Asthma and atopic diseases including food allergy, eczema, and rhinoconjunctivitis have increased exponentially over recent decades of industrialization and urbanization and are now the most common chronic medical conditions affecting children in the USA and are a global public health concern (1). Although heredity is a strong determinant of an allergic constitution, environmental factors, including a Western lifestyle, microbial exposures, and diet likely play a role in the rise of atopic diseases, which are characterized by Th2 responses. Cumulative data to date suggest that living on farms is associated with a major reduction in the risk of asthma and atopic diseases, including studies from Europe (2–4) and North America among the Amish (5, 6) and the Old Order Mennonites (OOM) (7–9). The individual factors that appear to be associated with this “farm-life effect” include consumption of unpasteurized farm milk (2, 10, 11) and exposure to farm animals, stables (2, 3) common in traditional one-family farms, but not in communal farms (6).

It is less appreciated that the temporal window for the immunomodulatory effects of a farming lifestyle extends from early postnatal to the prenatal period. Maternal microbial exposures during pregnancy have a strong and independent protective impact on atopic sensitization and allergic disease in their children (2–4, 12). This protection was associated with maternal exposure to farm animals and unboiled farm milk (3, 10), increased T regulatory cells (Tregs) in cord blood (13), and altered expression of receptors of the innate immunity (TLR2, TLR4, CD14) in school-aged children (3). These data suggest a maternal farmlife effect on protection against allergic diseases, but to date, only a single study has assessed the maternal, postnatal farming lifestyle effect related to human milk composition. In the European “Protection against Allergy-Study in Rural Environments” (PASTURE) cohort, IgA levels were higher in the milk of mothers with contact to farm animals and cats than in those unexposed during pregnancy (14).

Human milk contains immunologically active factors such as immune cells, cytokines, chemokines and hormones, immunoglobulins, critical growth factors, active enzymes including peroxidases and lysozymes, lactoferrin, saturated fatty acids, poly-unsaturated fatty acids (PUFAs) and additional secretory components, soluble CD14, TLR2, and tumor necrosis factor (TNF) receptor, along with maternal diet-derived food antigens (15). The role for maternal milk components in regulation of breastfed immunity is best demonstrated in mouse models, in which secretory breast milk IgA plays a role in the longterm gut homeostasis by regulating microbiota and host gene expression (16), and IgG immune complexes are shown to induce food-specific tolerance in offspring (17). Transforming growth factor (TGF)-β in murine milk plays a role in development of tolerance in the offspring (18). Epidermal growth factor (EGF) not only prevents dissemination of a gut-resident pathogen by inhibiting goblet cell-mediated bacterial translocation (19) but may also prevent food allergen uptake during breastfeeding, which may have implication for development of tolerance (20). Using human samples and infant cohorts, we have shown that human milk IgA controls excessive intestinal uptake of food antigens, which is associated with protection against food allergies in the offspring (21), as are higher levels of TGF-β1, along with IL-10 and IL-6 (22). In addition to providing immunostimulatory factors for the breastfed infant, breastfeeding significantly influences the development of the infant microbiome composition (23, 24), which influences gut dysbiosis in infancy. Early childhood precedes the development of multisensitization and atopic disease (25), including food allergy (26, 27) and asthma (28). One way breastfeeding impacts infant’s gut microbiota composition includes human milk oligosaccharides (HMOs), complex glycans that provide the main substrate for infant’s gut microbiota, particularly for Bifidobacteria and some Bacteroides (29, 30), and HMO compositional differences were detected in children protected against food allergy (31–33) and IgE-associated eczema (34). In addition, human milk contains microbiota, but the data are conflicting to which extent human milk may act as a source of bacterial species that colonize the infant gut (35–41). Human milk is also a rich source of short-chain fatty acids (SCFAs) (42), the function of which is unknown.

Breastfeeding provides continued exposure to the mother’s immune system during the crucial window of the first few months of life, when the infant’s immune system is continuously developing. However, the immunomodulatory composition of human milk characterized so far demonstrates a great deal of variability between mothers, which implies that the benefit of breastfeeding to the breastfed infants may differ between dyads. For the first time, we sought to characterize the differences in the multiple maternally derived immune and metabolic factors in human milk, including a broad characterization of specificity of IgA₁ and IgA₂, comparing farming and nonfarming lifestyles. Specifically, we here use the OOM as a model population of a lifestyle dating back 100 years. We hypothesized that because their lifestyle includes exposure to farm animals, homegrown food and unpasteurized milk, home births, and large family sizes and is limited in exposure to modern commodities such as cars and use of antibiotics, their human milk would have a different composition. Assessment of human milk composition can serve as a proxy for maternal immunity, the impact of which on allergic sensitization in the offspring starts in utero and expands postnatally through breastfeeding.

To our knowledge, this is the first multicomponent characterization and network model of human milk composition spanning antibodies, cytokines, human milk oligosaccharides, metabolites, and gut microbiome. It is also the first of such characterization among mother-infant pairs with different lifestyles and risk for allergic diseases. We also performed the most comprehensive assessment of IgA₁ and IgA₂ antibodies to date. We show that OOM milk is more abundant in many immune components, especially those involved in IgA immunity, and LNnT which enhances Bifidobacterium enrichment. Maternal use of antibiotics was associated with differences in several HMO concentrations. These data support the concept that maternal lifestyle and antibiotic use are regulators of human milk composition, which may have downstream implications for postnatal developmental programming of infant’s gut microbiome and immune system. Considering that the OOM lifestyle dates back about a century, our data may also imply that there has been a change in human milk composition along with its protective properties over time. Although the function of all human milk components is not well known, many of them have downstream implications for postnatal developmental programming of infant’s gut microbiome and immune system suggesting potential benefit to infants born to the OOM community.

While the regulation of human milk composition of immune factors is poorly known, a few prior studies have documented differences in human milk IgA levels between mothers in different countries (14, 55, 56) and those avoiding or consuming particular foods (21), implying that maternal exposures impact milk composition. Consistent with an earlier farmlife study showing higher levels of IgA in farming lifestyle mothers’s milk, we showed upregulation of specific IgA antibodies in the OOM milk. Higher IgA response in the OOM milk was seen specifically to peanut, egg ovalbumin of hens, dust mite, and S. equi, a horse respiratory pathogen; whereas Rochester milk had higher IgA response only to a single antigen, namely E. cloacae, which is a gut commensal. These findings could be attributed to specific antigenic exposures between the OOM and Rochester mothers, for example, the higher levels of egg-specific IgA could reflect that eggs are a staple in their daily diet of the OOM who commonly keep chicken; however, elevated antibody levels against milk of cows or peanut are hardly due to more common exposures in the OOM. Furthermore, aeroallergen-specific IgA responses were elevated to a broad array of aeroallergens in the OOM (although only dust mite reached statistical significance), however, exposure to such inhalant antigens is unlikely to be more common in the OOM who as an example do not allow pets inside the home. These data may imply another mechanism, aside from higher exposures, boosting mucosal antibody production in the OOM. The ultimate function for these specific IgA antibodies is unknown, although we and others have previously reported that higher levels of IgA and specific IgA are associated with protection against milk allergy of cows in an infant (21, 57, 58). While mechanism of protection is unknown, IgA was shown by us not only to decrease the uptake of specific food antigens through the gut epithelium but also to enhance targeted antigen uptake to gut-associated lymphoid tissues in an antibody-dependent manner (21, 59). The presence or the role of aeroallergen- or commensal-specific IgA in human milk has not been previously assessed, although secretory IgA in breast milk plays a role in longterm gut homeostasis by regulating microbiota and host gene expression (16).

In this novel study, we report on the presence of both IgA₁ and IgA₂ to a broad selection of food, aeroantigens, and commensals. IgA₁ antibodies appeared more commonly detectable than IgA₂, and for example, commensal-specific IgA₂ responses were undetectable. Our data are consistent with few previous studies suggesting that protein antigens such as BLG and tetanus toxoid predominantly induce IgA₁; however, our data do not support a predominant IgA₂ responses to polysaccharide antigens as reported with pneumococcal, meningococcal, and Hemophilus influenzae (60, 61). Clustering analyses showed that aeroallergens clustered together; however, they also clustered with peanut, which may be explained by the crossreactivity between peanut and pollen. Similarly, commensal IgA responses clustered together. Interestingly, milk and egg antigens were not clustered with either of the two larger clusters, neither were they clustered with each other. This may imply an exposure route that is different from that of the other antigens and from each other, such as the skin or a specific part of the GI tract based on the differential stability of these food antigens in the digestive tract.

The cytokines measured here are of interest because a few prior studies have documented differences in these human milk cytokine levels between geographic locations and countries. Higher levels of IL-10 were seen in Estonia mothers than in Swedish mother (55) and that of TGF-β1 in Finland compared with other European countries (14). Another study showed differences in cytokines and growth factor levels between developing and developed world countries (62, 63). Here, we found IFN-λ₃ and TGF-β₂ at higher levels in the OOM. Interestingly, a study performed in Sweden among mothers who had immigrated from a developing country early on had higher levels of IL-6, IL-8, and TGF-β₁ in their milk suggesting that early life exposures have an impact on the immune system that sustains into adulthood and impacts human milk composition (64). In addition, we found several cytokines involved in IgA production clustered together, such as IL-6, TGF-β₁, and TGF-β₂, which clustered with TGF-β₃ and APRIL and TSLP. This may indicate common pathways involved in cytokine production for the benefit of inducing upregulation of IgA levels in human milk. The cytokines measured here are of special interest, because we had previously identified those in higher concentrations associated with protection against food allergy (22). It may therefore be plausible that milk cytokines and IgA function in concert to support the infant against immune-mediated diseases.

Several large studies have confirmed the role of breastfeeding in modulating gut microbiome composition (24), showing that breast milk selects for intestinal microbiome dominated by Bifidobacterium for the first year of life (65). This is due to HMOs that are complex glycans and the third largest solid component in human milk. They are typically nondigestible by humans but provide the main substrate for infant’s gut microbiota, particularly, for Bifidobacterium and some Bacteroides (29, 30). Therefore, when breastfeeding ceases, signficant shifts in gut microbiome composition are seen with Clostridium and Bacteroides species replacing Lactobacilli-, Bifidobacteria-, and Enterobacteriaceae-dominated microbiota (66–68). We recently reported that HMO composition was related to maternal probiotic exposures (33). In the current report, we identified specific HMO levels associated with maternal antibiotics, suggesting a role for maternal microbiome in regulation of HMO composition. In this study, we found few differences in single HMOs between the OOM and Rochester mothers (LNnT, LFPIII, and DFLNT). However, because metabolic transitions between HMOs are not necessarily known, we sought to investigate whether certain conversions are different between OOM and Rochester groups. The path starting from LNH to FDSLNH were significantly higher in Rochester mothers; however, we found no specific HMO composition in relation to atopic disease development. This is in contrast with prior studies that have associated specific HMO profiles with atopic diseases (31–33), possibly explained by the small sample size.

Of particular interest in human milk is the presence of fatty acids, especially short chain fatty acids (SCFA), which are solely metabolized by gut bacteria from otherwise indigestible carbohydrates of fiber-rich diets. There is one known study so far indicating lower levels of acetate and butyrate in atopic mothers (42). SCFAs in human milk are likely produced by the maternal gut microbiota and distributed to the mammary gland via the circulation (42). We found an association between milk butyrate and specific taxa such as Prevotella, which is a known producer of butyrate (69), which may provide the first current evidence that SCFA in humans be produced by the human milk microbiota. Alternatively, maternal diet may contribute to SCFA levels in human milk. We also found higher levels of tricosanoic acid and palmitic acid in the OOM milk, but the implication of this unclear. FAs such as palmitic acid in human milk (70, 71) has been associated with changes in infant gut microbiota, but causality is unclear.

In this cohort, development of atopic disease in early childhood was more common in Rochester and associated with lower levels of IgA to dust mite as well as TSLP, but larger longitudinal studies are needed to conclusively assess whether OOM milk composition may provide benefit to their infants against development of allergic diseases. Assessing the relationships between human milk components and development of atopic outcomes in the children will only provide correlative data and causal relationship cannot be drawn. Human milk is only one of the many factors that can affect the development of atopic outcomes by 3 years of age. Not all the factors have been assessed or controlled for in this study. Further limitations include a small sample size of this pilot study and that all the assays were not performed on every sample due to limitation of volume. Allergic outcomes were limited to self-report of physician diagnosis or symptoms of atopic disease, although assessment was done by a pediatric allergist by telephone follow-up. Strenghts include access to a unique population living a traditional, agrarian lifestyle, with lifestyle representative of that commonly encountered a century ago, when allergic diseases were rare.

Boosting immunomodulatory properties of human milk might present an opportunity to modulate infant’s gut microbiome and risk for allergic diseases. Differences in human milk composition may have implications for developmental programming of the infant immune system postnatally by human milk but are also likely reflective of the maternal immune environment with implications to prenatal developmental programming. More studies are clearly needed to understand the role of maternally derived prenatal and postnatal factors on allergic diseases.

The datasets presented in this study can be found in online repositories and in Supplementary Material . The names of the repository/repositories and accession number(s) can be found in the article.

The studies involving human participants were reviewed and approved by University of Rochester Institutional Review Board. Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin.

AS, JL, CM, and KJ conceived the overall study and supervised the research. AS, RC, CP, RP, SF, PR, JS, CY, LB, KB, ST, EP, DW, MA, RL, JC, JT, and KJ contributed to the literature search, data collection, and data analysis. AS, JT, JC, and KJ provided the figures and drafted the manuscript, with additional input from all authors. All authors contributed to the article and approved the submitted version.

The project described was supported by Grant Numbers K08 AI091655 (KJ), U01 AI131344 from the National Institute of Infectious and Allergic Diseases (KJ), R21 TR002516 from the National Center for Advancing Translational Sciences, Founders’ Distinguished Professorship in Pediatric Allergy (KJ), and by Pilot Award (P30 ES001247) from Environmental Health Sciences Center (JL, CM), National Institute of Environmental Health Sciences, and Rochester CTSA award UL1 TR000042 from the National Center for Advancing Translational Sciences (JL, CM) of the National Institutes of Health. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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