Farming lifestyle and human milk: modulation of the infant microbiome and protection against allergy

Abstract

There has been an increased prevalence of several allergic manifestations such as food allergy, atopic eczema, allergic rhinitis, and asthma. Several explanations have been proposed why this has occurred, but one of the main contributing factors may be the gradual loss of microbial exposures over time in regions where allergy is prevalent. Such exposures occur in individuals who practice a traditional farming lifestyle and are protected against allergy. Infant consumption of human milk, more commonly practiced in these farming communities may provide an alternative in combatting allergy, as it known to be beneficial to infant health. In this review we cover human milk and its role in shaping the gut microbiome promoting the growth of beneficial bacteria like Bifidobacterium, as well as the downstream impact of the farming lifestyle, human milk and Bifidobacterium has on developing infant immunity.

Emergence of allergic disease and protection of the farming lifestyle

There has been a rise in the prevalence of allergic manifestations such as food allergy, atopic eczema, allergic rhinitis, and asthma within the last century. Initially thought to be a burden of industrialized nations, allergic disease has started to be recognized as a growing problem in developing regions as well.(1) Food allergy is one of the earliest manifestations of allergic diseases, originating from abnormal responses in the gut upon antigen encounter leading to sensitization. However, sensitization may occur at other epithelial surfaces such as the skin, with subsequent antigen exposure in the gut triggering an allergic response.(2) Hen’s egg and cow’s milk are the most common allergenic foods; however, other top food allergens include peanut, tree nut, and shellfish.

The growing prevalence of food allergy has put an enormous burden on the quality of life of individuals with food allergy and their families. Currently there is no effective therapy, so food avoidance is the only strategy to circumvent morbidities associated with an allergic response. The rapid rise in food allergy cannot be explained by genetic predisposition; however, a change in lifestyles or environment has been suggested as the contributor.(3) For example, studies from Europe and in America have shown a decreased prevalence of allergic diseases, including food allergy, in individuals who reside on farms.(4, 5) Mechanism(s) of this protection are potentially numerous and are not fully understood. One hypothesis is that farm living provides enhanced and diverse environmental exposures conferring a level of protection against the development of allergy.(6) For example, several studies have demonstrated that infant consumption of unpasteurized farm milk and contact with animals and their stables are protective against sensitization and allergy.(5) Moreover, this protection can extend prenatally as similar maternal farm exposures during pregnancy can also confer protection to the infant.(7, 8) Alternatively, infant consumption of human milk, which is more common and of longer duration in farming communities may provide another pathway of maternal influence on infant health outcomes.(4)

Human milk and infant microbiome

Human milk is a highly complex and important resource for developing infants. There is continued debate whether consumption of human milk provides protection against development or asthma and atopic diseases.(9) Yet, the recent updated report from the American Academy of Pediatrics suggest that infant consumption of human milk for at least 3 months has shown to be protective in relation to the development of atopic eczema and wheeze, there is a lack of data on food allergy to conclude whether feeding human milk is protective.(9) However, given the strong association between eczema and food allergy and the protective effect of human milk against eczema, human milk may also play a role in protection against food allergy, but this has yet to be tested in large, well-designed observational studies (randomized trials of breastfeeding are unethical).(10) Human milk contains numerous components including proteins, lipids, minerals, and hormones; in particular, microbiota and immunoglobulins, the majority of which being IgA that is transferred into the infant gut. These factors can contribute to early gut microbiome development, shaping early gut microbial communities and their function.(11) Human milk composition and its association with allergy has been examined, focused on components like secretory IgA and cytokines/chemokines.(11) The data on human milk composition in the context of the farming lifestyle is more limited; elevated TGF-β and IL-10 both cytokines associated with anti-inflammatory responses were found elevated in farm-exposed mothers.(12) We recently observed differences in human milk composition in the mothers of the Old Order Mennonite (OOM) community, who live a traditional farming lifestyle and are at a low risk of developing allergic disease compared to mothers living in an urban setting.(4, 13) In particular, we found differences in IgA specific responses, cytokines, and fatty acids in the human milk of OOM others compared to mothers not living on a farm (Seppo et al, Front Immunol, accepted for publication). In the European PASTURE study, soluble IgA levels in human milk were found to be associated with farm animal contact and inversely associated with the development of atopic dermatitis up to age 2.(14)

Human milk oligosaccharides (HMOs) are a major class of non-digestible carbohydrates (>200 structures) and the third most abundant solid found in human milk. HMO composition has been associated with the development of allergy in infants.(15) We found the level of lacto-N-neotetraose to be elevated in OOM mothers compared to mothers not living on the farm.(13) Humans lack the enzymes necessary to digest HMOs; therefore, they pass through the stomach and upper small intestine intact and exert a number of functions in the infant gut. In infants who are fed human milk, HMOs can bind pathogens directly, halting their ability to invade the intestinal epithelium.(16) Pathogen-epithelial interactions are mediated through glycan-lectin sugar receptors found on many intestinal cells and HMOs can mimic these receptors to act as a decoy to pathogens. Besides the local effect in the gut, HMOs can be absorbed into the bloodstream where they can exert effects systemically interacting with immune cells, modulating cytokine production, proliferation, and adhesion.(16)

Though indigestible by humans, HMOs also act as a prebiotic for select members of the intestinal microbiota genus Bifidobacterium, one of the few that can efficiently metabolize HMOs. Bifidobacterium are gram-positive, heterofermentative, anaerobic bacteria that are one of the first to colonize the infant gut within days after birth.(17) Bifidobacterium longum subspecies infantis (B. infantis) has been shown to be one of the few native bacterial subspecies that is able to digest HMOs most efficiently when compared to its other subspecies such as Bifidobacterium longum subspecies longum or Bifidobacterium longum subspecies breve.(16) This is due to unique gene clusters found in B. infantis that encode for various transport proteins and enzymes like glycosyl hydrolases that make it able to digest HMOs.(17) Early in the development of the gut microbiome, B. infantis is not the most abundant species; however, if the infant is colonized, B. infantis can become the predominate bacteria over the course of lactation.(17) Given the high concentration of HMOs and their selective stimulation of a small number of bacteria taxa, HMOs play a major role in shaping the infant gut microbiota. Few studies have examined the relationship between Bifidobacterium abundance and allergic disease, mainly focusing on the protection against the development of asthma.(18, 19) We recently found B. infantis enriched in the stool of OOM infants at a median of 2 months of age compared to non-farming infants.(13) Children from the OOM were also found to have less self-reported food allergy and atopic dermatitis, as well as an increased rate and longer duration of infant consumption of human milk compared to non-farming children.(4, 13) There has been a gradual loss of abundance of B. infantis (measured by fecal pH) in human milk-fed infants in developed regions, whereas in infants from less developed regions B. infantis is still the predominate microbe.(20) This gradual loss parallels the rise of allergic manifestations seen in developed regions. Instead of B. infantis, developed regions’ infant gut microbiome predominantly consists of B. longum and B. breve, which are more efficient at plant-derived carbohydrate metabolism.(20) The changes in infant gut microbiome composition, its function and the metabolic profile generated can potentially have downstream effects on the host, which can contribute to disease susceptibility.

Bifidobacterium and infant immunity

There is an intimate relationship between the gut microbiome and the host immunity. Alterations of the microbiome, which may affect both host immunity and its response to antigens are thought to be one of the major contributors to allergy development.(3) B. infantis in the gut has been shown to modulate numerous functions of host immunity (Table 1). In one study, IL-8 and IL-6 production were reduced in immature intestinal tissue treated with B. infantis supernatant in response to LPS and IL-1β.(17) Additionally, these supernatant-treated tissues also had decreased gene expression of TLR2 and TLR4, suggesting that the presence of B. infantis can act as an anti-inflammatory mediator dampening responses following inflammatory challenge.(17) Another study showed that IL-10 mRNA expression is increased in Caco-2 cells (intestinal epithelial cell line) treated with B. infantis grown in the presence of HMOs compared with B. infantis supplied with lactose.(17) Besides limiting inflammatory responses in the intestinal epithelium, B. infantis can also support intestinal epithelial barrier function.(17) In infants who were supplemented with B. infantis EVC001, they had decreased expression of fecal pro-inflammatory cytokines compared to control fed infants.(21) Human dendritic cells (DC) were found to produce more IL-10 compared to IL-12p70 following incubation with B. infantis; a cytokine profile that was not seen with other Bifidobacterium species (Bifidobacterium globosum, Bifidobacterium animalis).(22) Furthermore, the authors found that B. infantis induced a regulatory DC phenotype that enhanced the induction of regulatory T cells (Treg).(22) Recently, Henrick et. al. demonstrated differential immune states of infants based on colonization with Bifidobacterium.(21) Infants lacking Bifidobacterium had a more pro-inflammatory Th2/Th17 profile while infants with abundant Bifidobacterium had a more anti-inflammatory profile.(21) Early colonization of Bifidobacterium has also been shown to modulate B cells responses. Infants with early Bifidobacterium colonization were found to have higher levels of memory B cells at 4 and 18 months(23) and also increased salivary secretory IgA at 6 months.(24) It appears that the presence of Bifidobacterium leads to B cell activation, maturation and eventually antibody production. IgA can be beneficial as it can limit translocation of microorganisms and other antigens across mucosal barriers, thus limiting immune activation and sensitization.

Table 1.

Summary of B. infantis impact on host immune function

Cells/Tissue type	Effect of B. infantis	Reference
Immature intestinal tissue	Decreased IL-8 and IL-6 production Decreased TLR 2 and TLR4 mRNA expression	17
Intestinal epithelial cells (Caco-2)	Increased IL-10 mRNA expression	17
Dendritic Cells	Increased production of IL-10 compared to IL-12p70 Promotes a regulatory DC phenotype leading to increased regulatory T cells	22
Gut (colonization)	Increased levels of salivary secretory IgA Increased memory B cells Regulatory/anti-inflammatory immune profile Decreased fecal pro-inflammatory cytokines Increased fecal lactate and acetate	13, 21, 23, 24, 27

Gut microbes including B. infantis have a wide range of effects on host immunity, but how this effect is mediated is still being explored. Microbial metabolite production serves as one mechanism by which the microbiome modulates host immunity to influence health. Short-chain fatty acids (SCFA) are by-products of bacterial fermentation and are the most abundant microbial metabolite present in the colon. Acetate, propionate, and butyrate are the most abundant SCFA. Low fecal concentrations of these SCFA and abundance of genes related to their production have been associated with the development of allergic disease (asthma, food allergy, eczema, allergic rhinitis.(18, 25) SCFA can act in the host by direct binding on G-coupled protein receptors or modulating gene expression in cells functioning as a histone deacetylase inhibitor. SCFA have been shown to have effects on host immunity in the gut, mainly in the colon where they are predominantly reabsorbed. However, some SCFA are reabsorbed into systemic circulation where they can be redistributed to other organs. SCFAs have been shown to enhance intestinal epithelial integrity as well as modulate several immune cell populations.(26) They have been reported to modulate dendritic cell maturation, Treg differentiation, and antibody production.(26) B. infantis, previously discussed, was found to be elevated in OOM infants and is a known producer of acetate and lactate, the latter of which was found to be elevated in these infants.(13) Moreover, increased fecal lactate and acetate was found in infants supplemented with B. infantis EVC001.(27) Acetate and lactate can independently exert effects in the gut and also serve as substrates for other bacteria in the gut to synthesize to butyrate through cross-feeding, generating a special niche. In another study, farm exposure was characterized by a more mature microbiome including enrichment with known butyrate producing bacteria including Coprococcus and Roseburia at 12 months.(19)

Farming lifestyle and infant immunity

Differences in lifestyle exposures appear to induce differential infant immune responses. Farm exposure (unpasteurized milk consumption, farm animal contact) has been shown to associate with differential gene expression of TLRs and CD14 in children.(7) Additionally, stimulation of cord blood cells found more robust IFNγ and TNFα production in farm infants compared to non-farm infants.(8) Greater production of IFNγ, a cytokine associated with Th1 responses, may point to a shift of farm-exposed infants promoting more Th1-like responses. The robust and differential innate responses are possibly suggestive of enhanced microbial exposure due to farm exposure that may appear already before birth.(7) Similar to data from innate cells, increased frequency and function of Tregs were found in farm-exposed infants.(28) Increased Tregs can function to limit aberrant response that could develop into sensitization during antigen exposure. B cells have also been examined as well. In the Swedish FARMFLORA study, they found that the proportion of immature CD5⁺ B cells at birth associated with allergic disease (eczema, food allergy, allergic rhinoconjunctivitis, and/or asthma) development at 18 and 36 months of life.(29) However, living on a dairy farm did not appear to be a contributing factor. In a follow-up study, maturation promoting cytokine B-cell activating factor (BAFF) was found to elevated at birth in farm infants, and found to be associated with an increased proportion of CD27⁺ memory B cells(30), suggesting that differences in B cell maturation may be induced by farm exposure.

Future research needs

The gut microbiome during infancy influences host health through the effect of metabolites on host immunity, which may lead to protection from aberrant intestinal response and subsequent development of food allergy and other allergic diseases in childhood. What is not clear at this point is if the immune profile that has been described so far in farm-exposed infants/children is associated with the enrichment with specific microorganisms, such as B. infantis and/or increased levels of SCFA. As mentioned previously, only recently has the microbiome been explored in farming populations with the use of 16S rRNA gene sequencing.(13, 19) While it has started to provide information on the bacterial communities present in infants, this technique generally only achieves genus-level resolution and does not provide information on functional capacity of the microbiome. Future studies should be focusing on shotgun metagenomic sequencing, which can achieve taxonomic classification to the species and strain levels as well as insight into the metabolic capabilities of the microbial community.

Conclusion

Allergic diseases, including food allergy, continue to be a health burden on millions of individuals around the globe. It is apparent that aspects of the farm lifestyle offer a level of protection against allergy; one being the enhanced microbial exposures from living on the farm. Ingestion of human milk, and its diverse composition among people who live on farms has the potential to modulate the development of the infant gut microbiota and impact host immunity, appears to be a pathway that may induce a protective infant phenotype (Figure 1). Besides atopic eczema and wheeze, the relationship between human milk and additional allergic outcomes remains unclear. Reports have ranged from demonstrating a protective effect, increased risk, or no benefit of human milk consumption and the development of allergic outcomes.(9) Reasons for divergent outcomes vary but may include differences in criteria used to define human milk feeding, use of human milk exclusively and duration, and the interconnected nature of the allergic manifestations.(9) With food allergy, there is a lack of carefully designed studies to properly evaluate this relationship. Improvement in study design along with inclusion of additional parameters, such as human milk composition, infant microbiome, metabolomics, and infant immune response simultaneously and longitudinally are necessary to effectively investigate the impact of human milk and infant immunity.

Figure 1. The farming lifestyle along with human milk impact on infant gut microbiome and immunity and protection against allergy.

Compared to a non-farming lifestyle (A), one characteristic of a farming lifestyle (B) is the commonly practiced consumption of human milk which contains a diverse array of components (antibodies, microbiome, cytokines, fatty acids, etc.) that can shape the developing gut microbiome, potentially positively impacting infant immunity lowering the risk of allergy development.

Key Notes:

• Changes in environment such as the loss of certain microbial exposures over time is a contributor to the global rise of allergic diseases

• Infant consumption of human milk may provide a pathway in modulating infant microbiome and immunity possibly inducing protective responses against asthma and atopic dermatitis

• Individuals who live on farms are an example to further elucidate the mechanisms of protection against allergic disease development

Biographies