Breastfeeding, a personalized Medicine with Influence on Short- and Long-Term Immune Health

Author(s):
Valerie Verhasselt.

Summary

Compared to adulthood, early postnatal life is a period that is characterized by rapid changes. The neonate’s tissues are constantly changing due to the growth process and are exposed to multiple new antigens, which are found in the environment, are present in the diet, or are associated with gut microbiota colonization (Fig. 1). The neonatal immune system has its own reactivity, which profoundly differs from that of the adult. It is neither that of a “small adult” nor is it an immature or tolerance-prone immune system. The neonatal immune system is a different one with specific requirements for activation and regulation. Breast milk is most probably a key condition for physiological (and optimal) functioning and imprinting of the immune system in early life (Fig. 1). Similar to the immune system and environment, which are constantly changing in early life, breast milk composition is constantly evolving. Volume, macronutrients, micronutrients, immunological factors, microbiota, and microbiota-shaping molecules are changing with lactation stages, and are also affected by infant growth and
environmental immune challenges [1]. Here, we will focus on factors in breast milk that we – and others – extensively studied and found to actively influence their immune trajectory and long-term immune health. More specifically, we will review the importance of TGF-β, vitamin A, immunoglobulins, and allergens in mucosal immunity in both early life and long-term allergic disease susceptibility. There is strong evidence from rodent studies and epidemiological data that oral exposure to allergens in early
life, out of the context of breast milk, is not inducing immune tolerance but, instead, is priming for allergic responses [2]. Nonbreastfed infants are exposed to only a few allergens, such as β-lactoglobulin, which occur at high concentrations. In contrast, breastfed infants are exposed to a wide variety of breast milk allergens that are found at concentrations that are at least 1 million times lower [3]. Importantly, there is evidence from rodent studies that the neonatal immune system requires very-low-dose antigen



exposure for appropriate immune reactivity. Early life is also characterized by a relative lack of TGF-β in the mucosal tissue, a physiological deficiency in vitamin A, and a low mucosal and systemic immunoglobulin secretion, which contribute to the lack of oral tolerance induction in early life in the absence of breast milk. Breast milk is providing infants with these cofac- tors, which will affect gut epithelium barrier integrity, antigen transfer, and antigen presentation for successful regulatory immune response induction [4]. This will result in a decreased risk for allergic diseases in the long term, as shown for egg allergy both in an experimental mouse model as well as in humans [5].
We are starting to decipher the specific requirements for the neonatal immune system to function optimally, and we are discovering how breast milk fulfills these requirements and guides immune trajectories from early life. Answering these questions will provide the infant with preventive and curative approaches that are tailored to this very specific period of life and will ensure long-term immune health.

References
1 Ballard O, Morrow AL: Human milk composition: nutrients and bioactive factors.
Pediatr Clin North Am 2013;60:49–74.
2 Strobel S: Immunity induced after a feed of antigen during early life: oral tolerance v.
sensitisation. Proc Nutr Soc 2001;60:437–442
3 Munblit D, Verhasselt V: Allergy prevention by breastfeeding: possible mechanisms and evidence from human cohorts. Curr Opin Allergy Clin Immunol 2016;16:427–433.
4 Turfkruyer M, Verhasselt V: Breast milk and its impact on maturation of the neonatal
immune system. Curr Opin Infect Dis 2015;28:199–206.
5 Verhasselt V, Genuneit J, Metcalfe JR, et al: Ovalbumin in breastmilk is associated with a decreased risk of IgE-mediated egg allergy in children. Allergy 2020;75:1463–1466.

Abstract

The neonatal immune system has its own reactivity, constraints, and challenges, which profoundly differ from the adult. Breast milk is most probably a key requirement both for optimal immune function in early life and for imprinting of the immune system for long-term immune health. Here, we will highlight how breast milk fills the needs and the gaps of the developing immune system and thereby represents the unbeatable way to prevent infectious disease. We will further focus on some factors in breast milk that we extensively studied and found to actively influence the immune trajectory and long-term immune health. More specifically, we will review how the presence of allergens in breast milk together with maternal milk cofactors such as TGF-β, vitamin A, and immunoglobulins influence mucosal immunity in early life with long-term effects on allergic disease susceptibility. We will see that, depending on the content and the nature of allergens in breast milk as well as the presence of immune modulators, very different outcomes are observed, ranging from protection to an increased allergy risk. We are starting to decipher the specific requirements for the neonatal immune system to function optimally. We are discovering how breast milk fulfills these requirements and guides immune trajectories from early life. Answering these questions will provide the infant with preventive and curative approaches that are tailored to this very specific period of life and will ensure long-term immune health.

Genetic Programming, Environment, and Growth Render Infancy a Period of High Susceptibility to Infectious and Allergic Diseases

As compared to adulthood, early postnatal life is a period that is characterized by
rapid changes and multiple immune challenges. The neonates’ tissues are constantly
changing due to the growth process. Therefore, the immune system has to respect growth constraints in this period of life, and strong inflammatory immune responses, such as Th1 immune responses, should be avoided as they may lead to scars. Furthermore, tissue development requires the secretion of cytokines such as IL-33, which are also involved in the differentiation of Th2 immunity and allergic immune responses [1]. Thus, in early life, tissue growth results in the predisposition to mount low inflammatory and pro-Th2 immune responses, which can explain the propensity of the neonate to be susceptible to infectious diseases (lack of strong inflammatory responses) and to allergic diseases (bias towards Th2 immune responses). Another characteristic of early life is that the immune system is developing, and gut microbiota, which are a major promotor of immune development [2, 3], are establishing. Genetically programmed immune system development and lack of microbiota in early life result in immune deficiency, where the levels of mucosal antibodies and immune cells in the tissues are much lower than in the adult [4, 5]. The neonate is also exposed to multiple
new antigens, which are found in the environment, are present in the diet, or are associated with gut microbiota colonization. With all these antigens being new to the immune system, immune memory in early life is much lower than in the adult [6–8]. Naïve immune responses are known to be slower and less intense than memory ones, which also contribute to higher susceptibility to infection. Finally, yet importantly, requirements for immune activation in early life are different from adulthood. Elegant studies demonstrated that neonates are fully able to mount a cytotoxic immune response towards a viral infection when exposed to a dose that is 10,000 times lower than in the adult [9]. This has important implications for vaccination strategies and highlight the necessity of good hygiene especially in early life to prevent infection. Altogether, these observations indicate that for multiple reasons, early-life immune responses tend to be much less efficient in the fight against pathogens than in adulthood, and neonatal infection remains a common tragedy, with about 7 million cases and 700,000 deaths per year, accounting for 40% of mortality in those under 5 years of age [10]. Immune homeostasis also requires immune tolerance, an immune response that is characterized by the absence of inflammation and largely mediated by regulatory T cells. The induction of regulatory immune responses towards innocuous antigens, such as self-antigens and exogenous antigens derived from the diet, is critical
to avoid autoimmune and allergic diseases, respectively. A pioneer study by Billingham et al. [11] demonstrated that immune tolerance is easily induced upon systemic antigen exposure in utero and early postnatal life, and 70 years later, studies are still ongoing to decipher the mechanisms underlying these characteristics

[12]. However, the high prevalence of allergic diseases in infancy also indicates that mechanisms of tolerance towards exogenous innocuous antigens upon exposure through the skin and/or the mucosa are defective [13]. Instead, Th2 immune responses are preferentially induced [13]. As discussed here above, the liberation of mediators necessary for tissue growth contributes to this predisposition. The exposure to allergens through a skin barrier that is not fully developed can also favor allergic sensitization [14]. Furthermore, there is strong evidence from mouse models that the neonatal period is refractory to oral tolerance induction [15–19], the process by which immune tolerance to an antigen is acquired following its exposure through the digestive tract [20]. The spontaneous resolution of the majority of infant food allergies suggests a physiological maturation of the mechanisms of oral immune tolerance during the first years of life
[13, 21]. Gut colonization with microbiota plays certainly a major role in the setup of immune tolerance and the inhibition of allergic responses. This is clear from studies in germ-free mice, which have elevated levels of serum IgE (a hallmark of allergic immune responses) and are refractory to oral tolerance [22, 23].

Importantly, gut colonization with microbiota must occur in early postnatal life to efficiently regulate IgE immune responses [23], highlighting the concept of a window of opportunity to influence long-term immune responses [24]. There is accumulating evidence that the specific composition and the metabolites produced by the gut microbiota are critical for the generation of regulatory T cells and oral tolerance to be fully effective [2, 3, 20, 25]. In summary, the immune system in early life is not the one of a “small adult” and neither immature nor tolerance prone. The neonatal immune system is different from the adult one with specific requirements for activation and regulation. In the absence of specific interventions, the infant is highly susceptible to
both infectious and allergic diseases. Infant vaccination to elicit memory immune
responses and maternal vaccination during pregnancy to increase the levels of circulating antibodies during the first months are possible intervention targets, which are successful to a certain extent, to decrease morbidity and mortality due to infectious diseases in early childhood [10, 26]. Changes in allergy prevention guidelines and earlier introduction of potential allergens in the diet are promising strategies for food allergy prevention, with some limitations as we will discuss later on. A natural intervention does also exist, i.e., breastfeeding. We can distinguish the influence of breastfeeding on short-term immune outcomes, i.e. its effects while the infant is breastfed, and on long-term immune outcomes, i.e., months to years after breastfeeding has ceased (Fig. 1).

Breast Milk as a Physiological and Critical Strategy to Prevent Infectious Diseases in the Short Term

For years, breast milk was considered mainly as a source of nutrients for the developing
child. The extensive observations that breastfeeding affords protection towards infectious diseases and could reduce the mortality rate of common infections by more than half have added another key role to breastfeeding [27]. A recent meta-analysis concludes that scaling up breastfeeding to a near universal level could prevent 823,000 annual deaths in children younger than 5 years, mostly due to infectious disease. This protection relies in great part on the transfer of mucosal immunity through breast milk, which importantly relies on multiple, various, and adapting mechanisms (Fig. 1). Breast milk contains potent antibiotics, such as lactoferrin, lysozyme, and antimicrobial peptides [27, 28].

The presence of prebiotics such as human milk oligosaccharides [29] and probiotics
[30, 31] interferes with pathogenic bacterial expansion and invasion. Growth factors such as EGF and TGF-β contribute to maintain and repair potential pathogen-induced mucosal barrier breaks. Importantly, secretory IgA (SIgA) delivers personalized medicine. SIgA is present at high concentrations in colostrum (∼10 g/L) and at somewhat lower concentrations in mature breast milk (∼1 g/L). SIgA is specific for intestinal and respiratory pathogens in the maternal environment due to the selective migration of B cells originating from the maternal mu-cosa to the mammary gland [32]. They are thus providing mucosal immunity against pathogens, which are specifically found in the environment of the child. By their non-antigen-specific part, maternal SIgA will also contribute to the establishment of infant gut microbiota and protect the child form pathogens [33].

Breast Milk as a Key Player in the Education of the Immune System and Long-Term Susceptibility to Immune-Dependent Diseases

In addition to the provision of passive immunity and compensation for neonatal
immune deficiencies, breast milk actively influences the development of the immune
system (Fig. 1). Thereby, breast milk can guide immune trajectories and long-term susceptibility to diseases, which have an immune component in their physiopathology. There is evidence that breastfeeding decreases the risk of obesity and metabolic complications associated with obesity, such as type 2 diabetes [34]. However, there is lack of consistency regarding the possibility of long-term prevention of infectious diseases [35] and allergy [34, 36–40] by breastfeeding. Despite this, there are accumulating data indicating that breast milk has the potential to prevent these diseases by at least two major ways: by shaping the infant microbiota and by exposing the neonatal immune system to microbial antigens and allergens. The gut microbiota, and more specifically the microbiota-derived metabolites, are key for influencing the balance between health and disease [41]. In particular, gut microbiota influences susceptibility to infection and efficacy of vaccination [42], as well as susceptibility to allergy as we recently reviewed [2].

By the presence of prebiotics, probiotics, and antigen-specific and non-antigenspecific
antimicrobial compounds, breastfeeding plays a major role in the initial seeding of the infant gut microbiota and in its constant evolution in postnatal life [2]. The major event being known to affect microbiota composition is cessation of breastfeeding [43]. Therefore, we can speculate that the various concentrations of microbiota-shaping compounds in each mother’s breast milk will contribute to the heterogeneity in gut microbiota found in children and thereby influence their susceptibility to disease [2]. Finding which microbiota is best adapted to each environment and how to influence microbiota-shaping molecules in breast milk will open up new avenues for long-term immune health.

Breast milk also contains exogenous antigens, and our research in the last decade has been aimed at deciphering the long-term outcomes of the presence of allergens in breast milk on allergy susceptibility in the offspring. Recently, we expanded this research to infectious disease prevention. In the last part of this review, we will synthesize our main findings related to the presence of exogenous antigens in breast milk and discuss their implications for long-term immune health.

Allergy is a rising public health issue, with respiratory and food allergy affecting up to 20% of children (the Global Asthma Report 2018; http://
globalasthmareport.org/) [13]. After an era of avoidance, there has been a paradigm
shift, and new strategies for allergy prevention aim now at inducing tolerance
by antigen exposure. Following impressive results in large randomized clinical trials, the early introduction of eggs and peanuts in the diet is now recommended for food allergy prevention [13]. However, recent studies also demonstrated a significant proportion of infants already have egg sensitization and clinical reactivity (including anaphylaxis) prior to the first introduction of eggs into their solid food diet [13, 44–46]. This underscores the necessity to identify earlier and safer ways to promote oral tolerance development to food allergens in young infants, which may be particularly challenging knowing the refractoriness of early life to oral tolerance. We proposed the hypothesis that early oral
exposure to exogenous allergens in the presence of maternal milk immune modulators would alleviate oral tolerance induction in early life. Our experiments in mouse experimental settings demonstrated the key findings: 1. Mice exposed to only low amounts (ng/mL) of the egg-derived allergen ovalbumin (OVA) through breastfeeding are protected from allergic reactions to OVA when reexposed in adulthood via the oral (as a model of food allergy) [47] or respiratory route (as a model for respiratory allergy) [48–50]. This echoes to observations referenced here above indicating that the early-life immune system reacts to very low amounts of antigens. Importantly, other reports have shown the presence of dietary antigens in breast milk in the same range at very low concentrations (ng/mL), such as cow milk β-lactoglobulin [51–53], peanut allergens
(Ara h 1 and Ara h 2) [54, 55], or wheat antigen (gliadin) [56]. By comparison, it
is worth noting that allergen levels in cow’s milk are found in the range of mg/ mL. Thus, through breast milk, infants are exposed to a wide variety of dietary allergens at low concentrations while formula-fed infants are only exposed to cow milk-derived allergens and at much higher concentrations (unless hydrolyzed formulas are used). We can speculate this will lead to very different outcomes in terms of oral tolerance induction and allergy prevention in the offspring.

Our recent observation in a human birth cohort demonstrated that the risk of egg allergy in children was reduced by a factor of 4 at 2.5 years when comparing children exposed to breast milk with or without egg antigen [57]. To fully confirm that OVA in breast milk is responsible for a decreased egg allergy risk in children, further randomized controlled trials will need to be conducted. The next steps will be to identify maternal interventions leading to the consistent presence of OVA in breast milk in order to move towards a successful prevention of egg allergy by breastfeeding. 

2. Successful oral tolerance and allergy prevention upon OVA exposure through breast milk required the concomitant presence of maternal immunomodulatoryfactors. We identified TGF-β, OVA-specific IgG, and vitamin A to stimulate oral tolerance induction in the offspring. Each cofactor was shown to act by different mechanisms of action. TGF-β promoted the induction of OVAspecific Th1 cells, which counteract allergic Th2 responses [48, 58]. Vitamin A accelerated gut epithelium maturation resulting in a stronger barrier in the first week of life [50]. It also promoted the expression of RALDH in small-intestine dendritic cells, which was associated with their increased efficiency at activating T lymphocytes [50]. Maternal vitamin A supplementation resulted in the possibility to induce oral tolerance to OVA in breast milk in the offspring from birth, which otherwise is only efficient from the third week of life [50]. OVAspecific IgG in breast milk was necessary for a protected transfer of OVA though the neonatal gut barrier and the induction of a prolonged and strong protection from allergy, which was found to be mediated by FoxP3 Tregs [59]. Recently, another group confirmed these findings and showed that OVA-specific IgG also promoted antigen presentation by neonatal dendritic cells [60]. In other words, early life is characterized by a relative lack of TGF-β in mucosal tissue, a physiological deficiency in vitamin A, and low mucosal and systemic immunoglobulin secretion, which contribute to the lack of oral tolerance induction in early life in the absence of breast milk. Breast milk is providing infants with
these cofactors, which will affect gut epithelium barrier integrity, and antigen transfer and presentation for successful regulatory immune response induction.

This will result in a long-term low risk for allergic disease as we showed for egg allergy both in an experimental mouse model and in a human birth cohort. Altogether, these data suggest that, according to the levels of immune modulators in breast milk, oral tolerance to dietary antigens in breast milk will be induced with more or less efficiency, which will condition long-term susceptibility to allergy.

3. Unexpectedly, we found allergens from respiratory sources such as from the house dust mites Dermatophagoides pteronyssinus (Der p) and Blomia tropicalis in breast milk in similar amounts as dietary antigen [61, 62]. Since we detected Der p 1 in the digestive fluid of healthy adults [63], we propose that respiratory allergens are ingested by being trapped in the oropharynx or pulled back by the mucociliary epithelium, and follow the same route as dietary antigens to the mammary gland. In contrast to the observation with OVA, the transfer of Der p 1 through breast milk induced Th2 immune response priming and increased susceptibility to allergic disease in adult mice [64]. Importantly, in a human birth cohort, there was a positive association between allergic sensitization and respiratory allergies in children and the presence of Der p 1 levels in breast milk [61]. This observation stresses that not all the antigens in breast milk induce oral tolerance, even though they reach the gut mucosa together with breast milk-tolerogenic factors. Our most recent findings further showed that Der p allergen in maternal milk abolished the capacity of neonatal mice to mount oral tolerance to bystander OVA egg antigen, which resulted in an increased risk of egg food allergy in the long term [65]. Importantly, human data showed heterogeneity in breast milk content in Der p and OVA. Based on the presence of Der p and/or OVA in breast milk, we could
identify groups of lactating mothers which mirror the ones found in mice responsible for the different egg allergy risk [65]. These observations stress the need to identify how to counteract deleterious actions of some allergens in breast milk, such as those derived from house dust mite allergens, which prime for allergic sensitization to themselves and break oral tolerance induction to bystander ones.

We have started to identify some potential targets as we showed that protease from
Der p allergens are key players for the induction of gut Th2 mucosal immune imbalance
in mice breastfed by mothers inhaling Der p [65]. Ongoing research is aimed at identifying how to modulate Der p levels and their enzymatic activity in breast milk and/or in the breastfed child. Ultimately, this should contribute to ensure food allergy prevention in children.

4. Based on the findings that house dust mite allergens in breast milk could prime immune reactivity in the long term, we proposed that novel strategies of early life prevention of infectious disease may take advantage of the possibility to stimulate antigen-specific immune responses through breast milk. Microbial antigen transfer through breast milk would be a way to naturally vaccinate the infant [26, 66]. Some evidence in the literature supports this hypothesis, such as the observation that maternal HIV infection of noninfected breastfed children is associated with infant stimulation of IgG and IgA secretion in their gut mucosa [67] and HIV-specific interferon-γ-secreting PBMC are found in 50% of cases [68]. Recently, we addressed the original hypothesis that the presence of malaria antigen in breast milk may stimulate antimalarial immune defenses and reduce the malaria risk in breastfed infants [69]. As a first critical step to address this hypothesis, we investigated whether Plasmodium falciparum histidine- rich protein 2 (pHRP-2) and lactate dehydrogenase (pLDH) are detectable in breast milk of mothers from Uganda, a country with endemic malaria
[70]. We found that 15% of breast milk samples from mothers with asymptomatic
malaria do contain malaria antigens. Our preliminary data indicate that blood levels of malaria antigens determine their levels in breast milk. These landmark findings may have significant implications for susceptibility to malaria in children from endemic countries since malaria antigens in breast milk may strongly influence the immune responses to natural malaria infections and to malaria vaccines in breastfed children [26].

Concluding Remarks and Perspectives

The way breast milk composition constantly adapts to the needs of each infant in each setting is fascinating. This results in a major success for the prevention of many infectious diseases in breastfed infants. There is also evidence that breastfeeding could have long-term protective effects on infections, even after breastfeeding has ceased, and this may open new avenues for infant vaccination. Unfortunately, and maybe unexpectedly, breastfeeding does not provide a consistent protection from allergy. A hypothesis behind this lack of protection may be that the lifestyle of modern societies has resulted in changes in breast milk composition, which is not suitable anymore for allergy prevention. Research conducted to identify which factors in breast milk condition protection versus susceptibility is providing new clues, which ultimately will result in the protection of breastfed children from allergy.

Disclosure Statement
Valérie Verhasselt has no conflict of interest with regard to the writing of this chapter.

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