Demystifying Dysbiosis: Can the Gut Microbiome Promote Oral Tolerance Over IgE-mediated Food Allergy?

Author(s): Tom Marrs*, Kathleen Sim

Journal Name: Current Pediatric Reviews

Volume 14 , Issue 3 , 2018

Become EABM
Become Reviewer
REVIEW ARTICLE

Demystifying Dysbiosis: Can the Gut Microbiome Promote Oral Tolerance Over IgE-mediated Food Allergy?

Tom Marrs1, 2, * and Kathleen Sim3

1Department of Paediatric Allergy, School of Immunology & Microbial Sciences, King’s College London, London, UK;
2Children’s Allergies Department, Guy’s and St Thomas’ NHS Foundation Trust, St Thomas’ Hospital, Westminster Bridge Road, London, UK;
3Department of Medicine, Section of Paediatrics, Imperial College London, St. Mary’s Hospital Campus, Norfolk Place, UK

Abstract: The growing burden of food allergy is being driven by environmental exposures and the potential role of gut micro-organisms (or ‘microbiota’) is hotly debated. Early culture-based studies outlined that imbalances between commensal gut constituents (‘dysbiosis’) early in life may raise the risk of developing allergic disease. A number of studies using animal models describe mechanisms by which specific bacterial taxa within the gut microbiota, their diversity and dietary substrates such as fibre may promote oral tolerance. Next-generation sequencing now allows the detailed characterization of the microbiota in relation to epidemiological exposures and clinical food allergy status in humans. Faecal samples from one birth cohort characterized for food allergy status have been sequenced and showed less gut microbiota richness amongst three month infants who later developed food sensitization at one year. A large cross-sectional survey of young children with milk allergy showed that greater gut microbiota diversity and enrichment of Clostridia and Firmicutes phyla during early infancy is associated with greater likelihood of out-growing milk allergy by eight years of age. Case control studies are limited to selecting participants from amongst hospital patients and have only allowed comparison of heterogeneous groups. To assess whether infants’ gut microbiota may predispose towards the development of food allergy, cohort studies must be undertaken to evaluate gut microbiota development from early in infancy and prospectively characterise patterns according to whether challenge proven food allergy later develops, whilst adjusting for atopic dermatitis, dietary and antibiotic exposures.

Keywords : Food allergy, dysbiosis, microbiome, microbial, oral tolerance, IgE, atopy, allergy.


Article Information

Identifiers and Pagination:

Year: 2018
Volume: 14
Issue: 3
First Page: 156
Last Page: 163
Publisher Id: CPR-14-3-156
DOI: 10.2174/1573396314666180507120424

Article History:

Received Date: July 13, 2017
Revised Date: August 19, 2017
Accepted Date: April 25, 2018

* Address correspondence to this author at the Department of Paediatric Allergy, School of Immunology & Microbial Sciences, King’s College London, London, UK; E-mail: thomas.marrs@kcl.ac.uk

1. INTRODUCTION

The growing burden of food allergy suggests that environmental factors are driving its rising prevalence. The community of symbiotic micro-organisms residing in our gastro-intestinal tract (the gut ‘microbiota’) reflect aspects of our environmental exposure and may have the capacity to mediate some drivers towards the development of food allergy. Observational studies from the turn of the century highlighted that infants who carry a significant imbalance of micro-organisms amongst their gut microbiota (termed ‘dysbiosis’) may be at risk of developing atopic dermatitis and allergic sensitization [1]. Animal models have elucidated several mechanisms by which gut microbiota may influence the development of allergic disease. An increasing number of studies in humans report altered gut microbiota characteristics amongst children in relation to their food allergy status, however, none have utilized population-based designs or adjusted for clinical confounders such as atopic dermatitis or microbial confounders such as antibiotic exposure. Here, we review whether current studies are able to support the hypothesis that gut microbiota dysbiosis primes their host towards developing food allergy and look at how future work may more robustly answer this question.

1.1. Box “Glossary of terms”

16S rRNA gene      This gene encodes the 16S ribosomal RNA component of the 30S small bacterial and archaeal ribosomal subunit. The gene includes hypervariable regions that contain signature sequences which are useful for the identification of bacterial and archaeal taxa [2].

Diversity    A summary statistic for community structure, reflecting richness (total number) and evenness (skew in abundance) of comprising constituents. Shannon’s and Simpson’s indices of diversity are commonly reported and considered robust [3]. Further aspects of microbiota samples may be derived from these summary measures, for instance Pielous’s evenness quantifies skew and is based upon Shannon’s index [4].

Dysbiosis    Imbalance between micro-organisms comprising a community, which may indicate disrupted homeostasis between microbiota and host.

Genome    The entire hereditary information of an organism that is encoded by its DNA (or RNA for some viruses)

Metagenome   The collection of genomes and genes from the members of a microbiota.

Metagenomics     The process of shot-gun sequencing of DNA extracted from a sample and assemblage used to characterize the metagenome, from which information on the potential function of the microbiota can be gained [5].

Microbiota    The totality of Bacteria, Archaea, Eukarya, and their viruses that occupy a defined anatomic niche, for instance in the gastro-intestinal tract.

Microbiome     The entire habitat, their genomes and surrounding environmental or clinical data.

Next generation sequencing   The term encompasses a broad range of sequencing technologies which require preparation of templates (recombinant DNA combining adaptor with the target sequences), sequencing, genome alignment and assembly. These methodologies may be run in parallel on a large scale, making their sequencing affordable and more efficient than the ‘first generation’ Sanger sequencing technology [6].

Taxon (pl. –a)    A group of populations of organisms.

2. THE BURDEN OF FOOD ALLERGY

Children and adults living with immunoglobulin E (IgE) mediated food allergy risk life threatening reactions if they eat their respective food allergens. IgE-mediated food allergy, therefore, necessitates on-going rigorous avoidance of food allergens, the carriage of emergency medication at all times and living with the prospect of suffering a serious allergic reaction. These factors impair the quality of life of children with food allergy more than those living with either rheumatoid arthritis or diabetes [7 - 9].

The prevalence of IgE-mediated food allergy is rising amongst economically advantaged countries, with the greatest prevalence being demonstrated amongst young children. Population-based studies show that 7-10% of children in their first three years demonstrate signs of food allergy when challenged, and that this falls towards 4.2% amongst school-aged children as a proportion of milk and egg allergy resolves [10 - 12]. Clinical activity relating to food allergy has been rising amongst Western countries, with a six to seven fold increase in admission frequencies due to food allergy and anaphylaxis being reported over 20 years in the UK [13, 14]. In Australia, fatal food anaphylaxis has increased 9.7% per year (p = 0.04) with food-induced anaphylaxis admissions increasing 10% per year between 1997 to 2013 [15]. Therefore, the prevalence of food allergy has grown to an appreciable public health problem amongst western countries [16].

The rising burden of food allergy is also likely driven by the increasing prevalence of atopic dermatitis [18]. A systematic review has established that eczema increases the risk of children developing food allergy [17]. Dermatitis arises before food allergy, and more severe disease is associated with greater risk of food sensitization showing a clear dose response effect [10, 18]. It is therefore crucial that studies investigating relationships between the microbiota and food allergy examine whether this relationship is confounded by atopic dermatitis.

3. THE HYGIENE HYPOTHESIS HAS PROMOTED GREATER AWARENESS OF EXPOSURE TO MICRO- ORGANISMS

The hygiene hypothesis was born out of two studies highlighting inverse relationships between allergic disease and micro-organism exposure. The first was published in 1976 and found lower prevalence of asthma, eczema and urticaria amongst the Métis families of northern Saskatchewan in Canada who showed greater carriage of helminthes [19]. The more widely acknowledged study from the British National Child Development Study reported that the risk of hay fever and atopic dermatitis were inversely associated with the number of siblings [20]. At the time, David Strachan postulated that children living in larger families were protected from developing atopic dermatitis and allergies by greater exposure to pathogens.

This hypothesis led to a large body of work exploring how the prevalence of atopic dermatitis, asthma and rhinitis varied with respect to ‘hygiene-related’ epidemiological factors, such as mode of delivery (caesarean section versus normal vaginal delivery), living with older siblings, attending communal childcare facilities, antibiotic exposure and farming. For instance, living on a farm and attending communal child care facilities were shown to demonstrate associations with a reduced risk of asthma, rhinitis and atopic dermatitis [21 - 26]. Some studies have used molecular microbiological techniques to identify pathogens which may have a role in raising the risk of children developing allergic disease. For example, quantitative real-time Polymerase Chain Reaction was used to analyse faceal samples collected at one month of age from the 952 participants of the Child, Parent and Health: Lifestyle and Genetic Constitution (KOALA) Birth Cohort Study in the Netherlands. The authors demonstrated that colonisation with and increasing numbers of Escherichia coli were associated with a greater risk of parent reported atopic dermatitis at 2 years (Ptrend 0.02), whilst colonization with Clostridium difficile was significantly associated with atopic dermatitis, recurrent wheeze and atopic sensitization [27].

Around the turn of this century, some culture-based studies introduced the idea that an equilibrium between key constituents was of more importance than the presence of pathogens alone. Atopic Estonian and Swedish children demonstrated less frequent colonization with enterococci during their first month (72% vs. 96%, p < 0.05) and with bifidobacteria during the first year of life (17 to 39% vs 42 to 69%, p < 0.05) [6]. Such observations prompted Graham Rook to postulate the ‘Old Friends Hypothesis’, suggesting that the less-harmful agents associated with rural, hunter-gatherer lifestyle provide immunological protection from allergic disease [28. 29].

‘Next-generation’ sequencing technologies now allow a hypothesis-free approach to surveying the relative abundance and potential function of constituent identities amongst microbiome samples. Next-generation sequencing has become increasingly affordable over the last ten years, and allows parallel sequencing of millions of DNA reads extracted from microbiota samples. ‘16S’ techniques rely on amplification of bacterial DNA strands identified by universal bacterial primers annealing to well conserved 16S rRNA gene regions within the bacterial genome. With this technology, complementary strands of DNA are built from these 16S rRNA gene primers by successively adding individual fluorescently labelled nucleotides, alongside which a camera records which order of coloured nucleotides were incorporated to determine the sequence [30]. The major alternative non-16S rRNA gene sequencing approach aims to sequence all the genetic material within a sample (whether of human, bacterial, archaeal, eukaryotic or viral origin). These metagenomic methodologies such as shot-gun sequencing allow comparison of relative proportions of gene groups, enabling relative abundance comparisons of DNA reads indicating micro-organism identities and also functional capabilities of microbiome samples [31].

Next-generation sequencing technology allows the identification of around 80% more bacterial strains in faecal microbiota than conventional culture-based methods, highlighting its complexity and diversity [32]. Strong associations have been demonstrated between gut microbiota characteristics and insulin sensitivity and obesity [33 - 35]. Partly through this novel technology, the hygiene hypothesis has metamorphosed into the ‘biodiversity hypothesis’, which proposes that the diversity of the gut microbiota is of greater importance than identifying individual bacterial strains and that this aids the development of a regulatory T cell, cytokine and complement network, which protects against the development of allergic disease [36].

These hypotheses postulate that different routes and characteristics of micro-organism exposure may influence the development of food allergy. However, can we be sure that environmental exposures significantly influence the gut microbiota in the first place?

4. ENVIRONMENTAL FACTORS CAN INFLUENCE GUT MICROBIOTA CHARACTERISTICS

Studies using next-generation sequencing technology have shown that mode of delivery influences newborn colonization patterns, with those born by Caesarean carrying maternal skin-origin microbiota across oral, nasopharyngeal, skin and intestinal compartments [37]. Antibiotic studies have shown that microbiota manifest a recovery after therapy, however with difference in relative abundance of taxa [38]. Porcine animal models demonstrated striking differences in their gut microbiota and host-immune response, depending on whether they were sow-reared, indoors or outdoors [39]. The introduction of new food into the infant diet supports the development and diversification of gut microbiota, which in turn alters the immunological function of gut epithelia and the host-immune response as a whole [40, 41]. This is particularly pertinent in the light of recent randomised trials showing that early complementary feeding with food allergens such as peanut and egg have the capacity to prevent their respective food allergies [10, 42]. Thus, a range of environmental factors are associated with alteration of host gut microbiota characteristics.

5. MICROBIOTA-RELATED ENVIRONMENTAL FACTORS ARE ASSOCIATED WITH THE DEVELOPMENT OF FOOD ALLERGY

A systematic review has collated a range of evidence reporting microbiota-related environmental factors which are associated with the development of food allergy [43] . The largest case control study investigated 16,237 cases of cow’s milk allergy with age- and sex-matched controls, using a Finnish registry of state-funded hypoallergenic formula prescription. The authors reported that being born by caesarean section was associated with an increase in milk allergy diagnosis (adjusted Odds Ratio 1.18 [1.10–1.27]) [44]. The same study also showed that having a greater number of siblings was associated with protection from food allergy (ptrend < 0.01 across six categories of previous number of deliveries). A second population-based birth cohort of 5,276 one year old infants that diagnosed egg allergy using a challenge-based algorithm also demonstrated a significant protective association with having siblings (adjusted Odds Ratio 0.72 [0.62–0.83] per sibling) [45]. These relationships support the hypothesis that microbiota-related environmental factors influence the development of food allergy. However, have mechanisms been described by which the gut microbiota may mediate these associations?

6. DO ANIMAL STUDIES DEMONSTRATE POSSIBLE MECHANISMS FOR GUT MICROBIOTA DRIVING FOOD ALLERGY?

There is good animal model data elucidating different mechanisms by which the gastro-intestinal microbiota may influence the development of food allergy.

Germ-free mice, grown without any environmental exposure to microbes, have a strong disposition towards developing food allergy. These germ-free mice show that lacking exposure to environmental microbiota results in a profoundly under-developed immune system, with less local and lymphoid IgA and a paucity of plasma cells in their gastro-intestinal mucosa. They also suffer a dramatic reduction in their secondary lymphoid tissue, with smaller mesenteric lymph nodes that have no germinal centres and fewer Peyer’s patches [46]. The intraperitoneal administration of ovalbumin promotes sensitization and allergy to ovalbumin amongst mouse models. Regular oral feeding with ovalbumin before this intraperitoneal administration amongst wild-type mice with a normal gut microbiota prevents sensitization, and assures oral tolerance to ovalbumin. However, sensitised germ-free mice are unable to produce the Interferon-γ and IgG2a associated with tolerance induction, and instead uniformly produce high levels of IgE and much higher levels of Interleukin-4 (IL-4) in particular on ovalbumin challenge [47]. It therefore appears that the gut microbiota facilitates the development of oral tolerance.

A critical view may suggest that germ-free mice are not adequately representative of human pathophysiology. Antibiotic exposure may be considered a reasonable surrogate for an environmental factor associated with reduced gut microbiota abundance. Numerous studies show that rearing wild-type mice with commensal microbiota, treating them with a combination of broad-spectrum antibiotics followed by peanut sensitization and challenge procedures causes increased IgE levels and increased population of circulating basophils [48 - 50]. Both gradual re-population of commensal gut microbiota after antibiotic treatment and the introduction of clostridia strains by gavage are associated with attenuation of this allergic immune-phenotype [48, 50]. Colonisation with a consortium of clostridia (predominantly composed of members of Clostridium clusters XIVa, XIVb, and IV) has been shown to induce the production of IL-22 in the colon of antibiotic-treated mice, block food allergen sensitization and prevent the clinical responses analogous to peanut-induced anaphylaxis amongst pathogen-free murine models [48].

Some groups have demonstrated that the generation of colonic regulatory T cells are specifically promoted by clostridia species from the human gut microbiota, and that these cells allow attenuation of the allergic response [51, 52]. Others have demonstrated successful tolerance induction and regulatory B cell promotion using allergen immunotherapy amongst pathogen-free murine models when combined with administration of Clostridium butyricum [53]. It may be that clostridia inhibit the development of allergy by inhibiting the systemic absorption of key food allergens such as peanut, lowering its availability in the blood stream [48]. These models suggest that clostridia species may fulfil the ‘Old Friends’ Hypothesis for best supporting the development of oral tolerance to food allergens.

Alternatively, dietary substrates or their resulting metabolites such as short-chain fatty acids may influence the development of food allergy, rather than the presence of specific groups of bacteria such as clostridia species. Diets high in fibre, for instance, appear to impact on the relative balance of different constituent groups amongst the developing gut microbiota. This has been reported when comparing gut microbiota from children across different countries and also when adults and mice take part in dietary intervention studies [54 - 56]. Fibre is fermented into short-chain fatty acids by the intestinal microbiota, which interact with G-protein receptors on both colonic epithelial and immune cells [57]. Murine models of inflammatory bowel disease have shown that clostridia species are able to ferment dietary fibre and increase the luminal concentration of short-chain fatty acids such as butyrate proprionate, which in turn stimulate the production of colonic T-regulatory cells [58]. Dietary fibre has the ability to induce tolerance to peanut when compared to a diet without any fibre. Murine models fed fibre demonstrated a greater proportion of CD103+ dendritic cells and gut homing T-regulatory cells in their mesenteric lymph nodes, and did not develop sensitization or symptoms of anaphylaxis on peanut challenge [59]. The group showed that their dietary fibre increased the ability of CD103+ dendritic cells to covert vitamin A to retinoic acid, thereby promoting the differentiation of naive T cells into T-regulatory cells.

Animal model work has also shown that more diverse gut microbiota facilitates oral tolerance induction. A diverse gut microbiota stimulates T-regulatory cells to produce IL-10 and TGF-β, and has been shown to repress the development of Th2 allergic responses [60, 61]. The microbiota may induce these immunological responses through specific immune modulators. For example, Bacteroides fragilis, a common commensal in the human gut microbiota, carries Polysaccharide A, a key molecule capable of stimulating CD4 + CD45Rb and down-regulating inflammatory cytokine cascades [62, 63]. On the other hand, other teams have shown that the diversity of gut microbiota influences the capacity of gut microbiota to facilitate oral tolerance, rather than the carriage of particularly immunologically active moieties. The introduction of eight altered Schaedler commensal gut microbiota strains to five wild-type germ-free mouse strains induces de novo generation of mucosal CD4+CD25+ Foxp3+ cells, allowing the local production IL-10 [64]. By removing these T-regulatory cells from a model of oral tolerance, we see a relapse into allergy. Specific pathogen-free mice receiving PC61 anti-CD25 monoclonal antibody are no longer able to support tolerance after oral β-lactoglobulin gavage, and instead demonstrate raised β-lactoglobulin specific IgE and reduced ability to suppress IL-5 and IL-13 production from splenic preparations [65]. Perhaps most importantly, the capacity for mucosal tolerance induction in mice depends upon the diversity of commensal constituents amongst their gut microbiota. Germ-free mice, and those inoculated with single or dual commensal strains manifest raised IgE levels. However, increasing the diversity of their commensal gut microbiota reduces this predisposition, such that just less than half of animals inoculated with eight species have undetectable IgE, and all of those administered 40 commensal strains demonstrate undetectable IgE [66].

These models show that specific characteristics of bacteria, their diversity amongst the gut microbiota and specific dietary substrates may all support the development of oral tolerance. However, such animal models are considerably less complex than the gut microbiota associated with infants. So we next evaluate whether these hypotheses may be further substantiated amongst young children.

7. IS THERE GOOD EVIDENCE THAT GUT MICROBIOTA INFLUENCES THE DEVELOPMENT OF FOOD ALLERGY IN HUMANS?

There are now eight studies exploring associations between the gut microbiota and food allergy or sensitization in children, five of which utilised next-generation sequencing data. Unfortunately, none of the birth cohort studies were large enough to examine associations with challenge proven food allergy and they resorted to reporting food sensitization alone.

Three prospective birth cohort studies have been able to collect faecal samples in early infancy and follow up participants to determine their clinical status at a later point, however each using food sensitization as a clinical outcome rather than diagnosis of food allergy itself. The highest quality study in this area investigated the largest number of participants, with a combined total of 324 infants recruited from Sweden, England and Italy, to ascertain associations between food sensitization and gut microbiota characteristics across Europe. Six faecal samples were collected from each participant throughout their first year for conventional aerobic and anaerobic culture. No significant relationship was demonstrated between the age of faecal colonization with any cultured species and food sensitization at 18 months, and diversity of the microbiota was not assessed [67]. The second study originated from the Dutch KOALA cohort mentioned previously in which Bifidobacterium species, Escherichia coli, Clostridium difficile, Bacteroides fragilis group and Lactobacillus species were quantified from stool samples at 1 month of age using real-time Polymerase Chain Reaction and were tested for food sensitization at 1, 2 and 6-7 yrs of age. Clostridium difficile was more often found in the faecal samples of those sensitized to food later in childhood, but only significantly so amongst those with a family history of allergic disease (1.68 (1.07-2.65), p<0.05) [68]. The most recent prospective study was published by the team running the Canadian Healthy Infant Longitudinal Development (CHILD) cohort, who requested faecal samples from 166 children in their cohort at three and 12 months of age, and assessed participants’ food sensitization at 1 year. The authors were careful to exclude two infants who already demonstrated food sensitization at the time of their faecal sample at 3 months to ensure the analysis was entirely prospective rather than cross-sectional. However, the CHILD group also excluded participants with ‘microbiota-disrupting exposures’ which included any children who had received antibiotics, formula or were born by caesarean, leaving only 38 children in the analysis. Each quartile increase in overall gut microbiota richness at 3 months was associated with a 55% reduction in risk for food sensitization (adjusted Odds Ratio 0.45, 0.23–0.87) [69].

Four studies have used cross-sectional designs to investigate the gut microbiota of children with established food allergy, and one further assessing those with food sensitization. Two studies originated from Spanish infants aged two to 12 months who were diagnosed with IgE-mediated cow’s milk allergy by milk challenge at a tertiary referral centre, matched to healthy age/sex-matched controls attending hospital for routine appointments. The first study ascertained through conventional aerobic and anaerobic culture-based techniques that infants with cow’s milk allergy demonstrated a higher number of total colony-forming units of bacteria (p=0.002) and of anaerobes (p=0.002), whereas a lower number of yeasts (p=0.001) [70]. The same study team used 10 fluorescent in situ hybridization probes to characterise their relative abundance of faecal microbiota constituents. Clostridium coccoides (p<0.01) and Atopobium cluster species (p<0.01) were significantly more common amongst faecal microbiota from infants with cow’s milk allergy, but there were no differences with regard to Bifidobacteria, Lactobacilli and Bacteroides [71]. Furthermore, the children with milk allergy yielded increased faecal concentrations of short-chain fatty acids such as butyrate. None of these results support the animal model work suggesting clostridia taxa and short chain fatty acids may protect against the food allergy phenotype.

Two cross-sectional studies have used next-generation sequencing to assess faecal microbiota of children with established milk allergy. The first to be published showed that infants with cow’s milk allergy have significantly more diverse faecal microbiota (Shannon’s index, healthy=1.7±0.8 vs milk allergy=2.6±0.4, p<0.0001) and also significantly more even (Pielou’s evenness; healthy = 0.52 ± 0.2 versus milk allergy = 0.6 ± 0.3, p<0.05) faecal microbiota than age-matched healthy controls [72]. The large cross-sectional study amongst children with established milk allergy assessed faecal samples from 226 American children and compared the diversity of their faecal microbiota according to whether their milk allergy resolved by 8 years. Faecal samples from milk allergic children enrolling between 3 and 6 months of age demonstrated a greater diversity and enrichment of Clostridia and Firmicutes phyla if they grew out of their milk allergy by 8 years, supporting the putative benefits of clostridia [73].

A further two cross-sectional studies have assessed gut microbiota by next-generation sequencing amongst food allergy patient case series. The first enrolled 79 infants from clinical practice, 34 of whom were diagnosed with food allergy through skin and serum testing for sensitization and oral food challenge and 45 demonstrated negative test results and were controls. This clinical study showed no difference in diversity between the two groups, although their control population was selected from a similar cohort as their patients [74]. At the family level of classification, Clostridiaceae type 1 organisms were more prevalent amongst infants with food allergy than controls (p<0.02). The second study examined the gut microbiota amongst 45 children aged between 6 and 24 months of age according to their food sensitization measured by any specific IgE over 0.35 kUA/ml to milk, egg white, wheat, peanut, soy or gluten. Food sensitised children carried a lower Shannon diversity of both the total microbiota (p=0.01) and the bacterial phylum Bacteroidetes (p=0.02), and significantly lower overall gut microbiota richness (p=0.04) as measured by the Chao1 diversity index [75]. However, the class of clostridia and the majority of clostridia derived genera were more abundant in the children with food sensitization than controls (p<0.01). Interestingly, this work supports diversity hypothesis over the clostridia line of enquiry.

These eight studies do not provide good support for current animal model data, but neither are they robust in their clinical design. One of the prospective cohort studies suggests that lesser gut microbiota richness may predispose towards food sensitization, although two of the cross-sectional studies of established milk and food allergy respectively report increased diversity amongst allergic cases. Furthermore, a number of these studies find increased clostridia groups of constituents or species amongst cases with food allergy, which is counter to the animal model literature. However, thesse studies amongst children have significant shortcomings. None assess infants’ gut microbiota before they have developed allergy diagnosed by food challenges. The case control studies assess participants’ gut microbiota at the time of clinical diagnosis, conflating correlation with causation. Any priming of the gut microbiota towards food allergy must occur well before the relevant children present to specialist allergy centres. None of these studies control for potential confounders such as dietary choices, antibiotic exposure or mode of delivery, each of which may influence the development of allergic disease and are likely to have independent relationships with gut microbiota characteristics. Studies comparing diversity indices, relative abundance of clostridia or short chain fatty acid concentrations without parallel investigation of potential bias may be significantly flawed. It is therefore too early to draw together threads of cohesive support amongst the studies of children where consistency is currently lacking.

8. WHAT FURTHER STEPS ARE NEEDED TO ASSESS WHETHER GUT MICROBIOTA MAY FACILITATE ORAL TOLERANCE TO FOOD ALLERGENS?

Future studies should use a population-based approach to recruit infants early in life, and prospectively assess their dermatitis, sensitization and challenge-proven food allergy status, alongside likely confounders such as antibiotic usage and dietary intake. Gut microbiota characteristics would be most sensitively explored within a study capable of adjusting for the relative strength of associations between epidemiological exposures, rather than amending their sample size post-hoc according to assumed ‘microbiota-disrupting exposures’ or comparing highly heterogeneous groups of children [69, 73]. Observational studies are underway amongst well known randomised control trials such as the Enquiring About Tolerance (EAT) Study, which will help understand the influence of the microbiome in the context of population-based research [10]. Commercial treatment trials have the opportunity to assess potential confounders such as atopic dermatitis and microbiological confounders such as antibiotic usage to increase the rigour of their findings and develop greater understanding for designing better treatments. Thus far, microbiome studies in humans have not been able to adequately support or refute the respective mechanisms proposed by experimental models linking the gut microbiota to food allergy phenotypes. Nonetheless, next-generation sequencing is becoming more affordable for their use amongst large scale studies. More robust insights will be achieved through using more than one microbiological technique amongst study participants. In particular, researchers should consider utilizing both metagenomic and 16S rRNA gene sequencing in parallel with additional assessment of relevant bio-markers, to raise the integrity of research in this area.

Forthcoming consensus statements relating to the methodological design of microbiome studies will forge progress in this field, although the application to population-scaled research for young infants continues to require investigation. [Costea PI, et al. Nat Biotechnol. 2017 - standards in fecal metagenomic processing].

CONCLUSION

Our understanding of the gut microbiome is growing. Early studies suggest it has the capacity to influence the development of atopic outcomes and mechanistic data offers insights about its potential impact. However, studies amongst children vary greatly in design and the resulting data amongst infants do not yet consolidate a single pathway of relevance. Through careful investigation using a number of microbiological methodologies and judicious assessment of co-factors, the role of the gut microbiota may be further evaluated and its role in the development of food allergy be fully appreciated.

CONSENT FOR PUBLICATION

Not applicable.

CONFLICT OF INTEREST

The authors declare no conflict of interest, financial or otherwise.

ACKNOWLEDGEMENTS

Declared none.

REFERENCES

[1]
Björkstén B, Sepp E, Julge K, Voor T, Mikelsaar M. Allergy development and the intestinal microflora during the first year of life. J Allergy Clin Immunol 2001; 108(4): 516-20.
[2]
Medini D, Serruto D, Parkhill J, et al. Microbiology in the post-genomic era. Nat Rev Microbiol 2008; 6(6): 419-30.
[3]
Cox MJ, Cookson WO, Moffatt MF. Sequencing the human microbiome in health and disease. Hum Mol Genet 2013; 22(R1): R88-94.
[4]
R-Project; R-Project Pielou's measure of species evenness. 1-9-2017. .
[5]
Marchesi JR, Ravel J. The vocabulary of microbiome research: a proposal. Microbiome 2015; 3: 31.
[6]
Metzker ML. Sequencing technologies - the next generation. Nat Rev Genet 2010; 11(1): 31-46.
[7]
Cummings AJ, Knibb RC, Erlewyn-Lajeunesse M, King RM, Roberts G, Lucas JS. Management of nut allergy influences quality of life and anxiety in children and their mothers. Pediatr Allergy Immunol 2010; 21(4 Pt 1): 586-94.
[8]
Avery NJ, King RM, Knight S, Hourihane JO. Assessment of quality of life in children with peanut allergy. Pediatr Allergy Immunol 2003; 14(5): 378-82.
[9]
Primeau MN, Kagan R, Joseph L, et al. The psychological burden of peanut allergy as perceived by adults with peanut allergy and the parents of peanut-allergic children. Clin Exp Allergy 2000; 30(8): 1135-43.
[10]
Perkin MR, Logan K, Tseng A, et al. Randomized Trial of Introduction of Allergenic Foods in Breast-Fed Infants. N Engl J Med 2016; 374(18): 1733-43.
[11]
Osborne NJ, Koplin JJ, Martin PE, et al. Prevalence of challengeproven IgE-mediated food allergy using population-based sampling and predetermined challenge criteria in infants. J Allergy Clin Immunol 2011; 127(3): 668-76.e1, 2.
[12]
Roehr CC, Edenharter G, Reimann S, et al. Food allergy and non-allergic food hypersensitivity in children and adolescents. Clin Exp Allergy 2004; 34(10): 1534-41.
[13]
Gupta R, Sheikh A, Strachan DP, Anderson HR. Time trends in allergic disorders in the UK. Thorax 2007; 62(1): 91-6.
[14]
Turner PJ, Gowland MH, Sharma V, et al. Increase in anaphylaxis-related hospitalizations but no increase in fatalities: an analysis of United Kingdom national anaphylaxis data, 1992-2012. J Allergy Clin Immunol 2015; 135(4): 956-63.e1.
[15]
Mullins RJ, Wainstein BK, Barnes EH, Liew WK, Campbell DE. Increases in anaphylaxis fatalities in Australia from 1997 to 2013. Clin Exp Allergy 2016; 46(8): 1099-110.
[16]
Williams H, Stewart A, von Mutius E, Cookson W, Anderson HR. Is eczema really on the increase worldwide? J Allergy Clin Immunol 2008; 121(4): 947-54.e15.
[17]
Tsakok T, Marrs T, Mohsin M, et al. Does atopic dermatitis cause food allergy? A systematic review. J Allergy Clin Immunol 2016; 137(4): 1071-8.
[18]
Flohr C, Perkin M, Logan K, et al. Atopic Dermatitis and Disease Severity are the Main Risk Factors for Food Sensitization in Exclusively Breastfed Infants. J Invest Dermatol 2013; 134(2): 345-50.
[19]
Gerrard JW, Geddes CA, Reggin PL, Gerrard CD, Horne S. Serum IgE levels in white and metis communities in Saskatchewan. Ann Allergy 1976; 37(2): 91-100.
[20]
Strachan DP. Hay fever, hygiene, and household size. BMJ 1989; 299(6710): 1259-60.
[21]
Roduit C, Wohlgensinger J, Frei R, Bitter S, Bieli C, Loeliger S, et al. Prenatal animal contact and gene expression of innate immunity receptors at birth are associated with atopic dermatitis. J Allergy Clin Immunol 2011; 127(1): 179-85.
[22]
Westergaard T, Rostgaard K, Wohlfahrt J, Andersen PK, Aaby P, Melbye M. Sibship characteristics and risk of allergic rhinitis and asthma. Am J Epidemiol 2005; 162(2): 125-32.
[23]
Nicolaou NC, Simpson A, Lowe LA, Murray CS, Woodcock A, Custovic A. Day-care attendance, position in sibship, and early childhood wheezing: a population-based birth cohort study. J Allergy Clin Immunol 2008; 122(3): 500-6.e5.
[24]
Kinra S, Davey Smith G, Jeffreys M, Gunnell D, Galobardes B, McCarron P. Association between sibship size and allergic diseases in the Glasgow Alumni Study. Thorax 2006; 61(1): 48-53.
[25]
Gern JE, Reardon CL, Hoffjan S, et al. Effects of dog ownership and genotype on immune development and atopy in infancy. J Allergy Clin Immunol 2004; 113(2): 307-14.
[26]
Riedler J, Braun-Fahrländer C, Eder W, et al. Exposure to farming in early life and development of asthma and allergy: A cross-sectional survey. Lancet 2001; 358(9288): 1129-33.
[27]
Penders J, Stobberingh EE, van den Brandt PA, Thijs C. The role of the intestinal microbiota in the development of atopic disorders. Allergy 2007; 62(11): 1223-36.
[28]
Rook GA, Brunet LR. Old friends for breakfast. Clin Exp Allergy 2005; 35(7): 841-2.
[29]
Bloomfield SF, Rook GA, Scott EA, Shanahan F, Stanwell-Smith R, Turner P. Time to abandon the hygiene hypothesis: new perspectives on allergic disease, the human microbiome, infectious disease prevention and the role of targeted hygiene. Perspect Public Health 2016; 136(4): 213-24.
[30]
Caporaso JG, Lauber CL, Walters WA, et al. Ultra-high-throughput microbial community analysis on the Illumina HiSeq and MiSeq platforms. ISME J 2012; 6(8): 1621-4.
[31]
Lozupone CA, Stombaugh J, Gonzalez A, et al. Meta-analyses of studies of the human microbiota. Genome Res 2013; 23(10): 1704-14.
[32]
Eckburg PB, Bik EM, Bernstein CN, et al. Diversity of the human intestinal microbial flora. Science 2005; 308(5728): 1635-8.
[33]
Pedersen HK, Gudmundsdottir V, Nielsen HB, et al. Human gut microbes impact host serum metabolome and insulin sensitivity. Nature 2016; 535(7612): 376-81.
[34]
Le Chatelier E, Nielsen T, Qin J, et al. Richness of human gut microbiome correlates with metabolic markers. Nature 2013; 500(7464): 541-6.
[35]
Turnbaugh PJ, Ley RE, Mahowald MA, Magrini V, Mardis ER, Gordon JI. An obesity-associated gut microbiome with increased capacity for energy harvest. Nature 2006; 444(7122): 1027-31.
[36]
Haahtela T, Holgate S, Pawankar R, et al. The biodiversity hypothesis and allergic disease: world allergy organization position statement. World Allergy Organ J 2013; 6(1): 3.
[37]
Dominguez-Bello MG, Costello EK, Contreras M, et al. Delivery mode shapes the acquisition and structure of the initial microbiota across multiple body habitats in newborns. Proc Natl Acad Sci USA 2010; 107(26): 11971-5.
[38]
Costello EK, Stagaman K, Dethlefsen L, Bohannan BJ, Relman DA. The application of ecological theory toward an understanding of the human microbiome. Science 2012; 336(6086): 1255-62.
[39]
Mulder IE, Schmidt B, Stokes CR, et al. Environmentally-acquired bacteria influence microbial diversity and natural innate immune responses at gut surfaces. BMC Biol 2009; 7: 79.
[40]
Koenig JE, Spor A, Scalfone N, et al. Succession of microbial consortia in the developing infant gut microbiome. Proc Natl Acad Sci USA 2011; 108(Suppl. 1): 4578-85.
[41]
Francavilla R, Calasso M, Calace L, et al. Effect of lactose on gut microbiota and metabolome of infants with cow’s milk allergy. Pediatr Allergy Immunol 2012; 23(5): 420-7.
[42]
Du Toit G, Sayre PH, Roberts G, et al. Effect of Avoidance on Peanut Allergy after Early Peanut Consumption. N Engl J Med 2016; 374(15): 1435-43.
[43]
Marrs T, Bruce KD, Logan K, et al. Is there an association between microbial exposure and food allergy? A systematic review. Pediatr Allergy Immunol 2013; 24(4): 311-320.e8.
[44]
Metsälä J, Lundqvist A, Kaila M, Gissler M, Klaukka T, Virtanen SM. Maternal and perinatal characteristics and the risk of cow’s milk allergy in infants up to 2 years of age: a case-control study nested in the Finnish population. Am J Epidemiol 2010; 171(12): 1310-6.
[45]
Koplin JJ, Dharmage SC, Ponsonby AL, et al. Environmental and demographic risk factors for egg allergy in a population-based study of infants. Allergy 2012; 67(11): 1415-22.
[46]
Smith K, McCoy KD, Macpherson AJ. Use of axenic animals in studying the adaptation of mammals to their commensal intestinal microbiota. Semin Immunol 2007; 19(2): 59-69.
[47]
Sudo N, Sawamura S, Tanaka K, Aiba Y, Kubo C, Koga Y. The requirement of intestinal bacterial flora for the development of an IgE production system fully susceptible to oral tolerance induction. J Immunol 1997; 159(4): 1739-45.
[48]
Stefka AT, Feehley T, Tripathi P, et al. Commensal bacteria protect against food allergen sensitization. Proc Natl Acad Sci USA 2014; 111(36): 13145-50.
[49]
Hill DA, Siracusa MC, Abt MC, et al. Commensal bacteria-derived signals regulate basophil hematopoiesis and allergic inflammation. Nat Med 2012; 18(4): 538-46.
[50]
Bashir ME, Louie S, Shi HN, Nagler-Anderson C. Toll-like receptor 4 signaling by intestinal microbes influences susceptibility to food allergy. J Immunol 2004; 172(11): 6978-87.
[51]
Atarashi K, Tanoue T, Shima T, et al. Induction of colonic regulatory T cells by indigenous Clostridium species. Science 2011; 331(6015): 337-41.
[52]
Atarashi K, Tanoue T, Oshima K, et al. Treg induction by a rationally selected mixture of Clostridia strains from the human microbiota. Nature 2013; 500(7461): 232-6.
[53]
Shi Y, Xu LZ, Peng K, et al. Specific immunotherapy in combination with Clostridium butyricum inhibits allergic inflammation in the mouse intestine. Sci Rep 2015; 5: 17651.
[54]
De Filippo C, Cavalieri D, Di Paola M, et al. Impact of diet in shaping gut microbiota revealed by a comparative study in children from Europe and rural Africa. Proc Natl Acad Sci USA 2010; 107(33): 14691-6.
[55]
Cotillard A, Kennedy SP, Kong LC, et al. Dietary intervention impact on gut microbial gene richness. Nature 2013; 500(7464): 585-8.
[56]
Turnbaugh PJ, Bäckhed F, Fulton L, Gordon JI. Diet-induced obesity is linked to marked but reversible alterations in the mouse distal gut microbiome. Cell Host Microbe 2008; 3(4): 213-23.
[57]
Maslowski KM, Vieira AT, Ng A, et al. Regulation of inflammatory responses by gut microbiota and chemoattractant receptor GPR43. Nature 2009; 461(7268): 1282-6.
[58]
Furusawa Y, Obata Y, Fukuda S, et al. Commensal microbe-derived butyrate induces the differentiation of colonic regulatory T cells. Nature 2013; 504(7480): 446-50.
[59]
Tan J, McKenzie C, Vuillermin PJ, et al. Dietary Fiber and Bacterial SCFA Enhance Oral Tolerance and Protect against Food Allergy through Diverse Cellular Pathways. Cell Reports 2016; 15(12): 2809-24.
[60]
Maeda Y, Noda S, Tanaka K, et al. The failure of oral tolerance induction is functionally coupled to the absence of T cells in Peyer’s patches under germfree conditions. Immunobiology 2001; 204(4): 442-57.
[61]
Ishikawa H, Tanaka K, Maeda Y, et al. Effect of intestinal microbiota on the induction of regulatory CD25+ CD4+ T cells. Clin Exp Immunol 2008; 153(1): 127-35.
[62]
Mazmanian SK, Liu CH, Tzianabos AO, Kasper DL. An immunomodulatory molecule of symbiotic bacteria directs maturation of the host immune system. Cell 2005; 122(1): 107-18.
[63]
Mazmanian SK, Round JL, Kasper DL. A microbial symbiosis factor prevents intestinal inflammatory disease. Nature 2008; 453(7195): 620-5.
[64]
Geuking MB, Cahenzli J, Lawson MA, et al. Intestinal bacterial colonization induces mutualistic regulatory T cell responses. Immunity 2011; 34(5): 794-806.
[65]
Adel-Patient K, Wavrin S, Bernard H, Meziti N, Ah-Leung S, Wal JM. Oral tolerance and Treg cells are induced in BALB/c mice after gavage with bovine β-lactoglobulin. Allergy 2011; 66(10): 1312-21.
[66]
Cahenzli J, Köller Y, Wyss M, Geuking MB, McCoy KD. Intestinal microbial diversity during early-life colonization shapes long-term IgE levels. Cell Host Microbe 2013; 14(5): 559-70.
[67]
Adlerberth I, Strachan DP, Matricardi PM, et al. Gut microbiota and development of atopic eczema in 3 European birth cohorts. J Allergy Clin Immunol 2007; 120(2): 343-50.
[68]
van Nimwegen FA, Penders J, Stobberingh EE, et al. Mode and place of delivery, gastrointestinal microbiota, and their influence on asthma and atopy. J Allergy Clin Immunol 2011; 128(5): 948-55.
[69]
Azad MB, Konya T, Guttman DS, et al. Infant gut microbiota and food sensitization: associations in the first year of life. Clin Exp Allergy 2015; 45(3): 632-43.
[70]
Thompson-Chagoyan OC, Vieites JM, Maldonado J, Edwards C, Gil A. Changes in faecal microbiota of infants with cow’s milk protein allergy--a Spanish prospective case-control 6-month follow-up study. Pediatr Allergy Immunol 2010; 21: e394-400.
[71]
Thompson-Chagoyan OC, Fallani M, Maldonado J, et al. Faecal microbiota and short-chain fatty acid levels in faeces from infants with cow’s milk protein allergy. Int Arch Allergy Immunol 2011; 156(3): 325-32.
[72]
Berni Canani R, Sangwan N, Stefka AT, et al. Lactobacillus rhamnosus GG-supplemented formula expands butyrate-producing bacterial strains in food allergic infants. ISME J 2016; 10(3): 742-50.
[73]
Bunyavanich S, Shen N, Grishin A, et al. Early-life gut microbiome composition and milk allergy resolution. J Allergy Clin Immunol 2016; 138(4): 1122-30.
[74]
Ling Z, Li Z, Liu X, et al. Altered fecal microbiota composition associated with food allergy in infants. Appl Environ Microbiol 2014; 80(8): 2546-54.
[75]
Chen CC, Chen KJ, Kong MS, Chang HJ, Huang JL. Alterations in the gut microbiotas of children with food sensitization in early life. Pediatr Allergy Immunol 2015; 27(3): 254-62.