Shaping infant development from the inside out: Bioactive factors in human milk

Abstract

Human breast milk is the optimal nutrition for all infants and is comprised of many bioactive and immunomodulatory components. The components in human milk, such as probiotics, human milk oligosaccharides (HMOs), extracellular vesicles, peptides, immunoglobulins, growth factors, cytokines, and vitamins, play a critical role in guiding neonatal development beyond somatic growth. In this review, we will describe the bioactive factors in human milk and discuss how these factors shape neonatal immunity, the intestinal microbiome, intestinal development, and more from the inside out.

Introduction

Human milk is more than nutrition for growing human infants. This rich liquid is a personalized, biological system containing myriad bioactive factors, including nutrients, human and bacterial cells, immunomodulators, and extracellular vesicles (EVs), which all build and shape the health and development of the smallest and most vulnerable recipient. This biological fluid generated by a mother’s own body is custom-made for her growing infant, matching not only its nutritional needs through fat content or nutritional balance, but also immunological needs through antigen matching and providing for the infant’s developing intestinal microbiome, through bacterial seeding and nutrients in the form of human milk oligosaccharides (HMOs). This review summarizes the vast literature describing the numerous bioactive components of human milk, including how they serve the developing infant and how they are impacted by the mother¹ and the environment² (Figure 1).

Figure 1. Human milk is a dynamic and customized biological system fueling infant growth and development.

Human milk is a superfood teaming with proteins, peptides, cytokines, growth factors, lipids, vitamins, oligosaccharides and glycans, bacteria, immune cells, immunoglobulins, hormones, and extracellular vesicles all produced, absorbed, or packaged within the mammary gland. This diet is matched to the infant’s needs as best as the mother is able, based on her nutritional status, health, genetics, and the environment. Figure created with https://biorender.com.

Probiotics

Until the early 2000s, human milk was considered sterile^3–5. Since 2003 the number of studies describing the “human milk microbiome” has skyrocketed. This term refers to the collective bacteria and their genetic material present within a biological tissue or system⁶, in this case, human milk. The human milk microbiome is diverse and complex, containing a wide variety of bacterial species⁷. The bacterial community is generally stable over time, although variation between mothers results from differences in the maternal diet, body mass index (BMI), delivery mode, and overall maternal health, including the use of certain medications^{2, 8}, and preterm delivery^{2, 9}. Many of the bacteria present within human milk are the first colonizers of the infant intestinal microbiome, with a proportion of the genera present within the infant fecal microbiota matching those present within human milk¹⁰. Some of the core bacterial genera found within human milk include Pseudomonas, Staphylococcus, Streptococcus, Bifidobacterium, Lactobacillus, and Propionibacterium^{7, 8, 10–14} and include genera found on the skin, within the infant mouth¹⁵, the mother’s intestinal tract^16–19, and the mammary gland^{8, 10}. The composition of the milk microbiome is influenced by feeding mode and changes with feeding pumped human milk or tube feeding compared to breastfeeding, demonstrating the role of an infant’s oral microbiota in shaping the microbial composition of the milk²⁰. These early colonizers stimulate appropriate immune system development and function in the infant^21–23 and provide competition for other pathogenic species that otherwise may colonize the infant intestinal tract, thereby protecting the infant from enteric diseases such as necrotizing enterocolitis (NEC)²⁴. Notably, studies using germ-free mice (lacking all microbes) suggest that the timing of colonization in early development is key to preventing later disease through appropriate immune cell priming²⁵. Establishing a healthy intestinal microbiome from birth is associated with long-term benefits to overall health, reducing the risk of allergies^{21, 26, 27}, asthma²¹, inflammatory bowel diseases²¹, obesity^{28, 29}, and metabolic disease³⁰. Understanding which bacterial species confer beneficial effects on infant health and development over the short and long-term is crucial when developing infant formulas or probiotic supplements. Additionally, defining the nutrient sources these bacteria require to survive will be vital for promoting their colonization in the infant gut.

Human milk oligosaccharides (HMOs)

HMOs are natural prebiotics found within human milk. The term prebiotic indicates that these molecules are not digestible by the infant; instead, they are utilized by the host microbiota and selectively cultivate the growth of key microbial communities within the infant intestine^{31, 32}. HMOs are composed of five basic monosaccharide components: glucose, galactose, N-acetylglucosamine, fucose, and sialic acid⁹. These building blocks are arranged and linked in different ways to produce around 200 unique HMOs within human milk³³. HMOs are the third most abundant macronutrient in human milk, following lactose and lipids³⁴. These complex carbohydrates vary between mothers and are influenced by geographical location, lactation stage, and the mother’s genetics^34–36. Although these structures cannot be digested and utilized by the infant, they serve a critically important role in shaping the health and long-term outcomes of the newborn^{34, 37, 38}. HMOs are a food source for the developing intestinal microbiota^{9, 39, 40}, and they prevent bacterial and viral infection^{34, 41–43}, including direct adherence to or invasion of the intestinal cells^44–47, neutralizing bacterial toxins⁴⁸ and stimulation of immune priming⁴⁹. HMOs also directly influence intestinal epithelial cell (IEC) gene expression and response to injury in a structurally-specific manner, demonstrating that all HMOs are not created equal^{50, 51}.

Each bacterial species possesses specific enzymes capable of cleaving the different linkages within each HMO and thereby self-selecting for growth on specific substrates⁹. One elegant example is the bacterial species Bifidobacterium longum subsp. infantis (B. infantis), which is endowed with all the metabolic machinery required to break down HMOs with the lacto-N-tetraose [LNT] or lacto-N-neo-tetraose [LNnT] structure found solely in human milk-fed infants^{39, 40}. Notably, other Bifidobacterium subspecies associated with the adult microbiome lack the enzymatic activity to utilize HMOs for fuel⁹. B. infantis is one of a very small number of bacteria with this unique ability. Most bacteria can cleave one or two HMO linkages and therefore work in concert with other species to fully utilize HMO substrates, a phenomenon termed cross-feeding. A recent study identified lower levels of the HMO disialyllacto-N-tetraose in mom’s milk of infants with NEC. These infants also had lower levels of Bifidobacterium longum, indicating a link between human milk HMOs, the infant microbiome, and severe intestinal disease⁵². Understanding the bacterial-independent benefits of HMOs, as well as which bacteria are capable of utilizing specific linkages and the subsequent functional benefits of these species, is critical when attempting to mimic human milk for nutrition. Introducing substrates that unwanted or few bacterial species can utilize may impair microbial diversity or skew it toward a pathogenic state.

Extracellular vesicles (EVs)

Human milk is packed with extracellular vesicles⁵³. EVs are lipid bilayer-encased particles released from cells that carry biological cargo to send intercellular signals⁵⁴. In the case of human milk, these signals are transmitted from mother to baby. Almost all organisms, from bacteria to animals, produce EVs⁵⁵. For a more detailed discussion of the origin and initial studies on EVs, see⁵⁶.

Milk EV cargo are reflective of the cell type and physiological state of the secreting cell. The cells of origin for milk EVs could be mammary epithelial cells, as they exhibit a similar protein profile⁵⁷, local immune cells, or distant cell types whose EVs travel to the breast via the blood or lymphatic circulation. Milk EVs carry myriad proteins^57–60, lipids⁶¹, and non-coding RNAs, including miRNA and long non-coding RNA (lncRNA)^62–65. Bovine and porcine milk EVs are reported to carry mRNA; however, there are no reports of mRNA within human milk EVs to date^{66, 67}.

In vitro simulated digestion studies suggest that EVs can survive in low pH environments and in the presence of select digestive enzymes^{68, 69}, indicating that they should survive to the intestine to exert their functional effects. Studies indicate that milk EVs are absorbed by intestinal epithelial cells (IEC) via endocytosis^68–71, where their cargo can act locally or be transported to distant organs, including the brain, heart, liver, and spleen^72–74. In animal models of NEC, direct IEC-EV contact was important for beneficial effects on intestinal function compared to intraperitoneal EV administration⁷⁵.

Milk EV cargo can have anti-inflammatory, immunomodulatory, or neurodevelopmental effects and improve the barrier function of the intestinal epithelium^{56, 59, 61, 76–82}. For example, miRNAs found within milk EVs can alter the gene expression of immune cells⁸³ or reduce viral transmission^{84, 85}. Human milk EVs reduce inflammatory damage and promote intestinal regeneration in animal models of NEC^{58, 86}. They can also improve oral epithelial barrier function and dampen T cell activation in the absence of tolerance induction⁸⁷, suggesting that EVs can regulate immune responses, which is critical during immune development.

Similar to many other bioactive factors within human milk, EVs are affected by gestational age^{58, 88}, lactation peroid^{89, 90}, and maternal diet and health, including BMI, smoking, and stress^{76, 91}. Milk EVs and their cargo are sensitive to freezing⁹², microwave heating⁹³, and pasteurization⁹⁴; therefore, methods of human milk storage and processing should be carefully considered as studies continue to map the role of EVs and their cargo on infant health and development. Further, the role of EVs and their individual or collective cargo should be considered when manufacturing infant diets. More research is needed to determine the mechanisms behind the beneficial effects of individual EV cargo.

Proteins/Peptides

The protein composition of human milk is complex, encompassing greater than 1000 individual proteins, grossly divided into wheys, which constitute approximately 60–80% of milk proteins depending on the stage of lactation, and caseins, comprising the remaining protein content^95–99. Included in the caseins are proteins with well-recognized bioactivities in their intact forms: immunoglobulins, antibacterial proteins such as lactoferrins, growth factors, hormones, immunomodulators, enzymes (proteases, bile salt-sensitive lipase), and more^100–102. In addition, milk EV cargo also includes proteins, as described above. Whey proteins are more highly proteolyzed and thus provide a substantial portion of the enterally-absorbed amino acids for anabolism, while caseins are less proteolyzed during digestion. While some proteins in undigested milk have defined bioactivities, whether the activity of a given bioactive protein survives digestion in vivo remains to be comprehensively explored. From studies of in vivo digestion¹⁰³ and milk proteins in stool¹⁰⁴, it is known that select milk proteins can survive gastrointestinal proteolysis, examples being lactoferrin¹⁰⁴ and milk immunoglobulins^104–107.

During digestion, human milk proteins are proteolyzed into constituent peptides. Many of these peptides, in particular, those from whey, continue to be further proteolyzed into oligopeptides and amino acids, but some persist and are excreted in stool¹⁰⁸. From in silico studies¹⁰⁹ and in vitro digestions, milk proteins are comprised of hundreds of cryptic bioactive peptides with activities relevant to infant health. These activities include bacterial growth inhibition^{110, 111}, commensal bacterium growth stimulation¹¹², immune modulation^{110, 113, 114}, promoting cellular proliferation¹¹⁵, and stimulation of mucin production¹¹⁶. The in vivo presence of bioactive milk protein-derived peptides is being explored^{114, 117–119}. There is evidence from intestinal contents of infants that there are peptides that possess anti-inflammatory activity on monocyte cell lines in vitro¹¹⁹ and can modulate bacterial growth and survival¹¹⁴. Thus, the presence, roles, and identities of milk protein-derived bioactive peptides are not yet well defined and add another exciting dimension to the study of milk as a complex biological system.

Immunomodulators

Immunoglobins

Immunoglobins (Igs) are an important immunomodulatory factor found in human milk, which provides significant passive immunity to infants. The five different types of immunoglobulins include IgA, of which secretory IgA is the most predominant Ig in human milk, IgG, which can cross the placenta and is associated with long-term systemic immunity, IgM, which mediates resistance against bacterial and viral infections, as well as IgE and IgD, which are less studied and their role in infants is not well understood^120–122. The various roles that breast milk Igs play in disease prevention have been elegantly reviewed in ¹²³. Importantly, maternal IgA impacts the infant microbiome and mediates protection against NEC¹²⁴. Prematurity and gestational age can significantly impact the breast milk composition of immunoglobulins¹²⁵, but factors including maternal living conditions, lifestyle, and environmental factors can also affect the Ig composition¹²⁶. Maternal Ig transfer remains a critical component in the development of neonatal immunity, and the precise composition of Igs in breast milk, along with their impact on neonatal and pediatric diseases, is an important area of ongoing investigation.

Growth Factors

Epidermal Growth Factor

Human milk contains several growth factors, including epidermal growth factor (EGF) that are critical to intestinal development¹²⁷ maturation of the gut barrier¹²⁸, and intestinal epithelial homeostasis¹²⁹. EGF is abundant in human milk and colostrum and has been shown to provide protection against NEC^130–133, as well as late-onset sepsis¹³⁴. Described mechanisms mediating this protection include inhibition of the pro-inflammatory signaling pathway of toll-like receptor 4¹³¹, enhancing IEC proliferation¹³⁵, increased mucus production and goblet cell density¹³², as well as inhibition of bacterial translocation from pathogens in the gut¹³⁴. Together these studies demonstrate the potential of EGF as a preventative therapy in high-risk premature infants.

Heparin-binding epidermal growth factor-like growth factor

Another member of the EGF family is the Heparin-binding epidermal growth factor-like growth factor (HB-EGF), which is present in both amniotic fluid and human milk, suggesting a potential role in intestinal development¹³⁶. Similar to EGF, HB-EGF also has been studied in the context of preventing NEC, intestinal ischemia/reperfusion, and hemorrhagic shock in pre-clinical studies¹³⁷. HB-EGF also promotes IEC proliferation and migration important for tissue repair and regeneration^{138, 139}. In pre-clinical studies of intestinal inflammation, HB-EGF decreased the incidence of experimental NEC^{140, 141}, reduced intestinal cell apoptosis¹⁴⁰, and increased intestinal blood flow¹⁴². The clinical significance of HB-EGF was studied by evaluating infants with NEC compared to controls, and investigators found an HB-EGF single nucleotide polymorphism associated with NEC, and noted that plasma HB-EGF levels were lower in patients with NEC compared to those without¹⁴³.

Insulin-like growth factor-1

A key mediator of growth in infants is insulin-like growth factor-1 (IGF-1), and in a large study of Finnish mothers, this growth factor was found to be highly abundant in human milk¹⁴⁴. Higher human milk concentrations of IGF-1 were associated with higher weight after a year of age, but lower weight at 3 and 5 years of age¹⁴⁵, demonstrating the lasting effects that human milk and its components have on the growth and development of infants.

Cytokines

Many cytokines are present in human milk and provide immunomodulation to the immune system of the infant^{102, 146}. Specifically, interleukin (IL)-1β, IL-2, IL-6, IL-8, IL-10, IL-12, IL-18, interferon (IFN)-γ, and tumor necrosis factor (TNF)-α have physiologic relevance to the infant (reviewed in ^{102, 147}). Decreased immunomodulating components in the colostrum and breast milk from the mothers of premature infants compared to term infants^{146, 148} demonstrate that cytokine concentrations can change throughout different stages of lactation¹⁴⁹. One particular cytokine, transforming growth factor-β2 (TGF-β2), is a multi-functional cytokine present in high concentrations in human milk and is associated with immunomodulatory effects in infants^{150, 151}. These immunomodulatory effects include the production of immunoglobulins, richness in the diversity of the composition of neonatal gut microbes¹⁵², and TGF-β2 can dampen the intestinal macrophage inflammatory responses in the developing intestine¹⁵³ and attenuate intestinal injury¹⁵⁴. In clinical studies, investigators have shown that lower blood and tissue concentrations of TGF-β were associated with NEC in very low birth weight infants^{153, 155}. The origin of cytokines present in breast milk is not well understood but may be related to the leukocytes or epithelial cells present in human milk, which can support the immune system development in the infant¹⁴⁹. Additional studies are needed to determine how specific cytokines can shape neonatal immunity and whether an optimal concentration of these cytokines can be supplemented to infants as a personalized nutrition strategy.

Vitamins and micronutrients

Human milk is a rich source of vitamins and micronutrients. Vitamins are biological molecules that must be supplied by the diet, as they are not produced in the body but are required for growth and biological processes. Human milk contains water-soluble B vitamins (B₁, B₂, B₃, B₆, B₉, B₁₂), vitamin C, and lipid-soluble vitamins A and E^156–158. The levels of these vitamins present in maternal milk can vary based on the birth weight of the infant¹⁵⁹. Collectively, the B vitamins are coenzymes in metabolic processes and are crucial for neurotransmitter synthesis¹⁵⁶. Vitamin B₁ (thiamine) deficiency is uncommon in the US but occurs more frequently in developing countries such as Cambodia or Laos, where mothers receive inadequate thiamine in their diets^{160, 161}. Vitamin B₁ deficient infants are then at risk for beriberi, which is quickly fatal if left untreated^{160, 161}. Thiamine deficiency can also have long-term consequences on cognitive development¹⁶². Vitamins C and E are important antioxidants protecting developing tissues from oxidative stress and damage early in the postnatal period¹⁶³. Vitamin A is critical for vision, cell growth and development, and immune system function^164–167. Human milk and colostrum are excellent sources of vitamins E and A^{168, 169}. Vitamin E deficiency and subsequent oxidative stress can be linked to respiratory disease, congenital malformation, retinopathy, affect the central nervous system, and increased mortality^{170, 171}; however, high dose supplementation can lead to sepsis in premature infants¹⁷⁰. Many vitamins enter breast milk from the maternal diet. In one study, vitamin A and E levels correlated with self-reported vitamin supplementation¹⁶⁹, suggesting that dietary modulation could increase levels in breast milk. Thiamine levels can also be modulated through maternal supplementation¹⁶². However, this is not the case with vitamin D¹⁵⁶.

Human milk is not a good source of vitamins D or K¹⁵⁶, which are supplemented to infants. The hydroxylated derivatives of vitamin D (25-OH and 1,25-OH₂ vitamin D) are responsible for intestinal and renal calcium and phosphorus absorption, which support bone health, while vitamin K plays a role in blood clotting pathways¹⁵⁶. In the US, infants receive a vitamin K injection at birth, which is sufficient supplementation to prevent intracranial hemorrhage due to hemorrhagic disease of the newborn¹⁵⁶. Vitamin D can be supplemented to breastfed infants. Although the necessity of this supplementation is somewhat controversial, it remains recommended in the US¹⁷². Human milk is also a source of carotenoids (β-carotene, β-cryptoxanthin, lutein, zeaxanthin, and lycopene); these play critical roles in brain and eye development^173–175.

It is critical to understand vitamin requirements for premature infants. These vulnerable neonates normally receive their vitamins from the mother through the placenta¹⁶⁴; however, this supply is not available in the setting of prematurity. Vitamin levels change with the lactation stage, generally starting higher in the first few weeks after birth and then dropping to a steady-state thereafter¹⁵⁸. This decline in vitamin levels indicates that preterm infants will be receiving lower vitamin levels when matched for gestational age with their term counterparts¹⁵⁸. More information about appropriate vitamin requirements for developing infants is needed to make clear suggestions for vitamin supplementation, especially in preterm infants.

Conclusions

While much is known about the composition and beneficial effects of human milk, there is still more to learn. There is a need to understand how maternal nutrition and health influence human milk to improve the immunomodulatory components for infants. We need to improve our understanding of how individual HMOs select for specific bacteria and which bacteria, in turn, can metabolize specific HMOs. A deeper understanding of the role of individual bacterial species in infant health is needed as we begin to manipulate infant microbiomes with pre- and probiotics. How information from EVs is transferred to infants and downstream mechanistic effects of individual cargo are largely unknown. Finally, the influence of digestion on milk bioactive factors and the generation of milk protein-derived bioactive peptides remains largely undefined. A better understanding of each of these components of human milk will allow us to optimize the full benefits of human milk to all infants.