Specific-sized hyaluronan fragments promote expression of human β-defensin 2 in intestinal epithelium

Abstract

Hyaluronan (HA) is a glycosaminoglycan polymer found in the extracellular matrix of virtually all mammalian tissues. Recent work has suggested a role for small, fragmented HA polymers in initiating innate defense responses in immune cells, endothelium, and epidermis through interaction with innate molecular pattern recognition receptors, such as TLR4. Despite these advances, little is known regarding the effect of fragmented HA at the intestinal epithelium, where numerous pattern recognition receptors act as sentinels of an innate defense response that maintains epithelial barrier integrity in the presence of abundant and diverse microbial challenges. Here we report that HA fragments promote expression of the innate antimicrobial peptide human β-defensin 2 (HβD2) in intestinal epithelial cells. Treatment of HT-29 colonic epithelial cells with HA fragment preparations resulted in time- and dose-dependent up-regulated expression of HβD2 protein in a fragment size-specific manner, with 35-kDa HA fragment preparations emerging as the most potent inducers of intracellular HβD2. Furthermore, oral administration of specific-sized HA fragments promotes the expression of an HβD2 ortholog in the colonic epithelium of both wild-type and CD44-deficient mice but not in TLR4-deficient mice. Together, our observations suggest that a highly size-specific, TLR4-dependent, innate defense response to fragmented HA contributes to intestinal epithelium barrier defense through the induction of intracellular HβD2 protein.

FIGURE 1.

A, representative confocal micrographs show HβD2 expression in confluent cultures of HT-29 cells that were treated with medium alone or containing HA-35 (1 μm) for 12 h. Cells were fluorescently immunostained for HβD2 protein (red), and nuclei were stained with DAPI (blue). NS, immunostaining control in which no α-HβD2 antibody was utilized. B, mean secreted HβD2 as measured by ELISA in the culture medium of HT-29 cells treated for 9 h with medium alone or containing HA-35 (10 μm). No significant difference in secreted HβD2 peptide was detected between media or HA-treated cultures. C, 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 HA-35 (1 μm) at 3-h time intervals (0–24 h) in each experiment. Significance of differences in normalized HβD2 expression was evaluated by comparison of each time point with control treatment using Student's t test (***, p < 0.001). D, representative Western blots demonstrating HβD2 protein expression relative to GAPDH in the whole cell lysates of HT-29 cells treated with medium alone, medium containing HA-35 (10 μm), and medium containing HA-35 (10 μm) that was predigested with Streptococcus dysgalactiae hyaluronidase at 0.025 unit/ml for 16 h at 37 °C. Error bars, S.E.

FIGURE 2.

A, representative confocal micrographs of HT-29 cell cultures that were treated with medium alone, medium containing HA-35 (10 μm), or medium containing LPS (1 μg/ml) for 9 h. HβD2 protein is immunostained (red), and nuclei are stained with DAPI (blue). B, representative Western blot of HβD2 protein expression relative to GAPDH protein expression in whole cell lysates of HT-29 cells treated with medium alone, media supplemented with HA-35 (10 μm), or media containing LPS (1 μg/ml). C, average densitometric quantification of Western blot results of three experiments in which HT-29 cultures were treated with media alone, HA-35 (10 μm), or LPS (1 μg/ml) for 9 h. HβD2 protein expression is normalized to GAPDH protein in whole cell lysates. Significance of differences in normalized HβD2 expression was evaluated by comparison of each treatment with medium treatment using Student's t test (*, p < 0.05). Error bars, S.E.

FIGURE 3.

A, representative confocal micrographs of HT-29 cells treated with medium alone or equal mass (100 μg/ml) concentrations of HA-4.7, HA-35, or HA-74 for 6 h. Cells were fluorescently immunostained for HβD2 protein (red), and nuclei were stained with DAPI (blue). B, representative Western blot showing HβD2 protein relative to GAPDH protein expression in whole cell lysates of HT-29 cells treated with HA-4.7, HA-16, HA-35, or HA-74 at equal molar concentrations (10 μm). C, average densitometric quantification of immunoblots from four separate experiments in which HβD2 protein expression was evaluated relative to GAPDH protein expression in whole cell lysates of HT-29 cells. Confluent cultures of HT-29 cells were treated for 9 h with medium alone, or a range of concentrations (0.01–100 μm) of HA fragment preparations of different average molecular weight (HA-4.7, HA-16, HA-35, and HA-74). The solid black horizontal line indicates the mean HβD2/GAPDH ratio in medium-treated cells, and the surrounding gray-shaded region denotes the S.E. among replicate medium-treated samples. D, average densitometric quantification of immunoblots from four separate experiments in which HβD2 protein expression was evaluated relative to GAPDH protein expression in whole cell lysates of HT-29 cells. Confluent cultures of HT-29 cells were treated for 9 h with medium alone or containing HA-35 or HA-2M at equal mass concentrations (350 μg/ml). E, average densitometric quantification of immunoblots from two separate experiments, each with four replicates of each treatment group, in which HβD2 protein expression was evaluated relative to GAPDH protein expression in whole cell lysates of HT-29 cells. Confluent cultures of HT-29 cells were treated for 9 h with medium alone, medium containing equal molar concentrations (10 μm) of HA4.7 alone or HA-35 alone, a combination of HA-4.7 and HA-35 (10 μm each), HA-2M at equal mass concentration (350 μg/ml) relative to HA-35 alone (350 μg/ml = 10 μm for HA-35), or a combination of HA-2M and HA-35 (350 μg/ml each) for 8 h. Significance of differences in normalized HβD2 expression was evaluated by comparison of each treatment and dosage with medium treatment using Student's t test (*, p < 0.05; **, p < 0.01; ***, p < 0.001). N.S., not significant. F, molecular weight distribution and relative quantity of HA polymers in commercial HA fragment preparations used for in vitro experiments. The blue-shaded region indicates the portion of HA-35 fragment distribution that is enriched relative to other HA fragment preparations evaluated (∼20–45 kDa). Error bars, S.E.

FIGURE 4.

Representative confocal micrographs of distal colon cross-sections from adult mice provided with standard drinking water or drinking water supplemented with HA-35 (1 μm) ad libitum for 7 days. The mouse orthologue of HβD2, MuβD3, is fluorescently immunolabeled (red), and nuclei are stained with DAPI (blue). Bottom panels represent a more highly magnified portion of the lower power (top) field (as indicated by white boxes).

FIGURE 5.

A, representative fluorescent micrographs of epithelium of proximal colon sections from adult C57BL/6 wild-type mice. The mice were fed single daily doses of HA-4.7, HA-16, HA-35, HA-74, or HA-2M (300 μg/0.25 ml), and controls were given an equivalent volume of water alone by oral gavage for 3 consecutive days. MuβD3 is immunolabeled (shown in red), and nuclei are blue as a result of DAPI staining. NS, an immunostaining control in which no MuβD3 antibody was utilized. B, average scored MuβD3 staining intensity of proximal colon tissue sections from wild-type mice fed single daily doses of HA-4.7, HA-16, HA-35, HA-74, or HA-2M or an equivalent volume of water alone once daily for 3 consecutive days. Average MuβD3 staining intensity score represents 3 mice/group, with 3 stained sections/mouse, as judged by a blinded panel of four researchers on a scale of 0–4, with a score of 4 corresponding to peak MuβD3 staining for this data set. Significance of differences in mean MuβD3 staining intensity was evaluated using Student's t test in a pair-wise manner as indicated in the figure (*, p < 0.05). Error bars, S.E.

FIGURE 6.

A, representative fluorescent micrographs of epithelium of proximal colon sections from adult C57BL/6 wild-type, TLR4^−/−, and CD44^−/− mice. The mice were fed single daily doses of HA-28 (300 μg/0.25 ml), and controls were given an equivalent volume of water alone by gavage for 3 consecutive days. MuβD3 is immunolabeled (red), and nuclei are blue. B, average scored MuβD3 staining intensity of proximal colon tissue sections from wild-type, TLR4^−/−, and CD44^−/− mice fed HA-28 (300 μg) or an equivalent volume of water alone by oral gavage once daily for 3 consecutive days. Average MuβD3 staining intensity score represents 5 mice/group, with 3 stained sections/mouse, as judged by a blinded panel of four researchers on a scale of 0–4, with a score of 4 corresponding to peak MuβD3 staining. The mean staining intensity score of nonspecifically stained sections (1 section/mouse) was 0.24 ± 0.09. Significance of differences in mean MuβD3 staining intensity was evaluated using Student's t test in a pair-wise manner as indicated in the figure (*, p < 0.05; **, p < 0.01; ***, p < 0.001). N.S., not significant. Error bars, S.E.