Immunol. 17), and include prebiotic function (11C14), antiadhesive antimicrobial activity (18C20), and intestinal epithelial cell modulation (21C24). Induction of altered gene expression in intestinal epithelium by human milk oligosaccharides results in enhanced protection from pathogenic infection through modulation of epithelial cell surface glycans (21), and milk lactose induces the expression of antimicrobial peptide LL-37 in cultured epithelium (24), suggesting that direct effects of human milk glycans on intestinal epithelial cells may contribute significantly to the WEHI-539 hydrochloride protection from gastrointestinal infection associated with breast-feeding. Among the known glycan components of both human and bovine milk are abundant glycosaminoglycans (GAGs),2 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 (25). A recent study determined that HA is one of the GAGs contained in milk (26). 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 (27). 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 (28). Current evidence demonstrates that fragmented HA polymers generated in damaged or inflamed tissue act as endogenous danger signals, or damage-associated molecular patterns (29C31), triggering localized WEHI-539 hydrochloride 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) (31, 32). HA fragments play a role in enhancing innate epithelial defense independent of the proinflammatory immunomodulation characteristic of macrophage (33), chondrocyte (34), or endothelial cell activation (35) by low molecular weight HA or the stimulation of TLR4 by bacterial pathogen-associated molecular patterns (36). A polydispersed HA WEHI-539 hydrochloride 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 (37) or from the epithelium-depleting effects of radiation (38). 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 (39), skin (40), and vagina (41). 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 (42, 43). 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 (44). Interestingly, microbes stimulate the expression of inducible -defensins 2, 3, and 4 in epithelium through the interaction of a variety of pathogen-associated molecular patterns PRKACG with TLRs (45C49). TLR4 regulates the expression of human -defensin 2 (HD2) in epithelium following stimulation with LPS (48). The same cell surface receptor, TLR4, mediates the induction of HD2, without an accompanying increase in inflammatory cytokine production, in human keratinocytes exposed to low molecular weight HA (40) and vaginal epithelium (41). Our group has recently demonstrated the TLR4-dependent induction of murine HD2 ortholog in colonic epithelium following the administration of synthetic, specific sized HA (39). Therefore, it is becoming increasingly clear that low WEHI-539 hydrochloride to intermediate molecular weight HA is an endogenous ligand capable of promoting enhanced antimicrobial defense of epithelial barriers through TLR4-dependent pathways. WEHI-539 hydrochloride Despite the growing evidence of the significant role of HA in bolstering.