Human milk hyaluronan enhances innate defense of the intestinal epithelium

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.

Keywords: Cd44; Defensins; Epithelium; Host Defense; Human Milk; Hyaluronate; Salmonella Infection; Toll-like Receptors (TLR).

Figures

FIGURE 1.

Distribution of milk HA concentration by postpartum day in 1710 human milk samples collected from 44 unique donors during the first 6 months after delivery. A fourth degree polynomial regression curve was fitted to the entire data set and is indicated by the solid black line, with the 95% confidence interval of the curve represented by the dashed blue lines (R2 = 0.17, p = 6.1 × 10−67).

FIGURE 2.

A, average densitometric quantification of immunoblots from four individual experiments in which the abundance of HβD2 protein relative to GAPDH protein was evaluated in whole cell lysates of HT-29 cells. Replicate cultures were treated with 0.5 μg/ml milk HA for the time intervals indicated (0–48 h). B, representative Western blot demonstrating HβD2 protein expression relative to GAPDH in the whole cell lysates of HT-29 cells treated for 24 h with medium alone, medium containing milk HA isolate containing 0.5 μg/ml HA, or medium containing 0.5 μg/ml milk HA predigested with S. hyalurolyticus hyaluronidase at 0.25 unit/ml for 16 h at 60 °C. C, average densitometric quantification of immunoblots from four individual experiments, each separately using three donor-unique milk HA isolates in which the abundance of HβD2 protein relative to GAPDH protein was evaluated in whole cell lysates of HT-29 cells. Replicate cultures were treated for 24 h with medium alone, 0.5 μg/ml milk HA, or medium containing 0.5 μg/ml milk HA predigested with S. hyalurolyticus hyaluronidase. D, combined real-time quantitative PCR results from four individual experiments, each separately using three donor-unique milk HA isolates in which the abundance of DEFB4 mRNA relative to 18 S rRNA was evaluated in whole cell lysates of HT-29 cells. Replicate cultures were treated for 24 h with medium alone, 0.5 μg/ml milk HA, or medium containing 0.5 μg/ml milk HA predigested with S. hyalurolyticus hyaluronidase. Significance of differences in normalized HβD2 or DEFB4 expression was evaluated by comparison of each time point with control treatment, unless otherwise indicated by brackets, using Student's t test. *, p < 0.05; **, p < 0.01; ***, p < 0.001. E, average densitometric quantification of immunoblots from six individual experiments in which the abundance of HβD2 protein relative to GAPDH protein was evaluated in whole cell lysates of HT-29 cells. Replicate cultures were treated for 24 h with 0.001–5 μg/ml milk HA or medium containing 0.001–5 μg/ml milk HA predigested with S. hyalurolyticus hyaluronidase. Sigmoidal dose-response relationships are indicated, with the solid black curve corresponding to milk HA isolate treatment (EC50 = 0.03 μg/ml) and the dashed black line corresponding to hyaluronidase-digested milk HA treatment (EC50 = 5.94 μg/ml), with the observed difference between treatment responses being statistically significant by two-sided Student's t test analysis (p = 0.04). Error bars, S.E.

FIGURE 3.

A, representative fluorescent micrographs of epithelium in proximal colonic cross-sections from adult C57BL/6 wild-type mice. The mice were given single daily doses of 1 μg of milk HA in 0.25 ml of water (Milk HA) or 1 μg of milk HA that had been predigested with S. hyalurolyticus hyaluronidase in 0.25 ml of water (Milk HA + HAse) or water alone (Control) for 3 consecutive days. B, representative fluorescent micrographs of epithelium in intestinal cross-sections from newborn (day 1), nursing (day 10), or weaned (28 days) mice. The mouse ortholog of HβD2, MuβD3, is fluorescently immunolabeled (red), and nuclei are stained with DAPI (blue). NS, an immunostaining control in which no MuβD3 antibody was utilized.

FIGURE 4.

A, representative fluorescent micrographs of epithelium in proximal colon cross-sections from adult C57BL/6 wild-type, TLR4−/−, or CD44−/− mice. The mice were given single daily doses of milk HA containing 1 μg of HA in 0.25 ml of water or water alone (Control) by oral administration for 3 consecutive days. MuβD3 is fluorescently immunolabeled (red), and nuclei are stained with DAPI (blue). B, average scored MuβD3 staining intensity of proximal colon tissue sections from wild-type, TLR4−/−, or CD44−/− mice given single daily doses of 1 μg of milk HA in 0.25 ml of water or water alone (Control) by oral administration for 3 consecutive days. Average MuβD3 staining intensity score represents 5 mice/group, with 4 stained sections/mouse, as judged by a blinded panel of five researchers on a scale of 0–4 with a score of 4 corresponding to peak MuβD3 staining. Mean staining intensity score ± S.E. (error bars) of nonspecifically stained sections (1 section/mouse) was 0.26 ± 0.06. Significance of differences in mean MuβD3 staining intensity was evaluated using a two-tailed Student's t test. **, p < 0.01 for the comparison indicated by the bracket; ## and ###, p < 0.01 or p < 0.001, respectively, for the comparison with wild-type mice receiving milk HA.

FIGURE 5.

Cellular localization of CD44 and TLR4 in cultured HT-29 cells following 30-min HA treatment. Representative fluorescent micrographs of cultured HT-29 epithelium after 30-min treatment with medium alone, medium containing 0.5 μg/ml milk HA or 0.5 μg/ml milk HA that had been predigested with S. hyalurolyticus hyaluronidase, or medium containing 350 μg/ml hyaluronan with an average molecular mass of 35 kDa (HA-35). CD44 is fluorescently immunolabeled in red, and TLR4 is indicated by the green staining, whereas the nuclei are stained with DAPI (blue).

FIGURE 6.

A, average densitometric quantification of immunoblots from four individual experiments in which the abundance of HβD2 protein relative to GAPDH protein was evaluated in whole cell lysates of HT-29 cells. Replicate cultures were treated with medium alone, medium containing 350 μg/ml of a synthetic HA preparation with an average molecular mass of 35 kDa (HA-35), or medium containing 0.5 μg/ml milk HA for 24 h. Where indicated, HT-29 cultures were pretreated for 1 h with 2 μg/ml nonspecific mouse immunoglobulin (IgG) or 2 μg/ml mouse anti-human CD44 IgG (CD44) prior to the addition of medium, HA-35, or milk HA. B, relative quantification of total phosphorylated ERK1 and ERK2 by ELISA in cell lysates of HT-29 cells treated for 30 min with medium alone, medium containing 350 μg/ml HA-35, 0.5 μg/ml milk HA, or medium containing 0.5 μg/ml milk HA predigested with S. hyalurolyticus hyaluronidase. C, relative quantification of total phosphorylated JNK1, JNK2, and JNK3 by ELISA in cell lysates of HT-29 cells treated for 30 min with medium alone, medium containing 350 μg/ml HA-35, 0.5 μg/ml milk HA, or medium containing 0.5 μg/ml milk HA predigested with S. hyalurolyticus hyaluronidase. D, relative quantification of total phosphorylated p38 MAPK by ELISA in cell lysates of HT-29 cells treated for 30 min with medium alone, medium containing 350 μg/ml HA-35, 0.5 μg/ml milk HA or medium containing 0.5 μg/ml milk HA predigested with S. hyalurolyticus hyaluronidase. Significance of differences in protein expression was evaluated by comparison of each time point with control treatment (medium alone), unless otherwise indicated by brackets, using Student's t test. *, p < 0.05; **, p < 0.01. Error bars, S.E.

FIGURE 7.

A box plot representing the number of colony-forming units (CFU) of S. enterica serovar Typhimurium SL1344 collected from host HT-29 epithelium subject to pretreatment for 24 h with 0.5 μg/ml milk HA or 0.5 μg/ml milk HA predigested with S. hyalurolyticus hyaluronidase or medium alone.Horizontal lines indicate median colony-forming units with interquartile range denoted by the shaded box and the full range of observations represented by the error bars. Significance of differences in colony-forming units was evaluated as indicated by the brackets using Student's t test. ***, p < 0.001.