1. Volume 45, Issue 4, Pages 802-816 (October 2016)
Bile Acids Control Inflammation and Metabolic Disorder through Inhibition of NLRP3 Inflammasome 
Chuansheng Guo, Shujun Xie, Zhexu Chi, Jinhua Zhang, Yangyang Liu, Li Zhang, Mingzhu Zheng, Xue Zhang, Dajing Xia, Yuehai Ke, Linrong Lu, Di Wang 
Immunity 
Volume 45, Issue 4, Pages (October 2016)
DOI: /j.immuni
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2. Immunity 2016 45, 802-816DOI: (10.1016/j.immuni.2016.09.008)
Copyright © 2016 Elsevier Inc. Terms and Conditions

3. Figure 1
Bile Acids Suppress NLRP3 Inflammasome-Mediated Caspase-1 Activation and IL-1β Secretion
(A) LPS-primed BMDMs were treated with the indicated BAs at 50 μM and then stimulated with nigericin for 45 min. Supernatants were analyzed by ELISA for IL-1β release.
(B–G) LPS-primed BMDMs were treated with different doses of LCA (B–D) and TLCA (E–G) and then stimulated with nigericin for 45 min. Supernatants (SN) and cell extracts (Lysate) were analyzed by immunoblotting (B and E). Supernatants were analyzed by ELISA for IL-1β (C and F) and IL-18 (D and G) release.
(H) LPS-primed BMDMs were treated with LCA (30 μM) and then stimulated with ATP, nigericin, Alum, or MSU. Supernatants (SN) and cell extracts (Lysate) were analyzed by immunoblotting.
(I and J) LPS-primed BMDMs were treated with LCA (30 μM) and then stimulated with nigericin, Salmonella typhimurium (Salmonella) infection, anthrax Lethal Toxin (Lethal toxin), or poly (dA:dT) transfection. Supernatants (SN) and cell extracts (Lysate) were analyzed by immunoblotting (I). Supernatants were analyzed by ELISA for IL-1β (J).
(K and L) Pam3CSK4-primed BMDMs were treated with LCA (30 μM) and then stimulated with LPS transfection. Supernatants (SN) and cell extracts (Lysate) were analyzed by immunoblotting (K). Supernatants were analyzed by ELISA for IL-1β (L).
∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < Values are mean ± SEM. Data are representative of three independent experiments.
See also Figure S1.
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4. Figure 2 Bile Acids Inhibit NLRP3 Inflammasome Activation via TGR5
(A–F) LPS-primed BMDMs were treated with different doses of INT-777 (A–C) and betulinic acid (D–F) and then stimulated with nigericin. Supernatants (SN) and cell extracts (Lysate) were analyzed by immunoblotting (A and D). Supernatants were analyzed by ELISA for IL-1β (B and E) and IL-18 (C and F) release.
(G and H) LPS-primed BMDMs from Gpbar1+/+ and Gpbar1−/− mice were treated with 20 μM LCA and then stimulated with nigericin. Supernatants (SN) and cell extracts (Lysate) were analyzed by immunoblotting (G). Supernatants were analyzed by ELISA for IL-1β release (H).
(I) LPS-primed BMDMs were treated with 30 μM INT-777 and then stimulated with nigericin. ASC oligomerization in cross-linked cytosolic pellets was analyzed by immunoblotting.
(J) Representative immunofluorescence images and quantification of ASC speck formation in LPS-primed BMDMs stimulated with nigericin in the presence of 30 μM INT-777. Scale bars represent 200 μm (panel) and 20 μm (inset).
(K) LPS-primed BMDMs were treated with 30 μM INT-777 and then stimulated with nigericin. The NLRP3-ASC interaction was analyzed by immunoprecipitation and immunoblotting.
∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < Values are mean ± SEM. Data are representative of three independent experiments.
See also Figure S2.
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5. Figure 3
Bile Acids Inhibit NLRP3 Inflammasome Activation via cAMP-PKA signaling
(A–D) LPS-primed BMDMs were treated with different doses of forskolin (A and B), isobutylmethylxanthine (IBMX) (C and D), or (R)-(-)-Rolipram (C and D) as indicated, and then stimulated with nigericin for 45 min. Supernatants (SN) and cell extracts (Lysate) were analyzed by immunoblotting (A and C). Supernatants were analyzed for IL-1β by ELISA (B and D).
(E and F) LPS-primed BMDMs were treated with different doses of KH7 for 30 min and then stimulated with nigericin in the presence of 30 μM INT-777. Supernatants (SN) and cell extracts (Lysate) were analyzed by immunoblotting (E). Supernatants were analyzed by ELISA for IL-1β (F).
(G–J) LPS-primed BMDMs were treated with different doses of H89 (G and H) or PKI (I and J) for 30 min before stimulation with nigericin in the presence of 30 μM INT-777. Supernatants (SN) and cell extracts (Lysate) were analyzed by immunoblotting (G and I). Supernatants were analyzed by ELISA for IL-1β (H and J).
∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < Values are mean ± SEM. Data are representative of three independent experiments.
See also Figure S3.
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6. Figure 4 Bile Acids and TGR5 Signaling Promote NLRP3 Ubiquitination
(A–C) LPS-primed BMDMs were treated with 30 μM LCA (A), INT-777 (A and C), or INT-747 (B) for 30 min. NLRP3 immunoprecipitates were analyzed for ubiquitination (A and B), K48-ubiquitination, and K63-ubiquitination (C).
(D and E) LPS-primed BMDMs were treated with 30 μM LCA in the presence of different doses of MG132 as indicated (D) or 100 nM bafilomycin A1 (E) for 30 min. NLRP3 immunoprecipitates were analyzed for ubiquitination.
(F) LPS-primed BMDMs were treated with 100 μM forskolin for 30 min. NLRP3 immunoprecipitates were analyzed for ubiquitination.
(G) HEK293T cells expressing Flag-NLRP3 and HA-Ubiquitin were treated with different doses of forskolin as indicated for 30 min. Flag-NLRP3 immunoprecipitates were analyzed for ubiquitination.
(H) HEK293T cells expressing Flag-NLRP3, Myc-PRKACA, and HA-Ubiquitin, HA-Ubiquitin K48-only, or HA-Ubiquitin K63-only were immunoprecipitated with anti-Flag for ubiquitination.
(I) HEK293T cells expressing Myc-PRKACA, HA-Ubiquitin, and Flag-NLRP3 PYD, NACHT, or LRR were immunoprecipitated with anti-Flag for ubiquitination.
Data are representative of three independent experiments.
See also Figure S4.
Immunity  , DOI: ( /j.immuni )
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7. Figure 5
TGR5-cAMP-PKA Signaling Promotes Ser 291 Phosphorylation of NLRP3
(A and B) LPS-primed BMDMs were treated with 30 μM LCA (A) or 30 μM INT777 (A and B) for 30 min. NLRP3 immunoprecipitates were analyzed for PKA phosphorylation (A and B) and ubiquitination (B).
(C and D) LPS-primed BMDMs were treated with different doses of INT777 (C) or forskolin (D) in the presence or absence of H89. NLRP3 expression was analyzed by SDS-PAGE with (+) or without (–) Phos-tag.
(E) HEK293T cells were transfected with Flag-NLRP3 and different doses of Myc-PRKACA. Flag-NLRP3 expression was analyzed by SDS-PAGE with Phos-tag.
(F and G) HEK293T cells expressing Flag-NLRP3, HA-Ubiquitin, and different doses of Myc-PRKACA (F) or Myc-PRKACA K72H (G), Flag-NLRP3 was immunoprecipitated with anti-Flag for PKA-induced phosphorylation and ubiquitination.
(H) Purified PRKACA directly phosphorylated purified Flag-NLRP3 with ATP-γ-S in vitro. The extent of phosphorylation was assessed by western blot using the anti-Thiophosphate ester antibody.
(I) HEK293T cells expressing Myc-PRKACA and Flag-NLRP3 ΔPYD, ΔNACHT, or ΔLRR were immunoprecipitated with anti-Flag for PKA-induced phosphorylation.
(J) HEK293T cells were transfected with Myc-PRKACA and different Flag-tagged NLRP3 mutants as indicated. Flag immunoprecipitates were analyzed for PKA-induced phosphorylation.
(K) HEK293T cells were transfected with Myc-PRKACA and Flag-NLRP3 S291A. Flag-NLRP3 S291A expression was analyzed by SDS-PAGE with Phos-tag.
(L) Alignment of NLRP3 orthologs. The depth of purple shading indicates the degree of residue conservation. Asterisk, mouse Ser 291.
(M) HEK293T cells expressing Myc-PRKACA and Flag-NLRP3 S291A were immunoprecipitated with anti-Flag for PKA-induced phosphorylation and ubiquitination.
(N) ELISA of IL-1β secreted by HEK293T cells 48 hr after reconstitution by transfection of HA-ASC, Myc-pro-caspase-1, pro-IL-1β, and Flag-NLRP3, Flag-NLRP3 S291A, Flag-NLRP3 S219D, or Flag-NLRP3 S291E as indicated.
(O) ELISA of IL-1β secreted by THP1-defNLRP3 cells stably reconstituted with NLRP3 wild-type, NLRP3 S291A, NLRP3 S219D, or NLRP3 S291E as indicated.
(P) HEK293T cells were transfected with Myc-PRKACA, HA- ubiquitin, and different CAPS-associated Flag-tagged NLRP3 mutants as indicated. Flag immunoprecipitates were analyzed for PKA-induced phosphorylation and ubiquitination.
∗∗p < 0.01, NS p > Values are mean ± SEM. Data are representative of three independent experiments.
See also Figure S5.
Immunity  , DOI: ( /j.immuni )
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8. Figure 6
Bile Acids and TGR5 Signaling Mitigate LPS-Induced Sepsis and Alum-induced Peritonitis via Suppression of the NLRP3 Inflammasome
(A–C) ELISA of IL-1β (A), IL-18 (B), and TNF-α (C) in serum from Nlrp3+/+ and Nlrp3−/− mice intraperitoneally injected with LPS (20 mg/kg body weight) with or without TLCA (30 mg/kg body weight).
(D–F) ELISA of IL-1β (E), IL-18 (F), and TNF-α (G) in serum from Gpbar1+/+ and Gpbar1−/− mice intraperitoneally injected with LPS (20 mg/kg body weight) with or without TLCA (30 mg/kg body weight).
(G) Wild-type, Nlrp3−/−, and Gpbar1−/− mice were intraperitoneally injected with LPS (20 mg/kg body weight) with or without TLCA (30 mg/kg body weight) for 4 hr. NLRP3 immunoprecipitates were analyzed for PKA-induced phosphorylation and ubiquitination.
(H–J) ELISA of IL-1β (A), IL-18 (B), and TNF-α (C) in serum from Nlrp3+/+ and Nlrp3−/− mice intraperitoneally injected with LPS (20 mg/kg body weight) with or without INT-777 (50 mg/kg body weight).
(K–M) ELISA of IL-1β (E), IL-18 (F), and TNF-α (G) in serum from Gpbar1+/+ and Gpbar1−/− mice intraperitoneally injected with LPS (20 mg/kg body weight) with or without INT-777 (50 mg/kg body weight).
∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, NS p > Values are mean ± SEM of five mice per group. Data are representative of three independent experiments.
See also Figure S6.
Immunity  , DOI: ( /j.immuni )
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9. Figure 7
Bile Acids and TGR5 Signaling Suppress NLRP3-Dependent Insulin Resistance in HFD-induced T2D
Nlrp3+/+ and Nlrp3−/− mice were fed with a normal diet (ND) or a high-fat diet (HFD) for 14 weeks with or without INT-777 treatment. Glucose tolerance (A and B) and insulin tolerance (C and D) were tested as indicated. Adipose tissue (WAT) (E–G) and liver (H–J) were isolated and cultured for 24 hr, and supernatants were analyzed by ELISA for the release of IL-1β (E and H), IL-18 (F and I), and TNF-α (G and J). Caspase-1 activation in WAT was analyzed by immunoblotting as indicated (K).
∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, NS p > Values are mean ± SEM of five mice per group. Data are representative of three independent experiments.
See also Figure S7.
Immunity  , DOI: ( /j.immuni )
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