NNIW94 - Milk, Mucosal Immunity & the Microbiome: Impact on the Neonate

The neonatal immune system has its own reactivity, constraints, and challenges, which pro
foundly 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 in-creased 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.

Secretory immunoglobulin A (SIgA) is intimately involved in the transfer of maternal immu-
nity to the newborn breastfed infant. Recent research demonstrates the significance of SIgA in the initial development of the newborn’s microbiota and in the establishment of a tolero-genic immunologic disposition towards nonpathogenic organisms and environmental an-tigens. SIgA has long been known to prevent pathogen binding to the host epithelium through immune exclusion involving numerous mechanisms. This process primarily involves T-cell-dependent, somatically hypermutated monoclonal antibodies with high specificity towards pathogen surface antigens, and the success of the immune response is dependent upon the specific antigen recognition. Whereas this role is important, there is an alternate, dual role for SIgA in the health of the host – protection and promotion of commensal colo-nization and maintenance of homeostatic immunity. This latter role is primarily dependent upon N- and O-glycan moieties lining the secretory component and heavy chain of the SIgA dimer, with interactions independent of immunoglobulin specificity. These SIgA molecules are nonspecific polyclonal antibodies generated from plasma cells activated by dendritic cell sampling of luminal contents in the absence of inflammation. Breast milk is the primary sup-ply of such polyclonal polyreactive SIgA in the initial stages of neonatal colonization, and it provides vital pathogen resistance while promoting colonization of commensal microbiota.

Osteopontin (OPN) is an acidic phosphorylated glycoprotein involved in a wide range of
biological activities, such as cell proliferation and differentiation, as well as immunomodula-tory functions. OPN contains integrin and CD44 binding sites, and it exerts its multiple func-tions by binding to its receptors on the cell membrane to trigger various cellular signaling pathways. It is generated by a variety of cell types, including epithelial cells and immune cells. OPN appears in most body fluids, such as milk and blood, and is present at a high con-centration in human milk but not in bovine milk. Milk OPN is relatively resistant to digestion, and orally ingested OPN can enter the circulatory system. Milk OPN may, therefore, play essential roles in the development in early life. The impact of milk OPN on development has been investigated using cell models, animal models, and randomized clinical trials. Recent OPN studies strongly suggest that milk OPN plays important roles in intestinal proliferation and maturation, brain myelination, and neurodevelopment, as well as immune development.

Newborn infants, especially those born preterm, are at risk of infections in early life. In pre-
term infants, necrotizing enterocolitis (NEC), a devastating inflammatory gut condition, and late-onset sepsis (LOS) are important causes of serious morbidity and are the commonest reasons for death after the first week of life. Fresh breast milk from the infant’s mother re-duces the risks of these serious pathologies in a dose-dependent fashion. Considerable effort has been expended to better understand which specific components of human milk are likely to exert the greatest functional benefits, particularly those that have immune modulatory or anti-infectious properties. Lactoferrin is a whey glycoprotein present in especially high concentrations in colostrum and early milk. Studies show that lactoferrin impacts on immune function and, through a multitude of mechanisms, reduces the risk of viral, fungal, and bac-terial infections. Supplemental enteral bovine lactoferrin has been tested in a series of randomized clinical trials, many of which suggested important reductions in LOS in preterm or low-birth-weight infants. However, the largest trial to date – the Enteral Lactoferrin in Neonates (ELFIN) trial – recruited 2,203 infants and failed to show any significant reductions in LOS or NEC. Challenges in conducting clinical research and the translational relevance of these studies for clinical practice will be considered.

Dietary supplementation with bovine milk fat globule membrane (MFGM) concentrates has
recently emerged as a possible means to improve the health of infants and young children, or defense against infections. We identified 5 double-blind, randomized, controlled trials (DBRCT) exploring the effects of supplementing the diet of infants and children with bovine MFGM concentrates on infections. We reviewed 3 studies which found a protective effect against infections at different ages during infancy and early childhood. Two of them have reported effects on the metabolome, and 1 study also on the microbiome and lipidome. MFGM supplementation had moderate, albeit interesting, effects on the oral and fecal  microbiome, fecal and serum/plasma metabolome, and serum and erythrocyte membrane lipidome, which also are reviewed. We conclude that studies on MFGM supplementation during infancy and childhood indicate positive effects on the defense against infections  and other outcomes, but more high-quality DBRCTs with well-defined MFGM fractions and outcome measures are needed before firm conclusions can be drawn.

Norovirus infections belong to the most common causes of human gastroenteritis world-
wide, and epidemic outbreaks are responsible for hundreds of thousands deaths annually. Strikingly, no antiviral treatment is available due to the difficulty in cultivating virions or in generating a vaccine, and due to the fact that their infection mechanisms are poorly under-stood. However, there is consent that noroviruses bind to histo-blood group antigens  (HBGAs) on their way through the digestive tract. The HBGA profiles vary individually, making people more or less susceptible to different norovirus strains. In our current work, we tried to decipher the HBGA specificity of the most prevalent and clinically relevant norovirus GII.4 subfamily (Sydney 2012, JX459908) and its preferences for human milk oligosaccharides (HMOs) as potential anti-infectives. The structural evidence provided can explain at the molecular level why individuals with certain blood groups are at higher risk of infection, and how these infections may be prevented and treated by application of food additives. A cen-tral finding was that low-affinity binding of HMOs is surpassed by high-avidity binding of multivalent oligo and polyfucoses as found in algal polysaccharides (fucoidans). Insight into structural details of fucoidans and their impact on noroviral-blocking efficiency is provided and discussed.

Oligosaccharides are a group of complex glycans that are present in the milk of most mam-
mals. However, human milk is unique as the concentrations of human milk oligosaccharides (HMOs) are much higher than those of other mammals, and their structural composition is more complex and varies between women. These observations prompt several questions: (i) Why are humans unique when it comes to milk oligosaccharides? (ii) Which maternal  genetic and environmental factors drive the interindividual variation in HMO composition? (iii) What are the short- and long-term health benefits for the infant – and potentially also the mother? The combination of genome-wide association studies, milk transcriptomics, in vitro gene editing, and in silico pathway modeling allows us to reconstruct HMO biosyn-thetic pathways. Using new data mining approaches and leveraging samples and metadata from large mother-infant cohorts enable us to identify associations between HMO composi-tion and infant and maternal health outcomes. Suitable preclinical models and clinical inter-vention studies allow us to corroborate the established associations for causal relationships and test for in vivo efficacy in humans. Knowledge generated from these different approaches will help us establish true structure-function relationships and provide the rigorous evidence required to improve infant health and development.

Necrotizing enterocolitis (NEC) is a devastating inflammatory condition of the intestine,
which affects premature infants and causes untold damage. Its pathogenesis has to do with how colonizing bacteria interact with the immature newborn intestine. An immature innate immune response with increased TLR-4 on the cell surface and increased signaling molecules, such as NF-κB, can cause excessive inflammation. This is in conjunction with a decrease in the appearance of regulatory molecules which effect the control of innate re-sponses. This condition is so devastating that it must be prevented and not treated. Fortu-nately, breast milk and probiotics can affect the condition leading to reduced inflammation. How does this effect work? We have shown that breast milk tryptophan and Bifidobacterium infantis result in a metabolite (indole-3-lactic acid) response, which is anti-inflammatory via inhibition of the aryl hydrocarbon receptor transcription factor which stimulates an IL-8 response. We have also shown that breast milk complex carbohydrates interacting with Bacteroides fragilis can cause short-chain fatty acids which exert anti- inflammatory effects on the newborn intestine. These breast milk metabolites could  help prevent NEC if shown to be effective clinically.

Contrary to common belief, the human neonate is often born with a nonsterile gastrointes-
tinal tract, suggesting fetal colonization. This has been substantiated by numerous studies showing microbes in meconium. Shortly after birth, the infant is further colonized by mi-crobes that reflect the diet, which in the newborn consists of milk. When fed milk from the mother’s breast, the infant derives a set of live microbes that have the capability of coloniz-ing the gastrointestinal tract. This milk also provides a source of enzymes, such as lipase and alkaline phosphatase. Milk also provides a multitude of proteins, microRNAs, and other components that putatively interact with the host intestinal innate mucosal immune system to control infection, modulate intestinal inflammation, and provide signaling to distal sites for the development of adaptive immunity as well as growth and communication with the cen-tral nervous system. Colostrum differs from transitional and mature milk by being particu-larly rich in immunoglobulins as well as leukocytes. Live microbes found in fresh mother’s milk may be personalized for her infant and thus provide an impetus for either ensuring delivery of this personalized milk to the infant or, if that is not possible, to develop the  means to personalize donor milk or formula.

Human milk microbes play an important role in infant health and disease. Emerging evidence shows that human milk viruses are also transmitted from the mother to the infant via breast-feeding. These viruses include eukaryotic viruses, bacterium-infecting viruses called bacteriophages, and other viral particles. Human milk viruses are instrumental in shaping the  infant gut virome and microbiome. Eukaryotic DNA and RNA viruses contribute to pathogenic challenges and protection. Bacteriophages have the ability to kill bacteria or supply them with potentially beneficial gene functions, thereby shaping the microbiome. The early infant virome is dominated by bacteriophages that likely contribute to a highly dynamic microbiome in the early life. There is a critical window of early childhood growth with rapid maturation of metabolic, endocrine, neural, and immune pathways. The colonization of  microbes in the infant body during this time plays an important role in the establishment and maturation of these pathways. The virome transmitted via breastfeeding may also be particularly important at these critical time points of immune development. More longitudinal studies of mother-infant pairs will help to better define the human milk virome and their functional impact on the development of the growing infant.

At the beginning of the 21st century, some pioneer studies provided evidence of the exis-
tence of a site-specific human milk microbiota. Hygienically collected milk samples from healthy women contain a relatively low bacterial load, which consist mostly of Staphylococ-cus, Streptococcus, lactic acid bacteria, and other gram-positive bacteria (Corynebacterium, Propionibacterium, and Bifidobacterium). DNA from strict anaerobic bacteria is also detected in human milk samples. The origin of human milk bacteria still remains largely unknown. Although the infant’s oral cavity and maternal skin may provide microbes to milk, selected bacteria of the maternal digestive microbiota may access the mammary glands through oral- and enteromammary pathways involving interactions with immune cells. In addition, when milk is collected using external devices, such as breast pumps, some microorganisms may arise from unhygienic handling as well as from the water used to clean and rinse the devices, for example. The human milk microbiota has a wide spectrum of potential uses. Most of them have been focused on the infant (including the preterm ones), but some bac-terial strains present in human milk have also a big potential to be used to improve the mother’s health, mainly through the prevention or treatment of infectious mastitis during

Breastfeeding confers the infant short- and long-term health benefits and significantly modulates the developing infant gut microbiome. A specific human milk microbiome has relatively recently been discovered, but its origin remains poorly understood. Data from experimental and clinical studies suggest that the bacteria in milk may originate in the maternal gut and be transported via a specific enteromammary pathway, the details of which have not been elucidated yet. The milk microbiome is affected by the maternal metabolic state, antibiotic use, as well as the mode of delivery. We are only in the initial stages of understanding the biological function of the milk microbiome and its potential contribution to infant gut colonization. Several clinical studies indicate, however, that despite considerable differences in the overall composition of the milk and infant gut microbiomes, specific bacteria are detectable both in human milk and infant feces, and that the bacteria in milk are a source of microbes colonizing the neonatal gut. If the microbes in human milk are discovered  to contribute to the beneficial effects of breastfeeding, modulating or mimicking the milk microbiome may provide a novel means of improving child health.

The neonatal immune system has its own reactivity, constraints, and challenges, which pro
foundly 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 in-creased 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.

The human mammary gland is an integral effector component of the common mucosal im-
mune system. However, from physiological and immunological aspects, it displays several unique features not shared by other mucosal sites. The development, maturation, and activ-ity of the mammary gland exhibits a strong hormonal dependence. Furthermore, in com-parison 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.

The development of the mammary glands and the process of lactation is an integral com-
ponent of mammalian evolution, and suckling has been essential for the survival of the neo-nates of most mammalian species. The colostrum and milk, the major products of lactation, contain a wealth of biologically active products derived from the immunologic and micro-biological experiences in the maternal circulation and in the maternal mucosal surfaces. These include major immunoglobulin isotypes in the maternal circulation, secretory IgA, a variety of soluble proteins, casein, nutritional components, hormones, a large number of cellular elements and their secreted functional products (cytokines and chemokines), sev-eral peptides, lipids, polysaccharides and oligosaccharides, and a diverse spectrum of micro-organisms. During the past few decades, significant new information has become available about the evolutionary biology of mammalian lactation, the functional characterization of antibody and cellular immunologic products, the role of oligosaccharides and other proteins and peptides, and about the distribution and biologic functions of the microbiome observed in human products of lactation. This workshop explores this information in some detail in a series of presentations. A brief overview of the earlier observations on the immunologic as-pects of lactation is presented here, and detailed reviews of more recent observations are reported in subsequent presentations in this workshop.

Lactation is a defining characteristic of all mammals, and, indeed, mammals draw their name from mammae, or mammary glands. The evolution of mammary glands has been the subject of debate since Charles Darwin. The purpose of this brief review is not to examine all past theories of mammary evolution but to consider the evolution of the mammary gland in rela-tion to (1) modern paleobiology, giving special attention to the mammaliaforms which had many mammalian features, including delayed tooth development suggestive of milk intake. (2) Comparative aspects of mammary development in monotremes, marsupials, and euthe-rians, which reveal the close developmental relation of mammary glands to other skin glands and hair follicles. (3) The evolution of caseins, which are now known to derive from secre-tory calcium-binding phosphoproteins, which have a long history in regulating biomineral-ization. (4) The evolution of lipid secretion, and especially the evolutionary incorporation of immune system components (such as xanthine oxidoreductase) into the fat globule mem-brane. (5) The evolution of lactose synthesis, and especially the synthesis of the wide array of oligosaccharides found in some milks, including monotremes, marsupials, caniform carnivores and humans.