Efficacy of sodium butyrate adjunct therapy in shigellosis: a randomized, double-blind, placebo-controlled clinical trial

Rubhana Raqib, Protim Sarker, Akhirunnesa Mily, Nur Haque Alam, Abu Saleh Mohammed Arifuzzaman, Rokeya Sultana Rekha, Jan Andersson, Gudmundur H Gudmundsson, Alejandro Cravioto, Birgitta Agerberth, Rubhana Raqib, Protim Sarker, Akhirunnesa Mily, Nur Haque Alam, Abu Saleh Mohammed Arifuzzaman, Rokeya Sultana Rekha, Jan Andersson, Gudmundur H Gudmundsson, Alejandro Cravioto, Birgitta Agerberth

Abstract

Background: Treatment of shigellosis in rabbits with butyrate reduces clinical severity and counteracts the downregulation of cathelicidin (CAP-18) in the large intestinal epithelia. Here, we aimed to evaluate whether butyrate can be used as an adjunct to antibiotics in the treatment of shigellosis in patients.

Methods: A randomized, double-blind, placebo-controlled, parallel-group designed clinical trial was conducted. Eighty adult patients with shigellosis were randomized to either the Intervention group (butyrate, n = 40) or the Placebo group (normal saline, n = 40). The Intervention group was given an enema containing sodium butyrate (80 mM), twice daily for 3 days, while the Placebo group received the same dose of normal saline. The primary endpoint of the trial was to assess the efficacy of butyrate in improving clinical, endoscopic and histological features of shigellosis. The secondary endpoint was to study the effect of butyrate on the induction of antimicrobial peptides in the rectum. Clinical outcomes were assessed and concentrations of antimicrobial peptides (LL-37, human beta defensin1 [HBD-1] and human beta defensin 3 [HBD-3]) and pro-inflammatory cytokines (interleukin-1β [IL-1β] and interleukin-8 [IL-8]) were measured in the stool. Sigmoidoscopic and histopathological analyses, and immunostaining of LL-37 in the rectal mucosa were performed in a subgroup of patients.

Results: Compared with placebo, butyrate therapy led to the early reduction of macrophages, pus cells, IL-8 and IL-1β in the stool and improvement in rectal histopathology. Butyrate treatment induced LL-37 expression in the rectal epithelia. Stool concentration of LL-37 remained significantly higher in the Intervention group on days 4 and 7.

Conclusion: Adjunct therapy with butyrate during shigellosis led to early reduction of inflammation and enhanced LL-37 expression in the rectal epithelia with prolonged release of LL-37 in the stool.

Trial registration: ClinicalTrials.gov, NCT00800930.

Figures

Figure 1
Figure 1
Flow diagram for enrollment, allocation, follow-up and analysis of study patients according to CONSORT.
Figure 2
Figure 2
Rectal tissues from adult patients with shigellosis representing histology (A-C) and LL-37 immunostaining (D-F). (A) SE was completely eroded at the onset of disease (arrow heads). Huge appearance of blood (deep pink) due to hemorrhage was also evident in LP. (B) Partial recovery (arrow) of epithelial erosion (arrow head) and hemorrhage on day 7 following placebo treatments. (C) The epithelial erosion (arrows) and hemorrhage were completely healed by day 7 after treatment with butyrate. (D) Low expression of LL-37 in the SE (arrow heads) on day 1. (E) LL-37 expression in SE was still low (arrow heads) on day 7 after placebo treatment. (F) Treatment with butyrate led to higher and intense expression of LL-37 in SE (arrow heads) on day 7. SE: surface epithelium; LP: lamina propria. In figures A, B, C and F, bars equal to 100 μm. In figures D and E, bars equal to 50 μm.
Figure 3
Figure 3
Levels of Pro-inflammatory cytokines in the stool ofShigella-infected patients, treated with butyrate or placebo. Stool specimens were collected on indicated time points from Intervention (n = 39) and Placebo (n = 37) groups of patients. Concentrations of (A) IL-8 and (B) IL-1β in stool extracts were measured by enzyme linked immunosorbent assay (ELISA). Data are represented as mean ± SEM. Two-way repeated measure ANOVA was performed to determine significant interaction between butyrate and placebo therapy on different days, and when interaction was significant the Holm-Sidak post hoc comparison procedure was used to compare the effects of butyrate therapy on outcome measures. Significance: p≤ 0.05. Levels of IL-8 and IL-1β diminished significantly from day 1 to days 4 and 7 (≤ 0.001) in both groups. Diminution of IL-8 in the Intervention group was significantly higher than the Placebo group (p = 0.048). Attenuation of IL-1β in the Intervention group was higher but not significant compared with the Placebo group (p = 0.078). IL-8: interleukin-8; IL-1β: interleukin-1β.
Figure 4
Figure 4
LL-37 expression in the rectal mucosa of Intervention or Placebo groups of patients with shigellosis. Rectal biopsies were obtained on day 1 and day 7 from a subgroup of Shigella-infected patients, treated with butyrate (n = 15) or placebo (n = 11). Immunohistochemical detection of LL-37 was performed in paraffin sections. SE and LP were separately assessed for the quantification of LL-37 staining in each tissue section and the results were given as ACIA (Acquired Computerized Image Analysis) score. Lower and upper boundaries of the boxes and the horizontal bars in between indicate 25th percentile, 75th percentile and group median respectively. Single lines extending from the boxes represent lower and upper quartiles. Each circle above or below the boxes indicates one outlier. Two-way repeated measure ANOVA was performed to determine significant interaction between butyrate and placebo therapy on different days, and when interaction was significant the Holm-Sidak post hoc comparison procedure was used to compare the effects of butyrate therapy on outcome measures. Significance: p≤ 0.05. Expression of LL-37 in SE increased significantly from day 1 to day 7 in the Intervention group compared with the Placebo group (p = 0.04). There was no significant changes over time between groups in the expression of LL-37 in LP (p = 0.14). SE- surface epithelium; LP-lamina propria.
Figure 5
Figure 5
LL-37 level in the stool ofShigella-infected patients, treated with butyrate or placebo. Stool specimens were collected on indicated time points from Intervention (n = 39) or Placebo (n = 37) groups of patients. Level of LL-37 in stool extracts was measured by enzyme linked immunosorbent assay (ELISA). Data are represented as mean ± SEM. Two-way repeated measure ANOVA was performed to determine significant interaction between butyrate and placebo therapy on different days, and when interaction was significant the Holm-Sidak post hoc comparison procedure was used to compare the effects of butyrate therapy on outcome measures. Significance: p≤ 0.05. Concentration of LL-37 decreased significantly from day 1 to days 4 and 7 (< 0.001) in both groups. The decrease was significantly higher in the Placebo group than that in the Intervention group (p <0.001).

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