Breast Milk: Proactive Immunomodulation and Mucosal Protection Against Viruses and Other Pathogens

Chiara Cerini; Grace M Aldrovandi

Future Virology. 2013;8(11):1127-1134. 

Abstract and Introduction

Abstract

The majority of microorganisms infecting humans cross through the mucosal barrier. This is particularly true in infants who explore the world with their mouths while their immune system is still developing. Human milk not only supplies the nutritional needs of the newborn and protects the baby against infection by confering trophic protection to the intestinal mucosa, but additionally shapes the infant's gut microbiota and instructs immunomodulation. Reflecting maternal environmental exposition and in virtue of its multiple mechanism of action, secretory IgA in milk exerts a decisive role in direct and cross-protection against a variety of pathogens. Its active role in priming the infant's immune system is an intriguing hypothesis. From this perspective, breast milk antibodies produced by means of maternal immunization might represent protective and proactive factors able to shape and enhance the infant's immune responses. Strategies to optimize the benefits of maternal immunization include novel vaccine formulations and mucosal route of delivery.

Introduction

One of the key determinants of a successful pregnancy is mutual fetal and maternal immune tolerance. After birth, infant survival depends on transitioning from a tolerogenic state to a phase of immune activation, while both systemic and secretory immune functions mature.[1] During this period, infants are extremely vulnerable to infections and often have suboptimal responses to vaccination. Therefore, infants rely on maternal immune protection through antibodies initially acquired transplacentally and subsequently via breast milk.

Evolutionary biologists hypothesize that lactation evolved over 200 million years to provide infants with both immunologic and nutritional support.[2] This rich secretion contains antimicrobial, anti-inflammatory and immunomodulatory factors that serve to compensate for many aspects of the infant's functional immune immaturity.[3]

These developmental immune deficiencies include quantitative and qualitative delays in antibody production, suboptimal T-cell immunity, phagocytic function and complement cascade activation (reviewed in[4,5]). The immunologic benefit of breastfeeding is 'dose' dependent – that is, the benefit is proportional to the degree of its duration and exclusivity.[4] Exclusive breastfeeding refers to the practice of providing no other liquids or solid food to the infant – not even water – with the exception of oral rehydration solution, or drops/syrups of vitamins, minerals or medicines.[6] Although the protective effects of breastfeeding are most readily demonstrable in populations living in developing countries – where not only access to vaccines and healthcare, but also to potable water is scarce – several good-quality studies in industrialized countries have noted significant reductions in common infectious diseases of infancy (otitis media, respiratory tract diseases and nonspecific gastrointestinal infections) in breastfed infants. In addition, breastfeeding also has profound effects on the maturation and development of the infant's intestine and his/her systemic immunity.[5]

Infants are born with an essentially sterile intestinal tract, and over the course of the first 3 years of life environmental factors determine the colonizing organisms.[7] It has long been recognized that the stools of breastfed infants are distinct from those of formula-fed infants.[7] Emerging data on the critical role of the microbiota in immunity strongly suggest that these differences in the microbial composition may be responsible for the differences in autoimmunity and other diseases observed between breastfed and formula-fed infants.[8–12]

In this paper, we will review factors in breast milk that mediate infant protection from viral and other microbial pathogens. Additionally, we will discuss maternal immunization as a strategy to protect infants.

Breast Milk Antibodies

Breast milk is a remarkably 'altruistic' secretion; that is, its contents are directed at protecting the infant with minimal or no benefit to the mother. Not only is the concentration of secretory antibodies (mainly IgA) in breast milk remarkably high (10–100-fold higher than in serum),[13] milk antibodies also possess a broad range of specificities, reflecting both maternal immunologic memory and antibodies directed towards pathogens that do not infect breast tissues, such as rotavirus.[14] Secretory antibodies in milk mirror maternal antigenic stimulation of mucosa-associated lymphoid tissue (MALT) both in the gut and the airways. Given the symbiosis between the breastfed infant and his/her mother during the first weeks of life, the microorganisms in the mother's environment are likely the same as those encountered by the infant. Intriguingly, milk composition changes (i.e., increase in the total number of white blood cells and higher TNF-α levels) have been documented in relation to active infection in the nursing infant.[15]

Mothers may thus be considered as immune 'factories', providing infants with antibodies that prevent, postpone or attenuate diseases caused by pathogens in their environment. In contrast to most therapeutics and immunizations, breast milk displays the unique potential to adapt itself to the requirements of the infant. Timely immune defenses are tapped from its constituents by immune regulation, modulation and immune acceleration to stimulate novel substances; these ad hoc modifications provide defense even in the face of evolving organisms.[15]

Antibodies in milk are either transferred from plasma by transudation or locally produced by cells that migrate to the mammary gland from other mucosal sites.[16]

During the latter stages of pregnancy, hormones, chemotactic factors and cellular addressins induce T and B cells homing from inductive sites (the gut and bronchial-associated tissues) to the lactating breast.[17,18] Although all classes of immunoglobulins can be detected in milk, over 90% are IgA; IgM and IgG are less abundant.[19] By using sensitive measuring techniques, IgD and IgE can also be detected.[20] Human colostrum contains more than 1 g/l IgA, and during the first year of lactation, concentrations are maintained at approximately 0.5 g/day.[21] By contrast, less than 20% of maternal serum immunoglobulin is IgA; most are IgG.[22]

IgA in milk is principally in the form of secretory IgA (sIgA), which serves as a first line of mucosal defense. Maternal supply of sIgA is important as infant intestinal IgA production does not begin until several months of age, and even at 1 year of age serum IgA levels are only 20% of adult levels.[22] Although the extent to which antibodies are absorbed in early life remains controversial, it is most likely modest, except perhaps in preterm infants.[23–25] Unlike other antibody isotypes, secretory IgA is resistant to degradation in the protease-rich external environments of mucosal surfaces, and the majority of ingested sIgA survives passage through the intestinal tract intact for at least the first year of life, providing mucosal protection in spite of the increasing surface area of the GI tract.[26,27]

Other Factors in Milk

In addition to antibodies, breast milk contains more than 100,000 constituents, which can influence the growth, development and immune status of the infant. Human colostrum is packed with living leukocytes (105–107 cells/ml) that enhance the immature infant's immune system. Usually leukocytes in breast milk are 40–50% macrophages, 40–50% polymorphonuclear neutrophils and 5–10% lymphocytes.[28]

Antigen-specific memory B cells, plasmablasts and plasma cells in breast milk have been identified and characterized.[29] Breast milk B cells are strikingly different from their blood counterpart: they mostly display a phenotype of IgD memory B cells and have a particular profile of adhesion molecules (CD44+, CD62L, α4β7 +/− and α4β1 +), suggesting that these cells may originate from the gut-associated lymphoid tissue. Because of their low concentration, breast milk plasmablasts and plasma cells probably contribute only a small portion of antibodies secreted in milk.

Many cytokines (e.g., IL-1, IL-8, TGF-β and TNF), chemokines (e.g., MCP-1, MIP-1a and RANTES), growth factors (e.g., EGF, lactoferrin, cortisol, IGF, polyamines and nucleotides), hormones (e.g., erythropoietin, prolactin, insulin and leptin), vitamins (vitamin A, B1, B2, B6, B12 and D), neuropeptides (e.g., neurotensin and somatostatin), enzymes, mucins (MUC1 and MUC4) and antioxidants are present in milk at higher levels than those in the circulation. Generally speaking, there is considerable functional redundancy and many of these molecules act synergistically to compensate for the immunologic immaturity of the infant.[3]

Mechanisms of Infant Protection

Passive Protection

Secretory antibodies in milk exert their protection via multiple mechanisms.[30] First, sIgA acts as a barrier limiting access of microbes and noxious antigens to the intestinal epithelium. This is accomplished by a combination of agglutination, mucus entrapment and mucociliary clearance, and has been termed immune exclusion. Second, sIgA binds via its carbohydrate moieties to lectin-like bacterial adhesins, thereby blocking bacterial adhesion to receptors on epithelial cells. sIgA also appears to provide an internal mucosal barrier beneath the mucosal lining; incoming pathogens may be intercepted within epithelial cell vesicular compartments through the polymeric immunoglobulin receptor-mediated transport, translocated through the epithelium and thereby excreted.[31,32] Alternatively, sIgA may mediate export of pathogens back into the gut lumen.[27,33] Singularly or in combination, these mechanisms appear to mediate protection against a variety of gastrointestinal pathogens, including bacteria (Shigella, Salmonella, Campylobacter, Vibrio cholerae and Clostridium botulinum), as well as many parasites and fungi.[26,34] sIgA directly neutralizes enteroviruses,[35] respiratory syncytial virus,[36] rubella[37] and rotavirus.[14] In addition, sIgA can modify virulence factors, such as toxins of enterotoxigenic Escherichia coli,[38] the surface protein A (SpsA) of Streptococcus pneumoniae[39] and Clostridium difficile toxin A.[39] Given its opsonizing properties, sIgA favors cytotoxic pathways through interaction with FcαRI (primarily expressed on immune effector cells) and triggers antibody-dependent cellular cytotoxicity (ADCC), promoting antigen presentation to dendritic cells.[40,41] Furthermore, sIgA in the intestine is critically important for regulating bacterial communities in the gut lumen by both Fab-dependent and Fab-independent mechanisms.[42] These microbial communities are key regulators of immune activation of intestine cells. Indeed, recent studies have defined a trialogue between IgA, intestinal epithelial cells and the microbiota.[43] The net effect of these interactions determines host immune responsiveness to antigens.

Active Stimulation & Modulation of Infant Response

Breast milk represents the immunological integration of mother and child, not only by providing passive transfer of antibodies and other immune factors, but also by priming the infant immune system, facilitating the development of tolerance, regulating the inflammatory response, and promoting the humoral and cellular response to vaccines. The positive effects of breastfeeding – including its nutritional value – during the development of the infant's immune system apparently persist in the long term.[44,45] Data from European and US studies show that breastfed infants have higher antibody levels than formula-fed infants after receiving oral and parenteral vaccines.[46,47] Following MMR immunization, Pabst et al. documented a Th1-type response in breastfed children, which was not evident in the formula-fed group.[9]

Murine studies have clearly identified the microbiota as a fundamental force priming immune responses.[48] As noted above, breastfeeding profoundly shapes infant stool composition. In addition to sIgA, other factors in milk regulate the establishment of microbial communities. Breast milk is not sterile and contains many bacteria, particularly bifidobacteria and lactobacilli. Oligosaccharides are the third most abundant component in human milk. These human milk oligosaccharides act as prebiotics –that is, nondigestible components that promote the growth of commensal bacteria.[49,50] Indeed, mother's milk fosters the growth of healthy bacteria in the newborn's intestinal tract, which not only aids nutrient absorption, but also boosts the immune system. In addition, these factors constituting the human milk glycobiome can inhibit pathogen adhesion and directly influence intestinal maturation and integrity.[51] Notably, higher levels of non-3'-sialyllactose human milk oligosaccharides appear to play a role in HIV breast milk transmission.[52]

Numerous milk constituents have been implicated in modulating infant immunity and facilitating the development of tolerance.[53] Nucleotides and growth factors such as granulocyte colony-stimulating factor and macrophage colony-stimulating factor enhance the activity of T cells, natural killer cells and macrophages. Milk is enriched with antigen-specific T cells, which can survive passage through the GI tract.[54] In animal models, these cells can traverse the suckling's intestinal tract and are functional, but human studies in this regard are controversial. Nevertheless, in humans maternal–fetal microchimerism in tolerance has been implicated.[54] A variety of anti-inflammatory factors in breast milk aid to promote tolerance, including immunosuppressive cytokines (i.e., TGF-β and IL-10)[44] and long-chain polyunsaturated fatty acids.[55] Among other innate immunoregulatory factors in breast milk, soluble CD14 should be mentioned.[56] Innate sensing molecules such as TLR2 are also present in high concentrations in breast milk. TLR2 appears to inhibit the production of proinflammatory cytokines during bacterial ligand exposure.[57] In addition, breast milk contains naturally occurring anti-idiotypic antibodies, which display specificity against other autologous antibodies and thereby modulate the response to the antigen the idiotype is directed against.[58]

The tolerogenic state induced by breastfeeding is supported by evidence from both human and animal data. In large epidemiologic studies, breastfed infants have a lower incidence of several autoimmune diseases[53] and are more tolerant to renal transplants.[59–61] Intriguingly, the beneficial immunomodulatory effects of breastfeeding both on humoral and cellular responses appear to extend well beyond weaning. In rodent models, perinatal antigen exposure via mother's milk has been shown to prime the suckling animal's immune responses in a manner so profound that the effects can still be detected two generations later.[58,62] Compared with formula-fed pairs, breastfed children show enhanced antibody response to Haemophilus influenzae type b polysaccharide, diphtheria and tetanus toxoids, oral poliovirus, and pneumococcal serotypes 6B and 14 conjugate vaccines.[63,64] Enhanced Th1 responses, as well as significant changes in CD8+ T cells, natural killer cells and mitogen-induced IFN-γ production, have been documented in breastfed but not in formula-fed children who received MMR vaccination.[9]

Enhanced infant T- and B-cell development in breastfed infants has been associated with a number of cytokines, including IL-7, IL-8, IFN-γ and thymic stromal lymphoprotein.[57,65,66]

Maternal Immunization

Maternal immunization (before or during pregnancy, or immediately after delivery – known as the 'cocoon effect') has emerged as a simple, safe and cost-effective strategy to protect the neonate against illness, bypassing the well-known immunological obstacles associated with neonatal vaccination. This approach has been extremely successful in efforts to eliminate neonatal tetanus and is even being advocated as a strategy for the control of infant pertussis, influenza and other diseases.[67–69]

Most maternal vaccination strategies have focused on antenatal immunization, relying on effective transplacental antibody transfer and persistence of maternal antibodies in the infant for approximately 2 months. However, protective levels of maternal antibodies differ widely depending on initial levels, specificity and avidity.[70] For some diseases the levels of specific antibodies required for protection are not known (i.e., for pertussis). Interestingly, antenatal maternal vaccination appears to positively influence the production of specific antibodies transferred to the infant via breast milk for at least 5–6 months postpartum.[71–73] Assessing the amount of pneumococcal antibody transmitted to the infants after maternal pneumococcal immunization, Shahid et al. found that the half-life of the breast milk IgA for some serotypes (19F) were significantly higher in vaccine recipients up to 5 months after delivery.[71]

Deubzer et al. documented that maternal vaccination with polyvalent pneumococcal polysaccharide vaccine boosts the capacity of colostrum to inhibit the adherence of pneumococci to pharyngeal epithelial cells.[74] In a randomized, placebo-controlled trial during the RSV season, higher vaccine-specific anti-F IgA and IgG concentrations in breast milk were found in mothers who received the anti-RSV PFP-2 vaccine.[75]

These observations may be of greater importance in preterm babies, who receive less transplacentally acquired immunoglobulin. Interestingly, Mother Nature may have already accounted for this deficiency, as higher concentrations of sIgA are found in the breast milk of mothers who deliver preterm infants.[76] The implications for these differences on vaccine strategies remain to be elucidated.

Although data on postnatal maternal immunization are sparse, it appears to be beneficial. A randomized, placebo-controlled trial of 23-valent pneumococcal polysaccharide immunization in Papua New Guinea reported a nonsignificant decrease in the number of acute lower respiratory tract illness over a 3-year period in children whose mothers were vaccinated antepartum (78.5% in children whose mothers had received placebo vs 67.8% in children whose mothers had received vaccine; p = 0.1). However, among infants who were 1–17 months of age at the time of maternal immunization, there was a significant reduction in acute lower respiratory tract illness episodes over that same period (91.6% in placebo vs 76.2% in the vaccine arm; p = 0.02).[66] A significant transfer of pertussis antigen-specific IgA to breast milk in vaccinated postpartum mothers has also been documented.[77]

While maternal vaccination may substantially improve child health, it also poses challenges, including blunting of the infant's responses to her/his own vaccinations due to passively acquired maternal antibodies.[78] Lower seroconversion rates and antibody levels were first described for the measles vaccine[79] and have been observed with other vaccines.[80–82] However, these suppressive effects are highly variable and inconsistent not only between vaccines, but also for the same vaccine in different studies. Dose of vaccine and route of administration appear to be key factors in mediating the effect of transplacental maternal antibodies on infant vaccine response. Interestingly, blunting of infant antibody response does not appear to inhibit infant T-cell responses and/or infant priming.[83–86] T-cell priming in the presence of maternal antibodies has been associated with boosted high avidity antibody responses when the infant is subsequently challenged. Given the importance of T-cell responses that can be induced by a vaccine, particularly against viral pathogens, the significance of lower antibody levels is uncertain. In addition, how postpartum immunization and breast milk-mediated protection would impact infant immune response to vaccination remains largely unexplored.

Conclusion

Breast milk is a complex dynamic secretion that adapts to meet the infant's immune ontogeny. Its remarkable ability to protect against pathogens is evident even in industrial countries where hygiene and access to state-of-the-art medicine have markedly decreased infant morbidity and mortality. Immunologic immaturity often hinders newborns and young infants from responding to vaccines. During this vulnerable period, maternal immunity plays a critical role in ensuring the child's health. Maternal antibodies transmitted in utero or through breast milk are major elements in this protection. Maternal immunization aimed at increasing transplacental antibody transfer and persistence has proven to be of benefit against a variety of pathogens. However, this approach can be a double-edged sword, since maternal antibodies may be associated with inhibition of the infant's B-cell activation and antibody production. On the other hand, sIgA in breast milk has been shown to provide specific protection against many bacterial and viral infections, and appears to allow priming of the offspring's immune system. Strategies to optimize the protective effects of maternal immunization include novel vaccine formulations and mucosal routes of delivery. In breastfeeding mothers, stimulating mucosal immunity via oral or nasal vaccination may result in more robust protective responses and should be considered a strategy to protect the young infant. Further studies aimed at understanding the unique relationship in the mother–infant dyad and the mechanisms by which maternal immunity influences infant immune response are needed.

Future Perspective

Due to the pressing need to control infectious diseases (such as rubella, measles, tetanus, HB and seasonal influenza), routine vaccination of pregnant mothers is recommended. However, many questions remain about both the benefits and/or side effects of maternal immunization, and further studies are needed. Areas for inquiry include examining whether different types of vaccines and routes of administration influence breast milk antibody production, and examining whether time and type of maternal immunization influences the infant's active immune responses.

Sidebar

Executive Summary

Breast Milk Antibodies

  • Breast milk contains high concentration of secretory antibodies, mainly IgA, with a broad range of specificities reflecting both maternal immunologic memory and antigenic stimulation of maternal mucosal associated lymphoid tissue.

  • Secretory IgA is resistant to degradation and provides passive protection via multiple mechanisms.

Other Factors in Milk

  • Breast milk contains more than 100,000 constituents that influence the baby's growth, development and immune status.

Mechanisms of Infant Protection

  • Breast milk immune factors – mainly sIgA – act either as protective and proactive factors and sustain the development of the infant's immune system.

  • The active stimulation of the infant's immune system by breastfeeding shapes the baby's gut microbiota, facilitates the development of tolerance, regulates the inflammatory response and promotes responses to vaccines.

Maternal Immunization

  • Antenatal maternal immunization has been extremely successful in controlling some diseases; however concerns have arisen about blunting the infant's response to his/her own vaccinations due to passively acquired maternal antibodies. Postnatal maternal immunizations aimed to enhance breast milk antibodies production might be a promising approach.

Conclusion

  • In addition to being the best source of nutrition for the offspring, human milk is a complex and dynamic secretion that contains a myriad of protective factors against infectious disease and may influence the infant's immune system development.

  • Further studies are needed to understand the types of vaccines and routes of administrations that promote the production of protective antibodies in breastfeeding mothers.

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