. measured for 8 days. The feces were collected every 2 days for 8 days. Ileal and fecal lipids were extracted as described in the Supplemental Methods. Triglycerides were measured with a triglyceride colorimetric assay kit (Bioassay Systems). Free fatty acids (FFAs) were measured using reagents from Wako.Statistics. Experimental values are presented as the mean ± SD. Statistical analyses were performed using the 2-tailed Student's t test and 1-way ANOVA with Tukey's confirmation. Weighted UniFrac analysis to assess changes in bacterial abundance was performed on the Galaxy web-based platform. Statistical models including PCA, PLS-DA, and OPLS-DA were established to represent the major latent variables in the data matrix. P values of less than 0.05 were considered significant. Study approval. All animal studies were performed in accordance with the Institute of Laboratory Animal Resources guidelines and approved by the IACUC of the NCI.

Cholestasis is a common complication of sepsis, and the increased plasma levels of bile acids are predictive of sepsis-associated mortality. However, the exact mechanism by which cholestasis aggravates sepsis development remains elusive. Here, we show that bile acids are danger-associated molecular patterns (DAMPs) that can activate both signal 1 and 2 of the NLRP3 inflammasome in inflammatory macrophages. Mechanistically, bile acids induce a prolonged calcium influx and activate the NLRP3 inflammasome synergistically with ATP. Experimental cholestasis sensitizes, while cholestyramine, a bile acid sequestrant, protects mice from LPS-induced sepsis. FXR negatively regulates the NLRP3 inflammasome via physical interaction with NLRP3 and caspase 1. Fxr-null mice are more sensitive, while FXR-overexpressing mice are more resistant, to endoxemia shock. These findings suggest that bile acids and FXR play pivotal roles in sepsis via controlling the NLRP3 inflammasome, and that targeting FXR may represent a therapeutic strategy for cholestasis-associated sepsis.

Bile acids are synthesized from cholesterol in the liver and subjected to multiple metabolic biotransformations in hepatocytes, including oxidation by cytochromes P450 (CYPs) and conjugation with taurine, glycine, glucuronic acid, and sulfate. Mice and rats can hydroxylate chenodeoxycholic acid (CDCA) at the 6β-position to form α-muricholic acid (MCA) and ursodeoxycholic acid (UDCA) to form β-MCA. However, MCA is not formed in humans to any appreciable degree and the mechanism for this species difference is not known. Comparison of several Cyp-null mouse lines revealed that α-MCA and β-MCA were not detected in the liver samples from Cyp2c-cluster null (Cyp2c-null) mice. Global bile acid analysis further revealed the absence of MCAs and their conjugated derivatives, and high concentrations of CDCA and UDCA in Cyp2c-null mouse cecum and feces. Analysis of recombinant CYPs revealed that α-MCA and β-MCA were produced by oxidation of CDCA and UDCA by Cyp2c70, respectively. CYP2C9-humanized mice have similar bile acid metabolites as the Cyp2c-null mice, indicating that human CYP2C9 does not oxidize CDCA and UDCA, thus explaining the species differences in production of MCA. Because humans do not produce MCA, they lack tauro-β-MCA, a farnesoid X receptor antagonist in mouse that modulates obesity, insulin resistance, and hepatosteatosis.