Throughout history, physicians have noted that infants that are not breastfed had a greater incidence of disease relative to those that are breastfed. For example, a study in Chicago in the 1920s of more than 20,000 mother-infant dyads reported that infants that were not breastfed had 7-fold, 2-fold, and 4-fold greater mortality caused by gastrointestinal, respiratory, and other diseases, respectively (Grulee et al., 1934). Protection by breastfeeding had been attributed to its nutritional suitability for infants, but more specifically, to human milk components classified into 3 major functions: prebiotics, secretory antibodies, and multifunctional agents.
Most of the known prebiotics are glycans. A glycan is an oligosaccharide or other glycoconjugate, such as glycoprotein, starch, cellulose, glycolipid, glycosaminoglycan, mucin, or other structural carbohydrate. A prebiotic is a food component, generally a glycan that is not digestible by an animal, that confers a health benefit to the host through modification of the gut microflora, presumably by functioning as a food source for the microbiota (Macfarlane and Macfarlane, 1997). The microbiota of breastfed babies were noted to have a preponderance of Bifidobacterium bifidum and lactobacillae, in contrast to infants who were not breastfed, whose microbiota had a composition more typical of adults (Tissier, 1905). This causes the gut contents of breastfed infants to be more acidic, which can inhibit colonization by many pathogens. Human milk is rich in glycans, and a glycan was isolated from human milk that stimulated the growth of B. bifidum (Gyorgy et al., 1974).
Human milk is rich in secretory IgA (Hanson, 1961). When a lactating mother is exposed to an enteric pathogen, her mucosal surveillance system (Peyer’s patches and dendritic cells) samples its antigens, which, when presented to B cells, result in antibody production. As antibodies traverse the mammary epithelial cell, they form the secretory IgA complex that is secreted into milk. When consumed, the secretory IgA is resistant to digestion in the gut of the infant, where it binds to the enteric pathogen, thereby protecting the breastfed infant. This had been considered the major protective component in human milk for some time (Hanson et al., 1991).
Milk macronutrients, especially their partial digestion products, can also play protective roles (Newburg, 2005). These multifunctional components include peptide partial digestion products of milk proteins, such as lactoferrin and its digestion product, lactoferricin. Free fatty acids and monoglycerides result from partial digestion of human milk triglycerides. At concentrations found in stomach contents of breastfed infants, FFA and monoglycerides destroy enveloped viruses, such as vesicular stomatitis virus.
In this review, we argue that the milk glycans, including oligosaccharides, in addition to being prebiotic, have another important protective function. Milk glycans include components that directly protect the young mammal from pathogens. Because of their powerful protective activity, these specific glycans should be added to this list of protective factors in human milk (Morrow et al., 2005). In aggregate, all of these protective components exhibit activities that constitute an innate immune system of milk, whereby the mother protects her immature infant from the common pathogens to which it is exposed.
CELL SURFACE GLYCANS ARE THE BINDING TARGETS OF MANY ENTEROPATHOGENS
The intestinal mucosa is among the most heavily glycosylated tissues in the body. The cells that line the lumen of the gut are covered with glycoproteins, glycolipids, mucins, glycosaminoglycans, and other glycans (Newburg and Walker, 2007). Major functions of the cell surface glycans include mediating communication with the extracellular environment, including cell-cell communication, and binding to hormones and other signaling agents. Many enteric pathogens use specific cell surface glycan epitopes of the intestinal epithelial cells as their target for binding, which is the first obligatory step in their pathogenesis. The ability of a host cell to express specific glycan epitopes defines the organ and species specificity of each pathogen. If a soluble glycan were to have this epitope as part of its structure, it could inhibit the ability of a pathogen to dock to its receptor, and thereby spare the target cell from infection. We hypothesized that human milk glycans could contain such epitopes, and thereby protect the breast-fed infant against enteric pathogens (Newburg, 1999).
HUMAN MILK OLIGOSACCHARIDES INCLUDE STRUCTURES THAT MAY PROTECT AGAINST PATHOGENS
After lactose, the next most predominant glycans in human milk are the oligosaccharides. The oligosaccharide fraction is the third largest solid component in milk. Oligosaccharides are indigestible by the infant gut and therefore are not used as a nutrient. Because oligosaccharides are approximately 1% of human milk (Newburg and Neubauer, 1995), they represent on the order of 10% of the caloric content of human milk. This indicates important functions other than nutritional, such as protection of the infant against disease. The human milk oligosaccharides, as shown in the examples in Figure 1, have a lactose on the reducing end, have a core structure of polylactosamine (galactose-N-acetyl-glucosamine), and usually contain fucose and some sialic acid at the nonreducing terminus (Newburg and Neubauer, 1995). In contrast, bovine milk has an order of magnitude less concentration of oligosaccharides, mostly in colostrum, and the oligosaccharides are mainly sialylated. As is the nature of all glycans, a multiplicity of possible linkages between its component sugars and the multiplicity of possible sugars combine to create a vast number of potential structures and structural isomers. When the neutral human milk oligosaccharide fraction was analyzed by mass spectrometry, the molecular weights (m/z ratios) indicated structures of up to 32 sugars containing up to 15 fucoses (Stahl et al., 1991). The structural isomerism seen in the known human milk oligosaccharides indicated that these molecular weights represented many thousands of structures. Moreover, the terminal sugars of the known human milk oligosaccharides are homologs of the Lewis blood group structures, fucosylated glycan moieties with high biological activity in many important systems related to development and immune functions (Stahl et al., 2001). Therefore, it seemed likely that the vast number of potential structures in the human milk oligosaccharide fraction could contain components with structural homology to cell surface glycans, and, considering the elevated concentration of this fraction, would therefore be able to inhibit binding of pathogens to their receptors in the mucosa and protect the infant from disease. This hypothesis was tested in models of infection by human pathogens.
STABLE TOXIN OF ESCHERICHIA COLI IS INHIBITED BY A HUMAN MILK OLIGOSACCHARIDE
Enterotoxigenic E. coli is a major cause of diarrhea among travelers in Central America, the Middle East, and southern Asia. Its pathogenesis is associated with 2 toxins, labile toxin and stable toxin (STa). Labile toxin is homologous with cholera toxin; STa is an 18-AA peptide containing 3 disulfide bonds, making the molecule stable to heat and organic solvents. Stable toxin inhibits chloride transport from the gut to the intestinal epithelial cell, thereby inhibiting electrolyte and water resorption from the gut, resulting in secretory diarrhea in susceptible humans, including travelers and infants. An animal model for STa pathology is the suckling mouse, which is sensitive to this toxin between 2 and 4 d of life. Mice fed the toxin develop secretory diarrhea, which is lethal within several hours. Mice fed STa along with human milk have a significantly reduced rate of death (Cleary et al., 1983; Newburg et al., 1990). The only fraction of human milk (when tested at its concentration in milk) that prevented STa-induced death in mice was the oligosaccharide fraction, and it was the neutral, but not the acidic, fraction that conferred protection (Newburg et al., 1990). The active oligosaccharide was purified from the native oligosaccharide mixture, and was found to be a large oligosaccharide containing α1,2-linked fucose. It was active at its original concentration found in milk, at approximately 30 μg/L. Thus, one of the minor oligosaccharides had a potent ability to protect infants against a human pathogen.
CAMPYLOBACTER PYLORI BINDING IS INHIBITED BY 2′-FUCOSYLLACTOSE
Campylobacter pylori infection is the major cause of bacterial diarrhea in humans and is also associated with diarrhea in farm animals, especially birds. Thus, in settings where humans live in proximity to domestic fowl, children have a high rate of campylobacter-associated diarrhea. Breastfed infants have a reduced rate of diarrhea compared with those not breastfed. To investigate whether human milk glycans inhibit campylobacter, the binding specificity of campylobacter was investigated (Ruiz-Palacios et al., 1990). Campylobacter binds to the H-2 epitope (Fucα1,2Galβ1,4GlcNAc) expressed on the surface of mammalian cells in vitro, and to human intestinal epithelium. This binding is inhibited by human milk oligosaccharides and, specifically, by 2′-fucosyllactose (2′-FL), the major oligosaccharide in most human milk, and whose structure is an H-2 analog because it includes the Fucα1,2Gal moiety. To confirm that 2′-FL could inhibit campylobacter infection in vivo, a model was developed for the overexpression of H-2 in animal milk, which is normally low in H-2. Mice were transfected with the human fucosyltransferase I gene controlled by the whey acidic protein promoter to cause the gene to be expressed only in the lactating mammary gland. Thus, transfected dams produced increased H-2 epitope in their milk, whereas the milk of wild-type (i.e., nontransfected) dams did not. Suckling mice were infected with campylobacter, and those nursing the wild-type dams remained infected throughout the 15 d of the experiment (Table 1). In contrast, infant mice infected with campylobacter and nursing the transfected dams cleared the infection, even at increased inocula of campylobacter. This demonstrates that the human milk oligosaccharides are capable of inhibiting binding by campylobacter to its host cell glycans, and that this binding inhibition is capable of protecting mammals from enteric infection by virulent strains of campylobacter.
The ability of Fucα1,2 glycans to protect against multiple pathogens leads to the hypothesis that the human milk glycans could be an important component of an innate immune system of human milk (Newburg et al., 2004). This hypothesis generates 3 testable postulates: 1) that the glycans are constitutively produced; 2) that their expression varies by maternal genotype, and 3) that the human milk glycans protect human infants from human pathogens.
EXPRESSION OF GLYCANS IN HUMAN MILK VARIES BY MATERNAL GENOTYPE
The constitutive expression of human milk glycans is apparent by the continuity of expression of individual glycans over the course of lactation, which seems independent of environmental influences but that depends on the stage of lactation (Chaturvedi et al., 2001). The dependence of lactation on maternal genotype is apparent in secretor and nonsecretor mothers. Secretors have an active gene for fucosyltransferase II, whereas nonsecretors are homozygous recessive for this gene. Therefore, nonsecretors do not express normal fucosyltransferase II, and thus are unable to secrete α1,2-linked glycans into their secretions. This results in pronounced differences in the relative expression of α1,2-linked fucose to α3,4-linked fucose in the milk of secretors and nonsecretors. Lewis blood group types represent the interactions of the FUC2 (expresses fucosyltransferase II, which catalyzes the formation of α1,2-linked fucose) and FUC3 (expresses fucosyltransferase III, which catalyzes the formation of α1,3/4-linked fucose). Even among secretors, the 2 remaining secretor Lewis blood group types secrete different concentrations of α1,2-linked fucose in their milk. This reinforces the dependence of milk oligosaccharide expression on the genotype of the mother (Newburg et al., 2004). This variation in expression of α1,2-linked fucose in milk also provides a means to test whether human milk glycans protect human infants from human pathogens.
FUCOSYLATED GLYCANS IN HUMAN MILK PROTECT INFANTS
The individual variation in expression of glycans containing α1,2-linked fucose provides the opportunity to test the relationship between glycan expression in the milk of individuals and disease outcome in their infants. In a cohort of breastfeeding dyads, the relationship between concentrations of oligosaccharides in milk samples and disease outcomes in the breastfeeding infants consuming the milk was tested (Morrow et al., 2004).
The in vivo laboratory results discussed above predict that milk oligosaccharides should protect against the diarrheagenic activity of STa of E. coli without influencing E. coli colonization. Consistent with this prediction, Figure 2 shows that those breastfeeding infants who were positive for E. coli in their stools and who exhibited symptoms of diarrhea were consuming milk with significantly lower ratios of fucosylated to nonfucosylated oligosaccharides compared with infants who were colonized but did not exhibit symptoms of diarrhea. This implies that the fucosylated glycans in human milk protect against STa-E. coli diarrhea by inhibiting toxin.
In vitro, the fucosylated human milk oligosaccharide 2′-FL inhibited campylobacter binding to its receptors, and in vivo 2′-FL inhibited campylobacter infection. Consistent with these findings, the risk of campylobacter diarrhea in breastfed infants was inversely related to the concentrations of 2′-FL in milk samples, indicating that 2′-FL in milk protects against campylobacter (Figure 3; Morrow et al., 2004). Another fucosylated milk oligosaccharide is lactodifucohexaose-1 (LDFH-I), which contains a Lewis b moiety that binds to norovirus in vitro. The concentrations of LDFH-I in milk are inversely related to the risk of norovirus-associated diarrhea in breastfeeding infants, indicating that LDFH-I in milk protects breastfed infants from norovirus-induced diarrhea. Thus, specific glycans in human milk can protect against specific pathogens.
The composite total concentration of all α1,2-linked fucosyloligosaccharides in milk samples was a strong predictor of diarrhea of all causes; the total fucosyloligosaccharide concentrations were inversely related to risk of diarrhea in breastfed infants. These results lead to the conclusion that the human milk α1,2-linked fucosyloligosaccharides, by binding to specific pathogens, prevent these pathogens from binding to their host cell receptors in the intestinal mucosa, thereby protecting the infant from developing moderate to severe diarrhea.
SIALYL GLYCANS OF HUMAN MILK PROTECT AGAINST ROTAVIRUS
Protection of nursing infants by human milk glycans extends beyond the α1,2 fucosyloligosaccharides, as exemplified by the glycoprotein lactadherin. This 46-kDa glycoprotein is associated with the mucin on the milk fat globule membrane, contains sialic acid, and binds to rotavirus (Yolken et al., 1992). Rotavirus is the single largest cause of diarrhea in humans, is a major cause of diarrhea in infants, and is also a major pathogen in farm animals (Nakagomi and Nakagomi, 2002; Lepage and Vergison, 2007). A common model for infection by human rotavirus is MA104 cells, an immortal line of green monkey kidney cells, which form plaques after inoculation with rotavirus. Lactadherin inhibits the infection of MA104 cells by rotavirus in vitro (Figure 4). The inhibition is dose dependent. The inhibition is lost when the sialic acid is removed from the molecule, indicating that the glycans of the molecule, especially moieties containing sialic acid, are necessary for inhibition (Yolken et al., 1992). In breastfed infants who exhibited rotavirus in their feces (Figure 5), those who exhibited symptoms of diarrhea were consuming milk that was reduced (P = 0.05) in lactadherin concentrations compared with those who were asymptomatic (Newburg et al., 1998). Thus, this acidic glycoprotein in milk protects against rotavirus, indicating that many classes of human milk glycans have the potential to protect against pathogens.
GLYCANS INHIBIT RESPIRATORY DISEASE
Along with reports of less risk of diarrhea in breast-fed infants are reports of less risk of respiratory infection. We tested whether human milk glycans might also contribute to protection of the respiratory tract. The concentration of lacto-N-fucopentaose II (LNF-II) was measured in milk samples from wk 2 of lactation as a representative glycan. The cumulative risk of respiratory disease at wk 6 and 12 was measured, and high concentrations of lacto-N-fucopentaose II in milk were significantly related to decreased risk of respiratory disease in breastfed infants (Stepans et al., 2006). Thus, the protection afforded by human milk oligosaccharides to the breastfed infant is not limited to diarrhea; protection of the respiratory tract indicates that protection by human milk glycans may extend to other mucosal tissues.
SUMMARY AND CONCLUSIONS
The human milk glycans that we had shown to inhibit common human pathogens and have discussed in this review, including STa of E. coli, C. pylori, rotavirus, and respiratory pathogens, are representative of a growing number of human milk glycans that have been identified as having biological activities (Table 2). These bio-active glycans, each of which may inhibit 1 pathogen or 1 family of pathogens, collectively inhibit a wide array of pathogens. Therefore, in aggregate, the human milk glycans should be considered a major component of a human milk immunological defense system.
The benefits of human milk go beyond provision of a balanced source of nutrients that meet infant dietary requirements. The proposed innate immune system of human milk consists of several components. Human milk secretory IgA, a product of the adaptive immune system, and leukocytes are already recognized as having potential protective functions. Human milk also contains multifunctional components, molecules that are a source of nutrients after digestion, but whose partial digestion products or original structures also have protective biological functions. Examples of multifunctional components include lactoferrin, triglycerides, α-lactalbumin, and peptides released from milk proteins during digestion. Immunomodulatory components of milk include cytokines, chemokines, soluble cytokine receptors, nucleic acids, and other agonists and antagonists of mucosal signaling molecules. Human milk glycans are a principal part of this proposed innate immune system of human milk; some glycans inhibit pathogen binding, modulate mucosal functions, and act as prebiotics to promote the development and maintenance of healthy intestinal microbiota (Newburg and Walker, 2007).
Milk of other species also contains glycans, some in appreciable quantities. These glycans can be analogous, but not identical to, the human glycans, and presumably protect the young of those species from pathogens. The further study of the milk glycans seems a promising approach toward developing novel agents for the prevention and treatment of enteric and respiratory diseases.
|Oligosaccharides||Streptococcus pneumoniae||Andersson et al. (1986)|
|Enteropathogenic Escherichia coli (EPEC)||Cravioto et al. (1991)|
|Listeria monocytogenes||Coppa et al. (2003)|
|Fucosylated oligosaccharides||Campylobacter jejuni||Ruiz-Palacios et al. (2003)|
|Vibrio cholerae||Ruiz-Palacios et al. (2003)|
|Stable toxin||Newburg et al. (1990)|
|Macromolecule-associated glycans||Noroviruses, Pseudomonas aeruginosa||Jiang et al. (2004); Lesman-Movshovich et al. (2003)|
|Sialyllactose||Cholera toxin||Idota et al. (1995)|
|E. coli||Stins et al. (1994); Virkola et al. (1993)|
|P. aeruginosa||Devaraj et al. (1994)|
|Aspergillus fumigatus conidia||Bouchara et al. (1997)|
|Influenza virus||Gambaryan et al. (1997); Matrosovich et al. (1993)|
|Polyomavirus||Stehle et al. (1994)|
|Helicobacter pylori||Mysore et al. (1999)|
|Mannosylated glycopeptide||Enterohemorrhagic E. coli (EHEC)||Ashkenazi et al. (1991)|
|Mucin||S-fimbriated E. coli||Schroten et al. (1992)|
|Chondroitin sulfate||Human immunodeficiency virus (HIV)||Newburg et al. (1995)|
|Sulfatide||HIV||Viveros-Rogel et al. (2004)|
|Gb3||Shiga toxin||Newburg et al. (1992)|
|GM1||Labile toxin, cholera toxin||Otnaess et al. (1983)|
|GM3||EPEC||Idota and Kawakami (1995)|