Fidaxomicin and OP-1118 Inhibit Clostridium difficile Toxin A- and B-Mediated Inflammatory Responses via Inhibition of NF-κB Activity

Abstract

Clostridium difficile causes diarrhea and colitis by releasing toxin A and toxin B. In the human colon, both toxins cause intestinal inflammation and stimulate tumor necrosis factor alpha (TNF-α) expression via the activation of NF-κB. It is well established that the macrolide antibiotic fidaxomicin is associated with reduced relapses of C. difficile infection. We showed that fidaxomicin and its primary metabolite OP-1118 significantly inhibited toxin A-mediated intestinal inflammation in mice in vivo and toxin A-induced cell rounding in vitro We aim to determine whether fidaxomicin and OP-1118 possess anti-inflammatory effects against toxin A and toxin B in the human colon and examine the mechanism of this response. We used fresh human colonic explants, NCM460 human colonic epithelial cells, and RAW264.7 mouse macrophages to study the mechanism of the activity of fidaxomicin and OP-1118 against toxin A- and B-mediated cytokine expression and apoptosis. Fidaxomicin and OP-1118 dose-dependently inhibited toxin A- and B-induced TNF-α and interleukin-1β (IL-1β) mRNA expression and histological damage in human colonic explants. Fidaxomicin and OP-1118 inhibited toxin A-mediated NF-κB phosphorylation in human and mouse intestinal mucosae. Fidaxomicin and OP-1118 also inhibited toxin A-mediated NF-κB phosphorylation and TNF-α expression in macrophages, which was reversed by the NF-κB activator phorbol myristate acetate (PMA). Fidaxomicin and OP-1118 prevented toxin A- and B-mediated apoptosis in NCM460 cells, which was reversed by the addition of PMA. PMA reversed the cytoprotective effect of fidaxomicin and OP-1118 in toxin-exposed human colonic explants. Fidaxomicin and OP-1118 inhibit C. difficile toxin A- and B-mediated inflammatory responses, NF-κB phosphorylation, and tissue damage in the human colon.

Keywords: Clostridium difficile infection; antibiotics; signaling.

FIG 1

Fidaxomicin and OP-1118 inhibit toxin A- and B-induced TNF-α mRNA expression in human colonic explants. (A) Dose response of toxin A- and B-induced TNF-α mRNA expression. DMSO was used as a solvent for fidaxomicin and OP-1118. (B) Dose response of fidaxomicin and OP-1118 in toxin A- and B-induced TNF-α mRNA expression. (C) Dose response of toxin A- and B-induced TNF-α protein expression. (D) Dose response of fidaxomicin and OP-1118 in toxin A- and B-induced TNF-α protein expression. Each group consisted of 6 fresh human colonic explants. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

FIG 2

Fidaxomicin and OP-1118 inhibit toxin A- and B-induced IL-1β mRNA expression in human colonic explants. (A) Dose response of toxin A- and B-induced IL-1β mRNA expression. (B) Dose response of fidaxomicin and OP-1118 in toxin A-and B-induced IL-1β mRNA expression. (C) Dose response of toxin A-and B-induced IL-1β protein expression. (D) Dose response of fidaxomicin and OP-1118 in toxin A- and B-induced IL-1β protein expression. Each group consisted of 6 fresh human colonic explants. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

FIG 3

Fidaxomicin and OP-1118 inhibit toxin A- and B-induced NF-κB phosphorylation in human colonic explants and mouse ileal loops. The phosphorylated NF-κB signal in human colonic tissues (A) and mouse ileal loops (B) is shown in brown. (A) Each group consisted of 6 fresh human colonic explants. (B) Each group consisted of 6 mice. Magnification, ×200. Black bars, 100 μm.

FIG 4

Fidaxomicin inhibits toxin A-mediated TNF-α expression via NF-κB inhibition. (A and B) Western blot showing the protein signals of phosphorylated NF-κB (pNF-κB), total NF-κB, and GAPDH in RAW264.7 macrophages. (C and D) TNF-α levels in conditioned medium of RAW264.7 macrophages. The results are pooled from three separate experiments. **, P < 0.01; ***, P < 0.001.

FIG 5

Fidaxomicin inhibits toxin A- and B-mediated epithelial cell damage in fresh human colonic explants via NF-κB inhibition. (Top) The histological structure of the treated human colonic tissues was evaluated by H&E staining at a ×200 magnification. Luminal epithelial cell linings were damaged in human colonic explants exposed to toxin A or toxin B. Arrows indicate the locations of epithelial damage. Black bars, 100 μm. (Bottom) Histology scores for human colonic explants. C. difficile toxins caused damage to the colonic mucosal epithelial layer. The data are pooled from 6 patients per group. ***, P < 0.001.