, identify gene categories that influence the host, and reveal conservation at the level of gene function even in cases where those genes are derived from unrelated organisms.^19,20 Much current work is aimed at extending these techniques to understand gene expression at the RNA (transcriptional profiling) and protein (proteomics) levels, and to understand how microbial communities affect the flux of metabolites (metabolomics) in the host.²¹

The Microbiome and Immune Development

Early microbial colonization plays an important role the development of both the innate and the adaptive immune systems, ²² and there are several proposed mechanisms to explain how alterations in microbiome could lead to the development of allergic disease. Experimental, germ-free (gnotobiotic) mouse models have demonstrated that gut-associated lymphoid tissues (GALT) fail to develop when microbial colonization is delayed, leading to a Th2 skewed immune response.²³ Secretory IgA produced by resident B cells in GALT tissue may also promote oral tolerance by binding allergens in the gut and preventing their uptake.²⁴ Microbial colonization has been shown to be important in the development of Th1^25,26 and regulatory T cells (Tregs),^27–31 which are necessary to maintain immunologic balance and promote tolerance. Microbiota may also influence epigenetic modifications of genes. It is known that various forms of epigenetic changes, such as DNA methylation and histone modifications play an important role in immune development and regulation,³² and microbial metabolites butyrate and propionate have been shown to have inhibitory effects on histone deacetylases that may promote the development of peripherally induced Tregs.^33,34 Lastly, the gut microbiota plays a significant role in the development and maintenance of barrier function^35,36 and it is thought that a breakdown of this epithelial barrier may lead to allergic sensitization.^37,38

The impact of the microbiome on human development, nutritional needs, and even psychological variations has become evident with advances in our ability to study these complex communities of microorganisms.^39,40 There is also a growing appreciation for the role of the microbiome in immune regulation, and it is plausible that changes in the commensal microbiota may influence the development of food allergy and other allergic diseases.³⁶ When considering various determinants that may influence the unique bacterial families that constitute the microbiome, there are several factors to consider, including: environmental setting, mode of delivery, birth order, antibiotic exposure, and diet.⁴¹ In this review, we will explore the relationship between the gastrointestinal microbiota and IgE-mediated food allergy and other allergic diseases.

The Influence of the Microbiome in Allergic Disease

The potential impact of the microbiome on allergic disease was first studied in Europe using cross-sectional surveys to examine the prevalence of allergic diseases in children. The authors found that children living in farming environments had a significantly decreased frequency of hay fever, asthma and eczema compared to children living in urban areas.^42,43 This relationship was further explored in the GABRIELA and PARSIFAL cohorts, which confirmed previous observations that children living on farms had decreased rates of allergic disease compared to urban children.^44,45 Although most studies have focused on the impact of postnatal environmental exposure, there is increasing evidence that prenatal exposure may also be important.^46–48 Epidemiological studies examining the effect of prenatal exposures on the development of allergic disease have shown that maternal exposure to farming environments during pregnancy is associated with decreased rates of asthma, allergic rhinitis and eczema in their children.^49,50

Animal Exposure

Recent studies suggest that the protective association between farming and the development of allergic disease may be due to differences in microbial exposure. Using single-strand conformational polymorphism DNA analysis to examine house dust in the same GABRIELA and PARSIFAL cohorts, Ege et al. found that the diversity of microbial exposure was inversely associated with the prevalence of asthma even after controlling for farming status.⁵¹ Moreover, investigators examining the gut microbiota in children using 16S rDNA sequencing have shown that decreased microbial diversity early in life is associated with the development of asthma,⁵² allergic rhinitis,⁵³ and atopic dermatitis.⁵⁴ Similarly, pet ownership has also been shown to increase the diversity of the microbial composition of house dust⁵⁵ and infant fecal samples,⁵⁶ and early life pet ownership has been associated with a decreased risk for asthma and other atopic diseases.^57–59 Specifically, several studies have shown that infants who develop allergic disease later in life tended to have less Bacteroides, Bifidobacteria and Enterococci, but more Clostridiae comprising their microbiome early in life (Table 1).^54,60–62 It appears that a diverse microbial exposure both perinatally and early in life modifies both the innate⁴⁹ and adaptive⁶³ immune system resulting in a significantly decreased risk of allergic disease.

Table 1.

Summary of various early-life environmental exposures and specific gut microbiota associated with the development of allergic disease

Exposure	Bacteria	Risk
Less Animal Exposure	Bacteroides, Bifidobacteria, and Enterococci Clostridiae	Asthma, Allergic Rhinitis, and Eczema +/− Food Allergy
Delivery by Caesarean-section	Bacteroides, Bifidobacteria, and E. coli Klebsiella, Enterobacter, Enterococcus	Asthma, Allergic Rhinitis, and Eczema +/− Food Allergy
Decreased Siblingship	Clostridia, Lactobacillus, and Bacteroides	Eczema (Clostridia) ? Asthma, Allergic Rhinitis, and Food Allergy
Perinatal Antibiotic Use	Bifidobacteria and Lactobacillus Proteobacteria and Enterobacteriaceae	Asthma and Eczema +/− Food Allergy – Allergic Rhinitis
Bottle Feeding	Staphylococcus Clostridium difficile, Bacteroides, Enterococci, and Enterobacteriaceae	Asthma and Eczema (high-risk pts.) +/− Food Allergy and Allergic Rhinitis

Mode of Delivery

Another factor that has been implicated in altering the human microbiome is birth by Caesarean-section. Instead of traveling through the birth canal where colonization by maternal microbiota would typically occur, the baby is delivered through a sterile surface.⁶⁴ Subsequently, delivery by Caesarean-section has been shown to delay the development of the gut microbiota and shape its colonization to patterns similar to the maternal skin.⁶⁵ Studies examining the impact of this difference in microbiome and the development of allergic disease have found that children born by Caesarean-section had decreased microbial diversity and reduced Th1 responses during the first two years of life.²⁶ Other studies have shown an association between Caesarean-section delivery and the development of asthma, allergic rhinitis, and eczema.^62,66–68 Specifically, the microbiome of babies born by Caesarean-section showed a reduced abundance of Bacteroides, Bifidobacteria, and E. coli but increased amounts of Klebsiella, Enterobacter, Enterococcus, and clostridia (Table 1).^26,62,69,70

Birth Order and Family Size

An observation that infants with higher numbers of siblings had a decreased incidence of allergic disease was one driving principles behind Strachan’s proposal of the initial hygiene hypothesis.⁹ Since then, several studies have reproduced the inverse relationship between sibling number and asthma, allergic rhinitis, and eczema.⁷¹ This association was initially thought to arise from an increased exposure to infections during childhood. There is now evidence that birth order and family size may also mediate their protective effects through alterations in the gut microbiome.⁷⁰ Penders and colleagues showed that infants with an increased number of older siblings had decreased colonization rates of clostridia and increased rates of Lactobacillus and Bacteroides. Moreover, colonization with clostridia was associated with an increased risk of developing atopic dermatitis (Table 1).⁷⁰

Antibiotic Exposure

It is well known that early life antibiotic exposure can influence an infant’s microbiome. Data from a population-based study done by the CDC from 2003–2004 reported that 32% of laboring women received intrapartum antibiotics for group B Streptococcus infection prevention, maternal pyrexia, prematurity, and other factors.⁷² One of the most frequent reasons for early antibiotic use is prematurity, and it has been shown that bacterial microbiota colonization can be delayed in children who have a prolonged neonatal hospital course.⁷³ Studies using qPCR and 16S rRNA sequencing to specifically examine microbiome changes in preterm infants as a result of perinatal antibiotic exposure have found that infants receiving antibiotics had a lower bacterial diversity and higher abundance of Enterobacter.^74,75 Similar studies on full term infants receiving perinatal antibiotics also showed that antibiotic treatment as associated with less bacterial diversity along with higher proportions of Proteobacteria and Enterobacteriaceae and lower proportions of Bifidobacterium and Lactobacillus (Table 1).^76,77 When considering the relationship between the development of allergic disease and early-life antibiotic exposure, there appears to be an association between both prenatal^78,79 and postnatal^79–81 antibiotic exposure and asthma. It should be noted, however, that many studies might be confounded by an increased treatment of respiratory infections at an early age when manifestations of asthma may be indistinguishable from infection.^82,83 There may also be an association between postnatal but not prenatal antibiotic exposure and the development of atopic dermatitis,⁸⁴ but a significant relationship between early-life antibiotic use and allergic rhinitis has not been established.^85,86

Diet

A final area that has been shown to have significant effects on the microbiome is diet.⁸⁷ One specific dietary aspect that has been extensively studied in regard to its effects on gut microbial composition and the development of allergic disease is bottle versus breast-feeding. It has been demonstrated that breast milk may contain small oligosaccharides that promote the colonization of beneficial bacteria such as Bifidobacteria,⁸⁸ Most studies over the last 30 years, however, have only shown minor differences in gut microbiota between breast and formula-fed infants.⁶⁹ One of the most reproducible differences between breast and formula-fed infants is that formula fed infants have higher amounts of Clostridium difficile that make up their gut microbiome.^89–92 Some studies also suggest that Bacteroides, Enterococci, and Enterobacteriaceae may be more common in the microbiome of formula fed infants, where staphylococci tend to be more prevalent in breast fed infants (Table 1).⁶⁹ When evaluating the impact of breast-feeding on the development of allergic disease, breast-fed infants appear to have a lower rate of early wheezing and asthma, but this affect seems to diminish with age.^93–96 Studies examining the association between breast-feeding and the development of allergic rhinitis and eczema have been inconclusive, though there may a protective effect for eczema in high risk infants.^95,97

More generally, it is well known that westernized countries have a higher prevalence of allergic disease,⁹⁸ and modern western diets have been associated with differences in the gut microbiome.⁸⁷ Studies have also shown that differences in consumption of animal fat, carbohydrates, and fiber can cause changes in gut microbiota that can have profound affects on the immune system.^99,100 A Recent study further demonstrated that microbial metabolism of dietary fiber and subsequent production of short-chain fatty acids influenced Th2 inflammation and allergic airway disease in mice.¹⁰¹ Work directly addressing the influence of dietary factors and the development of allergic disease in humans are lacking however.

The Influence of the Microbiome in Food Allergy

In contrast to other allergic diseases, there is significantly less literature specifically evaluating the impact of the microbiome on the development of food allergy, and a majority of studies have been done using mouse models. Gnotobiotic and antibiotic treated mice that are reconstituted with well-characterized populations of gut microbiota can provide a particularly useful insight into the role that the microbiome plays in the maintenance of oral tolerance.^23,102,103

Murine Models

One of the first studies investigating the impact of the gut microbiota on oral tolerance induction showed that gnotobiotic mice had Th2 skewing and increased IL-4 production with OVA challenge that was abrogated with intestinal microbiota reconstitution of the bacteria Bifidobacterium infantis.²³ The authors demonstrated that the Bifidobacterium reconstitution was only effective when performed during the neonatal period, but not in older mice suggesting that there may be a window of time during immune development in which the commensal microbiota plays an important regulatory role.²³ In a similar gnotobiotic mouse model, antibiotic treated mice were shown to have an increased susceptibility to peanut sensitization characterized by increased peanut specific IgE and anaphylactic symptoms with peanut challenge.^102,103 Moreover, colonizing antibiotic-treated mice with a Clostridia-enriched microbiota, which has been previously shown to induce colonic Tregs,^28,104 confers a food allergy protective phenotype in an IL-22 dependent mechanism by affecting intestinal barrier function and reducing the amount of peanut allergen in the bloodstream after intragastric gavage.¹⁰³ Another study demonstrated that colonization of gnotobiotic mice with Bifidobacterium spp. and Bacteroides spp. from the fecal microbiota of healthy infants was protective in a mouse model of cow’s milk allergy.¹⁰⁵ Commensal microbiota may also impact the development of food allergy is through its activation of toll-like receptors (TLR) of intestinal epithelial cells (IECs).¹⁰⁶ TLR4 deficient mice have been shown to have a Th-2 skewed immune response and an increased susceptibility to food allergy that is abrogated with a TLR9 ligand.¹⁰² Furthermore, peripheral blood mononuclear cells (PBMCs) from food allergic patients were shown to have a negative effect on barrier function of IECs in vitro that was prevented with TLR9 activation.¹⁰⁷ Finally, a specific microbiota signature was recently linked to mice carrying a gain-of-function mutation in the IL-4 receptor α chain that results in an increased susceptibility to oral allergic sensitization and anaphylaxis.¹⁰⁸ The authors demonstrate that germ-free, wild-type mice reconstituted with this microbiota rendered these animals more prone to developing food allergy.

Human Studies

There have been a few epidemiological studies that have examined the relationship between environmental factors that are known to alter the gut microbiome and food allergy. One important aspect to consider in many of these studies is how the diagnosis of food allergy is established. Many studies rely on self-reported diagnosis of food allergy or evidence of IgE sensitization with either skin-prick testing (SPT) or serology, which is notoriously inaccurate compared to the gold standard of oral food challenge (OFC).^109,110

Mode of delivery has been the most widely studied environmental factor thought to contribute to the development of food allergy. Overall, there is evidence that Caesarean-section delivery increases the risk of developing IgE sensitization to food allergens,¹¹¹ but studies utilizing OFC proven food allergy have shown mixed results.^112–115 Although several studies have addressed the effect of farming environment and animal exposure on the development of other allergic diseases, far less have examined this environmental exposure and food allergy. In an Australian infant cohort of 5276 infants, J.J. Koplin et al. found that the presence of a dog in the home was inversely associated with the diagnosis of egg allergy at 1 year of age (aOR 0.72),¹¹⁵ however, this is the only study with OFC confirmed food allergy and more research needs to be done.¹¹⁶ The same authors used this infant cohort to also examine the influence of birth order on food allergy and found that children with older siblings had a significantly reduced risk of egg allergy at 1 year of age.¹¹⁵ Similar results have been found in studies evaluating siblingship size and cow milk allergy (CMA).¹¹⁴ Antibiotic exposure has shown conflicting results when assessed as a risk factor for developing food allergy. Although one study found that both pre and postnatal antibiotic exposure was associated with an increased risk of CMA,¹¹⁷ other studies have not demonstrated a statistically significant association.^112,115,118 A final area that has been evaluated in association with the development of food allergy is bottle versus breast-feeding. Aside from the fact that many studies that have examined this relationship rely on the presence of sensitization as a marker for food allergy, another obstacle in interpreting this literature is that the extent and duration of breast-feeding varies substantially between studies. Considering these limitations, there is insufficient data at this time to suggest whether breast-feeding is a protective factor in the development of food allergy.^95,119,120

Only a few studies have assessed the specific microbiota within the human gut that have been associated with the development of food allergy. Using conventional culturing techniques, a Spanish cohort of 46 patients with CMA demonstrated a greater total bacterial count and more anaerobes in the feces of allergic patients at diagnosis compared to matched controls, but no difference in the percentage of bacterial species.¹²¹ In a follow-up study of the same patient cohort, the authors better characterize the fecal microbiota using 10 fluorescent in situ hybridization and find that CMA patients had significantly more Clostridium coccoides and Atopobium cluster species compared to non-allergic controls, but there were no differences in Bifidobacteria, Lactobacilli, or Bacteroides genera.¹²² Using 16SrRNA sequencing, a separate study found increased levels of Clostridium sensu stricto and Anaerobacter but decreased levels of Bacteroides and Clostridium XVIII in the feces of 17 Chinese infants with IgE mediated food allergy.¹²³ Finally, using 16S rRNA sequencing to examine the gut microbiota in cohort of Canadian infants, M.B. Azad et al. showed that the 12 infants with food sensitization on skin prick testing had increased amounts of Enterobacteriaceae and less Bacteroidaceae in their feces.¹²⁴

Conclusions

Early microbial colonization plays an important role the development of both the innate and the adaptive immune systems, and there are several proposed mechanisms to explain how alterations in microbiome could lead to the development of allergic disease. Although some studies have identified notable relationships between the gastrointestinal microbiota and the development of asthma, allergic rhinitis, and eczema, specific studies examining the microbiome in human food allergy are lacking. Animal models suggest that the microbiome, particularly early in life, may play a crucial role in the susceptibility to food sensitization and food allergy, however, more work is required to confirm these findings. As technology and knowledge of the microbiome advances, discoveries in food allergy and atopic disease will likely provide insight into primary prevention and treatment strategies.

Key Points.

• Early microbial colonization plays an important role in the development of both the innate and the adaptive immune systems, and there are several proposed mechanisms to explain how alterations in microbiome could lead to the development of allergic disease.

• Although some studies have identified notable relationships between the gastrointestinal microbiota and the development of asthma, allergic rhinitis, and eczema, specific studies examining the microbiome in human food allergy are lacking.

• As technology and knowledge of the microbiome advances, discoveries in food allergy and atopic disease will likely provide insight into primary prevention and treatment strategies.