Bile Acid Administration Elicits an Intestinal Antimicrobial Program and Reduces the Bacterial Burden in Two Mouse Models of Enteric Infection

Abstract

In addition to their chemical antimicrobial nature, bile acids are thought to have other functions in the homeostatic control of gastrointestinal immunity. However, those functions have remained largely undefined. In this work, we used ileal explants and mouse models of bile acid administration to investigate the role of bile acids in the regulation of the intestinal antimicrobial response. Mice fed on a diet supplemented with 0.1% chenodeoxycholic acid (CDCA) showed an upregulated expression of Paneth cell α-defensins as well as an increased synthesis of the type-C lectins Reg3b and Reg3g by the ileal epithelium. CDCA acted on several epithelial cell types, by a mechanism independent from farnesoid X receptor (FXR) and not involving STAT3 or β-catenin activation. CDCA feeding did not change the relative abundance of major commensal bacterial groups of the ileum, as shown by 16S analyses. However, administration of CDCA increased the expression of ileal Muc2 and induced a change in the composition of the mucosal immune cell repertoire, decreasing the proportion of Ly6G⁺ and CD68⁺ cells, while increasing the relative amount of IgGκ⁺ B cells. Oral administration of CDCA to mice attenuated infections with the bile-resistant pathogens Salmonella enterica serovar Typhimurium and Citrobacter rodentium, promoting lower systemic colonization and faster bacteria clearance, respectively. Our results demonstrate that bile acid signaling in the ileum triggers an antimicrobial program that can be potentially used as a therapeutic option against intestinal bacterial infections.

Keywords: Citrobacter rodentium; Salmonella enterica; bile acids; intestinal antimicrobial peptides; mucins; mucosal immunity.

FIG 1

Bile acids induce the expression of AMPPs in ileal explants. Shown are the relative transcript levels of Defa genes in ileal explants treated with various bile acids. TCA, taurocholic acid; TCDCA, taurochenodeoxycholic acid; CA, cholic acid; CDCA, chenodeoxycholic acid; DCA, deoxycholic acid; LCA, lithocholic acid. The expression levels in explants treated with vehicle controls are set at 1 and indicated by a dotted line. Data were obtained by qPCR. n = 6 to 8 samples per group. Statistically significant differences are shown by asterisks (*, P < 0.05).

FIG 2

CDCA induces the synthesis of AMPPs in ileal explants independently of TLR4 activation. Shown are the relative transcript levels of AMPP genes in ileal explants treated with 5 μM CDCA, 10 nM E. coli LPS, or a combination of both. Data were obtained by qPCR. n = 12 to 14 samples per group. Statistically significant differences are shown by asterisks (*, P < 0.05; **, P < 0.01; ****, P < 0.0001).

FIG 3

CDCA induces the synthesis of ileal AMPPs in vivo. (A) Relative transcript levels of AMPP genes in the ileum of animals fed on a 0.1% CDCA-supplemented diet in comparison to animals fed on a normal diet for 16 h. Data were obtained by qPCR. n = 11 to 13 samples per group. Statistically significant differences are shown by asterisks (*, P < 0.05; **, P < 0.01). (B) Reg3b and Reg3g Western blots of total ileum lysates from two animals each fed with CDCA or on a normal diet (ND). (C to E) Immunofluorescent microscopy of ileal tissues from animals fed CDCA or normal diet stained for Reg3g (C), Reg3b (D), or lysozyme (E). Scale bars are 25 μm.

FIG 4

CDCA does not induce inflammation but alters the relative abundance of major immune cell types of the ileal mucosa. Animals were fed on a CDCA-supplemented diet or a normal diet (ND) for 16 h. (A) H&E staining of ileal cross sections. Scale bars are 100 μm. (B) FACS dot plots of CD68⁺, Ly6G⁺, CD4⁺, and IgGκ⁺ cells from the ileal mucosa; the results shown are from one representative animal. (C) Percentage of CD68⁺, Ly6G⁺, CD4⁺ and IgGκ⁺ cells from the ileal mucosa (CDCA, n = 5 animals; normal diet, n = 4 animals). Statistically significant differences are shown by asterisks (*, P < 0.05; **, P < 0.01).

FIG 5

Alcian blue staining of ileal sections from mice fed on a normal diet (ND) (A) or a CDCA-supplemented diet (B). Scale bars are 100 μm. (C) Relative transcript levels of ileal Muc2 in animals fed with the CDCA-supplemented diet in comparison to animals fed a normal diet. Data were obtained by qPCR. n = 8 to 9 samples per group (*, P < 0.05).

FIG 6

CDCA feeding attenuates the bacterial burden of enteric infections. (A and B) Bacterial counts (CFU per milligram of tissue) in the liver (A) and spleen (B) of mice fed with CDCA-supplemented diet or on a normal diet (ND) and orally infected with Salmonella Typhimurium SL1344. Counts were taken at day 3 postinfection. n = 12 to 15 mice/group. (C) Bacteria counts in the feces (CFU per milligram of feces) of mice fed the CDCA-supplemented or normal diet and orally infected with Citrobacter rodentium DS100. Counts were taken up to day 28 postinfection. n = 10 to 11 mice/group (*, P < 0.05).