Reduced Severity of Necrotizing Enterocolitis
Human milk oligosaccharide (hMOS) is a major component of human milk, similar in quantity to protein.
The structure of hMOS is distinct from other commercially available oligosaccharide or prebiotics. hMOSs are structural homologues to oligosaccharide components of glycoproteins in the infant gut and contain fucose and sialic acid.
Bovine milk (and most infant formulas) contains sparse quantities of acidic oligosaccharide that lack fucose.
There is variation in the hMOS content of human milk among mothers and over the course of lactation.
Infants do not directly digest hMOS, but some of the hMOS consumed is absorbed into the circulation, and some of it is metabolized by mutualist bacteria, which produces beneficial metabolites.
The functions of hMOS include prebiotic effects, inhibition of pathogen binding, neurodevelopment, and immunomodulation. After intestinal challenge, hMOS reduces gut inflammation while enhancing infant immunity, intestinal adaptation, and growth recovery.
Major hMOSs of human milk are being tested as additives to infant formula. Preclinical studies and clinical trials have established their safety, with no effects on normal growth. Consumption of hMOS through infant formula confers immune and metabolic benefits.
Human milk oligosaccharide (hMOS) is a major fraction of human milk. This fraction is composed of carbohydrate chains ranging in size from 3 to 32 sugars that use lactose as a core molecule. hMOS in nutrition was overlooked for many years, but the number of publications indexed on hMOS in PubMed per year nearly tripled between 2009 and 2016, indicating that hMOS has emerged as a major focus of investigation. Heightened attention to hMOS is likely the result of the emerging evidence of its effects on health and development, as well as the distinctive composition of the oligosaccharides of human milk and their homology to gut glycans. Technical developments have now led to commercial biosynthesis of major individual hMOS molecules, enabling preclinical and clinical studies. Several companies have obtained U.S. Food and Drug Administration approval of synthetically produced hMOS, 2’-fucosyllactose (2’-FL) and lacto- N -neotretraose (LNnT), as “generally recognized as safe”. Initial human trials have demonstrated safety and potential health benefits of 2’-FL in combination with other oligosaccharides in infant formula. In this chapter, we examine the structure and composition of human milk, the effects of hMOS on the infant gut microbial community, and other probable health effects of hMOS.
Structure and Composition
Human milk oligosaccharides are distinct in structure from commercially available oligosaccharides—fructo-oligosaccharide and galacto-oligosaccharide (GOS)—that were synthesized and first added to formula as substitutes for hMOS more than 10 years ago ( Fig. 4.1 ). Remarkably, the oligosaccharide is more abundant and complex in human milk compared with the milk of most other mammalian species and comprises more than 150 individual molecules. The absolute quantity of hMOS in mature milk may be as high as 5 to 20 g/L, with higher levels in early milk; the quantity of hMOS declines about 30% over the course of lactation. The quantity of hMOS in early preterm milk is about 10% higher than that in term milk within the first 14 days after delivery. The reported quantity of hMOS in mother’s milk is generally similar to that of protein. However, there is significant variation in the hMOS content of milk among mothers, over the course of lactation, and by maternal genetics. Measurement of hMOS content is also influenced by differences in methods, including milk collection, hMOS isolation, and analytic techniques.
From two thirds to three quarters of total hMOS are neutral. The majority of the neutral oligosaccharides comprises molecules containing one or more fucose moieties. A minority of the neutral oligosaccharide fraction consists of precursor molecules (e.g., lacto- N -tetraose [LNT], LNnT, and lacto- N -hexaose [LNH]) that lack fucose. Maternal ability to synthesize fucosylated hMOS depends on encoded enzymes made by specific fucosyltransferase ( FUT ) genes. Synthesis of hMOS containing the Fuc-α1,2-Gal linkage requires the FUT2 (“secretor”) enzyme. This class of hMOS may constitute half of the hMOS fraction when the mother is FUT2 + (“secretor”). Individual oligosaccharides containing this linkage include 2’-FL and lacto- N -fucopentaose I (LNFP I). However, nearly 25% of the U.S. population and many other populations worldwide are homozygous recessive for the FUT2 gene and therefore are incapable of synthesizing this class of hMOS. Mothers who are secretors have significantly higher hMOS content of milk compared with mothers who are nonsecretors.
Another strong genetic influence on neutral hMOS phenotype is the FUT3 (“Lewis”) enzyme, which is required for synthesis of Fuc-α1,4-Gal linkages. Some major hMOSs containing this linkage include lacto- N -difucohexaose I (LNDFH I) and LNFP II. But the FUT3 gene can also have inactivating mutations. As a result, about 10% of mothers cannot make Lewis hMOS. About two thirds of the population are both FUT2 + and FUT3 + and are thus capable of synthesizing all hMOSs. However, the remaining third of the population is nonsecretors, Lewis negative, or both and therefore lack the corresponding hMOS. Mothers with polymorphisms in FUT2 and FUT3 genes (“nonsecretor” and Lewis negative) typically have lower total hMOS concentrations, despite compensatory increase in acidic or precursor hMOS that do not require the FUT2 or FUT3 encoded enzymes.
Sialic-acid containing hMOSs range from about 25% to 40% of the hMOS fraction. The highest proportions are found in early lactation, with the percentage declining rapidly over the first few months of lactation. Synthesis of sialylated hMOS requires sialyltransferase (ST)3Gal enzymes to produce sialic acid–containing α2,3 linkages (which includes 3’-sialyllactose [3’-SL]) and ST6Gal gene enzymes to produce sialic acid-containing α2,6 linkages (which includes 6’-sialyllactose [6’-SL]).
Although human milk contains hundreds of distinct hMOSs, as much as 75% of the hMOS fraction of human milk consists of 12 individual hMOSs ( Table 4.1 and Fig. 4.2 ), including six fucosylated neutral hMOSs, two precursor nonfucosylated hMOSs, and four sialylated (acidic) hMOSs. As shown in Fig. 4.2 , the relative abundance of these different hMOS shifts over the course of lactation. The most abundant neutral oligosaccharide over the course of lactation is typically 2’-FL, but depending on maternal genetics and the timing of lactation, there are other major abundant neutral hMOSs, including the neutral precursor molecules LNT, and LNnT and the fucose-containing molecules LNFP I, LNDFH I, LNFP II, 3-fucosyllactose (3-FL), and lacto-difucotetraose. Among the many acidic oligosaccharides found in milk, the most abundant are disialyl lacto- N -tetraose (DSLNT), the two trisaccharide molecules 6’-SL and 3’-SL, and monofucosylmonosialylacto- N -hexaose, a molecule that contains both fucose and sialic acid.
|Oligosaccharide (abbreviation)||Structure||Type and size|
|2’-fucosyllactose (2’-FL)||Fucose α 1,2 γαλαχτoσεβ 1,4 glucose||Fucosylated, neutral, |
|Lacto- N -fucopentaose I (LNFPI)||Fucose α 1,2 -Gal β 1,3 -GlcNAc β 1,3 -Gal β 1,4 -Glc,||Fucosylated, neutral, |
|Lacto- N -difucohexose I (LNDFHI)||Fucose α 1,2 -Gal β 1,3 -(Fuc-α 1,4 )- GlcNAc β 1,3 -Gal β 1,4 -Glc||Difucosylated, neutral, hexaose|
|Lacto- N -fucopentose II (LNFPII)||Gal β 1,3 -(Fuc α 1,4 )-GlcNAc β 1,3 -Gal β 1,4 -Glc||Fucosylated, neutral, |
|3-Fucosyllactose (3-FL)||Fuc α 1,3 -(Gal β 1,4 )-Glc||Fucosylated, neutral|
|Lactodifucotetraose (LDFT)||Fucα 1-2 Galβ 1-4 (Fucα 1-3 )Glc||Difucosylated, neutral, tetraose|
|Disialyllacto- N -tetraose (DSLNT)||Neu5Ac α 2,3 -Gal β 1,3 -(Neu5Ac α 2,6 )-GlcNAc β 1,3 -Gal β 1,4 -Glc||Disialylated, acidic, hexaose|
|3’- sialyllactose (3’-SL)||Neu5Ac α 2,3 -Gal β 1,4 -Glc||Sialyl, acidic, triose|
|6’- sialyllactose (6’-SL)||Neu5Ac α 2,6 -Gal β 1,4 -Glc||Sialylated, acidic, triose|
|Monofucosylmonosialyllacto- N -hexaose (MFMSLNH)||Neu5Ac α 2,6 -(Gal β 1,3 )-GlcNAc β 1,3 -(Gal β 1,4 -[Fuc α 1,3 -] GlcNAc β 1,6 -)Gal β 1,4 -Glc||Sialylated and fucosylated, acidic octaose|
|Lacto- N -tetraose (LNT)||Gal β 1,3 -GlcNAc β 1,3 -Gal β 1,4 -Glc||Nonfucosylated, neutral, tetraose|
|Lacto- N -neotetraose (LNnT)||Gal β 1,4 -GlcNAc β 1,3 -Gal β 1,4 -Glc||Nonfucosylated, neutral, tetraose|
The relative abundance of different hMOSs shifts over the course of lactation. For example, the total quantity of hMOSs reduces over the course of lactation, and the quantity of sialic acid-containing hMOSs declines more rapidly than neutral hMOS. The biologic significance of these shifts in hMOS composition is not known.
Digestion and Absorption
Infants lack the enzymes necessary to hydrolyze or digest hMOS, and thus in a strict sense, hMOS is not a human nutrient, but a form of dietary fiber. Some hMOSs are absorbed in the small intestine, found intact in the infant’s circulation, and excreted in urine. More of the hMOSs are fermented by bifidobacteria and other mutualist bacteria in the colon; this fermentation produces short-chain fatty acids and small organic acids. However, a large proportion of the hMOS fraction is found intact in the infant’s stool. These findings are consistent with the multiple functions that have been identified for hMOSs, as described later.
Oligosaccharides are among the most abundant bioactive molecules of human milk and have multiple functions, including metabolic, antiinfective, and immunomodulatory functions. The individual hMOS molecules appear to have some common, or redundant, functions, but there is increasing evidence that individual hMOS molecules also have distinct effects (see Box 4.1 ).
In infancy, human milk feeding shapes the composition of microbiota, favoring colonization by bifidobacteria. The bifidogenic impact of human milk was first observed by the eminent Dr. Paul Gyorgy, whose pioneering work on human milk led to his discovery of a “bifidus factor” in human milk, that is, hMOS and hMOS bound to glycoprotein. Indeed, the predominance of bifidobacteria in the microbiota of exclusively breastfed infants is largely shaped by the hMOS fraction of human milk.
The hMOS fraction and the individual hMOS molecules are effective prebiotics, defined as dietary carbohydrates that are indigestible by humans but utilized by gut bacteria for anaerobic fermentation. hMOS is avidly utilized by the mutualist microbes Bifidobacterium longum infantis and Bacteroides spp., but not by the nonmutualists Campylobacter jejuni , Clostridium perfringens , or Escherichia coli . Mutualist bacteria ferment hMOS into small organic acids and short-chain fatty acids that acidify the gut and inhibit pathogens. However, even in this general function, specific bacteria differ in their ability to hydrolyze and metabolize specific hMOS molecules, depending on their genomic capacities, resulting in distinct patterns of bacterial growth and metabolic products with different combinations of hMOS.
Human milk oligosaccharides protect infants against enteric, urinary, and respiratory pathogens through several different protective mechanisms. The most highly studied are the hMOSs that protect against enteric pathogens. The protection that hMOSs offer against enteric infection may be attributed to multiple mechanisms, including their prebiotic effects. However, the primary mechanism of protection may be through competitive inhibition of pathogen binding to homologous gut receptors. The intestinal tract displays an abundant quantity of oligosaccharides lining the mucosal surface. The oligosaccharides located on terminal end of membrane-bound gut glycans can be used as binding sites by enteric pathogens, which then infect gut enterocytes. Competitive inhibition of this binding by hMOS is thought to depend on the degree of homology between milk and gut oligosaccharide structures. Nevertheless, the specificity of pathogen binding varies, allowing some hMOSs to inhibit multiple pathogens through competitive binding.
The fucose of fucosylated glycans, and the sialic acid ( N -acetylneuraminic acid) of acidic glycans, are common components of bacterial and viral mucosal receptors. Human milk acidic glycans include mucins, glycoproteins, and the gangliosides GM1, GD3, and GM3. MUC1 and MUC4 are the principal mucins of human milk. These human milk mucins (MUCs) are heavily glycosylated (i.e., contain copious oligosaccharides) and contain serine, threonine, and proline repeats and terminal cysteine-rich domains as part of their structures. Protection of mucosa through competitive binding of mucin to pathogens may often depend on the relative quantity of fucosylated and sialylated moieties available to bind the pathogen, thereby inhibiting binding to the infant’s mucosal glycan receptor. MUC1 and MUC4 are able to inhibit infection by Salmonella typhimurium . MUC1 also inhibits infection by rotavirus, and binding by norovirus, E. coli , and human immunodeficiency virus (HIV).
Acidic human milk glycans include the glycolipid gangliosides GM1and GM3, which also bind to pathogens. The ganglioside GM1 limits the adhesion of enterotoxigenic E. coli to Caco-2 cells to less than 20% of the positive control. GM3 depresses adhesion of enterotoxigenic E. coli and enteropathogenic E. coli in vitro. The most prevalent acidic hMOSs are the sialyllactoses, which inhibit E. coli , Pseudomonas aeruginosa , Aspergillus fumigatus conidia , and Helicobacter pylori .
Human milk secretory immunoglobulin A (sIgA) was recognized early as a milk component that protects infants against human pathogens. When sIgA binds an antigen, it renders the pathogen less infective. Oligosaccharides on the surface of sIgA play general structural and functional roles and appear to increase the binding of sIgA. Human milk bile salt–stimulated lipase (BSSL) binds to dendritic cell–specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN), a nonenzymatic function, and inhibits HIV type 1 transfer to the CD4+ class of T cells. Human milk lactoferrin is a multifunctional glycoprotein that displays innate antibacterial, antivirus, antifungal, and antiprotozoan activity and can block cell–virus interaction and disrupt bacterial cell membranes.
Many enteric pathogens use α 1,2 -fucosylated gut glycan structures for binding—this includes C. jejuni , Vibrio cholerae , enteropathogenic E. coli , human rotaviruses, and most major noroviruses, including GII.4, GII.17 and GII.10 strains. This predilection for pathogen binding to α 1,2 -fucosylated glycans explains the finding that acute diarrheal disease was more prevalent in FUT2 + secretors than in nonsecretors in population-based studies, including a recent genome wide association study (GWAS). Consistent with that finding, norovirus is inhibited by the large milk glycoproteins, mucin and BSSL, only when their source is milk from a secretor mother. Among “secretor-binding pathogens,” however, there are differences in the ability of simple oligosaccharide structures or large glycans of human milk to effectively inhibit binding to the gut receptor.
Some simple hMOSs bind to specific pathogens, competing with the ability of the pathogen to bind its homologous carbohydrate moiety on the host cell surface receptor. For example, C. jejuni is the most common agent of bacterial diarrhea worldwide. The receptor of C. jejuni contains an H-2-fucosylated critical determinant: C. jejuni has high avidity for the fucosylated antigen H-2 (Fucα1,2-Galβ1,4-GlcNAc); monoclonal antibodies against the H-2 epitope inhibit C. jejuni binding. Conversely, overexpression of H-2- fucosylated antigen on Chinese hamster ovary cells transforms these cells from C. jejuni nonbinding cells to C. jejuni binding cells. Ligands that bind to H-2 epitope, such as Ulex europaeus agglutinin and Lotus tetragonolobus lectins, inhibit C. jejuni adhesion. 2’-FL, the most prevalent of the milk oligosaccharides, is a major source of H-2 epitope in human milk. 2’-FL competes with H-2 epitopes of the host cell surface receptors for binding to C. jejuni . Thus 2’-FL inhibits C. jejuni binding to fucosylated H-2 epitopes on the apical surface of epithelial cells of the intestinal mucosa, thereby preventing C. jejuni adhesion to the host.
The clinical relevance of these in vitro and in vivo laboratory results was tested in a cohort of 93 breastfeeding mother–infant pairs. C. jejuni diarrhea occurred significantly less in infants consuming milk that contained high levels of 2’-FL but was unrelated to levels of other hMOSs, strongly supporting clinical relevance of protection by 2’-FL consumption. Similarly, the hMOS LDFH-I binds to Norwalk virus in vitro and specifically inhibits the ability of the virus to infect cells. In the aforementioned clinical study, only LDFH-I levels in milk consumed by the infants was inversely related to the risk of norovirus-associated diarrhea; levels of other hMOSs were not related. More recently, mice infected with a human cutivar of virulent Campylobacter exhibited the pathobiology, morbidity, and mortality of the human disease. Feeding physiologic human milk levels of 2’FL during the campylobacter infection reduced the pathobiology to the extent that the sick but treated mice resembled uninfected controls.
Thus the hMOSs and glycoproteins are strongly implicated as responsible for a major portion of the protection of human milk against infant diarrhea.
Human milk oligosaccharides regulate immune processes in the intestinal epithelium and in the central circulation. For example, in vitro, hMOSs reduce platelet-neutrophil complex formation, which decreases neutrophil β 2 -integrin expression. At physiologically plausible concentrations, an acidic hMOS fraction reduced platelet–neutrophil complex formation up to 20%, and neutrophils showed a dose-dependent decrease in β 2 -integrin expression. The neutral hMOS fraction had no effect.
In a variety of inflammatory diseases, excessive leukocyte infiltration causes severe tissue damage. This initial step of leukocyte extravasation is mediated by selectin binding to the glycan moiety of glycoconjugate ligands. Monocytes, lymphocytes, and neutrophils isolated from human peripheral blood, when passed over activated human umbilical vein endothelial cells under hemodynamic shear stress, adhere to the endothelial cells. Within physiologic concentrations, the acidic hMOS fraction significantly inhibited leukocyte rolling and adhesion. Individual hMOSs may serve as antiinflammatory components that contribute to the lower incidence of inflammatory diseases in human milk–fed infants.
Relative to the mature intestinal mucosa, that of the immature intestine overexpresses inflammatory genes and underexpresses feedback regulatory genes, making neonatal epithelial cells prone to exaggerated responses to proinflammatory stimuli. Moreover, the T helper cell 2 (Th2) bias that remains from prenatal immune regulation renders the neonatal mucosa hyperresponsive to bacterial infection and susceptible to food allergy. The oligosaccharides of human colostrum curb inflammatory genes and cytokine expression in the neonatal intestine. These genes are involved in the major immunologic signal pathways: immune cell trafficking, hematologic system development, promotion of Th1 cell activation and function, and suppression of Th2 cell activation and function. Colostrum hMOS modulates Toll-like receptor 3 (TLR3), TLR5 and interleukin-1 (IL-1β)–dependent pathogen-associated molecular pattern signaling pathways, depressing acute phase inflammatory cytokine protein expression. 3’-galactosyllactose, an oligosaccharide found in especially high concentrations in colostrum relative to mature milk, specifically quenches polyinosine–polycytidylic acid (the TLR3 ligand)–induced IL-8 levels.
The human milk oligosaccharide 2’-FL modulates CD14 expression through decreasing CD14 messenger RNA (mRNA) transcription and decreases the amount of CD14 bound to membrane. Modulation of CD14 expression and binding quenches inflammation elicited by type I pili E. coli infection in human enterocytes. Feeding 2’-FL to mice modified their gene expression, changed microbiota species, and increased survival during dysbiosis (DSN, personal communication). Other examples of modulating the neonatal immune system through hMOS have also been reported. LNFP III, a human milk oligosaccharide containing the Lewis X (Lex) epitope, strongly promotes a Th2 response in vivo. LNFP III induces recruitment of suppressor macrophages and accelerates maturation of dendritic cells. Sialyl α 2,3 -lactose (3’-SL) arouses mesenteric lymph node CD11c+ dendritic cells and causes release of cytokines that expand Th1 and Th17 T lymphocytes. Although the precise mechanism of many other antiinflammatory effects of hMOS in diverse inflammatory disease conditions has not been elucidated, such putative antiinflammatory functions of hMOS could contribute to the lower incidence of inflammatory diseases in breastfed versus formula-fed infants.
A number of studies in diverse populations have examined the relationship between administration of hMOS and infant growth. Before reviewing these studies, we first consider biologic plausibility. In the “as consumed” form, hMOSs are not directly a human nutrient, given that human infants (and adults) lack the enzymatic repertoire to digest hMOS. Given this, what is the potential mechanism by which hMOS could influence infant growth? One possibility, as previously noted, is the potential for hMOS to reduce infections and gut inflammation, which could, in turn, improve growth by making the nutrient content of human milk more bioavailable to the infant. Another potential explanation pertains to the prebiotic role of hMOS. Providing hMOS can shift the microbiota beneficially, supporting the growth of mutualist bacteria and reducing the burden of potentially pathogenic bacteria. The fermentation of hMOS by bacteria in the small intestine and colon can produce metabolites, including small-chain fatty acids and small organic acids, that can beneficially influence bacterial metabolism in the gut and the metabolites found in the infant’s circulation. Furthermore, provision of hMOS can restore gut architecture after intestinal insult. These several putative mechanisms provide ample justification for studying the role of hMOS in infant growth.
Because hMOS composition varies significantly among mothers, it is possible to examine the association between varied composition and infant health outcomes using epidemiology. Several longitudinal studies of mother-infant pairs have reported that variation in hMOS composition affects infant growth, but the findings appear inconsistent. In a longitudinal study of 25 term infants in Oklahoma, the growth of breastfed infants was measured at 1 and 6 months of postnatal life. Higher diversity and evenness of hMOSs measured in mothers’ milk was associated with lower infant fat mass. Individual hMOSs showed varying relationships to growth: LNFP I was associated with lower infant weight and lower lean mass, whereas LNnT was associated with lower body fat. Other individual hMOS molecules—DSLNT and LNFP II—were associated with greater fat mass. A longitudinal study of 33 Gambian mother–infant pairs at 4, 16, and 20 weeks postpartum also reported that the relative abundance of other hMOSs in mothers’ milk was associated with growth. But the pattern differed from that reported in infants in Arizona. The Gambian study found that infant weight-for-age Z-score growth was positively associated with the abundance of 3’-SL, whereas there was a negative association with LSTc abundance in mother’s milk. Several other hMOSs—LNFP I/III and DFLNHa—were positively associated with infant height-for-age Z-scores. The findings of a third longitudinal study of 50 infants in Singapore measured hMOSs at a single time point, and examined growth at 30, 60, and 120 days of postnatal life. In the Singapore infants, no significant differences were found in body weight, body length, or body mass index in relation to hMOS concentrations.
In addition to these observational studies in breastfed term infants, two randomized, controlled trials (RCTs) were conducted in the United States and Europe, and these studies compared the growth of infants given standard formula with those given formula with hMOS. Neither trial found a significant difference in the growth of hMOS-fed and non–hMOS-fed control infants.
Taken together, the results of these studies do not support a consistent association between total or relative intake of specific hMOSs and growth in healthy, term infants. The two observational studies that reported growth effects related to individual hMOSs were conducted in distinct populations and environments and could potentially have been influenced by particular microbe–hMOS interactions found within each study population. Alternatively, the findings in those two studies might have been influenced by chance, given the multiple comparisons made within each study.
However, several preclinical studies support a role for hMOS in growth recovery after infection, malnutrition, or gut insult. Mouse models of growth recovery after intestinal resection, after dextran sulfate sodium (DSS)–induced colitis, and in mice recovering from necrotizing enterocolitis (NEC) all showed that 2’-FL was associated with significantly greater growth recovery. These studies are reviewed in greater depth in the following sections.
Reduced Severity of Necrotizing Enterocolitis
Nearly 7% of very low–birth weight preterm infants (infants born <33 weeks gestational age) are affected by NEC. NEC, which is a major gastrointestinal emergency in preterm infants, results in significant mortality. Although the exact cause of NEC is not known, the condition has a sudden onset and represents an immature, hyperinflammatory response to intestinal stimuli, resulting in necrosis and ischemia. Dysbiosis appears to be a major contributing factor, with evidence of a surge in microbes of the family Enterobacteriaceae, although other forms of dysbiosis have also been reported.
Feeding human milk is one of the best-evidenced clinical interventions to reduce the risk and severity of NEC. The epidemiology of NEC and the results of human milk feeding trials have raised the possibility that the oligosaccharide fraction of human milk may explain some of this protective effect.
Several studies using a rat model of NEC have demonstrated that hMOSs prevent morbidity and mortality caused by NEC. These preclinical studies found that the strongest and most consistent protective effect occurs with the disialylated (acidic) oligosaccharide DSLNT but that the neutral hMOS fraction of milk, and 2’-FL specifically, also protects against NEC. In a multisite epidemiologic study of NEC in preterm infants, it was found that human milk lacking DSLNT was significantly predictive of NEC in human milk–fed infants.
Several other preclinical studies suggest the potential for 2’-FL to reduce risk or severity of NEC. He et al tested 2’-FL in T84 and H4 cells and found that exposure to 2’-FL attenuates inflammatory response to uropathogenic E. coli (UPEC) and other pathogenic E. coli . The relevance of this to the neonatal gut was supported by the finding of Ward et al. that UPEC is a major contributor to NEC in some populations. In a neonatal mouse model of NEC, Good et al. tested 2’-FL and found that it significantly reduced the severity of NEC, helped preserved intestinal mucosal architecture, and modestly enhanced growth in mice with NEC. The mechanism thought to be responsible for these effects were that 2’-FL upregulated the vasodilatory molecule endothelial nitric oxide synthase (eNOS) and thereby restored intestinal perfusion. Administration of 2’-FL to eNOS-deficient mice or to mice that received eNOS inhibitors did not protect against NEC. Although 16S analysis indicated that 2’-FL also shifted the microbiota of the neonatal mouse gut, these changes did not seem to explain the observed reduction in NEC severity. In cultured endothelial cells, 2’-FL treatment induced eNOS, linking eNOS and hMOS in the endothelium. Thus 2’-FL protects against NEC, in part through maintaining mesenteric perfusion via increased eNOS expression. This suggests that 2’-FL found in human milk may mediate some of the protective benefits of breast milk against more severe NEC or recovery from NEC.
However, providing 2’-FL has not shown effectiveness in all preclinical models. Cilieborg et al. tested 5 g/L of 2’-FL in a neonatal preterm pig model of NEC. They found no significant difference in NEC lesion scores between pigs given 2’-FL-fortified formula versus regular formula. Although the 2’-FL–fed pigs tended to have less anaerobic bacteria in cecal contents, no significant difference between groups was found in gut microbiota, as measured by fluorescence in situ hybridization and 454 pyrosequencing.
Taken as a whole, the evidence for hMOS protection against NEC is compelling. Human milk trials indicate protection by human milk feeding against NEC, and preclinical studies and one epidemiologic study have indicated protection by one or more hMOSs. To date, DSLNT appears to be the strongest candidate oligosaccharide for reduction of NEC risk in animal models, with some evidence that 2’-FL could contribute to reducing NEC severity through restoration of intestinal perfusion and potentially other mechanisms. Although additional clinical epidemiology of DSLNT as a predictor of NEC risk should be conducted, the depth of evidence to date is such that testing DSLNT as a protective molecule in clinical trials may be warranted. However, few hMOSs are currently available for clinical testing, as noted at the end of this chapter. Thus additional effort is needed toward the synthesis and commercial manufacturing of a larger repertoire of hMOSs for clinical testing and use.
Normal intestinal adaptation occurs between intrauterine life and extrauterine life, and is related to gut glycosylation. hMOS may support intestinal adaptation under circumstances of intestinal failure and reduce the burden of extrauterine growth retardation associated with intestinal failure.
At birth, the gut mucosa of mice is heavily sialylated but becomes heavily fucosylated at weaning, with a decrease in sialylation. Germ-free mice do not develop highly fucosylated mucosa, but fucosylation is rapidly induced by bacterial colonization. Likewise, minimizing the colonization of conventional mice by treatment with a cocktail of antibiotics reduces gut fucosylation while depleting the microbiota restores gut fucosylation, fut2 mRNA, and fucosyl transferase enzyme. TLR4 is essential for transcellular signaling that leads to fucosylation. TLR4 and MyD88 knockout mice are unable to induce fut2 upon colonization. Extracellular signal-regulated kinase (ERK) and Jun kinase (JNK) signal transduction pathways downstream from TLR4 and MyG88 are activated by restitution of bacterial colonization. Drugs that specifically inhibit ERK and JNK activation strongly inhibit colonization-dependent fut2 induction, confirming the direct involvement of these pathways in signaling. The nuclear transcription factors activated by the ERK and JNK pathways, activating transcriptor factor-2 and c-jun, together activate genetic control element AP1 domains, two of which are contained in the control region of the fut2 gene. Goto et al. demonstrated that fucosylation is induced by type 3 innate lymphoid cells via IL-22 signaling in a bacteria-dependent manner. The ability of microbes to induce fucosylation in their host to create a more favorable niche without inducing an inflammatory response is a clear form of mutualism: The bacteria benefit from fucosylation, and the host benefits from the presence of mutualist bacteria.
The benefit of gut glycosylation to the host was tested in a mouse model of DSS-induced colitis. Conventionally colonized mice with highly fucosylated mucosa successfully recovered from mucosal injury, but with minimally fucosylated mucosa as a result of bacterial depletion by antibiotics, recovery was impaired. Replacement of the bacterial community, or just monocolonization with fucose-utilizing Bacteriodes fragilis , induced mucosal fucosylation and supported recovery from injury. In contrast, colonization by a mutant B. fragilis unable to utilize fucose did not promote recovery. Thus colonization, signaling, and fucosylation central to this type of mutualism are intrinsic to resilience and return to homeostasis by the gut.
The previous data suggest that through the provision of hMOS, feeding human milk could enhance the availability of oligosaccharides to the growing infant gut, and augment the capacity of the gut to adapt and recover from insult. This view is supported by three preclinical studies conducted by different teams studying different forms of enteric insult or injury. In a mouse model of NEC, described previously, Good et al. demonstrated that mouse pups that developed NEC grew better and had less severe disease if concurrently fed 2’-FL. The effect was attributed to upregulation of eNOS and restoration of intestinal perfusion. In a mouse model of DSS-induced colitis, Weiss et al. tested two hMOSs–2’-FL and 3-FL. In mice fed 2’-FL, recovery from DSS challenge was faster compared with controls: mice fed 2’-FL recovered weight more quickly, had lower postchallenge fecal calprotectin levels, and had a more diverse microbial community with increased abundance of Barnesiella organisms that can metabolize 2’-FL. In a mouse model of intestinal adaptation following ileocecal resection (ICR), Mezoff et al. tested 2’-FL in half the mice, which were supplemented postoperatively compared with mice given their regular feeds. All ICR mice steadily increased weight gain over the postoperative study period of 56 days. But the 2’-FL-supplemented mice had a significantly greater weight recovery after 21 days, a greater crypt depth at 56 days compared with control ICR mice, increased microbial alpha diversity measured from small bowel luminal content following resection, and a bloom in organisms of the genus Parabacteroides (this organism is crypt-dwelling and can metabolize 2’-FL). Transcriptional analysis of the intestine revealed enriched ontologies and pathways related to antimicrobial peptides, metabolism, and energy processing. Thus 2’-FL supplementation assisted recovery and intestinal adaptation after several different forms of gut injury or insult.
The gut microbiota–brain axis has become a major scientific focus because of the recent observations that the gut microbiota influence neurobehavior, including anxiety, depression, and aggressive behavior. Gut microbes may influence neurodevelopment by several different mechanisms: by modifying inflammation or the immune system, via gut metabolites absorbed into circulation, or via the enteric nervous system.
An increasingly compelling body of evidence links human milk feeding to improved neurodevelopment. Much of the effect of human milk on neurodevelopment may be attributable to hMOS. Historically, most studies of hMOS and neurodevelopment have focused on the impact of sialylated hMOS for enrichment of sialic acid in gangliosides, which constitute a critical component of the nervous system. Several recent in vivo studies have tested two dominant forms of sialyllactose, 6’-SL and 3’-SL, for their impact on neurobehavior. Mice fed for 2 weeks were then exposed to a social disruption stressor. Exposure to the stressor significantly changed gut microbial composition and resulted in anxiety-like behavior under controlled conditions. But in mice fed hMOS, the stressor exposure did not significantly change microbial community structure. Further, 3’SL and 6’SL helped maintain normal behavior on tests of anxiety. In another study, the two sialyllactose isomers were studied to determine the impact of ingestion of these hMOS on brain sialic acid content, and on modulation of the microbiome of neonatal piglets, which were randomly allocated to the following diets for 21 days: control, 2 or 4 g 3’-SL/L, 2 or 4 g 6’-SL/L, or 2 g polydextrose and 2 g GOS/L). Dietary sialyllactose did not affect feed intake, growth, or fecal consistency. Ganglioside-bound sialic acid in the corpus callosum of pigs fed 2 g 3’-SL or 6’-SL/L increased by 15% in comparison with control pigs. Similarly, ganglioside-bound SA in the cerebellum of pigs fed 4 g 3’-SL/L increased by 10% in comparison with control pigs. Significant microbiome differences were observed in the proximal and distal colons of piglets fed control formula compared with those fed sialyllactose. Thus supplementation of formula with 3’-SL or 6’-SL can enrich ganglioside sialic acid in the brain and modulate gut-associated microbiota in neonatal pigs.
In addition, recent studies of 2’-FL have indicated that this hMOS may have a distinct role in neurodevelopment. In a rodent model, Vazquez et al. showed that ingested 2’-FL enhances learning, memory, and hippocampal long-term potentiation and impacts a variety of brain molecular markers. A subsequent study using the same model aimed to determine whether oral 2’-FL has an effect on the development of newborn brain and cognitive skills later in life. Rat pups received an oral supplementation of 2’-FL or water during the lactation period. 2’-FL fed rats performed significantly better in learning tasks compared with controls. Long-term potentiation was more intense and longer lasting in the rats supplemented with 2’-FL than in control animals, both in young and adult animals. Oral administration of 2’-FL exclusively during lactation also enhanced cognitive abilities in adulthood. These findings indicate the impact of 2’-FL on brain development. The influence of 2’-FL on neurobehavior could be direct or through indirect mechanisms, such as via gut microbial composition and metabolism.
2’-FL can also affect neuronally dependent gut motor function. Using a standard ex vivo colonic preparation, Bienenstock et al. examined the acute effects of a variety of fucosylated and sialylated hMOSs, and determined that only 2’-FL and a related oligosaccharide, 3-FL, decreased colonic smooth muscle contractions. Specifically, 2’-FL and 3-FL reduced the amplitude, velocity and frequency of migrating motor complexes within 10 to 15 minutes after administration, whereas other hMOS molecules, GOS, and lactose elicited no response. These data suggest that 2’-FL and 3-FL contribute to functional regulation of gut motility, which may be especially important to the healthy gut maturation and function of preterm infants.
Randomized Controlled Trials in Healthy, Term Infants
Human milk contains a higher concentration and a greater structural diversity of oligosaccharides than are found in the milk of many other species. Bovine milk is the source from which most infant formulas are produced. Bovine colostrum contains low levels of acidic oligosaccharides, and bovine milk is particularly lacking in fucosylated oligosaccharide. Thus manufacturers of formula milk have considerable interest in advancing infant feeding through the addition of hMOS to infant formula.
To date, several RCTs have reported on hMOS added to infant formula. The first such trial was a growth and tolerance study conducted in 424 formula-fed healthy, singleton infants born at term in the United States. Infants were enrolled by day 5 of life and followed up for 4 months. All were fed a standard formula containing a total oligosaccharide quantity of 2.4 g/L. The three formula study groups each tested a specific combination of 2’-FL and GOS, respectively: group 1—0 g/L and 2.4 g/L; group 2—0.2 g/L and 2.2 g/L; and group 3—1.0 g/L and 1.4 g/L. The trial also included a human milk–fed reference group. In the formula groups, each study dose combination was well tolerated over the 4-month study period. Study groups did not differ in weight, length, head circumference growth, average stool consistency, number of stools per day, or the occurrence of reflux. In formula-fed infants, similar to breastfed infants, 2’-FL was detected in plasma and urine samples. A substudy was conducted for analysis of blood samples drawn at 6 weeks of age. In the plasma samples analyzed, breastfed infants and infants fed one of the formulas with 2’-FL did not differ from one another, but they had significantly lower concentrations of inflammatory cytokines compared with the control group fed regular formula. In ex vivo respiratory syncytial virus-stimulated peripheral blood mononuclear cell cultures, breastfed infants and infants fed one of the formulas with 2’-FL again did not differ from one another but had lower concentrations of inflammatory cytokines compared with infants fed the control formula.
A separate RCT was conducted in 175 formula-fed European infants to compare the effects of infant formula supplemented with two different hMOSs on infant growth, tolerance, and morbidity. Healthy infants, 0 to 14 days old, were randomized to an intact-protein, cow’s milk–based infant formula or the same formula with addition of 1.0 g/L 2’-FL and 0.5g/L LNnT, which were fed for the first 6 months of life. All study infants received standard formula without hMOS from 6 to 12 months of life. Weight gain did not differ between study groups. Digestive symptoms and behavioral patterns were also typically similar between groups, but the intervention groups had softer stool ( P = .021) and fewer nighttime wake-ups at 2 months. Infants receiving test (versus control) had significantly fewer parental reports of bronchitis, lower rates of respiratory tract infections, and less antibiotic use through 12 months of age (42% versus 60.9%).
These trials of hMOS added to infant formula represent a promising step in narrowing the compositional gap between human milk and infant formula. But it is unclear whether the addition of only one or two hMOSs found in breast milk will recapitulate the complexity of actions exerted by the complex mixture of hMOS ingested by breastfed infants. Thus as more individual hMOS molecules become commercially available, we anticipate that more oligosaccharides will be added to infant formula as mixtures, either hMOS alone or in combination with other prebiotics.