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Human Milk Hyaluronan Enhances Innate Defense of the Intestinal Epithelium*

Open AccessPublished:August 15, 2013DOI:https://doi.org/10.1074/jbc.M113.468629

      Background:

      Human milk contains hyaluronan (HA).
      Results: Milk HA concentration is highest immediately after delivery. Treatment of epithelium with physiologic levels of milk-derived HA increases intracellular expression of β-defensin (in vitro and in vivo) and resistance to Salmonella.
      Conclusion: Milk HA enhances functional antimicrobial defense mechanisms of the intestinal epithelium.
      Significance: Milk HA may be a mediator of maternal protection of newborns.
      Breast-feeding is associated with enhanced protection from gastrointestinal disease in infants, mediated in part by an array of bioactive glycan components in milk that act through molecular mechanisms to inhibit enteric pathogen infection. Human milk contains hyaluronan (HA), a glycosaminoglycan polymer found in virtually all mammalian tissues. We have shown that synthetic HA of a specific size range promotes expression of antimicrobial peptides in intestinal epithelium. We hypothesize that hyaluronan from human milk also enhances innate antimicrobial defense. Here we define the concentration of HA in human milk during the first 6 months postpartum. Importantly, HA isolated from milk has a biological function. Treatment of HT-29 colonic epithelial cells with human milk HA at physiologic concentrations results in time- and dose-dependent induction of the antimicrobial peptide human β-defensin 2 and is abrogated by digestion of milk HA with a specific hyaluronidase. Milk HA induction of human β-defensin 2 expression is also reduced in the presence of a CD44-blocking antibody and is associated with a specific increase in ERK1/2 phosphorylation, suggesting a role for the HA receptor CD44. Furthermore, oral administration of human milk-derived HA to adult, wild-type mice results in induction of the murine Hβ D2 ortholog in intestinal mucosa and is dependent upon both TLR4 and CD44 in vivo. Finally, treatment of cultured colonic epithelial cells with human milk HA enhances resistance to infection by the enteric pathogen Salmonella typhimurium. Together, our observations suggest that maternally provided HA stimulates protective antimicrobial defense in the newborn.

      Introduction

      Numerous positive health outcomes are associated with breast-feeding in infants (
      • Howie P.W.
      • Forsyth J.S.
      • Ogston S.A.
      • Clark A.
      • Florey C.D.
      Protective effect of breast feeding against infection.
      ,
      • Klement E.
      • Cohen R.V.
      • Boxman J.
      • Joseph A.
      • Reif S.
      Breastfeeding and risk of inflammatory bowel disease. A systematic review with meta-analysis.
      ,
      • Anderson J.W.
      • Johnstone B.M.
      • Remley D.T.
      Breast-feeding and cognitive development. A meta-analysis.
      ,
      • Newburg D.S.
      • Walker W.A.
      Protection of the neonate by the innate immune system of developing gut and of human milk.
      ), particularly regarding gastrointestinal infection. Grulee et al. (
      • Grulee C.G.
      • Sanford H.N.
      • Herron P.H.
      Breast and artificial feeding. Influence on morbidity and mortality of twenty thousand infants.
      ) conducted the first major evaluation of morbidity and mortality among 20,061 breast-fed and artificially fed infants in 1934, reporting as much as 50% reduction in gastrointestinal infection incidence among breast-fed infants. Modern epidemiologic studies reinforced and expanded upon these findings (
      • Howie P.W.
      • Forsyth J.S.
      • Ogston S.A.
      • Clark A.
      • Florey C.D.
      Protective effect of breast feeding against infection.
      ,
      • Morrow A.L.
      • Guerrero M.L.
      • Shults J.
      • Calva J.J.
      • Lutter C.
      • Bravo J.
      • Ruiz-Palacios G.
      • Morrow R.C.
      • Butterfoss F.D.
      Efficacy of home-based peer counselling to promote exclusive breastfeeding. A randomised controlled trial.
      ), indicating that breast-feeding confers remarkably enhanced protection from both gastrointestinal and respiratory infections, including Salmonella infection (
      • Jones T.F.
      • Ingram L.A.
      • Fullerton K.E.
      • Marcus R.
      • Anderson B.J.
      • McCarthy P.V.
      • Vugia D.
      • Shiferaw B.
      • Haubert N.
      • Wedel S.
      • Angulo F.J.
      A case-control study of the epidemiology of sporadic Salmonella infection in infants.
      ).
      In addition to nutrients, breast-feeding supplies a wide array of bioactive components that enhance both innate and adaptive immunity in the neonatal gastrointestinal tract. Milk components act as critical stimuli in the ontology of intestinal immune education and microflora development (
      • Newburg D.S.
      • Walker W.A.
      Protection of the neonate by the innate immune system of developing gut and of human milk.
      ), supplying passive defense mediators (
      • Ward P.P.
      • Uribe-Luna S.
      • Conneely O.M.
      Lactoferrin and host defense.
      ,
      • Brandtzaeg P.
      Mucosal immunity. Integration between mother and the breast-fed infant.
      ), growth hormones (
      • Martin L.J.
      • Woo J.G.
      • Geraghty S.R.
      • Altaye M.
      • Davidson B.S.
      • Banach W.
      • Dolan L.M.
      • Ruiz-Palacios G.M.
      • Morrow A.L.
      Adiponectin is present in human milk and is associated with maternal factors.
      ), prebiotics (
      • Asakuma S.
      • Hatakeyama E.
      • Urashima T.
      • Yoshida E.
      • Katayama T.
      • Yamamoto K.
      • Kumagai H.
      • Ashida H.
      • Hirose J.
      • Kitaoka M.
      Physiology of consumption of human milk oligosaccharides by infant gut-associated bifidobacteria.
      ,
      • Marcobal A.
      • Barboza M.
      • Sonnenburg E.D.
      • Pudlo N.
      • Martens E.C.
      • Desai P.
      • Lebrilla C.B.
      • Weimer B.C.
      • Mills D.A.
      • German J.B.
      • Sonnenburg J.L.
      Bacteroides in the infant gut consume milk oligosaccharides via mucus-utilization pathways.
      ,
      • Sela D.A.
      • Li Y.
      • Lerno L.
      • Wu S.
      • Marcobal A.M.
      • German J.B.
      • Chen X.
      • Lebrilla C.B.
      • Mills D.A.
      An infant-associated bacterial commensal utilizes breast milk sialyloligosaccharides.
      ,
      • Yu Z.-T.
      • Chen C.
      • Kling D.E.
      • Liu B.
      • McCoy J.M.
      • Merighi M.
      • Heidtman M.
      • Newburg D.S.
      The principal fucosylated oligosaccharides of human milk exhibit prebiotic properties on cultured infant microbiota.
      ), and immunomodulators (
      • M'Rabet L.
      • Vos A.P.
      • Boehm G.
      • Garssen J.
      Breast-feeding and its role in early development of the immune system in infants. Consequences for health later in life.
      ).
      The best characterized protective milk component is soluble IgA (
      • M'Rabet L.
      • Vos A.P.
      • Boehm G.
      • Garssen J.
      Breast-feeding and its role in early development of the immune system in infants. Consequences for health later in life.
      ). However, milk also contains an abundant and extraordinarily diverse array of glycans, including oligosaccharides, glycolipids, glycoproteins, mucins, glycosaminoglycans, and other complex carbohydrates, which provide infant protection (
      • Newburg D.S.
      • Walker W.A.
      Protection of the neonate by the innate immune system of developing gut and of human milk.
      ,
      • Newburg D.S.
      Neonatal protection by an innate immune system of human milk consisting of oligosaccharides and glycans.
      ,
      • Bode L.
      Human milk oligosaccharides. Every baby needs a sugar mama.
      ). The ways in which human milk glycans shape innate gastrointestinal defense are diverse (
      • Newburg D.S.
      Neonatal protection by an innate immune system of human milk consisting of oligosaccharides and glycans.
      ,
      • Bode L.
      Human milk oligosaccharides. Every baby needs a sugar mama.
      ), and include prebiotic function (
      • Asakuma S.
      • Hatakeyama E.
      • Urashima T.
      • Yoshida E.
      • Katayama T.
      • Yamamoto K.
      • Kumagai H.
      • Ashida H.
      • Hirose J.
      • Kitaoka M.
      Physiology of consumption of human milk oligosaccharides by infant gut-associated bifidobacteria.
      ,
      • Marcobal A.
      • Barboza M.
      • Sonnenburg E.D.
      • Pudlo N.
      • Martens E.C.
      • Desai P.
      • Lebrilla C.B.
      • Weimer B.C.
      • Mills D.A.
      • German J.B.
      • Sonnenburg J.L.
      Bacteroides in the infant gut consume milk oligosaccharides via mucus-utilization pathways.
      ,
      • Sela D.A.
      • Li Y.
      • Lerno L.
      • Wu S.
      • Marcobal A.M.
      • German J.B.
      • Chen X.
      • Lebrilla C.B.
      • Mills D.A.
      An infant-associated bacterial commensal utilizes breast milk sialyloligosaccharides.
      ,
      • Yu Z.-T.
      • Chen C.
      • Kling D.E.
      • Liu B.
      • McCoy J.M.
      • Merighi M.
      • Heidtman M.
      • Newburg D.S.
      The principal fucosylated oligosaccharides of human milk exhibit prebiotic properties on cultured infant microbiota.
      ), antiadhesive antimicrobial activity (
      • Ruiz-Palacios G.M.
      • Cervantes L.E.
      • Ramos P.
      • Chavez-Munguia B.
      • Newburg D.S.
      Campylobacter jejuni binds intestinal H(O) antigen (Fucα1, 2Galβ1, 4GlcNAc), and fucosyloligosaccharides of human milk inhibit its binding and infection.
      ,
      • Morrow A.L.
      • Ruiz-Palacios G.M.
      • Altaye M.
      • Jiang X.
      • Guerrero M.L.
      • Meinzen-Derr J.K.
      • Farkas T.
      • Chaturvedi P.
      • Pickering L.K.
      • Newburg D.S.
      Human milk oligosaccharides are associated with protection against diarrhea in breast-fed infants.
      ,
      • Liu B.
      • Yu Z.
      • Chen C.
      • Kling D.E.
      • Newburg D.S.
      Human milk mucin 1 and mucin 4 inhibit Salmonella enterica serovar Typhimurium invasion of human intestinal epithelial cells in vitro.
      ), and intestinal epithelial cell modulation (
      • Angeloni S.
      • Ridet J.L.
      • Kusy N.
      • Gao H.
      • Crevoisier F.
      • Guinchard S.
      • Kochhar S.
      • Sigrist H.
      • Sprenger N.
      Glycoprofiling with micro-arrays of glycoconjugates and lectins.
      ,
      • Kuntz S.
      • Rudloff S.
      • Kunz C.
      Oligosaccharides from human milk influence growth-related characteristics of intestinally transformed and non-transformed intestinal cells.
      ,
      • Kuntz S.
      • Kunz C.
      • Rudloff S.
      Oligosaccharides from human milk induce growth arrest via G2/M by influencing growth-related cell cycle genes in intestinal epithelial cells.
      ,
      • Cederlund A.
      • Kai-Larsen Y.
      • Printz G.
      • Yoshio H.
      • Alvelius G.
      • Lagercrantz H.
      • Strömberg R.
      • Jörnvall H.
      • Gudmundsson G.H.
      • Agerberth B.
      Lactose in human breast milk an inducer of innate immunity with implications for a role in intestinal homeostasis.
      ). Induction of altered gene expression in intestinal epithelium by human milk oligosaccharides results in enhanced protection from pathogenic Escherichia coli infection through modulation of epithelial cell surface glycans (
      • Angeloni S.
      • Ridet J.L.
      • Kusy N.
      • Gao H.
      • Crevoisier F.
      • Guinchard S.
      • Kochhar S.
      • Sigrist H.
      • Sprenger N.
      Glycoprofiling with micro-arrays of glycoconjugates and lectins.
      ), and milk lactose induces the expression of antimicrobial peptide LL-37 in cultured epithelium (
      • Cederlund A.
      • Kai-Larsen Y.
      • Printz G.
      • Yoshio H.
      • Alvelius G.
      • Lagercrantz H.
      • Strömberg R.
      • Jörnvall H.
      • Gudmundsson G.H.
      • Agerberth B.
      Lactose in human breast milk an inducer of innate immunity with implications for a role in intestinal homeostasis.
      ), suggesting that direct effects of human milk glycans on intestinal epithelial cells may contribute significantly to the protection from gastrointestinal infection associated with breast-feeding.
      Among the known glycan components of both human and bovine milk are abundant glycosaminoglycans (GAGs),
      The abbreviations used are: GAG
      glycosaminoglycan
      HA
      hyaluronan
      HβD2
      human β-defensin 2
      TLR
      Toll-like receptor
      ELSA
      enzyme-linked sorbent assay
      MuβD3
      murine β-defensin 3
      HBSS
      Hanks' balanced salt solution.
      large linear polysaccharide polymers containing amino sugars. Hyaluronan (HA) is a GAG usually found as a high molecular weight polymer and consists of repeating disaccharides of N-acetyl-β-d-glucosamine and β-d-glucuronic acid. Unlike other GAGs, HA is synthesized without a protein core and is not sulfated, nitrosylated, or phosphorylated in vivo (
      • Stern R.
      • Asari A.A.
      • Sugahara K.N.
      Hyaluronan fragments. An information-rich system.
      ). A recent study determined that HA is one of the GAGs contained in milk (
      • Coppa G.V.
      • Gabrielli O.
      • Buzzega D.
      • Zampini L.
      • Galeazzi T.
      • Maccari F.
      • Bertino E.
      • Volpi N.
      Composition and structure elucidation of human milk glycosaminoglycans.
      ). Milk GAGs may play a significant role in enhancing intestinal defense against pathogens, as suggested by inhibition of HIV engagement with host receptor CD4 by chondroitin sulfate derived from human milk (
      • Newburg D.S.
      • Linhardt R.J.
      • Ampofo S.A.
      • Yolken R.H.
      Human milk glycosaminoglycans inhibit HIV glycoprotein gp120 binding to its host cell CD4 receptor.
      ). However, the specific function of milk HA has not been reported previously.
      HA is found in every tissue of the body, primarily in the form of high molecular weight polymers (∼107 Da), and plays a fundamental role in tissue homeostasis (
      • Laurent T.C.
      • Laurent U.B.
      • Fraser J.R.
      Functions of hyaluronan.
      ). Current evidence demonstrates that fragmented HA polymers generated in damaged or inflamed tissue act as endogenous “danger signals,” or “damage-associated molecular patterns” (
      • Beck-Schimmer B.
      • Oertli B.
      • Pasch T.
      • Wüthrich R.P.
      Hyaluronan induces monocyte chemoattractant protein-1 expression in renal tubular epithelial cells.
      ,
      • Chen G.Y.
      • Nuñez G.
      Sterile inflammation. Sensing and reacting to damage.
      ,
      • Taylor K.R.
      • Yamasaki K.
      • Radek K.A.
      • Di Nardo A.
      • Goodarzi H.
      • Golenbock D.
      • Beutler B.
      • Gallo R.L.
      Recognition of hyaluronan released in sterile injury involves a unique receptor complex dependent on Toll-like receptor 4, CD44, and MD-2.
      ), triggering localized innate defense responses. Endogenous fragmented HA is thought to be recognized in much the same way as the conserved pathogen-associated molecular patterns, such as LPS and peptidoglycan, via Toll-like receptors (TLRs) (
      • Taylor K.R.
      • Yamasaki K.
      • Radek K.A.
      • Di Nardo A.
      • Goodarzi H.
      • Golenbock D.
      • Beutler B.
      • Gallo R.L.
      Recognition of hyaluronan released in sterile injury involves a unique receptor complex dependent on Toll-like receptor 4, CD44, and MD-2.
      ,
      • Scheibner K.A.
      • Lutz M.A.
      • Boodoo S.
      • Fenton M.J.
      • Powell J.D.
      • Horton M.R.
      Hyaluronan fragments act as an endogenous danger signal by engaging TLR2.
      ).
      HA fragments play a role in enhancing innate epithelial defense independent of the proinflammatory immunomodulation characteristic of macrophage (
      • Noble P.W.
      • McKee C.M.
      • Cowman M.
      • Shin H.S.
      Hyaluronan fragments activate an NF-κB/I-κB α autoregulatory loop in murine macrophages.
      ), chondrocyte (
      • Campo G.M.
      • Avenoso A.
      • Campo S.
      • D'Ascola A.
      • Nastasi G.
      • Calatroni A.
      Small hyaluronan oligosaccharides induce inflammation by engaging both Toll-like-4 and CD44 receptors in human chondrocytes.
      ), or endothelial cell activation (
      • Taylor K.R.
      • Trowbridge J.M.
      • Rudisill J.A.
      • Termeer C.C.
      • Simon J.C.
      • Gallo R.L.
      Hyaluronan fragments stimulate endothelial recognition of injury through TLR4.
      ) by low molecular weight HA or the stimulation of TLR4 by bacterial pathogen-associated molecular patterns (
      • O'Neill L.A.
      Signal transduction pathways activated by the IL-1 receptor/Toll-like receptor superfamily.
      ). A polydispersed HA fragment preparation of polymers of less than 750 kDa injected intraperitoneally protects wild-type mice in a TLR4-dependent manner from a microflora-mediated epithelial damage model of colitis (
      • Zheng L.
      • Riehl T.E.
      • Stenson W.F.
      Regulation of colonic epithelial repair in mice by Toll-like receptors and hyaluronic acid.
      ) or from the epithelium-depleting effects of radiation (
      • Riehl T.E.
      • Foster L.
      • Stenson W.F.
      Hyaluronic acid is radioprotective in the intestine through a TLR4 and COX-2-mediated mechanism.
      ). Low molecular weight HA has been also been shown to induce elevated expression of antimicrobial defensin proteins that may contribute to enhanced epithelial defense in the intestine (
      • Hill D.R.
      • Kessler S.P.
      • Rho H.K.
      • Cowman M.K.
      • de la Motte C.A.
      Specific-sized hyaluronan fragments promote expression of human β-defensin 2 in intestinal epithelium.
      ), skin (
      • Gariboldi S.
      • Palazzo M.
      • Zanobbio L.
      • Selleri S.
      • Sommariva M.
      • Sfondrini L.
      • Cavicchini S.
      • Balsari A.
      • Rumio C.
      Low molecular weight hyaluronic acid increases the self-defense of skin epithelium by induction of β-defensin 2 via TLR2 and TLR4.
      ), and vagina (
      • Dusio G.F.
      • Cardani D.
      • Zanobbio L.
      • Mantovani M.
      • Luchini P.
      • Battini L.
      • Galli V.
      • Diana A.
      • Balsari A.
      • Rumio C.
      Stimulation of TLRs by LMW-HA induces self-defense mechanisms in vaginal epithelium.
      ).
      Defensins are small cationic peptides that play a critical role in the preservation of epithelial barrier integrity in the presence of continuous microbial challenges. These antimicrobial peptides are expressed by gastrointestinal, urogenital, and pulmonary epithelium, skin, and the ocular surface (
      • Selsted M.E.
      • Ouellette A.J.
      Mammalian defensins in the antimicrobial immune response.
      ,
      • Garreis F.
      • Schlorf T.
      • Worlitzsch D.
      • Steven P.
      • Bräuer L.
      • Jäger K.
      • Paulsen F.P.
      Roles of human β-defensins in innate immune defense at the ocular surface. Arming and alarming corneal and conjunctival epithelial cells.
      ). Defensins have direct antimicrobial activity against a wide range of human pathogens and commensals, including both Gram-positive and Gram-negative bacteria, virus, fungi, and protozoa (
      • Zasloff M.
      Antimicrobial peptides of multicellular organisms.
      ). Interestingly, microbes stimulate the expression of inducible β-defensins 2, 3, and 4 in epithelium through the interaction of a variety of pathogen-associated molecular patterns with TLRs (
      • O'Neil D.A.
      Regulation of expression of β-defensins. Endogenous enteric peptide antibiotics.
      ,
      • Wada A.
      • Mori N.
      • Oishi K.
      • Hojo H.
      • Nakahara Y.
      • Hamanaka Y.
      • Nagashima M.
      • Sekine I.
      • Ogushi K.
      • Niidome T.
      • Nagatake T.
      • Moss J.
      • Hirayama T.
      Induction of human β-defensin-2 mRNA expression by Helicobacter pylori in human gastric cell line MKN45 cells on cag pathogenicity island.
      ,
      • Ogushi K.
      • Wada A.
      • Niidome T.
      • Mori N.
      • Oishi K.
      • Nagatake T.
      • Takahashi A.
      • Asakura H.
      • Makino S.
      • Hojo H.
      • Nakahara Y.
      • Ohsaki M.
      • Hatakeyama T.
      • Aoyagi H.
      • Kurazono H.
      • Moss J.
      • Hirayama T.
      Salmonella enteritidis FliC (flagella filament protein) induces human β-defensin-2 mRNA production by Caco-2 cells.
      ,
      • Vora P.
      • Youdim A.
      • Thomas L.S.
      • Fukata M.
      • Tesfay S.Y.
      • Lukasek K.
      • Michelsen K.S.
      • Wada A.
      • Hirayama T.
      • Arditi M.
      • Abreu M.T.
      β-Defensin-2 expression is regulated by TLR signaling in intestinal epithelial cells.
      ,
      • Froy O.
      Regulation of mammalian defensin expression by Toll-like receptor-dependent and independent signalling pathways.
      ). TLR4 regulates the expression of human β-defensin 2 (HβD2) in epithelium following stimulation with LPS (
      • Vora P.
      • Youdim A.
      • Thomas L.S.
      • Fukata M.
      • Tesfay S.Y.
      • Lukasek K.
      • Michelsen K.S.
      • Wada A.
      • Hirayama T.
      • Arditi M.
      • Abreu M.T.
      β-Defensin-2 expression is regulated by TLR signaling in intestinal epithelial cells.
      ). The same cell surface receptor, TLR4, mediates the induction of HβD2, without an accompanying increase in inflammatory cytokine production, in human keratinocytes exposed to low molecular weight HA (
      • Gariboldi S.
      • Palazzo M.
      • Zanobbio L.
      • Selleri S.
      • Sommariva M.
      • Sfondrini L.
      • Cavicchini S.
      • Balsari A.
      • Rumio C.
      Low molecular weight hyaluronic acid increases the self-defense of skin epithelium by induction of β-defensin 2 via TLR2 and TLR4.
      ) and vaginal epithelium (
      • Dusio G.F.
      • Cardani D.
      • Zanobbio L.
      • Mantovani M.
      • Luchini P.
      • Battini L.
      • Galli V.
      • Diana A.
      • Balsari A.
      • Rumio C.
      Stimulation of TLRs by LMW-HA induces self-defense mechanisms in vaginal epithelium.
      ). Our group has recently demonstrated the TLR4-dependent induction of murine HβD2 ortholog in colonic epithelium in vivo following the administration of synthetic, specific sized HA (
      • Hill D.R.
      • Kessler S.P.
      • Rho H.K.
      • Cowman M.K.
      • de la Motte C.A.
      Specific-sized hyaluronan fragments promote expression of human β-defensin 2 in intestinal epithelium.
      ). Therefore, it is becoming increasingly clear that low to intermediate molecular weight HA is an endogenous ligand capable of promoting enhanced antimicrobial defense of epithelial barriers through TLR4-dependent pathways.
      Despite the growing evidence of the significant role of HA in bolstering innate defense of the intestine (
      • Zheng L.
      • Riehl T.E.
      • Stenson W.F.
      Regulation of colonic epithelial repair in mice by Toll-like receptors and hyaluronic acid.
      ,
      • Riehl T.E.
      • Foster L.
      • Stenson W.F.
      Hyaluronic acid is radioprotective in the intestine through a TLR4 and COX-2-mediated mechanism.
      ,
      • Hill D.R.
      • Kessler S.P.
      • Rho H.K.
      • Cowman M.K.
      • de la Motte C.A.
      Specific-sized hyaluronan fragments promote expression of human β-defensin 2 in intestinal epithelium.
      ), an endogenous source of HA responsible for mediating innate defense remains unknown. In light of the recent report that human milk contains HA (
      • Coppa G.V.
      • Gabrielli O.
      • Buzzega D.
      • Zampini L.
      • Galeazzi T.
      • Maccari F.
      • Bertino E.
      • Volpi N.
      Composition and structure elucidation of human milk glycosaminoglycans.
      ) and our finding that that HA promotes expression of the antimicrobial peptide HβD2 in intestinal mucosa (
      • Hill D.R.
      • Kessler S.P.
      • Rho H.K.
      • Cowman M.K.
      • de la Motte C.A.
      Specific-sized hyaluronan fragments promote expression of human β-defensin 2 in intestinal epithelium.
      ), we hypothesized that innate epithelial antimicrobial defense is enhanced in the intestinal epithelium by HA supplied in breast milk. We have determined the concentration range of HA in human breast milk collected from a cohort of 44 mothers who provided multiple samples during the first 6 months after delivery. Our data confirm the previous finding that human milk contains HA (
      • Coppa G.V.
      • Gabrielli O.
      • Buzzega D.
      • Zampini L.
      • Galeazzi T.
      • Maccari F.
      • Bertino E.
      • Volpi N.
      Composition and structure elucidation of human milk glycosaminoglycans.
      ,
      • Coppa G.V.
      • Gabrielli O.
      • Zampini L.
      • Galeazzi T.
      • Maccari F.
      • Buzzega D.
      • Galeotti F.
      • Bertino E.
      • Volpi N.
      Glycosaminoglycan content in term and preterm milk during the first month of lactation.
      ) and demonstrate that milk HA concentration is highest in the critical first weeks after birth and decreases in the population to a steady-state level over the next 2 months. Accordingly, using physiologic levels of HA, we demonstrate two independent parameters of enhanced epithelial antimicrobial defense that are specifically enhanced by HA purified from human milk: 1) HA-dependent induction of the antimicrobial peptide HβD2 in cultured human colonic epithelial cells and in murine colonic mucosa following oral administration of a milk HA preparation and 2) HA-dependent protection from intracellular infection by Salmonella typhimurium in cultured intestinal epithelial cells pretreated with milk-derived HA. Furthermore, the in vivo induction of the murine HβD2 ortholog is dependent upon expression of the cell surface receptors TLR4 and CD44.

      DISCUSSION

      Our recent report indicating that HA promotes expression of the antimicrobial peptide HβD2 in intestinal epithelium (
      • Hill D.R.
      • Kessler S.P.
      • Rho H.K.
      • Cowman M.K.
      • de la Motte C.A.
      Specific-sized hyaluronan fragments promote expression of human β-defensin 2 in intestinal epithelium.
      ) and the observation that human milk contains HA (
      • Coppa G.V.
      • Gabrielli O.
      • Buzzega D.
      • Zampini L.
      • Galeazzi T.
      • Maccari F.
      • Bertino E.
      • Volpi N.
      Composition and structure elucidation of human milk glycosaminoglycans.
      ) resulted in our hypothesis that HA supplied in human breast milk enhances innate intestinal epithelial antimicrobial defense. Our data support the finding of Coppa et al. (
      • Coppa G.V.
      • Gabrielli O.
      • Buzzega D.
      • Zampini L.
      • Galeazzi T.
      • Maccari F.
      • Bertino E.
      • Volpi N.
      Composition and structure elucidation of human milk glycosaminoglycans.
      ) that human milk contains HA while further defining the time-dependent change in milk HA concentration from the start of lactation through the first 6 months postpartum. In addressing our hypothesis, we have demonstrated two parameters of epithelial antimicrobial defense that are greatly augmented by HA supplied in breast milk. First, cultured human colonic epithelial cells or the intestinal mucosa of wild-type mice treated with human milk HA at physiologically relevant concentrations exhibit dramatic induction of HβD2 expression that is significantly reduced by predigestion with a substrate-specific hyaluronidase and is specifically associated with increased ERK1/2 phosphorylation. Similar induction of murine HβD2 ortholog is observed in the intestinal mucosa of nursing mice relative to newborn or weaned animals. The induction of murine HβD2 ortholog by milk HA is dependent upon both HA cell surface receptors TLR4 and CD44 in vivo. Second, cultured colonic epithelial cells exhibit enhanced resistance to infection with the enteric pathogen Salmonella following pretreatment with milk HA isolates, an effect that is also abolished by hyaluronidase digestion. Taken together, our data indicate that HA naturally supplied in human breast milk contributes significantly to the induction of antimicrobial defense in intestinal epithelium.
      Analysis of 1710 unique milk samples collected from a cohort of 44 mothers revealed high concentrations of HA in the initial weeks of lactation (Fig. 1). Despite wide variation between individual donors, HA concentrations in milk consistently decreased over the first 8 weeks postpartum, reaching a steady-state concentration by the third month after birth. Importantly, all samples evaluated contained detectable levels of HA, adding to the recent findings of Coppa et al. (
      • Coppa G.V.
      • Gabrielli O.
      • Buzzega D.
      • Zampini L.
      • Galeazzi T.
      • Maccari F.
      • Bertino E.
      • Volpi N.
      Composition and structure elucidation of human milk glycosaminoglycans.
      ,
      • Coppa G.V.
      • Gabrielli O.
      • Zampini L.
      • Galeazzi T.
      • Maccari F.
      • Buzzega D.
      • Galeotti F.
      • Bertino E.
      • Volpi N.
      Glycosaminoglycan content in term and preterm milk during the first month of lactation.
      ), who utilized pooled milk samples collected from a limited cohort to suggest that HA is a universally expressed component of human milk. The use of a competitive ELSA that was highly specific to HA but independent of HA polymer size3 allowed for reproducible quantification of HA in whole milk samples without biochemical manipulation of the sample. Numerous bioactive components of human milk are present in high concentrations in the early stages of lactation, including other GAGs (
      • Coppa G.V.
      • Gabrielli O.
      • Zampini L.
      • Galeazzi T.
      • Maccari F.
      • Buzzega D.
      • Galeotti F.
      • Bertino E.
      • Volpi N.
      Glycosaminoglycan content in term and preterm milk during the first month of lactation.
      ), sIgA (
      • Brandtzaeg P.
      Mucosal immunity. Integration between mother and the breast-fed infant.
      ), and fatty acids (
      • Bokor S.
      • Koletzko B.
      • Decsi T.
      Systematic review of fatty acid composition of human milk from mothers of preterm compared to full-term infants.
      ). Given the critical nature of the immediate postnatal period and the high susceptibility of newborns to infection (
      • Howie P.W.
      • Forsyth J.S.
      • Ogston S.A.
      • Clark A.
      • Florey C.D.
      Protective effect of breast feeding against infection.
      ,
      • Jones T.F.
      • Ingram L.A.
      • Fullerton K.E.
      • Marcus R.
      • Anderson B.J.
      • McCarthy P.V.
      • Vugia D.
      • Shiferaw B.
      • Haubert N.
      • Wedel S.
      • Angulo F.J.
      A case-control study of the epidemiology of sporadic Salmonella infection in infants.
      ), it is perhaps unsurprising to find that HA in milk is also present at higher concentrations in the first weeks after birth. Maternal genetics and nutrition probably contribute to the intradonor variation in HA content we have observed.
      Milk HA treatment exhibited both time-and dose-dependent induction of HβD2 protein in cultured HT-29 epithelium (Figs. 2, A and E), with maximal activity at 500 ng/ml, the approximate mean concentration of HA present in milk within the first 30 days postpartum (Fig. 1). In addition, transcription of the gene encoding the HβD2 peptide, DEFB4, was specifically and significantly up-regulated by milk HA at physiologic concentration (Fig. 2), indicating that the increased expression of HβD2 peptide is mediated by transcriptional activation. Numerous reports have indicated that the signaling properties of HA, including the induction of HβD2 (
      • Hill D.R.
      • Kessler S.P.
      • Rho H.K.
      • Cowman M.K.
      • de la Motte C.A.
      Specific-sized hyaluronan fragments promote expression of human β-defensin 2 in intestinal epithelium.
      ), are highly size-specific (
      • Stern R.
      • Asari A.A.
      • Sugahara K.N.
      Hyaluronan fragments. An information-rich system.
      ). Size analysis of the HA we isolated from milk indicates broad polydispersity, containing 104- to 106-Da polymers and with polymer size distribution varying widely between individuals.3 Future studies may be required to determine the size-specific bioactivity of naturally occurring milk HA. Importantly, the milk HA preparations isolated from unique donors independently exhibited similar HβD2- and DEFB4-inducing activity at similar concentrations (Fig. 2), indicating that the expression of bioactive HA in milk is likely to be widespread among the general population. In addition, although previous work suggests that HA acts as a specific inducer of HβD2 in intestinal epithelium independent of other ligands (
      • Hill D.R.
      • Kessler S.P.
      • Rho H.K.
      • Cowman M.K.
      • de la Motte C.A.
      Specific-sized hyaluronan fragments promote expression of human β-defensin 2 in intestinal epithelium.
      ), we cannot exclude the possibility that additional contaminant milk glycans contribute to or potentiate induction of HβD2 by milk HA. Analysis of the milk HA preparations indicates that undersulfated chondroitin is the predominant non-HA component in the isolates (Table 1). Indeed, hyaluronidase-treated milk preparations promoted some, albeit greatly reduced, induction of HβD2 protein and DEFB4 transcription at physiologically relevant concentrations (Fig. 2). However, substantially increased doses of hyaluronidase-treated milk HA isolates were required to achieve effects comparable with treatment with milk preparations containing intact HA. The observed EC50 of hyaluronidase-treated milk isolates is 200-fold higher than the HA-containing milk preparation (Fig. 2E). Clearly, the presence of HA in milk isolates contributes significantly to HβD2 induction. In addressing our hypothesis that HA in milk enhances innate intestinal epithelial antimicrobial defense, evaluating induction of antimicrobial peptide HβD2 by milk HA presented in context with other milk carbohydrates has merit in that it perhaps more nearly replicates the physiologic setting of milk HA presentation to the neonatal intestinal mucosa.
      The intestinal mucosa of nursing mice expresses enhanced MuβD3 peptide relative to newborn or weaned animals (Fig. 3C), suggesting that milk components contribute to induction of MuβD3. We have previously detected HA in mouse milk,3 and together these observations, this indicates that mice are a relevant animal model for the evaluation of the hypothesis that milk HA specifically enhances epithelial antimicrobial MuβD3 expression in vivo. Oral administration of human milk HA to adult C57BL/6 mice resulted in substantial induction of the murine HβD2 ortholog in the mucosa of the proximal colon relative to administration of control or hyaluronidase degraded human milk preparations (Fig. 3, A and B), recapitulating the induction of MuβD3 observed in nursing mice (Fig. 3C). Consistent with our in vitro observations, induction of murine HβD2 ortholog in vivo by the human milk HA preparation is substantially reduced in the absence of intact hyaluronan, suggesting that the presence of HA in the milk contributes significantly to defensin induction. In addition, up-regulated expression of MuβD3 in the murine intestinal epithelium is seen distally in the transverse colonic mucosa (data not shown), suggesting that human milk HA retains bioactivity within the digestive tract in the context of both murine and microflora-driven catabolic activity. Previous work by Balogh et al. (
      • Balogh L.
      • Polyak A.
      • Mathe D.
      • Kiraly R.
      • Thuroczy J.
      • Terez M.
      • Janoki G.
      • Ting Y.
      • Bucci L.R.
      • Schauss A.G.
      Absorption, uptake and tissue affinity of high-molecular weight hyaluronan after oral administration in rats and dogs.
      ) has demonstrated that 87–96% of radiolabeled HA is recovered in the feces of rats following oral administration of a high molecular weight HA preparation, and the diffusion of HA through the paracellular junctions of cultured intestinal epithelium decreases rapidly with increasing molecular weight (
      • Hisada N.
      • Satsu H.
      • Mori A.
      • Totsuka M.
      • Kamei J.
      • Nozawa T.
      • Shimizu M.
      Low-molecular weight hyaluronan permeates through human intestinal Caco-2 cell monolayers via the paracellular pathway.
      ). In light of these reports and our own observation that milk HA promotes HβD2 expression in isolated cultures of human colonocytes (FIGURE 2, FIGURE 5), it appears highly unlikely that the effect of oral administration of milk HA is mediated through the circulation and instead involves a direct interaction at the luminal surface of the intestinal epithelium.
      Induction of the murine HβD2 ortholog following oral administration of milk HA is dependent upon expression of cell surface receptors TLR4 and CD44. Expression of MuβD3 was significantly enhanced in the intestinal mucosa of wild-type animals following oral administration of human milk HA. In contrast, MuβD3 expression was similar to control treatment in both TLR4- and CD44-deficient animals following administration of the milk HA preparation for 3 days (Fig. 4). Prior work in animal models has principally implicated TLR4 in HA-dependent modulation of intestinal defense (
      • Zheng L.
      • Riehl T.E.
      • Stenson W.F.
      Regulation of colonic epithelial repair in mice by Toll-like receptors and hyaluronic acid.
      ,
      • Riehl T.E.
      • Foster L.
      • Stenson W.F.
      Hyaluronic acid is radioprotective in the intestine through a TLR4 and COX-2-mediated mechanism.
      ), particularly following oral administration of HA (
      • Hill D.R.
      • Kessler S.P.
      • Rho H.K.
      • Cowman M.K.
      • de la Motte C.A.
      Specific-sized hyaluronan fragments promote expression of human β-defensin 2 in intestinal epithelium.
      ,
      • Asari A.
      • Kanemitsu T.
      • Kurihara H.
      Oral administration of high molecular weight hyaluronan (900 kDa) controls immune system via Toll-like receptor 4 in the intestinal epithelium.
      ), and the induction of HβD2 in cultured keratinocytes is dependent upon TLR4 (
      • Gariboldi S.
      • Palazzo M.
      • Zanobbio L.
      • Selleri S.
      • Sommariva M.
      • Sfondrini L.
      • Cavicchini S.
      • Balsari A.
      • Rumio C.
      Low molecular weight hyaluronic acid increases the self-defense of skin epithelium by induction of β-defensin 2 via TLR2 and TLR4.
      ). Our results suggest that TLR4 is an essential cell surface receptor for the induction of murine HβD2 ortholog by milk HA (Fig. 4), a finding that is consistent with our previous report that induction of MuβD3 by synthetic HA is TLR4-dependent (
      • Hill D.R.
      • Kessler S.P.
      • Rho H.K.
      • Cowman M.K.
      • de la Motte C.A.
      Specific-sized hyaluronan fragments promote expression of human β-defensin 2 in intestinal epithelium.
      ). However, CD44-dependent induction of defense effectors has been reported in monocytes and macrophages (
      • Taylor K.R.
      • Yamasaki K.
      • Radek K.A.
      • Di Nardo A.
      • Goodarzi H.
      • Golenbock D.
      • Beutler B.
      • Gallo R.L.
      Recognition of hyaluronan released in sterile injury involves a unique receptor complex dependent on Toll-like receptor 4, CD44, and MD-2.
      ,
      • Jiang D.
      • Liang J.
      • Fan J.
      • Yu S.
      • Chen S.
      • Luo Y.
      • Prestwich G.D.
      • Mascarenhas M.M.
      • Garg H.G.
      • Quinn D.A.
      • Homer R.J.
      • Goldstein D.R.
      • Bucala R.
      • Lee P.J.
      • Medzhitov R.
      • Noble P.W.
      Regulation of lung injury and repair by Toll-like receptors and hyaluronan.
      ,
      • Jiang D.
      • Liang J.
      • Noble P.W.
      Hyaluronan as an immune regulator in human diseases.
      ) and renal epithelium (
      • Beck-Schimmer B.
      • Oertli B.
      • Pasch T.
      • Wüthrich R.P.
      Hyaluronan induces monocyte chemoattractant protein-1 expression in renal tubular epithelial cells.
      ); CD44 is required for the induction of murine HβD2 ortholog following oral administration of milk HA (Fig. 4); and anti-CD44 antibodies specifically abrogate the induction of HβD2 in cultured human epithelial cells (Fig. 6A). To our knowledge, this is the first report to suggest a role for CD44 in the regulation of defensin expression. Thus, both TLR4 and CD44 are required in the response to milk HA in vivo, and genetic deletion of either putative HA receptor is sufficient to abrogate the induction of MuβD3.
      These findings indicate differing regulation of the induction of HβD2 by milk HA or synthetic HA-35, because genetic deletion of CD44 (
      • Hill D.R.
      • Kessler S.P.
      • Rho H.K.
      • Cowman M.K.
      • de la Motte C.A.
      Specific-sized hyaluronan fragments promote expression of human β-defensin 2 in intestinal epithelium.
      ) or application of anti-CD44 antibody (Fig. 6A) did not significantly inhibit the induction of HβD2 by HA-35. Differences in receptor membrane dynamics or the differing activation of signal transduction mediators by the two HA preparations may account for the apparent differences in CD44 dependence. Previous work by Taylor et al. (
      • Taylor K.R.
      • Yamasaki K.
      • Radek K.A.
      • Di Nardo A.
      • Goodarzi H.
      • Golenbock D.
      • Beutler B.
      • Gallo R.L.
      Recognition of hyaluronan released in sterile injury involves a unique receptor complex dependent on Toll-like receptor 4, CD44, and MD-2.
      ) suggests a mechanism by which TLR4 and CD44 complexes, colocalized in the cell membrane, cooperatively regulate the macrophage response to HA fragments generated in sterile injury through a shared signal transduction pathway. Recognition of milk HA and subsequent signal transduction resulting in MuβD3 expression may utilize similar TLR4-CD44 receptor complexes expressed on the intestinal epithelial surface. However, analysis of CD44 and TLR4 localization in the cell membrane of HT-29 cells treated with milk HA or HA-35 resulted in no observable difference in the relative distribution of CD44 and TLR4. Interestingly, aggregation of CD44 epitopes within the epithelial cell membrane appeared to be increased in the presence of milk HA but not HA-35 (Fig. 5). An alternate explanation of the observed phenomena may include separate signal transduction pathways independently activated by TLR4 and CD44 capable of acting in a complementary manner to enhance defensin expression in the presence of broadly polydispersed milk HA. Although TLR4-dependent induction of HβD2 following stimulation with bacterial pathogen-associated molecular patterns acts through the canonical TLR signal transduction mediators IRAK, TRAF6, and JNK to promote translocation of the transcription factor AP-1 and engagement of the DEFB4 promoter (
      • O'Neill L.A.
      Signal transduction pathways activated by the IL-1 receptor/Toll-like receptor superfamily.
      ,
      • Froy O.
      Regulation of mammalian defensin expression by Toll-like receptor-dependent and independent signalling pathways.
      ), HβD2 expression is also regulated by other ligands via the MAPK/ERK signal transduction pathway (
      • Froy O.
      Regulation of mammalian defensin expression by Toll-like receptor-dependent and independent signalling pathways.
      ,
      • Moon S.-K.
      • Lee H.-Y.
      • Li J.-D.
      • Nagura M.
      • Kang S.-H.
      • Chun Y.-M.
      • Linthicum F.H.
      • Ganz T.
      • Andalibi A.
      • Lim D.J.
      Activation of a Src-dependent Raf-MEK1/2-ERK signaling pathway is required for IL-1α-induced upregulation of β-defensin 2 in human middle ear epithelial cells.
      ). Engagement of HA with CD44 has repeatedly been demonstrated to result in specific MAPK/ERK activation (
      • Meran S.
      • Luo D.D.
      • Simpson R.
      • Martin J.
      • Wells A.
      • Steadman R.
      • Phillips A.O.
      Hyaluronan facilitates transforming growth factor-β1-dependent proliferation via CD44 and epidermal growth factor receptor interaction.
      ,
      • Puré E.
      • Assoian R.K.
      Rheostatic signaling by CD44 and hyaluronan.
      ) in a mechanism that is potentially dependent on HA size (
      • Yang C.
      • Cao M.
      • Liu H.
      • He Y.
      • Xu J.
      • Du Y.
      • Liu Y.
      • Wang W.
      • Cui L.
      • Hu J.
      • Gao F.
      The high and low molecular weight forms of hyaluronan have distinct effects on CD44 clustering.
      ), and milk HA may induce HβD2 expression in a CD44-dependent manner through this pathway. Analysis of HT-29 cell lysates for the relative abundance of phosphorylated isoforms of ERK1/2, JNK1/2/3, and p38 MAPK using an ELISA panel (Fig. 6, B–D) revealed an increase in total phosphorylated ERK1/2 following treatment with milk HA, but not HA-35 or hyaluronidase-digested milk HA (Fig. 6D). Future studies will be required to distinguish between cooperative and complementary signal transduction mechanisms regulating the TLR4- and CD44- dependent response to milk HA; however, our results indicate that ERK1/2 activation is a specific consequence of the application of broadly polydispersed milk HA.
      Treatment of cultured colonic epithelium with human milk HA enhances resistance to infection by the enteric pathogen Salmonella (Fig. 7), providing functional evidence in support of the hypothesis that endogenous human milk HA enhances antimicrobial defense of intestinal epithelium. Although numerous examples of the direct inhibition of pathogen-host binding by interaction with human milk glycans have been reported (
      • Newburg D.S.
      Neonatal protection by an innate immune system of human milk consisting of oligosaccharides and glycans.
      ,
      • Ruiz-Palacios G.M.
      • Cervantes L.E.
      • Ramos P.
      • Chavez-Munguia B.
      • Newburg D.S.
      Campylobacter jejuni binds intestinal H(O) antigen (Fucα1, 2Galβ1, 4GlcNAc), and fucosyloligosaccharides of human milk inhibit its binding and infection.
      ,
      • Morrow A.L.
      • Ruiz-Palacios G.M.
      • Altaye M.
      • Jiang X.
      • Guerrero M.L.
      • Meinzen-Derr J.K.
      • Farkas T.
      • Chaturvedi P.
      • Pickering L.K.
      • Newburg D.S.
      Human milk oligosaccharides are associated with protection against diarrhea in breast-fed infants.
      ,
      • Liu B.
      • Yu Z.
      • Chen C.
      • Kling D.E.
      • Newburg D.S.
      Human milk mucin 1 and mucin 4 inhibit Salmonella enterica serovar Typhimurium invasion of human intestinal epithelial cells in vitro.
      ,
      • Newburg D.S.
      • Linhardt R.J.
      • Ampofo S.A.
      • Yolken R.H.
      Human milk glycosaminoglycans inhibit HIV glycoprotein gp120 binding to its host cell CD4 receptor.
      ), experimental conditions included the removal of medium containing human milk HA prior to the introduction of Salmonella, suggesting that the observed reduction in Salmonella infection occurs due to modification of host epithelial cells. Infection resistance resulting from modulation of epithelium by human milk glycans has not been extensively studied; however, previous studies suggest at least two potential mechanisms for milk HA-induced antimicrobial protection. First, treatment of cultured Caco-2 epithelium with sialyllactose, a human milk oligosaccharide, results in the down-regulated expression of specific glycan epitopes on the surface of the host epithelium correlating with a reduction in the binding of pathogenic E. coli (
      • Angeloni S.
      • Ridet J.L.
      • Kusy N.
      • Gao H.
      • Crevoisier F.
      • Guinchard S.
      • Kochhar S.
      • Sigrist H.
      • Sprenger N.
      Glycoprofiling with micro-arrays of glycoconjugates and lectins.
      ). Milk HA may act similarly to enhance resistance to Salmonella in HT-29 epithelium through the modulation of surface receptor expression.
      Second, peak milk HA-dependent expression of antimicrobial HβD2 peptide occurred at the same time point as peak milk HA-dependent protection against Salmonella. This implies a potential role for HβD2 in the observed resistance to intracellular infection. Accumulation of intracellular defensins has been demonstrated to inhibit replication of the obligate intracellular pathogen Listeria monocytogenes in macrophages (
      • Arnett E.
      • Lehrer R.I.
      • Pratikhya P.
      • Lu W.
      • Seveau S.
      Defensins enable macrophages to inhibit the intracellular proliferation of Listeria monocytogenes.
      ), and a similar mechanistic link between HβD2 and resistance to intracellular Salmonella may exist in milk HA-treated intestinal epithelium. Although questions regarding the mechanism of milk HA-mediated Salmonella infection resistance remain, our results suggest that milk HA may contribute to the reduced incidence of enteric Salmonella infection associated with breast-feeding in human infants (
      • Jones T.F.
      • Ingram L.A.
      • Fullerton K.E.
      • Marcus R.
      • Anderson B.J.
      • McCarthy P.V.
      • Vugia D.
      • Shiferaw B.
      • Haubert N.
      • Wedel S.
      • Angulo F.J.
      A case-control study of the epidemiology of sporadic Salmonella infection in infants.
      ). Numerous prior reports have implicated HA in modulation of the immune response (
      • Stern R.
      • Asari A.A.
      • Sugahara K.N.
      Hyaluronan fragments. An information-rich system.
      ,
      • Taylor K.R.
      • Yamasaki K.
      • Radek K.A.
      • Di Nardo A.
      • Goodarzi H.
      • Golenbock D.
      • Beutler B.
      • Gallo R.L.
      Recognition of hyaluronan released in sterile injury involves a unique receptor complex dependent on Toll-like receptor 4, CD44, and MD-2.
      ,
      • Jiang D.
      • Liang J.
      • Noble P.W.
      Hyaluronan as an immune regulator in human diseases.
      ); however, this is the first report to our knowledge to suggest a role for endogenous HA in resistance to enteric infection through direct effects on epithelium.
      Our observations are consistent with prior publications indicating that human milk contains HA (
      • Coppa G.V.
      • Gabrielli O.
      • Buzzega D.
      • Zampini L.
      • Galeazzi T.
      • Maccari F.
      • Bertino E.
      • Volpi N.
      Composition and structure elucidation of human milk glycosaminoglycans.
      ,
      • Coppa G.V.
      • Gabrielli O.
      • Zampini L.
      • Galeazzi T.
      • Maccari F.
      • Buzzega D.
      • Galeotti F.
      • Bertino E.
      • Volpi N.
      Glycosaminoglycan content in term and preterm milk during the first month of lactation.
      ) while providing the first evidence that this HA has biological functions at physiologic concentrations. At least two previous reports have indicated that oral administration of HA results in altered function of innate (
      • Hill D.R.
      • Kessler S.P.
      • Rho H.K.
      • Cowman M.K.
      • de la Motte C.A.
      Specific-sized hyaluronan fragments promote expression of human β-defensin 2 in intestinal epithelium.
      ) or adaptive (
      • Yang C.
      • Cao M.
      • Liu H.
      • He Y.
      • Xu J.
      • Du Y.
      • Liu Y.
      • Wang W.
      • Cui L.
      • Hu J.
      • Gao F.
      The high and low molecular weight forms of hyaluronan have distinct effects on CD44 clustering.
      ) components of intestinal defense; however, it was previously unclear if these observations reflected an endogenous physiologic process. The role of human milk glycans, including HA, in generating intestinal homeostasis in breast-fed infants in the presence of diverse and persistent microbial challenges is incompletely understood. Milk contains a diverse bacterial community (
      • Hunt K.M.
      • Foster J.A.
      • Forney L.J.
      • Schütte U.M.
      • Beck D.L.
      • Abdo Z.
      • Fox L.K.
      • Williams J.E.
      • McGuire M.K.
      • McGuire M.A.
      Characterization of the diversity and temporal stability of bacterial communities in human milk.
      ), seeding the developing gastrointestinal microbiome along with maternally and environmentally acquired species and interacting with the antigen-naive adaptive mucosal immune system (
      • Brandtzaeg P.
      The mucosal immune system and its integration with the mammary glands.
      ). The combined effect of milk probiotics, prebiotics, antibiotics, immunomodulators, and microbial transfer is evident in the observation that breast-fed infants exhibit dramatically different gastrointestinal microbial communities in comparison with artificially fed infants (
      • Penders J.
      • Thijs C.
      • Vink C.
      • Stelma F.F.
      • Snijders B.
      • Kummeling I.
      • van den Brandt P.A.
      • Stobberingh E.E.
      Factors influencing the composition of the intestinal microbiota in early infancy.
      ,
      • Dominguez-Bello M.G.
      • Costello E.K.
      • Contreras M.
      • Magris M.
      • Hidalgo G.
      • Fierer N.
      • Knight R.
      Delivery mode shapes the acquisition and structure of the initial microbiota across multiple body habitats in newborns.
      ,
      • Palma G.D.
      • Capilla A.
      • Nova E.
      • Castillejo G.
      • Varea V.
      • Pozo T.
      • Garrote J.A.
      • Polanco I.
      • López A.
      • Ribes-Koninckx C.
      • Marcos A.
      • García-Novo M.D.
      • Calvo C.
      • Ortigosa L.
      • Peña-Quintana L.
      • Palau F.
      • Sanz Y.
      Influence of milk-feeding type and genetic risk of developing coeliac disease on intestinal microbiota of infants. The PROFICEL study.
      ). Given the robust correlation between breast-feeding and gastrointestinal health (
      • Howie P.W.
      • Forsyth J.S.
      • Ogston S.A.
      • Clark A.
      • Florey C.D.
      Protective effect of breast feeding against infection.
      ,
      • Newburg D.S.
      • Walker W.A.
      Protection of the neonate by the innate immune system of developing gut and of human milk.
      ,
      • Grulee C.G.
      • Sanford H.N.
      • Herron P.H.
      Breast and artificial feeding. Influence on morbidity and mortality of twenty thousand infants.
      ,
      • Newburg D.S.
      Neonatal protection by an innate immune system of human milk consisting of oligosaccharides and glycans.
      ), the role of breast milk in establishing the microbial-epithelial interface may be of critical importance to lifelong gastrointestinal health (
      • Le Huërou-Luron I.
      • Blat S.
      • Boudry G.
      Breast- v. formula-feeding. Impacts on the digestive tract and immediate and long-term health effects.
      ), potentially accounting in part for the reduced lifetime risk of inflammatory bowel disease (
      • Klement E.
      • Cohen R.V.
      • Boxman J.
      • Joseph A.
      • Reif S.
      Breastfeeding and risk of inflammatory bowel disease. A systematic review with meta-analysis.
      ), obesity (
      • Owen C.G.
      • Martin R.M.
      • Whincup P.H.
      • Smith G.D.
      • Cook D.G.
      Effect of infant feeding on the risk of obesity across the life course. A quantitative review of published evidence.
      ,
      • Oddy W.H.
      Infant feeding and obesity risk in the child.
      ), and allergic disease (
      • Gdalevich M.
      • Mimouni D.
      • David M.
      • Mimouni M.
      Breast-feeding and the onset of atopic dermatitis in childhood. A systematic review and meta-analysis of prospective studies.
      ,
      • van Odijk J.
      • Kull I.
      • Borres M.P.
      • Brandtzaeg P.
      • Edberg U.
      • Hanson L.A.
      • Høst A.
      • Kuitunen M.
      • Olsen S.F.
      • Skerfving S.
      • Sundell J.
      • Wille S.
      Breastfeeding and allergic disease. A multidisciplinary review of the literature (1966–2001) on the mode of early feeding in infancy and its impact on later atopic manifestations.
      ) associated with breast-feeding. The bioactivity of milk components relative to the function of intestinal epithelium, such as the induction of HβD2 in intestinal epithelium by milk HA, could have a profound effect in shaping intestinal microbial ecology through the modulation of epithelial substrates and the generation of selective niches (
      • Costello E.K.
      • Stagaman K.
      • Dethlefsen L.
      • Bohannan B.J.
      • Relman D.A.
      The application of ecological theory toward an understanding of the human microbiome.
      ). Altered expression of α-defensins in the intestine has been shown to result in significant shifts in microbial species distribution (
      • Salzman N.H.
      • Hung K.
      • Haribhai D.
      • Chu H.
      • Karlsson-Sjöberg J.
      • Amir E.
      • Teggatz P.
      • Barman M.
      • Hayward M.
      • Eastwood D.
      • Stoel M.
      • Zhou Y.
      • Sodergren E.
      • Weinstock G.M.
      • Bevins C.L.
      • Williams C.B.
      • Bos N.A.
      Enteric defensins are essential regulators of intestinal microbial ecology.
      ), and our data demonstrate that the effect of milk HA on intestinal epithelium alters the interaction of host epithelium with at least one relevant component of the human microbiome, S. enterica. Future studies will probably enhance understanding of the complex dynamic regulating host epithelium and microbial interactions and the role of dietary components in shaping the relationship between enterocyte and bacterium. Of more immediate practical relevance, the supplementation of artificial formulas with HA among other glycan components of human milk may reduce the incidence of enteric infection and diarrhea among susceptible infants (
      • Newburg D.S.
      Neonatal protection by an innate immune system of human milk consisting of oligosaccharides and glycans.
      ).

      Acknowledgments

      We thank the study participants and the staff of the Cleveland Clinic Children's Hospital, particularly Shirley Leaser, RN, IBCLC, for generous and enthusiastic assistance in the recruitment of new mothers for our study cohort. Dr. John Peterson is greatly appreciated for microscopy assistance, and we thank Dr. Mark Lauer for assisting with carbohydrate analysis. Finally, we thank Artin Soroosh, Melissa Michaud, and Ryan Verbic for diligently documenting and processing numerous donated human milk samples.

      REFERENCES

        • Howie P.W.
        • Forsyth J.S.
        • Ogston S.A.
        • Clark A.
        • Florey C.D.
        Protective effect of breast feeding against infection.
        BMJ. 1990; 300: 11-16
        • Klement E.
        • Cohen R.V.
        • Boxman J.
        • Joseph A.
        • Reif S.
        Breastfeeding and risk of inflammatory bowel disease. A systematic review with meta-analysis.
        Am. J. Clin. Nutr. 2004; 80: 1342-1352
        • Anderson J.W.
        • Johnstone B.M.
        • Remley D.T.
        Breast-feeding and cognitive development. A meta-analysis.
        Am. J. Clin. Nutr. 1999; 70: 525-535
        • Newburg D.S.
        • Walker W.A.
        Protection of the neonate by the innate immune system of developing gut and of human milk.
        Pediatr Res. 2007; 61: 2-8
        • Grulee C.G.
        • Sanford H.N.
        • Herron P.H.
        Breast and artificial feeding. Influence on morbidity and mortality of twenty thousand infants.
        JAMA. 1934; 103: 735-738
        • Morrow A.L.
        • Guerrero M.L.
        • Shults J.
        • Calva J.J.
        • Lutter C.
        • Bravo J.
        • Ruiz-Palacios G.
        • Morrow R.C.
        • Butterfoss F.D.
        Efficacy of home-based peer counselling to promote exclusive breastfeeding. A randomised controlled trial.
        Lancet. 1999; 353: 1226-1231
        • Jones T.F.
        • Ingram L.A.
        • Fullerton K.E.
        • Marcus R.
        • Anderson B.J.
        • McCarthy P.V.
        • Vugia D.
        • Shiferaw B.
        • Haubert N.
        • Wedel S.
        • Angulo F.J.
        A case-control study of the epidemiology of sporadic Salmonella infection in infants.
        Pediatrics. 2006; 118: 2380-2387
        • Ward P.P.
        • Uribe-Luna S.
        • Conneely O.M.
        Lactoferrin and host defense.
        Biochem. Cell Biol. 2002; 80: 95-102
        • Brandtzaeg P.
        Mucosal immunity. Integration between mother and the breast-fed infant.
        Vaccine. 2003; 21: 3382-3388
        • Martin L.J.
        • Woo J.G.
        • Geraghty S.R.
        • Altaye M.
        • Davidson B.S.
        • Banach W.
        • Dolan L.M.
        • Ruiz-Palacios G.M.
        • Morrow A.L.
        Adiponectin is present in human milk and is associated with maternal factors.
        Am. J. Clin. Nutr. 2006; 83: 1106-1111
        • Asakuma S.
        • Hatakeyama E.
        • Urashima T.
        • Yoshida E.
        • Katayama T.
        • Yamamoto K.
        • Kumagai H.
        • Ashida H.
        • Hirose J.
        • Kitaoka M.
        Physiology of consumption of human milk oligosaccharides by infant gut-associated bifidobacteria.
        J. Biol. Chem. 2011; 286: 34583-34592
        • Marcobal A.
        • Barboza M.
        • Sonnenburg E.D.
        • Pudlo N.
        • Martens E.C.
        • Desai P.
        • Lebrilla C.B.
        • Weimer B.C.
        • Mills D.A.
        • German J.B.
        • Sonnenburg J.L.
        Bacteroides in the infant gut consume milk oligosaccharides via mucus-utilization pathways.
        Cell Host Microbe. 2011; 10: 507-514
        • Sela D.A.
        • Li Y.
        • Lerno L.
        • Wu S.
        • Marcobal A.M.
        • German J.B.
        • Chen X.
        • Lebrilla C.B.
        • Mills D.A.
        An infant-associated bacterial commensal utilizes breast milk sialyloligosaccharides.
        J. Biol. Chem. 2011; 286: 11909-11918
        • Yu Z.-T.
        • Chen C.
        • Kling D.E.
        • Liu B.
        • McCoy J.M.
        • Merighi M.
        • Heidtman M.
        • Newburg D.S.
        The principal fucosylated oligosaccharides of human milk exhibit prebiotic properties on cultured infant microbiota.
        Glycobiology. 2013; 23: 169-177
        • M'Rabet L.
        • Vos A.P.
        • Boehm G.
        • Garssen J.
        Breast-feeding and its role in early development of the immune system in infants. Consequences for health later in life.
        J. Nutr. 2008; 138: 1782S-1790S
        • Newburg D.S.
        Neonatal protection by an innate immune system of human milk consisting of oligosaccharides and glycans.
        J. Anim. Sci. 2009; 87: 26-34
        • Bode L.
        Human milk oligosaccharides. Every baby needs a sugar mama.
        Glycobiology. 2012; 22: 1147-1162
        • Ruiz-Palacios G.M.
        • Cervantes L.E.
        • Ramos P.
        • Chavez-Munguia B.
        • Newburg D.S.
        Campylobacter jejuni binds intestinal H(O) antigen (Fucα1, 2Galβ1, 4GlcNAc), and fucosyloligosaccharides of human milk inhibit its binding and infection.
        J. Biol. Chem. 2003; 278: 14112-14120
        • Morrow A.L.
        • Ruiz-Palacios G.M.
        • Altaye M.
        • Jiang X.
        • Guerrero M.L.
        • Meinzen-Derr J.K.
        • Farkas T.
        • Chaturvedi P.
        • Pickering L.K.
        • Newburg D.S.
        Human milk oligosaccharides are associated with protection against diarrhea in breast-fed infants.
        J. Pediatr. 2004; 145: 297-303
        • Liu B.
        • Yu Z.
        • Chen C.
        • Kling D.E.
        • Newburg D.S.
        Human milk mucin 1 and mucin 4 inhibit Salmonella enterica serovar Typhimurium invasion of human intestinal epithelial cells in vitro.
        J. Nutr. 2012; 142: 1504-1509
        • Angeloni S.
        • Ridet J.L.
        • Kusy N.
        • Gao H.
        • Crevoisier F.
        • Guinchard S.
        • Kochhar S.
        • Sigrist H.
        • Sprenger N.
        Glycoprofiling with micro-arrays of glycoconjugates and lectins.
        Glycobiology. 2005; 15: 31-41
        • Kuntz S.
        • Rudloff S.
        • Kunz C.
        Oligosaccharides from human milk influence growth-related characteristics of intestinally transformed and non-transformed intestinal cells.
        Br. J. Nutr. 2008; 99: 462-471
        • Kuntz S.
        • Kunz C.
        • Rudloff S.
        Oligosaccharides from human milk induce growth arrest via G2/M by influencing growth-related cell cycle genes in intestinal epithelial cells.
        Br. J. Nutr. 2009; 101: 1306-1315
        • Cederlund A.
        • Kai-Larsen Y.
        • Printz G.
        • Yoshio H.
        • Alvelius G.
        • Lagercrantz H.
        • Strömberg R.
        • Jörnvall H.
        • Gudmundsson G.H.
        • Agerberth B.
        Lactose in human breast milk an inducer of innate immunity with implications for a role in intestinal homeostasis.
        PLoS ONE. 2013; 8: e53876
        • Stern R.
        • Asari A.A.
        • Sugahara K.N.
        Hyaluronan fragments. An information-rich system.
        Eur. J. Cell Biol. 2006; 85: 699-715
        • Coppa G.V.
        • Gabrielli O.
        • Buzzega D.
        • Zampini L.
        • Galeazzi T.
        • Maccari F.
        • Bertino E.
        • Volpi N.
        Composition and structure elucidation of human milk glycosaminoglycans.
        Glycobiology. 2011; 21: 295-303
        • Newburg D.S.
        • Linhardt R.J.
        • Ampofo S.A.
        • Yolken R.H.
        Human milk glycosaminoglycans inhibit HIV glycoprotein gp120 binding to its host cell CD4 receptor.
        J. Nutr. 1995; 125: 419-424
        • Laurent T.C.
        • Laurent U.B.
        • Fraser J.R.
        Functions of hyaluronan.
        Ann. Rheum Dis. 1995; 54: 429-432
        • Beck-Schimmer B.
        • Oertli B.
        • Pasch T.
        • Wüthrich R.P.
        Hyaluronan induces monocyte chemoattractant protein-1 expression in renal tubular epithelial cells.
        J. Am. Soc. Nephrol. 1998; 9: 2283-2290
        • Chen G.Y.
        • Nuñez G.
        Sterile inflammation. Sensing and reacting to damage.
        Nat. Rev. Immunol. 2010; 10: 826-837
        • Taylor K.R.
        • Yamasaki K.
        • Radek K.A.
        • Di Nardo A.
        • Goodarzi H.
        • Golenbock D.
        • Beutler B.
        • Gallo R.L.
        Recognition of hyaluronan released in sterile injury involves a unique receptor complex dependent on Toll-like receptor 4, CD44, and MD-2.
        J. Biol. Chem. 2007; 282: 18265-18275
        • Scheibner K.A.
        • Lutz M.A.
        • Boodoo S.
        • Fenton M.J.
        • Powell J.D.
        • Horton M.R.
        Hyaluronan fragments act as an endogenous danger signal by engaging TLR2.
        J. Immunol. 2006; 177: 1272-1281
        • Noble P.W.
        • McKee C.M.
        • Cowman M.
        • Shin H.S.
        Hyaluronan fragments activate an NF-κB/I-κB α autoregulatory loop in murine macrophages.
        J. Exp. Med. 1996; 183: 2373-2378
        • Campo G.M.
        • Avenoso A.
        • Campo S.
        • D'Ascola A.
        • Nastasi G.
        • Calatroni A.
        Small hyaluronan oligosaccharides induce inflammation by engaging both Toll-like-4 and CD44 receptors in human chondrocytes.
        Biochem. Pharmacol. 2010; 80: 480-490
        • Taylor K.R.
        • Trowbridge J.M.
        • Rudisill J.A.
        • Termeer C.C.
        • Simon J.C.
        • Gallo R.L.
        Hyaluronan fragments stimulate endothelial recognition of injury through TLR4.
        J. Biol. Chem. 2004; 279: 17079-17084
        • O'Neill L.A.
        Signal transduction pathways activated by the IL-1 receptor/Toll-like receptor superfamily.
        Curr. Top Microbiol. Immunol. 2002; 270: 47-61
        • Zheng L.
        • Riehl T.E.
        • Stenson W.F.
        Regulation of colonic epithelial repair in mice by Toll-like receptors and hyaluronic acid.
        Gastroenterology. 2009; 137: 2041-2051
        • Riehl T.E.
        • Foster L.
        • Stenson W.F.
        Hyaluronic acid is radioprotective in the intestine through a TLR4 and COX-2-mediated mechanism.
        Am. J. Physiol. Gastrointest Liver Physiol. 2012; 302: G309-G316
        • Hill D.R.
        • Kessler S.P.
        • Rho H.K.
        • Cowman M.K.
        • de la Motte C.A.
        Specific-sized hyaluronan fragments promote expression of human β-defensin 2 in intestinal epithelium.
        J. Biol. Chem. 2012; 287: 30610-30624
        • Gariboldi S.
        • Palazzo M.
        • Zanobbio L.
        • Selleri S.
        • Sommariva M.
        • Sfondrini L.
        • Cavicchini S.
        • Balsari A.
        • Rumio C.
        Low molecular weight hyaluronic acid increases the self-defense of skin epithelium by induction of β-defensin 2 via TLR2 and TLR4.
        J. Immunol. 2008; 181: 2103-2110
        • Dusio G.F.
        • Cardani D.
        • Zanobbio L.
        • Mantovani M.
        • Luchini P.
        • Battini L.
        • Galli V.
        • Diana A.
        • Balsari A.
        • Rumio C.
        Stimulation of TLRs by LMW-HA induces self-defense mechanisms in vaginal epithelium.
        Immunol. Cell Biol. 2011; 89: 630-639
        • Selsted M.E.
        • Ouellette A.J.
        Mammalian defensins in the antimicrobial immune response.
        Nat. Immunol. 2005; 6: 551-557
        • Garreis F.
        • Schlorf T.
        • Worlitzsch D.
        • Steven P.
        • Bräuer L.
        • Jäger K.
        • Paulsen F.P.
        Roles of human β-defensins in innate immune defense at the ocular surface. Arming and alarming corneal and conjunctival epithelial cells.
        Histochem. Cell Biol. 2010; 134: 59-73
        • Zasloff M.
        Antimicrobial peptides of multicellular organisms.
        Nature. 2002; 415: 389-395
        • O'Neil D.A.
        Regulation of expression of β-defensins. Endogenous enteric peptide antibiotics.
        Mol. Immunol. 2003; 40: 445-450
        • Wada A.
        • Mori N.
        • Oishi K.
        • Hojo H.
        • Nakahara Y.
        • Hamanaka Y.
        • Nagashima M.
        • Sekine I.
        • Ogushi K.
        • Niidome T.
        • Nagatake T.
        • Moss J.
        • Hirayama T.
        Induction of human β-defensin-2 mRNA expression by Helicobacter pylori in human gastric cell line MKN45 cells on cag pathogenicity island.
        Biochem. Biophys. Res. Commun. 1999; 263: 770-774
        • Ogushi K.
        • Wada A.
        • Niidome T.
        • Mori N.
        • Oishi K.
        • Nagatake T.
        • Takahashi A.
        • Asakura H.
        • Makino S.
        • Hojo H.
        • Nakahara Y.
        • Ohsaki M.
        • Hatakeyama T.
        • Aoyagi H.
        • Kurazono H.
        • Moss J.
        • Hirayama T.
        Salmonella enteritidis FliC (flagella filament protein) induces human β-defensin-2 mRNA production by Caco-2 cells.
        J. Biol. Chem. 2001; 276: 30521-30526
        • Vora P.
        • Youdim A.
        • Thomas L.S.
        • Fukata M.
        • Tesfay S.Y.
        • Lukasek K.
        • Michelsen K.S.
        • Wada A.
        • Hirayama T.
        • Arditi M.
        • Abreu M.T.
        β-Defensin-2 expression is regulated by TLR signaling in intestinal epithelial cells.
        J. Immunol. 2004; 173: 5398-5405
        • Froy O.
        Regulation of mammalian defensin expression by Toll-like receptor-dependent and independent signalling pathways.
        Cell. Microbiol. 2005; 7: 1387-1397
        • Coppa G.V.
        • Gabrielli O.
        • Zampini L.
        • Galeazzi T.
        • Maccari F.
        • Buzzega D.
        • Galeotti F.
        • Bertino E.
        • Volpi N.
        Glycosaminoglycan content in term and preterm milk during the first month of lactation.
        Neonatology. 2012; 101: 74-76
        • Calabro A.
        • Benavides M.
        • Tammi M.
        • Hascall V.C.
        • Midura R.J.
        Microanalysis of enzyme digests of hyaluronan and chondroitin/dermatan sulfate by fluorophore-assisted carbohydrate electrophoresis (FACE).
        Glycobiology. 2000; 10: 273-281
        • Ohya T.
        • Kaneko Y.
        Novel hyaluronidase from streptomyces.
        Biochim. Biophys. Acta. 1970; 198: 607-609
        • Shimada E.
        • Matsumura G.
        Degradation process of hyaluronic acid by Streptomyces hyaluronidase.
        J. Biochem. 1980; 88: 1015-1023
        • Aksamitiene E.
        • Hoek J.B.
        • Kholodenko B.
        • Kiyatkin A.
        Multistrip Western blotting to increase quantitative data output.
        Electrophoresis. 2007; 28: 3163-3173
        • de la Motte C.A.
        • Hascall V.C.
        • Drazba J.
        • Bandyopadhyay S.K.
        • Strong S.A.
        Mononuclear leukocytes bind to specific hyaluronan structures on colon mucosal smooth muscle cells treated with polyinosinic acid. Polycytidylic acid inter-α-trypsin inhibitor is crucial to structure and function.
        Am. J. Pathol. 2003; 163: 121-133
        • Bhilocha S.
        • Amin R.
        • Pandya M.
        • Yuan H.
        • Tank M.
        • LoBello J.
        • Shytuhina A.
        • Wang W.
        • Wisniewski H.G.
        • de la Motte C.
        • Cowman M.K.
        Agarose and polyacrylamide gel electrophoresis methods for molecular mass analysis of 5–500 kDa hyaluronan.
        Anal. Biochem. 2011; 417: 41-49
        • Bals R.
        • Wang X.
        • Meegalla R.L.
        • Wattler S.
        • Weiner D.J.
        • Nehls M.C.
        • Wilson J.M.
        Mouse β-defensin 3 is an inducible antimicrobial peptide expressed in the epithelia of multiple organs.
        Infect. Immun. 1999; 67: 3542-3547
        • Jiang D.
        • Liang J.
        • Fan J.
        • Yu S.
        • Chen S.
        • Luo Y.
        • Prestwich G.D.
        • Mascarenhas M.M.
        • Garg H.G.
        • Quinn D.A.
        • Homer R.J.
        • Goldstein D.R.
        • Bucala R.
        • Lee P.J.
        • Medzhitov R.
        • Noble P.W.
        Regulation of lung injury and repair by Toll-like receptors and hyaluronan.
        Nat. Med. 2005; 11: 1173-1179
        • Jiang D.
        • Liang J.
        • Noble P.W.
        Hyaluronan as an immune regulator in human diseases.
        Physiol. Rev. 2011; 91: 221-264
        • Meran S.
        • Luo D.D.
        • Simpson R.
        • Martin J.
        • Wells A.
        • Steadman R.
        • Phillips A.O.
        Hyaluronan facilitates transforming growth factor-β1-dependent proliferation via CD44 and epidermal growth factor receptor interaction.
        J. Biol. Chem. 2011; 286: 17618-17630
        • Puré E.
        • Assoian R.K.
        Rheostatic signaling by CD44 and hyaluronan.
        Cell. Signal. 2009; 21: 651-655
        • Yang C.
        • Cao M.
        • Liu H.
        • He Y.
        • Xu J.
        • Du Y.
        • Liu Y.
        • Wang W.
        • Cui L.
        • Hu J.
        • Gao F.
        The high and low molecular weight forms of hyaluronan have distinct effects on CD44 clustering.
        J. Biol. Chem. 2012; 287: 43094-43107
        • Homer C.R.
        • Richmond A.L.
        • Rebert N.A.
        • Achkar J.-P.
        • McDonald C.
        ATG16L1 and NOD2 interact in an autophagy-dependent antibacterial pathway implicated in Crohn's disease pathogenesis.
        Gastroenterology. 2010; 139 (1641.e1–2): 1630-1641
        • Bokor S.
        • Koletzko B.
        • Decsi T.
        Systematic review of fatty acid composition of human milk from mothers of preterm compared to full-term infants.
        Ann. Nutr. Metab. 2007; 51: 550-556
        • Balogh L.
        • Polyak A.
        • Mathe D.
        • Kiraly R.
        • Thuroczy J.
        • Terez M.
        • Janoki G.
        • Ting Y.
        • Bucci L.R.
        • Schauss A.G.
        Absorption, uptake and tissue affinity of high-molecular weight hyaluronan after oral administration in rats and dogs.
        J. Agric. Food Chem. 2008; 56: 10582-10593
        • Hisada N.
        • Satsu H.
        • Mori A.
        • Totsuka M.
        • Kamei J.
        • Nozawa T.
        • Shimizu M.
        Low-molecular weight hyaluronan permeates through human intestinal Caco-2 cell monolayers via the paracellular pathway.
        Biosci. Biotechnol. Biochem. 2008; 72: 1111-1114
        • Asari A.
        • Kanemitsu T.
        • Kurihara H.
        Oral administration of high molecular weight hyaluronan (900 kDa) controls immune system via Toll-like receptor 4 in the intestinal epithelium.
        J. Biol. Chem. 2010; 285: 24751-24758
        • Moon S.-K.
        • Lee H.-Y.
        • Li J.-D.
        • Nagura M.
        • Kang S.-H.
        • Chun Y.-M.
        • Linthicum F.H.
        • Ganz T.
        • Andalibi A.
        • Lim D.J.
        Activation of a Src-dependent Raf-MEK1/2-ERK signaling pathway is required for IL-1α-induced upregulation of β-defensin 2 in human middle ear epithelial cells.
        Biochim. Biophys. Acta. 2002; 1590: 41-51
        • Arnett E.
        • Lehrer R.I.
        • Pratikhya P.
        • Lu W.
        • Seveau S.
        Defensins enable macrophages to inhibit the intracellular proliferation of Listeria monocytogenes.
        Cell. Microbiol. 2011; 13: 635-651
        • Hunt K.M.
        • Foster J.A.
        • Forney L.J.
        • Schütte U.M.
        • Beck D.L.
        • Abdo Z.
        • Fox L.K.
        • Williams J.E.
        • McGuire M.K.
        • McGuire M.A.
        Characterization of the diversity and temporal stability of bacterial communities in human milk.
        PLoS One. 2011; 6: e21313
        • Brandtzaeg P.
        The mucosal immune system and its integration with the mammary glands.
        J. Pediatr. 2010; 156: S8-S15
        • Penders J.
        • Thijs C.
        • Vink C.
        • Stelma F.F.
        • Snijders B.
        • Kummeling I.
        • van den Brandt P.A.
        • Stobberingh E.E.
        Factors influencing the composition of the intestinal microbiota in early infancy.
        Pediatrics. 2006; 118: 511-521
        • Dominguez-Bello M.G.
        • Costello E.K.
        • Contreras M.
        • Magris M.
        • Hidalgo G.
        • Fierer N.
        • Knight R.
        Delivery mode shapes the acquisition and structure of the initial microbiota across multiple body habitats in newborns.
        Proc. Natl. Acad. Sci. U.S.A. 2010; 107: 11971-11975
        • Palma G.D.
        • Capilla A.
        • Nova E.
        • Castillejo G.
        • Varea V.
        • Pozo T.
        • Garrote J.A.
        • Polanco I.
        • López A.
        • Ribes-Koninckx C.
        • Marcos A.
        • García-Novo M.D.
        • Calvo C.
        • Ortigosa L.
        • Peña-Quintana L.
        • Palau F.
        • Sanz Y.
        Influence of milk-feeding type and genetic risk of developing coeliac disease on intestinal microbiota of infants. The PROFICEL study.
        PLoS One. 2012; 7: e30791
        • Le Huërou-Luron I.
        • Blat S.
        • Boudry G.
        Breast- v. formula-feeding. Impacts on the digestive tract and immediate and long-term health effects.
        Nutr. Res. Rev. 2010; 23: 23-36
        • Owen C.G.
        • Martin R.M.
        • Whincup P.H.
        • Smith G.D.
        • Cook D.G.
        Effect of infant feeding on the risk of obesity across the life course. A quantitative review of published evidence.
        Pediatrics. 2005; 115: 1367-1377
        • Oddy W.H.
        Infant feeding and obesity risk in the child.
        Breastfeed Rev. 2012; 20: 7-12
        • Gdalevich M.
        • Mimouni D.
        • David M.
        • Mimouni M.
        Breast-feeding and the onset of atopic dermatitis in childhood. A systematic review and meta-analysis of prospective studies.
        J. Am. Acad. Dermatol. 2001; 45: 520-527
        • van Odijk J.
        • Kull I.
        • Borres M.P.
        • Brandtzaeg P.
        • Edberg U.
        • Hanson L.A.
        • Høst A.
        • Kuitunen M.
        • Olsen S.F.
        • Skerfving S.
        • Sundell J.
        • Wille S.
        Breastfeeding and allergic disease. A multidisciplinary review of the literature (1966–2001) on the mode of early feeding in infancy and its impact on later atopic manifestations.
        Allergy. 2003; 58: 833-843
        • Costello E.K.
        • Stagaman K.
        • Dethlefsen L.
        • Bohannan B.J.
        • Relman D.A.
        The application of ecological theory toward an understanding of the human microbiome.
        Science. 2012; 336: 1255-1262
        • Salzman N.H.
        • Hung K.
        • Haribhai D.
        • Chu H.
        • Karlsson-Sjöberg J.
        • Amir E.
        • Teggatz P.
        • Barman M.
        • Hayward M.
        • Eastwood D.
        • Stoel M.
        • Zhou Y.
        • Sodergren E.
        • Weinstock G.M.
        • Bevins C.L.
        • Williams C.B.
        • Bos N.A.
        Enteric defensins are essential regulators of intestinal microbial ecology.
        Nat. Immunol. 2010; 11: 76-83