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Human milk bioactives and roles in the GI tract
Key points
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In addition to nutrients, human milk contains myriad bioactive components that promote development of neonatal intestinal immunity.
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A range of bioactive proteins in human milk function in protection against infection, immunomodulation, and nutrient utilization.
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Human milk growth factors and hormones modulate growth and development of the intestines and have immunomodulatory effects.
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Human milk oligosaccharides promote a healthy intestinal microbiome and protect infants from infection.
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Human milk exposes infants to maternal bacteria and their metabolites, which have immune-modulating effects.
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Research on human milk extracellular vesicles and miRNA is relatively new; however, emerging data suggest these components play important roles in programming intestinal immune ontogeny.
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Bioactive factors in human milk are particularly important for preterm infants, for whom prenatal intestinal development is interrupted.
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Human milk bioactive factors are unique and many are lacking and/or inactive in bovine milk and infant formula.
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Milk processing techniques, such as pasteurization, impact the activity of various milk bioactive substances.
Introduction
In addition to providing the gold standard of nutrition for infants, human milk also contains numerous components that are considered bioactive. These are not utilized directly for nutritional purposes and instead provide for specialized needs of the infant, such as immunoprotection and immunomodulation, metabolic homeostasis, digestion, regulatory activities, and microbial colonization. These factors are usually not found in commercial formulas unless specifically added. Additionally, many are not found in banked donor milk due to inactivation during pasteurization or storage. Given the personalized nature of human milk and the communicative role of milk in the co-adapting mother-infant-milk triad, bioactive factors in donor milk may not be dynamically interactive with the infant. Thus mother’s own milk (MoM) plays an important role in guiding early intestinal development.
This chapter summarizes some of the important and well-known bioactive components found in human milk, how they synergize with the intrinsically programmed development of the intestine during neonatal life, and how they are affected by maternal and environmental factors. We apply a special focus on preterm infants, born before 37 weeks of gestational age, and how they differ from those born at term.
Ontogeny of intestinal immunity
The development of the intestinal immune system begins in utero ( Fig. 8.1 ). This prior immune development is critical given that, at birth, the neonate is challenged with extraordinary bacterial and antigenic exposures. Several maturational events occur during fetal development, providing the neonate with an armamentarium of immunologic weapons that protect the intestinal mucosal surface from invasion by pathogens and allow immunomodulation for tolerance to potentially beneficial microorganisms and antigenic nutrients. While the hallmarks of a well-functioning intestinal immune system, including mononuclear leukocytes in the lamina propria and organized gut-associated lymphoid tissue, are identifiable by 200 days gestation, these do not reach functional maturity until 4 to 6 months after birth. Many of the mechanisms through which this maturation occurs are intrinsic to the intestine; however, extrinsic stimuli, such as those provided by human milk, augment the intrinsic mechanisms. This is particularly important for infants born preterm, who have not had the full complement of in utero development.
Human milk promotes the development of the intestinal immune system and protects preterm infants against adverse consequences. These include intestinal injuries and dysfunction (often placed under the umbrella term necrotizing enterocolitis or NEC ), chronic lung disease, infection, sepsis, and retinopathy of prematurity. Interactions between human milk bioactive components and the developing neonate also play a major role in later neurodevelopment, food allergy, inflammatory bowel disease, metabolic syndrome, and even transgenerational outcomes.
What’s special about the preterm infant
Many aspects of the innate immune system are present at the time of term birth, but many are lacking when the infant is born preterm. At birth, even in infants born at term, the adaptive immune system is not yet trained and hence relies on transplacental passage of maternal IgG during the last three months of gestation in addition to maternal antibodies provided via breastfeeding. The immune response to gut pathogens in term newborns requires protein components from mother’s milk to efficiently fulfill toll-like receptor (TLR) binding and signal transduction. Innate immune functions in the fetus are developmentally regulated during pregnancy; hence, the preterm infant has a high propensity to be at risk of developing severe infections.
The significantly higher risk of infection for preterm infants is influenced by several factors ( Fig. 8.2 ). Infants born preterm have reduced proportions of peripheral blood lymphocytes, fewer Paneth cells, reduced production of antimicrobial peptides, a patchy intestinal mucus layer, higher permeability of the epithelial tight junctions, and lower counts of phagocytic peripheral monocytes. Additionally, passive immunity transferred to the fetus via the placenta increases with gestational age.
Breastfeeding
Breastfeeding has been shown to reduce infant mortality by 12% compared to formula feeding and to be greatly beneficial in both short- and long-term disease prevention. Numerous mechanisms related to immune, nutritional, and bioactive components of human milk have been reported to account for this beneficial effect.
Human milk is a dynamic biofluid that is responsive to infant and mammary health status. Temporal changes to human milk composition occur over the course of lactation (from colostrum to mature milk), over a day (circadian rhythms), and even over the course of a feed. Some bioactive factors of human milk are at a higher concentration in colostrum (the “first milk”) due to open tight junctions between mammary epithelial cells during this period ( Table 8.1 ). Among these, immunoglobulins and immune cells provide passive immunity transfer from the mother. The high concentration of these protective factors in colostrum provides early protection during the vulnerable neonatal period. Transfer of maternal cells is also thought to help with immune responses and tissue repair.
Table 8.1
Bioactive Components of Human Milk and Their Function a
| Human Milk Component | Function | Concentration in Colostrum | Concentration in Mature Milk |
|---|---|---|---|
| Lactoferrin | Immunomodulation, antiviral, antibacterial, iron metabolism | 5.8 g/L | 2 g/L |
| Lysozyme | Antibacterial and antiviral | 0.36 g/L | 0.4 g/L |
| Microbes | Infant microbiome colonization, infant immune training | 10 6 cells/mL | 10 6 cells/mL |
| Exosomes | Cell-to-cell communication, carriers of “cargo” such as nucleic acids and proteins | 1.62 particles/mL | 0.4 particles/mL |
| Immunoglobulins | Transfer of immunity from mother to infants, aggregation of bacteria for immune exclusion, entrapment of bacteria via hydrophily, reduction of bacterial motility via binding to flagella |
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| Enzymes | Digestion, absorption, mitigation of inflammation |
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| Growth factors | Maturation of intestinal mucosa, neuronal growth, stimulation of cell proliferation and maturation, angiogenesis |
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| Oligosaccharides | Prebiotic stimulation of bacterial growth, decoy pathogen receptors, immune modulation | 20–30 g/L | 5–15 g/L |
| miRNAs | Modulation of immune pathways, microbial colonization, oxidative stress, inflammation, development |
|
|
EGF , Epidermal growth factor; VEGF , vascular endothelial growth factor.
a Many of these compounds are targets of active research for addition to commercial formulas due to their potentially beneficial activities.
Human milk bioactive components
Human milk contains a variety of bioactive factors such as hormones, cytokines, leukocytes, immunoglobulins, lactoferrin, lysozyme, stem cells, oligosaccharides (HMOs), microbiota, metabolites, and microRNAs ( Table 8.1 ). Understanding the roles of human milk bioactive factors on immune function provides a scientific basis upon which to build breastfeeding recommendations. It may also enhance subsequent health in those infants who can only formula feed by providing modifications to formulas that provide some of the benefits incurred by human milk. However, there is still much to learn about interactions between the various human milk components and the mechanisms underlying their health benefits. Here we will discuss the major bioactive components of human milk and their role in intestinal ontogeny and immune development.
Proteins and peptides
It is well recognized that breastfed infants have fewer infections than formula-fed infants. Several human milk proteins have been shown to be involved in protecting against infection ( Fig. 8.3 ). , These are discussed in the subsections that follow.
Lactoferrin
After whey and casein, lactoferrin is the most abundant protein in breast milk. Its role is multifaceted ( Fig. 8.4 ): it plays a major role in immunity of the infant, prevents infection, is involved in iron metabolism, has antiinflammatory properties, and is an antioxidant.
In the early 1970s, human milk was found to have bacteriostatic activity against Escherichia coli . The active agent, lactoferrin, was found to be a protein with high affinity for iron. Lactoferrin can sequester environmental iron required for pathogen survival. This action confers a bacteriostatic effect that inhibits the growth or reproduction of bacteria. As a corollary, the growth of those bacteria that do not require much iron is enhanced. Many of these taxa that do not require iron are beneficial to humans, such as Lactobacillus and Bifidobacterium . Lactoferrin also has a direct bactericidal function. With its high positive ionic charge, lactoferrin can form a complex with lipopolysaccharide (LPS), a highly negatively charged bacterial cell wall component, creating holes in the outer membrane of Gram-negative bacteria. These membrane breaches can be fatal for bacteria and can allow penetration of another human milk protein, lysozyme, which degrades the proteoglycan matrix and kills the bacterium.
In addition to its antibacterial activities, lactoferrin also has important antiviral properties. The antiviral action of lactoferrin is mediated through its ability to block viral entry into host cells. It does this by binding to heparan sulfate glycosaminoglycan cell receptors or by binding directly to viral particles. As such, lactoferrin’s antiviral action is important during the initial phase of infection. Highly relevant to the recent COVID-19 pandemic is evidence that lactoferrin can bind to at least some of the receptors used by coronaviruses and thereby block their entry. Thus lactoferrin may contribute to protection from COVID-19 infection in breastfed infants.
The iron-binding capacity of lactoferrin increases intestinal iron absorption. Each lactoferrin molecule can bind two iron ions. Lactoferrin molecules enter intestinal epithelial cells via their own receptor, then release their bound iron into the cell for transportation to the circulation via transferrin. Lactoferrin thereby aids in iron uptake from human milk, which is notably low in iron.
Lactoferrin levels change as milk matures. Colostrum has higher concentrations in both term and preterm milk. , Preterm milk tends to maintain higher levels of lactoferrin over time, providing prolonged protection from infection for these vulnerable infants. Concentrations of lactoferrin similar to those found in human milk increase intestinal cell proliferation, whereas lower concentrations increase intestinal cell differentiation. , The implications for this finding are that the intestinal mucosa of breastfed infants is more developed than that of formula-fed infants. Increased mucosal development caused by lactoferrin may therefore increase the mucosal surface and enhance the uptake of iron as well as other nutrients.
Lactoferrin is also present in cow’s milk; however, studies in vitro have shown that human milk is more effective in preventing bacterial growth than cow’s milk. Studies investigating the addition of cow’s milk lactoferrin to breast milk, donor human milk, and infant formula have shown no differences in late outcomes of preterm infants between treated and control infants.
Lysozyme
Widely distributed in tears, saliva, and milk, , lysozyme plays important roles as a nonspecific immune factor. Lysozyme , first described by Alexander Fleming in 1922, is an enzyme that catalyzes the hydrolysis of 1,4 beta linkages between sugar molecules found in peptidoglycan, which is the major component of the Gram-positive bacterial cell wall. This hydrolysis of peptidoglycan compromises the integrity of bacterial cell walls causing lysis (or disintegration) of the bacteria, hence its name. In addition to its bactericidal effects on Gram-positive bacteria, it acts synergistically with lactoferrin to destroy Gram-negative bacteria. Following penetration of the cell wall by lactoferrin, lysozyme can enter the cell and disrupt cellular respiration.
The lysozyme content of human milk ranges from 3 to 3000 μg/mL, and the typical concentration is about 200–400 μg/mL. , Cow’s milk comparatively contains only minute quantities of lysozyme and has a lower activity compared to human milk. , Reduced lysozyme sulfate levels have been associated with bronchopulmonary dysplasia in preterm newborns. The relevance of this association between lysozyme, which is found in high concentrations in human milk, and a lower incidence of bronchopulmonary dysplasia seen in preterm infants is of interest, but causality has not yet been proven.
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