Regulation of antibacterial defense in the small intestine by the nuclear bile acid receptor

Abstract

Obstruction of bile flow results in bacterial proliferation and mucosal injury in the small intestine that can lead to the translocation of bacteria across the epithelial barrier and systemic infection. These adverse effects of biliary obstruction can be inhibited by administration of bile acids. Here we show that the farnesoid X receptor (FXR), a nuclear receptor for bile acids, induces genes involved in enteroprotection and inhibits bacterial overgrowth and mucosal injury in ileum caused by bile duct ligation. Mice lacking FXR have increased ileal levels of bacteria and a compromised epithelial barrier. These findings reveal a central role for FXR in protecting the distal small intestine from bacterial invasion and suggest that FXR agonists may prevent epithelial deterioration and bacterial translocation in patients with impaired bile flow.

Fig. 1.

FXR is expressed in the intestine. (A) Total RNA was prepared from the indicated tissues of WT mice (n = 5), and FXR mRNA concentrations were measured by RTQ-PCR by using cyclophilin as the internal control. Data represent the mean ± SEM and are plotted as fold change relative to mRNA levels in duodenum. (B) In situ hybridization analysis was performed with an ³⁵S-labeled FXR antisense riboprobe and transverse sections of distal ileum from WT mice. Bright-field (Left) and dark-field (Right) images are shown. (Scale bar: 50 μm.)

Fig. 2.

Effects of BDL and GW4064 on serum bilirubin concentrations and FXR-regulated genes. (A) Total bilirubin was measured in serum from WT and FXR-KO mice (n = 5–9) subjected to sham operation and vehicle (Veh) treatment, BDL and vehicle treatment, or BDL and GW4064 (GW) treatment. The presence of different lowercase letters indicates statistical significance (P < 0.01) within each strain. (B–H) Total RNA was prepared from ileum of WT and FXR-KO mice (n = 5–9) subjected to sham operation and vehicle (Veh) treatment, BDL and vehicle treatment, or BDL and GW4064 (GW) treatment. Expression of the indicated genes was measured by RTQ-PCR by using cyclophilin as the internal control. Data represent the mean ± SEM and are plotted as fold change relative to mRNA levels in sham-operated mice treated with vehicle. The presence of different lowercase letters indicates statistical significance (P < 0.05) within each strain.

Fig. 3.

FXR activation blocks bacterial overgrowth and translocation. Aerobic (A, C, and E) and anaerobic (B, D, and F) bacteria were quantified in ileum (A and B), cecum (C and D), and mesenteric lymph node complexes (E and F) of WT and FXR-KO mice (n = 5–9) subjected to sham operation and vehicle (Veh) treatment, BDL and vehicle treatment, or BDL and GW4064 (GW) treatment. Data represent the mean ± SEM. The presence of different lowercase letters indicates statistical significance (P < 0.05) within each strain. Asterisks indicate statistical significance (P < 0.05) between sham-operated, vehicle-treated FXR-KO mice and sham-operated, vehicle-treated WT mice. CFU, colony-forming units.

Fig. 4.

FXR activation blocks mucosal injury. (A and B) Transverse sections of terminal ileum are shown for WT mice subjected to sham operation and vehicle treatment (WT/Sham/Veh), BDL and vehicle treatment (WT/BDL/Veh), or BDL and GW4064 treatment (WT/BDL/GW) and FXR-KO mice subject to sham operation and vehicle treatment (FXR-KO/Sham/Veh). The sections were immunostained with anti-occludin antisera (A) or stained with hematoxylin and eosin (H&E) (B). Occludin immunostaining (dark brown) is detected in the enterocytes of the villus epithelium. Notable examples of diminished occludin immunostaining are indicated by blue arrowheads in the WT/BDL/Veh section. Occludin immunostaining is drastically reduced in ileum from the FXR-KO/Sham/Veh mice. Edema and dilated lymphatic vessels are indicated by yellow arrowheads in hematoxylin and eosin (H&E)-stained sections. (Scale bars: 50 μm.) (C–F) Electron microscopy performed with sections of terminal ileum prepared from BDL WT mice (C and D) and sham-operated FXR-KO mice (E and F). C–E are oriented with the epithelial brush border at the top. Edema is indicated in C–E by yellow arrowheads. Bacteria in the mucosa are indicated in D and E by white arrowheads. (E Inset) A higher magnification of the bacterium and the surrounding edema. (F) Bacteria in the lymphatic vessels of a sham-operated FXR-KO mouse. (Inset) A higher magnification view of the bacterium indicated by the white arrowhead. (Scale bars: 2 μm.) (G) Neutrophils were quantified in transverse sections of terminal ileum prepared from WT and FXR-KO mice subjected to sham operation and vehicle treatment (Veh), BDL and vehicle treatment, or BDL and GW4064 (GW) treatment. Neutrophils were counted in 10 randomly chosen villi from four mice in each treatment group. The presence of different lowercase letters indicates statistical significance (P < 0.05) within each strain. The asterisk indicates statistical significance (P < 0.01) between sham-operated, vehicle-treated FXR-KO mice and sham-operated, vehicle-treated WT mice.