Modifying the Balance – What is the Role of the Gut Microbiome in Allergic Disease?
Christina E. West
The immune system is a system of cells and tissues that protects us against invading pathogens. It must learn how to provide tolerance to “non threats” such as food components, commensal microbiota and to the organism itself. So it must actually learn how to distinguish “harmless” from “dangerous” – and this is an active process.
Immune System Development
Immune cells and organs proliferate rapidly in the first trimester and the development of secondary lymphoid organs is nearly complete at birth. However, quite recent work indicates that these organs, particularly the GALT, remain highly responsive to environmental stimuli (antigens and allergens) throughout life.
Declining biodiversity and aberrant intestinal colonization is a main theme in the “allergy epidemic.” Complex microbial communities in the gastrointestinal tract have a symbiotic relationship with the host,and microbial exposures in the perinatal period seem to be critical for the ontogeny of both innate and adaptive immunity.
Colonization of the mucosal surfaces appears to be vital for the establishment of tolerance. Delay or disruption in the colonization of the gastrointestinal tract may result in dysregulated immune responses. This has been clearly shown in animal models and there is also indirect evidence in humans. For example, delivery by cesarean section is associated with disturbed intestinal colonization patterns and increased risk of both allergic and autoimmune disease. But we do not yet know exactly how the mechanism of tolerance establishes itself in humans.
Gut microbiota have also been demonstrated to enhance the integrity and functions of the gut barrier. Gut microbiota will increase secretory IgA production locally and also induce the goblet cells to produce mucin, thereby enhancing the gut barrier. The gut microbiota can also be involved in crosstalk with distant organs as bacterial ligands, bacterial metabolites and immune cells may enter the systemic circulation and seed distant organs e.g. the lung.
Toll-like receptors (TLR) are believed to be ancient gatekeepers in innate immunity; they are expressed on a number of epithelial and endothelial cells, leukocyte subsets in blood. TLR activation can either increase or decrease the suppressor activity of Tregulatory cells, thus providing an important link between innate and adaptive immunity.
In a study within a high-risk cohort in Perth, where blood was first sampled at birth and afterwards during the first five years of life, infants that later developed allergic disease showed increased inflammatory responses following TLR activation in early life compared with healthy children. However, these responses failed to develop into mature Th1 type responses in allergic children (Tulic MK et al., JACI 2011).
Sterile womb paradigm versus in utero colonization hypothesis
The sterile womb paradigm asserts that the fetus and the placenta are sterile and that gut microbiota is acquired after birth. Vaginal delivery will obviously have a huge influence of this process.
The emerging in utero colonization hypothesis posits that the placenta harbors its own microbiota and that the colonization of the gut begins already in utero. More research is needed to confirm the latter hypothesis.
Building on the results from the high-risk cohort in Perth, where babies who went on to develop allergic disease had exaggerated innate immune responses (Tulic MK et al., JACI 2011), we hypothesized that this immune dysfunction might be dependent on microbial stimuli very early in life. So in another high-risk cohort, in which atopic mothers were included, we sampled feces from the mothers in the 3rd trimester and then afterwards from their infants at 1 week, 1 mo and 12 mo of age. Blood was drawn at 6 mo of age and PBMC were activated with microbial ligands. We studied microbial development in babies that had eczema, plus evidence of sensitization or had food allergy. We paired the despite their heredity, were non-sensitized and had no eczema (Chart 1).
There were both maternal effects and also postnatal effects. In pregnancy, there was reduced diversity microbial development to healthy controls, which of the main phylum Bacteridetes in mothers whose infants developed IgE-associated eczema. Although this was not seen in their infants, it could be consistent with results from a Swedish study that found reduced Bacteroides in infants who developed IgE-associated eczema (Abrahamsson TR et al., JACI 2012). In our study, we found reduced Ruminococcaceae in infants that developed IgE-associated eczema. Notably, the underrepresentation of Ruminococcus in these infants was associated with exagerrated innate inflammatory immune responses (West CE, et al. Clin Exp Allergy 2015). Interestingly, underrepresentation of Ruminococcaceae was also a feature in food sensitized 1-year-olds in a Canadian cohort (Azad MB et al., Clin Exp Allergy 2015).
Very recently, we studied the longitudinal gut microbial development in allergic disease in more detail as we had collected stool samples in a probiotic allergy prevention trial in infancy. We did a follow up at 8 years of age including a very thorough clinical assessment and collection of biological specimens (West CE et al., Allergy 2013). We clinically phenotyped the children at 8 years of age and found 21 children which had IgE-associated allergic disease. Seventy-two children served as healthy controls as they were neither IgE sensitized nor did they show any other allergic manifestations.
Because we could study these children longitudinally from infancy to school age, we could identify both temporal and consistent underrepresentation of specific taxa in allergic diseases (Sjödin KS et al., Allergy in press). Ruminoccous was transiently underrepresented whereas Prevotella, Coprococ- cus and Bacteroides were consistenly underrepresented. Another novel finding is that Faecalibacterium, which is suggested as a bacterial biomarker for gut health, correlated with the T cell regulatory markers IL10 and FOXP3 in allergic children at 8 years of age. In addition, we found transient and minor effects of probiotic intake so, during the intervention, there was a higher abundance of Lactobacillus at 6 mo of age; when we looked at 8 years of age there were no effects on the global microbiota nor were there any long-lasting effects on the Lacto- bacillus population.
Importance of Bacteroidetes
One of the functions of Bacteroidetes is that they stimulate epithelial mucin production and, together with Prevotella, Coprococcus and Bacteroides, they have the capacity to ferment carbohydrates to produce butyrate, with both nutritive and an- ti-inflammatory effects. Very recently, Bacteroides-derived fecal sphingolipids were negatively associated with food allergy (Lee-Sarwar K et al., JACI 2018).
In summary, this is the first study to have actually looked at the longitudinal development of gut microbiota in relation to IgE associated diseases (Sjödin KS et al., Allergy in press). Although there was a temporal underrepresentation of Ruminococcus, one important finding is that it was actually consistent underrepresentation of Bacteroides, Prevotella and Coprococcus.
The dysbiosis seen in a number of inflammatory non-communicable diseases has aroused huge interest and prompted researchers to use various microbiota modulation techniques to try to favorably modulate gut microbiota to improve immunological and metabolic outcomes and also influence the gut- brain axis. We do have to keep in mind that nutrition and gut microbiota are interrelated, so there are a number of dietary exposures that can have effects both on immunological programming and the gut microbiota. These include pre- and probiotics, dietary fibers, folate and antioxidants, when and how we introduce allergenic foods and n3-PUFA. So the aim of optimized intervention at this stage would be to try to induce the tolerogenic environment in the gut and also to promote tolerance acquisition.
Fiber is one option and I am involved in one study where dietary fiber is given in pregnancy to improve maternal gut microbiota with possible effects on a later colonization and immune outcomes in their children. Pre-, pro- and synbiotics are still of interest and it will be highly interesting to disentangle the effects of HMOs in tolerance acquisition in the human setting. In addition, we are becoming increasingly aware that environmental microbes also have a huge impact on our microbiota thereby giving rise to other interventions than diet.
I think that during the last two years, we have come a little bit closer toward understanding this process. We have to take into account the prenatal and postnatal period in order to promote establishment of oral tolerance (Chart 2).
- Our commensal gut microbiota is im port for the development of immune tolerance
- Dysbiosis is implicated in allergic diseases; more studies investigating the functional aspects of the gut microbiota are needed
- The gut microbiota is tunable
- Gut microbiota modulation strategies should aim to optimize both maternal and infant colonization patterns
Human Milk Oligosaccharides (HMO): Factors Affecting their Composition and their Physiological Significance
Human milk oligosaccharides (HMOs) are elongations of the milk sugar lactose by galactose, N-acetylglucosamine, fucose and sialic acid. The HMOs composition of brestmilk is strongly influenced by polymorphisms of the maternal fucosyltransferases, FUT2 and FUT3, and by stage of lactation. Clinical observational studies with breastfed
infant-mother dyads associate specific HMOs with infant gut microbiota, morbidity, infectious diarrhea and allergies.
Observational and basic research data suggest that HMOs influence the establishment of the early life microbiota and mucosal immunity and inhibit pathogens, thereby contributing to protection from infections. Clinical intervention trials with infant formula supplemented with the single HMO, 2’-fucosyllactose (2’FL) or with 2 HMOs, 2’FL and Lacto-N-neotetraose (LNnT), demonstrated that they allow for age appropriate growth and are well tolerated. A priori defined exploratory outcomes related feeding an infant formula with 2 HMOs to fewer reported lower respiratory tract illnesses and reduced need for antibiotics during the first year of life, compared to feeding a control formula. In parallel, the early life microbiota composition shifted towards that of breastfed infants. Together, HMOs likely contribute to immune protection in part through their effect on the early life gut microbiota, findings that warrant further clinical research to appreciate our understanding of HMO biology and significance for infant nutrition.
What are human milk oligosaccharides (HMOs)? What is their importance for infant nutrition? These questions have intrigued scientists and pediatricians alike for over a century. Advances in analytics as well as large-scale synthesis technologies stimulated great progress in recent years. These provided the materials and tools that enabled the detailed and accurate measurement of HMO quality and quantity, and the study of HMOs in basic research models, and through clinical observational studies and intervention trials.
“HMOs are not HMOs”, meaning that one specific HMO is not equal to another HMO, especially when considering structure function relations. Chemically, HMOs are elongations of the milk-specific sugar lactose in different linkages by one or a combination of the following monosaccharides: L-fucose (Fuc), D-galactose (Gal),
N-acetyl-D-glucosamine (GlcNAc) and N-acetylneuraminic acid (sialic acid, SA). Gal and GlcNAc generally elongate lactose as a disaccharide Gal-GlcNAc. The numerous and diverse HMOs produced might be categorized by specific structural features brought about by different glycosyltransferases involved in their synthesis. However, many HMOs combine different structural features.
Breastmilk, the recommended and naturally adapted nutrition for infants, is associated with a reduced risk for infection- related illnesses and possibly for diabetes and overweight, while the situation for allergies is less clear . This suggests that breastmilk-specific components such as HMOs and other bioactives, may contribute to such benefits.
Due to their structural similarity with mucosal glycans and their non-digestible nature, HMOs expectedly affect numerous glycan-mediated processes like the colonization of the early life microbiota and the infectivity of pathogens (Figure 1A). Based on clinical observational and basic research data, HMOs, act in a structure-function specific way helping the (i) establishment of mucous adapted microbiome, (ii) resistance to pathogens and (iii) reactivity of the mucosal barrier and immunity, thereby contributing to immune protection.
Here, we briefly review genetic and environmental factors affecting HMO composition in breastmilk and the physiological role of HMOs as supported by clinical observation studies, preclinical research on mode of action and insights from clinical intervention trials.
Maternal glycosyltransferase polymorphisms affect HMOs composition.
HMOs resemble the blood group antigens and further sialylated glycans that cover the human mucosa. The same glycosyltransferases are generally involved in the synthesis of mucosal cell glycans and mammary gland expressed HMOs. The fucosyltransferases FUT2 (Secretor gene) and FUT3 (Lewis gene) are the best described due to their natural polymorphisms in humans  (Figure 2A). Specific genetic polymorphisms abolish their respective enzyme activity. Thus, specific HMOs structures that depend on FUT2 or FUT3 can be identified. FUT2-dependent HMOs all contain alpha-1,2 linked fucose, for example 2’FL, lactodifucosyllactose (LDFT), Lacto-N-fucosylpentose-I (LNFP-I). Interestingly, trace amounts of 2’FL were found in breast milk of presumed FUT2 negative mothers in Asian populations [3, 4], indicating that the nature of these inactivating polymorphisms and thus the HMO profile may be population-specific . Typical FUT3 dependent HMOs are LNFP-II, lacto-N-difucosylhexose-I (LNDFH-I) containing alpha-1,4 linked fucose and to a lesser extent 3-fucosyllactose (3FL) and LDFT containing alpha-1,3 linked fucose. In breastmilk that does not contain any detectable LNFP-II, reduced amounts of HMOs with alpha-1,3 fucose on glucose and increased amounts of those with an alpha-1,3 fucose on GlcNAc are found. Hence, another FUT (e.g. FUT4, 5, 6, 7 or 9) is also involved in the formation of HMOs with an alpha-1,3 linked fucose on GlcNAc and glucose.
The absence of a functional FUT2 or FUT2 and FUT3 affects the concentration of total HMOs in milk, when expressed as the sum of all quantified HMOs  (Figure 2). While some HMOs increase when FUT2 is missing (e.g. LNT, 3FL), in the absence of fucosylation additional larger non-fucosylated HMOs might also be produced.
To date, no common genetic polymorphisms for sialylated HMOs are described, indicating that if inactivating polymorphisms in sialyltransferase genes exist, they are extremely rare. From mouse studies, the sialyltransferases ST6Gal1 and ST3Gal4 are involved in the synthesis of 6’-sialyllactose (6’SL) and 3’-sialyllactose (3’SL) respectively, with a further sialyltransferase, probably ST3Gal1, also making 3’SL .
Another mechanism affecting HMO composition is probably the donor and acceptor substrate availability as suggested by the increase of 3FL when the major fucosyl-HMO 2’FL decreases in concentration .
Interestingly, HMO concentrations change during stage of lactation with different HMOs showing different dynamics . HMOs like 6’SL or LNT decrease more rapidly during the first weeks of lactation, while 2’FL and 3’SL, for example, decrease more slowly over a longer time period and again others, like 3FL, actually increase in concentration with time of lactation (Figure 2B).
Such compositional changes due to the genetic background of mothers and stage of lactation can confound observations relating HMOs to clinical parameters in the breastfed infants and, therefore, need to be considered.
HMOs composition and infant gestational age, maternal diet and physiological state.
HMOs concentrations in colostrum, transitional and mature milk seem not to change between mothers giving birth to preterm (n=18; gestational age <37 weeks) and term (n=14; gestational age ≥37 weeks) infants . Further, fucosylated and sialylated HMOs were reported to be similar between preterm and term milk, although preterm milk seemed more variable in the expression of fucosylated HMOs .
Today, we do not know whether and how maternal diet might influence HMO composition. A recent observational study with 33 breastfeeding mothers and their infants from The Gambia, Africa, reported a significantly higher HMO content in milk at 20 weeks of lactation in the dry season (n=21) compared to the wet season (n=12) . The authors propose a possible link to the higher energy intake during the dry season. In two other African mother-infant cohorts from Malawi (n=88 and n=215), total HMOs and also sialyl-HMOs and fucosyl-HMOs, were lower at 6 months postpartum in breastmilk of mothers having severely stunted infants compared to those with normal size infants . These studies suggest that maternal nutritional and health status may affect HMO composition.
By analogy, higher maternal body mass index and gestational weight gain, generally reflecting an altered metabolic physiology, might affect HMO composition. Studies to this end are currently ongoing  (Binia et al. 2017 Abstract at FASEB SRC). Suitable studies are warranted to investigate possible alterations of HMOs composition due to maternal energy and specific nutrient intake.
The HMO composition is associated to the establishing gut microbiota in infants.
The early life microbiome has a major impact on the developing immune defenses, itself being an important element by providing pathogen colonization resistance, for example. Interestingly, the establishing intestinal microbiota also contributes, via an innate lymphoid cell-mediated process, to improved protection against respiratory tract infection . The pioneers of human milk and breastfeeding research observed a strong link between breastfeeding and immune protection to infectious morbidity and mortality. Breastfed infants were recognized to harbor an early gut microbiota dominated by bifidobacteria, not seen in formula fed infants, and a human milk specific “bifidofactor” was identified in the HMO fraction of breast milk .
From research on early life microbiota, we know that bifidobacteria can utilize and grow on different individual HMOs in a strain-specific way [13, 14]. Several studies observed an increased bacterial metabolic activity upon growth on HMOs, exemplified by the formation of the short chain fatty acid acetate [15, 16]. Noteworthy, numerous potentially pathogenic bacteria from the Enterobacteriaceae group were shown not to grow on individual HMOs as the sole carbon source , while growth of other pathogens, like Streptococcus agalactiae (group B Streptococcus, GBS) was shown to be inhibited by HMOs [18, 19].
Recently, LNnT in breastmilk was associated with Bifidobacterium longum ssp infantis abundance . In bi-associated gnotobiotic mice harboring only one
Bacteroides and one B. longum ssp infantis strain, LNnT lead to bifidobacteria dominance although both bacteria could actually use LNnT in vitro . In gnotobiotic mice humanized with seven human microbes, B. longum ssp infantis also showed higher abundance when these mice were fed 2’FL combined with LNnT as compared to LNT alone (Sprenger N. et al. unpublished observation), although B. longum ssp infantis is able to grow on many different HMOs, including LNT, as substrate .
Genomic and glycomic analyses in infants provided further evidence for a role of HMOs in shaping the early infant gut microbiome, revealing associations between individual HMOs and bacterial genera in infant stool [21-23]. A bifidobacteria dominated gut microbiota in breastfed infants (n=105) at 4 months of age was associated to breastmilk containing FUT2- HMOs . The FUT2 status of the infants and its possible confounding effects on the infant microbiota profile were not assessed, despite earlier data proposing the FUT2 status itself can influence the gut microbiota at least in adults . In another cohort, the analysis of a relatively small subgroup of 4 month exclusively breastfed infants (n=14) showed an association of maternal FUT2-positive status with higher bifidobacteria abundance up to 2-3 years of age . However, no statistically significant HMO effects on global bifidobacteria shifts were reported in another recent study of 33 Gambian mothers and infants , while the abundance of individual bifidobacteria like B. longum ssp infantis still correlated with LNnT concentrations in breastmilk. These first reports reveal the need for larger observational studies of similar design, including comprehensive breastmilk HMO analysis and infant FUT2 phenotyping to gain a more robust understanding of the link between HMO and infant gut microbiome composition.
Today, clinical observations in conjunction with basic research data suggest that FUT2-HMOs, like 2’FL and LNFP-I , but likely other non-FUT2 dependent HMOs, like LNnT for example, are involved in the establishment of a bifidobacteria dominated early life gut microbiota. In vitro studies help to understand HMO-related microbial metabolic capacities and strain specificities, while animal and human observational studies indicate that the interaction between bacteria and the gut mucosa reflect a more complex picture. Hence, with infant health in mind, it is central to gain a better understanding of HMO effects on the microbiome dynamics in their natural ecosystem through a holistic and ecology inspired approach.
HMO composition is linked to infection risk in infants
HMOs were studied in relation with infectious diarrhea incidence in a cohort of Mexican mothers and infants (n=93) [27, 28]. Higher breastmilk concentrations of α1-2 fucosylated HMOs were associated with lower incidence of all causes of moderate-to-severe diarrhea. The most frequently identified cause of diarrhea in the cohort was Cambylobacter jejuni followed by calicivirus and enteropathogenic Escherichia coli. Specifically, higher concentrations of 2’FL and LNFP-I in breastmilk related with a lower incidence of C. jejuni and calicivirus diarrhea respectively. These observations during the breastfeeding period did not persist in the post- breastfeeding period, indicating a possible transient HMO effect in the protection from infectious diarrhea. This fits their presumed role as antiadhesive antimicrobials. Experimental data from preclinical models also show protective effects of 2’FL from C. jejuni  and aggregating invasive E. coli . From these data, 2’FL and other FUT2-HMOs seem to act as soluble ligands blocking C. jejuni from adhering to gut epithelial cells, while the protection from E. coli might rather be due to an anti-inflammatory effect, possibly combined with the modulation of the gut microbiota composition.
Glycans containing α1-2-linked fucose expressed on epithelial cells of FUT2 positive infants could act as receptors for pathogen binding, conferring a risk to specific infectious diseases for this population . Genetic studies have shown that infants and children with a non-functional FUT2 gene have strain-specific protection against norovirus and Rotavirus [32, 33]. For specific rotavirus strains, susceptibility depends on FUT2, but also on FUT3 status . Experimentally, infectivity of some rotavirus strains was reduced by the FUT2 HMO 2’FL, while other viral strains were affected by sialylated HMO, namely 3’SL and 6’SL . Similarly, 2’FL also bound to specificn norovirus strains .
Besides interfering with pathogen attachment to the host mucosa, HMOs were recently reported to exert bacterial growth inhibitory activities on pathogenic group B Streptococcus (GBS) [18, 19, 37] a major cause of sepsis in preterm infants. Growth of GBS was specifically inhibited by LNT and LNFP-I, while sialylated HMOs or galactooligosaccharides (GOS) had no effect . Experimental data suggests a putative glycosyltransferase of GBS to be involved . Possibly pointing to a similar mechanism, HMOs from milk of a FUT2 negative mother were shown to have bacteriostatic properties via an alteration of biofilm formation . In an observation study of 183 Gambian infant mother pairs, FUT3 positive mothers were reported to be less likely carriers of GBS, as were their infants at birth . Interestingly, infants of FUT3 positive mothers were also more likely to clear GBS colonization from birth to 2-3 months of age compared to infants of FUT3 negative mothers.
In a pilot study of 49 mother-infant pairs, higher breastmilk concentrations of the FUT3 HMOs LNFP-II at 2 weeks were associated with a lower risk of respiratory and gastrointestinal illnesses at 6 and 12 weeks in infants . This association was no longer significant past the breastfeeding period. Similarly, in a nested case cohort study of 143 HIV exposed uninfected children from Zambia, higher concentrations of fucosylated HMOs in breastmilk at 1-month post-partum related to a lower risk of mortality up to 2 years of age . In another small mother-infant cohort from The Gambia (n=33), higher relative breastmilk concentrations of fucosylated HMOs (sum of LNFP-I and LNFP-III) and concomitant lower relative abundance of LNT was associated with lower risk of sickness up to 4 months of age .
For respiratory pathogens, direct exposure to HMO would appear less evident and thus any putative HMO-related protection may be mediated by the intestinal microbiome [11, 40]. Yet, experimentally, direct exposure of Streptococcus pneumoniae to LNnT and sialyl-LNnT and subsequent infection effectively blocked its colonization in the lung of a rabbit model . In a cell based assay, LNnT and 2’FL dose dependently reduced Influenza and Respiratory Syncytial Virus (RSV) concentrations within respiratory tract cells .
Observational studies together with findings from preclinical models have provided first evidence for an association between HMOs and the risk of infections, mostly in a structure function specific way. Mechanistically, HMOs may act through multiple functions, although preclinical models highlight specific individual functions. The current studies also provide directions to be considered in future observational studies such as timing of milk sampling and breastmilk intake, etiology of infections, quantitative versus categorical analysis of HMOs and finally mother and
HMO composition might be linked to allergy in infants
Numerous environmental, including nutrition, and genetic factors affect allergies. Among them are breastmilk bioactives, and possibly HMOs. In a cohort of 266 Finnish mother infant pairs with a hereditary allergy risk, 2’FL concentrations in early breastmilk associated with a lower risk to manifest IgE-associated eczema at 2 years of age only in C-section born infants . This observation suggests that 2’FL may influence IgE-associated eczema through the modulation of the early life gut microbiota, known to be different in C-section born infants compared to vaginal born infants. A possible relation of HMOs with cow milk allergy (CMA) was studied in another cohort of 39 mothers with infants who developed CMA by 18 months of age and 41 mothers with infants without CMA . An association was seen between the milk concentration of several individual HMOs (LNFP-III, 6’SL, LNFP-I, DSLNT) and HMO clusters with reduced risk of CMA, with LNFP-III providing the strongest association. Breastmilk sampling varied over the first 6 months after birth and therefore might have introduced a bias, because HMO concentrations change dramatically during this period. Mechanistically, the authors speculate that LNFP-III might act on the immune system via dendritic cells and DC-SIGN. In a preclinical food allergy model, 2’FL and 6’SL were tested and both reduced symptoms involving mast cell activity .
The observational studies to date have their limitations, but still provide valuable preliminary data on possible relationships between specific HMOs and risk of allergies. To appreciate such a proposed link requires replication in larger cohorts with harmonized milk sampling, stratification for mode of delivery and evaluation of infant FUT2 and FUT3 genotypes.
Insight from clinical intervention trials with specific HMOs.
Recent progress in industrial biotechnology has made available few individual HMOs, namely 2’FL and LNnT. Preclinical safety toxicity tests established their safety and both obtained approval as novel foods in the European Union and were generally recognized as safe in the USA.
In adults, both 2’FL and LNnT were studied alone or in combination at different doses from 5 to 20 g/day in a placebo controlled, blinded and randomized trial (n=100). Both HMOs were well tolerated and increased bifidobacteria abundance .
In infants, two placebo controlled, blinded and randomized clinical intervention trials showed the growth-safety and tolerance of 2’FL combined with either GOS or fructooligosaccharides (FOS) [47, 48] (Kajizer et al. 2016 FASEB J). Infants fed with an infant formula supplemented with 2’FL (0.2 or 1 g/L) combined with GOS or GOS alone showed similar growth as breastfed infants up to 4 months of age (n=314). In a subgroup of infants, immune markers were measured in plasma at baseline and upon stimulation of blood cells with RSV. Globally the immune profile resembled that of breastfed infants when the infant formula was supplemented with 2’FL at the lower or higher dose . Another randomized controlled infant trial showed that an infant starter formula supplemented with 2 HMOs, 2’FL and LNnT, (n=88) allowed for age appropriate growth of term born infants, and was well tolerated when compared to the same infant formula without HMO (n=87) . Interestingly, secondary exploratory findings showed an association between feeding the 2-HMO infant formula and less reported lower respiratory tract illnesses and medication use (especially antibiotics and antipyretics) during the first year of life and beyond the 6 months feeding period. At 3 months, the global microbiota profile shifted in the 2-HMO formula- fed infants away from the control formula fed infants and towards that observed in breastfed reference infants. This shift was mainly due to increases in Bifidobacterium concomitant with decrease in Escherichia and Peptostreptococcaceae . A significantly higher number of infants who were fed the 2-HMO supplemented formula showed a microbiota community structure typical for breastfed infants compared to control formula fed infants, who had primarily a different microbiota community structure. Interestingly, infants with a microbiota community structure typical for control formula fed infants had a 2 times higher risk to use antibiotics during the first year of life compared to those with a microbiota community typical for breastfed infants .
These first clinical intervention trials with specific HMOs demonstrate their growth-safety and digestive tolerance. Additionally, as suggested from basic research and observational data, 2’FL and LNnT might contribute to protection from infection-related illnesses and reduced need for antibiotics, possibly through the modulation of the establishing early life gut microbiota.
HMOs composition is affected most notably by the maternal FUT2 and FUT3 status. This is likely due to an evolutionary selective pressure imposed by pathogens or the microbiome at large. Stage of lactation alters HMO composition possibly indicating different infant needs at different extra uterine developmental stages. However, giving birth to a preterm or term infant, who are at different developmental stages, seems not to affect the HMO composition of breastmilk. Clinical observations corroborated by preclinical data and clinical intervention trials support a role for specific HMO in immune protection, primarily from infection related morbidity and use of antibiotics. Further clinical studies, well-designed observational and especially placebo-controlled interventions, are warranted to further substantiate and grow our understanding of the HMO biology and significance for infant nutrition.
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Human Milk Oligosaccharides: Factors Affecting Their Composition and Their Physiological Significance
Human Milk Oligosaccharides: Factors Affecting Their Composition and Their Physiological Significance
Norbert Sprenger, Aristea Binia, and Sean Austin
Breastfeeding is related to a lower risk of infections and possibly diabetes and overweight in later life, while the situation for allergies is less clear , which suggests that breast-milk-specific components may contribute to such benefits. Among them are the nondigestible human milk oligosaccharides (HMOs), the third largest solid breast milk component. HMOs are elongations of the milk sugar lactose by galactose, N-acetylglucosamine, fucose, and sialic acid, which results in structures similar to those on the mucosa. Most HMOs are not present in farmed-animal milks and are different from generic prebiotics such as galacto- and plant-derived fructo-oligosaccharides. Maternal fucosyltransferases FUT2 and FUT3, encoded by the Secretor and Lewis genes, respectively, followed by lactation stage, have the most striking impact on the HMO composition . The presence or absence of functional FUT2 and FUT3 not only affects the abundance of individual fucosyl-HMOs, but also the total HMO concentration in breast milk. The maternal nutritional and health status might influence HMO composition in the breast milk; however, today there are only circumstantial data to this end. Clinical observational studies in breastfed infant-mother dyads associate specific HMOs with infant gut microbiota, morbidity, infectious diarrhea, and allergies. Although observational studies do not establish causality, together with experimental data they suggest possible biological roles for HMOs. In particular, it is believed that they affect the (i) establishment of the early-life microbiota dominated by bifidobacteria, (ii) resistance to pathogens, and (iii) intestinal mucosal barrier and immunity, thereby contributing to immune protection (Fig. 1a) .
Clinical intervention trials with infant formula supplemented with 1 HMO (2'fucosyllactose, 2'FL) or 2 HMOs (2'FL with lacto-N-neotetraose) demonstrated that they allow for age-appropriate growth and are well tolerated [4, 5]. A priori defined secondary outcomes suggested that feeding an infant formula with 2 HMOs relates to fewer reported lower respiratory tract illnesses and reduced requirement for related medication (antibiotics and antipyretics) during the first year of life . In parallel, the early-life microbiota composition and community structure in infants fed the 2-HMO formula shifted towards that of breastfed infants. Formula containing 2 HMOs shifted the global microbiota profile towards that of breastfed infants, characterized by a Bifidobacterium dominance and lower abundance of Escherichia, for example. Interestingly, infants with a microbiota community structure typical for control-formula-fed infants had a 2 times higher risk to use antibiotics during the first year of life than those with a microbiota community typical for breastfed infants.
Fig. 1. Illustration of the different biological functions of HMO (a) and risk for infection-related illnesses and medication use in infants fed a formula supplemented with 2 HMOs (redrawn from Puccio et al. ). Illnesses and medication use were reported by parents and verified by a study physician (b). URT, upper respiratory tract; LRT, lower respiratory tract. Odds ratios with 95% confidence intervals are shown based on percent of infants with at least 1 event at 1 year of age (Fisher’s exact test: * p < 0.05, ** p < 0.001) .
Together, clinical observational studies corroborated by preclinical experimental data and clinical intervention trials support a role for specific HMOs in immune protection leading to the reduced use of antibiotics. Further clinical studies, well-designed observational and especially placebo-controlled interventions, are warranted to further substantiate and grow our understanding of the HMO biology and significance for infant nutrition.
- Victora CG, Bahl R, Barros AJ, et al: Breastfeeding in the 21st century: epidemiology, mechanisms, and lifelong effect. Lancet 2016;387:475–490.
- Kunz C, Meyer C, Collado MC, et al: Influence of gestational age, secretor, and Lewis blood group status on the oligosaccharide content of human milk. J Pediatr Gastroenterol Nutr 2017;64:789–798.
- Bode L: The functional biology of human milk oligosaccharides. Early Hum Dev 2015;91:619–622.
- Marriage BJ, Buck RH, Goehring KC, et al: Infants fed a lower calorie formula with 2'FL show growth and 2'FL uptake like breast-fed infants. J Pediatr Gastroenterol Nutr 2015;61:649–658.
- Puccio G, Alliet P, Cajozzo C, et al: Effects of infant formula with human milk oli gosaccharides on growth and morbidity: a randomized multicenter trial. J Pediatr Gastroenterol Nutr 2017;64:624–631.
- Ferrer-Admetlla A, Sikora M, Laayouni H, et al: A natural history of FUT2 polymorphism in humans. Mol Biol Evol 2009;26:1993–2003
Human milk oligosaccharides (HMOs) are elongations of the milk sugar lactose by galactose, N-acetylglucosamine, fucose; and sialic acid. The HMO composition of breast milk is strongly influenced by polymorphisms of the maternal fucosyltransferases, FUT2 and FUT3, and by the stage of lactation. Clinical observational studies with breastfed infant- mother dyads associate specific HMOs with infant gut microbiota, morbidity, infectious diarrhea, and allergies. Observational and basic research data suggest that HMOs influence the establishment of early-life microbiota and mucosal immunity and inhibit pathogens, thereby contributing to protection from infections. Clinical intervention trials with infant formula supplemented with the single HMO, 2′-fucosyllactose (2′FL), or with 2 HMOs, 2′FL and lacto-N-neotetraose (LNnT), demonstrated that they allow for age-appropriate growth and are well tolerated. A priori defined exploratory outcomes related feeding an infant formula with 2 HMOs to fewer reported illnesses of the lower respiratory tract and reduced need for antibiotics during the first year of life compared to feeding a control formula. In parallel, early-life microbiota composition shifted towards that of breastfed infants. Together, HMOs likely contribute to immune protection in part through their effect on early-life gut microbiota, findings that warrant further clinical research to improve our understanding of HMO biology and significance for infant nutrition.
What are human milk oligosaccharides (HMOs)? What is their importance for infant nutrition? These questions have intrigued scientists and pediatricians alike for over a century. Advances in analytics as well as large-scale synthesis technologies stimulated great progress in recent years. These provided the ma- terials and tools that enabled the detailed and accurate measurement of HMO quality and quantity, and the study of HMOs in basic research models, and through clinical observational studies and intervention trials.
“HMOs are not HMOs,” meaning that one specific HMO is not equal to another HMO, especially when considering structure-function relationships. Chemically, HMOs are elongations of the milk-specific sugar lactose in different linkages by one or a combination of the following monosaccharides: L-fucose (Fuc), D-galactose (Gal), N-acetyl-D-glucosamine (GlcNAc), and N-acetylneuraminic acid (sialic acid). Gal and GlcNAc generally elongate lactose as a disaccharide Gal-GlcNAc. The numerous and diverse HMOs produced might be categorized by specific structural features brought about by different glycosyltransferases involved in their synthesis. However, many HMOs combine different structural features.
Breast milk, the recommended and naturally adapted nutrition for infants, is associated with a reduced risk for infection-related illnesses and possibly for diabetes and overweight, while the situation for allergies is less clear . This suggests that breast-milk-specific components such as HMOs and other bioactives may contribute to such benefits.
Due to their structural similarity with mucosal glycans and their nondigestible nature, HMOs expectedly affect numerous glycan-mediated processes like the colonization of the early-life microbiota and the infectivity of pathogens (Fig. 1a). Based on clinical observational and basic research data, HMOs act in a structure-function-specific way helping the (i) establishment of a mucous-adapted microbiome, (ii) resistance to pathogens, and (iii) reactivity of the mucosal barrier and immunity, thereby contributing to immune protection.
Here, we briefly review genetic and environmental factors affecting HMO composition in breast milk and the physiological role of HMOs as supported by clinical observation studies, preclinical research on mode of action, and insights from clinical intervention trials.
Maternal Glycosyltransferase Polymorphisms Affect HMO Composition
HMOs resemble the blood group antigens and further sialylated glycans that cover the human mucosa. The same glycosyltransferases are generally involved in the synthesis of mucosal cell glycans and mammary gland-expressed HMOs. The fucosyltransferases FUT2 (secretor gene) and FUT3 (Lewis gene) are the best described due to their natural polymorphisms in humans  (Fig. 2a). Specific genetic polymorphisms abolish their respective enzyme activity. Thus, specific HMO structures that depend on FUT2 or FUT3 can be identified. FUT2- dependent HMOs all contain α1,2-linked fucose, for example 2′-fucosyllactose (2′FL), lactodifucosyllactose (LDFT), and lacto-N-fucosylpentose (LNFP)-I. In- terestingly, trace amounts of 2′FL were found in breast milk of presumed FUT2-negative mothers in Asian populations [3, 4], indicating that the nature of these inactivating polymorphisms and thus the HMO profile may be population specific . Typical FUT3-dependent HMOs are LNFP-II, lacto-N-difucosylhexose (LNDFH)-I containing α1,4-linked fucose, and to a lesser extent 3-fucosyllactose (3FL) and LDFT containing α1,3-linked fucose. In breast milk that does not contain any detectable LNFP-II, reduced amounts of HMOs with α1,3-linked fucose on glucose and increased amounts of those with an α1,3-linked Fuc on GlcNAc are found. Hence, another FUT (e.g., FUT4, FUT5, FUT6, FUT7, or FUT9) is also involved in HMO formation with an α1,3-linked Fuc on GlcNAc and glucose.
The absence of a functional FUT2 or FUT2 and FUT3 affects the concentra- tion of total HMOs in milk when expressed as the sum of all quantified HMOs  (Fig. 2). While some HMOs increase when FUT2 is missing (e.g., LNnT and 3FL), in the absence of fucosylation additional larger nonfucosylated HMOs might also be produced.
To date, no common genetic polymorphisms for sialylated HMOs have been described, indicating that if inactivating polymorphisms in sialyltransferase genes exist, they are extremely rare. From mouse studies, the sialyltransferases ST6Gal1 and ST3Gal4 are involved in the synthesis of 6′-sialyllactose (6′SL) and 3′-sialyllactose (3′SL), respectively, with a further sialyltransferase, probably ST3Gal1, also making 3′SL .
Another mechanism affecting HMO composition is probably the donor and acceptor substrate availability, as suggested by the increase in 3FL when the ma- jor fucosyl-HMO 2′FL decreases in concentration .
Interestingly, HMO concentrations change during the stage of lactation with different HMOs showing different dynamics . HMOs like 6′SL or LNnT decrease more rapidly during the first weeks of lactation, while 2′FL and 3′SL, for example, decrease more slowly over a longer time period, and again others, like 3FL, actually increase in concentration with time of lactation (Fig. 2b).
Such compositional changes due to the genetic background of mothers and stage of lactation can confound observations relating HMOs to clinical parameters in the breastfed infants and, therefore, need to be considered.
Fig. 1. Illustration of the different biological functions of human milk oligosaccharides (HMOs). a Risk for infection-related illnesses and medication use in infants fed a formula supplemented with 2 HMOs (redrawn from Puccio et al. ). b Upper (URT) and lower respiratory tract (LRT) infections and other diseases as well as medication use were re- ported by parents and verified by a study physician. ORs with 95% CIs are shown based on percent of infants with at least 1 event at 1 year of age (Fisher’s exact test: * p < 0.05,** p < 0.001) (replotted from Ferrer-Admetlla et al. ).
Fig. 2. Human milk oligosaccharide (HMO) composition 3 months postpartum by FUT2 and FUT3 status with schematic illustration of typical HMO in one or the other group (a). HMO dynamics at different stages of lactation (replotted from Austin et al. ) depicting mean concentrations with standard deviations (b).
HMO Composition and Maternal Diet, Gestational Age, and Physiological State of the Infant
HMO concentrations in colostrum, transitional milk, and mature milk seem not to change between mothers giving birth to preterm (n = 18; gestational age<37 weeks) and term (n = 14; gestational age ≥37 weeks) infants . Further, fucosylated and sialylated HMOs were reported to be similar between preterm and term milk, although preterm milk seemed more variable in the expression of fucosylated HMOs .
Today, we do not know whether and how maternal diet might influence HMO composition. A recent observational study including 33 breastfeeding mothers and their infants from the Gambia, Africa, reported a significantly higher HMO content in milk at 20 weeks of lactation in the dry season (n = 21) than the wet season (n = 12) . The authors propose a possible link to the high- er energy intake during the dry season. In 2 other African mother-infant cohorts from Malawi (n = 88 and n = 215), total HMOs and also sialyl- and fucosyl-HMOs were lower 6 months postpartum in the breast milk of mothers having severely stunted infants compared to those with normal-size infants . These studies suggest that maternal nutritional and health status may affect HMO composition.
By analogy, higher maternal body mass index and gestational weight gain, which generally reflects an altered metabolic physiology, might affect HMO composition. Studies to this end are currently ongoing [10; Binia et al.: abstract at FASEB Science Research Conferences in 2017]. Suitable studies are warranted to investigate possible alterations in HMO composition due to maternal energy and specific nutrient intake.
The HMO Composition Is Associated with the Gut Microbiota in Infants
The early-life microbiome has a major impact on the developing immune system, itself being an important element by providing pathogen colonization resistance, for example. Interestingly, the establishing intestinal microbiota also contributes, via an innate lymphoid cell-mediated process, to improved protection against respiratory tract infection . The pioneers of human milk and breastfeeding research observed a strong link between breastfeeding and immune protection to infectious morbidity and mortality. Breastfed infants were recognized to harbor an early gut microbiota domi- nated by bifidobacteria, not seen in formula-fed infants, and a human-milk- specific “bifidofactor” was identified in the HMO fraction of breast milk .
From research on early-life microbiota, we know that bifidobacteria can utilize and grow on different individual HMOs in a strain-specific way [13, 14]. Several studies observed an increased bacterial metabolic activity upon growth on HMOs, exemplified by the formation of the short-chain fatty acid acetate [15, 16]. Noteworthy, numerous potentially pathogenic bacteria from the Enterobacteriaceae group were shown not to grow on individual HMOs as the sole carbon source , while growth of other pathogens, like Streptococcus agalactiae (group B Streptococcus, GBS) was shown to be inhibited by HMOs [18, 19].
Recently, LNnT in breast milk was associated with Bifidobacterium longum ssp. infantis abundance . In bi-associated gnotobiotic mice harboring only 1 Bacteroides and 1 B. longum ssp. infantis strain, LNnT lead to bifidobacteria dominance although both bacteria could actually use LNnT in vitro . In gnotobiotic mice humanized with 7 human microbes, B. longum ssp. infantis also showed higher abundance when these mice were fed 2′FL combined with LNnT as compared to LNnT alone [Sprenger et al., unpubl. observation], although B. longum ssp. infantis is able to grow on many different HMOs, including LNnT, as substrate .
Genomic and glycomic analyses in infants provided further evidence for a role of HMOs in shaping the early infant gut microbiome, revealing associations between individual HMOs and bacterial genera in infant stool [21–23]. A Bifidobacterium-dominated gut microbiota in breastfed infants (n = 105) at 4 months of age was associated with breast milk containing FUT2-HMOs . The FUT2 status of the infants and its possible confounding effects on the infant microbiota profile were not assessed, despite earlier data proposing the FUT2 status itself can influence the gut microbiota at least in adults . In another cohort, the analysis of a relatively small subgroup of infants exclusively breastfed for 4 months (n = 14) showed an association of maternal FUT2-positive status with higher Bifidobacterium abundance up to 2–3 years of age . However, no statistically significant HMO effects on global Bifidobacterium shifts were reported in another recent study of 33 Gambian mothers and infants , while the abundance of individual bifidobacteria like B. longum ssp. infantis still correlated with LNnT concentrations in breast milk. These first reports reveal the need for larger observational studies of similar design, including comprehensive HMO analysis of breast milk and infant FUT2 phenotyping to gain a more robust understanding of the link between HMO and infant gut microbiome composition.
Today, clinical observations in conjunction with basic research data suggest that FUT2-HMOs, like 2′FL and LNFP-I, but likely also other non-FUT2-dependent HMOs, like LNnT for example, are involved in the establishment of a Bifidobacterium-dominated early-life gut microbiota. In vitro studies help to understand HMO-related microbial metabolic capacities and strain specificities, while animal and human observational studies indicate that the interaction between bacteria and the gut mucosa reflect a more complex picture. Hence, with infant health in mind, it is central to gain a better understanding of HMO effects on the microbiome dynamics in their natural ecosystem through a holistic and ecology-inspired approach.
HMO Composition Is Linked to Infection Risk in Infants
HMOs were studied in relation with infectious diarrhea incidence in a cohort of Mexican mothers and infants (n = 93) [27, 28]. Higher breast milk concentrations of α1,2-fucosylated HMOs were associated with a lower incidence of all-cause moderate-to-severe diarrhea. The most frequently identified cause of diarrhea in the cohort was Campylobacter jejuni followed by calicivirus and enteropathogenic Escherichia coli. Specifically, higher concentrations of 2′FL and LNFP-I in breast milk correlated with a lower incidence of C. jejuni and calici- virus diarrhea, respectively. These observations during the breastfeeding period did not persist in the period after breastfeeding, indicating a possible transient HMO effect in the protection from infectious diarrhea. This fits their presumed role as anti-adhesive antimicrobials. Experimental data from preclinical models also show protective effects of 2′FL from C. jejuni  and aggregating invasive E. coli . From these data, 2′FL and other FUT2-HMOs seem to act as soluble ligands blocking C. jejuni from adhering to gut epithelial cells, while the protection from E. coli might rather be due to an anti-inflammatory effect, possibly combined with the modulation of the gut microbiota composition.
Glycans containing α1,2-linked Fuc expressed on epithelial cells of FUT2- positive infants could act as receptors for pathogen binding, conferring a risk to specific infectious diseases for this population . Genetic studies have shown that infants and children with a nonfunctional FUT2 gene have strain-specific protection against norovirus and rotavirus [32, 33]. For specific rotavirus strains, susceptibility depends on FUT2 but also on FUT3 status . Experimentally, infectivity of some rotavirus strains was reduced by the FUT2 HMO 2′FL, while other viral strains were affected by sialylated HMO, namely 3′SL and 6′SL . Similarly, 2′FL also bound to specific norovirus strains .
Besides interfering with pathogen attachment to the host mucosa, HMOs were recently reported to exert bacterial-growth-inhibitory activities on pathogenic GBS [18, 19, 37], a major cause of sepsis in preterm infants. Growth of GBS was specifically inhibited by LNT and LNFP-I, while sialylated HMOs or galactooli- gosaccharides (GOS) had no effect . Experimental data suggest a putative gly- cosyltransferase of GBS to be involved . Possibly pointing to a similar mechanism, HMOs from milk of a FUT2-negative mother were shown to have bacterio static properties via an alteration in biofilm formation . In an observation study of 183 Gambian infant-mother pairs, FUT3-positive mothers were reported to be less likely carriers of GBS, as were their infants at birth . Interestingly, infants of FUT3-positive mothers were also more likely to clear GBS colonization from birth to 2–3 months of age compared to infants of FUT3-negative mothers.
In a pilot study of 49 mother-infant pairs, higher breast milk concentrations of the FUT3-HMO LNFP-II at 2 weeks were associated with a lower risk of respiratory and gastrointestinal illnesses at 6 and 12 weeks in infants . This association was no longer significant after the breastfeeding period. Similarly, in a nested case cohort study of 143 HIV-exposed uninfected children from Zambia, higher concentrations of fucosylated HMOs in breast milk 1 month postpartum related to a lower risk of mortality up to 2 years of age . In another small mother-infant cohort from the Gambia (n = 33), higher relative breast milk concentrations of fucosylated HMO (sum of LNFP-I and LNFP-III) and concomitant lower relative abundance of LNT was associated with a lower risk of sickness up to 4 months of age .
For respiratory pathogens, direct HMO exposure would appear less evident, and thus any putative HMO-related protection may be mediated by the intestinal microbiome [11, 40]. Yet, experimentally, direct exposure of Streptococcus pneumoniae to LNnT and sialyl-LNnT and subsequent infection effectively blocked its colonization in the lung of a rabbit model . In a cell-based assay, LNnT and 2′FL dose-dependently reduced influenza and respiratory syncytial virus concentrations within respiratory tract cells .
Observational studies together with findings from preclinical models have provided first evidence for an association between HMOs and the risk of infections, mostly in a structure-function-specific way. Mechanistically, HMOs may act through multiple functions, although preclinical models highlight specific individual functions. The current studies also provide directions to be considered in future observational studies, such as timing of milk sampling and breast milk intake, etiology of infections, quantitative versus categorical HMO analysis and finally mother and infant genetics.
HMO Composition Might Be Linked to Allergy in Infants
Numerous environmental, including nutrition, and genetic factors affect allergies. Among them are breast milk bioactives and possibly HMOs. In a cohort of 266 Finnish mother-infant pairs with a hereditary allergy risk, 2′FL concentrations in early breast milk associated with a lower risk to manifest IgE-associated eczema at 2 years of age only in C-section born infants . This observation suggests that 2′FL may influence IgE-associated eczema through the modulation of the early-life gut microbiota, known to be different in C-section-born infants compared to vaginal-born infants. A possible relation of HMOs with cow milk allergy (CMA) was studied in another cohort of 39 mothers with infants who developed CMA by 18 months of age and 41 mothers with infants without CMA . An association was seen between the milk concentration of several indi- vidual HMOs [LNFP-III, 6′SL, LNFP-I, and DSLNT (DiSialyllacto-N-tetraos)] and HMO clusters with reduced risk of CMA, with LNFP-III providing the strongest association. Breast milk sampling varied over the first 6 months after birth and this was taken into account in the statistical analysis, because HMO concentrations change dramatically during this period. Mechanistically, the authors speculate that LNFP-III might act on the immune system via dendritic cells and DC-SIGN (Dendritic Cell-Specific Intercellular adhesion molecule- 3-Grabbing Non-integrin). In a preclinical food allergy model, 2′FL and 6′SL were tested and both reduced symptoms involving mast cell activity .
The observational studies to date have their limitations, but still provide valuable preliminary data on possible relationships between specific HMOs and risk of allergies. To appreciate such a proposed link requires replication in larger cohorts with harmonized milk sampling, stratification for mode of delivery, and evaluation of infant FUT2 and FUT3 genotypes.
Insight from Clinical Intervention Trials with Specific HMOs
Recent progress in industrial biotechnology has made available few individual HMOs, namely 2′FL and LNnT. Preclinical safety toxicity tests established their safety, and both obtained approval as novel foods in the European Union and were generally recognized as safe in the USA.
In adults, both 2′FL and LNnT were studied alone or in combination at different doses from 5 to 20 g/day in a placebo-controlled, blinded, randomized trial (n = 100). Both HMOs were well tolerated and increased bifidobacterial abundance .
In infants, 2 placebo-controlled, blinded, randomized, clinical intervention trials showed the growth safety and tolerance of 2′FL combined with either GOS or fructooligosaccharides [47, 48; Kajizer et al., unpubl.]. Infants fed with an infant formula supplemented with 2′FL (0.2 or 1 g/L) combined with GOS or GOS alone showed similar growth as breastfed infants up to 4 months of age (n = 314). In a subgroup of infants, immune markers were measured in plasma at baseline and upon stimulation of blood cells with respiratory syncytial virus. Globally the immune profile resembled that of breastfed infants when the infant formula was supplemented with 2′FL at the lower or higher dose . Another randomized controlled infant trial showed that an infant starter formula supplemented with 2 HMO, 2′FL, and LNnT (n = 88) allowed for age-appropriate growth of term born infants and was well tolerated when compared to the same infant formula without HMO (n = 87) . Interestingly, secondary exploratory findings showed an association between feeding the 2-HMO infant formula and less-re- ported lower respiratory tract illnesses and medication use (especially antibiotics and antipyretics) during the first year of life and beyond the 6-month feeding period. At 3 months, the global microbiota profile shifted in the 2-HMO-formula-fed infants away from the control-formula-fed infants and towards that ob- served in breastfed reference infants. This shift was mainly due to increases in Bifidobacterium concomitant with decreases in Escherichia and Peptostrepto- coccaceae . A significantly higher number of infants who were fed the 2-HMO-supplemented formula showed a microbiota community structure typical for breastfed infants compared to control-formula-fed infants, who had primarily a different microbiota community structure. Interestingly, infants with a microbiota community structure typical for control-formula-fed infants had a 2 times higher risk to use antibiotics during the first year of life than those with a microbiota community typical for breastfed infants .
These first clinical intervention trials with specific HMOs demonstrate their growth safety and digestive tolerance. Additionally, as suggested from basic re- search and observational data, 2′FL and LNnT might contribute to the protection from infection-related illnesses and reduce the need for antibiotics, possibly through the modulation of the establishing early-life gut microbiota.
HMO composition is affected most notably by the maternal FUT2 and FUT3 status. This is likely due to an evolutionary selective pressure imposed by pathogens or the microbiome at large. Stage of lactation alters HMO composition possibly indicating different infant needs at different extrauterine developmental stages. However, giving birth to a preterm or term infant, who are at different developmental stages, seems not to affect the HMO composition of breast milk. Clinical observations corroborated by preclinical data and clinical intervention trials support a role for specific HMOs in immune protection, primarily from infection-related morbidity and use of antibiotics. Further clinical studies, well- designed observational studies, and especially placebo-controlled interventions are warranted to further substantiate and grow our understanding of the HMO biology and significance for infant nutrition.
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SummaryThe information summarized in the various comprehensive presentations in this workshop represented a diverse spectrum of historical, evolutionary, and functional aspects of mammalian lactation and the process of breastfeeding. This workshop was dedicated to Prof. Lars A. Hanson (MD, PhD) for his outstanding contributions to the understanding of the biology of milk and the dissemination of knowledge on breastfeeding to advance current practices of breastfeeding in the contemporary human society worldwide. The dedication ceremony was followed by scientific presentations in session I of the workshop with the keynote addressed by Olav T. Oftedal. Oftedal provided an elegant perspective of the evolution of lactation in different mammalian species. Based on studies on synapsids (ancestral to mammals, which appear to have diverged from sauropsids [ancestral to crocodiles, lizards, and birds]), he proposed that lactation may have first evolved as a source of moisture and antimicrobial compounds for parchment-shelled eggs, followed by the evolution of some skin secretions, which eventually became milk. It was suggested that among basal animals (monotremes), each mammary gland develops as a triad in association with a hair follicle and sebaceous gland as a mammopilo- sebaceous unit (MPSU).
In other mammalian species, such as marsupials, there is a similar triad, but the hair follicles are shed during development. In the diverse group of eutherian mammals, some show no association with the mammary hair, while others, such as the horse, develop as MPSU with mammary hair and sebaceous glands present in the mammary gland.
The MPSU also bears significant resemblance to apocrine glands (APSU), suggesting that mammary glands may have also evolved from an APSU-type structure. Recent studies have suggested that most constituents of mammalian milk are unique and found only in mammary secretions. He proposed that if a milk protein occurs in the milk of monotremes, marsupials, and eutherians, the major mammalian taxa, then the protein must have evolved before the groups diverged and are inherited from the ancestral taxa. These observations have provided unique and new insights into the genetic origin and functions of specific mammary constituents in the products of lactation. Oftedal briefly alluded to the 4 primary types of caseins, members of the secretory calcium-binding phosphoproteins (SCPPs), as an evolutionary challenge because of their diversity and the large size of the micelles in milk. These proteins have an ancient history in the evolution of mineralized tissues. Based on related SCPP genes, caseins may have evolved as protolacteal secretion that delivered calcium to eggs. Finally, his presentation discussed briefly the evolution of the milk fat globule membrane, lactose,
and other neutral and acidic oligosaccharides. The next presentation provided a brief historical overview of the immunology of milk and mammalian lactation. This presentation served as an introduction to the subsequent specific topics discussed in this workshop. Mother’s milk has been considered a complete food for the infant from times immemorial, and it has been associated with unique healing powers and beneficial effects.
These include cure for insomnia, loss of appetite, ascites, piles, skin disorders, sexual dysfunction, muscle weakness, contraception, and prevention of cancer and infections. Breastfeeding developed a spiritual and religious importance in the Middle Ages in Europe, as evidenced by the deep faith and respect for Nursing Madonna, Virgin Mary, and the breastfed Jesus. The modern history of breast milk immunology can be traced to a publication by Paul Ehrlich as early as 1892 and subsequent demonstration of specific maternal antibody transport to the colostrum and milk. The immunologic composition of human milk and its biologic linkage to mucosa-associated lymphoid tissue was initially recognized by Gugler and Von Muralt, and by Lars Hanson. These elegant studies were followed by the identification of secretory IgA in human external secretions by Chodirkar and Tomasi, and Bienenstock and Tomasi, and in the human milk by Hanson and Johansson. Subsequent studies by Beer and Bellingham, Ogra et al., Mohr, and Okamoto and others identified several cellular and soluble immunologic factors in the human milk and their transport to the suckling neonate via the process of breastfeeding. It is now known that human colostrum and milk contains a wealth of immunologically active products derived from the innate and adoptive immunologic, microbiologic, dietary, and other maternal experiences in the maternal mucosal surfaces, especially the gut, and the maternal circulation. This historical review was dedicated to the memory of Dr. S.S. Ogra, the principal investigator of most milk related research carried out in her laboratory in the early 1970s and 1980s in the School of Medicine at the University at Buffalo. This presentation briefly reviewed lactation performance and the presence and function of diverse soluble elements detected in mammalian colostrum and milk to date. These included: secretory IgA and other immunoglobulin isotypes, anti secretory factor, soluble CD14, and soluble Toll-like receptors, as well as several cytokines and lymphokines. It also introduced the role of colostrum- and milk-associated cellular components, such as leukocytes, macrophages, epithelial cells, stem cells, and T lymphocytes, and cell-mediated immune responses. This overview also summarized earlier studies on the transfer of tuberculin-specific maternal cellular immunity to the neonate via breastfeeding and more recent investigations on the transfer of maternal cellular immunity and engulfment of maternal
DNA via the transfer of leukocytes and stem cells. Finally, the risks and benefits of the colostrum and milk to the neonate and the developing infant were briefly considered here. Detailed discussion of the issues identified here follows in subsequent presentations in this session and sessions II and III of this workshop. Jiri Mestecky reviewed in some detail the evidence for the existence of mucosa- associated lymphoid tissue and common mucosal immune sites for effective immunization in the mucosal system, and the importance of mammary glands as an integral component of the common mucosal immune system. He discussed recent studies on the structure, biologic activities, and the spectrum of antibodies of the IgA isotype specific for microbial, dietary, and other environmental antigens and macromolecules in the colostrum and milk. He concluded his presentation by identifying possible directions for future investigations in the immunobiology of the mammary gland and lactation. These include the routes for the most effective induction of IgA responses in milk, the identification of phenotypes of B lymphocytes that express homing receptors for the
mammary gland, and the determination of effective timing for maternal immunization to provide optimal levels of protective immune reactivity in the colostrum and the milk for the neonate.
Helena Tlaskalová-Hogenová, Miloslav Kverka, and Jiří Hrdý introduced the wide spectrum of immunomodulatory components present in human milk and colostrum, including those of innate and adaptive immunity, and factors influencing the composition and colonization of newborn gut microbiota. They discussed the nature of autoantibodies and the spectrum of newly detected cytokines and lymphokines in human milk. The presentation was completed with an overview of different cellular components of and cytokine gene expression on colostral cells in healthy and allergic mothers. Studies carried out to date have identified over 35 cytokines in the colostrum and milk, and some of them have been identified for the first time in human milk. Their possible functions include the development of intestinal lymphoid tissue, functional development of the gut structure, angiogenesis, central and enteric nervous system development, and establishment of immunologic homeostasis in the mammary gland as well as in the breastfeeding neonate.
Valerie Verhasselt discussed the influence of breastfeeding on the development
of immunologic health in the breastfed neonate and infant. She began with the examination of the unique specificities of the neonatal immune system, including unique limitations to the development of immune responses after postnatal exposure to environmental antigens. She reviewed the role of TGF-β, vitamin A, several environmental allergens, and specific antibodies in the context of early life and long-term allergic disease susceptibility. Based on controlled epidemiological data and several experimental studies, she proposed that early-life oral exposure to allergens does not induce tolerance but may prime for allergic responses. It has been suggested that non breastfed infants are exposed to only few allergens, but in very high concentrations, such as β-lactoglobulins. On the other hand, breastfed infants are exposed to a wide variety of allergens in the maternal milk and colostrum but in extremely low concentrations. Additionally, breast milk provides the infant with significant amounts of TGF-β, vitamin A, and other cofactors which affect the integrity of
the barrier of gut epithelium and regulate antigen transfer and presentation to the mucosa-associated lymphoid tissue. As a result, breastfeeding results in a low risk for allergic disorders in the long term. Such conclusions are supported by recent studies in human birth cohorts and by studies carried out in her laboratories with the induction of egg allergy in experimental (mouse) animal models.
Carine Blanchard made the final presentation in session I. Due to certain unavoidable
circumstances, she could not provide a full-length manuscript of her presentation. Therefore, a more detailed summary of her talk is presented here. Her presentation focused on the immunologic evaluation of human milk oligosaccharides (HMO) with respect to disease expression in the neonate after exposure to allergens and infectious agents. She discussed in some detail the enzyme fucosyltransferase (FUT) and its genotypes FUT2 and FUT3. These enzymes are expressed on blood groups ABH and Lewis, intestinal mucosa, and other human body fluids. Recent studies have suggested that the early trajectory of neonatal microbial colonization is significantly influenced by the number of environmental factors. These include gestational age of the neonate, method of delivery, use of antibiotics, geographic location of birth, genetics, maternal stage of lactation, maternal diet, and specific immunologic components delivered to
the neonate via breastfeeding. These factors appear to determine the outcome of
colonization and the composition of the neonatal microbiome as healthy or aberrant
based on the metabolites generated by the microbiome. An aberrant microbiome
has been associated with the development of sustained inflammation, induction of asthma, atopy, obesity, inflammatory bowel disease, and other disease states. Employing FUT2 and FUT3 genotypes as proxy for the HMOs, several ongoing investigations have provided important information on the role of HMOs in human milk and colostrum;
1. 2′-Fucosylated HMOs in human milk alleviate the negative effects of cesarean section on infant gut microbiota.
2. HMOs in infant formula significantly improve the outcome of infections in infants.
3. Maternal FUT2- and FUT3-positive status is related to a lower risk of respiratory infections during the first 6 months of neonatal life.
4. Maternal HMOs are associated with the prevention of colonization and growth of the pathogenic microbiome.
5. Elevated serum IgE levels are associated with the absence of gut microbiota in experimental models of infection.
She summarized the results of the LIFE child cohort studies from Leipzig (Germany) and Bangkok (Thailand) and other investigations involving cholera toxin/ovalbumin-induced food allergy in experimental animal models These studies have also demonstrated that the use of HMO and FUT2/FUT3 genotypes in infant feeding is associated with significantly decreased allergic sensitization. The mechanisms underlying such protection appear to be related to the modulation of regulatory T-cell function by HMOs and independent of the regular prebiotic effects associated with milk oligosaccharides and other soluble products in the milk.
Based on the information summarized above from the presentations in session I of the workshop, it is apparent that we have come a long way in understanding the evolutionary biology of mammalian lactation, the presence and role of specific innate and adaptive immunologic mechanisms associated with human milk, and the impact of breastfeeding on the systemic and mucosal immunologic development in the neonate. Recent information about the microbiology of the milk and lactation and its influence on gut colonization, presented in session II, and studies about the role of HMOs and other soluble components of the colostrum and milk, presented in session III, are summarized next by W. Allan Walker and Bo Lönnerdal, respectively. It is gratifying to note the wealth of new information presented in this workshop, and it is hoped to generate further interest in exploring many unanswered questions related to the mammary glands and lactation and its impact on the neonate.
AbstractThe development of the mammary glands and the process of lactation is an integral component of mammalian evolution, and suckling has been essential for the survival of the neonates of most mammalian species. The colostrum and milk, the major products of lactation, contain a wealth of biologically active products derived from the immunologic and microbiological experiences in the maternal circulation and in the maternal mucosal surfaces. These include major immunoglobulin isotypes in the maternal circulation, secretory IgA, a variety of soluble proteins, casein, nutritional components, hormones, a large number of cellular elements and their secreted functional products (cytokines and chemokines), several peptides, lipids, polysaccharides and oligosaccharides, and a diverse spectrum of microorganisms. During the past few decades, significant new information has become available about the evolutionary biology of mammalian lactation, the functional characterization of antibody and cellular immunologic products, the role of oligosaccharides and other proteins and peptides, and about the distribution and biologic functions of the microbiome observed in human products of lactation. This workshop explores this information in some detail in a series of presentations. A brief overview of the earlier observations on the immunologic aspects of lactation is presented here, and detailed reviews of more recent observations are reported in subsequent presentations in this workshop.
IntroductionThese two quotes reflect the depth of human interest in breastfeeding for over 2,500 years [1, 2]. Milk and other lactational products of all mammals, including humans, have been associated with unique healing powers and beneficial effects. Mother’s milk has been considered a complete food for the infant in many ancient scriptures. With the evolution of agricultural civilization, long before the development of commercial milk formula foods, milks from buffalo, cow, sheep, camel, donkey, horse, elephant, and goat were highly recommended for the treatment of insomnia, loss of appetite, ascites, piles, infestations by worms, skin disorders, muscle weakness, dysfunctions of sexual activity, and a large variety of other human ailments . In addition, breastfeeding also developed a religious and spiritual importance in the Middle Ages in Europe, as evidenced by the deep faith and respect for Nursing Madonna, the Virgin Mary
breastfeeding the infant Jesus . During the upsurge of Marian theology in Europe, milk was viewed as the processed blood, and the milk of the Virgin paralleled the role of the blood of Christ . This is best exemplified by “the miracle of the lactation of St. Bernard,” based on a vision concerning St. Bernard of Clairvaux in France being hit with a squirt of milk traveling an impressive distance from the breast in the statue of the Virgin nursing the infant Jesus . Such blessed milk is believed to have given him great wisdom and cured an infection in his eye.
The modern history of immunology of mammalian lactation can be traced to as early as 1892 with observations by Paul Ehrlich that nonsuckling frequently resulted in death in the newborn foals, lambs, or piglets [7–9]. About the same time, observations by Escherich  provided for the first time evidence for exquisite sensitivity of intestinal microflora to human milk. Subsequently, the association of certain milk proteins such as immune lactoglobulin with specific
It has been shown that during placentation of mammals, different maternal
immunologic components are transported selectively via the placenta or breastfeeding in different primates. For example, in rabbits, rodents, and some carnivores, maternal IgG is actively transported to the fetus in large amounts from the maternal serum across the placenta. On the other hand, such effective placental transport does not occur in horses, cattle, swine, and other mammals, as reviewed in detail by Butler and Kehrli  and summarized briefly in Table 1.
The immunologic composition of human milk and its biologic linkage to mucosal immunity was initially recognized by the identification of major classes of immunoglobulin in the milk by Gugler and von Muralt , and Hanson . These elegant studies were followed by the identification of the unique immunoglobulin, the secretory IgA (SIgA), in human milk by Hanson and Johansson . Subsequent studies by Beer et al. , Ogra and Ogra , Ogra et al. , Mohr , and Okamoto et al.  identified several cellular and soluble immunologic elements in the human milk and their possible transport to the suckling neonate via the process of breastfeeding. Finally, it is of interest that Ehrlich  demonstrated for the first time that maternal immunization and subsequent breastfeeding induced significant protection in suckling mice against the toxic effects of subsequently ingested ricin and abrin. His imaginative studies also raised the possibility of protection against infections such as syphilis, mumps, typhus, and measles via the process of breastfeeding [7–9].
During the past 3 decades, significant information has been obtained to suggest that the immunologic activity inherent in the products of lactation represents, to a major extent, the effector functional elements of the common mucosal immune system. Following the discovery of IgA in the serum by Heremans et al. , and of secretory IgA in the milk by Hanson and Johansson , the presence of SIgA was also demonstrated in other mucosal secretions by Chodirkar and Tomasi , Tomasi and Zigelbaum , and Bienenstock and Tomasi . These observations were followed by the identification of antibacterial, antiviral, and antiparasitic activity in the milk associated with SIgA and other immunoglobulin classes, demonstration of a number of specific cellular elements and cellmediated immune responses, and detection of cytokines and other immunoregulatory factors in milk. The relationship of the immunologic activity in the milk and mammary glands to other mucosal surfaces was documented conclusively by several elegant studies which identified intestinal and respiratory tracts, and the sublingual tissues as the primary induction sites of specific IgA-committed B cells and their active migration to the mammary glands [26–29]. Since the discovery of Bifidobacterium bifidum subspecies in 1953, it is now clear that this organism predominates in the feces of the breastfed infants. Specific factors stimulating the growth of this organism are uniquely present in human but not in cow’s milk.
A significant biologic database is now available to support the clinical observations dating back from antiquity to the last few centuries, which have suggested a strong association between breastfeeding and protection against a variety of infectious and noninfectious disease processes in humans. These include protection against infectious diarrheal diseases, fertility and childbearing, and immunomodulation of mucosal and systemic immune responses. This information has been extensively reviewed in many recent publications [30–34]. It is now clear that human milk contains a wealth of biologically active products.
These include soluble proteins, hormones, a number of cellular elements and their functional products (cytokines, chemokines, and hormones), several peptides, proteins, lipids, oligosaccharides, and numerous microorganisms . This information is briefly reviewed in Table 2. Significant new information has also become available about the following specific areas of milk: (a) evolution of lactation in mammalian species; (b) further characterization of immunologic
Immunology of Milk and Lactation
Lactational Performance: Secretion of Colostrum and Milk.
mucosal sites. Colostrum is the first postpartum product of lactation. It is dense in protein and fat, and it contains the highest amounts of soluble as well as cellular immunologic components compared to transitional or mature milk. Successful lactation
with continued contribution of the transported or locally synthesized products in mature milk is also determined by continued contribution of neural, endocrine, and other maternal-infant interactions activated at the time of delivery. Early and frequent breast contact by the nursing infant is also important for continued stimulation of neural pathways to maintain prolactin and oxytocin release. Lactation often ceases when suckling stops.
The levels of IgA are usually 4–5 times higher than those of IgM and about 26–30
times higher than IgG levels . As lactation progresses, the levels of IgA and
IgM in the mature milk decline rapidly. However, this decline is compensated by
the increase in the total volume of milk produced (Table 3). It is estimated that a
fully breastfed neonate may consistently receive about 1 g of IgA each day and
approximately 1% of this amount for IgM and IgG . Comparative studies of
immunoglobulin activity in the feces of breastfed infants have suggested that the
fecal content of IgA may be 15–20 fold higher after human milk feeding compared
to bovine IgG after feeding of bovine immunoglobulin products .
fluid secretion, and it has been used to treat infectious diarrheas, antiinflammatory bowel disease, and other inflammatory conditions . Human milk is also rich in other anti-inflammatory components. These include vitamins, especially A, C, E, as well as the enzymes catalase and glutathione peroxidase .
Soluble CD14 and Soluble Toll-Like Receptor
important nonbinding protein found in human milk.
cellular elements. These include epithelial cells, activated neutrophils, macrophages,
stem cells, and B and T lymphocytes. In addition, recent observations have suggested that human milk is rich in bacteria and other cellular and subcellular
living organisms .
Activated milk macrophages exhibit enhanced phagocytosis and have been
found to have a receptor for SIgA. Its role in the mechanism of protection in the breastfeeding neonate remains to be defined.
Lymphocytes and Cell-Mediated Immunity
Interaction of Cellular Elements with the Neonatal Immune System
possible functions in the breastfeeding infant. During the course of normal breastfeeding, millions of viable cellular elements are ingested by the infant. Earlier studies have suggested that milk cells are taken up in the neonatal mucosal surface and may transfer to the neonate, with varying degrees of specific immunologic information [17, 19, 20, 53]. Studies carried out by Ogra et al.  in 1977 in a group
of formula-fed infants after a single-feed administration of human colostrum have clearly demonstrated the uptake and transfer of IgA. In another group of infants of tuberculin-positive mothers, these investigators also demonstrated the transfer of in vitro correlates of T cells mediated against tuberculin after prolonged breastfeeding. The tuberculosis-specific T-cell response in such infants were short lived and undetectable after 10–12 weeks in spite of continued breastfeeding. Subsequently, several experimental animal studies have demonstrated the engraftment of maternal DNA via milk leukocytes in infant tissue [44–46]. Although these observations have been linked to lymphocytes, such transfer may also occur with epithelial and stem cells. In other experimental investigations, the transfer of maternal T cells and HLA antigens appears to be associated with the development of immunologic tolerance to maternal HLA antigens. Breastfed infants also express a lower frequency of precursors of cytotoxic T lymphocytes reacting with maternal HLA than nonbreastfed infants [54, 55]. Recent observations have also shown that maternal cytotoxic T lymphocytes localize in the Peyer’s patches of breastfed infants . Such localization may serve to compensate for the immature adaptive immune response functions in the neonate.
Risks and Benefits of Breastfeeding
homeostasis and the survival of the infant, breastfeeding offers significant benefits to the mother as well. Feeding of about 6 sucklings per 24-h period has been found to provide significant contraceptive benefit for the mother. It has been proposed that more conceptions are prevented by breastfeeding than by all other contraceptive approaches and family planning programs in many parts of the world . Such an important maternal influence has also been shown to contribute to the reduction in infant mortality correlated with reduced crowding in the family, with less risk of infection and improved availability of food and nutrition to the infant and the mother. An interpregnancy space of less than 2 years has been shown to increase the risk of infant mortality by over 50% before 5 years of age .
maturation of the mucosal immune system. The development of intestinal mucosal
integrity is to a large extent determined by the maturation of mucosa- associated lymphoid tissue and other tissue sites, and the establishment of the mucosal microbiome. Recent investigations have demonstrated that SIgA antibody and other soluble immunologic products in the breast milk promote long term gut homeostasis by regulating the acquisition of the mucosal microbiome and host gene expression [60, 61].
Exclusive breastfeeding in the first 6 months is clearly a major determinant of the prevention of diarrheal disease in infants, especially with Escherichia coli, Shigella, Vibrio cholera, Campylobacter, some parasitic infestations (Giardia lamblia), viruses (rotavirus), and possibly other mucosal infections. Case-control studies have suggested that breastfeeding and specific antibody activity in the colostrum and milk more often provide protection against severe disease and hospitalization rather than total prevention of colonization and infection [60, 61]. A number of studies have clearly demonstrated a beneficial role of human milk in preventing or modifying the severity of necrotizing enterocolitis in premature infants [32, 34]. Similarly, anti-infection benefits related to milk-associated antibodies, soluble cytokines, or other protective features have been observed in breastfed infants against several genitourinary respiratory infections, otitis media in childhood, neonatal sepsis, and possibly sudden infant death
syndrome of unexplained origin .
Breastfeeding and the immunologic components of human milk have been shown to confer long-lived protection against reactive airway disease and bronchial asthma, eczema, and other atopic and allergic states. The protective effects may reflect multiple synergistic mechanisms, including maturation of gut and airway mucosa by growth factors in human milk, reduction in the absorption of allergens and other antigens by modulation of the mucosal microbiome, and induction of specific mucosal tolerance (oral tolerance), and immune exclusions.
Combined with other protective factors in the gut, SIgA can impede allergen sensitization by blocking the transport of foreign macromolecules across the immature neonatal gut epithelia and modulating the development of specific antibodies or immune complexes. It should also be pointed out that cow’s milk protein and other food antigens ingested by the lactating mothers have been observed on colostrum and milk. Other studies have suggested that early breastfeeding may be associated with decreased serum antibody responses to cow milk proteins and other maternal dietary antigens in the breastfed infant.
From an evolutionary biology perspective, it would seem that breastfeeding should provide only beneficial effects to the neonate. However, there is considerable debate regarding the protective “immune-mediated effects” of breastfeeding on the development of atopy and allergy. Some investigations have proposed the act of breastfeeding itself, regardless of the constituents of the breast milk, may be more or equally important defense mechanisms for the infant. An interesting study has suggested no protective effect of indirect breastfeeding (breast milk fed by the bottle) compared to infants receiving direct breastfeeding .
Existing information about the potential risks and benefits of the milk microbiome
and other factors in human colostrum and milk will be explored in further detail in subsequent studies of this workshop. In conclusion, it may be worthwhile to recapitulate that from an evolutionary standpoint, human colostrum and milk continue to remain the single most important vehicle for the transport of all maternal immunologic experiences via breastfeeding to the neonate for its survival and well-being throughout its life
The author declares to have no conflict of interest.
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Human milk oligosaccharides (HMO) are abundant in human milk (5-20 g/L) (1) and exert numerous beneficial effects (2-4). The two predominant HMO are lacto-N-tetraose (LNT) and 2´-fucosyllactose (2´-FL), although 2´-FL presence depends upon maternal secretor type (1). Bovine milk oligosaccharide content and composition are lower and less complex (5), thus infant formulae are nearly devoid of oligosaccharides (6). Most contain prebiotics, but large scale production has enabled the recent addition of 2´-FL and LNnT to formula (6). Note that LNT type 1 and LNnT type 2 cores differ, which affects their recognition (7) and utilization (8), thus their functionality is likely not identical.
Two randomized clinical trials have investigated adding HMO to formula (9, 10). In the first, infants were fed control formula (CF) or formula containing 0.2 or 1.0 g/L 2´-FL for the first 4 months of life and were compared to a breast-fed (BF) reference (9). All formulae also contained galactooligosaccharides, which was reduced in the 2´-FL formulae to maintain a total oligosaccharide content of 2.4 g/L. Growth, stool consistency or adverse events were similar across treatments (9). Immune outcomes were assessed in these infants using blood samples collected on day of life 42 (11).
Infants fed the 2´-FL formulae did not differ from BF, but had 29 to 83% lower plasma proinflammatory cytokine concentrations than CF-fed infants. In terms of immune cells, BF infants had higher total T-cell and cytotoxic (CD8+) T-cells than CF-fed infants. Total T-cells in infants fed 2´-FL were intermediate between BF and CF, and CD8+ T cells in infants fed 1.0 g/L 2´-FL were intermediate between BF and CF (11).
In the second study, infants received a CF or a formula with 1.0 g/L 2´-FL and 0.5 g/L LNnT for 6 months, after which all were fed the CF until 12 months of age (10). Weight gain and digestive symptoms were similar in both groups, except infants fed HMO had softer stool and fewer nighttime wake-ups at 2 months. Secondary outcomes showed that consuming HMO-supplemented formula reduced parent- reported morbidity (particularly bronchitis) and antipyretics and antibiotics use (10). In addition, the HMO formula shifted the microbiome composition to be more similar to that of BF and increased Bifidobacterium abundance (12). Taken together, supplementing formula with 2´-FL or 2´-FL+LNnT affected infant immunity, reduced infection and medication use, and increased bifidobacteria abundance, thus narrowing the gap between breast-and formula-fed infants.
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5. Zivkovic AM, Barile D. Bovine milk as a source of functional oligosaccharides for improving human health. Adv Nutr. 2011; 2:284-9.
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7. Bohari MH, Yu X, Zick Y, Blanchard H. Structure-based rationale for differential recognition of lacto- and neolacto- series glycosphingolipids by the N-terminal domain of human galectin-8. Sci Rep. 6: 39556. doi: 10.1038/srep39556
8. Özcan E, Sela DA. Inefficient metabolism of the human milk oligosaccharides Lacto-N-tetraose and Lacto-N-neotetraose shifts Bifidobacterium longum subsp. infantis physiology. Front Nutr. 2018; 5:46. doi: 10.3389/fnut.2018.00046
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- The microbial trajectory across pregnancy and early life coincides with key neurodevelopmental periods.
- Diet, drugs and stress modulate early-life microbial colonization.
- Early-life interventions with prebiotics and probiotics could modulate the microbiota and neurodevelopment.
Microbiota · Neuropsychiatry · Gut-brain axis · Brain development · Early life · Stress · Diet · Nutrition
Pregnancy and early life are characterized by marked changes in body microbial composition. Intriguingly, these changes take place simultaneously with neurodevelopmental plasticity, suggesting a complex dialogue between the microbes that inhabit the gastrointestinal tract and the brain. The purpose of this chapter is to describe the natural trajectory of microbiota during pregnancy and early life, as well as review the literature available on its interaction with neurodevelopment. Several lines of evidence show that the gut microbiota interacts with diet, drugs and stress both prenatally and postnatally. Clinical and preclinical studies are illuminating how these disruptions result in different developmental outcomes. Understanding the role of the microbiota in neurodevelopment may lead to novel approaches to the study of the pathophysiology and treatment of neuropsychiatric disorders.
The connection between the brain and the gastrointestinal tract has been extensively studied, but the existence of a bidirectional microbiota-gut-brain axis has only received attention in the last decade [1, 2]. The individual microorganisms that live in our body, the microbiota, and their collective genomes, the microbiome, exert considerable influence over host brain and behaviour [3, 4] (Table 1). Variations in microbiota composition have been linked to neuropsychiatric disorders, including autism, stress, anxiety and major depressive disorder [3, 5].
Almost 30 years ago, it was proposed that prenatal and postnatal environmental factors interact with genetics to program health and disease in adulthood [6, 7]. Building on Barker’s hypothesis, it was recently proposed that the microbiota could play an important role in programming adult brain health and disease . Whether diet or other factors, such as stress and drugs, interact with the microbiota in early life to program brain health is currently being addressed by clinical and preclinical studies. This chapter reviews the natural trajectory of the composition of the microbiota during pregnancy and early life and outlines the current knowledge on the interaction be- tween the microbiota and neurodevelopment.
Early-Life Neurodevelopmental Plasticity and the Microbiota
Dramatic structural and functional changes in the brain are characteristic of the first years of life. This neurodevelopmental plasticity requires timely and adequate migration, division and differentiation of neuronal and glial precursors . Neuronal migration and axonal guidance establish short- and long-range connections that enable the recruitment of multiple brain areas for the execution of complex behaviours [10, 11]. Differentiated oligodendrocytes insulate neuronal axons with a myelin sheath to guarantee proper conductance of neuronal signals . A growing emphasis is now placed on the role of astrocytes and microglia in facilitating synaptic pruning during early life through adolescence, allowing later in life the fine tuning of complex circuits . Plasticity is a key feature of the standard neurodevelopmental trajectory and modulates the dynamics of synaptic connections and neural circuitry formation. Deviations from the neurodevelopmental trajectory can account for increased susceptibility to brain diseases later in life.
There is a growing appreciation of the link between neurodevelopment and intestinal microbiota. Studies in germ-free mice have shown abnormal brain development, especially in male mice [14–16]. More recent studies in these microbiota-deficient mice have shown altered expression of genes implicated in neurophysiology processes, such as neurotransmission, neuronal plasticity, metabolism and morphology in the amygdala  and hippocampus . Hypermyelination in the pre-frontal cortex and abnormal microglia maturation characterize the glia profile of these animals [19–23]. Furthermore, they showed increased blood-brain barrier permeability . Functionally, such changes translate to increased stress response [14, 16], changes in anxiety  and fear recall , cognitive deficits , social changes [21, 28] and visceral pain responses . Thus, the complete absence of microbial colonization in early life has dramatic effects on offspring’s brain development and function.
Dynamics of the Maternal Microbiota during Pregnancy
Pregnancy is a unique period in human life, and both the gut and vaginal microbiome have evolved to follow an optimum trajectory to support the mother and the developing fetus and allow for the ideal handover of microbiome at birth, informing maternal and child health outcomes.
The human female gut microbiota undergoes dynamic compositional changes across gestation [30–32]. As pregnancy progresses, a reduction in the diversity of the intestinal microbiota takes place, characterized by an enrichment in Proteobacteria . This natural shift in the bacterial populations is functional to the increased metabolic demands by the developing fetus. The Proteobacteria expansion can help the body with the increased energetic requirement that is characteristic of the third trimester . Interestingly, when gut microbiota from this time period was transferred to microbiota-depleted rats, they showed increased adiposity, reduced glucose tolerance and inflammation, signs of metabolic syndrome . This suggests that the changes in gut microbiota composition during pregnancy have an adaptive role for maternal and newborn health.
The vaginal microbiota composition also changes during pregnancy towards a less diverse configuration [34, 35]. As with gastrointestinal microbiota, the change in vaginal microbiota has a specific role during pregnancy. An in- crease in the presence of Lactobacilli helps maintain a low pH, limiting bacterial growth opportunity for other bacteria . Furthermore, vaginal microbiota composition is critical in the context of vertical transmission of microbial populations . Whether interventions in the physiological trajectory of maternal microbiota could alter the prenatal environment and, in turn, deviate normal brain development is a key question in neuroscience that is starting to be addressed both in preclinical and clinical areas.
Preclinical Models of Early-Life Microbiota Trajectory
Similar to humans, mice and rat intestinal and vaginal microbiota go through compositional changes during pregnancy, providing a robust preclinical model for studying the link between maternal gut environment and offspring brain development [37–40]. Early gestation is characterized by a transitional increase in the relative abundance of Akkermansia and Bifidobacterium, which in late pregnancy decrease to levels seen in non-pregnant mice. In contrast, Bacteroides remain relatively elevated throughout pregnancy . Interestingly, microbiota compositional changes also occur post-partum. The relative abundance of Actinobacteria increases early post-partum, while the one of Bacteroidetes decreases .
The vaginal microbiota has its own trajectory in pregnant mice. After the first week of pregnancy, there is an increase in bacterial diversity characterized by a growth of the Firmicutes and Bacteroidetes phyla [40, 41]. The changes seen in mice gut microbiota during pregnancy and post-partum make it a solid approach to the study of interventions in the maternal microbiota and the impact on offspring’s neurodevelopment.
External Challenges to Maternal Microbiota Dynamics
Given the importance of early-life microbiota in neurodevelopment, any factor that affects its composition has the potential to influence brain health. Indeed, a variety of exogenous factors affect the trajectory of microbiota composition during pregnancy. Diet, drugs, infection, hospitalization, prematurity and stress are among the influences that divert maternal microbiota from its natural course and impact on offspring’s brain, immune system and the hypothalamic-pituitary-adrenal axis (HPA) development.
Diet and Maternal Microbiota
Diet is one of the major sculptors of the diversity and abundance of the intestinal microbiota . Inadequate intake of macronutrients or micronutrients during pregnancy has been related to altered maternal microbiota  and offspring’s poor neurocognitive outcome (Table 2) . This association suggests a role for the maternal microbiota in brain prenatal programming.
One of the most common macronutrient consumption imbalances during pregnancy is the consumption of high-fat diets. Maternal overweight has been associated in humans with increased risk of poor neurodevelopmental outcomes . In rodents, consumption of a high-fat or Western diet prior and during pregnancy impairs the trajectory of maternal and offspring’s microbiota [37, 46]. This alteration was associated with a neuroinflammatory profile in the hippocampus and amygdala of the offspring, resulting in juvenile impaired social behaviour and anxiety-like phenotype . Interestingly, a high-fat diet prior to and during pregnancy impairs maternal HPA axis plasticity and the offspring’s hypothalamic gene response to stress [48, 49]. However, caution is required when interpreting the literature on the neurobiological changes induced by diets rich in fat and sugar in rodents as the content of the control diets regarding fibre and other nutrients needs to be taken into account [50, 51]. Nevertheless, preclinical studies on maternal high-fat and Western diets (see  for an extensive review) support the idea of a role for diet-induced microbiota changes in brain programming.
During fetal development, micronutrients are required for neurological development. Deficiency in B vitamins, folate or ions, such as iron and zinc, exerts detrimental effects on neurocognitive development in humans and rodents [52, 53]. Folate deficiency is paradigmatic of the impact of micronutrient deficit on offspring neurodevelopment. Mammalian cells are unable to synthetize this vitamin; thus, humans depend on food or supplements to compensate for their requirement . Failure to achieve normal serum folate levels during pregnancy has been associated with increased neural tube defects in the off- spring . Conveniently, bacteria residing in our colon can produce many vitamins of the B group, including folate. In mice, a loss-of-function mutation in an intestinal folate transporter can account for folate malabsorption, suggesting that bacterial produced folate plays a major role in host metabolism . In humans, consumption of a vegetarian diet during early pregnancy was associated with a distinctive microbial composition rich in biosynthesis pathways for fatty acids, lipids and folate .
Prebiotics and Probiotics
Research on the effect of prebiotic and probiotic administration during pregnancy is at an early stage (Table 3). Current reports indicate that the administration of prebiotics or probiotics to pregnant women is not associated with an increase or decrease in the risk of preterm birth or other infant and maternal adverse pregnancy outcomes . Researchers are beginning to shed light on their effects on offspring’s brain and immune development .
Prebiotics promote the growth of beneficial bacteria and include indigestible fibres that are fermented by colonic bacteria to produce short-chain fatty acids and provide a health benefit . In humans, the effects of maternal intake of prebiotics on neurodevelopment have not been well studied, and there is uncertainty about their effects on allergy risk [60, 61]. Galacto-oligosaccharide (GOS) and inulin administration to pregnant mice modulated the gut microbiota and prevented immune activation and intestinal permeability in the offspring . More-over, it has recently been shown that the addition of inulin to a mouse maternal high-fat diet abrogated the negative metabolic effect of the high-fat diet on offspring .
Probiotics are beneficial strains of bacteria that confer a health benefit to the host . There is lack of research on the prenatal impact of probiotics on neurodevelopment in humans and rodents. Administration of probiotics to pregnant women impact on immunity, reducing the risk of atopy but not of asthma [65, 66]. More preclinical and clinical research must be conducted to determine the impact of prenatal probiotics on the maternal and off- spring microbiota.
Antibiotics are widely used during pregnancy, but little is known about their effects on the trajectory of the maternal microbiome . Preclinical models are starting to shed light on the effect of antibiotic exposure on offspring neurodevelopment. Administration of antibiotics to pregnant rats caused impairments in social behaviour and pre-pulse inhibition of the offspring . In mice, administration of non-absorbable antibiotics during pregnancy reduced the exploratory behaviour in the offspring . These results warrant further research on the effect of microbiota.
Recently, Maier et al.  showed that a large amount of non-antibiotic human-targeted drugs have antimicrobial properties. Among them, drugs that can be prescribed during pregnancy, such as proton pump inhibitors, were found to disturb the growth of commensal bacteria (Table 2). Interestingly, psychotropic medications also influence the composition of gut bacteria in rodents [70, 71]. Selective serotonin uptake inhibitors, tricyclic antidepressants and antipsychotics negatively impact bacterial growth [71–73]. Looking at the effects on post- natal development, prenatal exposure to fluoxetine induces an anxiety-like phenotype in rats . Also, in rodents, valproic acid administration during pregnancy disturbs the microbiome of the offspring and results in impairment of the social behaviour of the offspring [75,76]. Owing to the prevalence of psychotropic administration during pregnancy, it is crucial to characterize the interaction between maternal health, microbiota and off- spring neurodevelopment.
Stress and the Maternal Microbiota
In humans, prenatal and postnatal maternal stress has been associated with young adult offspring behavioural and depressive symptoms  and aberrant infant intestinal microbiota development (Table 2) [78, 79]. In rodents, prenatal stress shifts maternal gut and vaginal bacterial community and induces long-lasting alterations in the gut microbiota composition of the offspring [40, 80]. Moreover, this alteration was shown to occur in a sex- specific manner, and it correlates with hyper-reactivity of the HPA axis .
The Microbiota in Transition: from Prenatal to Postnatal
When the first contact with the microbiota occurs re- mains controversial. The sterility of the uterus during pregnancy is one of the paradigms that research on the microbiome is revisiting. Bacteria have been found in the placenta [81, 82], amniotic fluid and meconium of humans [83, 84]. Moreover, the presence of specific bacteria in utero has been associated with pregnancy risks, including higher rates of preterm delivery . Nevertheless, the reliability of these findings is widely debated in the context of whether it is contamination or not [86, 87]. The existence of germ-free mice models further dismisses the idea of a prenatal microbiome . It is generally accepted that the moment of birth is the first opportunity for large-scale bacterial colonization of the newborn. Thus, the mode of delivery has a tremendous impact on the establishment of the microbiota of infants.
Early-Life Microbiota and Birth Mode
A large number of studies associate the mode of delivery to a distinctive trajectory of microbiota development in the newborn [35, 36, 66, 88–99]. Unexposed to the birth canal, Caesarean section (C-section)-born babies elude mother-neonate vertical vaginal transmission of bacteria and viruses [36, 89, 100]. In turn, the microbiota resembles skin and environment microbiota, suggesting that C-section first colonizers come from diverse sources (Table 2) [35, 89].
That said it is worth reinforcing that mode of delivery- induced changes in microbiota composition are transitory. Vaginally delivered infants have significantly higher microbiota richness and diversity than C-section-born infants as early as 3 days after birth [88, 100–102]. Nevertheless, the early decline in Proteobacteria and the late Firmicutes expansion occur timely over the first year of life of C-section-born infants .
The time course of these microbiota alterations overlaps with a critical period for neuro- and immune development (see  for extensive review). It has been suggested that C-section-distinctive microbiota composition plays a functional role in predisposing these infants to a greater relative risk of neonatal infections, allergy, asthma, obesity and type 1 diabetes [35, 101, 104–108]. Pre-clinical models of C-section suggest that the mode of delivery could impact on early neuronal maturation [109, 110]. Whether modifying the initial colonizing microbiota induces directly or indirectly different trajectories in brain development has yet to be deciphered.
Epidemiology studies have shown that C-section-induced changes in terms of brain health and school performance later in life are subtle at best [111, 112] and, in the case of autism, do not withstand correcting for familial confounding .
Various strategies have been designed to restore the normal trajectory of the microbiota . Although controversial, artificial vaginal microbiota transference was performed to C-section-born infants to mimic vertical transmission . Other interventions, including supplementation with probiotics and prebiotics, were proposed to decrease the impact of delivery mode on the microbiota.
Early Postnatal Perturbations of the Microbiota
Early postnatal life entails an intrinsic sensitivity to environmental factors. As with the maternal microbiome, infant exposure to differences in diet, drugs and stress can interfere with the trajectory of the microbiota and neuro-development in a manner that is characteristic of this developmental period.
Mode of Nutritional Provision in Early Life
The stability and composition of the early-life gut microbiota community is also dependent on diet . Ac- cumulating evidence suggests that breastfeeding and formula-based nutrition leave a distinctive fingerprint in the intestinal microbiota (Table 2). Gut bacterial composition of infants exclusively breastfed is characterized by higher relative abundance of Bacteroides and Bifidobacterium compared to the one from formula-fed infants [108, 116]. Furthermore, breastfeeding had a positive effect on myelination and increased general, verbal and non-verbal cognitive abilities during childhood . The implications of these findings are still unclear, but longitudinal studies are starting to shed light on the effect of early-life nutrition on the temporal course of microbiota maturation.
Human breast milk has a unique composition that interacts with the developing gut microbiota. Culture-dependent and -independent techniques revealed that it is a source of bacteria . Interestingly, the human milk microbiome can be influenced by maternal body mass index and mode of delivery . The other main components of breast milk are human milk oligosaccharides, which act as prebiotics [120, 121]. Supplementation of infant formula with GOS increases the abundance of Bi- fidobacteria and Lactobacilli to levels reported in breast-fed infants [122, 123]. Both breast milk microbes and prebiotics play a role in the standard gut microbial develop- mental trajectory.
Later in life, feeding transitions drive important changes in composition and functionality of the intestinal microbiota [36, 89, 124]. From breastfeeding to solid food, the microbiome transitions from being enriched in genes associated with digestion of sugars from breast milk, vitamin production and iron transport to degradation of starch and high sugars . Further-more, the microbiota continues to undergo change; at 7–12 years of age, the composition and function of the microbiota remains significantly different from the one of adults , suggesting a role of the microbiome in the neurodevelopmental changes associated with adolescence.
Probiotics and Prebiotics
Most of the evidence available on the effect of early-life exposure to pre-and probiotics comes from preclinical studies. Early-life prebiotic administration in humans has shown effects on reducing the risk of atopy, an autoimmune disease , but neurodevelopmental outcomes have not been studied yet. In preclinical studies, oligosaccharides have been shown to modulate the gut-brain axis, highlighting the role of breastfeeding in neurodevelopment. Administration of the human milk oligosaccharides 3’Sialyllactose (3’SL) or 6’Sialyllactose (6’SL) to mice exposed to social disruption prevented stress-induced colonic microbial disruption and anxiety-like behavior . Furthermore, fructo-oligosaccharide (FOS) and GOS administration attenuated corticosterone release in response to an acute stressor and protected the mice from the impact of chronic stress on the microbiota .
Preliminary clinical trials of probiotic interventions have yielded promising results with regard to reducing the risk for gastrointestinal problems, sepsis, allergies and even autism spectrum disorder and attention deficit hyperactivity disorder [129–134]. Several groups have now shown that early probiotic interventions mitigate the effects of early-life stress, maternal high-fat diet and maternal immune activation on infant outcomes [47, 135–138]. Oral administration at weaning of Bifidobacterium fragilis ameliorates the abnormal stereotyped and anxiety-like behaviours of the maternal immune activation mouse model of autism . Probiotic administration during adolescence restores social interaction-induced long- term potentiation in an animal model of social impairment by maternal high-fat diet exposure . In maternally separated rat pups, a combination of Lactobacillus rhamnosus and Lactobacillus helveticus reduced pup corticosterone responses to stress and normalized fear behaviour [135, 137, 138]. Another probiotic, Bifidobacterium infantis, normalized behavioural deficits in adult rats exposed to maternal separation .
Although clinical evidence on the role of pre-and probiotics for neurodevelopment is still lacking, preclinical research gives cause for a focus on early-life microbiota interventions.
Drugs: Antibiotics and Beyond in a Paediatric Setting
Antibiotics are commonly prescribed during the first years of life, yet the effect on brain health programming is unknown. Longitudinal clinical studies support the idea that early-life exposure to antibiotics perturbs the natural trajectory of the microbial communities by altering their stability . Furthermore, neonatal exposure to antibiotics in rodents not only altered the microbiota but also induced increased visceral sensitivity and long-lasting changes in brain cytokines and behaviour [141, 142].
The interaction between early-life exposure to psychotropics, neurodevelopment and the microbiota is currently unknown. Not only exposure to psychotropics mediated by breastfeeding but direct administration of these drugs early in life could impact the developing microbiota. Serotonin uptake inhibitors and atypical antipsychotics indicated for the treatment of paediatric psychiatric disorders are among the non-antibiotic drugs known to change the microbiome composition [70, 71]. Atypical antipsychotics indicated for the treatment of the irritability associated with autism spectrum disorders have been shown to inhibit gut bacteria . At the same time, the composition of the microbiota of autistic patients was shown to be altered [143–147]. Whether there is an interaction between microbiota populations, psychotropic drugs and behaviour has yet to be determined.
The impact of stress on the development of the HPA axis has been shown to contribute to the programming of brain health in later life . Interestingly, evidence from preclinical studies shows that early-life stress also alters the microbiota. Maternal separation during early life disrupted the microbiota of the offspring of rhesus monkeys and rats [149, 150]. Interestingly, a diet containing prebiotics in combination with live Lactobacillus rhamnosus GG attenuated the effects of early-life maternal separation on anxiety-like behaviour and hippocampal-dependent learning . Germ-free mice were more vulnerable to restraint stress, resulting in higher adrenocorticotropic hormone and corticosterone in plasma [14, 16], a reduction in glucocorticoid receptor mRNA and an increased stress response . Remarkably, these effects were rescued with microbiota transplantation during adolescence but not adulthood .
Pregnancy and the first years of life are unique stages of plasticity for the intestinal microbiota. In both cases, there is a dynamic trajectory of the intestinal microbiota composition that is functional to the requirements of the host. Although plasticity represents an opportunity for adaptation, it is also a vulnerable stage. As we have reviewed, clinical and preclinical studies suggest that diet, stress and drugs can interact with the natural trajectory of the microbiota and play a part in programming brain health (Fig. 1). However, the evidence is still scarce, and further research is needed to understand the functional implications of these interactions.
The nervous system and the microbiota show concurrent developmental trajectories, offering a unique opportunity for intervention. There is potential for the development of early-life-targeted interventions of the microbiome, aiming to reduce the risk of disease later in life. Further research is needed on the characterization of critical windows to modulate the microbiota and the consequences of early intervention.
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Physiological Effects of Feeding Infants and Young Children Formula Supplemented with Milk Fat Globule Membranes
Physiological Effects of Feeding Infants and Young Children Formula Supplemented with Milk Fat Globule Membranes
Olle Hernell, Magnus Domell, Tove Grip, Bo Lnerdal, and Niklas Timby
An increasing number of studies have reported different health benefits from oral supplementation with bovine milk fat globule membrane (MFGM) to infants and children (Table 1) [1, 2]. MFGM is a biologically active milk fraction that contains a large proportion of milk phospho-lipids, sphingomyelins, and gangliosides together with several hundred identified proteins, including mucins, butyrophilin, lactoferrin, and lactadherin (Fig. 1). Formula-fed infants are of special interest with respect to MFGM supplementation since they have a lower intake of MFGM components compared to breast-fed infants because, traditionally, the MFGM fraction is discarded with the milk fat when this is replaced by vegetable oils as the fat source in infant formulas.
Clinical Studies on the Effects of MFGM Concentrates Fed to Infants and Children
Table 1. Double-blind, randomized controlled trials exploring the effects of milk fat globule membrane (MFGM) supplementation to the diet of infants or children
In the first double-blind, randomized, controlled trial (DBRCT) in 550 healthy, primarily breast-fed 6- to 11-month-old infants, supplementation with an MFGM-enriched protein fraction reduced diarrheal morbidity . In another DBRCT in 70 infants, supplementation with bovine milk gangliosides, provided as a complex bovine milk lipid fraction from 2–8 until 24 weeks of age, increased hand-eye coordination, performance, and general IQ after adjustment for socioeconomic background variables . A third DBRCT including 253 preschool children aged 2.5–6 years evaluated a daily intake of a formula enriched with 500 mg of phospho-lipids with the addition of a phospholipid-rich MFGM concentrate for 4 months, and found reduced days with fever and less behavioral problems during the intervention . In an Indian DBRCT, 450 infants between 8 and 24 months of age were randomized to a daily dose of milk powder supplemented with 2 g of a spray-dried ganglioside concentrate or milk powder only for 12 weeks . There was no difference between the groups either in the primary outcome rotavirus diarrhea or in the secondary outcomes, including all-cause diarrhea. However, the authors noted that the incidence of rotavirus diarrhea during the study period was lower than expected, making the study underpowered compared to the intention of the design. In a Swedish DBRCT in 160 formula-fed healthy term infants, supplementation with a protein-rich MFGM fraction from <2 until 6 months of age improved cognitive scoring in Bayley III . Further, a reduced incidence of acute otitis media, a reduced antipyretic use, lower concentrations of serum IgG against pneumococci after vaccination, and a lower prevalence of Moraxella catarrhalis in the oral microbiota suggested an infection-protective effect of MFGM supplementation [8, 9]. In a non-inferiority DBRCT in 199 healthy term infants from 14 days to 4 months of age, a formula enriched with lipids and a formula with a protein-rich bovine MFGM fraction yielded a noninferior weight gain with no serious adverse events compared with a standard formula .
Fig. 1. Schematic drawing of the release of the milk fat globules and composition of the MFGM. Illustration by Erik Domellöf. Reproduced from Hernell et al.  with permission.
Studies investigating the effect of bovine MFGM-supplemented diets on infants and children have shown promising results regarding both neurodevelopment and defense against infections. However, the scientific base of knowledge for MFGM supplementation to infants and children is still limited. The number of studies published on MFGM provided to infants and children is small, and the interventions are heterogeneous: different MFGM concentrates have been given for different durations at different infant/child ages and with different main outcomes. However, MFGM supplementation seems safe down to the age of the first week of life in term infants, as no serious adverse effects have been reported. Infant formulas supplemented with bovine MFGM concentrates have already been launched on many markets, but before firm conclusions can be drawn on the likely health benefits of supplementing the diet of infants and children with MFGM ,more high quality DBRCTs are needed.
- Hernell O, Timby N, Domellöf M, Lönnerdal B: Clinical benefits of milk fat globule membranes for infants and children. J Pediatr 2016;173(suppl):S60–S65.
- Timby N, Domellöf M, Lönnerdal B, Hernell O: Supplementation of infant formula with bovine milk fat globule membranes. Adv Nutr 2017;8:351–355.
- Zavaleta N, Kvistgaard AS, Graverholt G, et al: Efficacy of an MFGM-enriched complementary food in diarrhea, anemia, and micronutrient status in infants. J Pediatr Gastroenterol Nutr 2011;53:561–568.
- Gurnida DA, Rowan AM, Idjradinata P, et al: Association of complex lipids containing gangliosides with cognitive development of 6-month-old infants. Early Hum Dev 2012;88:595–601.
- Veereman-Wauters G, Staelens S, Rombaut R, et al: Milk fat globule membrane (INPULSE) enriched formula milk decreases febrile episodes and may improve behavioral regulation in young children. Nutrition 2012;28:749–752.
- Poppitt SD, McGregor RA, Wiessing KR, et al: Bovine complex milk lipid containing gangliosides for prevention of rotavirus infection and diarrhoea in northern Indian infants. J Pediatr Gastroenterol Nutr 2014;59:167–171.
- Timby N, Domellöf E, Hernell O, et al: Neurodevelopment, nutrition, and growth until 12 mo of age in infants fed a low-energy, low-protein formula supplemented with bovine milk fat globule membranes: a randomized controlled trial. Am J Clin Nutr 2014;99:860–868.
- Timby N, Hernell O, Vaarala O, et al: Infections in infants fed formula supplemented with bovine milk fat globule membranes. J Pediatr Gastroenterol Nutr 2015;60:384 389.
- Timby N, Domellöf M, Holgerson PL, et al: Oral microbiota in infants fed a formula supplemented with bovine milk fat globule membranes – a randomized controlled trial. PLoS One 2017;12:e0169831.
- Billeaud C, Puccio G, Saliba E, et al: Safety and tolerance evaluation of milk fat globule membrane-enriched infant formulas: a randomized controlled multicenter non-inferiority trial in healthy term infants. Clin Med Insights Pediatr 2014;8:51–60.
- Timby N, Lönnerdal B, Hernell O, Domellöf M: Cardiovascular risk markers until 12 mo of age in infants fed a formula supplemented with bovine milk fat globule membranes. Pediatr Res 2014;76:394–400.
Dietary supplementation with bovine milk fat globule membrane (MFGM) concentrates has recently emerged as a possible means to improve the health of infants and young children. Formula-fed infants are of special interest since infant formulas traditionally have lower concentrations of biologically active MFGM components than human milk. We iden- tified 6 double-blind randomized controlled trials (DBRCT) exploring the effects of supple- menting the diet of infants and children with bovine MFGM concentrates. Two studies found a positive effect on cognitive development in formula-fed infants. Three studies found a protective effect against infections at different ages during infancy and early child- hood. We conclude that supplementation with MFGM during infancy and childhood appears safe, and the studies indicate positive effects on both neurodevelopment and defense against infections, especially in formula-fed infants. However, due to the small number of studies and the heterogeneity of interventions and outcomes, more high-quality DBRCTs are needed before firm conclusions can be drawn on the likely health benefits of MFGM supplementation to infants and children.
An increasing number of studies have reported various health benefits from oral supplementation with bovine milk fat globule membrane (MFGM) to humans of different ages, including infants and children [1, 2]. The MFGM is formed during the release of milk fat from the endothelial cell of the lactating mammary gland and is composed of a phospholipid and cholesterol triple layer which contains proteins and glycoproteins  (Fig. 1). Milk phospholipids, sphingomyelins, and gangliosides are largely located on the MFGM, although phospholipids are also secreted as smaller vesicles devoid of a triglyceride core, which typically separate from the whey fraction [3, 4]. The proteome of the human MFGM is very complex with several hundred proteins identified, including mucins, butyrophilin, lactoferrin, and lactadherin [5, 6]. Bovine MFGM-rich fractions contain approx- imately the same number of proteins . MFGM is also rich in sialic acid as part of gangliosides  and glycosylated proteins. The genes regulating MFGM syn- thesis are conserved across species suggesting a functional benefit of this fraction in milk , even if the detailed MFGM composition varies among species .
Breastfed infants have a higher intake of MFGM components than formula- fed infants because, traditionally, the MFGM fraction is discarded with the milk fat which is replaced by blends of vegetable oils as the source of fat in infant formulas. Resulting from advances in dairy technology, bovine MFGM concentrates are now commercially available and possible to use as a supplement to foods, including infant formulas.
Physiological Effects of Single Components of the Milk Fat Globule Membrane
Dietary gangliosides , sialic acid , and sphingomyelin  have been shown to be important for optimal brain development and function in different animal models. However, it should be noted that some of these models are disease models or models with inhibited de novo synthesis, which is far from supplementing a healthy infant or child. In a small study on premature infants with a birth weight <1,500 g, infants receiving formula with high sphingomyelin content (20 vs. 13% of all phospholipids in milk) to cover shortages of breast milk performed better than those fed the lower content at neurobehavioral follow-up between 6 and 18 months corrected age . Further, oral sphingomyelin , as well as a bovine MFGM concentrate , increased maturation of the intestine in rats. Gangliosides have also been suggested to play an important role in the development of intestinal microbiota composition, gut immunity, and, consequently, in the defense against infections . Other components of MFGM are also involved in the defense against infections, e.g., the glycoproteins butyrophilin, lactadherin, and mucins , which all have antimicrobial effects, and the lipid fraction of bovine MFGM has antiviral effects in vitro . Both lipid and protein components of MFGM have anticancer effects in vitro , and intake of MFGM in early life has also been suggested to protect against obesity later in life .
Illustration by Erik Domellöf. Reproduced from Hernell et al.  with permission.
Clinical Studies on Milk Fat Globule Membrane Concentrates Fed to Infants and Children
In a literature search (August 31, 2017), we identified 6 double-blind randomized controlled trials (DBRCT) exploring the effects of supplementing the diet of infants or children with MFGM (Table 1):
In a Peruvian DBRCT, 550 healthy, primarily breastfed 6- to 11-month-old infants consumed 40 g/day of an instant complementary food fortified with 1 recommended dietary allowance of multiple micronutrients and a protein source for 6 months. They were randomized to the protein source being either an MFGM-enriched protein fraction (Lacprodan® MFGM-10; Arla Foods Ingredients, Viby, Denmark) or skim milk powder (control group) . There was no difference between the groups in the incidence of diarrhea, but longitudinal prevalence of diarrhea was significantly lower in the MFGM group compared to the control group (3.84 vs. 4.37%, p < 0.05). In a multivariate model adjusted for initial anemia and potable water facilities, the incidence of bloody diarrhea was lower in the MFGM group, with an adjusted OR of 0.59 (95% CI 0.34–1.02, p = 0.025).
In a DBRCT performed in Indonesia, 70 term infants were randomized to a control formula or an infant formula enriched with bovine milk gangliosides, provided as a complex bovine milk lipid fraction (AnmumInfacare; Fonterra Cooperative Group, Auckland, New Zealand) . A breastfed reference group (BFR) (n = 40) was also recruited. The intervention started between 2 and 8 weeks and continued until 24 weeks of age. After adjustment for socioeconomic background variables, the hand-eye coordination IQ (129.5 vs. 122.0, p =0.006), performance IQ (131.1 vs. 123.2, p < 0.001), and general IQ (125.4 vs.120.6, p = 0.041) measured with the Griffiths Mental Developmental Scale were higher in the ganglioside-supplemented group than in the control group, and the ganglioside-supplemented group did not differ from the BFR group.
In a Belgian DBRCT, 253 preschool children aged 2.5–6 years received 200 mL of a chocolate formula milk daily for 4 months . They were randomized to a formula without phospholipids (placebo group) or enriched with 500 mg of phospholipids with the addition of 2.5% of a phospholipid-rich MFGM concentrate (Inpulse; Büllinger SA, Büllingen, Belgium) (intervention group). The intervention group had fewer days with fever (mean ± SD: 1.71 ± 2.47 vs.2.60 ± 3.06, p = 0.028), and lower parental scoring of internal (p < 0.003), external (p < 0.005), and total (p < 0.002) behavioral problems measured by the Achenbach System of Empirically Based Assessment (ASEBA). However, ASEBA scoring was only performed after the intervention but not at baseline, and differences were not confirmed when the children’s teachers made the scoring.
In an Indian DBRCT, 450 infants between 8 and 24 months of age were randomized to a daily dose of milk powder supplemented with 2 g of a spray-dried ganglioside concentrate (Fonterra Cooperative Ltd,) or milk powder only (control group) for 12 weeks . There was no difference between the groups, nor in the primary outcome rotavirus diarrhea, or in secondary outcomes including all-cause diarrhea. However, the authors noted that the incidence of rotavirus diarrhea during the study period was lower than expected, making the study under-powered as compared to the intention of the design.
In a Swedish DBRCT, 160 formula-fed healthy term infants were randomized to receive an experimental formula (EF) supplemented with a protein-rich MFGM fraction (Lacprodan® MFGM-10; Arla Foods Ingredients) or standard formula (SF) from <2 to 6 months of age. The EF had lower energy (60 vs. 66 kcal/100 mL) and protein (1.20 vs. 1.27 g/100 mL) densities, and MFGM-proteins made up 4% (wt/wt) of the total protein content in the formula. In addition, a BFR group including 80 infants was also studied. The formula-fed infants regulated their ingested volumes by increasing meal size, resulting in no differences in energy intake, protein intake, blood urea nitro- gen (BUN), serum insulin level, or growth, including body fat percentage, up to 12 months of age . The surprisingly high level of self-regulation for the bottle-fed infants might be explained by a low level of parental control in the study population .
At 12 months of age, the EF group achieved higher scores (mean ± SD) in the cognitive domain of Bayley III (105.8 ± 9.2) than the SF group (101.8 ± 8.0, p = 0.008) and did not differ from the BFR group (106.4 ± 9.5, p = 0.73) . During the intervention, the EF group had a lower incidence of acute otitis media than the SF group (1 vs. 9%, p = 0.034), a lower incidence and longitudinal prevalence of antipyretic use, lower concentrations of serum IgG against pneumococci after vaccination and a lower prevalence of Moraxella catarrhalis in the oral microbiota, all suggesting an infection-protective effect of EF [26, 27]. During the intervention, the EF group gradually reached higher serum cholesterol concentrations than the SF group, and there was no significant difference between the EF and BFR group at 6 months of age .
In a multicenter noninferiority DBRCT, 199 healthy term infants were randomized to 3 different formulas from 14 days to 4 months of age; a SF (control), a formula enriched with lipids (MFGM-L; Fonterra Cooperative Group Ltd), and a formula with a protein-rich (MFGM-P, Lacprodan® MFGM-10, Arla Foods Ingredients) bovine MFGM fraction, respectively . Weight gain was noninferior in the MFGM-L and MFGM-P groups compared with the control group. Adverse events and morbidity rates were similar across groups except for a higher rate of eczema in the MFGM-P group (13.9 vs. 1.4% in the MFGM-L group and 3.5% in the control group, p = 0001). It is, however, not clear how and when eczema was diagnosed, and the number of infants diagnosed were few in the MFGM-L and control groups (1 and 2, respectively). The authors also concluded that care must be taken in interpreting the exploratory endpoints. A higher risk of skin rash was not confirmed in a Swedish study  which studied the same MFGM-P fraction.
Studies on the supplementation of bovine MFGM to the diet of infants and children have shown promising results regarding both neurodevelopment and defense against infections. These findings are supported by known effects of individual components of MFGM mostly based on in vitro and/or animal studies. However, the scientific base of knowledge for MFGM supple- mentation to infants and children is still limited. The number of published studies on MFGM supplementation to infants and children is small, and the interventions are heterogeneous: different MFGM concentrates have been given for different durations at different ages and with different main out- comes. However, MFGM supplementation seems safe down to the age of the first week of life in term infants, as no serious adverse effects have been reported.
Infant formulas supplemented with bovine MFGM concentrates have already been launched on many markets, but before any general recommendations or guidelines of MFGM use in infants and children can be given, more high-quality DBRCTs are needed.
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