First, GW4064 did not decrease aerobic or anaerobic bacteria counts in the ileum or cecum of FXR-KO mice ( Fig. Two important differences were seen between WT and FXR-KO mice. To determine whether FXR is required for the antibacterial actions of GW4064, the same set of experiments was performed in FXR-KO mice. Because both iNos and Il18 are induced by proinflammatory stimuli, including lipopolysaccharide and various cytokines ( 32, 33), this likely reflects regulation of these genes by additional signaling pathways that are activated by BDL. 5), there was a trend toward increased expression in BDL mice that occurred in an FXR-independent fashion ( Fig. Although both genes are induced by FXR (Fig. In contrast, the iNos and Il18 genes showed a more complex expression pattern ( Fig. None of these genes was induced by GW4064 in FXR-KO mice. GW4064 administration increased expression of these genes in BDL mice to levels above those seen in sham-operated animals. In WT mice, expression of Fgf15, Shp, Ang1, Car12, and Ibabp was reduced by BDL ( Fig. BDL also had marked effects on genes regulated by FXR in ileum. GW4064 administration had no effect on total serum bilirubin concentrations in BDL mice. Based on these data, the possibility that Il18 induction by FXR is pharmacological rather than physiological cannot be excluded.Īs expected, BDL caused jaundice manifested by increased total serum bilirubin concentrations in both WT and FXR-KO mice ( Fig. In contrast, FXR activation induced Il18 but did not contribute to its basal expression (Fig. For example, activation of FXR had little or no effect on Ibabp or iNos expression in WT mice, but its elimination caused a marked decrease in the basal expression of these genes (Fig. Notably, FXR had different effects on different genes. 5, which is published as supporting information on the PNAS web site). The regulation of all of these genes by FXR was confirmed by RTQ-PCR by using WT, CYP27-KO, and FXR-KO mice administered either GW4064 or vehicle (Fig. Carbonic anhydrase 12 (CAR12) is a member of a family of proteins involved in the maintenance of pH and ion balance ( 24). Although the function of RNASE4 is not established, ANG1 is part of the acute phase response to infection and has potent antibacterial and antimycotic actions ( 23).

Angiogenin (ANG1) and RNase A family member 4 (RNASE4) are closely related proteins that arise by differential splicing from a common gene locus ( 22).

The proinflammatory cytokine IL18 stimulates resistance to an array of pathogens, including intracellular and extracellular bacteria and mycobacteria, and appears to have a protective role during the early, acute phase of mucosal immune response ( 20, 21). NO, the product of inducible NO synthase (iNOS), has direct antimicrobial effects and regulates many different aspects of the innate immune response, including mucus secretion, vascular tone, and epithelial barrier function ( 18, 19).

Interestingly, several of the other genes in Table 1 have established roles in mucosal defense in the intestine. Among these were the established FXR target genes small heterodimer partner ( Shp), fibroblast growth factor 15 ( Fgf15), and ileal bile acid binding protein ( Ibabp), which encode proteins involved in bile acid homeostasis ( 9– 12, 16, 17). Microarray analysis yielded a list of 15 genes whose expression was changed ≥2-fold by GW4064 administration ( Table 1). RNA was prepared from ileal mucosa of CYP27-KO mice administered either the potent, synthetic FXR agonist GW4064 ( 15) or vehicle for 14 h. The CYP27-knockout (KO) mice produce only low levels of bile acids and thus are essentially devoid of endogenous FXR agonists ( 14). To gain insight into the function of FXR in the small intestine, transcriptional profiling experiments were done by using mice lacking the gene encoding sterol 27-hydroxylase (CYP27), a key enzyme for bile acid synthesis.

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