In this video you will be able to watch key Q&A from NNI symposium as schedule for 6th WCPGHAN on “HMOs: A window of opportunity or a strong weapon in strengthening immunity”.
Mother's milk has been long touted for its salutary effects on the newborn and its ability to shield infants from certain infections. Now research from Harvard Medical School conducted in mice shows that at least part of its protective effects come from a surprising source: the microbes residing in maternal intestines.
Although rotavirus vaccination programs are now widespread, rotavirus-associated diarrhea persists in many developing countries and rotavirus continues to be a major viral pathogen in areas where vaccination is not common.
- Human milk oligosaccharides (HMO) are a predominant component of human milk and are comprised of diverse structures that are neutral or acidic and some forms are sialylated or fucosylated, which contributes to their biological functions.
- HMO protect the infant from pathogenic infections, facilitate the establishment of the gut microbiota, promote intestinal development, and stimulate immune maturation.
- Some types of HMO are now commercially available and are being added to infant formula alone or in combination with other prebiotics.
Human milk oligosaccharides, Immunity, Infant
The immune system of the infant is functionally immature and naïve. Human milk contains bioactive proteins, lipids, and carbohydrates that protect the newborn and stimulate innate and adaptive immune development. This review will focus on the role human milk oligosaccharides (HMO) play in neonatal gastrointestinal and systemic immune develop ment and function. For the past decade, intense research has been directed at defining the complexity of oligosaccharides in the milk of many species and is beginning to delineate their diverse functions. These studies have shown that human milk contains a higher concentration as well as a greater structural diversity and degree of fucosylation than the milk oligosaccharides in other species, particularly bovine milk from which many infant formulae are produced. The commercial availability of large quantities of certain HMO has furthered our understanding of the functions of specific HMO, which include protecting the infant from pathogenic infections, facilitating the establishment of the gut microbiota, promoting intestinal development, and stimulating immune maturation. Many of these actions are exerted through carbohydrate-carbohydrate interactions with pathogens or host cells. Two HMOs, 2’-fucosyllactose (2’FL) and lacto-N-neotetraose (LNnT), have recently been added to infant formula. Although this is a first step in narrowing the compositional gap between human milk and infant formula, it is unclear whether 1 or 2 HMO will recapitulate the complexity of actions exerted by the complex mixture of HMO ingested by breastfed infants. Thus, as more HMO become commercially available, either isolated from bovine milk or chemically or microbially synthesized, it is anticipated that more oligosaccharides will be added to infant formula either alone or in combination with other prebiotics.
The human infant enters the world with a functionally naïve immune system affecting both adaptive and innate immune responses  , which leaves the newborn at high risk for common infections. Postnatal immune maturation is stimulated by antigenic exposures and host-microbe interactions [1, 2]. How and what the infant is fed influences the development and competence of the immune system [3–5]. Human milk protects the infant during this vulnerable period by providing bioactive components that protect the infant from pathogenic infection, support intestinal development, promote barrier function, stimulate immune development, facilitate immune tolerance, and feed gut microbes [2–5]. Thus, human milk supplies multiple layers of protection for the infant ( Fig. 1 ).
Breastfeeding, particularly exclusive breastfeeding for 6 months or more, relative to formula-feeding, decreases the incidence and/or severity of infectious diseases . Many diseases with infectious and immune components in their etiology, including diarrhea, respiratory and urinary tract infections, otitis media, bacteremia, and necrotizing enterocolitis occur less often in breast than formula-fed infants [6, 7]. Breastfeeding has also been implicated in reducing the incidence of other diseases involving the immune system and immune tolerance, such as inflammatory bowel disease, celiac disease, asthma, allergy, type 1 diabetes, as well as acute lymphoblastic and acute myeloblastic leukemias [6, 8]. These benefits may be mediated in part through effects of breastfeeding on the intestinal microbiota [8, 9] , which in turn stimulates maturation and specificity of the neonatal mucosal and systemic immune systems .
The immune benefit of breastfeeding has been attributed in part to the diverse bioactive components in human milk [2–5]. A strong case can be made for a key role of human milk oligosaccharides (HMO) in neonatal immune defense and maturation. As will be described below, HMO are present in high concentrations in human milk, exist in an incredible structural diversity [10–13], and confer host protection and mediate immune responses through a number of mechanisms [14, 15].
HMO Content and Composition
HMO are complex soluble glycans that are predominantly present in free form in milk. These glycans are synthesized from 5 basic monosaccharides: galactose, glucose, N-acetylglucosamine, fucose, and the sialic acid derivative N-acetylneuraminic acid [10, 11]. With few exceptions, all HMO carry lactose (Galβ1–4Glc) at the reducing end, which can be elongated in β1–3 or β1–6 linkage by 2 different disaccharides, either Galβ1–3GlcNAc (type 1 chain) or Galβ1–4GlcNAc (type 2 chain) .
The HMO content has been reported in the range of 1–10 g/L in mature milk and 15–23 g/L in colostrum [10–13]. In term breast milk, ~35–50% of HMO are fucosylated, 12–14% are sialylated, and 42–55% are nonfucosylated neutral HMO [10–13] ( Table 1 ). However, HMO composition is influenced by maternal genetics, including secretor and Lewis Blood Group status [10, 11]. HMO fucosylation is mediated by the 2 fucosyltransferases FUT2 (secretor gene) and FUT3 (Lewis gene). Nonsecretor mothers, who lack a functional FUT2 enzyme and represent about 30% of women worldwide, produce milk lacking in α1-2-fucosylated oligosaccharides like 2α -fucosyllactose (2’ FL) and lacto-N-fucopentaose (LNFP) I [10, 11]. The absence of these compounds may have functional consequences. For example, infants consuming milk produced by women who are nonsecretors exhibit delayed colonization of bifidobacteria, higher abundance of Streptococcus taxa, and have functional differences in the metabolic activity of their microbiota . Infants fed milk from nonsecretor mothers are at higher risk for diarrheal diseases .
HMO and the Microbiome
The development of the infant gut microbiota is a sequential process that begins in utero and continues during the first 2–3 years of life. Microbial composition and diversity is shaped by host genetics and multiple environmental factors, of which diet is a principal contributor [8, 9]. Studies conducted over the past decade have shown that specific Bacteroides and Bifidobacterium species that commonly colonize breastfed infants efficiently utilize HMO as carbon sources. This is particularly true of B. longum ssp. infantis (B. infantis), which is a predominant gut microbe in most breastfed infants . The discovery of a genomic island in B. infantis that encodes specific enzymes for the metabolism of HMO supports an adaptation of this species to the intestinal milieu of the breastfed infant [18, 19]. Indeed, a recent study in human infants fed formula supplemented with 2’ FL (1 g/L) and LNnT (0.5 g/L) demonstrated that the global microbiota composition of infants fed formulae with 2’ FL and LNnT was significantly different to that of infants fed nonsupplemented formula (p < 0.001) at the genus level and closer to that of breastfed infants at 3 months of age . In addition, Bifidobacterium was more abundant (p < 0.01), whereas Escherichia and unclassified Peptostreptococcaceae were less abundant in infants fed formula with 2’ FL and LNnT compared to infants fed nonsupplemented formula, and these levels were closer to those observed in breastfed infants . Furthermore, the concentrations of several important metabolites in stool (propionate, butyrate, and lactate) in infants fed the HMO-supplemented formula were more similar to those of breastfed infants .
Previously, we have shown that HMO fermentation by neonatal pig microbiota produced short-chain fatty acids and promoted the growth of beneficial bacteria in vitro  and in vivo . Gut bacteria and the immune response, particularly the gastrointestinal immune response, are tightly interrelated . Thus, in this animal model, HMO-induced changes in the gut bacterial populations of the pigs could alter the course of an intestinal infection  which in turn would alter the immune response . Alternatively, the change in the gut bacteria could directly affect the immune system of these animals . Additional ways whereby HMO may mediate neonatal immunity are summarized in the following section.
HMO as Immune Modulators
Summarized in Figure 2 are the results of an accumulating body of evidence showing that HMO indirectly and directly influence infant mucosal and systemic immune function. In general, intestinal health and barrier function are considered a first line of defense in innate immunity. Cell proliferation takes place in the crypts, and cells differentiate as they migrate up the villus-crypt axis, with the exception of Paneth cells, which migrate down to the base of the crypt. HMO reduce intestinal crypt cell proliferation [25, 26] , increase intestinal cell maturation  , and increase barrier function  (indicated by 1–3 in Fig. 2 ). A layer formed by mucus glycoproteins or mucins produced by goblet cells acts as a lubricant and a protective physical barrier between the intestinal epithelium and the luminal contents (indicated by 4 in Fig. 2 ). HMO may influence goblet cell function, as has been shown for galacto-oligosaccharides (GOS) . HMO affects epithelial immune gene expression both directly [28–30] and indirectly through the microbiota  (indicated by 5 and 6 in Fig. 2 , respectively). As noted above, HMO serve as prebiotics to promote the growth of healthy bacteria, including Bifidobacteria and Bacteroides genera  (indicated by 7 in Fig. 2 ), and HMO inhibit infections by bacteria and viruses by either binding to the pathogen in the lumen or by inhibiting binding to cell-surface glycanreceptors [14–15, 22] (indicated by 8 in Fig. 2 ). Additionally, dietary oligosaccharides decorate the intestinal lining contributing to the intestinal glycan repertoire . HMO also contribute to epithelial barrier function by supporting the growth of B. infantis in the infant gut [10, 18]. B. infantis produces peptides that have been shown to normalize intestinal permeability through enhanced tight junction protein expression in a mouse model of colitis . It is likely that HMO support other bacterial species that are important for the maintenance of gut integrity. These changes in intestinal barrier function would, in turn, alter both the local and systemic immune system . HMO affect immune cell populations and cytokine secretion [22, 36] (indicated by 9 in Fig. 2 ). Some HMO are also absorbed into the blood stream [37–39] (indicated by 10 in Fig. 2 ), where they exert systemic effects by binding of monocytes, lymphocytes, and neutrophils to endothelial cells  (indicated by 11 in Fig. 2) and formation of platelet-neutrophil complexes  (indicated by 12 in Fig. 2 ). Readers are referred to a recent review by Kulinich and Liu  for additional discussion of this topic.
Carbohydrate Binding as a Potential Mechanism of HMO in the Immune System
Carbohydrates and carbohydrate-binding proteins play an important role in immune responses. Cells have unique glycan signatures made from combinations of specific glycan motifs that are engaged when a cell contacts another cell or other components of its environment [42, 43]. However, many of the glycan motifs found on mammalian cells are also found on microbes and in food, including human milk. These similarities provide opportunities for host-microbe-HMO interactions.
Lectins are carbohydrate-binding proteins on the surfaces of mammalian cells that translate recognition of specific motifs and the spatial presentation of those motifs into action. Lectins are grouped according to their carbohydrate recognition domains (CRD) [42, 43].
There are at least a dozen CRD identified in mammals, but 3 classes of lectins related to the influence of HMO on immune responses are C-type lectins, siglecs (sialic acid-binding Ig-like lectins), and galectins.
C-type lectins require calcium to function and include selectins, mannose-binding lectin, and dendritic cell-specific intercellular adhesion molecule 3-grabbing non-integrin (DC-SIGN). C-type lectin receptors on the surface of dendritic cells (DC) determine whether the cell will induce tolerance rather than lymphocyte activation . DC-SIGN is of particular interest with regard to mechanisms by which HMO can influence immunity because it has a CRD specific for fucose units. Furthermore, DC-SIGN is expressed by cells in the gastrointestinal tracts of infants . These intestinal cells are likely antigen-presenting cells as DC-SIGN is expressed by antigen-presenting cells, specifically DC . Although interactions between fucosylated ligands and DC-SIGN contribute to immune tolerance, the cellular response ultimately depends upon the other ligand-receptor reactions occurring simultaneously 
Siglecs are sialic acid-binding lectins most commonly found on subsets of immune cells . There are at least 16 siglecs expressed by different leukocyte populations, which include sialoadhesin (siglec-1), CD22 (siglec-2), myelin-associated glycoprotein (MAG, siglec-4), siglec-15, and CD33-related siglecs. Siglec specificity derives from differences in secondary binding sites . Siglecs are endocytic cell surface receptors that carry cargo between the cell surface and intracellular vesicles; these receptors are mainly expressed on cells involved in antigen processing and presentation . Furthermore, sialic acid-containing molecules can gain entry to macrophages by binding to siglecs on the cell surface . On mammalian cells, some sialic acid-containing glycans function as self-associated molecular patterns and prevent immune responses to nonpathogenic stimuli. Ligation of particular siglecs stimulates the production of the immunoregulatory cytokine interleukin (IL)-10 .
Galectins are important for cell turnover and immune regulation. The CRD of galectins is specific for β-galactosides. When cells are desialylated, the density of exposed galactose moieties on the cell surface increases. For example, naïve T cells express CD45 with an α-2,6-linked sialic acid. The amount of α-2,6-linked sialic acid is reduced following T-cell activation. The decrease in α-2,6-linked sialic acid renders the activated T cells susceptible to galectin-1- mediated apoptosis . Thus, binding of sialyated HMO to cells may prevent apoptosis.
HMO as Modulators of Mucosal Immunity
Intestinal cell lines have been used to determine effects of HMO on immune-related gene expression and protein production. These cells have been co-incubated with oligosaccharides  , bacteria  , or lipopolysaccharides (LPS) to model a bacterial infection . Co-incubation of Bifidobacterium with cells of the Caco-2 intestinal cell line and HMO resulted in downregulation of intestinal cell genes related to chemokine activity compared to co-incubation with glucose or lactose . Conversely, in the absence of a bacterial co-stimulant, HMO increased expression of several chemokines by the HT-29 cell line . Additional work in T84 and HCT8 intestinal cell lines showed that complex mixtures of HMO as well as 2’ FL reduced signatures of intestinal inflammation .
HMO have been demonstrated to affect the course of a gastrointestinal viral infection. In an acute rotavirus (RV) infection model where a 21-day-old piglet’s ileum was isolated in situ, intestinal loops co-treated with HMO and RV had reduced non-structural protein-4 (NSP-4) mRNA ex-pression indicating that HMO can reduce RV replication . Intestinal cytokine and chemokine expression, however, was not affected. Both neutral and acidic HMO decreased NSP4 intestinal mRNA expression in the in situ model, whereas only acidic HMO effectively inhibited RV infectivity in an in vitro model .
HMO as Modulators of Systemic Immunity and Protection from Infection
HMO are detected in the plasma of infants fed human milk at concentrations of 1–133 mg/L [37, 39] , suggesting the potential for dietary HMO to directly affect immune cells circulating in the blood. As discussed above, many immune receptors recognize the oligosaccharide structures of their glycoprotein ligands [14, 15]. Because a subset of HMO is structurally similar to selectin ligands  , it is likely that HMO can bind directly to immune cells and trigger signaling that results in changes to immune cell populations and functions. For instance, the P- and E-selectins recognize sialyl-Lewis x (sLeX), a glycan moiety of several HMO . Additionally, fucosylation and sialylation, 2 enzymatic modifications common to HMO, enable binding to selectins . HMO- induced disruption of immune protein-carbohydrate interactions reduced neutrophil rolling  and activation . HMO directly affect immune cell proliferation and cytokine production in ex vivo experiments with peripheral blood mononuclear cells (PBMC) from neonatal pigs .
Stimulation with isolated HMO stimulated production of the regulatory cytokine IL-10 . Others observed that the acidic HMO induce IL-10 production; additionally, they found that acidic HMO induce IFN-γ from ex vivo stimulated human cord blood mononuclear cells . Isolated HMO enhanced proliferation of PBMC stimulated with a T-cell mitogen, phytohemagglutinin (PHA), and sialylated HMO enhanced proliferation of PBMC stimulated with the B-cell mitogen LPS . In contrast, 2’ FL inhibited proliferation of unstimulated PBMC cultured for 3 days. Thus, the response to HMO may depend on the state of the infant. In the unstimulated state, HMO dampen proliferation, whereas HMO enhance proliferation in response to a mitogenic stimuli.
To date, very few studies have fed HMO and analyzed immune outcomes [22, 29, 30, 51–54] ( Table 2 ). Piglets  have been fed 2’ FL, but only growth and toxicological outcomes were reported. A recently published paper described immune outcomes in human infants fed formula containing 2.4 g/L GOS, 2.2 g/L GOS + 0.2 g/L 2’ FL, or 1.4 g/L GOS + 1.0 g/L 2’FL compared to a breastfed reference control . Infants were fed the formula from 5 days to 4 months of age, and blood samples were obtained at 6 weeks of age for cytokine analysis, immune cell phenotyping, and ex vivo stimulation of isolated PBMC. Breastfed infants and infants fed either formula with 2’ FL were similar and had lower plasma inflammatory cytokines than infants fed the control formula. In addition, cytokine secretion by PBMC from breastfed infants and infants fed either 2’ FL-containing formula that were stimulated ex vivo with respiratory syncytial virus was similar and secreted less tumor necrosis factor-α and interferon-γ and tended to have lower IL-1Ra, IL-6, and IL-1β than cells from infants fed the control formula .
Another recent study in human infants evaluated the effect of formula supplemented with both 2’ FL (1.0 g/L) and LNnT (0.5 g/L) compared to an unsupplemented formula. Infants were fed the formulae from 14 days to 6 months of age, after which they were switched to a stan-dard follow-on formula and followed until 12 months of age. Infants fed the HMO- supplemented formula had significantly fewer parental reports (p = 0.004–0.047) of: bronchitis through 4 months (2.3 vs. 12.6%), 6 months (6.8 vs. 21.8%), and 12 months (10.2 vs. 27.6%); lower respiratory tract infection (AE cluster) through 12 months (19.3 vs. 34.5%); antipyretics use through 4 months (15.9 vs. 29.9%); and antibiotics use through 6 months (34.1 vs. 49.4%) and 12 months (42.0 vs. 60.9%) than those fed the standard formula .
Several studies in animal models support the reduced incidence of infection in human infants fed formula with HMO. In mice infected with Escherichia coli , once daily oral gavage with 100 mg, 2’ FL prevented body weight loss and reduced colonization with adherent-invasive E. coli , colonic inflammation, crypt cell CD14 expression, as well as IL-6, IL-17, and tumor necrosis factor-α production in response to adherent-invasive E. coli infection compared to mice treated with vehicle . Mice fed 2’ FL and subjected to ileocecal resection gained more weight and had greater crypt depth and villus height at the site of transection than nonsupplemented mice . The 2’ FL-fed mice also had upregulated mucosal immune response genes in the distal small bowel . The studies where pigs and human infants were fed the HMO have focused on 2’ FL, which is readily available in large quantities at reasonable cost, and fucosylated oligosaccharides have been shown to feed specific beneficial classes of bacteria during intestinal inflammatory events . Given what is known about the effects of other HMO, these compounds also should be used in feeding studies when available in sufficient quantities.
Only 1 in vivo study used a complex mixture of HMO and assessed immune outcomes. In that report, neonatal pigs fed a diet containing 4 g/L HMO, consisting of 40% 2’ FL, 10% 6’-sialyllactose (6’ SL), 35% lacto-N-neote-traose (LNnT), 5%, 3‘-sialyllactose (3’ SL), and 10% free sialic acid, had a reduced duration of diarrhea, in response to RV infection to 48.8 ± 9.8 h versus 80.6 ± 4.5 h in pigs fed nonsupplemented formula . Ileal tissue from the pigs fed HMO contained greater IFN-γ (produced by Th1 cells) and IL-10 (an anti-inflammatory cytokine) mRNA than that from pigs fed formula .
In a mouse model of food allergy, 2’ FL and 6’ SL administered via oral gavage reduced symptoms in mice sensitized to ovalbumin, an egg protein . Specifically, oval-bumin-stimulated splenocytes from mice treated with 6’SL produced more IL-10 and less IFN-γ than those from untreated mice. Furthermore, 2’ FL- or 6’ SL-treated mice had more regulatory immune cells in their intestinal immune tissues than untreated mice. Interestingly, neither 2’ FL nor 6’SL affected intestinal T regulatory cells when administered to nonsensitized mice . This exemplifies the necessity of identifying an appropriate challenge model to assess the effects of dietary compounds on the immune system. In mice, the milk oligosaccharides LNFP III and LNnT are
Th2-biasing and suppress Th1 responses . Recently, it has been reported that human infants who were fed human milk with low LNFP III concentrations (<60 μ M ) were 6.7-times (95% CI 2.0–22) more likely to become affected with cow’s milk allergy when compared to infants receiving milk with high LNFP III concentrations  .
Another approach using knockout mice showed that SL-containing compounds can directly affect gastrointestinal mucosal immunity [52, 59]. In one study, the presence of 3’SL in the milk increased the number of immune cells infiltrating the gut in IL-10 null mice . Furthermore, supplementation with 3’SL increased colitis severity in newborn IL-10 and St3gal4 (the enzyme that synthesizes 3’SL) null mice, and cross-fostering wildtype mice to deficient dams reduced colitis severity. One caveat of this work is that it was conducted in the absence of endogenous IL-10 production, whereas other in vivo studies have demonstrated that some HMO increase intestinal IL-10 [22, 51]. 3’ SL is a product of several pathogenic bacteria  and the conformation (α2,3-link between sialic acid and galactose) on pathogenic bacteria and in human milk is the same. 3’ SL is recognized by DC and generates an immune response through the TLR4 signaling pathway . These results suggest that the presence of 3’ SL increases the inflammatory response through direct effects on DC. When TLR4 was absent, 3’ SL was less effective at inducing DC activation. However, those DC also demonstrated a minimal increase in CD40 expression suggesting that at least one other 3’ SL-sensing mechanism, albeit much less efficient than the TLR4 pathway, exists on DC. TLR4 is the receptor for E. coli LPS. Another link between 3’ SL and TLR4 is ex-plained in a newer paper, where it is demonstrated that 3’ SL stimulates the proliferation of the intestinal E. coli population and that this overgrowth of E. coli is responsible for exacerbation of dextran sulfate sodium colitis through release of proinflammatory cytokines from intestinal DC .
These examples demonstrate the complexity of the relationships between oligosaccharides, the gut bacteria and the immune system.
The rich diversity of HMO has the potential to modulate both innate and adaptive neonatal immunity. Findings from in vitro experiments and animal models show that HMO directly interact with gastrointestinal epithelial cells as well as with mucosal and systemic immune cells to modulate immune function. HMO also beneficially shape the microbiome of the breastfed infant. The increased availability of HMO from commercial sources as well as accumulating evidence demonstrating that formula supplemented with HMO is safe and may confer benefits for human infants have led to the recent addition of 2’ FL alone or in combination with LNnT to infant formulae. In addition, due to their beneficial effects on immune function and host defense, HMO may also be beneficial for other segments of the population who are immune compromised or at high risk for infection. There are limited studies in which animals or humans have been fed HMO. Additionally, few studies have assessed the effects of feeding complex mixtures of HMO on the immune response. Thus, future research is needed to delineate mechanisms and to fully realize the potential for HMO to benefit infant immune function.
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The human microbiome is a collection of bacteria, archaea, fungi, and viruses, all residing within our bodies, including their genetic material. In recent years, research has revealed multiple essential roles of these microorganisms in metabolism, immunity, endocrinology, and overall health. Numerous physiological and disease states, including obesity and the metabolic syndrome, have been correlated with microbial changes, termed dysbiosis. Our microbiomes change in response to our environment, diet, weight, hormones, and other factors. It is, therefore, not surprising that during pregnancy, when dramatic weight gain and metabolic and immune changes occur, there are also significant changes in the microbiome. Throughout pregnancy, alterations in the microbiota composition occur at a variety of body sites, including the gut, vagina, oral cavity, and placenta. Yet, there remains much to be discovered regarding the precise microbial alterations during pregnancy, their timing, and, potentially, their further effects. Understanding the roles of the microbiome throughout pregnancy in health and disease is of great importance for opening new research avenues and suggesting new therapeutic approaches. This includes questions regarding the safety and effects of antibiotics and probiotics during pregnancy on the fetal and offspring health.
The major changes that occur in gut microbiota during pregnancy include an increase in the bacterial load and profound alterations in the composition of the gut microbiota, mostly during late pregnancy [1, 2]. These dramatic changes are characterized by reduced individual richness (alpha diversity), increased between-subject diversity (beta diversity), increased abundance of Actinobacteria and Proteobacteria phyla, and reduced abundance of Faecalibacterium and other short-chain fatty acid producers. Transfer of third-trimester microbiota into germ-free mice was shown to cause increased weight gain, insulin resistance, and a greater inflammatory response compared to the first-trimester microbio ta . The vaginal microbiome undergoes significant changes during pregnancy as well , including a significant decrease in overall diversity, increased stability, and increased abundance of Lactobacillus species .
The main change occurring in the oral microbiota during pregnancy is an incr ease in the microbial load, including higher levels of the pathogenic bacteria Porph yromonas gingivalis and Aggregatibacter actinomycetemcomitans, and Candida . Antibiotics administered during pregnancy were shown to affect the m icrobiome composition and diversity, as well as to have some effects on the offspring.
Unlike in the case of disease states, we believe that the microbial alterations observed during pregnancy are vital for a healthy pregnancy. While more research in this field is required to reveal specific mechanisms and pathways regulating these alterations, the microbial changes during pregnancy are likely coordinated with the immune, endocrine, and metabolic states. High progesterone and estrogen levels may affect the microbiome as it has been previously shown that microbial components can respond to and regulate host hormones, and that host hormones influence bacterial growth. Additionally, significant immune changes occur during pregnancy in order to protect the fetus and mother from infection on the one hand, while enabling fetal immune development. These are likely to affect the microbial components as well. Finally, metabolic changes occur during pregnancy, including changes in energy homeostasis, fat storage, and hormonal regulation. Many of these metabolic processes somewhat resemble states of the metabolic syndrome, obesity, and diabetes, which have all been correlated with microbial changes.
Future research will likely reveal the interactions and pathways linking the various physiological changes to the microbial changes, thereby explaining the significance of each change observed. Such studies may also have clinical relevance in terms of recommendations for antibiotic treatments, probiotics, and potential therapies for pregnancy complications.
- Collado MC, Isolauri E, Laitinen K, Salminen S: Distinct composition of gut microbiota during pregnancy in overweight and normal-weight women. Am J Clin Nutr 2008;88:894–899.
- Koren O, Goodrich JK, Cullender TC, et al: Host remodeling of the gut microbiome and metabolic changes during pregnancy. Cell 2012;150:470–480.
- Nuriel-Ohayon M, Neuman H, Koren O: Microbial changes during pregnancy, birth, and infancy. Front Microbiol 2016;7:1031.
- Aagaard K, Riehle K, Ma J, et al: A metagenomic approach to characterization of the vaginal microbiome signature in pregnancy. PLoS One 2012;7:e36466.
- Fujiwara N, Tsuruda K, Iwamoto Y, et al: Significant increase of oral bacteria in the early pregnancy period in Japanese women. J Investig Clin Dent DOI: 10.1111/ jicd.12189.
In recent years, microbiome research has revealed multiple essential roles of the microorganisms residing within the human body in host metabolism, immunity, and overall health. Numerous physiological and pathological states, including obesity and the metabolic syndrome, have been correlated with microbial changes, termed dysbiosis. Our microbiomes change in response to our environment, diet, weight, hormones, and other factors. It is, therefore, not surprising that there are also significant changes in the microbiome during pregnancy when dramatic weight gain and metabolic and immunological changes occur. In this review, we summarize the known changes in microbial composition throughout pregnancy at a variety of body sites, including the gut, vagina, oral cavity, and placenta, and we describe several studies that have linked pregnancy complications with microbial changes. Unlike the case of certain disease states, such as obesity, where dysbiosis is considered to have negative effects, we believe that the microbial alterations observed during pregnancy are vital for a healthy pregnancy. While more research in this field is required to reveal specific mechanisms and pathways regulating these alterations, the microbial changes during pregnancy are likely coordinated with the immune, endocrine, and metabolic states.
The human microbiome includes hundreds of different microbial species residing within and on us, playing essential roles in our metabolism and immune and endocrine system. These microbial populations have been shown to change during our lifetime, from infancy to childhood, adulthood, and old age. The microbial populations are also highly affected by weight gain, diet, and immune and hormonal changes. Therefore, it is not surprising that there are distinct alterations in the microbiota at multiple sites within the body during pregnancy. Pregnancy, a complex physiological process, is associated with simultaneous hormonal changes, weight gain, and immune system modulations, which must all be synchronized to preserve the health of both the mother and the offspring  . In this review, we describe the pronounced microbial changes that occur in the pregnant female. We hypothesize that an appropriate microbiota is essential for the healthy early development of the fetus and pregnancy maintenance. Moreover, we suggest that the microbial changes during pregnancy are highly correlated with other physiological changes, including those in hormones, immunity, and metabolism. Understanding the roles of the microbiome throughout pregnancy in health and disease is of great importance for opening new research avenues and suggesting new therapeutic approaches. Yet, there remains much to be discovered regarding the precise microbial alterations during pregnancy, their timing, and, potentially, their further effects.
Studying the microbiota in pregnancy opens another fascinating question of whether the fetus is exposed to microbes, and, if so, at what stage of development. While it has been thought for over a century that we are born germ free  , numerous pieces of evidence now cast doubt on this hypothesis and suggest that a bacterial presence already exists in the fetoplacental unit [3, 4] . The enigma of whether a placental microbiota exists as well is still not fully resolved.
The Healthy Microbiome
The human microbiome is a collection of bacteria, archaea, fungi, and viruses, all residing within our bodies and including their genetic material. Within each one of us, there are trillions of microbial cells representing hundreds of different species. Together, they play important roles in host metabolism, immunity, endocrinology  , and overall health. The microbial compositions vary between people and are greatly affected by diet, additional environmental factors, weight gain, and immune state, etc. Different body sites harbor different microbial populations due to varying levels of pH, oxygen, nutrients, humidity, and temperature  . Therefore, the gut, oral cavity, and vagina each harbor distinct bacterial communities, potentially playing different beneficial roles. In this review, we mainly discuss alterations during pregnancy in bacterial communities of the microbiome, as these are the best studied to date. It is important to remember that pregnancy is a healthy physiological process in which beneficial microbial alterations are expected. This is in contrast to disease states such as obesity, inflammatory bowel disease (IBD), diabetes, and the metabolic syndrome, in which an unhealthy shift in microbiota composition, termed dysbiosis, may occur  .
Factors Affecting the Microbiota during Pregnancy
One group of initial changes that occur during pregnancy is hormonal changes. Most importantly, progesterone and estrogen levels rise dramatically, with numerous physiological effects. These hormonal levels are likely to affect the microbiome composition since it has previously been shown that microbial components can respond to and regulate host hormones, and that host hormones influence bacterial growth. On the other hand, the microbiota can also produce and secrete hormones, emphasizing the bidirectional nature of the interplay between microbiota and hormones. Nonetheless, direct effects of progesterone and estrogen on the microbiota, and the effects of the microbiota on these hormones, have not yet been proven  .
Additionally, significant immune changes occur during pregnancy, and these are likely to affect the microbiota. The immune changes are complex in order to protect the fetus and mother from infection on the one hand, while nevertheless enabling fetal immune development and preventing fetal rejection by the maternal immune system. The microbial components are crucial players in this immune modulation; however, they themselves are also affected by immune changes.
Finally, metabolic changes occur during pregnancy, including changes in energy homeostasis, fat storage, and hormonal regulation. In many ways, the metabolic changes associated with pregnancy are similar to those that occur in the metabolic syndrome, including weight gain, elevated fasting blood glucose levels, insulin resistance, glucose intolerance, low-grade inflammation, and changes in metabolic hormone levels  . While the microbiota plays active roles in these metabolic processes, it is also highly affected by host metabolism, as seen by dysbiosis in obesity, the metabolic syndrome, and diabetes. Therefore, the metabolic changes occurring during pregnancy are expected to influence the microbiota composition.
Pregnancy Leads to Changes in the Gut Microbiota
Several alterations in the gut microbiota have been associated with pregnancy progression. In general, pregnancy is characterized by an increase in the bacterial load and profound alterations in the composition of the gut microbiota  . Most of the changes relative to nonpregnant women are seen in late pregnancy. These dramatic changes are characterized by reduced individual richness (α diversity), increased between-subject diversity (β diversity), and alterations in abundance of certain species  . Increased abundance of members of the Actinobacteria and Proteobacteria phyla are observed at the expense of reduced abundance of Faecalibacterium and other short-chain fatty-acid producers . Faecalibacterium is a butyrate-producing bacterium with anti-inflammatory activities, which is depleted in metabolic syndrome patients  . This is of particular interest, since pregnancy shares some characteristics with the metabolic syndrome, including weight gain, insulin insensitivity, and higher levels of low-grade inflammatory markers  . However, in contrast to metabolic syndrome patients, in the context of pregnancy, these parameters are normal requirements for healthy fetal development. Further comparisons between the microbial signatures of pregnancy and disease states such as the metabolic syndrome may highlight common as well as unique pathways and microbial involvement in each condition.
When starting to dissect the roles of the gut microbiota during pregnancy, the third-trimester microbiota was shown to cause increased weight gain, insulin resistance, and a greater inflammatory response compared to the first-trimester microbiota when transferred to germ-free mice  . These findings demonstrate that the microbial components actively contribute to changes in host immunology as well as metabolism. Gut microbiota have also been suggested to play a role in host weight gain during pregnancy via increased absorption of glucose and fatty acids, increased fasting-induced adipocyte factor secretion, induction of catabolic pathways, and stimulation of the immune system [8, 10] .
Since the microbial communities are greatly affected by the host diet and initial weight, the microbiota of pregnant women differ accordingly  . Overweight pregnant women exhibit significantly higher levels of gut Bacteroides and Staphylococcus than pregnant women of normal weight  . While most studies show significant alterations in gut microbiota during pregnancy, DiGiulio et al.  did not detect any changes in gut or vaginal microbiota composition including richness indexes during gestation.
Antibiotics administered during pregnancy were shown to affect the microbiome composition and diversity, as well as to promote weight gain in rodents  . It is especially intriguing to understand the consequences of the maternal microbiome composition during pregnancy on the offspring in terms of weight gain, immunity, and infant health  . Recently, the maternal microbiota was shown to shape the offspring’s immune system in terms of immune gene expression and numbers of innate cells  , and it was also hypothesized that microbial exposure during pregnancy may be of great importance for preventing allergic disease in the offspring 
Pregnancy Leads to Changes in the Vaginal Microbiota
The vaginal microbiome undergoes significant changes during pregnancy as well  , including a significant decrease in overall diversity, increased stability, and increased abundance of Lactobacillus species  . Lactobacillus species are lactic acid-producing bacteria that normally dominate the vaginal microbiota. Their increase during pregnancy correlates with a decrease in the vaginal pH, creating a barrier against pathogenic bacteria and viral infections, and an increase in vaginal secretions [17, 18] . The major changes in the vaginal microbial compositions occur in early pregnancy, while the communities at the later stages of pregnancy resemble those of the nonpregnant state  . In pregnancy, there is often a dominant Lactobacillus species, although the specific species varies according to the ethnic group [19, 20] . As expected, during the postpartum period, the vaginal microbiome gradually reverts to baseline characteristics, including a decrease in Lactobacillus species abundance, an increase in α diversity, and enrichment of bacteria associated with vaginosis, such as Actinobacteria .
Pregnancy Leads to Changes in the Oral Microbiota
The main change occurring in the oral microbiota during pregnancy is an increase in the microbial load. A study which compared the abundance of 7 common bacterial species in the oral cavity of nonpregnant women and women at different stages of pregnancy found that the total viable microbial counts were higher during pregnancy, as were levels of the pathogenic bacteria Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans, and Candida  .
It remains to be discovered how pregnancy leads to changes in the oral composition. There have also been several studies that found correlations between oral infections and pregnancy complications, further suggesting mechanisms connecting the oral microbiome with the state of pregnancy. One explanation for the increased oral microbial load may be the overall immune changes (suppression) during pregnancy.
Debate over the Placental Microbiota
Until recently, the fetoplacental unit was considered to be germ free, and the first exposure to microbes was assumed to occur during delivery. Accordingly, any signs of bacteria in the placenta or embryonic fluids were considered to be a result of contamination originating from the lower genital tract and posing a potential danger to the pregnancy  . However, with the recent leap in understanding the complexity of our microbiota and its important roles in healthy states, multiple studies have tried to examine whether a healthy placental microbiota exists. Several findings using both culture and metagenomic techniques suggest the presence of bacteria in the healthy placenta. First, bacteria have been cultured from placentas of healthy women without chorioamnionitis  . Furthermore, using whole-genome shotgun sequencing of samples from 320 subjects, Aagaard et al.  reported that the placenta contains a unique microbiome, somewhat resembling the oral one. This similarity between the oral and placental microbiota may be less surprising than it initially appears, since periodontal infections have previously been linked to an increased risk of pregnancy complications  . One hypothesis is that bacteria may pass from the oral cavity to the placenta through an unknown mechanism. In contrast, other studies highly doubt the presence of a placental microbiome, since even when such colonization is found, it is of extremely low biomass and may therefore represent contamination rather than a real phenomenon. It was in fact shown that low-biomass samples many times resemble contamination controls due to commercial reagents, as opposed to unique placental microbiota  . Such technical contaminations are especially prevalent when placental samples are received following natural births, when contaminations from the birth canal are likely.
As for amniotic fluid, a number of studies have shown the presence of microbes in healthy women using cultivation and PCR techniques  . Additionally, it was claimed that the bacterial populations detected in meconium might represent prenatal bacterial exposure. This may explain differences in microbial compositions found in meconium between infants of mothers who received probiotics during pregnancy compared to controls  .
The debate over the placental microbiome remains an intriguing, open question. If indeed future research will verify the existence of these populations, further studies should investigate the potential roles of microbiota in gestation and fetal development.
Pregnancy Complications Are Correlated with Dysbiosis and Infections
Pregnancy complications occur commonly (usually with unknown etiology), are observed in approximately 1 in every 6 pregnancies, and pose a serious risk for both maternal and fetal health and survival  . The most common pregnancy complications include preeclampsia, eclampsia, intrauterine growth restriction, and preterm birth. While some bacterial infections have been correlated with pregnancy complications, precise causal mechanisms are, as yet, unknown  . Several more recent studies have attempted to test for correlations between the microbial communities present during pregnancy and pregnancy complications  .
Two studies demonstrated a correlation between high α (within-individual) diversity in gut microbiota and preterm birth  , while a third study did not  . Additionally, certain vaginal communities in early pregnancy stages were significantly associated with an elevated rate of preterm birth  . These communities include higher abundances of Gardnerella and Ureaplasma , lower abundances of Lactobacillus sp., and higher α diversity. Additionally, the presence of certain vaginal fungi, even when asymptomatic, such as Candida albicans , is correlated with higher rates of preterm birth  .
Finally, oral infections have been reported as risk factors for pregnancy complications such as preterm birth [31, 32] . There are several theories regarding potential mechanisms for this effect, including the direct contact with microbiota via the placenta or more systemic inflammation and hormone production leading to preterm birth [4, 33, 34]
In this review, we discuss the dramatic changes observed in the microbiome composition at multiple sites (gut, vagina, oral cavity, and placenta) during healthy pregnancy and in complicated pregnancy. We try to present these changes in the context of the overall unique physiology during pregnancy. Pregnancy is a natural process of growth and development, in which many physiological changes occur, including changes in body composition, weight gain, hormonal levels, inflammation, and metabolic states. These multiple changes have distinct effects on the microbiota, which is altered accordingly. These changes are finely synchronized to ensure the healthy development of the embryo and fetus, and to meet the growing needs of the fetus up to delivery.
The debate over the sterile fetus has not been resolved. While some studies provide evidence for the presence of bacteria in the placenta, others contradict this, claiming that the bacteria observed were introduced by contamination. Additional studies are, therefore, required to resolve this issue. The possibility of early microbial colonization of the fetus suggests that from the very beginning of development, there may be reciprocal interactions between the developing host and microbiota, and that maternal microbial components may be transferred very early in development.
Future research will likely reveal the interactions and pathways linking the various physiological changes to the microbial changes, thereby explaining the significance of each change observed. Such studies may also have clinical relevance in terms of recommendations for antibiotic treatments, probiotics, and potential therapies for pregnancy complications.
The authors declare that no financial or other conflict of interest exists in relation to the contents of the chapter.
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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.
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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.
The neonatal immune system has its own reactivity, constraints, and challenges, which profoundly differ from the adult. Breast milk is most probably a key requirement both for optimal immune function in early life and for imprinting of the immune system for long-term immune health. Here, we will highlight how breast milk fills the needs and the gaps of the developing immune system and thereby represents the unbeatable way to prevent infectious disease. We will further focus on some factors in breast milk that we extensively studied and found to actively influence the immune trajectory and long-term immune health. More specifically, we will review how the presence of allergens in breast milk together with maternal milk cofactors such as TGF-β, vitamin A, and immunoglobulins influence mucosal immunity in early life with long-term effects on allergic disease susceptibility. We will see that, depending on the content and the nature of allergens in breast milk as well as the presence of immune modulators, very different outcomes are observed, ranging from protection to an increased allergy risk. We are starting to decipher the specific requirements for the neonatal immune system to function optimally. 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.
Genetic Programming, Environment, and Growth Render Infancy a Period of High Susceptibility to Infectious and Allergic Diseases
As compared to adulthood, early postnatal life is a period that is characterized by
rapid changes and multiple immune challenges. The neonates’ tissues are constantly
changing due to the growth process. Therefore, the immune system has to respect growth constraints in this period of life, and strong inflammatory immune responses, such as Th1 immune responses, should be avoided as they may lead to scars. Furthermore, tissue development requires the secretion of cytokines such as IL-33, which are also involved in the differentiation of Th2 immunity and allergic immune responses . Thus, in early life, tissue growth results in the predisposition to mount low inflammatory and pro-Th2 immune responses, which can explain the propensity of the neonate to be susceptible to infectious diseases (lack of strong inflammatory responses) and to allergic diseases (bias towards Th2 immune responses). Another characteristic of early life is that the immune system is developing, and gut microbiota, which are a major promotor of immune development [2, 3], are establishing. Genetically programmed immune system development and lack of microbiota in early life result in immune deficiency, where the levels of mucosal antibodies and immune cells in the tissues are much lower than in the adult [4, 5]. The neonate is also exposed to multiple
new antigens, which are found in the environment, are present in the diet, or are associated with gut microbiota colonization. With all these antigens being new to the immune system, immune memory in early life is much lower than in the adult [6–8]. Naïve immune responses are known to be slower and less intense than memory ones, which also contribute to higher susceptibility to infection. Finally, yet importantly, requirements for immune activation in early life are different from adulthood. Elegant studies demonstrated that neonates are fully able to mount a cytotoxic immune response towards a viral infection when exposed to a dose that is 10,000 times lower than in the adult . This has important implications for vaccination strategies and highlight the necessity of good hygiene especially in early life to prevent infection. Altogether, these observations indicate that for multiple reasons, early-life immune responses tend to be much less efficient in the fight against pathogens than in adulthood, and neonatal infection remains a common tragedy, with about 7 million cases and 700,000 deaths per year, accounting for 40% of mortality in those under 5 years of age . Immune homeostasis also requires immune tolerance, an immune response that is characterized by the absence of inflammation and largely mediated by regulatory T cells. The induction of regulatory immune responses towards innocuous antigens, such as self-antigens and exogenous antigens derived from the diet, is critical
to avoid autoimmune and allergic diseases, respectively. A pioneer study by Billingham et al.  demonstrated that immune tolerance is easily induced upon systemic antigen exposure in utero and early postnatal life, and 70 years later, studies are still ongoing to decipher the mechanisms underlying these characteristics
. However, the high prevalence of allergic diseases in infancy also indicates that mechanisms of tolerance towards exogenous innocuous antigens upon exposure through the skin and/or the mucosa are defective . Instead, Th2 immune responses are preferentially induced . As discussed here above, the liberation of mediators necessary for tissue growth contributes to this predisposition. The exposure to allergens through a skin barrier that is not fully developed can also favor allergic sensitization . Furthermore, there is strong evidence from mouse models that the neonatal period is refractory to oral tolerance induction [15–19], the process by which immune tolerance to an antigen is acquired following its exposure through the digestive tract . The spontaneous resolution of the majority of infant food allergies suggests a physiological maturation of the mechanisms of oral immune tolerance during the first years of life
[13, 21]. Gut colonization with microbiota plays certainly a major role in the setup of immune tolerance and the inhibition of allergic responses. This is clear from studies in germ-free mice, which have elevated levels of serum IgE (a hallmark of allergic immune responses) and are refractory to oral tolerance [22, 23].
Importantly, gut colonization with microbiota must occur in early postnatal life to efficiently regulate IgE immune responses , highlighting the concept of a window of opportunity to influence long-term immune responses . There is accumulating evidence that the specific composition and the metabolites produced by the gut microbiota are critical for the generation of regulatory T cells and oral tolerance to be fully effective [2, 3, 20, 25]. In summary, the immune system in early life is not the one of a “small adult” and neither immature nor tolerance prone. The neonatal immune system is different from the adult one with specific requirements for activation and regulation. In the absence of specific interventions, the infant is highly susceptible to
both infectious and allergic diseases. Infant vaccination to elicit memory immune
responses and maternal vaccination during pregnancy to increase the levels of circulating antibodies during the first months are possible intervention targets, which are successful to a certain extent, to decrease morbidity and mortality due to infectious diseases in early childhood [10, 26]. Changes in allergy prevention guidelines and earlier introduction of potential allergens in the diet are promising strategies for food allergy prevention, with some limitations as we will discuss later on. A natural intervention does also exist, i.e., breastfeeding. We can distinguish the influence of breastfeeding on short-term immune outcomes, i.e. its effects while the infant is breastfed, and on long-term immune outcomes, i.e., months to years after breastfeeding has ceased (Fig. 1).
Breast Milk as a Physiological and Critical Strategy to Prevent Infectious Diseases in the Short Term
For years, breast milk was considered mainly as a source of nutrients for the developing
child. The extensive observations that breastfeeding affords protection towards infectious diseases and could reduce the mortality rate of common infections by more than half have added another key role to breastfeeding . A recent meta-analysis concludes that scaling up breastfeeding to a near universal level could prevent 823,000 annual deaths in children younger than 5 years, mostly due to infectious disease. This protection relies in great part on the transfer of mucosal immunity through breast milk, which importantly relies on multiple, various, and adapting mechanisms (Fig. 1). Breast milk contains potent antibiotics, such as lactoferrin, lysozyme, and antimicrobial peptides [27, 28].
The presence of prebiotics such as human milk oligosaccharides  and probiotics
[30, 31] interferes with pathogenic bacterial expansion and invasion. Growth factors such as EGF and TGF-β contribute to maintain and repair potential pathogen-induced mucosal barrier breaks. Importantly, secretory IgA (SIgA) delivers personalized medicine. SIgA is present at high concentrations in colostrum (∼10 g/L) and at somewhat lower concentrations in mature breast milk (∼1 g/L). SIgA is specific for intestinal and respiratory pathogens in the maternal environment due to the selective migration of B cells originating from the maternal mu-cosa to the mammary gland . They are thus providing mucosal immunity against pathogens, which are specifically found in the environment of the child. By their non-antigen-specific part, maternal SIgA will also contribute to the establishment of infant gut microbiota and protect the child form pathogens .
Breast Milk as a Key Player in the Education of the Immune System and Long-Term Susceptibility to Immune-Dependent Diseases
In addition to the provision of passive immunity and compensation for neonatal
immune deficiencies, breast milk actively influences the development of the immune
system (Fig. 1). Thereby, breast milk can guide immune trajectories and long-term susceptibility to diseases, which have an immune component in their physiopathology. There is evidence that breastfeeding decreases the risk of obesity and metabolic complications associated with obesity, such as type 2 diabetes . However, there is lack of consistency regarding the possibility of long-term prevention of infectious diseases  and allergy [34, 36–40] by breastfeeding. Despite this, there are accumulating data indicating that breast milk has the potential to prevent these diseases by at least two major ways: by shaping the infant microbiota and by exposing the neonatal immune system to microbial antigens and allergens. The gut microbiota, and more specifically the microbiota-derived metabolites, are key for influencing the balance between health and disease . In particular, gut microbiota influences susceptibility to infection and efficacy of vaccination , as well as susceptibility to allergy as we recently reviewed .
By the presence of prebiotics, probiotics, and antigen-specific and non-antigenspecific
antimicrobial compounds, breastfeeding plays a major role in the initial seeding of the infant gut microbiota and in its constant evolution in postnatal life . The major event being known to affect microbiota composition is cessation of breastfeeding . Therefore, we can speculate that the various concentrations of microbiota-shaping compounds in each mother’s breast milk will contribute to the heterogeneity in gut microbiota found in children and thereby influence their susceptibility to disease . Finding which microbiota is best adapted to each environment and how to influence microbiota-shaping molecules in breast milk will open up new avenues for long-term immune health.
Breast milk also contains exogenous antigens, and our research in the last decade has been aimed at deciphering the long-term outcomes of the presence of allergens in breast milk on allergy susceptibility in the offspring. Recently, we expanded this research to infectious disease prevention. In the last part of this review, we will synthesize our main findings related to the presence of exogenous antigens in breast milk and discuss their implications for long-term immune health.
Allergy is a rising public health issue, with respiratory and food allergy affecting up to 20% of children (the Global Asthma Report 2018; http://
globalasthmareport.org/) . After an era of avoidance, there has been a paradigm
shift, and new strategies for allergy prevention aim now at inducing tolerance
by antigen exposure. Following impressive results in large randomized clinical trials, the early introduction of eggs and peanuts in the diet is now recommended for food allergy prevention . However, recent studies also demonstrated a significant proportion of infants already have egg sensitization and clinical reactivity (including anaphylaxis) prior to the first introduction of eggs into their solid food diet [13, 44–46]. This underscores the necessity to identify earlier and safer ways to promote oral tolerance development to food allergens in young infants, which may be particularly challenging knowing the refractoriness of early life to oral tolerance. We proposed the hypothesis that early oral
exposure to exogenous allergens in the presence of maternal milk immune modulators would alleviate oral tolerance induction in early life. Our experiments in mouse experimental settings demonstrated the key findings: 1. Mice exposed to only low amounts (ng/mL) of the egg-derived allergen ovalbumin (OVA) through breastfeeding are protected from allergic reactions to OVA when reexposed in adulthood via the oral (as a model of food allergy)  or respiratory route (as a model for respiratory allergy) [48–50]. This echoes to observations referenced here above indicating that the early-life immune system reacts to very low amounts of antigens. Importantly, other reports have shown the presence of dietary antigens in breast milk in the same range at very low concentrations (ng/mL), such as cow milk β-lactoglobulin [51–53], peanut allergens
(Ara h 1 and Ara h 2) [54, 55], or wheat antigen (gliadin) . By comparison, it
is worth noting that allergen levels in cow’s milk are found in the range of mg/ mL. Thus, through breast milk, infants are exposed to a wide variety of dietary allergens at low concentrations while formula-fed infants are only exposed to cow milk-derived allergens and at much higher concentrations (unless hydrolyzed formulas are used). We can speculate this will lead to very different outcomes in terms of oral tolerance induction and allergy prevention in the offspring.
Our recent observation in a human birth cohort demonstrated that the risk of egg allergy in children was reduced by a factor of 4 at 2.5 years when comparing children exposed to breast milk with or without egg antigen . To fully confirm that OVA in breast milk is responsible for a decreased egg allergy risk in children, further randomized controlled trials will need to be conducted. The next steps will be to identify maternal interventions leading to the consistent presence of OVA in breast milk in order to move towards a successful prevention of egg allergy by breastfeeding.
2. Successful oral tolerance and allergy prevention upon OVA exposure through breast milk required the concomitant presence of maternal immunomodulatoryfactors. We identified TGF-β, OVA-specific IgG, and vitamin A to stimulate oral tolerance induction in the offspring. Each cofactor was shown to act by different mechanisms of action. TGF-β promoted the induction of OVAspecific Th1 cells, which counteract allergic Th2 responses [48, 58]. Vitamin A accelerated gut epithelium maturation resulting in a stronger barrier in the first week of life . It also promoted the expression of RALDH in small-intestine dendritic cells, which was associated with their increased efficiency at activating T lymphocytes . Maternal vitamin A supplementation resulted in the possibility to induce oral tolerance to OVA in breast milk in the offspring from birth, which otherwise is only efficient from the third week of life . OVAspecific IgG in breast milk was necessary for a protected transfer of OVA though the neonatal gut barrier and the induction of a prolonged and strong protection from allergy, which was found to be mediated by FoxP3 Tregs . Recently, another group confirmed these findings and showed that OVA-specific IgG also promoted antigen presentation by neonatal dendritic cells . In other words, early life is characterized by a relative lack of TGF-β in mucosal tissue, a physiological deficiency in vitamin A, and 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 cofactors, which will affect gut epithelium barrier integrity, and antigen transfer and presentation for successful regulatory immune response induction.
This will result in a long-term low risk for allergic disease as we showed for egg allergy both in an experimental mouse model and in a human birth cohort. Altogether, these data suggest that, according to the levels of immune modulators in breast milk, oral tolerance to dietary antigens in breast milk will be induced with more or less efficiency, which will condition long-term susceptibility to allergy.
3. Unexpectedly, we found allergens from respiratory sources such as from the house dust mites Dermatophagoides pteronyssinus (Der p) and Blomia tropicalis in breast milk in similar amounts as dietary antigen [61, 62]. Since we detected Der p 1 in the 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. 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, there was a positive association between allergic sensitization and respiratory allergies in children and the presence of Der p 1 levels in breast milk . This observation stresses that not all the antigens in breast milk induce oral tolerance, even though they reach the gut mucosa together with breast milk-tolerogenic factors. Our most recent findings further showed that Der p allergen in maternal milk abolished the capacity of neonatal mice to mount oral tolerance to bystander OVA egg antigen, which resulted in an increased risk of egg food allergy in the long term . Importantly, human data showed heterogeneity in breast milk content in Der p and OVA. Based on the presence of Der p and/or OVA in breast milk, we could
identify groups of lactating mothers which mirror the ones found in mice responsible for the different egg allergy risk . These observations stress the need to identify how to counteract deleterious actions of some allergens in breast milk, such as those derived from house dust mite allergens, which prime for allergic sensitization to themselves and break oral tolerance induction to bystander ones.
We have started to identify some potential targets as we showed that protease from
Der p allergens are key players for the induction of gut Th2 mucosal immune imbalance
in mice breastfed by mothers inhaling Der p . Ongoing research is aimed at identifying how to modulate Der p levels and their enzymatic activity in breast milk and/or in the breastfed child. Ultimately, this should contribute to ensure food allergy prevention in children.
4. Based on the findings that house dust mite allergens in breast milk could prime immune reactivity in the long term, we proposed that novel strategies of early life prevention of infectious disease may take advantage of the possibility to stimulate antigen-specific immune responses through breast milk. Microbial antigen transfer through breast milk would be a way to naturally vaccinate the infant [26, 66]. Some evidence in the literature supports this hypothesis, such as the observation that maternal HIV infection of noninfected breastfed children is associated with infant stimulation of IgG and IgA secretion in their gut mucosa  and HIV-specific interferon-γ-secreting PBMC are found in 50% of cases . Recently, we addressed the original hypothesis that the presence of malaria antigen in breast milk may stimulate antimalarial immune defenses and reduce the malaria risk in breastfed infants . As a first critical step to address this hypothesis, we investigated whether Plasmodium falciparum histidine- rich protein 2 (pHRP-2) and lactate dehydrogenase (pLDH) are detectable in breast milk of mothers from Uganda, a country with endemic malaria
. We found that 15% of breast milk samples from mothers with asymptomatic
malaria do contain malaria antigens. Our preliminary data indicate that blood levels of malaria antigens determine their levels in breast milk. These landmark findings may have significant implications for susceptibility to malaria in children from endemic countries since malaria antigens in breast milk may strongly influence the immune responses to natural malaria infections and to malaria vaccines in breastfed children .
Concluding Remarks and Perspectives
The way breast milk composition constantly adapts to the needs of each infant in each setting is fascinating. This results in a major success for the prevention of many infectious diseases in breastfed infants. There is also evidence that breastfeeding could have long-term protective effects on infections, even after breastfeeding has ceased, and this may open new avenues for infant vaccination. Unfortunately, and maybe unexpectedly, breastfeeding does not provide a consistent protection from allergy. A hypothesis behind this lack of protection may be that the lifestyle of modern societies has resulted in changes in breast milk composition, which is not suitable anymore for allergy prevention. Research conducted to identify which factors in breast milk condition protection versus susceptibility is providing new clues, which ultimately will result in the protection of breastfed children from allergy.
Valérie Verhasselt has no conflict of interest with regard to the writing of this chapter.
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Research suggests that HMOs support immunity in 4 main ways, as follows:
- Promoting the growth of beneficial gut bacteria1,2
- Preventing pathogens from binding to the intestinal wall, which reduces their ability to infect the infant1,3
- Assisting gut barrier function, which can help prevent pathogen binding to the intestinal cells1,3
- Directly modulating the immune system, which helps in educating the developing immune system1,4
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- Jantscher-Krenn E, Bode L. Human milk oligosaccharides and their potential benefits for the breast-fed neonate. Minerva Pediatr. 2012 Feb;64(1):83-99.
- Eiwegger T, Stahl B, Schmitt J, Boehm G, Gerstmayr M, Pichler J et al. Human milk-derived oligosaccharides and plant-derived oligosaccharides stimulate cytokine production of cord blood T-cells in vitro. Pediatr Res. 2004 Oct;56(4):536-40.
Inflammatory bowel disease (IBD) currently affects 1 in 200 people in the developed countries, and in recent decades the incidence has also been..