Article

Immunology of Human Milk and Lactation: Historical Overview

19.05.2020

40 min read

Summary

The information summarized in the various comprehensive presentations in this workshop represented a diverse spectrum of historical, evolutionary, and functional aspects of mammalian lactation and the process of breastfeeding. This workshop was dedicated to Prof. Lars A. Hanson (MD, PhD) for his outstanding contributions to the understanding of the biology of milk and the dissemination of knowledge on breastfeeding to advance current practices of breastfeeding in the contemporary human society worldwide. The dedication ceremony was followed by scientific presentations in session I of the workshop with the keynote addressed by Olav T. Oftedal. Oftedal provided an elegant perspective of the evolution of lactation in different mammalian species. Based on studies on synapsids (ancestral to mammals, which appear to have diverged from sauropsids [ancestral to crocodiles, lizards, and birds]), he proposed that lactation may have first evolved as a source of moisture and antimicrobial compounds for parchment-shelled eggs, followed by the evolution of some skin secretions, which eventually became milk. It was suggested that among basal animals (monotremes), each mammary gland develops as a triad in association with a hair follicle and sebaceous gland as a mammopilo- sebaceous unit (MPSU).

In other mammalian species, such as marsupials, there is a similar triad, but the hair follicles are shed during development. In the diverse group of eutherian mammals, some show no association with the mammary hair, while others, such as the horse, develop as MPSU with mammary hair and sebaceous glands present in the mammary gland.

The MPSU also bears significant resemblance to apocrine glands (APSU), suggesting that mammary glands may have also evolved from an APSU-type structure. Recent studies have suggested that most constituents of mammalian milk are unique and found only in mammary secretions. He proposed that if a milk protein occurs in the milk of monotremes, marsupials, and eutherians, the major mammalian taxa, then the protein must have evolved before the groups diverged and are inherited from the ancestral taxa. These observations have provided unique and new insights into the genetic origin and functions of specific mammary constituents in the products of lactation. Oftedal briefly alluded to the 4 primary types of caseins, members of the secretory calcium-binding phosphoproteins (SCPPs), as an evolutionary challenge because of their diversity and the large size of the micelles in milk. These proteins have an ancient history in the evolution of mineralized tissues. Based on related SCPP genes, caseins may have evolved as protolacteal secretion that delivered calcium to eggs. Finally, his presentation discussed briefly the evolution of the milk fat globule membrane, lactose,
and other neutral and acidic oligosaccharides. The next presentation provided a brief historical overview of the immunology of milk and mammalian lactation. This presentation served as an introduction to the subsequent specific topics discussed in this workshop. Mother’s milk has been considered a complete food for the infant from times immemorial, and it has been associated with unique healing powers and beneficial effects.

These include cure for insomnia, loss of appetite, ascites, piles, skin disorders, sexual dysfunction, muscle weakness, contraception, and prevention of cancer and infections. Breastfeeding developed a spiritual and religious importance in the Middle Ages in Europe, as evidenced by the deep faith and respect for Nursing Madonna, Virgin Mary, and the breastfed Jesus. The modern history of breast milk immunology can be traced to a publication by Paul Ehrlich as early as 1892 and subsequent demonstration of specific maternal antibody transport to the colostrum and milk. The immunologic composition of human milk and its biologic linkage to mucosa-associated lymphoid tissue was initially recognized by Gugler and Von Muralt, and by Lars Hanson. These elegant studies were followed by the identification of secretory IgA in human external secretions by Chodirkar and Tomasi, and Bienenstock and Tomasi, and in the human milk by Hanson and Johansson. Subsequent studies by Beer and Bellingham, Ogra et al., Mohr, and Okamoto and others identified several cellular and soluble immunologic factors in the human milk and their transport to the suckling neonate via the process of breastfeeding. It is now known that human colostrum and milk contains a wealth of immunologically active products derived from the innate and adoptive immunologic, microbiologic, dietary, and other maternal experiences in the maternal mucosal surfaces, especially the gut, and the maternal circulation. This historical review was dedicated to the memory of Dr. S.S. Ogra, the principal investigator of most milk related research carried out in her laboratory in the early 1970s and 1980s in the School of Medicine at the University at Buffalo. This presentation briefly reviewed lactation performance and the presence and function of diverse soluble elements detected in mammalian colostrum and milk to date. These included: secretory IgA and other immunoglobulin isotypes, anti secretory factor, soluble CD14, and soluble Toll-like receptors, as well as several cytokines and lymphokines. It also introduced the role of colostrum- and milk-associated cellular components, such as leukocytes, macrophages, epithelial cells, stem cells, and T lymphocytes, and cell-mediated immune responses. This overview also summarized earlier studies on the transfer of tuberculin-specific maternal cellular immunity to the neonate via breastfeeding and more recent investigations on the transfer of maternal cellular immunity and engulfment of maternal
DNA via the transfer of leukocytes and stem cells. Finally, the risks and benefits of the colostrum and milk to the neonate and the developing infant were briefly considered here. Detailed discussion of the issues identified here follows in subsequent presentations in this session and sessions II and III of this workshop. Jiri Mestecky reviewed in some detail the evidence for the existence of mucosa- associated lymphoid tissue and common mucosal immune sites for effective immunization in the mucosal system, and the importance of mammary glands as an integral component of the common mucosal immune system. He discussed recent studies on the structure, biologic activities, and the spectrum of antibodies of the IgA isotype specific for microbial, dietary, and other environmental antigens and macromolecules in the colostrum and milk. He concluded his presentation by identifying possible directions for future investigations in the immunobiology of the mammary gland and lactation. These include the routes for the most effective induction of IgA responses in milk, the identification of phenotypes of B lymphocytes that express homing receptors for the
mammary gland, and the determination of effective timing for maternal immunization to provide optimal levels of protective immune reactivity in the colostrum and the milk for the neonate.

Helena Tlaskalová-Hogenová, Miloslav Kverka, and Jiří Hrdý introduced the wide spectrum of immunomodulatory components present in human milk and colostrum, including those of innate and adaptive immunity, and factors influencing the composition and colonization of newborn gut microbiota. They discussed the nature of autoantibodies and the spectrum of newly detected cytokines and lymphokines in human milk. The presentation was completed with an overview of different cellular components of and cytokine gene expression on colostral cells in healthy and allergic mothers. Studies carried out to date have identified over 35 cytokines in the colostrum and milk, and some of them have been identified for the first time in human milk. Their possible functions include the development of intestinal lymphoid tissue, functional development of the gut structure, angiogenesis, central and enteric nervous system development, and establishment of immunologic homeostasis in the mammary gland as well as in the breastfeeding neonate.

Valerie Verhasselt discussed the influence of breastfeeding on the development
of immunologic health in the breastfed neonate and infant. She began with the examination of the unique specificities of the neonatal immune system, including unique limitations to the development of immune responses after postnatal exposure to environmental antigens. She reviewed the role of TGF-β, vitamin A, several environmental allergens, and specific antibodies in the context of early life and long-term allergic disease susceptibility. Based on controlled epidemiological data and several experimental studies, she proposed that early-life oral exposure to allergens does not induce tolerance but may prime for allergic responses. It has been suggested that non breastfed infants are exposed to only few allergens, but in very high concentrations, such as β-lactoglobulins. On the other hand, breastfed infants are exposed to a wide variety of allergens in the maternal milk and colostrum but in extremely low concentrations. Additionally, breast milk provides the infant with significant amounts of TGF-β, vitamin A, and other cofactors which affect the integrity of
the barrier of gut epithelium and regulate antigen transfer and presentation to the mucosa-associated lymphoid tissue. As a result, breastfeeding results in a low risk for allergic disorders in the long term. Such conclusions are supported by recent studies in human birth cohorts and by studies carried out in her laboratories with the induction of egg allergy in experimental (mouse) animal models.

Carine Blanchard made the final presentation in session I. Due to certain unavoidable
circumstances, she could not provide a full-length manuscript of her presentation. Therefore, a more detailed summary of her talk is presented here. Her presentation focused on the immunologic evaluation of human milk oligosaccharides (HMO) with respect to disease expression in the neonate after exposure to allergens and infectious agents. She discussed in some detail the enzyme fucosyltransferase (FUT) and its genotypes FUT2 and FUT3. These enzymes are expressed on blood groups ABH and Lewis, intestinal mucosa, and other human body fluids. Recent studies have suggested that the early trajectory of neonatal microbial colonization is significantly influenced by the number of environmental factors. These include gestational age of the neonate, method of delivery, use of antibiotics, geographic location of birth, genetics, maternal stage of lactation, maternal diet, and specific immunologic components delivered to
the neonate via breastfeeding. These factors appear to determine the outcome of
colonization and the composition of the neonatal microbiome as healthy or aberrant
based on the metabolites generated by the microbiome. An aberrant microbiome
has been associated with the development of sustained inflammation, induction of asthma, atopy, obesity, inflammatory bowel disease, and other disease states. Employing FUT2 and FUT3 genotypes as proxy for the HMOs, several ongoing investigations have provided important information on the role of HMOs in human milk and colostrum;
1. 2′-Fucosylated HMOs in human milk alleviate the negative effects of cesarean section on infant gut microbiota.
2. HMOs in infant formula significantly improve the outcome of infections in infants.
3. Maternal FUT2- and FUT3-positive status is related to a lower risk of respiratory infections during the first 6 months of neonatal life.
4. Maternal HMOs are associated with the prevention of colonization and growth of the pathogenic microbiome.
5. Elevated serum IgE levels are associated with the absence of gut microbiota in experimental models of infection.

She summarized the results of the LIFE child cohort studies from Leipzig (Germany) and Bangkok (Thailand) and other investigations involving cholera toxin/ovalbumin-induced food allergy in experimental animal models These studies have also demonstrated that the use of HMO and FUT2/FUT3 genotypes in infant feeding is associated with significantly decreased allergic sensitization. The mechanisms underlying such protection appear to be related to the modulation of regulatory T-cell function by HMOs and independent of the regular prebiotic effects associated with milk oligosaccharides and other soluble products in the milk.

Based on the information summarized above from the presentations in session I of the workshop, it is apparent that we have come a long way in understanding the evolutionary biology of mammalian lactation, the presence and role of specific innate and adaptive immunologic mechanisms associated with human milk, and the impact of breastfeeding on the systemic and mucosal immunologic development in the neonate. Recent information about the microbiology of the milk and lactation and its influence on gut colonization, presented in session II, and studies about the role of HMOs and other soluble components of the colostrum and milk, presented in session III, are summarized next by W. Allan Walker and Bo Lönnerdal, respectively. It is gratifying to note the wealth of new information presented in this workshop, and it is hoped to generate further interest in exploring many unanswered questions related to the mammary glands and lactation and its impact on the neonate.

Author(s): Pearay L. Ogra

Immunology of Human Milk and Lactation: Historical Overview (full doc)

Immunology of Human Milk and Lactation: Historical Overview (abs doc)

Abstract

The development of the mammary glands and the process of lactation is an integral component of mammalian evolution, and suckling has been essential for the survival of the neonates 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 microbiological 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), several peptides, lipids, polysaccharides and oligosaccharides, and a diverse spectrum of microorganisms. 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 aspects of lactation is presented here, and detailed reviews of more recent observations are reported in subsequent presentations in this workshop.

Introduction

These two quotes reflect the depth of human interest in breastfeeding for over 2,500 years [1, 2]. Milk and other lactational products of all mammals, including humans, have been associated with unique healing powers and beneficial effects. Mother’s milk has been considered a complete food for the infant in many ancient scriptures. With the evolution of agricultural civilization, long before the development of commercial milk formula foods, milks from buffalo, cow, sheep, camel, donkey, horse, elephant, and goat were highly recommended for the treatment of insomnia, loss of appetite, ascites, piles, infestations by worms, skin disorders, muscle weakness, dysfunctions of sexual activity, and a large variety of other human ailments [3]. In addition, breastfeeding also developed a religious and spiritual importance in the Middle Ages in Europe, as evidenced by the deep faith and respect for Nursing Madonna, the Virgin Mary
breastfeeding the infant Jesus [4]. During the upsurge of Marian theology in Europe, milk was viewed as the processed blood, and the milk of the Virgin paralleled the role of the blood of Christ [5]. This is best exemplified by “the miracle of the lactation of St. Bernard,” based on a vision concerning St. Bernard of Clairvaux in France being hit with a squirt of milk traveling an impressive distance from the breast in the statue of the Virgin nursing the infant Jesus [6]. Such blessed milk is believed to have given him great wisdom and cured an infection in his eye.

The modern history of immunology of mammalian lactation can be traced to as early as 1892 with observations by Paul Ehrlich that nonsuckling frequently resulted in death in the newborn foals, lambs, or piglets [7–9]. About the same time, observations by Escherich [10] provided for the first time evidence for exquisite sensitivity of intestinal microflora to human milk. Subsequently, the association of certain milk proteins such as immune lactoglobulin with specific

protective functions was identified by Smith [11]. This protein is now referred to as the immunoglobulin G in the bovine colostrum. Furthermore, studies by Dixon et al. [12] demonstrated that IgG in the colostrum is actively transported to the colostrum from maternal serum during lactation. It is now well known that newborns of many mammalian species will die from infections if they fail to receive lactational products adequately via breastfeeding or the neonate fails to suckle. Interestingly, however, human neonates can survive without any breast milk feeding, and they can be raised normally on nonhuman milk-based formula feeds.

It has been shown that during placentation of mammals, different maternal
immunologic components are transported selectively via the placenta or breastfeeding in different primates. For example, in rabbits, rodents, and some carnivores, maternal IgG is actively transported to the fetus in large amounts from the maternal serum across the placenta. On the other hand, such effective placental transport does not occur in horses, cattle, swine, and other mammals, as reviewed in detail by Butler and Kehrli [13] and summarized briefly in Table 1.

The immunologic composition of human milk and its biologic linkage to mucosal immunity was initially recognized by the identification of major classes of immunoglobulin in the milk by Gugler and von Muralt [14], and Hanson [15]. These elegant studies were followed by the identification of the unique immunoglobulin, the secretory IgA (SIgA), in human milk by Hanson and Johansson [16]. Subsequent studies by Beer et al. [17], Ogra and Ogra [18], Ogra et al. [19], Mohr [20], and Okamoto et al. [21] identified several cellular and soluble immunologic elements in the human milk and their possible transport to the suckling neonate via the process of breastfeeding. Finally, it is of interest that Ehrlich [7] demonstrated for the first time that maternal immunization and subsequent breastfeeding induced significant protection in suckling mice against the toxic effects of subsequently ingested ricin and abrin. His imaginative studies also raised the possibility of protection against infections such as syphilis, mumps, typhus, and measles via the process of breastfeeding [7–9].
During the past 3 decades, significant information has been obtained to suggest that the immunologic activity inherent in the products of lactation represents, to a major extent, the effector functional elements of the common mucosal immune system. Following the discovery of IgA in the serum by Heremans et al. [22], and of secretory IgA in the milk by Hanson and Johansson [16], the presence of SIgA was also demonstrated in other mucosal secretions by Chodirkar and Tomasi [23], Tomasi and Zigelbaum [24], and Bienenstock and Tomasi [25]. These observations were followed by the identification of antibacterial, antiviral, and antiparasitic activity in the milk associated with SIgA and other immunoglobulin classes, demonstration of a number of specific cellular elements and cellmediated immune responses, and detection of cytokines and other immunoregulatory factors in milk. The relationship of the immunologic activity in the milk and mammary glands to other mucosal surfaces was documented conclusively by several elegant studies which identified intestinal and respiratory tracts, and the sublingual tissues as the primary induction sites of specific IgA-committed B cells and their active migration to the mammary glands [26–29]. Since the discovery of Bifidobacterium bifidum subspecies in 1953, it is now clear that this organism predominates in the feces of the breastfed infants. Specific factors stimulating the growth of this organism are uniquely present in human but not in cow’s milk.

A significant biologic database is now available to support the clinical observations dating back from antiquity to the last few centuries, which have suggested a strong association between breastfeeding and protection against a variety of infectious and noninfectious disease processes in humans. These include protection against infectious diarrheal diseases, fertility and childbearing, and immunomodulation of mucosal and systemic immune responses. This information has been extensively reviewed in many recent publications [30–34]. It is now clear that human milk contains a wealth of biologically active products.

These include soluble proteins, hormones, a number of cellular elements and their functional products (cytokines, chemokines, and hormones), several peptides, proteins, lipids, oligosaccharides, and numerous microorganisms [32]. This information is briefly reviewed in Table 2. Significant new information has also become available about the following specific areas of milk: (a) evolution of lactation in mammalian species; (b) further characterization of immunologic

components of human milk; (c) identification and functional characterization of the mucosal microbiome and its role in modulating the homeostasis of human biologic functions; (d) development and functional aspects of the microbiome and virome of human colostrum and milk, and (e) the role of milk oligosaccharides, other milk proteins, and peptides in the mechanism of protection induced by breastfeeding and ingestion of human milk.This workshop was designed to review this information in some detail. Recent information about the microbiological aspects of milk and lactation and its influences on the microbial colonization in the gut, and of other unique protective factors in the milk, is provided in the subsequent presentations in this workshop. A brief overview of the recent progress and highlights of earlier observations on the immunologic aspects of human milk are summarized below.

Immunology of Milk and Lactation

Lactational Performance: Secretion of Colostrum and Milk.

The immunologically active products observed in the mammary glands and the products of lactation are (a) derived from local synthesis within the mammary glands, (b) secondary to the transport of components from the maternal circulation and blood stream, and (c) selective and specific transport from the induction sites in the mucosa-associated lymphoid tissue in the gastrointestinal tract, nasopharynx, sublingual lymphoid tissue, and lymphoid tissue in bronchoepithelial
mucosal sites. Colostrum is the first postpartum product of lactation. It is dense in protein and fat, and it contains the highest amounts of soluble as well as cellular immunologic components compared to transitional or mature milk. Successful lactation
with continued contribution of the transported or locally synthesized products in mature milk is also determined by continued contribution of neural, endocrine, and other maternal-infant interactions activated at the time of delivery. Early and frequent breast contact by the nursing infant is also important for continued stimulation of neural pathways to maintain prolactin and oxytocin release. Lactation often ceases when suckling stops.

Soluble Components

Immunoglobulin. The major isotypes of immunoglobulin in the milk or colostrum is the 11SIgA. Other isotypes including 7SIgA, IgG, IgM, and IgD are also present in varying amounts, and IgE may be occasionally observed. The 11SIgA exists as a dimer of two 7SIgA molecules linked together by J-chain, a polypeptide chain associated with a secretory component, and a polymeric immunoglobulin receptor [35]. Immunoglobulin Activity. The studies on the temporal distribution of class specific immunoglobulins and different cellular components in the product of lactation by S. Ogra and her colleagues [18, 19, 21, 30] in the late 1970s established a sequential database for their levels in the colostrum and mature milk. Highest levels of SIgA and IgM are observed during the first 3–5 days of lactation.

The levels of IgA are usually 4–5 times higher than those of IgM and about 26–30
times higher than IgG levels [36]. As lactation progresses, the levels of IgA and
IgM in the mature milk decline rapidly. However, this decline is compensated by
the increase in the total volume of milk produced (Table 3). It is estimated that a
fully breastfed neonate may consistently receive about 1 g of IgA each day and
approximately 1% of this amount for IgM and IgG [36]. Comparative studies of
immunoglobulin activity in the feces of breastfed infants have suggested that the
fecal content of IgA may be 15–20 fold higher after human milk feeding compared
to bovine IgG after feeding of bovine immunoglobulin products [37].

Immunoglobulin Reactivity

Studies carried out with many microorganisms, including respiratory syncytial virus, Escherichia coli 083 and Streptococcus pneumoniae, have demonstrated the appearance of antibodies in the milk IgA independent of, and even in the absence of, any detected antibody activity in the serum. These and other elegant studies have clearly demonstrated that SIgA and its functional activity is derived from the initial exposure to specific antigens in the neonatal respiratory and intestinal mucosal lymphoid tissue and the transport of specific antibody producing IgA B cells to the mammary glands. These observations have firmly established the concept of the enteromammary and the bronchomammary axis of immunologic reactivity in the mammary glands [28, 38, 39].

Antisecretory Factor

An important molecule which binds like a lectin to certain polysaccharides (and inhibits gut fluid secretion induced by cholera toxin) has been described by Lönnroth and Lange [40] in 1984. This hormone- like factor is produced in many tissues, including the mammary glands. It has been found in samples of human milk in poor socioeconomic settings. It is possibly induced by exposure to enterotoxin-producing bacteria. The antisecretory factor seems to have a protective anti-inflammatory effect by reducing
fluid secretion, and it has been used to treat infectious diarrheas, antiinflammatory bowel disease, and other inflammatory conditions [34]. Human milk is also rich in other anti-inflammatory components. These include vitamins, especially A, C, E, as well as the enzymes catalase and glutathione peroxidase [32].

Soluble CD14 and Soluble Toll-Like Receptor

Colostrum and milk contain high concentrations of soluble CD14. This molecule helps lipopolysaccharides, a surface structure on gram-negative bacteria, to bind to TLR-4 receptor and activate phagocytes [41]. It appears that by means of milk CD14, phagocytes in the gut mucosa are activated by gram-negative as well as gram-positive organisms [34]. It has also been suggested that CD14 promotes differentiation and expansion of B cells and the anti-inflammatory activity of lactoferrin, another
important nonbinding protein found in human milk.

Cytokines

A large number (> 150) of cytokines, growth factors, and chemokines have been identified to date. These soluble complex molecules play a critical immunomodulating role in (1) stimulation of growth, differentiation, and immunoglobulin products of B cells, (2) enhancement of thymocyte proliferation, (3) inhibition of IL-2 production by T cells, and (4) suppression of IgE production. Cytokines such as IL-1β, TNF-α, IL-10, and TGF-β have been demonstrated in human milk and appear to be secreted by milk macrophages as well as by the epithelium of the mammary glands. In addition, IL-6 and interferon-α have also been described in human milk.

Chemokines

They are small cytokines with selective and discrete target cell functions. They are classified based on spacing between the cysteine residues (CXC, CC). Some chemokines, including IL-8, have been detected in human milk. Most chemokines serve as signals to mobilize phagocytic cells to an area of inflammation, and such activated phagocytic cells produce more cytokines.

Growth Factors

Finally, several growth factors, including IL-7, have been recovered from human milk [32]. It has been suggested that IL-7, which is a growth factor for T-cell progenitors for memory T cells, also supports thymic growth [42].

Cellular Components

Human colostrum and milk are endowed with a variety of maternally derived
cellular elements. These include epithelial cells, activated neutrophils, macrophages,
stem cells, and B and T lymphocytes. In addition, recent observations have suggested that human milk is rich in bacteria and other cellular and subcellular
living organisms [32].

Leukocytes

There are > 1–3 × 106 leukocytes/mL in colostrum and early milk. The number of such cells decreases gradually to < 1 × 106 /mL over the next 2–3 months of lactation [18]. Flow-cytometric analysis has indicated that lymphocytes constitute about 4% of the cell pool in the milk. It has been shown that by 4–6 months into lactation, epithelial cells constitute about 75–80% of milk cells [43]. Polymorphonuclear neutrophils represent about 40–50% of the total cell population in early colostrum [43]. In lactating mothers, the development of infection is often associated with an increase in milk leukocytes, and their numbers decline with resolution of infection. Experimental animal model data suggest that milk leukocytes cross the intestinal epithelial barrier in the infant and may engraft in different organs, including mesenteric lymph nodes, liver, and spleen. The leukocytes may provide anti-infective benefits to the mammary gland of the lactating mother and possibly also to the breastfeeding infant [44–46].

Macrophages

Human milk macrophages exhibit several phenotypic and functional characteristics, and they are able to produce many cytokines, such as IL-1β, IL-6, TNF-β, and GM-CSF, spontaneously. Milk macrophages are capable of antigen presentation and specific synthesis of prostaglandin E2, plasminogen activator, lysozyme, and C3 [47]. Milk macrophages have been shown to express the marker DC-SIGN which is a dendritic cell receptor for human immunodeficiency virus (HIV). As a result, it has been proposed that unstimulated milk macrophages may add to the risk of HIV transfer via the breast milk [48].

Activated milk macrophages exhibit enhanced phagocytosis and have been
found to have a receptor for SIgA. Its role in the mechanism of protection in the breastfeeding neonate remains to be defined.

Epithelial Cells

Human colostrum and milk contain both ductal and alveolar epithelial cells and myoepithelial cells. The majority of these cells are viable and can be propagated in vitro. It has been proposed that milk epithelial cells are detached from the stroma of mammary glands in an active process driven by differential gene expression. These cells appear in clusters and may serve a still to be defined useful biologic purpose [32].

Stem Cells

Some cells in human colostrum and milk have been found to express stem cell markers, CK5, nestin, P63 , and CD498. Milk cells expressing such markers were able to self-replicate and differentiate into luminal or myoepithelial cells. It has also been observed that such milk stem cells express pluripotency markers and may differentiate into cells of other lineages, including adipocytes, hepatocytes, and pancreatic β-cells. The implication of these findings to the neonatal immune system and cellular homeostasis remain to be defined [32].

Lymphocytes and Cell-Mediated Immunity

The overall number of lymphocytes in the human colostrum and milk is relatively small. Of the 1–3 × 106 leukocytes/ mL of colostrum during the first few days after birth, lymphocytes account for about 4–6% of total cells [20]. The B cells account for about 6% of the lymphocytes, and the bulk of the remaining cells are of T-lymphocyte phenotype (83%), with an additional small number of natural killer cells (8–10%). T Cells. Most T cells are CD8+ and CD4+ cells, with a relative predominance of CD8+ cells. Most cells are CD45RO+, a marker associated with activation and immunologic memory. The T cells are mainly αβ-receptor-bearing cells. Vδ1 and Vγ2 (but not Vδ2) marker-bearing cells are significantly overrepresented in milk T cells, possibly due to direct homing to the mammary glands. Milk lymphocytes share the CCR9 receptor from the thymus-expressed chemokine (TECK) in the epithelium, with all small intestinal CD4+ and CD8+ cells, and with other similar cells from tonsils, lungs, skin, synovium, and body fluids [49– 52]. Although the precise function of such cells in lactational products and mammary glands remains to be defined, they may represent an important mechanism specifically targeting the immune system.

Interaction of Cellular Elements with the Neonatal Immune System

In view of the large number and the diversity of cellular elements observed in the colostrum and milk and the large volume of milk consumed by the human infant during the neonatal period and early infancy, it is reasonable to postulate that these cells exert
possible functions in the breastfeeding infant. During the course of normal breastfeeding, millions of viable cellular elements are ingested by the infant. Earlier studies have suggested that milk cells are taken up in the neonatal mucosal surface and may transfer to the neonate, with varying degrees of specific immunologic information [17, 19, 20, 53]. Studies carried out by Ogra et al. [19] in 1977 in a group
of formula-fed infants after a single-feed administration of human colostrum have clearly demonstrated the uptake and transfer of IgA. In another group of infants of tuberculin-positive mothers, these investigators also demonstrated the transfer of in vitro correlates of T cells mediated against tuberculin after prolonged breastfeeding. The tuberculosis-specific T-cell response in such infants were short lived and undetectable after 10–12 weeks in spite of continued breastfeeding. Subsequently, several experimental animal studies have demonstrated the engraftment of maternal DNA via milk leukocytes in infant tissue [44–46]. Although these observations have been linked to lymphocytes, such transfer may also occur with epithelial and stem cells. In other experimental investigations, the transfer of maternal T cells and HLA antigens appears to be associated with the development of immunologic tolerance to maternal HLA antigens. Breastfed infants also express a lower frequency of precursors of cytotoxic T lymphocytes reacting with maternal HLA than nonbreastfed infants [54, 55]. Recent observations have also shown that maternal cytotoxic T lymphocytes localize in the Peyer’s patches of breastfed infants [56]. Such localization may serve to compensate for the immature adaptive immune response functions in the neonate.

Risks and Benefits of Breastfeeding

In his keynote, Prof. Oftedal elegantly outlined the evolutionary biology of the mammary gland and lactation. This presentation also implied that the origin of the mammary gland is buried deep in time, and many of its evolutionary novelties and specific nutritional products, such as caseins and other milk-specific proteins, and the methods of sugar synthesis appear to have originated more than 300 million years ago [57]. It is clear that the evolution of the mammary gland and lactation has become an important characteristic of mammalian reproduction, and an essential mech-

anism of defense and survival for the mammalian neonate. As a result, the relative benefits and risks of breastfeeding must be considered with regard to the immunologic, nutritional, and microbiological components of the products of lactation identified to date. This workshop was specifically designed to examine the recent progress made in these areas of lactation and maternal-neonatal interactions in some detail. The functional elements of the immunology of lactation and its benefits and risks are briefly summarized below. The contributions of the milk microbiome and virome and of nutritional and other protective factors in milk and their role in the mechanisms of host defense in the breastfed neonate are discussed in some detail in the presentations in sections II and III, respectively. The known benefits and potential risks of breastfeeding have been reviewed in extensive detail by Hanson [34] in a special publication in 2004 and more recently by Kim et al. [32] in 2015. An overview of these effects on the breastfeeding neonate as well as on the lactating mother is presented in Table 4.

Maternal Benefits

Although lactation is designed to mobilize the best maternal attributes for the
homeostasis and the survival of the infant, breastfeeding offers significant benefits to the mother as well. Feeding of about 6 sucklings per 24-h period has been found to provide significant contraceptive benefit for the mother. It has been proposed that more conceptions are prevented by breastfeeding than by all other contraceptive approaches and family planning programs in many parts of the world [58]. Such an important maternal influence has also been shown to contribute to the reduction in infant mortality correlated with reduced crowding in the family, with less risk of infection and improved availability of food and nutrition to the infant and the mother. An interpregnancy space of less than 2 years has been shown to increase the risk of infant mortality by over 50% before 5 years of age [59].

Neonatal Benefits

Immunologic benefits for the neonate associated with breastfeeding include
maturation of the mucosal immune system. The development of intestinal mucosal
integrity is to a large extent determined by the maturation of mucosa- associated lymphoid tissue and other tissue sites, and the establishment of the mucosal microbiome. Recent investigations have demonstrated that SIgA antibody and other soluble immunologic products in the breast milk promote long term gut homeostasis by regulating the acquisition of the mucosal microbiome and host gene expression [60, 61].

Exclusive breastfeeding in the first 6 months is clearly a major determinant of the prevention of diarrheal disease in infants, especially with Escherichia coli, Shigella, Vibrio cholera, Campylobacter, some parasitic infestations (Giardia lamblia), viruses (rotavirus), and possibly other mucosal infections. Case-control studies have suggested that breastfeeding and specific antibody activity in the colostrum and milk more often provide protection against severe disease and hospitalization rather than total prevention of colonization and infection [60, 61]. A number of studies have clearly demonstrated a beneficial role of human milk in preventing or modifying the severity of necrotizing enterocolitis in premature infants [32, 34]. Similarly, anti-infection benefits related to milk-associated antibodies, soluble cytokines, or other protective features have been observed in breastfed infants against several genitourinary respiratory infections, otitis media in childhood, neonatal sepsis, and possibly sudden infant death
syndrome of unexplained origin [34].

Breastfeeding and the immunologic components of human milk have been shown to confer long-lived protection against reactive airway disease and bronchial asthma, eczema, and other atopic and allergic states. The protective effects may reflect multiple synergistic mechanisms, including maturation of gut and airway mucosa by growth factors in human milk, reduction in the absorption of allergens and other antigens by modulation of the mucosal microbiome, and induction of specific mucosal tolerance (oral tolerance), and immune exclusions.

Combined with other protective factors in the gut, SIgA can impede allergen sensitization by blocking the transport of foreign macromolecules across the immature neonatal gut epithelia and modulating the development of specific antibodies or immune complexes. It should also be pointed out that cow’s milk protein and other food antigens ingested by the lactating mothers have been observed on colostrum and milk. Other studies have suggested that early breastfeeding may be associated with decreased serum antibody responses to cow milk proteins and other maternal dietary antigens in the breastfed infant.

From an evolutionary biology perspective, it would seem that breastfeeding should provide only beneficial effects to the neonate. However, there is considerable debate regarding the protective “immune-mediated effects” of breastfeeding on the development of atopy and allergy. Some investigations have proposed the act of breastfeeding itself, regardless of the constituents of the breast milk, may be more or equally important defense mechanisms for the infant. An interesting study has suggested no protective effect of indirect breastfeeding (breast milk fed by the bottle) compared to infants receiving direct breastfeeding [62].

Neonatal Risks

Because of changing mammalian and (especially) human environments, there are now several documented risks in breastfeeding. However, these risks are not immunologic in nature; they are rather related to the maternal use of chemicals, radionucleotides, and other medications or drugs, maternal metabolic disorders, and possibly hyperbilirubinemia associated with breastfeeding. A number of infectious agents have been recovered from human colostrum and milk, and the transmission of infection to the neonate has been observed frequently. However, the development of disease associated with such transmission is relatively infrequent. Infection in the breastfeeding infant has, however, been observed with active maternal Mycobacterium tuberculosis infection, active maternal sepsis or mastitis, and active infection with HIV, HBV, HSV, and CMV (Table 4).

Existing information about the potential risks and benefits of the milk microbiome
and other factors in human colostrum and milk will be explored in further detail in subsequent studies of this workshop. In conclusion, it may be worthwhile to recapitulate that from an evolutionary standpoint, human colostrum and milk continue to remain the single most important vehicle for the transport of all maternal immunologic experiences via breastfeeding to the neonate for its survival and well-being throughout its life
span.

Acknowledgments

This historical overview of the immunology of milk and lactation and the select laboratory data summarized here are largely based on the investigations carried out by the following collaborators and co-authors from 1970 to 2000: Drs. Y. Chiba; J. Cumella; L. Duffy; J. Freihorst; M. Fishaut; R. Garofalo; G. Losonsky; S.S. Ogra; Y. Okamoto; and D. Wong The secretarial assistance of Mrs. Judith Maurino, my colleague and my secretarial support for almost 5 decades, in the preparation of this manuscript is gratefully acknowledged.

Disclosure Statement
The author declares to have no conflict of interest.

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Pearay L. Ogra