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. 2017 Apr 4;25(4):856-867.e5.
doi: 10.1016/j.cmet.2017.03.007.

Farnesoid X Receptor Regulation of the NLRP3 Inflammasome Underlies Cholestasis-Associated Sepsis (V体育平台登录)

Haiping Hao  1 , Lijuan Cao  1 , Changtao Jiang  2 , Yuan Che  1 , Songyang Zhang  2 , Shogo Takahashi  3 , Guangji Wang  4 , Frank J Gonzalez  5
Affiliations

Farnesoid X Receptor Regulation of the NLRP3 Inflammasome Underlies Cholestasis-Associated Sepsis

Haiping Hao et al. Cell Metab. .

Abstract

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. VSports手机版.

Keywords: NLRP3 inflammasome; bile acids; cholestasis; damage-associated molecular patterns; farnesoid X receptor; protein-protein interaction; sepsis. V体育安卓版.

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Figures

Figure 1.
Figure 1.. Bile Acids Are Altered in LPS-Induced Sepsis
(A) Heatmap illustrating serum metabolite profiles in mice 1 hr after LPS treatment; the top 35 metabolite features were ranked by t test, distance measure was by Pearson correlation, and clustering was determined using the Ward algorithm. C1–C6 represents control mice and L1–L6 represents LPS-treated mice. (B) Three-dimensional principal component analysis (3D-PCA) of serum metabolome containing, overall, 77 markers 1 hr and 6 hr after LPS treatment in mice. (C) Variable importance in projection (VIP) plot identified by partial least squares discriminant analysis (PLS-DA) displaying the top 45 most important metabolite features in mice after 1 hr of LPS treatment. (D and E) Levels of the main bile acid species in serum (D) and PMs(E) in mice after 6 hr of LPS treatment determined by high-performance liquid chromatography-quadrupole time-of-flight tandem mass spectrometry (HPLC-triple/TOF-MS); data of PMs were normalized by protein concentrations. (F and G) mRNA levels of FXR and bile acid transporters in the liver of mice after 1 (F) or 6 hr(G)of LPS treatment; Gapdh mRNA was used as internal control (n = 6). Data are representative of two independent experiments and shown as mean ± SEM of six mice, **p < 0.01, *p < 0.05; two-tailed Student’s t tests. See also Figure S1.
Figure 2.
Figure 2.. Bile Acids Activate Both Signal 1 and 2 of the NLRP3 Inflammasome
(A-C) Bile acid activation of the NLRP3 inflammasome in PMs. Relative expression of Nlrp3 and Il-1β mRNAs (A). PMs were stimulated with 50 μM bile acids or 50 ng/mL LPS for 4 hr. Western blot analysis of caspase 1 and IL-1β in cells with (B) or without (C) LPS priming at 50 ng/mL for 2 hr. The concentration for all bile acids tested is 50 μM. (D-F) Synergistic effects of bile acids and ATP as assessed by western blot (D) and ELISA (E and F). LPS-primed PMs were stimulated with 25 μM bile acids for 4 hr with or without 0.5 mM ATP, which was spiked for 30 min. (G-J) ELISA analysis of IL-1β in BM D M s and peripheral blood mononuclear cells (PBMCs). Freshly prepared BM D M s (G) or differentiated BM D M s (H) were stimulated with bile acids (50 μM) for 4 hr, with or without LPS (500 ng/mL) priming for 2 hr. Human PBMCs (I) or human PBMC differentiated macrophages (J) were stimulated with bile acids (50 μM) for 4 hr, with or without LPS (500 ng/mL) priming for 2 hr. A total of 2 mM ATP treatment for 30 min was used as the positive control. GAPDH was used as internal standard/loading control in qPCR/immunoblot analyses. Data are representative of at least two independent experiments and expressed as mean ± SEM (n = 3). **p < 0.01, *p < 0.05 compared to control unless indicated otherwise; two-tailed Student’s t test. See also Figure S2.
Figure 3.
Figure 3.. Bile Acids Activate the NLRP3 Inflammasome in a Calcium Influx-Dependent Manner
(A-C) Intracellular calcium concentrations detected by a fluorescent probe Fluro-4AM together with probenecid; THP-1 cells were stimulated with 200 μM bile acids (A) and 2 mM ATP was spiked in 75 s before (B) or 150 s after bile acid treatment (C). Data are presented as mean value of n = 5; error bars were not shown for clarity. (D-F) LPS-prim edTHP-1 cells treated with 100 μM bile acid s for 4 hr with or with o u t 1 mM ATP spiked in at last30 min. Cells were pretreated with 50 μM BAPTA-AM or 2.5 μM ionomycin for 30 min. (G) Representative immunofluorescence images of calcium influx in THP-1 cells that were stimulated with 200 μM bile acids or 2 mM ATP for 5 min. MitoTracker and Fluro-4AM/probenecid were loaded 45 min prior to stimulation. (H and I) Representative immunofluorescence images of caspase 1 in THP-1 cells that were stimulated with 200 μM bile acids for 4 hr(H) or primed with LPS for 2 hr and then stimulated with 100 μM bile acids for 4 hr or 1 mM ATP for 30 min. Scale bars, 20 μm; one representative image of three independent experiments is shown. Data are representative of at least two independent experiments and expressed as mean ± SEM (n = 3). **p < 0.01, *p < 0.05 between indicated groups; n.s. represents no significance; two-tailed Student’s t test. See also Figure S3.
Figure 4.
Figure 4.. FXR and Bile Acid Axis Controls Septic Shock in Mice
(A) Total bile acids in serum of mice. (B, E, H, and K) Survival analyses of LPS-challenged mice, n = 10. Bile duct ligation-induced cholestasis (B), resin treatment (E), Fxr−/− (H), and Ad-FXR transfection (K). (C, F, I, and L) Serum levels of IL-1β. Bile duct ligation-induced cholestasis (C), resin treatment (F), Fxr−/− (I), and Ad-FXR transfection (L). (D, G, J, and M) Western blot analysis of lysates of PMs from mice. Bile duct ligation-induced cholestasis (D), resin treatment (G), Fxr−/− (J), and Ad-FXR transfection (M). GAPDH or β-actin was used as loading control (n = 3). Data are shown as mean ± SEM of six mice, **p < 0.01, *p < 0.05 compared to sham (A and C) or LPS (F and I) group; two-way ANOVA (A and C) or two-tailed Student’s t tests (F, I, and L). See also Figure S4.
Figure 5.
Figure 5.. The FXR Agonist GW-4064 Does Not Inhibit Septic Shock in Mice
(A) Survival analysis (n = 10). (B) Serum levels of IL-1β determined by ELISA. (C) Relative mRNA levels of Nlrp3, Pycard, Caspase-1, and Il-1β in PMs. (D) Western blot analysis of lysates of PMs. (E) Individual bile acids in serum. Mice were challenged with LPS at a dose of 30 mg/kg for6 hr with or without combination of 5 mg/kg GW −4064, which was intraperitoneally injected 30 min prior to LPS. Gapdh mRNA and GAPDH were used as internal standard/loading controls in qPCR and western blot analyses, respectively. Data are shown as mean ± SEM of six mice (B, C, and E), **p < 0.01; two-way ANOVA (C) or two-tailed Student’s t tests (B and E). Data are representative of two independent experiments. See also Figure S5.
Figure 6.
Figure 6.. FXR Negatively Regulates the NLRP3 Inflammasome
(A-C) Representative western blot analysis of Caspase-1 and IL-1β in PMs. PMs were stimulated with 0–200 μM D C A for 4 hr(A), or primed with 50 ng/mL LP S for 2 hr before treatment with 100 μM bile acids for an additional 4 hr (B), or treatment with 200 μM of various bile acids for 4 hr (C). (D) Representative immunofluorescence images of LPS-primed PMs that were stimulated with 50 μM bile acids for 4 hr or 0.5 mM ATP for 30 min. Scale bars, 20 μm. (E-G) The WT mice transplanted with the bone m arrow from WT and Fxr−/− mice. Mice were challenged with LPS at a dose of 20 mg/kg for 6 hr. (E) Diagnostic PCR for the genotype at the Fxr locus in the genomic DNA isolated from the circulating white blood cells and peripheral tissues. (F) The plasma levels of IL-1β. (G) Representative western blot analysis of IL-1β and Caspase-1 in PM s ex vivo. Data are shown as mean ± SEM, *p < 0.05; by two-tailed Student’s t tests, n = 7. Data are representative of two independent experiments; β-actin or tubulin was used as loading control in western blot analyses. See also Figure S6.
Figure 7.
Figure 7.. FXR Physically Interacts with CASPASE 1 and NLRP3
(A) Co-immunoprecipitation analysis of FXR interaction with NLRP3 inflammasome in THP-1 cells. (B) Influence of the FXR plasmid transfection. (C) Influence of FXR silencing. Proteins were immunoprecipitated with FXR-specific antibody or an IgG control. Precipitates were probed for FXR, NLRP3, ASC, and CASPASE 1; aliquots were blotted in parallel as a loading control. GAPDH was used as loading control in western blot analyses (n = 3). (D) Schematic illustration of the role and mechanism of FXR/bile acids in the regulation of NLRP3 inflammasome in cholestasis-associated sepsis.
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