THE ROLE OF HYALURONAN IN INNATE INTESTINAL DEFENSE

Hyaluronan (HA) is a glycosaminoglycan polymer found in the extracellular matrix of all mammalian tissues and a component of human milk. Breast-feeding is associated with enhanced protection from gastrointestinal infection in infants. However, the function of naturally produced milk hyaluronan relevant to antimicrobial function was unknown. Recent work has suggested a role for small, fragmented HA polymers in initiating innate defense responses through interaction with innate molecular pattern recognition receptors such as TLR4. It was hypothesized that hyaluronan from human breast milk enhances innate intestinal epithelial antimicrobial defense. Synthetically produced HA fragments promote expression of the innate antimicrobial peptide human ß-defensin 2 (HßD2) in intestinal epithelial cells. Treatment of HT-29 colonic epithelial cells with HA fragment preparations resulted in time- and dose-dependent upregulated expression of HßD2 protein in a fragment size-specific manner, with 35 kDa HA fragment preparations emerging as the most potent inducers of intracellular HßD2. Furthermore, oral administration of specific-sized HA fragments promotes the expression of an HßD2 ortholog in the colonic epithelium of both wild-type and CD44-deficient mice, but not in TLR4-deficient mice. Thus, a highly size-specific, TLR4-dependent, innate defense response to fragmented HA contributes to intestinal epithelium antimicrobial defense through the induction of intracellular HßD2 protein. Having examined the induction of antimicrobial defense by synthetically produced HA, functional antimicrobial defense was evaluated following treatment of epithelium with HA isolated from human milk. The expression of milk HA was defined during the first 6 months postpartum among a cohort of 44 healthy donors. Treatment of HT-29 epithelium with preparations of human milk HA at physiologic concentrations resulted in a time- and dose-dependent induction of the antimicrobial peptide HßD2 that was abrogated by digestion of milk HA with a specific hyaluronidase. In addition, oral administration of human milk-derived HA to adult, wild-type mice resulted in induction of murine HßD2 ortholog in intestinal mucosa. Finally, treatment of cultured colonic epithelium with human milk HA preparations significantly enhanced resistance to infection by the enteric pathogen Salmonella enterica. Together, these observations suggest that maternally provided HA mediates one of the multiple protective antimicrobial defense mechanisms delivered through milk to the newborn.