Allergy Prevention By Breastfeeding.docx Journal In Nur..docx

ALLERGY PREVENTION BY BREASTFEEDING Possible Mechanisms and Evidence From Human Cohorts

Daniel Munblit; Valérie Verhasselt Disclosures Curr Opin Allergy Clin Immunol. 2016;16(5):427-433. 

Abstract and Introduction

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How Breast Milk May Contribute to Allergy Prevention

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Conclusions

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References

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Sidebar

Key Points 

Human breast milk contains factors which are specifically suited to neonates' requirement for growth and immune maturation.

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Lack of evidence of prevention of allergy by breastfeeding suggests its composition may not fit modern life environment requirement.

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Breast milk contains molecules which can act on critical factors for allergy physio-pathogenesis: barrier dysfunction and defect in regulatory immune response.

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Maternal intervention during lactation period may impact on levels of critical factors in breast milk which are capable to counteract allergy development and thereby efficiently prevent allergy.

How Breast Milk May Contribute to Allergy Prevention Breast milk contains hundreds of bio-active molecules which are involved in infant growth, confer infant passive immune defense, actively modulate immune system, impact on cognitive development and metabolism.[3] The human milk composition is unique to each mother. It changes with infant age and is modulated by maternal environment as best known for microbial specific IgA levels which fluctuate with maternal infection.[4] We will examine in the following paragraphs the ways various breast milk factors may influence on its ability to prevent allergy and how breast milk composition can be modulated (Table 1).

Can Breastfeeding Promote an Antiallergic gut Barrier? Allergic diseases are targeting body barriers (skin for atopic dermatitis, lung mucosa for asthma and gut mucosa for food allergies). A primary defect in barrier functions is a suspected cause of the allergy physiopathology. This is best exemplified by atopic dermatitis genetic predisposition in patients with filaggrin gene mutation which is necessary for an adequate skin epithelial function. Gut barrier dysfunction is also considered to play an important role in allergic sensitization and severity of symptoms.[5] The adult gut barrier function is a result of many actors: first, secretory IgA in gut lumen

which have transcytosed across gut epithelial cells; second, a layer of mucus produced by goblet cells; third, antimicrobial peptides which are mainly produced by goblet and Paneth cells; and fourth, epithelial cells which are sealed by tight junctions. The gut barrier function is modulated by mediators secreted by cells in the underlying lamina propria. In particular, type 2 innate lymphoid cells (ILCs) produce IL-13 and amphiregulin which stimulate mucus secretion and tissue repair and ILC3 by their secretion of IL-22 which stimulate antimicrobial peptide secretion and tissue repair. [6] Altogether, these mechanisms provide control over potential allergen delivery, microbiota composition and gut epithelium anti-inflammatory properties which impacts allergic sensitization.[2] At birth, most of these actors are 'deficient': IgA secretion in neonate is very low, goblet and Paneth cells are rare, epithelial cell permeability and their proinflammatory cytokine secretion are high. [7] Very few data are available on ILC function in the neonate except for ILC3, also called LTi, which are known at this age to be involved in postnatal lymphoid organ development.[6] The presence of gut trophic factors, capable of actively stimulating crypt and villi formation of immature gut epithelium, in breast milk has been known for a long time.[3] Maternal milk was also found to be critical to decrease the permeability of intestinal epithelium to intact protein which is increased in the first week of life both in humans[8] and in rodents.[9] A recent analysis of exfoliated gut epithelial cells in stools samples from 3-month-old infants showed a total of 1214 genes differentially expressed between breastfed and formula fed.[10] Analysis of gene networks reflected broad differences with respect to signal transduction (WNT, NOTCH, TGF-b), cytoskeletal remodeling, cell adhesion and immune response. Milk growth factors such as epidermal growth factor (EGF), insulin growth factor (IGF), transforming growth factor beta (TGF-β) activate proliferation and differentiation of gut epithelial cells, and to stimulate tissue repair.[3] Molecules renowned for their antibacterial activity such as lactoferrin were also found to promote gut epithelium proliferation and differentiation.[11] In addition to maternal endogenous growth factors which level may be difficult to modulate, some other factors found at different quantities in maternal diet, and in their milk and may also affect neonatal gut barrier. Levels of vitamin A in breast milk are dependent on maternal intake[12] and it is a major epithelial cells differentiation factor.[13] Vitamin A and its precursors are found in high amounts in colostrum and lower amounts in later milk.[14] We recently observed that vitamin A supplementation in mice during lactation was sufficient to promote crypt formation and decrease gut permeability in the neonate.[15]Importantly, vitamin A supplementation did not affect expression of gut epithelial enzymes involved in milk digestion such as lactase and β-galactosidase.[15] A major developmental immaturity in the human and mouse small intestine is its propensity to respond to stimuli by mounting higher inflammatory response compared with adults. [7] As epithelium inflammatory

response plays a critical role in allergy development,[2] this neonatal specifics may favor allergic sensitization to dietary antigen in early life. TGF-β and other factors such hydrocortisone, fatty acids, and lactoferrin are known to dampen inflammatory cytokines secretion by epithelial cells.[16] Recently, the impact of human colostrum oligosaccharides on fetal human epithelial cells was assessed. Data demonstrated a significant decrease of acute phase inflammatory cytokine secretion whereas cytokine involved in tissue repair and homeostasis were increased. [17] Studies also showed the presence of breast milk factors capable of interfering with microbial signaling in gut epithelial cells such as sTLR and sCD14 which bind LPS.[18] Most recent work confirmed the ability of human milk oligosaccharides (HMOs) to downregulate CD14 expression in human enterocytes[19] and milk EGF to suppress TLR4 signaling, both actions attenuating LPS-induced inflammation.[20] These observations are critical because neonatal epithelial cells are not tolerant to LPS in contrast to adult.[7] Interestingly, more than a decade ago, Jones et al.[21] showed that low sCD14 levels in mature milk are associated with eczema development and then Savilahti et al.[22] reported similar trends for colostrum. Later studie,[23,24]however, failed to reproduce these results and did not report any protective effect of this soluble receptor on eczema. The controversy between the outcomes of the studies may be a consequence of a difference in CD14 genotype with breastfeeding being associated with a decreased risk of atopic sensitization in children with a CT/CC genotype.[25] Altogether, these observations perfectly illustrate that neonate gut barrier homeostasis is dependent on maternal milk factors. They suggest that low levels of certain factors in mother's milk, or absence of breastfeeding, may lead to low grade inflammation preventing tolerance to dietary antigens. This highlights the necessity to delineate which factors control the levels of critical molecules for the epithelial cell anti-inflammatory and barrier function such as oligosaccharides and growth factors.

Can Breastfeeding Promote Tolerance? Most allergic symptoms result from inappropriate Th2 immune response to antigen instead of Treg/Th1 responses observed in nonatopic patients.[2] In early life, immune system is prone to develop Th2 immune response and progressively matures towards Treg/Th1 immunity.[26] As discussed here above, breast milk can impact indirectly on infant immunity by its effects on gut barrier. In this section, we are assessing more specifically how breast milk can impact on neonate immune system itself and its maturation.

Breastfeeding and Infant Gut Microbiota One of the major factors known to affect immune regulation is the gut microbiota. This has been best studied in mice and exemplified in the extreme case of germ-free mice; those display a poorly developed immune system, deficient oral tolerance induction and a propensity to mount Th2 immune responses. Bacteria strains which specifically promote differentiation of Tregs (i.e. clostridia,bacteroides fragilis) or in

contrast proinflammatory Th17 lymphocytes (SFB) were further identified.[27] In parallel to mice studies, it was suggested that alterations in the intestinal microbiota composition during infancy might participate in the allergy 'epidemics'.[28] This hypothesis is supported by a recent prospective birth cohort showing that infants at risk of asthma exhibited transient gut microbial dysbiosis during the first 100 days of life. [29] Initial colonization occurs at birth and, during natural delivery, bacteria derived from the vagina and feces provide the founder population for the neonate. Thus, maternal microbiota composition and mode of delivery are shown to be important factors for infant microbiota establishment. Breast milk is thought to further shape the diversification of the microbiota. In particular, maternal IgA present in breast milk have been the focus of many studies which show that milk IgA are a critical link between maternal and infant microbiota composition. Maternal gut microbiota induces mucosal IgA which shape gut microbiota composition and are transferred trough breast milk; milk IgA will in turn shape neonate microbiota.[4] In addition, several studies have provided evidence for breast milk to contain approximately 103–104 cfu/ml; this finding suggests that milk microbiome may be regarded as constant source of commensal bacteria for the infant gut.[30] A recent trial attempted to modify human milk microbiome using a mixture of probiotic strains.[31] Authors found higher levels of both lactobacilli and bifidobacteria in women receiving probiotic supplements and who delivered vaginally.[31] Major advances in the identification of mechanisms by which microbiota ability to modulate immune system function give some new clues of breast milk action on protective effects of breast milk dependent microbiota. Works in the last years have highlighted that impact of microbiota on immune function is mainly mediated by microbiota metabolism of ingested nutrients. Fiber fermentation by certain bacteria species such as clostridia generates short chain fatty acids (SCFA) such as propionate, butyrate, and acetate which were shown to promote Treg differentiation and prevent allergy. Anti-inflammatory effects of SCFA have also been reported on other cell types such as epithelial and innate immune cells.[32]Reduced levels of fecal acetate were found in infants with increased allergy risk and dysbiosis.[29]Importance of SCFA on immune regulation has generated wide interest in the study of factors which are able to influence SCFA levels in breast milk and/or in factors stimulating SCFA producing bacteria. In this regard, HMOs may be critical candidates. The concentration of HMOs is between 10 and 15 g/l, which exceeds the levels found in bovine milk by 100–1000-fold, and concentrations are even higher in human colostrum.[33] HMOs function as prebiotics, serving as metabolic substrates for specific bacteria (e.g. Bifidobacterium longum subsp. Infantis) and stimulating their preferential growth.[33]Bifidobacterium longum are capable of metabolizing HMO into SCFA which exert beneficial effects as described above. Importantly, prebiotics are also able to modulate Bifidobacterium longum gene expression which are involved in carbohydrate degradation and cell adherence.[34] Furthermore, HMO can exert direct beneficial effects such as by inhibition of adhesion of pathogens on epithelial surfaces and induction of anti-inflammatory mediators secretion by epithelial cells

and immune cells.[33] Very elegant studies also demonstrated that maternal gut microbiota produces various metabolites during gestation which are transferred through breast milk and influence early postnatal immune system maturation and secretion of antimicrobial peptides by gut epithelial cells.[35] These effects were shown to be dependent on maternal milk immunoglobulins and to be independent of neonate microbial colonization. According to compelling evidence of immuno-modulatory effect by gut microbiota, many studies attempted to prevent allergy development by probiotic administration to pregnant mothers, the main rational being to modify maternal gut microbiota and subsequently that of their offspring. Although some studies found increased numbers of bifidobacteria in infant gut from mothers supplemented with bifidobacteria, others failed to reproduce these findings.[36] In addition to a possible probiotic direct effect on composition of maternal-child transferred microbiota, probiotics may affect the levels of immunomodulatory factors in breast milk. Prescott et al.[37] found higher levels of TGF-β1 and IgA levels in week one breast milk from the mothers receiving B. lactis HN019 probiotics, and higher IgA levels in those receiving L. rhamnosus HN001. Two other studies[38,39] reported no effect on TGF-β levels in breast milk from the mothers receiving probiotics during pregnancy with contrasting results shown by Rautava et al..[40] Zuccotti et al.[41] produced a meta-analysis, reviewing the ability of probiotics to prevent allergy development suggesting some protection against atopic dermatitis. At present, factors controlling breast milk oligosaccharides levels are unknown and the impact of direct prebiotics administration to infants on allergy development requires further research.[42]

Breastfeeding and Vitamins In addition to direct effect via microbiota metabolism, nutrients can directly impact on Tregs function. The role of vitamin D as an immuno-modulatory and protolerogenic agent has been the subject of many studies.[32] However, the most recent World Allergy Organization (WAO) guideline panel suggest not using vitamin D in pregnant women, breastfeeding mothers, or healthy term infants as a means of allergic disease prevention due to a very low certainty evidence.[43] Major advances have been brought recently on the impact of vitamin A on immune homeostasis (reviewed in[32]). We recently identified that neonatal mice are physiologically deficient in retinol; serum retinol levels then progressively increased and reached adult levels at 3 weeks.[15] Low vitamin A levels at birth were found to be responsible for a leaky gut barrier, deficient function of mesenteric lymph node CD103+ neonatal DC, resulting in inefficient T-cell activation and incapacity to induce oral tolerance in neonates for allergy prevention. [15]Importantly, maternal vitamin A supplementation during breastfeeding was sufficient to rescue neonatal gut barrier and immunological defects and provided allergy prevention from birth. Importantly, our observations also showed that vitamin A was involved in the maturation of neonates' immune responses towards Th1 immune responses; this adds a dietary factor to the genetically programmed and microbiota driven

neonate Th1 immune maturation.[15] Relevance of these data is supported by human studies showing low retinol levels in healthy infants from well-nourished countries and observational studies linking low retinol levels at birth with increased atopic risk in young adults. [44] We found only a single intervention trial assessing impact of vitamin A supplementation at birth on risk of atopy development. This trial performed in Guinea-Bissau reported association between neonatal vitamin A supplementation and increased atopy prevalence in girls.[45] Since vitamin A has been shown to strongly stimulate proinflammatory responses in case of mucosal inflammation, we propose that endemic enteric infectious diseases in Guinea-Bissau may be responsible for opposite effects of vitamin A in this setting. Intervention trials in developed country settings are needed to confirm that vitamin A supplementation has a beneficial role in lactating mothers for allergy prevention.

Breastfeeding and Oral Tolerance In recent years, the concept has emerged that early oral exposure to food antigens may be necessary for immune tolerance induction and long-term prevention of inappropriate immune reactions to food allergens.[46] The World Health Organisation (WHO) recommends exclusive breastfeeding for at least 6 months in all infants.[47] The recent research, however, challenged this approach showing that early food introduction may decrease the risk of food allergy development.[48] These findings highlight the importance of food protein transfer via breast milk as this is the first exposure to foods for the infant. In a mouse model, we found that mice exposed to a few nanograms of egg ovalbumin (OVA) antigen through breast milk were protected from OVA-induced allergic airway disease and that TGF-β from breast milk was critical for tolerance induction.[49] We found protection to be more profound when OVA was transferred through the breast milk of OVA-immunized mothers compared with OVA exposed nonimmunized mothers.[50] These data highlighted that variation in milk composition and in particular its allergen content, TGF-β and allergen-specific immunoglobulin may contribute to heterogeneity of results on allergy prevention by breastfeeding. There are limited data on food consumption and subsequent appearance of the food proteins in the milk. Antigens from maternal diet are found in concentrations in the range of the ng/ml in contrast to mg/ml for antigen in formula milk. Most studies show a lack of direct correlation between amount of food allergen consumption and its level in breast milk. [51] The ongoing QUEST study conducted by Dr Debbie Palmer is an intervention trial analyzing the impact of egg consumption in the diet of breastfeeding mothers on egg allergy development in infants. Results of this trial will bring more information on the possibility to stimulate oral tolerance through breast milk; this would indicate whether food diversification through dietary allergen exposure via breast milk may contribute to allergy prevention. Interestingly, a recent trial found that maternal cow's milk avoidance during lactation was associated with lower levels of mucosal-specific IgA levels and the development of cow's milk allergy in infants showing the possible direct and indirect effects of nutritional intervention in lactating mothers. [52]

Recently, we further proposed that inconsistencies in findings on food allergy prevention by breastfeeding may result from variations in duration of breastfeeding induced protection. In mice, we found that prevention of food allergy by egg antigen exposure through breast milk was of limited duration (Rekima et al. in revision). Nutritional intervention by TGF-β supplementation after weaning prolonged beneficial effects of breast milk on food allergy prevention. The need for exogenous TGF-β in early life may be related to insufficient endogenous TGF-β synthesis in infants. This is supported by data showing low expression of TGF-β in the small intestine of rats before weaning.[53] and by analyses of TGF-β in infant stools which show that levels of TGF-β between birth and 6 months of age are five times lower than those at 1 year.[54] From these data, we propose that in early life insufficient endogenous synthesis of TGF-β combined with insufficient exogenous sources may preclude long-term maintenance of tolerance and prevention of food allergy. The systematic review by Oddy and Rosales [55] assessed relationship between TGF-β in human milk and immunological outcomes in infants and children. Two-thirds of the studies selected for this review found an association between higher TGF-β1 or 2 levels in colostrum or HM and reduced risk of atopic outcomes in the infant. This hypothesis may underlie the protective effect of breast milk on allergy.[55] To define if breastfeeding could also impact on respiratory allergy disease by oral tolerance, we have been assessing the presence of respiratory allergens in breast milk. We showed that house dust miteDermatophagoides Pteronyssinus (Der p) 1 was present in human breast milk of mothers from three geographically diverse regions of the world.[56] We also found allergen from Blomia Tropicalis, Blo t 5, mite common in the tropical region to be present in milk from a Brazilian cohort.[57]Der p 1 in maternal milk was found in similar quantities as dietary egg antigen OVA and to be immunoreactive. [56] In contrast to the observation with OVA, we found that the transfer of Der p 1 through breast milk induced Th2 immune response priming and increased susceptibility to allergic disease in adulthood.[56] Importantly, in a human birth cohort, we found an increased allergic sensitization risk and respiratory allergies in children breastfed by mothers with increased Der p 1 levels in breast milk.[58] Compared with OVA, Der p displays strong intrinsic adjuvant properties[59] and our current work is assessing how Der p affects neonatal gut mucosal immune system maturation and how maternal milk factors could counteract their deleterious action.

Conclusions Although breast milk is strongly recommended for prevention of infectious disease, it does not fulfill public health expectations in regard to allergy prevention. Breast milk is a unique food for the developing infant; it contains numerous biological factors which may impact on various homeostatic processes such as tissue development and repair, immune regulation, and gut microbiota establishment. Furthermore, it possesses the capacity to be modulated by maternal environment. These characteristics make breast

milk a strong candidate for allergy prevention. In order to achieve this task, further studies are required to establish which maternal interventions should be encouraged to adapt milk composition to the new proallergenic, western life environment.

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