Review

published: 29 May 2017

doi: 10.3389/fimmu.2017.00584

I
Nicole Theresa Cacho1* and Robert M. Lawrence 2
1
 Division of Neonatology, Department of Pediatrics, University of Florida, Gainesville, FL, United States, 2 Division of Pediatric
Infectious Disease, Department of Pediatrics, University of Florida, Gainesville, FL, United States

Human milk is a dynamic source of nutrients and bioactive factors; unique in providing for
the human infant’s optimal growth and development. The growing infant’s immune system
has a number of developmental immune deficiencies placing the infant at increased risk
of infection. This review focuses on how human milk directly contributes to the infant’s
innate immunity. Remarkable new findings clarify the multifunctional nature of human
milk bioactive components. New research techniques have expanded our understand-
ing of the potential for human milk’s effect on the infant that will never be possible with
milk formulas. Human milk microbiome directly shapes the infant’s intestinal microbiome,
while the human milk oligosaccharides drive the growth of these microbes within the gut.
New techniques such as genomics, metabolomics, proteomics, and glycomics are being
used to describe this symbiotic relationship. An expanded role for antimicrobial proteins/
peptides within human milk in innate immune protection is described. The unique milieu
of enhanced immune protection with diminished inflammation results from a complex
Edited by:
interaction of anti-inflammatory and antioxidative factors provided by human milk to the
Joseph M. Bliss, intestine. New data support the concept of mucosal-associated lymphoid tissue and its
Women & Infants Hospital of Rhode
contribution to the cellular content of human milk. Human milk stem cells (hMSCs) have
Island, United States
recently been discovered. Their direct role in the infant for repair and regeneration is
Reviewed by:
Dina Weilhammer, being investigated. The existence of these hMSCs could prove to be an easily harvested
Lawrence Livermore National source of multilineage stem cells for the study of cancer and tissue regeneration. As
Laboratory (DOE), United States
Leonardo H. Travassos,
the infant’s gastrointestinal tract and immune system develop, there is a comparable
Federal University of Rio de Janeiro, transition in human milk over time to provide fewer immune factors and more calories
Brazil
and nutrients for growth. Each of these new findings opens the door to future studies of
*Correspondence:
human milk and its effect on the innate immune system and the developing infant.
Nicole Theresa Cacho
[email protected] Keywords: human milk, breast milk, innate immunity, colostrum, preterm

Specialty section:
This article was submitted to
INTRODUCTION
Microbial Immunology,
The innate immune system is the first line of defense against infection and is activated within
a section of the journal
Frontiers in Immunology
minutes, reacting in a nonspecific, preprogrammed, and patterned manner to various infectious or
foreign (non-self) stimuli (1). The infant’s immune system is immature at birth, and this immaturity
Received: 30 January 2017
is pronounced for the premature infant placing the infant at increased risk of infection (2). Important
Accepted: 01 May 2017
Published: 29 May 2017 developmental immune deficiencies at birth include incomplete physical and chemical barriers, poor
innate effector cell function, limited and delayed secretory immunoglobulin A (IgA) production,
Citation:
Cacho NT and Lawrence RM (2017)
incomplete complement cascade function, and insufficient anti-inflammatory mechanisms of the
Innate Immunity and Breast Milk. respiratory and gastrointestinal (GI) tracts.
Front. Immunol. 8:584. Human milk is the everchanging secretions of the human breast, an evolving composition of
doi: 10.3389/fimmu.2017.00584 nutrients and active factors. Just as nutrition and protection of the fetus occurs through the mutable

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Cacho and Lawrence Breast Milk Immunity

nature of the uterus, placenta, and amniotic fluid, the evolution of pylori, Reovirus, E. coli, and Burkholderia cepacia. The specific
human milk from colostrum through transitional milk to mature molecular mechanism of this binding by glycans and interference
milk provides nutrition and protection appropriate for the time- with infection by the pathogen requires additional clarification.
affected development of the infant (3). There is a large body of evi- Unraveling the complex glycoprotein–ligand interactions will
dence documenting the benefits of human breast milk for human require application of newer technologies such as nanosurface
infants, in diminishing morbidity and mortality and protecting plasmon resonance and glycan microarrays (20). Another chal-
against specific infections during the period of breastfeeding lenge is to demonstrate whether, how, and when this occurs in
(4–6). Additional data demonstrate long-term health benefits for a mother–infant dyad exposed to a specific pathogen through
the infant (and the mother) beyond the period of lactation (7, 8) changes in the glycan composition of the mother’s milk (21).
and led to the current recommendations for duration of exclusive Lactoferrin (LF), another glycoprotein abundant in colostrum
breastfeeding from the American Academy of Pediatrics (9) and and transitional milk and ubiquitously expressed in most exocrine
the World Health Organization. Although research into the spe- secretions, is one of the best studied glycoproteins in human milk.
cific factors in human breast milk, which lead to the remarkable LF has multiple functions in host defense through binding iron,
health benefits of exclusive breastfeeding, has been ongoing for binding to bacterial membranes, inhibition of tumor necrosis
decades; there are still intriguing mysteries of how human milk factor-alpha (TNF-α) and interleukin-1β (IL-1β), stimulating
contributes to the development and regulation of both the infant’s the activity, maturation of lymphocytes, and contributing to an
innate (10) and adaptive immune function (11–13). anti-oxidizing mileau (22). Peptide breakdown products of LF,
Elucidating the relationship between the innate immune sys- lactoferricin and lactoferrampin, have specific direct antibacterial
tem and human milk, as well as their individual and interactive and antifungal effects (23).
transition over time, remains challenging. Many components in There are numerous other HMGPs that have been described
milk are multifunctional, serving as enzymes, antimicrobial pro- but the extent and specificity of their immune function need to be
teins/peptides (AMPs), growth factors, chemokines, antioxidants, elucidated. Lactadherin can inactivate viruses and limits inflam-
anti-inflammatory elements, prebiotics, probiotics, and nutrients mation by increasing the effective phagocytosis of apoptotic cells.
for the growing infant (14, 15). The use of modern molecular The sialic acid component of lactadherin seems to directly inter-
approaches such as microbial genomics, metabolomics, proteom- act with rotavirus while the protein backbone of the molecule
ics, and glycomics has led to novel discoveries in both composi- demonstrates a proangiogenic effect on neovascularization. The
tion and function of human milk components. This review will list of bioactive glycoproteins in human milk is still expand-
focus on the current understanding of the critical interactions ing, and their individual multifunctional nature is just being
between human breast milk and the infant’s developing innate described. Butyrophilin, leptin, adiponectin, bile salt-stimulated
immune system (Figure 1) (12, 16–18). lipase lysozyme, lactoperoxidase (LP), xanthine dehydrogenase,
α-lactalbumin, κ-casein, and β-casein are just a few of these
CHEMICAL BARRIERS OF INNATE glycoproteins requiring additional study (19).
IMMUNITY
CELLULAR CONTRIBUTORS TO INNATE
The chemical barrier of the intestine is predominately the mucus IMMUNITY
layers lining the GI tract. These mucus layers minimize antigenic
contact between epithelial cells and commensal bacteria as well The cellular layers of the infant’s intestine include the barrier of
as potentially pathogenic bacteria. Antimicrobial peptides pro- epithelial cells with the tight junctions, specialized goblet, Paneth
duced by Paneth cells, released into the mucus layer, bolster this and microfold cells, the lamina propria and Peyer’s patches with
chemical barrier innate effect by neutralizing microbes via vari- macrophages, neutrophils, and dendritic cells. The immunity
ous mechanisms. The multifunctionality of individual human milk provided by these cellular interactions is partially innate and
factors adds another layer of complexity to the innate protection partially the beginning of adaptive immunity (24). Immaturity
effected within the intestinal mucus layers. of the intestinal barrier function, limitations in production of
Human milk oligosaccharides (HMOs) are the predominant AMPs, and ineffective response and action of epithelial cells and
glycans and important nutrients in human milk. They function phagocytes place the infant at risk for infection in the neonatal
in direct pathogen binding and as prebiotics facilitating the period (25, 26).
establishment of a healthy infant microbiome (2). The human Within fresh human milk, there is a remarkable repertoire of
milk glycoproteins (HMGPs) vary in size, structure, and amount heterogeneous living cells. Cells originating in the breast include
in human milk and can be classified based on their location lactocytes (secretory cells), myoepithelial cells from the ducts
relative to the cell (secreted or attached to the cell membrane) as well as progenitor cells and mammary stem cells, and a small
and the different mucus layers. Mucin 1 (MUC1) and Mucin 4 number of squamous epithelial cells from the nipple and skin of
(MUC4), gangliosides (GM1, GM3, and GD3), and glycoproteins the breast. Cells originating from the blood, which are in human
similarly function by binding pathogens and do so without stimu- milk, include immune cells (macrophages, neutrophils, and
lating an inflammatory response (19). MUC1 and MUC4 have lymphocytes), hematopoietic progenitor cells, and hematopoietic
been described as binding to specific pathogens including HIV, stem cells (27, 28). The role of live breast cells in human milk
rotavirus, Escherichia coli, and Salmonella. Other HMGPs have in the infant remains uncertain. New theories are being formu-
demonstrated binding to H. influenzae, Streptococci, Helicobacter lated to explain the fact that these breast milk epithelial cells are

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Cacho and Lawrence Breast Milk Immunity

Figure 1 | Summary of innate immunity and breast milk with groups including components of the innate immunity, maturation of breast milk over
lactation, and breast milk by gestation (preterm and term).

capable of motility and can in primary culture form functioning as these cells from human milk do establish a microchimeric state
mammospheres (29, 30). Hassiotou et al. (29) explain the fact that in the infant. Hassiotou et al. (29) have demonstrated the pleu-
these cells are in different stages of differentiation as a continuum ripotential differentiation nature of hMSCs. hMSCs were able to
of mammary development, albeit that does not answer the ques- differentiate into the three germ layer cell lineages, in in vitro test-
tion why they are in such large numbers in expressed human ing. Expression of octamer-binding transcription factor 4, related
breast milk. to “self-renewal” functions, is upregulated in hMSCs isolated from
There are large numbers of macrophages present in early human milk (33). Using milk from genetically modified mice,
lactation that decrease with the maturation of the milk Hassiotou et  al. (36) demonstrated the persistence of modified
(31, 32). These macrophages appear to function by phagocytizing milk stem cells within the brain, thymus, pancreas, liver, spleen,
pathogens without initiating a significant, unregulated inflam- and kidneys of non-modified mice (36). These data specifically
matory response. An increase in milk leukocytes, above the suggest a potential role for hMSCs in tissue regeneration in the
number usually present at the specific stage of lactation, occurs breastfed infant and perhaps regeneration of cells of the infant’s
with infection in either the mother or the infant and suggests innate immune system. Beyond that, the hMSCs in human milk
a functional role for milk leukocytes in protecting the infant may provide a ready source of patient-specific stem cells with a
(21, 33, 34). Breast milk from mothers of infants with severe bron- true multilineage potential for the study of such stem cells and
chiolitis demonstrated an increased number of live cells. Those variables related to breast cancer, tissue regeneration, and even
live cells produced a specific cytokine profile response when bioengineering.
stimulated with live respiratory syncytial virus, a common cause
of bronchiolitis (35). These data directly support the concept of MICROBIOTA
mucosal-associated lymphoid tissue and that cells and/or activat-
ing factors are transferred from the mother in her breast milk to In the past, human milk was considered sterile, but that is far
the infant at the time of infection or exposure to an infection. from the truth. Using culture techniques, the majority of bacteria
Additional studies measuring changes in the bioactive factors in identified as facultative anaerobes in human milk belong to the
human milk of mothers with sick infants or who are sick them- Staphylococcus and Streptococcus species and other species in
selves for various specific infections should provide more insight smaller numbers (Propionibacterium, Rothia, Enterococcus, and
into the essential bioactive factors in human milk for protection Lactobacillus species) (37, 38). Obligate anaerobic bacteria were
against specific infections. later identified, Bifidobacterium and Bacteroides species (39–41).
The human milk stem cells (hMSCs) likely play an important Culture-independent techniques (sequencing and metagenomics
role in the “regeneration” of the breast in preparation for lactation analysis) have demonstrated a significantly more complex and
and theoretically in the infant’s tolerance of maternal cell antigens diverse group of bacteria in human milk (42, 43). There is a large

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Cacho and Lawrence Breast Milk Immunity

degree of interindividual variability in the milk microbiome. effect of probiotics added to the diet of lactating women, which
Similar factors affecting the infant’s or the mother’s intestinal increased the levels of secretory immunoglobulin A (sIgA) in the
microbiomes also affect the milk microbiome (genetics, mode infant’s stool and on IL-6 mean values in colostrum and on IL-10
of delivery, geographic area, gestational age, maternal diet and and TGF-β1 mean values in mature breast milk. This demonstrates
nutrition, antibiotics, lactation stage, etc.) (42, 44). Nevertheless, the possibility of potentially beneficial microbes influencing the
most samples from healthy women appear to contain a “core” content of the woman’s breast milk and subsequently the infants’
microbiome (34, 45). Hunt et al. name nine bacterial groups and GI tract via specific bioactive factors reaching the infant. The
Jimenez et al. reported seven bacterial groups. These two analy- ultimate benefit to the infant remains to be studied (58). Human
ses shared just three common bacterial groups: Staphylococcus, milk is not simply adding additional bacteria to the infant’s gut
Streptococcus, and Propionibacterium (34, 45). Variability between and intestinal microbiome but providing both bacteria and prebi-
these two studies and others (43) can be attributed to the use of otics to function in a symbiotic relationship creating the milieu in
different primers, types of sequencing, comparison with different which the infant’s innate intestinal immunity functions and the
microbial reference libraries, possible variation due to geographic intestine develops. How that symbiotic interaction influences the
regions, and timing of the milk collection as well as technique and infant’s health in the future, as it relates to immune protection and
sterility of milk collection (43, 46, 47). immune reactivity (allergy or autoimmune disease) will require
The actual origin of the milk microbiota remains uncertain careful research involving not only genomics, metabolomics,
(48). Postulated origins include mammary gland itself, skin flora proteomics, and glycomics but also epigenetics and techniques
of the breast, an “entero-mammary pathway” where intestinal still need to be developed.
bacteria translocate and home to the breast and retrograde flow
from the infant’s mouth into the breast (12, 49–51). Tracing the INNATE MECHANISMS OF PATHOGEN
origins of milk microbiota and the relative influence of environ- RECOGNITION
mental factors on its makeup should inform our understanding of
the role of the common groups of bacteria or their interaction in There are specific TLRs present in human milk, including TLR2,
symbiosis with the infant’s innate immune system and developing TLR3, TLR5 as well as soluble CD14 (sCD14), and human
intestine. β-defensin-1 (hBD-1), which function as pattern recognition
Human milk directly contributes to the establishment of the receptors (PRRs) and AMPs (59). Chatterton et al. (59) discuss
intestinal microbiota and facilitates a symbiosis between that the potential role of PRRs in human milk affecting the protective
microbiota and the infant by providing essential nutrients, in response in the intestine balanced with other bioactive factors in
particular milk glycans or HMOs, for microbial metabolism milk affecting an anti-inflammatory mileau. There is evidence that
(52). Nanthakumar et al. (53) proposed that specific microbiota TLR responses in the infant are modified by soluble TLRs (sTLRs)
colonizing the gut early on in the infant induce the expression and sCD14. The interaction of both sTLRs and sCD14 with other
of intestinal epithelial cell (IEC) fucosyltransferase 2. This leads bioactive factors in human milk upregulates and downregulates
to fucosylation of surface markers on GI epithelial cells, which the action of various TLR-mediated inflammatory responses
enhances the growth of microbes utilizing fucose as part of their (60, 61). LeBouder et al. (61) demonstrated the effect of human
metabolism (53). Microorganisms utilize the available glycosami- milk on TLR-mediated microbial recognition. They describe
noglycans, glycoproteins, glycolipids, and oligosaccharides (milk specific responses on epithelial cells, monocytes, dendritic cells,
glycans) as prebiotics to facilitate growth (54). The intestinal and peripheral blood monocytic cells. The responses were differ-
microbiota limits the growth of pathogenic bacteria by competi- ent based on which TLRs were activated. Infant formulas did not
tion for nutrients and receptors. Specific microbes facilitate the exhibit such effects.
formation of the intestinal mucus layer and the development of Immunoglobulins are the most recognized immune protec-
the IEC barrier and submucosal lymphoid structures. Separately, tive component in human breast milk. As preformed Igs from
the HMOs bind to surface molecules of bacteria and viruses pre- the mother, they constitute a discrete group of proteins capable
venting binding to the intestinal epithelium and appear to dimin- of pathogen recognition. sIgA is the principal Ig in human milk
ish intestinal inflammation via signaling pathways (54). Newburg (>90% of the Ig fraction), immunoglobulin M (IgM) in the
and Morelli (55) describe the symbiosis of the microbiota and pentameric form is next most abundant. There is a small amount
the infant’s intestinal development as dependent on HMOs of immunoglobulin G (IgG) in colostrum and transitional milk,
in the mother’s milk. The “commensal” microbes, specifically with IgG becoming a much larger proportion of human milk
Bacteroides and Bifidobacterium, induce mucosal glycan produc- Igs in mature milk (62). Secretory IgA binds pathogens block-
tion, which further supports microbial growth, and the microbes ing infection without stimulating a significant inflammatory
convert indigestible milk glycans to absorbable short-chain fatty response. In a largely innate immune-like action, sIgA simply
acids (55). He et al. (56) describe two different HMOs (3′-galac- blocks the pathogens contact with the intestinal epithelial layer
tosyllactose and 2′-fucosyllactose) present in high amounts in and traps the pathogens within the mucin layers. The action of
colostrum. These HMOs affect pathogen-associated molecular sIgA in the extracellular space is different from sIgA’s intracellular
pattern signaling pathways [Toll-like receptor (TLR) 3, TLR5, neutralization of viruses and bacterial lipopolysaccharides within
IL-1β, or cluster of differentiation 14 (CD14) expression and epithelial cells. The glycan sugar component (galactose, fucose,
binding] decreasing cytokine production and the inflammatory and mannose) of sIgA contributes to sIgA resistance to proteoly-
response (56, 57). There are now even clinical trials examining the sis in the intestine and functions through a broad spectrum of

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Cacho and Lawrence Breast Milk Immunity

binding of pathogenic bacteria when compared with the antigen capacity of tissues (69). Enterocytes and immune cells produce
specific binding of the variable region of the Ig structure. The anti-inflammatory cytokines including transforming growth
broad spectrum binding related to the glycan sugar component factor-beta (TGF-β), IL-10, IL-11, and IL-13. These factors act in
of sIgA is more consistent with an innate immune response which an innate manner in the intestine.
would explain the absence of an increase in specific sIgA in the Human milk caseins, LF, LP, OPN, Igs, superoxide dis-
milk of mothers with sick infants (21). mutase (SOD), platelet-activating factor acetylhydrolase, and
Immunoglobulin M causes agglutination of recognized patho- alkaline phosphatase each have both infection protective and
gens and complement activation as well as innate immune-like anti-inflammatory effects. Specific hormones or growth factors
activities. Immunoglobulin G (IgG) activates phagocytosis with predominantly exert their anti-inflammatory effects on intestinal
antigen transport to the lamina propria for B-cell activation affect- innate immunity through their action on the proliferation and
ing the infant’s adaptive response. The list of pathogens (viruses, differentiation of IECs and immune cells (lymphocytes and mac-
bacteria, fungi, and parasites) recognized by human milk Igs is rophages) and modulating the inflammatory cytokine response.
extensive (63). Gao et al. (64) report data from proteomic analysis Transforming growth factors-β2 and -β1 upregulate tight
of human milk, which demonstrate increased amounts of sIgA junction proteins (caludin-1 and claudin-4) and downregulate
and IgM in transitional milk with IgG predominating in mature TNF-α and IL-1β. In addition to growth stimulation, TGF-β has
milk. They suggest that this transition fits with the infant’s devel- anti-inflammatory properties through stimulation of epithelial
oping immunity and evolving adaptive immunity to produce cell migration and repair of the epithelium after mucosal damage
increasing amounts of IgG. (70). Similarly, insulin-like growth factors, milk fat globule epi-
There are various other AMPs, in human milk, active in microbe dermal growth factor-8 (MFG-E8), and epidermal growth factor
killing, which can supplement the protection of the immature (EGF) influence growth and proliferation of IECs. Both MFG-E8
neonatal intestine (14, 65). hBD-1 is one example of an AMP and EGF diminish the activation of nuclear factor kappa-light-
in human milk, which affects pathogen membrane permeability chain enhancer of activated B cells (59). Trefoil factor 3 (TFF3)
and cytokine stimulation in the intestine (66). Other proteins is an effector molecule that is present in the intestine and in large
[lysozyme, LF (and peptide derivatives of it—lactoferricin and amounts in human breast milk. Generally, this molecule improves
lactoferrampin), α-lactalbumin, transferring, and osteopontin healing in the GI tract. The TFF3 present in breast milk produces
(OPN)] within human milk are recognized as important AMPs downregulation of cytokines and promotes hBDs expression
functioning via various mechanisms some of which enhance the in IECs (71). Glucagon-like peptide 1 (GLP-1) is secreted from
anti-inflammatory effects of human milk (59, 64, 67). LF and the enteroendocrine cell in the distal intestine and plays a role
its derivatives demonstrate a wide variety of actions on various in regulating glucose metabolism and food intake. GLP-1 likely
targets including iron deprivation, destabilization of microbial acts through vagal afferent pathway ultimately influencing feed-
membranes, binding microbial receptors, affecting chemokine ing behavior (72). Recently, the first study to report GLP-1 in
production, stimulating epithelial cell growth, stimulating T-cell human milk showed that it was higher in hindmilk compared to
growth and differentiation, and production of reactive oxygen foremilk and was correlated with infant weight gain during the
species (ROSs). LF also interacts with other components in first 6 months of life (73). Alternative forms of neonatal nutrition
human milk such as OPN, ceruloplasmin, and neutrophil per- such as formula and TPN do not contain these anti-inflammatory
oxidase, although the exact significance and function of these properties, which may put these infants at a disadvantage by
interactions remain uncertain (22, 68). Xanthine oxidoreductase, creating a relative deficiency of anti-inflammatory factors and
another protein found in large amounts in milk and upregulated activity.
in mature milk, affects mammary epithelium, generation of milk Any enteral feeding directly stimulates growth of the neonatal
fat droplet membranes, and adds to the bactericidal effect of gut. Human milk contains specific direct growth factors including
human milk by synthesizing ROS (64, 66). These different AMPs platelet-derived growth factor, hepatocyte growth factor (HGF),
do not simply act in microbial recognition and inactivation; each vascular endothelial growth factor, and insulin (74–76). Each of
have different secondary functions within the intestine. Equally these is important in angiogenesis, cell development, and tissue
important is the limited inflammatory response generated by proliferation. HGF is expressed in the intestinal tissues and is
these AMPs in the gut. present in human milk. HGF may play a role in mucosal growth
and repair (77). Animal studies have shown that HGF given after
ANTI-INFLAMMATORY FACTORS intestinal resection or colitis has improved gut proliferation and
AND EFFECTS nutrient transport (78, 79). Weiss et  al. (80) describe declining
levels of anti-inflammatory, proresolving lipoxin A4 (LXA4), and
Maintenance of a homeostasis between protective inflammation resolvin D1 (RvD1) and D2 (RvD2) in the lipid profile of human
and modulation of inflammation is essential to protecting the milk from colostrum through the first month of life. The average
infant against infection at the same time as limiting the tissue amount of LXA4 in human milk was two times the amount of
damage due to inflammation (59). Oxidative stress through the proinflammatory leukotriene B4 (LTB4) (80). This highlights the
production of free radicals does have some potentially beneficial importance of minimizing inflammation in the early period of
effects for the host in terms of antibacterial action, immune an infant’s life.
defense, and signal transduction. The oxidative activity within Specific antioxidants in human milk include vitamins A,
the infant must be maintained in equilibrium with antioxidant E, C, LF, lysozyme, glutathione peroxidase, SOD, catalase,

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Cacho and Lawrence Breast Milk Immunity

ceruloplasmin, coenzyme Q10, thioredoxin, leptin, adiponectin, 3–4 months, while the amount of lysozyme increases (87). Tregoat
and trace elements—iron, copper, zinc, and selenium (69). These et al. (88) described declining amounts of mannan-binding lectin
antioxidants act by reacting directly with a free radical before concentrations in transition from colostrum to mature milk.
damage occurs or by interfering with the ongoing oxidation More recent published studies using proteomic analysis of human
in liquid phase or in cell membranes. The total antioxidative milk (16, 64, 89) continue to facilitate our understanding of the
capacity of human milk is highest in colostrum and declines over complex nature of human milk and its role related to immune
lactation with variability from person to person and time to time function and intestinal development. Gao et al. (64) describe a
(69). Adiponectin, leptin, LF, and lysozyme each have antioxidant similar transition of the various Igs, from colostrum to mature
effects, and their concentrations in human milk also vary over milk using proteomic analysis of the milk. They describe a large
time (81). Vitamin A or its derivatives bind to radicals of oxygen, percentage of milk proteins having to do with immune function
thiol, or peroxide, limiting their oxidative damage on cells. There including complement factors and serine protease inhibitors
are higher concentrations of vitamin A in colostrum than mature important in regulating the complement system. The quantities
milk (82). Higher concentrations of α-tocopherol (the predomi- of these factors declined in transition from colostrum to mature
nant form of vitamin E) are in colostrum than transitional or milk. They also identified proteins associated with the extracel-
mature milk (83). Vitamin E forms part of the milk fat globule lular matrix including cytokines, fibronectin, tenascin, and
membrane and constitutes the major portion of antioxidative OPN. These proteins were more prevalent in transitional milk.
function of breast milk at 1  month of age (84). Vitamin C is a Glutathione and antioxidant activity-related proteins were more
hydrosoluble vitamin in human milk and an effective antioxidant common in mature milk. Overall, the quantity of immune factors
in extracellular fluids. and immune effects of human milk diminish over time in parallel
Nucleotides, nucleosides, and nucleic acids are essential to with the developing immune system of the infant. Nevertheless,
cellular metabolic function. They constitute approximately it will be essential to understand the specific roles of the various
15–20% of the non-protein nitrogen or total potentially available bioactive components of human milk and how the change in milk
nucleosides in human milk (85). Nucleotides function predomi- composition over time influences the evolving effects of human
nantly in cellular energy metabolism related to adenosine triphos- milk on the intestine and innate immunity.
phate, as messengers and coenzymes in metabolic pathways, and
in nucleic acid production and salvage. In the face of infection, DIFFERENCES IN PRETERM AND TERM
nucleotides are essential to the immune response (cellular activa-
HUMAN MILK
tion, proliferation and action, and cellular signal transduction)
and the repair of intestinal inflammation and damage. Brunser Preterm babies require additional nutrition and immune protec-
et al. (86) reported on the benefit of nucleotide supplementation tion compared to term infants. Interestingly, preterm breast milk
for infants less than 6 months old at decreasing the severity and has been found to contain increased nutrients such as protein
incidence of diarrhea. They postulated that the effect was due to (90) and higher concentrations of certain immune factors.
effects on the intestinal integrity and repair as an example of an Preterm human milk also has higher amounts of phagocytes and
anti-inflammatory effect. secretory IgA (24). These increased amounts may serve a protec-
tive role since premature infants have poorly functioning neu-
EVOLUTION OF BIOACTIVE FACTORS IN trophils, limited production of Igs, and lower levels of passively
HUMAN MILK OVER LACTATION acquired Igs.
A few studies reported a difference in breast milk microbiome
The composition of human milk is dynamic with significant when comparing preterm and term milk. Some trends include
change from colostrum, transitional to mature milk, between more Bifidobacterium in term milk (91) and more Enterococcus
preterm and term milk and with interindividual and intraindi- in preterm milk (91). A study looking at preterm infants, testing
vidual variation. Specifically, HMOs show interindividual varia- stool and breast milk samples, found a high proportion of anti-
tion relative to the total number of HMOs and individual HMOs biotic-resistant high-risk clones in both fecal and milk samples
varying with mother’s Lewis blood group and secretor status. during the neonatal intensive care unit (NICU) admission (92).
Colostrum is produced between birth through the first 5 days Differences are also seen in gut microbiome between term and
of lactation, transitional milk is from 5 days to 2 weeks postpar- preterm infants. Variations in microbiota of preterm infants have
tum and maturation of the milk continues until it is “fully mature” been described as predisposing to development of necrotizing
at 4–6 weeks postpartum. There is only a small volume of colos- enterocolitis (NEC) (93–97). Hormones and cytokines also vary
trum produced, rich in leukocytes, protein, HMOs, and bioactive by gestational age. EGF has anti-inflammatory properties and
factors—IgA, LF, EGF, TGF-β, colony-stimulating growth factor, is higher in preterm milk compared to full-term milk (24) The
and antioxidants (31). Transitional milk has decreasing amounts cytokine IL-10, with anti-inflammatory properties, was detected
of protein and Igs and increasing lactose and fat and water-soluble in lower amounts in breast milk for infants with increased risk of
vitamins resulting in a higher caloric density of the milk to meet NEC (98). Trend et al. reported that TGF-β2 concentrations in
the infant’s growth demands while the quantities of bioactive fac- human milk were significantly higher in the extreme premature
tors declines over time. The composition of mature milk remains infant group compared to the term infant group (99). Castellote
constant after 6  weeks through the remainder of the lactation et  al. also reported that TGF-β2 was higher in preterm infants
period. The amount of Igs and LF in milk decreases over the first compared with term infants (100). A study by Maheshwari et al.

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Cacho and Lawrence Breast Milk Immunity

demonstrated that this anti-inflammatory cytokine suppresses and term milk remain unknown. Interestingly, most immune
endotoxin-induced cytokine responses of gut macrophages in factors decrease over the first month regardless of gestational age,
the preterm infant in vitro and protects rat pups from gut injury thus term and preterm milk become more similar over time as the
in vivo (101). They also reported that percentages of IL-10 and chronological age of the baby increases.
TNF-α were lower in preterm milk compared to term milk.
Trend et al. found that AMPs can limit the in vitro growth of SUMMARY
bacteria associated with neonatal sepsis (52). This is particularly
important in preterm infants who are at increased risk of late-onset Feeding an infant human breast milk is not a matter of filling
sepsis. The hBD-1 was higher in preterm colostrum compared to the infant with an “appropriate” amount of important nutrients
term colostrum (99). Wang et al. (102) also found that hBD-1 and and a protective level of bioactive factors. Although the various
hBD-2 were in higher concentration in mature preterm milk than factors do complement and supplement the innate immunity of
mature term milk. Armogida et al. and Trend et al. both found the infant, they actively affect the ongoing development of the
that human α-defensin 5 levels were not affected by preterm birth, infant’s immunity and intestinal development. As Lars Bode et al.
which suggests that these defensins are differentially regulated declared, “Human milk is alive, …” (27). The cells, the microbes,
(99, 103). The amount of LF in human milk does not seem to be and the bioactive factors make milk alive, and the interactions of
affected by gestational age (99) but is more dependent on milk human milk with its natural host, the infant, create a symbiotic
volume expressed. There are conflicting results for concentrations commensal relationship. This is the challenge to explain and
of lysozyme in preterm and term milk. understand the complexity and dynamic relationship between
It has been proposed that the increased levels of immune factors the everchanging secretion, human breast milk and the develop-
in preterm milk may be a result of a compensatory mechanism ing, evolving human infant.
whereby in the mother during preterm labor, the breast shifts the
immune content of the milk to provide more protection. Another AUTHOR CONTRIBUTIONS
explanation suggested by Goldman et  al. (62) is that increased
immune factors in preterm milk may be due to increased maternal All authors have made substantial, direct, and intellectual
systemic inflammation, a postulated condition leading to preterm contribution to the work and approved it for publication. NC is
delivery. The mechanisms for the differences between preterm primarily responsible for Figure 1.

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Cacho and Lawrence Breast Milk Immunity

103. Armogida SA, Yannaras NM, Melton AL, Srivastava MD. Identification and Copyright © 2017 Cacho and Lawrence. This is an open-access article distrib-
quantification of innate immune system mediators in human breast milk. uted under the terms of the Creative Commons Attribution License (CC BY).
Allergy Asthma Proc (2004) 25(5):297–304. The use, distribution or reproduction in other forums is permitted, provided
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Frontiers in Immunology  |  www.frontiersin.org 10 May 2017 | Volume 8 | Article 584