The impact of gut microbiome on human development, nutritional needs, and disease has become evident with advances in the ability to study these complex communities of microorganisms, and there is 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. This article reviews 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.
Key points
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Early microbial colonization plays an important role in the development of 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.
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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.
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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.
Introduction
Food allergy, defined as an adverse, immune-mediated reaction to a food that is reproducible on a subsequent exposure, affects nearly 5% of all adults and up to 8% of children in the United States. Recent data from the US Centers for Disease Control and Prevention have found that the prevalence among children 0 to 17 years increased by 50% from 1999 to 2011. Even before this increase in prevalence, food allergies were the leading cause of anaphylaxis in patients presenting to the emergency department in the United States. Studies have also shown that a diagnosis of food allergy results in a significantly lower quality of life. 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. However, since its introduction, others have revisited this idea, suggesting 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.
Introduction
Food allergy, defined as an adverse, immune-mediated reaction to a food that is reproducible on a subsequent exposure, affects nearly 5% of all adults and up to 8% of children in the United States. Recent data from the US Centers for Disease Control and Prevention have found that the prevalence among children 0 to 17 years increased by 50% from 1999 to 2011. Even before this increase in prevalence, food allergies were the leading cause of anaphylaxis in patients presenting to the emergency department in the United States. Studies have also shown that a diagnosis of food allergy results in a significantly lower quality of life. 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. However, since its introduction, others have revisited this idea, suggesting 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.
The human microbiome
It has been estimated that the human gut is populated with up to 100 trillion microbes. 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. The ancient symbiotic relationship between multicellular animals and resident microbes has shaped the evolution of the immune system into its present state. Although the composition of the microbiota changes substantially from infancy to adulthood, most organisms come from the four phyla Actinobacteria, Bacteroidetes, Firmicutes, and Proteobacteria.
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 (ie, species) and for novel ones. 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 (eg, infection by Clostridium difficile ). Beyond 16S data, genome sequencing from microbial communities (ie, 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. 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.
The microbiome and immune development
Early microbial colonization plays an important role the development of 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. Experimental, germ-free (gnotobiotic) mouse models have demonstrated that gut-associated lymphoid tissues fail to develop when microbial colonization is delayed, leading to a Th2 skewed immune response. Secretory IgA produced by resident B cells in gut-associated lymphoid tissues may also promote oral tolerance by binding allergens in the gut and preventing their uptake. Microbial colonization has been shown to be important in the development of Th1 and regulatory T cells (Tregs), 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, 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. Lastly, the gut microbiota plays a significant role in the development and maintenance of barrier function and it is thought that a breakdown of this epithelial barrier may lead to allergic sensitization.
The impact of the microbiome on human development, nutritional needs, and even psychological variations has become evident with advances in the ability to study these complex communities of microorganisms. 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. 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. This article explores 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 with children living in urban areas. 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 with urban children. Although most studies have focused on the impact of postnatal environmental exposure, there is increasing evidence that prenatal exposure may also be important. Epidemiologic 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.
Animal Exposure
Recent studies suggest that the protective association between farming and the development of allergic disease may be caused by differences in microbial exposure. Using single-strand conformational polymorphism DNA analysis to examine house dust in the same GABRIELA and PARSIFAL cohorts, Ege and colleagues found that the diversity of microbial exposure was inversely associated with the prevalence of asthma even after controlling for farming status. 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, allergic rhinitis, and atopic dermatitis. Similarly, pet ownership has also been shown to increase the diversity of the microbial composition of house dust and infant fecal samples, and early life pet ownership has been associated with a decreased risk for asthma and other atopic diseases. 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 ). It seems that a diverse microbial exposure perinatally and early in life modifies the innate and adaptive immune system resulting in a significantly decreased risk of allergic disease.
| Exposure | Bacteria | Risk |
|---|---|---|
| Less animal exposure | ↓ Bacteroides, bifidobacteria, and enterococci ↑ Clostridiae | ↑ Asthma, allergic rhinitis, and eczema +/− Food allergy |
| Delivery by cesarean section | ↓ Bacteroides, bifidobacteria, and Escherichia 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 patients) +/− Food allergy and allergic rhinitis |
Mode of Delivery
Another factor that has been implicated in altering the human microbiome is birth by cesarean section. Instead of traveling through the birth canal where colonization by maternal microbiota would typically occur, the baby is delivered through a sterile surface. Subsequently, delivery by cesarean section has been shown to delay the development of the gut microbiota and shape its colonization to patterns similar to the maternal skin. Studies examining the impact of this difference in microbiome and the development of allergic disease have found that children born by cesarean section had decreased microbial diversity and reduced Th1 responses during the first 2 years of life. Other studies have shown an association between cesarean section delivery and the development of asthma, allergic rhinitis, and eczema. Specifically, the microbiome of babies born by cesarean section showed a reduced abundance of Bacteroides , bifidobacteria, and Escherichia coli but increased amounts of Klebsiella , Enterobacter , Enterococcus , and clostridia (see Table 1 ).
Birth Order and Family Size
An observation that infants with higher numbers of siblings had a decreased incidence of allergic disease was one driving principle behind Strachan’s proposal of the initial hygiene hypothesis. Since then, several studies have reproduced the inverse relationship between sibling number and asthma, allergic rhinitis, and eczema. 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. 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 (see Table 1 ).
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 Centers for Disease Control and Prevention from 2003 to 2004 reported that 32% of laboring women received intrapartum antibiotics for group B Streptococcus infection prevention, maternal pyrexia, prematurity, and other factors. 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. Studies using quantitative polymerase chain reaction 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 . 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 (see Table 1 ). When considering the relationship between the development of allergic disease and early life antibiotic exposure, there seems to be an association between prenatal and postnatal 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. There may also be an association between postnatal but not prenatal antibiotic exposure and the development of atopic dermatitis, but a significant relationship between early life antibiotic use and allergic rhinitis has not been established.
Diet
A final area that has been shown to have significant effects on the microbiome is diet. 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 breastfeeding. It has been demonstrated that breast milk may contain small oligosaccharides that promote the colonization of beneficial bacteria, such as bifidobacteria. Most studies over the last 30 years, however, have only shown minor differences in gut microbiota between breastfed and formula-fed infants. One of the most reproducible differences between breastfed and formula-fed infants is that formula-fed infants have higher amounts of C difficile that make up their gut microbiome. Some studies also suggest that bacteroides, enterococci, and Enterobacteriaceae may be more common in the microbiome of formula-fed infants, whereas staphylococci tend to be more prevalent in breastfed infants (see Table 1 ). When evaluating the impact of breastfeeding on the development of allergic disease, breastfed infants seem to have a lower rate of early wheezing and asthma, but this affect seems to diminish with age. Studies examining the association between breastfeeding and the development of allergic rhinitis and eczema have been inconclusive, although there may be a protective effect for eczema in high-risk infants.
More generally, it is well known that westernized countries have a higher prevalence of allergic disease, and modern western diets have been associated with differences in the gut microbiome. 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. 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. Work directly addressing the influence of dietary factors and the development of allergic disease in humans, however, is lacking.
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 most 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.
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 interleukin (IL)-4 production with OVA challenge that was abrogated with intestinal microbiota reconstitution of the bacteria Bifidobacterium infantis . 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 when the commensal microbiota plays an important regulatory role. 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. Moreover, colonizing antibiotic-treated mice with a clostridia-enriched microbiota, which has been previously shown to induce colonic Tregs, 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. 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 (CMA). Commensal microbiota may also impact the development of food allergy through its activation of toll-like receptors (TLR) of intestinal epithelial cells. TLR4-deficient mice have been shown to have a Th2 skewed immune response and an increased susceptibility to food allergy that is abrogated with a TLR9 ligand. Furthermore, peripheral blood mononuclear cells from food-allergic patients were shown to have a negative effect on barrier function of intestinal epithelial cells in vitro that was prevented with TLR9 activation. 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. 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 epidemiologic 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 or serology, which is notoriously inaccurate compared with the gold standard of oral food challenge (OFC).
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 cesarean section delivery increases the risk of developing IgE sensitization to food allergens, but studies using OFC-proved food allergy have shown mixed results. 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, Koplin and colleagues found that the presence of a dog in the home was inversely associated with the diagnosis of egg allergy at 1 year of age (adjusted odds ratio, 0.72); however, this is the only study with OFC-confirmed food allergy and more research needs to be done. 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. Similar results have been found in studies evaluating siblingship size and CMA. Antibiotic exposure has shown conflicting results when assessed as a risk factor for developing food allergy. Although one study found that prenatal and postnatal antibiotic exposure was associated with an increased risk of CMA, other studies have not demonstrated a statistically significant association. A final area that has been evaluated in association with the development of food allergy is bottle versus breastfeeding. 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 breastfeeding varies substantially between studies. Considering these limitations, there are insufficient data at this time to suggest whether breastfeeding is a protective factor in the development of food allergy.
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 patients with allergy at diagnosis compared with matched control subjects, but no difference in the percentage of bacterial species. 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 with control subject without allergy, but there were no differences in Bifidobacteria , Lactobacillus , or Bacteroides genera. 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. Finally, using 16S rRNA sequencing to examine the gut microbiota in a cohort of Canadian infants, Azad and colleagues showed that the 12 infants with food sensitization on skin prick testing had increased amounts of Enterobacteriaceae and less Bacteroidaceae in their feces.
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