Improved outcome in shigellosis associated with butyrate induction of an endogenous peptide antibiotic

Abstract

Shigella is a major cause of morbidity, mortality, and growth retardation for children in developing countries. Emergence of antibiotic resistance among Shigellae demands the development of effective medicines. Previous studies found that the endogenous antimicrobial peptide LL-37 is down-regulated in the rectal epithelium of patients during shigellosis and that butyrate up-regulates the expression of LL-37 in colonic epithelial cells in vitro and decreases severity of inflammation in experimental shigellosis. In this study, Shigella-infected dysenteric rabbits were treated with butyrate (0.14 mmol/kg of body weight) twice daily for 3 days, and the expression levels of the rabbit homologue to LL-37, CAP-18, were monitored in the colon. Butyrate treatment resulted in (i) reduced clinical illness, severity of inflammation in the colon, and bacterial load in the stool, (ii) significant up-regulation of CAP-18 in the surface epithelium, and (iii) disappearance of CAP-18-positive cells in lamina propria. The active CAP-18 peptide was released in stool from its proform by butyrate treatment. In healthy controls, CAP-18 expression was localized predominantly to the epithelial surface of the colon. In infected rabbits, CAP-18 expression was localized to immune and inflammatory cells in the colon, whereas the ulcerated epithelium was devoid of CAP-18 expression. The combination of CAP-18 and butyrate was more efficient in killing Shigella in vitro than CAP-18 alone. Our findings indicate that oral butyrate treatment in shigellosis may be of clinical value because of induction of the endogenous cathelicidin CAP-18 in the colonic epithelium, stimulation of the release of the active peptide CAP-18, and promoting elimination of Shigella.

Conflict of interest statement

Conflict of interest statement: No conflicts declared.

Figures

Fig. 1.

Kaplan–Meier survival curves of infected rabbits by time after treatment with butyrate or saline. Compared with infected rabbits, butyrate-treated rabbits had earlier reduction of RBCs in stool.

Fig. 2.

Immunohistochemical staining of CAP-18 in paraffin-embedded sections of rabbit colon. All sections were counterstained with hematoxylin. (A) Immunoreactive signals for CAP-18 (brown) in healthy rabbits were almost exclusively located in the SE. (B) In Shigella-infected rabbits, surface and crypt epithelia were almost devoid of CAP-18 staining (arrowheads); abundant CAP-18-expressing inflammatory cells (arrows) were seen in the LP. (C) Reappearance of CAP-18 staining in the SE and disappearance of CAP-18-expressing cells from the LP in infected rabbits treated with butyrate.

Fig. 3.

The relative expression of CAP-18 transcript in colonic biopsies from healthy, Shigella-infected, and butyrate-treated rabbits was measured by quantitative real-time PCR. Each bar represents data from one rabbit. For each rabbit, the first bar represents proximal colon, and the second bar represents distal colon. The last bar represents distal colon from only one rabbit. Under each bar, histological grades of inflammation are expressed as mild, 1; moderate, 2; severe, 3; and normal, 0 histology.

Fig. 4.

Western blot analysis of CAP-18 in stool extracts from healthy, Shigella-infected and butyrate-treated rabbits with the CAP-18-specific polyclonal antisera. Lanes 1 and 2 show a marker and the synthetic CAP-18 peptide, respectively. A single band corresponding to the pro-CAP-18 protein appears in the stool of healthy (lanes 3 and 4) and Shigella-infected (lane 5) rabbits 1 day postinfection. Two immunoreactive bands are visualized at day 2 postinfection (lane 6), showing presence of both pro-CAP-18 and active CAP-18 peptides in stool. Butyrate treatment resulted in release of the active CAP-18 peptide in stool (lanes 7–10); day-1 and day-2 stool specimens (lanes 7 and 8) also contained the pro-CAP-18 protein.

Fig. 5.

Inhibitory effects of various concentration of CAP-18 in presence (dotted line) or absence (solid line) of 40 mM butyrate (4.4 mg/ml) on the growth of Shigella bacterial colonies.