Low concentrations of human neutrophil peptide ameliorate experimental murine colitis

Abstract

Human neutrophil peptides (HNPs) not only have antimicrobial properties, but also exert multiple immunomodulatory effects depending on the concentration used. We have previously demonstrated that the intraperitoneal administration of high-dose HNP-1 (100 µg/day) aggravates murine dextran sulfate sodium (DSS)-induced colitis, suggesting a potential pro-inflammatory role for HNPs at high concentrations. However, the role of low physiological concentrations of HNPs in the intestinal tract remains largely unknown. The aim of this study was to examine the effects of low concentrations of HNPs on intestinal inflammation. We first examined the effects of the mild transgenic overexpression of HNP-1 in DSS-induced colitis. HNP-1 transgenic mice have plasma HNP-1 levels similar to the physiological concentrations in human plasma. Compared to wild-type mice treated with DSS, HNP-1 transgenic mice treated with DSS had significantly lower clinical and histological scores, and lower colonic mRNA levels of pro-inflammatory cytokines, including interleukin (IL)-1β and tumor necrosis factor (TNF)-α. We then injected low-dose HNP-1 (5 µg/day) or phosphate-buffered saline (PBS) intraperitoneally into C57BL/6N and BALB/c mice administered DSS. The HNP-1-treated mice exhibited significantly milder colitis with reduced expression levels of pro-inflammatory cytokines compared with the PBS-treated mice. Finally, we examined the in vitro effects of HNP-1 on the expression of cytokines associated with macrophage activation. Low physiological concentrations of HNP-1 did not significantly affect the expression levels of IL-1β, TNF-α, IL-6 or IL-10 in colonic lamina propria mononuclear cells activated with heat-killed Escherichia coli, suggesting that the anti-inflammatory effects of HNP-1 on murine colitis may not be exerted by direct action on intestinal macrophages. Collectively, our data demonstrated a biphasic dose-dependent effect of HNP-1 on DSS-induced colitis: an amelioration at low concentrations and an aggravation at high concentrations. Low concentrations of HNPs may contribute to the maintenance of intestinal homeostasis.

Figure 1

Human neutrophil peptide-1 (HNP-1) transgenic mice develop less severe dextran sulfate sodium (DSS)-induced colitis than wild-type mice. Wild-type mice and HNP-1 transgenic mice were treated with 2.5% DSS for 5 days (n=5/group). (A) Clinical disease activity index scores were assessed on day 5. (B) Representative photomicrographs of colon sections from (a) wild-type and (b) HNP-1 transgenic mice. Hematoxylin and eosin stain; ×100 magnification. (C) Histological scores of distal colon. Data are shown as the means ± SD. ^**P<0.01, significant differences relative to wild-type mice.

Figure 2

Dextran sulfate sodium (DSS)-treated human neutrophil peptide-1 (HNP-1) transgenic mice have lower colonic mRNA levels of pro-inflammatory cytokines than DSS-treated wild-type mice. The colonic expression levels of interleukin (IL)-1β, tumor necrosis factor (TNF)-α, IL-6 and monocyte chemoattractant protein (MCP)-1 in colon tissues of wild-type and HNP-1 transgenic mice (n=5/group) were examined by RT-qPCR. Data are shown as the means ± SD. *P<0.05, significant differences relative to wild-type mice.

Figure 3

Low-dose human neutrophil peptide-1 (HNP-1) ameliorates dextran sulfate sodium (DSS)-induced colitis in C57BL/6N and BALB/c mice. Phosphate-buffered saline (PBS) or 5 µg of HNP-1 was intraperitoneally administered to DSS-treated C57BL/6N mice from days 1 to 5 (n=5/group) and DSS-treated BALB/c mice from days 1 to 7 (n=6/group). (A) Clinical disease activity index scores assessed on day 5 in C57BL/6N mice and day 7 in BALB/c mice. (B) Representative photomicrographs of colon sections from PBS-treated and HNP-1-treated mice. Hematoxylin and eosin stain; ×100 magnification. (C) Histological scores of distal colon. Data are shown as the means ± SD. ^*P<0.05 and ^**P<0.01, significant differences relative to PBS-treated mice.

Figure 4

Low-dose human neutrophil peptide-1 (HNP-1) attenuates pro-inflammatory cytokines expression in the colon tissues of dextran sulfate sodium (DSS)-treated C57BL/6N and BALB/c mice. The colonic expression levels of interleukin (IL)-1β, tumor necrosis factor (TNF)-α, IL-6 and monocyte chemoattractant protein (MCP)-1 in phosphate-buffered saline (PBS)- and HNP-1-injected (A) C57BL/6N (n=5/group) and (B) BALB/c mice (n=6/group). The cytokine mRNA levels were examined by RT-qPCR. Data are shown as the means ± SD. ^*P<0.05 and ^**P<0.01, significant differences relative to PBS-treated mice.

Figure 5

In vitro effects of human neutrophil peptide (HNP) on cytokine expression and on the viability of colonic lamina propria mononuclear cells (LPMCs) activated with heat-killed Escherichia coli (E. coli). (A and C) The expression levels of interleukin (IL)-1β, tumor necrosis factor (TNF)-α, IL-6 and IL-10 in macrophage-enriched LPMCs incubated without or with heat-killed E. coli and various concentrations of (A) HNP-1 or (C) native HNPs for 6 h. The cytokine mRNA levels were measured by RT-qPCR. Data are the means ± SD from 3 experiments. ^*P<0.05 and ^**P<0.01, significant differences relative to E. coli-stimulated cells without HNP-1 treatment. (B and D) The viability of LPMCs incubated without or with heat-killed E. coli and various concentrations of (B) HNP-1 or (D) native HNPs for 24 h. The WST-8 assay was used to evaluate cell viability. Data are the means ± SD from 5 experiments. ^*P<0.05 compared with unstimulated conditions.