Human milk oligosaccharides (HMOs) comprise a group of structurally complex, unconjugated glycans that are highly abundant in human milk.
A new study by researchers at Loma Linda University Health has found that eating nuts on a regular basis strengthens brainwave frequencies associated with cognition, healing, learning, memory and other key brain functions.
An Oxford University study, published in Human Microbiome Journal, examined the composition and diversity of the human gut microbiome to determine if it is related to differences in personality.
A healthy system of gut bacteria, or microbiota, is crucial to health: Gut bacteria not only aid with digestion, but also play an important role in the body's immune response.
The human neonate is often born with a nonsterile gastrointestinal tract. The mechanism of this in utero colonization is poorly understood, but potential sources include ascending vaginal microbes, transloca- tion of microbes from the maternal intestine or skin, or hematogenous spread from the maternal oral cavity.
Furthermore, human milk is not sterile. Using both culture and non-culture-based techniques, this fluid has been found to be rich in microbes, and they will eventually colonize or at least be present in the intestine of the developing infant with the potential to elicit numerous immune responses via metabolite production, interaction with the
developing infant immune system, and induction of signals for host gene expression.
In addition to the milk microbes, this fluid also provides a source of enzymes such as lipase and alkaline phosphatase. Immunoglobulins, especially IgA, are found at relatively high concentrations. Milk also provides a multitude of biologically active proteins such as lactoferrin and lysozyme. Carbohydrates, including disaccharides such as lactose, when not digested and absorbed by the host can be utilized by microbes that metabolize this sugar to other highly biologically active agents such
as short-chain fatty acids, acetate, propionate, and butyrate. Oligosaccharides serve as nutrients for certain microbes that flourish in their presence and have the ability to interact with the intestinal mucosa of the host.
The cellular composition (including immune cells and stem cells) and siRNAs found in milk exosomes also appear to play important roles in terms of immunologic protection, immune modulation, and transcriptional regulation.
Although exciting, much of the information currently available is associational. These agents are present in milk; we have theories about their roles, without a strong body of mechanistic data. We have also the impression that just because they are present in human milk, they provide specific advantages to the infant who is not fed these components. However, it is clear that despite major efforts, not all babies will be able to receive and benefit directly from their own mothers’ milk. A better understanding not only of the composition of human milk – but also the mechanisms of how these components affect the host – should help us optimize milk for a larger number of infants.
Ardissone N, de la Cruz DM, Davis-Richardson AG, et al: Meconium microbiome analysis identifies bacteria correlated with premature birth. PLoS One 2014;9:e90784.
Cacho NT, Lawrence RM: Innate immunity and breast milk. Front Immunol 2017;8:584. Gomez-Gallego C, Garcia-Mantrana I, Salminen S, Collado MC: The human milk microbiome and factors influencing its composition and activity. Semin Fetal Neonatal Med 2016;21:400–405.
Hassiotou F, Geddes DT, Hartmann PE: Cells in human milk: state of the science. J Hum Lact 2013;29:171–182.
Rogier EW, Frantz AL, Bruno ME, et al: Lessons from mother: long-term impact of anti- bodies in breast milk on the gut microbiota and intestinal immune system of breast-
fed offspring. Gut Microbes 2014;5:663–668.
Compared to adulthood, early postnatal life is a period that is characterized by rapid changes. The neonate’s tissues are constantly changing due to the growth process and are exposed to multiple new antigens, which are found in the environment, are present in the diet, or are associated with gut microbiota colonization (Fig. 1). The neonatal immune system has its own reactivity, which profoundly differs from that of the adult. It is neither that of a “small adult” nor is it an immature or tolerance-prone immune system. The neonatal immune system is a different one with specific requirements for activation and regulation. Breast milk is most probably a key condition for physiological (and optimal) functioning and imprinting of the immune system in early life (Fig. 1). Similar to the immune system and environment, which are constantly changing in early life, breast milk composition is constantly evolving. Volume, macronutrients, micronutrients, immunological factors, microbiota, and microbiota-shaping molecules are changing with lactation stages, and are also affected by infant growth and
environmental immune challenges . Here, we will focus on factors in breast milk that we – and others – extensively studied and found to actively influence their immune trajectory and long-term immune health. More specifically, we will review the importance of TGF-β, vitamin A, immunoglobulins, and allergens in mucosal immunity in both early life and long-term allergic disease susceptibility. There is strong evidence from rodent studies and epidemiological data that oral exposure to allergens in early
life, out of the context of breast milk, is not inducing immune tolerance but, instead, is priming for allergic responses . Nonbreastfed infants are exposed to only a few allergens, such as β-lactoglobulin, which occur at high concentrations. In contrast, breastfed infants are exposed to a wide variety of breast milk allergens that are found at concentrations that are at least 1 million times lower . Importantly, there is evidence from rodent studies that the neonatal immune system requires very-low-dose antigen
exposure for appropriate immune reactivity. Early life is also characterized by a relative lack of TGF-β in the mucosal tissue, a physiological deficiency in vitamin A, and a low mucosal and systemic immunoglobulin secretion, which contribute to the lack of oral tolerance induction in early life in the absence of breast milk. Breast milk is providing infants with these cofac- tors, which will affect gut epithelium barrier integrity, antigen transfer, and antigen presentation for successful regulatory immune response induction . This will result in a decreased risk for allergic diseases in the long term, as shown for egg allergy both in an experimental mouse model as well as in humans .
We are starting to decipher the specific requirements for the neonatal immune system to function optimally, and we are discovering how breast milk fulfills these requirements and guides immune trajectories from early life. Answering these questions will provide the infant with preventive and curative approaches that are tailored to this very specific period of life and will ensure long-term immune health.
1 Ballard O, Morrow AL: Human milk composition: nutrients and bioactive factors.
Pediatr Clin North Am 2013;60:49–74.
2 Strobel S: Immunity induced after a feed of antigen during early life: oral tolerance v.
sensitisation. Proc Nutr Soc 2001;60:437–442
3 Munblit D, Verhasselt V: Allergy prevention by breastfeeding: possible mechanisms and evidence from human cohorts. Curr Opin Allergy Clin Immunol 2016;16:427–433.
4 Turfkruyer M, Verhasselt V: Breast milk and its impact on maturation of the neonatal
immune system. Curr Opin Infect Dis 2015;28:199–206.
5 Verhasselt V, Genuneit J, Metcalfe JR, et al: Ovalbumin in breastmilk is associated with a decreased risk of IgE-mediated egg allergy in children. Allergy 2020;75:1463–1466.
Early-Life Nutrition and Gut Immune Development
Lieke van den Elsen, Akila Rekima, and Valérie Verhasselt
Gut immune function conditions development of diseases that result from defects in immune regulation such as allergic and obesity-related disease . As epidemiological studies support the developmental origin of health and disease, the deciphering of the critical factors modulating gut immune development should allow the advance of primary prevention strategies specifically adapted to the early-life immune system. Here, we will emphasize how nutrition can shape microbiota composition and metabolite production with immune-modulatory properties. We will also focus on the role of dietary compounds recently demonstrated to be essential in immune development and function such as dietary antigens, vitamin A, and aryl hydrocarbon receptor (AhR) ligands.
Microbiota is necessary for lymphoid tissue development and immune differentiation such as IgA secretion, regulation of IgE responses, and differentiation of T cells subsets . Besides mode of delivery, nutrition is the key factor directing the early microbiota composition and function . Breast milk contains viable bacteria that will contribute to the establishment of the neonatal microbiota, and maternal IgA will alter colonization patterns in the neonate. Breast milk also contains nutrients specific for the growth of commensals, i.e. human milk oligosaccharides (HMO), which stimulate the growth of bifidobacteria and affect their metabolic function. In animals fed solid food, Clostridia can metabolize dietary fibers into short-chain fatty acids (SCFA), while in breastfed neonates, SCFA are derived from HMO metabolized by bifidobacteria (Fig. 1a). The role of SCFA in gut immunity in preweaned mice has not been assessed yet. In young weaned mice, they were found to stimulate regulatory T cell expansion, IgA and mucus secretion, gut epithelium barrier function, ILC3 function, and induce resistance to food allergy and gut inflammatory disease  (Fig. 1a). Some commensals, such as Lacto-bacillus, metabolize tryptophan, an essential amino acid that is a common constituent of protein-based foods (Fig. 1b). The metabolites bind AhR expressed in ILC3 and stimulate the postnatal formation of isolated lymphoid follicles and IL-22 secretion necessary for gut barrier function and protection from Citrobacter infection and colitis  (Fig. 1b). In breastfed infants, AhR ligands could originate from maternal microbiota or from maternal diet (Fig. 1b).
Mice studies have recently highlighted the regulatory function of diet-derived antigens in the small intestine  (Fig. 1c). In the human , food diversification in the first year of life was associated with decreased risk of allergies. The shaping of immune reactivity by induction of oral tolerance to specific antigens during the period of immune ontogeny may be possible in the case of egg (OVA) and peanut antigen. Additional TGF-ß, vitamin A, and IgG from maternal milk were critical for tolerance induction towards OVA transferred through breast milk in rodents  (Fig. 1c). TGF-ß is a growth factor for epithelium, and both vitamin A and IgG acted on antigen transfer through epithelium. Vitamin A also increased the function of dendritic cells involved in tolerance and Th1 differentiation (Fig. 1c). Our recent data showed that not all the antigens in breast milk induce oral tolerance. Antigen from house dust mite, Der p 1, is present in human breast milk and its presence increased the risk of allergy both in mice and in the humans. This stresses the need to identify how maternal milk factors could be modulated to counteract deleterious action of some allergens .
Fig. 1. Impact of food on immune ontogeny. a Short-chain fatty acids (SCFAs) stimulate regulatory T-cell expansion, IgA, and mucus secretion, gut epithelium barrier function, antimicrobial peptide secretion (AMP), and ILC3 function and induce resistance to food allergy and gut inflammatory disease. Human milk oligo-saccharides (HMO) are present in human milk and stimulate the growth of bifido-bacteria that can metabolize HMO into SCFA. After weaning, metabolic function of bifidobacteria changes, and they become able to metabolize complex sugars from dietary fibers similarly to clostridia found in microbiota of older children. b Aryl hydrocarbon receptor (AhR) ligands bind to AhR receptor expressed on ILC3. They stimulate the postnatal formation of isolated lymphoid follicles (ILF) and IL-22 secretion necessary for gut barrier function and protection from Citrobacter infection and colitis. AhR ligands are found in cruciferous vegetables such as broccoli and cabbage. They can also be produced by some commensals, such as Lactobacillus, which metabolize tryptophan from protein-based foods into indole derivatives. In breastfed infants, AhR ligands can originate from maternal microbiota metabolites with maternal milk immunoglobulin helping in the transfer of these metabolites to the neonate. Breast milk contributes also to AhR-mediated immune ontogeny by stimulating the growth of Lactobacillus. c After weaning, antigens derived from solid food are necessary for populating the small intestine with induced Tregs. Tregs specific to dietary antigens can be induced by oral exposure. Before weaning, oral tolerance can be induced to antigens from maternal diet present in breast milk. This requires the presence of additional cofactors in breast milk such as TGF-ß, vitamin A, and IgG. Vitamin A increases gut barrier function, the capacity of dendritic cells (DCs) to metabolize vitamin A into retinoic acid and Th1 differentiation. Antigens bound to IgG are better transported across the epithelium and induce FoxP3 Tregs that are responsible for potent and long-lasting tolerance. After weaning, Treg induction towards oral antigen is favored by SCFAs. These induce TGF-ß secretion from epithelium and stimulate retinoic acid formation from vitamin A by DCs.
In conclusion, before weaning, the physiological food for mammals is providing the neonate with the factors necessary for immune maturation, which the neonate would otherwise miss due to the lack of a diverse microbiota and solid food-derived molecules. Breast milk exposes the infant to a variety of food antigens, and it contains ligands that are critical for lymphoid tissue development and immune function such as AhR ligands and vitamin A. It provides HMO, as surrogates to fibers found in solid food, for commensals to produce SCFA. Breast milk also delivers a microbiota and food for commensal growth in the sterile neonate gut. After weaning, solid food-derived antigens and vitamins as well as food metabolites produced by the microbiota will continue to shape the immune system and dictate susceptibility to local and systemic immune-mediated disease.
- Belkaid Y, Harrison OJ: Homeostatic immunity and the microbiota. Immunity 2017;46:562–576.
- van Best N, Hornef MW, Savelkoul PH, Penders J: On the origin of species: factors shaping the establishment of infant’s gut microbiota. Birth Defects Res C Embryo Today 2015;105:240–251.
- Rooks MG, Garrett WS: Gut microbiota, metabolites and host immunity. Nat Rev Immunol 2016;16:341–352.
- Kim KS, Hong SW, Han D, et al: Dietary antigens limit mucosal immunity by inducing regulatory T cells in the small intestine. Science 2016;351:858–863.
- Palmer DJ, Prescott SL, Perkin MR: Early introduction of food reduces food allergy – pro and con. Pediatr Allergy Immunol 2017;28:214–221.
- Munblit D, Verhasselt V: Allergy prevention by breastfeeding: possible mechanisms and evidence from human cohorts. Curr Opin Allergy Clin Immunol 2016;16:427–433.
Gut immune function conditions the development of local and systemic diseases that result from defects in immune regulation, such as inflammatory bowel disease, allergy and obesity. As epidemiological studies support the developmental origin of health and disease, deciphering the critical factors modulating gut immune development should allow the advance of primary prevention strategies specifically adapted to the early-life immune system. Here, we will review gut mucosal immunity development and cover in more detail the recent understanding of the impact of early nutrition on this process. We will emphasize how nutrition can shape microbiota composition and metabolic function and thereby the production of metabolites with immune-modulatory properties. We will also focus on the role of dietary compounds recently demonstrated to be essential in immune development and function, such as dietary antigens, vitamin A, and aryl hydrocarbon receptor ligands. Finally, we will discuss that early-life physiologic food for mammals contains factors capable of compensating for neonatal immune deficiencies, but also factors that are decisive for immune maturation towards a maternal milk-independent and efficient immune system.
Early-life gut immune function can be qualified as deficient according to the high susceptibility of neonates and infants to enteric infections. The high incidence of food allergy in infants further highlights a deficient capacity to mount oral tolerance. Mucosal immunity will mature in postnatal life to evolve, ideally, to mount regulatory immune responses towards dietary and nonpathogenic microbial antigens and inflammatory responses against pathogens. In parallel to immune system maturation, the gut develops from a nearly sterile environment at birth to harboring a microbiota that is very simple and highly variable and eventually reaches a dense, complex, and stable microbiota around 3 years of life . Both epidemiological and experimental studies have linked perturbations in gut microbiota composition in early life with the risk of developing immune- mediated disease in later life [reviewed in 2]. Infants at risk of asthma show a reduction in the relative abundance of certain bacterial genera during their first 3 months of life, while no major difference in microbiota composition was found at older ages. Clinical studies also suggest that disturbances in the intestinal flora early in life, caused by cesarean section delivery or early antibiotic exposure, may contribute to the development of diseases such as food allergy, inflammatory bowel disease, and types 1 and 2 diabetes .
Here, we will review experimental evidence that nutrition can affect gut immune ontogeny directly and indirectly by shaping microbiota composition, with lifelong impact on immune homeostasis.
Gut Immune Ontogeny
The gut epithelium progresses from being flat and poorly proliferating towards a highly proliferative epithelium, with crypt and villus architecture dramatically expanding the absorptive surface. This development is completed at birth in humans while still developing postnatally in mice [reviewed in 3]. A high gut per- meability is found in the neonate, both in mice and humans. In mice, few goblet and Paneth cells are present at birth and there is a low secretion of IgA in the gut lumen. Neonatal enterocytes however produce CRAMP (cathelicidin-related antimicrobial peptide) that acts as an antimicrobial defense mechanism specific to the early-life period, as it is also found in breast milk and the vernix caseosa. The high susceptibility of preterm neonates to necrotizing enterocolitis shows that an immature gut epithelium is susceptible to highly inflammatory respons- es; however, regulatory mechanisms such as decreased Toll-like receptor (TLR), TLR3 and TLR4, expression and signaling are found in neonates born at term, which contribute to dampening inflammatory responses upon microbial colonization .
Peyer’s patches and mesenteric lymph nodes (MLNs) develop in utero while isolated lymphoid follicles (ILF) do so after birth . A common scheme operates for lymphoid tissue formation: a stromal cell produces chemokines (CXCL13) which will attract CXCR5-expressing lymphoid tissue inducer (LTi) cells of hematopoietic origin (more recently known as a subset of innate lymphoid cells [ILC], ILC3, expressing RAR-related orphan receptor γ [RORγ]t). Binding of lymphotoxin (LT) αβ secreted by LTi to LTβ receptors expressed by stromal cells induces chemokine secretion and adhesion molecule expression needed for the attraction and retention of additional hematopoietic cells, leading to lymph node growth . Peyer’s patches require CD11c+ cells expressing the receptor tyrosine kinase RET, in addition to LTi and stromal cells, for their for- mation. ILF originate from cryptopatches, which are a cluster of LTi in the lamina propria. These expand after birth in the presence of B cells and a few T cells to become ILF .
A comprehensive analysis of the emergence of immune cells in murine neonatal small intestine was recently undertaken [reviewed in 3]. This showed that myeloid cells are found in the lamina propria at birth, and their numbers remain stable postnatally. We further found that, although in similar frequency in neonatal MLNs as compared to the adult, the capacity of neonatal CD103+ dendritic cells (DCs) to metabolize retinol was significantly impaired . Importantly, we found this defect responsible for inefficient oral tolerance induction in the neonate . After a massive recruitment of CD4+ T cells and B lymphocytes into the gut mucosa during the first 2 days of life, B cells were found to continue to expand, while TCR-αβ lymphocytes did so only after weaning. To- row and Hornef  also found that, before weaning, CD4+ T lymphocytes were mostly found in Peyer’s patches, leaving the lamina propria relatively empty compared to the adult. They further uncovered that these lymphocytes exhibit a naïve phenotype until weaning, due to active suppression by FoxP3+ regulatory T cells (Tregs) and maternal milk IgA. Similar to mice, a recent analysis of T-cell representation and differentiation in tissues of infants from 2 months to 2 years of age revealed a higher proportion of naïve and regulatory T cells in all tissues compared to the adult . Except for the subset of ILC3 involved in lymphoid tissue morphogenesis, LTi, limited data are currently available addressing the representation and function of ILCs in neonatal gut mucosa.
In summary, early postnatal life gut mucosal immunity is characterized by a leaky barrier with poorly developed innate and adaptative effector mechanisms, which are kept under control by regulatory cells. Breastfeeding is therefore critical for the prevention of infectious diseases in young infants as it provides the breastfed infant with antigen-specific (IgA and IgG) and nonantigen-specific antimicrobial molecules (lysozyme, lactoferrin, oligosaccharides, and leukocytes) . Despite the high proportion of Tregs in the neonatal gut mucosa, oral tolerance can hardly be induced in neonates [reviewed in 7], and highly inflammatory reactionssuchas necrotizing enterocolitis cantake place intheneonatal gut. Here also, breast milk compensates for the lack of molecules involved in immunoregulation in the neonatal gut. TGF-β, epidermal growth factor, human milk oligosaccharides (HMOs), and maternal IgG and IgA present in breast milk were in- deed found to exert anti-inflammatory and immunoregulatory effects [7, 8]. In the next parts of this review, we will analyze how nutrition is involved in neonatal gut immune maturation towards an autonomous system, no longer dependent on breast milk to mount efficient regulatory and effector immune responses.
Impact of Nutrition on Gut Mucosal Immune Ontogeny through Microbiota Shaping
Gut Microbiota Ontogeny
Besides the mode of delivery, known to strongly affect colonization of the neonatal intestine within the first days after birth, nutrition is the key factor directing the early microbiota composition and function [reviewed in 1]. The gut flora in breastfed infants is usually dominated by Bifidobacterium and Lactobacillus species, while formula-fed infants harbor a more diverse gut microbiota with increased abundance of Escherichia coli, Clostridium, and Bacteroides. The microbiomes of newborns and young infants are enriched in genes required for the degradation of sugars from breast milk, such as HMOs. Upon weaning, the microbiota functionally maturates by a decrease in the relative abundance of genes involved in the degradation of HMO and enrichment of genes involved in the degradation of complex sugars and starch. Cessation of breastfeeding, not solid food introduction, is critical for this shift.
Mechanisms of Microbiota–Driven Immune Shaping
Germ-free (GF) mice represent an extreme situation that illustrates the necessity of microbiota for gut immune development. Gut microbiota was shown to be necessary for ILF formation in the small intestine and the development of MLN and Peyer’s patches. Differentiation of immune responses is also dependent on microbial colonization. Microbiota is necessary for IgA secretion, regulation of IgE responses, differentiation of Th17 and Th1 cells, and RORγt+ Treg expansion in the colon . Gut microbial composition exerts direct effects on the immune system through microorganism-associated molecular pattern (MAMP) signaling and indirectly via the production of metabolites. An example of a well-studied MAMP is polysaccharide A from Bacteroides fragilis that was found to promote Treg differentiation and MYD88-signaling involved in epithelium repair and antimicrobial peptide secretion . Fermentation of dietary fibers in the colon by anaerobic bacteria, such as clostridia and bifidobacteria, generate short-chain fatty acids (SCFAs) including butyrate, acetate, and propionate. SCFAs signal through G-protein-coupled receptors, such as GPR43, GPR41, and GPR109A, present on epithelial and immune cells, and via inhibition of histone deacetylases, with long-term consequences through epigenetic modification. Among many of their reported immune effects, SCFAs promote colonic Treg differentiation, mucus production, and IgA secretion (Fig. 1a). Commensals, particularly Lactobacillus, metabolize tryptophan, an essential amino acid that is a common constituent of protein-based foods such as eggs, fish, meat, and cheese (Fig. 1b). The metabolites, which are indole derivatives, bind aryl hydrocarbon receptor (AhR). AhR was found to be necessary for ILC3 function both in postnatal development of ILF and IL-22 secretion necessary for gut barrier function and protection from Citrobacter infection and colitis .
While most of the studies addressing the role of the gut microbiota on immune maturation have been performed in adults and observations extrapolated to the developing neonate, some recent publications have specifically addressed the impact of colonization in early life on immune ontogeny [reviewed in 2]. These have started to show clear specificities of early-life immune responses to microbiota colonization and highlight the concept of a window of opportunity to induce a lifelong effect on immune homeostasis by shaping the microbiota in early life. Thus, GF mice conventionalized during adult life exhibit a different transcriptional profile in jejunum and colon compared to conventionally raised mice. High IgE found in GF mice can only be normalized if colonization occurs before 4 weeks of age. Another detailed mechanistic study showed that colonization of GF mice with gut microbiota in the first 2 weeks of life, but not in adults, was sufficient to protect mice from increased susceptibility to colitis due to mucosal invariant natural killer T (iNKT) cell accumulation. The protective effect of colonization in early life could also be induced by B. fragilis monocolonization and depends on polysaccharide A signaling. Colonization with Clostridium during weaning in- duces colonic Treg and fecal IgA as well as IL-22 production by RORγt+ ILCs and T lymphocytes, promoting gut barrier function and resistance to food allergy . In contrast, neonatal colonization with specific strains of the commensal E. coli impairs oral tolerance induction by affecting intestinal permeability and the balance of tolerogenic DCs and Tregs through the production of a genotoxin .
Age-specific mechanisms of action of microbiota-driven immune shaping were also found in neonates. Gomez de Aguero et al.  found that maternal microbiota generates AhR-binding metabolites that are transferred in utero and postnatally through breast milk and induce mononuclear cells and ILC3 expansion. The latter effect was found to be increased by maternal antibodies in the milk. The strong impact of gut microbiota on immune function has stimulated research on the potential to promote infant microbiota development by oral ad- ministration of probiotics to induce health benefits. Despite an increase in probiotic administration, data supporting their efficacy is lacking . Another strategy is to promote the growth of beneficial bacteria using prebiotics.
Fig. 1. Impact of food on immune ontogeny. a Short-chain fatty acids (SCFAs) stimulate regulatory T-cell expansion, IgA, and mucus secretion, gut epithelium barrier function, an- timicrobial peptide secretion (AMP), and innate lymphoid cell (ILC)3 function and induce resistance to food allergy and gut inflammatory disease. Human milk oligosaccharides (HMO) are present in human milk and stimulate the growth of bifidobacteria that can me- tabolize HMO into SCFA. After weaning, the metabolic function of bifidobacteria changes, and they become capable of metabolizing complex sugars from dietary fibers, similarly to clostridia found in microbiota of older children. b Aryl hydrocarbon receptor (AhR) ligands bind to AhR receptor expressed on ILC3. They stimulate the postnatal formation of isolated lymphoid follicles (ILF) and IL-22 secretion necessary for gut barrier function and protec- tion from Citrobacter infection and colitis. AhR ligands are found in cruciferous vegetables such as broccoli and cabbage. They can also be produced by some commensals, such as Lactobacillus, which metabolize tryptophan from protein-based foods into indole deriva- tives. In breastfed infants, AhR ligandscan originate from maternal microbiota metabolites with maternal milk immunoglobulin helping in the transfer of these metabolites to the neonate. Breast milk may also contributes to AhR-mediated immune ontogeny by stimu- lating the growth of Lactobacillus. c After weaning, antigens derived from solid food are necessary to populate the small intestine with induced Tregs. Tregs specific to dietary an- tigens can be induced by oral exposure. Before weaning, oral tolerance can be induced to antigens from the maternal diet which are present in breast milk. This requires the pres- ence of additional cofactors in breast milk such as TGF-β, vitamin A, and IgG. Vitamin A in- creases gut barrier function, the capacity of dendritic cells (DCs) to metabolize vitamin A into retinoic acid and Th1 differentiation. Antigens bound to IgG are better transported across the epithelium and induce FoxP3 Tregs that are responsible for potent and long- lasting tolerance. After weaning, Treg induction towards oral antigen is favored by SCFAs. These induce TGF-β secretion from epithelium and stimulate retinoic acid formation from vitamin A by DCs.
Early Nutrition Driving Microbiota Composition and Function
Breast milk contains 102 to 104 viable bacteria/mL that will directly affect the establishment of the neonatal microbiota. It also contains prebiotic and immunologic compounds that can alter colonization patterns in the neonate. In particular, HMOs stimulate the growth of bifidobacteria, which metabolize these oligosaccharides into SCFAs that favor immune regulation (Fig. 1a). HMO also have the capacity to modify the gene expression involved in metabolic function in commensals and thereby their secretion of metabolites which can affect growth  and inflammation . Metabolized HMOs are also beneficial for other commensals that do not directly degrade HMOs . In this context, dietary and synthetic oligosaccharides are the object of studies to assess similar early-life immune regulation, with promising results . Breast milk secretory IgA antibodies are specific for an array of common intestinal pathogens and commensals due to the selective migration of B cells originating from the mucosal membranes to the mammary gland. In addition to providing excellent passive mucosal immunity to neonates, maternal IgA and other molecules found in breast milk with antimicrobial properties, such as lactoferrin and lysozyme, will shape the microbiota composition of the breastfed infant .
Direct Impact of Nutrition on Gut Mucosal Immune Ontogeny
Here, we will focus on the impact of breastmilk and selected nutrients that have recently been the focus of experimental studies in early life for their impact on immune ontogeny, that is, dietary antigens, vitamin A, RORγt, and AhR ligands.
Solid Food and Dietary Antigens
The role of dietary antigens on gut mucosal immunity ontogeny has been assessed by comparing mice that were free of specific pathogens, that were devoid of microbiota (GF mice), or that were devoid of both microbiota and dietary- derived antigen (antigen-free mice) from birth . Others also studied the impact of dietary antigen after weaning on immune ontogeny of specific pathogen- free mice . These studies demonstrate that, after weaning, dietary-derived antigens are necessary and sufficient to stimulate memory CD4+ T lymphocytes and peripherally induced Tregs to populate small-intestine lamina propria. They are required for controlling the Th2 immune response and susceptibility to food allergy and necessary for IgA and IgG secretion. Evidence that exposure to diet-derived proteins is important to induce immune regulation also arises from intervention studies analyzing the impact of food diversification in the first year of life . These observations, made both in mice and in humans, high- light that one should consider the administration of an extensively hydrolyzed formula to infants with allergies with care, as this nutritional approach may decrease regulatory function of the gut.
The shaping of immune reactivity by induction of oral tolerance to specific antigens during the period of immune ontogeny, was recently reviewed . Egg introduction between 4 and 6 months was able to prevent from egg allergy, and peanut introduction between 4 and 11 months was able to prevent from peanut allergy . However, the results obtained for the prevention of peanut allergy required adherence to the protocol that would hardly be achievable in the daily life . Furthermore, the early introduction of other allergens such as fish or milk did not prevent allergy, and early introduction of gluten did not reduce the risk of celiac disease . Overall, these data show that promoting tolerance by oral antigen exposure during immune ontogeny is possible, but that additional cofactors are required to enhance the chance of success. In this regard, the role of breast milk factors in promoting oral tolerance has attracted increased interest. Antigens from the maternal diet are found in breast milk at concentrations 1,000-fold lower (ng/ mL) than antigen levels in formula milk (mg/mL). Unexpectedly, we also found antigens of respiratory sources such as the house dust mite Dermatophagoides pteronyssinus (Der p) and Blomia tropicalis in breast milk, in similar amounts as dietary antigen [18, 19]. Since we detected Der p 1 in digestive fluid of healthy adults , we propose that respiratory allergens are ingested by being trapped in the oropharynx or pulled back by the mucociliary epithelium and follow the same route as dietary antigens to the mammary gland. We have specifically addressed the factors in breast milk that could improve the chance of oral tolerance induction to dietary and respiratory antigens in rodents. We found that mice exposed to a few nanograms of egg ovalbumin (OVA) antigen through breast milk were protected from OVA-induced allergic airway disease and food allergy [21, 22]. Importantly, TGF-β from breast milk was necessary for oral tolerance induction . We demonstrated protection to be more profound when OVA was transferred through the breast milk of OVA-immunized mothers than OVA-exposed nonimmunized mothers . OVA-specific IgG in milk was necessary for protection during transfer of OVA though the gut barrier and the induction of a pro- longed protection mediated by FoxP3 Tregs. We also identified a key role of vita- min A in breast milk in the process of neonatal gut immunity maturation (see below) . Our more recent data further indicated that the nature of antigen found in breast milk could dramatically affect immune outcome. In contrast to the observation with OVA, the transfer of Der p 1 through breast milk induced Th2 immune response priming and increased susceptibility to allergic disease in adult mice . Importantly, in a human birth cohort, the risk of allergic sensitization and respiratory allergies in children breastfed by mothers increased with Der p 1 levels in breast milk . This observation stresses that not all the antigens in breast milk induce oral tolerance, and that there is a need to identify how maternal milk factors could be modulated to counteract the deleterious actions of some allergens. Ongoing intervention studies will help to decipher whether antigens in breast milk impact on immune tolerance induction .
Vitamin A is found in animal-derived food such as milk, liver, and egg yolk, while its precursors, the carotenoids, are found in vegetables such as carrots and broccoli. Major advances have recently been made on the impact of vitamin A and its metabolite retinoic acid (RA) on immune homeostasis . Specifically, RA pro- motes CD4+ T cell differentiation, supports the generation of IgA-secreting B cells, and mediates the balance between ILC3 and ILC2. RA also imprints gut- homing specificity on T and B cells to the small intestine. Vitamin A in conjunction with SCFA derived from fibers metabolized by microbiota, promoted oral tolerance and prevented food allergy . We recently identified that neonatal mice are physiologically deficient in retinol; serum retinol levels then progressively increase and reach adult levels at 3 weeks due to breast milk vitamin A . Low vitamin A levels at birth were found to be responsible for a leaky gut barrier, deficient RALDH expression by MLN CD103+ neonatal DCs, resulting in inefficient T-cell activation and the incapacity to induce oral tolerance in neonates . Importantly, vitamin A supplementation was sufficient to accelerate gut epithelium differentiation in terms of architecture and barrier function while preserving the capacity of epithelial cells to digest milk sugars . It also promoted immune maturation and allowed tolerance induction from birth, as observed in 3-week-old mice. Our observations also showed that vitamin A was involved in the maturation of the neonate’s immune responses towards Th1 immunity; this adds a dietary factor to the genetically programmed and microbiota-driven neonatal Th1 immune maturation . Relevance of these data for the human is supported by reports on low retinol levels in healthy infants from well-nourished countries  and observational studies linking low retinol levels at birth with increased atopic risk in young adults . In early postnatal life, vitamin A may also be involved in immune ontogeny by acting on lymphoid tissue organogenesis and development. Indeed, RA was shown to be necessary both for CXCL13 secretion by stromal cells , the first step in lymphoid tissue organogenesis, and for LTi differentiation and lymph node development . This points to an important role of maternal intake of vitamin A and precursors during pregnancy in immune ontogeny and possibly in early postnatal life for lymph node development.
Aryl Hydrocarbon Receptor and RAR-Related Orphan Receptor-γt Ligands
RORγt is a master transcription factor for the development of lymphoid organs, Th17, and ILC3. Their presence in GF animals suggests that the microbiota is not a critical source of RORγt ligand . The natural ligand was recently identified as a derivative of cholesterol, indicating that sterol metabolism may be essential for proper lymphoid tissue development in utero and possibly in early postnatal life for ILF.
In addition to binding microbiota-derived metabolites of tryptophan and pollutants, AhR binds dietary ligands contained in cruciferous vegetables, such as broccoli, that may then impact on gut immunity development after weaning. During the lactation period, AhR ligands in breast milk could originate from the maternal diet as well as from maternal microbiota-derived metabolites .
Before weaning, breast milk supplies the neonate with antimicrobial and regulatory factors that complement its developing immune system. Breast milk is also providing the neonate with the factors necessary for immune maturation that the neonatal mammal would otherwise miss due to the lack of microbiota and solid-food-derived molecules. The maturating impact of breast milk was high- lighted in a recent analysis of exfoliated gut epithelial cells in stools of 3-month- old children that were breastfed versus those formula fed and that showed a total of 1,214 genes differentially expressed between breastfed and formula-fed children . Analysis of gene networks reflected broad differences with respect to signal transduction, cytoskeletal remodeling, cell adhesion, and immune response. Gut trophic factors such as epidermal growth factor, HMOs, and vita- min A found in human milk are most probably involved in these effects . Breast milk exposes the infant to a variety of food antigens, and it contains ligands that are critical for lymphoid tissue development and immune function such as AhR ligands and vitamin A. It provides HMOs, as surrogates to fibers found in solid food, for commensals to produce SCFAs. Breast milk also delivers microbiota and food for commensal growth in the sterile neonate gut.
While the impact of gut microbiota-derived antigens and metabolites on gut mucosal immunity has largely been demonstrated, there is growing experimental and clinical evidence that diet may be as important for immune ontogeny and function. Before weaning, maternal milk, the physiological food for mammals, will reassemble all the varied exogenous factors required for immune maturation. Solid-food-derived antigens, vitamins, lipids, as well as food metabolites produced by the microbiota will then continue to shape the immune system function and dictate long-term susceptibility to local and systemic immune- mediated disease.
- 1 van Best N, Hornef MW, Savelkoul PH, Penders J: On the origin of species: factors shaping the establishment of infant’s gut microbiota. Birth Defects Res C Embryo Today 2015;105:240–251.
- 2 Gensollen T, Iyer SS, Kasper DL, Blumberg RS: How colonization by microbiota in early life shapes the immune system. Science 2016; 352:539–544.
- 3 Torow N, Hornef MW: The neonatal window of opportunity: setting the stage for life-long host-microbial interaction and immune homeostasis. J Immunol 2017;198:557–563.
- 4 van de Pavert SA, Mebius RE: New insights into the development of lymphoid tissues. Nat Rev Immunol 2010;10:664–674.
- 5 Turfkruyer M, Rekima A, Macchiaverni P, et al: Oral tolerance is inefficient in neonatal mice due to a physiological vitamin A deficiency. Mucosal Immunol 2016;9:479–491.
- 6 Thome JJ, Bickham KL, Ohmura Y, et al: Ear- ly-life compartmentalization of human T cell differentiation and regulatory function in mucosal and lymphoid tissues. Nat Med 2016;22:72–77.
- 7 Turfkruyer M, Verhasselt V: Breast milk and its impact on maturation of the neonatal immune system. Curr Opin Infect Dis 2015;28: 199–206.
- 8 Koch MA, Reiner GL, Lugo KA, et al: Maternal IgG and IgA antibodies dampen mucosal T helper cell responses in early life. Cell 2016; 165:827–841.
- 9 Rooks MG, Garrett WS: Gut microbiota, metabolites and host immunity. Nat Rev Immunol 2016;16:341–352.
- 10 Stefka AT, Feehley T, Tripathi P, et al: Commensal bacteria protect against food allergen sensitization. Proc Natl Acad Sci USA 2014; 111:13145–13150.
- 11 Secher T, Payros D, Brehin C, et al: Oral tolerance failure upon neonatal gut colonization with Escherichia coli producing the genotoxin colibactin. Infect Immun 2015;83:2420–2429.
- 12 Gomez de Aguero M, Ganal-Vonarburg SC, Fuhrer T, et al: The maternal microbiota drives early postnatal innate immune development. Science 2016;351:1296–1302.
- 13 Charbonneau MR, O’Donnell D, Blanton LV, et al: Sialylated milk oligosaccharides promote microbiota-dependent growth in models of infant undernutrition. Cell 2016;164: 859–871.
- 14 Gonzalez R, Klaassens ES, Malinen E, et al: Differential transcriptional response of Bifidobacterium longum to human milk, formula milk, and galactooligosaccharide. Appl Environ Microbiol 2008;74:4686–4694.
- 15 Kim KS, Hong SW, Han D, et al: Dietary antigens limit mucosal immunity by inducing regulatory T cells in the small intestine. Science 2016;351:858–863.
- 16 Menezes JS, Mucida DS, Cara DC, et al: Stimulation by food proteins plays a critical role in the maturation of the immune system. Int Immunol 2003;15:447–455.
- 17 Palmer DJ, Prescott SL, Perkin MR: Early introduction of food reduces food allergy – pro and con. Pediatr Allergy Immunol 2017;28: 214–221.
- 18 Baiz N, Macchiaverni P, Tulic MK, et al: Early oral exposure to house dust mite allergen through breast milk: a potential risk factor for allergic sensitization and respiratory allergies in children. J Allergy Clin Immunol 2017;139:369–372.e10.
- 19 Macchiaverni P, Ynoue LH, Arslanian C, et al: Early exposure to respiratory allergens by placental transfer and breastfeeding. PLoS One 2015;10:e0139064.
- 20 Tulic MK, Vivinus-Nebot M, Rekima A, et al: Presence of commensal house dust mite allergen in human gastrointestinal tract: a potential contributor to intestinal barrier dysfunction. Gut 2016;65:757–766.
- 21 Rekima A, Macchiaverni P, Turfkruyer M, et al: Long-term reduction in food allergy susceptibility in mice by combining breastfeeding-induced tolerance and TGF-β-enriched formula after weaning. Clin Exp Allergy 2017;47:565–576.
- 22 Verhasselt V, Milcent V, Cazareth J, et al: Breast milk-mediated transfer of an antigen induces tolerance and protection from allergic asthma. Nat Med 2008;14:170–175.
- 23 Mosconi E, Rekima A, Seitz-Polski B, et al: Breast milk immune complexes are potent inducers of oral tolerance in neonates and prevent asthma development. Mucosal Immunol 2010;3:461–474.
- 24 Macchiaverni P, Rekima A, Turfkruyer M, et al: Respiratory allergen from house dust mite is present in human milk and primes for allergic sensitization in a mouse model of asthma. Allergy 2014;69:395–398.
- 25 Venter C, Brown KR, Maslin K, Palmer DJ: Maternal dietary intake in pregnancy and lactation and allergic disease outcomes in off- spring. Pediatr Allergy Immunol 2017;28: 135–143.
- 26 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 Rep 2016;15:2809–2824.
- 27 Pesonen M, Kallio MJ, Siimes MA, Ranki A: Retinol concentrations after birth are inversely associated with atopic manifestations in children and young adults. Clin Exp Allergy 2007;37:54–61.
- 28 van de Pavert SA, Ferreira M, Domingues RG, et al: Maternal retinoids control type 3 innate lymphoid cells and set the offspring immunity. Nature 2014;508:123–127.
- 29 Santori FR, Huang P, van de Pavert SA, et al: Identification of natural RORγ ligands that regulate the development of lymphoid cells. Cell Metab 2015;21:286–297.
- 30 Chapkin RS, Zhao C, Ivanov I, et al: Noninvasive stool-based detection of infant gastrointestinal development using gene expression profiles from exfoliated epithelial cells. Am J Physiol Gastrointest Liver Physiol 2010; 298:G582–G589.
Metabolomics in Human Milk Research
Carolyn M. Slupsky
The first years of a child’s life are critical for growth and development, and despite decades of research, we still do not understand how infant diet shapes a child for both short- and long-term health. The link between food and health is complex, and although breastfeeding is known to have short- and long-term benefits, the relationship between food and the developing neonate is not understood, primarily because in the past there has been a lack of analytical tools. Indeed, most infant studies rely on crude measures of child health such as growth, and absence of obvious disease. While these assessments can reveal rudimentary associations between dietary components and lack of adverse outcomes in the short-term, they do not directly address the impact of food or food components on metabolic health that may have long-term consequences.
Making matters more complex, analysis of food is not trivial. Although decades of research have gone into studying human milk, most research has focused on studying proteins, lipids, and micronutrients. It is now recognized that there are other factors in milk that may be important for infant health, including small-molecule metabolites that include a unique class of sugars known as oligosaccharides. Human milk oligosaccharides, which are complex in structure, act as both food for beneficial bacteria and decoys for pathogens , and it is now being shown that they can help build the immune system through modulating CD14 expression and altering plasma cytokine levels [2, 3]. Additionally, there are other metabolites present in milk, and their function is not fully understood, although their expression appears to be controlled through the mammary gland as well , and they may have important consequences for the developing neonate. Through the development of modern nuclear magnetic resonance- and mass spectrometry-based metabolomics techniques, we are now in an era where we can measure these small molecules in food, and this will help us understand how food impacts health in an unprecedented way.
Analysis of the infant metabolome has led to important revelations regarding how infant diet impacts development. Breastfed infants have been shown to have lower levels of plasma branched-chain amino acids (isoleucine, leucine, and valine), and urea, as well as higher levels of ketone bodies (acetone), acetate, and myo-inositol . Additionally, breastfed infants have lower insulin levels than their formula-fed counterparts 2 h after feeding . High levels of serum branched-chain amino acids and/or insulin activates mechanistic target of rapamycin (mTOR), a serine/threonine kinase that is a master regulator of cell metabolism. mTOR Complex 1 (mTORC1) signaling is particularly important for the control of growth and metabolism of bone, skeletal muscle, the central nervous system, the gastrointestinal tract, blood cells, and other organs. For formula-fed infants, enhanced activation of this pathway may have lasting impacts on overall metabolism and potentially health.
More study of human milk and infant metabolism that incorporates metabolic phenotype (measured through the metabolome of blood, urine, and feces), gut microbial composition and function, as well as genetic (and epigenetic) data will help us understand the purpose of specific milk components, the individual responses to diet, as well as how diet and genetics work together with the gut ecosystem to guide cognitive and metabolic development.
- Underwood MA, German JB, Lebrilla CB, et al: Bifidobacterium longum subspecies
infantis: champion colonizer of the infant gut. Pediatr Res 2015;77:229–235.
- Goehring KC, Marriage BJ, Oliver JS, et al: Similar to those who are breastfed, infants fed a formula containing 2'-fucosyllactose have lower inflammatory cytokines in a randomized controlled trial. J Nutr 2016;146:2559–2566.
- He Y, Liu S, Kling DE, et al: The human milk oligosaccharide 2'fucosyllactose modulates CD14 expression in human enterocytes, thereby attenuating LPS-induced inflammation. Gut 2016;65:33–46.
- Smilowitz JT, O’Sullivan A, Barile D, et al: The human milk metabolome reveals diverse oligosaccharide profiles. J Nutr 2013;143:1709–1718.
- Slupsky CM, He X, Hernell O, et al: Postprandial metabolic response of breast-fed infants and infants fed lactose-free vs regular infant formula: a randomized controlled trial. Sci Rep 2017;7:3640.
The link between food and health is complex, particularly for the developing neonate, as the period after birth is the time when long-term programming is occurring notably in the neurologic, immune, and metabolic regulatory systems. Breastfeeding is known to have short- and long-term benefits, and yet the intricate relationship of this unique food with the neonate is not fully understood. Application of multiomic approaches incorporating new bioinformatic tools will allow for better characterization of phenotypes over the traditional approaches that were limited to crude assessment of growth parameters and observation of clinical disease. Metabolomics has the capability of allowing for a relatively noninvasive assessment of phenotypes via the assessment of small molecules in biofluids such as serum or urine that provides an opportunity to assess metabolism systemically in the developing neonate. Metabolomics can also be used to assess the metabolic activities of gut microbes through measurement of microbial by-products in the stool. Understanding the composition of human milk, how its components work synergistically together, and how they change over time will provide insight into how immunity and metabolism is established in early life, and how it can potentially prevent the development of chronic diseases in later life.
The postnatal period for human infants is recognized as a key period for growth and development, particularly for the rapidly developing brain. All of the infant’s metabolic, immunologic, intestinal, and physiologic systems are also rapidly changing. The ability of milk to nourish, fuel, and supply substrates to this growth has been recognized . However, the role of milk in orchestrating the entire metabolic process is not clear. A new generation of tools are now available to interrogate both milk and its effects on infant metabolism. Here, metabolomics is defined as the science of analyzing metabolites as an ensemble with sufficient diversity to map those metabolites onto pathways and sufficient quantitative accuracy to estimate metabolite flows through those pathways.
Brain growth during infancy has been positively associated with intelligence [2, 3], and human milk helps promote that development, particularly white matter development . Indeed, the human brain volume increases between 8 and 15% every 3 months from birth until approximately 18 months of age. Brain metabolites also change with dramatic increases in N-acetylaspartate, creatine, and glutamate and decreases in myoinositol in the cerebral white matter and cortex during the first 3 postnatal months . Epidemiological evidence suggests that breastfeeding practice is positively associated with intelligence and educational attainment at age 30 , and negatively associated with obesity, cardiovascular disease , and diabetes  in adulthood. How human milk is able to fuel and shape metabolism and guide overall health and development is an ongoing area of active research, and clues are beginning to emerge that the microbiota within the infant gastrointestinal (GI) tract are important.
The period immediately after birth, and for the first 2–3 years of the child’s life, is a key period for the development of intestinal microbiota [reviewed in 9, 10]. In the GI tract resides an ecosystem where microbes must compete in order to survive and persist, and the host must shape the microbiota in order to foster a beneficial community . One theory states that for symbiont-directed control, where a microbe alters global host phenotype to increase its own fitness, there must be low microbial diversity and limited competition between microbes . Interestingly, until the weaning period, breastfed infants have lower microbial diversity than formula-fed infants [reviewed in 12]. Infancy is also a time of instability with respect to the gut microbiota as evidenced by greater inter individual variability compared with adults [reviewed in 13]. This instability represents an important window of opportunity for the development of the gut microbial community. A recent study using gnotobiotic mice demonstrated that there is a critical window of time for intestinal immune development, and if conventionalization does not occur during that period, immune development can- not be fully achieved . Evidence is accumulating that the immune system is linked inextricably with global metabolism . Thus, it stands to reason that immune development will have important consequences for metabolic development , and if both are not optimally established early in life, consequences may be realized later in life.
The complex nature of milk therefore means that it has important functions for optimizing the health and the microbiota of the infant. Understanding the intricacies of the system at each stage of childhood that are vital for establishing the eventual host phenotype, including immunity, metabolism, and brain health, will be important for understanding the role of milk and its components in shaping health. While we are not yet at a stage where these complex relationships have been elucidated, linking host genetics and microbial ecology with the environment, including diet (macro- and micronutrient composition), and phenotype (measured through metabolomic analysis of serum, urine, and feces) will help us move toward that goal.
Human Milk and the Human Milk Metabolome
During the first few months of life, human milk provides essential nutrients for the infant while helping to establish the bacterial species that will constitute the gut microbiome. In addition to providing the essentials for growth and development, proteins, fatty acids, carbohydrates, and micronutrients, human milk provides a variety of cytokines, inflammatory mediators, and signaling molecules [reviewed in 17, 18].
Human milk varies greatly throughout lactation, with the colostrum very rich with immune factors and oligosaccharides that tend to decrease as lactation progresses [reviewed in 17]. Interestingly, we and others [19–23] observed a number of metabolites that change in concentration over time in human milk that includes amino acids, sugars, fatty acids, and others (Table 1). In general, oligosaccharides tend to decrease over time, while increases in lactose, several amino acids, as well as short- and medium-chain free fatty acids are noted. What this means is that milk maturation is not stochastic. Its composition is carefully controlled by the mammary gland that is guided by maternal genetics, continuously changing to meet the nutrient requirements of the neonate and help guide microbial succession throughout the period of exclusive milk feeding and possibly beyond. It is interesting to point out that microbial and fecal metabolic profiles are more similar between formula-fed infants from different mothers than breastfed infants from different mothers [24, 25], which could be attributed to the differences in milk composition between mothers who are breastfeeding and driven by changes in milk composition over the lactation period. In contrast, the composition of formula does not change. These results imply that diet (breast- or formula feeding) is a key driver in selecting microbes to colonize the GI tract.
One example of how maternal genetics tightly controls milk composition may be observed through the correlation of 2 of the more abundant oligosaccharides: 2′-fucosyllactose (2′FL) and 3-fucosyllactose (3FL). The concentration of these two oligosaccharides is dictated by the expression levels of enzymes encoded by the fucosyltransferase 2 (FUT2) and fucosyltransferase 3 (FUT3) genes, both of which are located on chromosome 19 [19, 26]. Production of 2′FL or 3FL is highly correlated such that as the concentration of 2′FL increases, 3FL decreases (Pearson’s correlation r = –0.76; Fig. 1a). In women who are secretors (having a functional FUT2 gene), during the course of the first 3 months of lactation 2′FL decreases from approximately 8 mmol/L in colostrum to 3 mmol/L, whereas 3FL increases from approximately 0.3 mmol/L in colostrum to 1.6 mmol/L, where the concentrations of each oligosaccharide begin to level out [19, 20] (Fig. 1b). The reason for this correlated change in concentration is not fully understood. However, human milk oligosaccharides have been credited with developing the microbiota, and in particular selecting for and maintaining high levels of Bifidobacterium longum subsp. infantis (B. infantis) in the infant GI tract during the period of exclusive human milk feeding, in addition to helping to build the immune system [9, 10]. These 2 oligosaccharides have also been recognized as having antiviral properties through acting as decoys to prevent binding of viruses to the GI tract [27, 28]. Human milk oligosaccharides have been reported to be absorbed into the blood, and 2′FL has specifically been shown to decrease plasmacytokine levels . Additionally, 2′FL has been shown to modulate CD14 expression on human enterocytes to attenuate LPS-induced inflammation . Controlling systemic inflammation  may be particularly important in the first few months of life, as circulating levels of cytokines such as IL-6 may modulate fatty acid metabolism and induce insulin resistance [reviewed in 16], which could have important consequences on metabolic development.
Another interesting metabolite that increases over time in human milk is urea. We observed that it increases from 2.7 mmol/L in colostrum to 4.5 mmol/L on postpartum day 90 [19, 20]. This increase may be a way to help create a steady nitrogen source for the growing populations of microbes in the infant GI tract . While most of the free amino acids are low in concentration in human milk throughout lactation (<1 mmol/L), several do increase over time (Table 1). In particular, glutamate increases from 0.5 mmol/L in colostrum to 1.5 mmol/L in mature milk over the first 3 months [19, 20]. Dietary glutamate is extensively metabolized by the intestinal epithelium [reviewed in 31], and this increase may be important for maturation of the intestinal tract of infants; however, more re- search needs to be done to fully understand the role of milk-derived free glutamate in the developing neonate.
Table 1. Variation in milk metabolites over time in women who are secretors
Fig. 1. a Pearson correlation of 2′- fucosyllactose (2′FL) with 3FL. b Changes in the con- centration (mmol/L) of 2′FL and 3FL from early lactation to postpartum day 90. Data from Smilowitz et al.  and Spevacek et al. .
Human Milk and Infant Health
The impact of human milk on infant metabolism can be demonstrated through comparison of the metabolomes of infants that have been formula fed and breastfed. Breastfed infants have been reported to have higher total cholesterol and LDL-C than formula-fed infants . Additionally, breastfed infants have lower levels of short-chain unsaturated and higher levels of longer-chain poly- unsaturated fatty acids containing phosphatidylcholines . In contrast, breastfed infants have lower levels of polyunsaturated fatty acid-containing long-chain triglycerides, and higher levels of shorter-chain sphingomyelin and 16:0 and 20:4 cholesterol esters than formula-fed infants . Breastfed infants also have higher fasting levels of acetate, acetone, myoinositol, glutamine, proline, and formate, as well as lower levels of urea, creatine, essential amino acids, and their by-products (threonine and valine, 2-hydroxybutyrate and 3-hydroxy- isobutyrate), choline, and dimethyl sulfone than formula-fed infants (Table 2) . In the postprandial state, breastfed infants have higher acetate, acetone, myoinositol, formate, methanol, and betaine, and lower 2-hydroxybutyrate, 3-hydroxyisobutyrate, alanine, isoleucine, leucine, lysine, methionine, proline, threonine, tyrosine, valine, choline, creatine, dimethyl sulfone, and urea (Table 3) . Similar differences were observed in nonhuman primates .
Analysis of the urine metabolome of formula- and breastfed infants largely mirrors what is observed in the serum metabolome, with differences in metabolites related to protein and amino-acid metabolism, ketogenesis, and fatty-acid oxidation . The urine metabolome also reveals differences in gut microbial function, as was observed with lower TMAO (trimethylamine-N-oxide) in breastfed compared with formula-fed infants .
Analysis of the fecal metabolome can reveal functionality of the microbes within the GI tract of infants. For example, fecal metabolome analysis of breast- fed infants revealed evidence of lower protein fermentation  and lower levels of short-chain fatty acids (propionate, butyrate, and acetate) and free amino acids, as well as higher levels of lactate and fucosylated oligosaccharides compared with formula-fed infants [35, 37, 38]. Measurement of fucosylated oligosaccharides in the stool is directly related to the consumption of human milk, as bovine milk (which the majority of formulas are based from) does not contain fucosylated oligosaccharides. Differences in lactate and short-chain fatty acids between breastfed and formula-fed infants can also be correlated with the resident microbes in the GI tract of breastfed infants. Compared with formula-fed infants, breastfed infants have higher levels bacteria from the Bifidobacterium and Lactobacillus genera, which produce lactate and acetate as primary fermentation products . Acetate is higher in the plasma of breastfed infants (Tables 2, 3), which may suggest that acetate produced by these bacteria may be absorbed. Evidence of temporal changes in the fecal metabolome has also been reported , which implies important changes in microbial structure and function during development.
Although there are common metabolome differences reflected in the blood, urine, and feces between breastfed and formula-fed infants, it is important to note that the composition of formulas vary depending on the study. Indeed, formulas on the market vary widely with differences in macro- and micronutrient composition. Changing one component in the formula can result in profound changes to an infant’s metabolism, the structure and function of the gut microbiome, or both. For instance, a recent study revealed that replacing lactose in infant formula with corn-syrup solids resulted in a lowering of many amino ac- ids measured in the postprandial state (1–2 h) compared with infants fed lactose-based formula . While this seems to be desirable, 2 h after feeding, glucose, ketones, and nonesterified fatty acids were lower in the infants fed formula with corn-syrup solids compared with those fed lactose-based formula, and their insulin levels were significantly higher than in breastfed infants.
Other studies have looked at adding probiotics to infant formula [24, 39, 40]. For example, supplementation of nonhuman primates with a formula containing B. animalis subsp. lactis resulted in slightly increased serum BCAA, and a structuring of the microbiota that was different from the formula-fed infants at 3 months . Of potential concern was the marked increase in the fecal polyamines cadavarine and putrescine in the third month. Supplementation of human infants with a formula containing Bifidobacterium. bifidum, B. breve, B. longum, and B. infantis resulted in changes in both the structure and function (assessed through the fecal metabolome) of colonic bacteria during the period of supplementation but had no detectable long-term effects . Another study where infant formula was supplemented with a probiotic/prebiotic combination of B. animalis subsp. lactis CNCM I-3446 and bovine milk oligosaccharides revealed an α diversity that was similar to breastfed infants over the course of supplementation, in addition to higher levels of the Bifidobacterium genus in general compared with control formula , but no metabolome changes were measured and reported. The fact that changes in metabolic parameters were observed in 2 of these studies suggests that these early gut microbes impact metabolic development of the infant and further that a metabolic assessment should be considered an important outcome when evaluating changes in formula ingredients.
Table 2. Semifasted serum metabolite differences between breastfed and formula-fed infants
Table 3. Postprandial serum metabolite differences between breastfed and formula-fed infants
The Collaboration between Milk and the Developing Neonate
The components of human milk are unique and are specific to the growing neonate. Replacing human milk with milk from other animals has profound consequences for the developing neonate. For instance, the consistently observed in- creases in plasma amino acids in formula-fed infants may inhibit several early steps in the insulin signaling cascade , and the sustained increases could contribute to hepatic mitochondrial dysfunction that may be associated with future increased BMI, insulin resistance, and dyslipidemia .
Components of human milk may also interact directly with specific metabolic pathways, such as the mechanistic target of rapamycin (mTOR) pathway , to optimize development. mTOR complex 1 (mTORC1) is a nutrient-sensitive kinase that plays an important role in many aspects of cell growth, protein and lipid synthesis, as well as lipid accumulation and adipogenesis. It is particularly important in child development for the control of growth and metabolism of bone, skeletal muscle, the central nervous system, the GI tract, blood cells, and other organs [reviewed in 44]. Amino acids such as leucine can regulate mTORC1, and a correlation between the amount of leucine in the whey fraction and serum leucine levels in infants has been reported . Bacteria, such as Lactobacillus plantarum, have also been shown to act on the TOR-dependent host nutrient-sensing system in Drosophila . Interestingly, it was recently ob- served that L. plantarum might be vertically transmitted from mother to infant through breastfeeding . Thus, there may be a connection between the types of microbes colonizing the infant GI tract and expression of the mTOR pathway, although more studies are needed to confirm.
Diet is remarkable in both its ability to shape gut microbiota as well as metabolism and the immune system. The exquisite linkage of microbiota, host immunity, and host metabolism hints at the complexity of human milk, and how it has been optimized for neonatal development. The changes in human milk composition throughout lactation (increases and decreases in many metabolites) likely reflect the changing needs of the neonate and the gut microbiota. Analysis of the metabolome (either serum or urine or both) reflects the human phenotype and should be considered an essential component of any study that aims to capture the metabolic effects of diet. Analysis of the fecal metabolome can inform on the function of the gut microbes. Once a healthy baseline has been defined, the response to diet can be assessed through analysis of the serum, urine, and fecal metabolomes both in the short term and long term.
More studies on human milk should be done to assess how secretor status and Lewis blood type (both in the mother and the infant) affects infant development including immunity and metabolism, as well as other factors such as maternal health and environmental exposures. Incorporating additional data including genetic and epigenetic data will be important to understand individual responses to diet and microbial succession. Incorporating a nutrigenomic approach together with an analysis of microbial structure and function will also help us understand, on a deeper level, how human milk and its components affect gene expression either by themselves or through the gut microbiota. This approach will take many years but may ultimately allow us to understand the interplay between food, gut microbiota, and metabolism, and overall provide a better understanding on how to achieve optimal health through diet.
1 Hinde K, German JB: Food in an evolutionary context: insights from mother’s milk. J Sci FoodAgric 2012;92:2219–2223.
2 Gale CR, O’Callaghan FJ, Bredow M, et al: The influence of head growth in fetal life, in- fancy, and childhood on intelligence at the ages of 4 and 8 years. Pediatrics 2006;118: 1486–1492.
3 Gale CR, O’Callaghan FJ, Godfrey KM, et al: Critical periods of brain growth and cognitive function in children. Brain 2004;127:321–329.
4 Isaacs EB, Fischl BR, Quinn BT, et al: Impact of breast milk on intelligence quotient, brain size, and white matter development. Pediatr Res 2010;67:357–362.
5 Blüml S, Wisnowski JL, Nelson MD, et al: Metabolic maturation of the human brain from birth through adolescence: insights from in vivo magnetic resonance spectrosco- py. Cereb Cortex 2013;23:2944–2955.
6 Victora CG, Horta BL, Loret de Mola C, et al: Association between breastfeeding and intelligence, educational attainment, and income at 30 years of age: a prospective birth cohort study from Brazil. Lancet Glob Health 2015; 3:e199–e205.
7 Parikh NI, Hwang S-J, Ingelsson E, et al: Breastfeeding in infancy and adult cardiovascular disease risk factors. Am J Med 2009; 122:656–663.e1.
8 Horta BL, Loret de Mola C, Victora CG: Long-term consequences of breastfeeding on cholesterol, obesity, systolic blood pressure and type 2 diabetes: a systematic review and meta-analysis. Acta Paediatr 2015;104:30–37.
9 Smilowitz JT, Lebrilla CB, Mills DA, et al: Breast milk oligosaccharides: structure-function relationships in the neonate. Annu Rev Nutr 2014;34:143–169.
10 Underwood MA, German JB, Lebrilla CB, et al: Bifidobacterium longum subspecies infantis: champion colonizer of the infant gut. Pe- diatrRes 2015;77:229–235.
11 Foster KR, Schluter J, Coyte KZ, et al: The evolution of the host microbiome as an eco-system on a leash. Nature 2017;548:43–51.
12 Mueller NT, Bakacs E, Combellick J, et al: The infant microbiome development: mom matters. Trends Mol Med 2015;21:109–117.
13 Arrieta M-C, Stiemsma LT, Amenyogbe N, et al: The intestinal microbiome in early life: health and disease. Front Immunol 2014;5: 427.
14 El Aidy S, Hooiveld G, Tremaroli V, et al: The gut microbiota and mucosal homeostasis. Gut Microbes 2014;4:118–124.
15 Man K, Kutyavin VI, Chawla A: Tissue immunometabolism: development, physiology, and pathobiology. Cell Metab 2017;25:11–26.
16 Buck MD, Sowell RT, Kaech SM, et al: Metabolic instruction of immunity. Cell 2017;169: 570–586.
17 Bardanzellu F, Fanos V, Reali A: “Omics” in human colostrum and mature milk: looking to old data with new eyes. Nutrients 2017;9: 843.
18 Munblit D, Peroni DG, Boix-Amorós A, et al: Human milk and allergic diseases: an unsolved puzzle. Nutrients 2017;9:894.
19 Smilowitz JT, O’Sullivan A, Barile D, et al: The human milk metabolome reveals diverse oligosaccharide profiles. J Nutr 2013;143: 1709–1718.
20 Spevacek AR, Smilowitz JT, Chin EL, et al: Infant maturity at birth reveals minor differences in the maternal milk metabolome in the first month of lactation. J Nutr 2015;145: 1698–1708.
21 Sundekilde UK, Downey E, O’Mahony JA, et al: The effect of gestational and lactational age on the human milk metabolome. Nutrients 2016;8:E304.
22 Wu J, Domellöf M, Zivkovic AM, et al: NMR- based metabolite profiling of human milk: a pilot study of methods for investigating com- positional changes during lactation. Biochem Biophys Res Commun 2016;469:626–632.
23 Xu G, Davis JC, Goonatilleke E, et al: Absolute quantitation of human milk oligosaccha- rides reveals phenotypic variations during lactation. J Nutr 2017;147:117–124.
24 He X, Slupsky CM, Dekker JW, et al: Inte- grated role of Bifidobacterium animalis sub- sp. lactis supplementation in gut microbiota, immunity, and metabolism of infant rhesus monkeys.mSystems 2016;1:e00128-16.
25 O’Sullivan A, He X, McNiven EMS, et al: Ear- ly diet impacts infant rhesus gut microbiome, immunity, and metabolism. J Proteome Res 2013;12:2833–2845.
26 Austin S, De Castro CA, Bénet T, et al: Tem- poral change of the content of 10 oligosaccharides in the milk of Chinese urban mothers. Nutrients 2016;8:E346.
27 Koromyslova A, Tripathi S, Morozov V, et al: Human norovirus inhibition by a humanmilk oligosaccharide. Virology 2017;508:81– 89.
28 Weichert S, Koromyslova A, Singh BK, et al: Structural basis for norovirus inhibition by human milk oligosaccharides. J Virol 2016; 90:4843–4848.
29 Goehring KC, Marriage BJ, Oliver JS, et al: Similar to those who are breastfed, infants fed a formula containing 2′-fucosyllactose have lower inflammatory cytokines in a randomized controlled trial. J Nutr 2016;146:2559– 2566.
30 He Y, Liu S, Kling DE, et al: The human milk oligosaccharide 2′fucosyllactose modulates CD14 expression in human enterocytes, thereby attenuating LPS-induced inflammation. Gut 2016;65:33–46.
31 Burrin DG, Stoll B: Metabolic fate and function of dietary glutamate in the gut. Am J ClinNutr 2009;90:850S–856S.
32 Harit D, Faridi MMA, Aggarwal A, et al: Lip- id profile of term infants on exclusive breast- feeding and mixed feeding: a comparative study. Eur J Clin Nutr 2008;62:203–209.
33 Prentice P, Koulman A, Matthews L, et al: Lipidomic analyses, breast- and formula- feeding, and growth in infants. J Pediatr 2015; 166:276–281.e6.
34 Slupsky CM, He X, Hernell O, et al: Post- prandial metabolic response of breast-fed infants and infants fed lactose-free vs regular infant formula: a randomized controlled trial. Sci Rep 2017;7:3640.
35 Martin F-PJ, Moco S, Montoliu I, et al: Impact of breast-feeding and high- and low-protein formula on the metabolism and growth of infants from overweight and obese mothers. Pediatr Res 2013;75:535–543.
36 Chow J, Panasevich MR, Alexander D, et al: Fecal metabolomics of healthy breast-fed versus formula-fed infants before and during in vitro batch culture fermentation. J Proteome Res 2014;13:2534–2542.
37 Bridgman SL, Azad MB, Field CJ, et al: Fecal short-chain fatty acid variations by breast- feeding status in infants at 4 months: differences in relative versus absolute concentrations. Front. Nutr 2017;4:11.
38 Underwood MA, Gaerlan S, De Leoz MLA, et al: Human milk oligosaccharides in premature infants: absorption, excretion, and influence on the intestinal microbiota. Pediatr Res 2015;78:670–677.
39 Bazanella M, Maier TV, Clavel T, et al: Ran- domized controlled trial on the impact of early-life intervention with bifidobacteria on the healthy infant fecal microbiota and metabolome. Am J Clin Nutr 2017;106:1274– 1286.
40Simeoni U, Berger B, Junick J, et al: Gut microbiota analysis reveals a marked shift to bifidobacteria by a starter infant formula containing a synbiotic of bovine milk-derived oligosaccharides and Bifidobacterium animalis subsp. lactis CNCM I-3446. Environ Microbiol 2016;18:2185–2195.
41 Hyde R, Taylor PM, Hundal HS: Amino acid transporters: roles in amino acid sensing and signalling in animal cells. Biochem J 2003; 373:1–18.
42 Morán-Ramos S, Ocampo-Medina E, Gutier- rez-Aguilar R, et al: An amino acid signature associated with obesity predicts 2-year risk of hypertriglyceridemia in school-age children. Sci Rep 2017;7:5607.
43 Melnik BC: Milk – a nutrient system of mammalian evolution promoting mTORC1-dependent translation. Int J Mol Sci 2015;16: 17048–17087.
44 Semba RD, Trehan I, Gonzalez-Freire M, et al: Perspective: the potential role of essential amino acids and the mechanistic target of rapamycin complex 1 (mTORC1) pathway in the pathogenesis of child stunting. Adv Nutr 2016;7:853–865.
45 Melnik BC: Excessive leucine-mTORC1-signalling of cow milk-based infant formula: the missing link to understand early childhood obesity. J Obes 2012;2012:197653.
46 Storelli G, Defaye A, Erkosar B, et al: Lactobacillus plantarum promotes Drosophila systemic growth by modulating hormonal sig- nals through TOR-dependent nutrient sensing. Cell Metab 2011;14:403–414.
47 Murphy K, Curley D, O’Callaghan TF, et al: The composition of human milk and infant faecal microbiota over the first three months of life: a pilot study. Sci Rep 2017;7:40597.
The human mammary gland is an integral effector component of the common mucosal immune system [1–4]. However, from physiologi-cal and immunological aspects, it displays several unique features not shared by other mucosal sites [1, 2, 4]. The development, maturation and activity of the mammary gland exhibits a strong hormonal dependence [1, 4]. Furthermore, in comparison to the intestinal and respiratory tracts, the mammary gland is not colonized by high numbers of bacteria of enormous diversity and does not contain mucosal inductive sites analogous to the intestinal Peyer's patches [1, 4–6]. Consequently, when exposed to antigens, local or generalized immune responses are low or not present [2, 4, 5]. Comparative evaluations of various immunization routes effective in the induction of antibodies in human milk are limited [2, 4, 6]. Systemic immunization induces IgG antibodies in plasma, but due to the low levels of total IgG in human milk, their protective effect remains unknown [3, 5]. Oral or intranasal immunization or infection induces secretory IgA in milk as demonstrated in several studies [1, 2, 7]. Other routes of mucosal immunization such as sublingual or rectal exposure effective in the induction of antibodies in various external secretions have not been explored in the mammary gland.Because secretory IgA in milk displays protective functions [2, 3, 5], alternative immunization routes and antigen-delivery systems should be explored.
1 Butler JE, Rainard P, Lippolis J, et al: The mammary gland in mucosal and regional
immunity; in Mestecky J, Strober W, Russell MW, et al (eds): Mucosal Immunology, ed 4. Amsterdam, Elsevier/Academic Press, 2015, vol II, pp 2269–2306.
2 Mestecky J, Blair C, Ogra PL: Immunology of Milk and the Neonate. Adv Exp Med
Biol. New York, Plenum, 1991, vol 310, pp 1–488.
3 Ogra SS, Weintraub DI, Ogra PL: Immunologic Aspects of Human Colostrum and
Milk: Interaction with the Intestinal Immunity of the Neonate. Adv Exp Med Biol. New York, Plenum, 1978, vol 107, pp 95–112.
4 Brandtzaeg P: The secretory immune system of lactating human mammary glands
compared with other exocrine organs. Ann NY Acad Sci 1983;409:353–382.
5 Ogra PL Losonsky GA, Fishaut M: Colstrum-drived immunity and maternal-neonatal interaction. Ann NY Acad Sci 1983;409:82–95.
6 Ladjeva I, Peterman JH, Mestecky J: IgA subclasses of human colostral antibodies
specific for microbial and food antigens. Clin Exp Immunol 1989;78:85–90.
7 Pakkanen SH, Kantele JM, Moldoveanu Z, et al: Expression of homing receptors on
IgA1 and IgA2 plasmablasts in blood reflects differential distribution of IgA1 and IgA2 in various body fluids. Clin Vaccine Immunol 2010;17:393–401.
AbstractThe human mammary gland is an integral effector component of the common mucosal immune system. However, from physiological and immunological aspects, it displays several unique features not shared by other mucosal sites. The development, maturation, and activity of the mammary gland exhibits a strong hormonal dependence. Furthermore, in comparison to the intestinal and respiratory tracts, the mammary gland is not colonized by high numbers of bacteria of enormous diversity and does not contain mucosal inductive sites analogous to the intestinal Peyer’s patches. Consequently, when exposed to antigens, local or generalized immune responses are low or not present. Comparative evaluations of various immunization routes effective in the induction of antibodies in human milk are limited. Systemic immunization induces IgG antibodies in plasma, but due to the low levels of total IgG in human milk, their protective effect remains unknown. Oral or intranasal immunization or infection induces secretory IgA in milk, as demonstrated in several studies. Other routes of mucosal immunization, such as sublingual or rectal exposure effective in the induction of antibodies in various external secretions, have not been explored in the mammary gland. Because secretory IgA in milk displays protective functions, alternative immunization routes and antigen delivery systems should be explored.
IntroductionIn nature, milk is the external secretion which is essential for the survival of the offspring due to its nutritional value and, from the immunological view, as the source of passive protection against infection [1, 2]. Humans are the only mammals in which maternal milk is frequently substituted by milk proteins derived from other mammalian species or plants. Although breastfeeding is preferred to alternative means of nutrition, the prenatal transplacental transport of plasmaderived immunoglobulins into the fetal circulation provides in many species effective protection in the systemic compartment . In addition to the humoral factors of innate immunity, milk of various mammalian species contains high levels of immunoglobulin, which differ in their structural features and effector functions [1, 3]. In sharp contrast to the dominance of IgG in the milk of many mammals (e.g., pigs, cows, and horses), human milk contains more IgA in its
secretory form as SIgA than immunoglobulins of other isotypes [1, 3–5]. Due to the essential role of antibodies in the prevention of mucosally acquired infections , extensive efforts have been devoted to the design of vaccines effective in the induction of antibodies of desired specificity in external secretions, including the evaluation of routes of immunizations, forms of antigen delivery of relevant antigens, and possible use of mucosal adjuvants [7, 8].
Properties, Origin, and Biological Activities of Antibodies in MilkIn contrast to IgG antibodies, which dominate in the milk of many species and are derived from the circulatory pool , human milk contains IgA as the main immunoglobulin isotype . It is represented by SIgA in its dimeric (∼60%) and tetrameric (∼40%) forms, with small amounts of dimeric IgA lacking the secretory component (SC; see below) and trace amounts of monomeric IgA . The presence of SIgA in its dimeric and tetrameric forms is of functional importance due to 4 and 8 antigen binding sites. Furthermore, the characteristic distribution of SIgA subclasses reflects, to a certain degree, the origin of cells producing SIgA1 or SIgA2 as well as the specificity of these antibodies [9, 10]. Contrary to the earlier proposal in which monomeric IgA was polymerized within epithelial cells during the transcytotic pathway through the acquisition of the epithelial IgA receptor called SC, immunochemical analyses of milk SIgA clearly revealed that polymeric IgA (pIgA) dimers and tetramers are produced in these forms by IgA plasma cells adjacent to mucosal and glandular epithelial cells expressing a receptor (polymeric immunoglobulin receptor), which selectively binds pIgA and IgM and after epithelial transcytosis remains associated with polymeric im-
[6, 11]. Mucosal bacteria, particularly in the intestinal tract and oral cavity, are
in vivo coated with SIgA without harm . In fact, antibody coating inhibits adherence of bacteria to the receptors expressed on the surface of epithelial cells and participates in the formation of a bacterial biofilm of the same species at
bind to the corresponding bacterial glycan structures, thus preventing their adherence to epithelial receptors . However, SIgA in concert with humoral factors of innate immunity enhances (or focuses) their antimicrobial activities .
The inhibition of absorption of soluble but biologically inert antigens from food by SIgA has been well documented as a means to prevent the overstimulation of the entire immune system by the increased absorption of such antigens in the absence of specific antibodies . Furthermore, it should be stressed that due to the multivalency of milk SIgA (4 antigen binding sites in dimers and 8 in tetramers), the biological effectiveness for viral neutralization is enormously enhanced due to the bonus effect of multivalency [6, 12]. Specificity of Antibodies in Milk Reflects the Site of Antigenic Stimulation
at Various Inductive Sites The mammary gland as the effector site is populated by precursors of IgA-producing cells from remote inductive sites [7, 8, 10]. Consequently, the specificity of SIgA in milk depends on the encounter with antigens, dominantly in the gastrointestinal and respiratory tracts. As shown in Table 2, human colostrum and milk contain antibodies of the IgA isotype to a broad spectrum of environmental antigens of microbial and food origin. In response to the antigens encountered by the mother at the time of pregnancy and after the delivery, such antibodies provide the most relevant passive SIgA-mediated immunity [1–4, 13]. Although there is considerable individual variability in total and antigen-specific SIgA antibody levels among lactating mothers, antibodies to gram-positive and gramnegative bacteria, viruses, and food antigens are commonly found in all samples [4, 13]. Not surprisingly and in harmony with earlier results, antibodies to food antigens and antigens I/II of the oral bacterium Streptococcus mutans are associated with the IgA1 subclass, while those against lipopolysaccharides of gramnegative bacteria present in the large intestine; bacterial polysaccharides are
mostly IgA2 [9, 13]. Interestingly, the distribution of total IgA1 and IgA2 was ∼53 and 47%, respectively, with marked individual variability . In comparison to other external secretions, human milk is in this respect reminiscent of the IgA subclass distribution in the lower intestinal tract and markedly differs from the secretions of the upper respiratory and upper digestive tracts . There are, however, several interesting observations which remain unexplained. External secretions, including milk and sera obtained from HIV-infected individuals, display extremely low levels of HIV-specific antibodies of the IgA isotypes irrespective of the route of HIV infection . It appears that HIV, specifically its negative factor (nef), selectively suppresses IgA responses. Furthermore, milk collected from lactating mothers contains high titers of antisperm antibodies of the IgA isotype . Because the female genital tract is a poor inductive site, and various antigens administered intravaginally stimulate only minimal or no local
responses , it is possible to speculate that these antisperm antibodies are induced due to the exposure to sperm by alternative inductive sites, specifically through oral or anal receptive sexual encounter, both of which are effective in the induction of generalized mucosal immune responses .
Induction of Antibodies in Milk by Local or Systemic Immunization
could contribute significant quantities of IgG or IgA to external secretions, including the milk, has been explored in early studies . In animals whose milk contains IgG as the dominant isotype, systemic immunization is effective, and IgG of plasma origin is present in their milk . In contrast, in human milk, low levels of total IgG are present. Nevertheless, subcutaneous immunization with 3 selected rubella virus vaccines resulted in the induction of IgG-, IgM-, and IgAspecific antibodies in sera, but only minimal levels of IgG or IgM virus-specific antibodies were detectable in milk . However, IgA antibodies were detectable in the milk of all systemically immunized women 2–4 weeks after immunization, with peak responses at the 4th week. Although primary immunization with previously unencountered antigen does not stimulate mucosal immune responses, in women who had been previously sensitized by the mucosal route, systemic immunization could evince an SIgA response in milk, as demonstrated with cholera vaccine . In previously unexposed lactating Swedish women, systemically administered cholera vaccine did not induce SIgA antibodies in
milk. In contrast, in lactating Pakistani women, presumably naturally exposed
to Vibrio cholerae, such an immunization induced SIgA antibodies in milk . Thus, the initial mucosal exposure profoundly influenced the outcome of subsequent systemic immunization and may favor the induction of milk SIgA antibodies to some antigens.
Induction of Generalized Mucosal Responses and the Common Mucosal Immune System Pioneering studies performed in animals and later in humans helped to discover
the origin of antibody-forming cells in anatomically remote mucosal tissues and associated secretory glands, including the mammary, salivary, and tear glands, and had an enormous impact on the feasible approaches and design of mucosally administered vaccines . Oral administration of dinitrophenylated pneumococci to lactating rabbits led to the appearance of specific antibodies of the IgA isotype in milk . Subsequent extensive experiments with oral administration of various particulate or soluble antigens confirmed earlier observations and demonstrated that, in addition to milk, SIgA antibodies were present in secretions of other mucosal tissues and glands . The fundamental explanation for these observations of such importance was provided by earlier studies
should be stressed, however, that information relevant to the human mammary gland with respect to the origin of IgA precursor cells from various inductive sites has not been, for obvious ethical reasons, explored and remains controversial. Based on immunohistochemical staining of 2 samples of lactating mammary glands and spectra of antibodies, some authors speculate that the IgA precursors originate in the upper respiratory tract and Waldeyer’s ring, while others concerning the tissue origin of IgA-producing cells found in mucosal tissues and glands . Several investigators demonstrated that lymphocytes from the intestinal Peyer’s patches, bronchus-associated lymphoid tissue, and perhaps Waldeyer’s ring of the oropharynx are an enriched source of cells that express the potential to populate remote mucosal tissues and glands [19, 23]. Experiments performed primarily in mice led to the extended phenotyping of IgA precursor cells as surface IgA-positive cells that express surface receptors, later termed homing receptors, that are involved in interactions with specific ligands present on the surface of endothelial cells of postcapillary venules in mucosal
tissues . Importantly, for immunization studies and their physiologic interpretation,
cells from such inductive sites, including Peyer’s patches, bronchusassociated lymphoid tissue, Waldeyer’s ring, rectal tonsils, and sublingual tissue, differ in their expression of homing receptors, which interact with corresponding ligands and, therefore, lead to the tissue-selective distribution of cells from various inductive site to tissue-elective effector sites (Table 3) [19, 23, 24]. It should be stressed, however, that information relevant to the human mammary gland with respect to the origin of IgA precursor cells from various inductive sites has not been, for obvious ethical reasons, explored and remains controversial. Based on immunohistochemical staining of 2 samples of lactating mammary glands and spectra of antibodies, some authors speculate that the IgA precursors originate in the upper respiratory tract and Waldeyer’s ring, while others
2. Determine the phenotypes of specific antibody-secreting cells in the mammary gland, as compared to the peripheral blood, with respect to the immunoglobulin isotypes and expression of homing receptors.
3. Determine the levels and duration of humoral immune responses of specific antibodies with regard to the immunization route, immunoglobulin isotypes, and the antigen delivery systems in human milk.
5. Are there marked racially related differences (developed and developing countries) in the magnitude and specificity of immune responses to relevant antigens?
6. Determine the most effective timing of maternal mucosal immunization to provide optimal levels of protective antibodies in milk after birth.
7. Continue efforts in the exploration of effectiveness of currently evaluated mucosally administered adjuvants in the induction of specific antibodies in milk.
The author declares that he has no relevant or material financial interest that relates to
the research described in this paper.
1. Butler JE, Rainard P, Lippolis J, et al: The mammary gland in mucosal and regional immunity; in Mestecky J, Strober W, Russell MW, et al (eds): Mucosal Immunology, ed 4. Amsterdam, Elsevier/ Academic Press, 2015, vol II, pp 2269–2306.
2. Sharma D, Hanson LA, Korotkova M, et al: Human milk: its components and their immunobiologic functions; in Mestecky J, Strober W, Russell MW, et al (eds): Mucosal Immunology, ed 4. Amsterdam, Elsevier/Academic Press, 2015, vol II, pp 2307–2344.
3. Mestecky J, Blair C, Ogra PL: Immunology of milk and the neonate. Adv Exp Med Biol 1990; 310: 1–488.
4. Ogra SS, Weintraub DI, Ogra PL: Immunologic aspects of human colostrum and milk: interaction with the intestinal immunity of the neonate. Adv Exp Med Biol 1978; 107: 95–112.
5. Jackson S, Moldoveanu Z, Mestecky J: Appendix I: collection and processing of human mucosal secretions: in Mestecky J, Strober W, Russell MW, et al (eds): Mucosal Immunology, ed 4. Amsterdam, Elsevier/Academic Press, 2015, vol II, pp 2345–2344.
6. Mestecky J: Protective activities in mucosal antibodies, in Kiyono H, Pascual D (eds): Mucosal Vaccines: Innovation for Preventing Infectious Disease, ed 2. Amsterdam, Elsevier/Academic Press, 2019, pp 71–84.
7. Russell MW, Mestecky J: Mucosal vaccines: an overview: in Mestecky J, Strober W, Russell MW, et al (eds): Mucosal Immunology, ed 4. Amsterdam, Elsevier/Academic Press, 2015, vol II, pp 1039–1046.
8. Boyaka PN, McGhee JR, Czerkinsky C, Mestecky J: Mucosal vaccines: an overview: in Mestecky J, Bienenstock J, Lamm ME, et al (eds): Mucosal Immunology, ed 3. Amsterdam, Elsevier/Academic Press, 2005, vol I, pp 855–874.
9. Woof JM, Mestecky J: Mucosal immunoglobulins; in Mestecky J, Strober W, Russell MW, et al (eds): Mucosal Immunology, ed 4. Amsterdam, Elsevier/Academic Press, 2015, pp 287–324.
10. Pakkanen SH, Kantele JM, Moldoveanu Z, et al: Expression of homing receptors on IgA1 and IgA2 plasmablasts in blood reflects differential distribution of IgA1 and IgA2 in various body fluids. Clin Vaccine Immunol 2010; 17: 393–401.
11. Baker K, Blumberg RS, Kaetzel CS: Immunoglobulin transport and immunoglobulin receptors; in Mestecky J, Strober W, Russell MW, et al (eds): Mucosal Immunology, ed 4. Amsterdam, Elsevier/ Academic Press, 2015, pp 349–409.
12. Mestecky J, Russell MW: Specific antibody activity, glycan heterogeneity and polyreactivity contribute to the protective activity of S-IgA at mucosal surfaces. Immunol Lett 2009; 124: 57–62.
13. Ladjeva I, Peterman JH, Mestecky J: IgA subclasses of human colostral antibodies specific for microbial and food antigens. Clin Exp Immunol 1989; 78: 85–90.
14. Mestecky J, Tamaras G: HIV-1/SIV humoral responses in external secretions. Curr Immunol Rev 2019; 15: 49–62.
15. Ulcova-Gallova Z, Mestecky J, Fialova P: Antispermatozoal antibodies in human colostrum and milk. Zentralbl Gynäkol 1994; 116: 636–638.
16. Russell MW, Mestecky J: Tolerance and protection against infection in the genital tract. Immunol Invest 2010; 39: 500–525.
17. Kantele A, Hakkinen M, Moldoveanu Z, et al: Differences in immune responses induced by oral and rectal immunization with Salmonella typhi Ty21a: evidence for compartmentalization within the common mucosal immune system in humans. Infect Immun 1998; 66: 5630–5635.
18. Brandtzaeg P: The secretory immune system of lactating human mammary glands compared with other exocrine organs. Ann NY Acad Sci 1983; 409: 353–382.
19. Mestecky J: The common mucosal immune system and current strategies for induction of immune responses in external secretions. J Clin Immunol 1987; 7: 265–276.
20. Losonsky GA, Fishaut JM, Strussenberg J, et al: Effect of immunization against rubella on lactation products. I. Development and characterization of specific immunologic reactivity in breast milk. J Infect Dis 1982; 145: 654–660.
21. Montgomery PC, Cohn J, Lally ET: The induction and characterization of secretory IgA antibodies. Adv Exp Med Biol 1974; 45: 453–462.
22. Craig SW, Cebra JJ: Peyer’s patches: an enriched source of precursors for IgA-producing immunocytes in the rabbit. J Exp Med 1971; 134: 188–200.
23. Brandtzaeg P: The mucosal B cell system. in Mestecky J, Strober W, Russell MW, et al (eds): Mucosal Immunology, ed 4. Amsterdam, Elsevier/ Academic Press, 2015, pp 623–667.
24. Mikhak Z, Agace WW, Luster AD: Lymphocyte trafficking to mucosal tissues; in Mestecky J, Strober W, Russell MW, et al (eds): Mucosal Immunology, ed 4. Amsterdam, Elsevier/Academic Press, 2015, pp 805–839.
25. Mestecky J, McGhee JR, Arnold RR, et al: Selective induction of an immune response in human external secretions by ingestion of bacterial antigen. J Clin Invest 1978; 61: 731–737.
26. Czerkinsky C, Prince SJ, Michalek SM, et al: IgA antibody-producing cells in peripheral blood after antigen ingestion: evidence for a common mucosal immune system in humans. Proc Natl Acad Sci USA 1987; 84: 2449–2453.
27. Goldblum RM, Ahlstedt S, Carlsson B, et al: Antibody- forming cells in human colostrum after oral immunization. Nature 1975; 257: 797–799.
28. Gregory RL, Schöller M, Filler SJ, et al: IgA antibodies to oral and ocular bacteria in human external secretions. Protides Biol Fluids 1985; 32: 53–56.
29. Moro I, Crago SS, Mestecky J: Localization of IgA and IgM in human colostral elements using immunoelectron microscopy. J Clin Immunol 1983; 3: 382–391.
30. Ogra PL, Losonsky GA, Fishaut M: Colostrumderived immunity and maternal-neonatal interaction. Ann NY Acad Sci 1983; 409: 82–95.