Abstract
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.
Highlights
In light of the recent report that human milk contains HA [26] and our finding that that HA promotes expression of the antimicrobial peptide human -defensin 2 (HD2) in intestinal mucosa [39], we hypothesized that innate epithelial antimicrobial defense is enhanced in the intestinal epithelium by HA supplied in breast milk
Milk HA Enhances Phosphorylation of ERK1/2 in Vitro—Regulation of HD2 expression at the level of signal transduction occurs predominantly through ERK, JNK, and MAPK [49], and these pathways have been associated with HA signaling (TLR4 through JNK and MAPK; CD44 through ERK) (40, 60 – 62).We examined whether milk HA and HA35 signal HBD2 expression via similar pathways
Our recent report indicating that HA promotes expression of the antimicrobial peptide HD2 in intestinal epithelium [39] and the observation that human milk contains HA [26] resulted in our hypothesis that HA supplied in human breast milk enhances innate intestinal epithelial antimicrobial defense
Summary
In light of the recent report that human milk contains HA [26] and our finding that that HA promotes expression of the antimicrobial peptide HD2 in intestinal mucosa [39], we hypothesized that innate epithelial antimicrobial defense is enhanced in the intestinal epithelium by HA supplied in breast milk. Using physiologic levels of HA, we demonstrate two independent parameters of enhanced epithelial antimicrobial defense that are enhanced by HA purified from human milk: 1) HA-dependent induction of the antimicrobial peptide HD2 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 milkderived HA. The in vivo induction of the murine HD2 ortholog is dependent upon expression of the cell surface receptors TLR4 and CD44
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