Signal-induced NLRP3 phase separation initiates inflammasome activation

Abstract

NLRP3 inflammasome is activated by diverse stimuli including infections, intracellular and environmental irritants. How NLRP3 senses these unrelated stimuli and what activates NLRP3 remain unknown. Here we report that signal-dependent NLRP3 phase separation initiated its activation, in which the palmitoyltransferase ZDHHC7-mediated tonic NLRP3 palmitoylation and an IDR region in the FISNA domain of NLRP3 play important roles. Moreover, three conserved hydrophobic residues in the IDR critically mediate multivalent weak interactions. NLRP3-activating stimuli including K⁺ efflux and NLRP3-interacting molecules imiquimod, palmitate, and cardiolipin all cause NLRP3 conformational change and induce its phase separation and activation in cells and/or in vitro. Surprisingly, amphiphilic molecules like di-alcohols used to inhibit biomolecular phase separation and chemotherapeutic drugs doxorubicin and paclitaxel activate NLRP3 independently of ZDHHC7 by directly inducing NLRP3 phase separation. Mechanistically, amphiphilic molecules decrease the solubility of both palmitoylated and non-palmitoylated NLRP3 to directly induce its phase separation and activation while NLRP3 palmitoylation reduces its solubility to some extent without activation. Therefore, ZDHHC7-mediated NLRP3 palmitoylation in resting cells licenses its activation by lowering the threshold for NLRP3 phase separation in response to any of the diverse stimuli whereas NLRP3 solubility-reducing molecules like di-alcohols and chemotherapeutic drugs activate NLRP3 directly. The signal-induced NLRP3 phase separation likely provides the simplest and most direct mechanistic basis for NLRP3 activation.

Fig. 1. ZDHHC7-mediated NLRP3 palmitoylation is required for NLRP3 activation.

a Enriched genes by the genome-wide CRISPR-Cas9 screening. Genes in NF-κB pathway are shown as green spots, genes known in NLRP3 inflammasome are shown as red spots, and genes (red font) with no known function in NLRP3 pathway are shown as red spots. b NLRP3 activation in WT or ZDHHC7^–/– THP-1 cells. Cells were pretreated with 10 ng/mL Pam2CSK4 for 3 h, followed by 4 μM nigericin treatment for another 1 h. c NLRP3 activation in THP-1 cells pretreated with 2-BP or not before immunoblotting. Cells were pretreated with 1 μg/mL LPS for 2 h and then with the 2-BP (2.5, 5, 10 μM) for 1 h, followed by 4 μM nigericin treatment for another 1 h. d NLRP3 activation in the WT, ZDHHC7^–/– or ZDHHC7^–/– THP-1 cells reconstituted with ZDHHC7- or ZDHHS7-mCherry. ZDHHS7, S-acyltransferase catalytic mutant C¹⁶⁰S. Cells were treated as in (b). e LDH analysis in (d). f, g Palmitoylation of Flag-hNLRP3 in HEK293T cells with ZDHHC7 expression was detected by ABE (f) or APE assay (g). h HeLa cells stably expressing Flag-hNLRP3 were treated overnight with 2-BP (100 μM) as indicated. The hNLRP3 palmitoylation level was detected by ABE assay. i Palmitoylation of endogenous NLRP3 in WT or ZDHHC7^–/– THP-1 cells detected by ABE assay. j B16 cells expressing Flag-mNLRP3 with indicated combined mutations (Supplementary information, Fig. S3e). The mNLRP3 palmitoylation level was detected by APE assay. k Palmitoylation of Flag-hNLRP3-WT, Flag-hNLRP3-C¹³⁰S, or Flag-hNLRP3-C²⁶¹S in HEK293T cells with ZDHHC7 expression was detected by ABE assay. l NLRP3 activation in NLRP3^–/– or NLRP3^–/– THP-1 cells reconstituted with indicated NLRP3. Cells were pretreated with 1 μg/mL LPS for 3 h, followed by 4 μM nigericin treatment for another 1 h. m mNLRP3 activation in WT or Nlrp3^–/– immortalized BMDM (iBMDM) cells reconstituted with indicated Flag-mNLRP3. Cells were pretreated with 1 μg/mL LPS for 3 h, followed by 6 μM nigericin treatment for another 1 h. Statistical significance was indicated as follows: ns, not significant, ***P < 0.001.

Fig. 2. ZDHHC7 is required for NLRP3 inflammasome activation in vivo.

a, b mNLRP3 activation in WT or Zdhhc7^−/− BMDMs by ATP (a) or nigericin (b) treatment. Cells were pretreated with 1 μg/mL LPS for 3 h, followed by 5 mM ATP or 6 μM nigericin treatment for another 1 h. c, d Aim2 (c) or Nlrc4 (d) activation in WT and Zdhhc7^−/− BMDMs by poly(dA:dT) transfection or S. Typhimurium infection. Cells were pretreated with 1 μg/mL LPS for 3 h, followed by 1 μg/mL poly(dA:dT) transfection for 4 h or S. Typhimurium (MOI = 0.5) infection for 1 h. e, f PI staining (e) and percentage of PI-positive cells (f) in WT and Zdhhc7^−/− BMDMs treated as in (a–d). g WT or Zdhhc7^–/– mice (n = 5) were intraperitoneally injected with PBS or LPS (22.5 mg/kg) for 12 h. IL-1β and TNF-α in sera were detected by enzyme-linked immunosorbent assay (ELISA) analysis. h WT or Zdhhc7^–/– mice (n = 12) were intraperitoneally injected with LPS (22.5 mg/kg); survival was calculated using the Mantel–Cox test. i WT mice (n = 5) were intraperitoneally injected with vehicle or 2-bromopalmitate (2-BP, 50 mg/kg) twice 24 h or 1 h before experiments, then subjected to intraperitoneal injection of PBS or LPS (22.5 mg/kg) for 12 h. IL-1β, TNF-α, and IL-6 in sera were detected by ELISA analysis. j WT mice (n = 12) were intraperitoneally injected with 2-BP and LPS as in (i), and survival was calculated using the Mantel–Cox test. Statistical significance was indicated as follows: ns, not significant, **P < 0.01, ***P < 0.001.

Fig. 3. ZDHHC7-mediated NLRP3 palmitoylation is required for NLRP3 aggregation.

a Images of HeLa cells stably expressing mNG-NLRP3 were taken at the indicated times after 8 μM nigericin treatment. Red arrows represent NLRP3 aggregates and white triangles represent vesicles. Scale bars, 4 μm. b Images or statistical histogram of HeLa cells stably expressing Flag-hNLRP3 pretreated with 50 μM 2-BP overnight followed by 8 μM nigericin treatment for another 1 h, and immunostained with anti-Flag antibody. Scale bar, 10 μm. The percentage of cells with NLRP3 condensates was quantified from at least 100 cells (n = 3, mean ± s.d., two-sided t-test). ND not detectable. c Images (left) and colocalization analysis (right) of WT, ZDHHC7^−/−, and PYCARD^−/− THP-1 cells pretreated with 1 μg/mL LPS for 3 h alone, or followed by 4 μM nigericin treatment for another 1 h, immunostained with anti-hNLRP3 or anti-TGN46 antibody. Scale bar, 10 μm. Quantitative analysis of co-localization along a white line was shown. White arrows, colocalized TGN46 and hNLRP3. Yellow arrows, NLRP3-NEK7-ASC specks. ZDHHC7 and ASC expression was analyzed by western blotting assay using antibodies against ZDHHC7, ASC and GAPDH (right). d HeLa WT, ZDHHC7^–/–, and ZDHHC7^–/– cells reconstituted with ZDHHC7-mCherry (ZDC7) or ZDHHS7-mCherry (ZDS7) were stimulated with 8 μM nigericin for 1 h, and immunostained with anti-Flag antibody before imaging. e Percentage of cells with hNLRP3 aggregates in (d). f Palmitoylation of Flag-hNLRP3 in WT, ZDHHC7^–/–, or ZDHHC7^–/– HeLa cells reconstituted with ZDHHC7- or ZDHHS7-mCherry was detected by ABE assay. g Images (left) or percentage of cells with hNLRP3 aggregates (right) in HeLa cells stably expressing indicated Flag-hNLRP3 after nigericin treatment. Cells were treated with 8 μM nigericin for 1 h, immunostained with anti-Flag antibody. h Images (right) or percentage of cells with mNLRP3 aggregates (left) in HeLa cells stably expressing indicated Flag-mNLRP3 mutants after nigericin treatment. Cells were treated with 8 μM nigericin for 1 h, and immunostained with anti-Flag antibody. i Images (left) and statistical diagram (right) of mNG-hNLRP3 aggregates in WT and ABHD13^−/− HeLa cells left untreated (Con) or treated with 8 μM nigericin for 1 h before live cell imaging. Statistical significance was indicated as follows: ns, not significant, *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 4. Condensation but not vesicle localization is required for NLRP3 activation.

a Live cell images of HeLa cells stably expressing mNeonGreen (mNG)-hNLRP3 or mNeonGreen-hNLRP3-3K/A mutant after 1 h treatment with 8 μM nigericin. Scale bars, 10 μm. b, c Images (b) and colocalization analysis (c) of HeLa cells stably expressing mNG-hNLRP3 after 8 μM nigericin or 40 μg/mL imiquimod for 1 h, immunostained with anti-TGN46 antibody. Quantitative analysis of co-localization along a white line was shown. d, e The same as (b, c) except HeLa cells stably expressing mNG-hNLRP3-3K/A were used. f Sequence information of mNLRP3-KKEE-2^nd, mNLRP3-KEKE-2^nd and mNLRP3-KEEE-2^nd mutants. g Live cell images of HeLa cells stably expressing indicated mNLRP3 mutants left untreated or treated with 8 μM nigericin for 1 h. h mNLRP3 activation in Nlrp3^–/– iBMDM cells reconstituted with indicated mNLRP3 mutants. Cells were pretreated with 1 μg/mL LPS for 3 h, followed by 6 μM nigericin for another 1 h. i Sequence information of hNLRP3, mNLRP3, Chimeric mNLRP3-4K/A-1 and Chimeric mNLRP3-4K/A-2. j Live cell images of HeLa cells stably expressing indicated Chimeric mNLRP3-4K/A in i. Cells were treated with 8 μM nigericin for 1 h. k mNLRP3 activation in Nlrp3^–/– iBMDM cells reconstituted with the indicated mNLRP3 mutants. Cells were pretreated with 1 μg/mL LPS for 3 h, followed by 6 μM nigericin for another 1 h. l Palmitoylation of the WT, C¹²⁶S, 4 K/A, Chim-1 or Chim-2 mNLRP3 in B16 cells detected by the ABE assay. m Images of HeLa cells stably expressing mNG-hNLRP3, mNG-hNLRP3 and dox-induced mScarlet-ASC, or mNG-hNLRP3-C²⁶¹S and dox-induced mScarlet-ASC at the indicated times after 8 μM nigericin treatment. Cells were treated with 1 μg/mL dox for 6 h before imaging. Scale bar, 10 μm. n Images of HeLa cells stably expressing mNG-hNLRP3 and mScarlet-hASC or mScarlet-hASC^PYD (1–91 AA). Cells were left untreated (Con, left) or treated with 8 μM nigericin (right) for 1 h before live cell imaging. Scale bar, 10 μm.

Fig. 5. NLRP3 phase separates upon activation.

a Images of HeLa cells stably expressing mNG-hNLRP3-3K/A were taken at the indicated times after 8 μM nigericin treatment. Red and yellow arrows represent the fusion and fission of NLRP3 aggregates, respectively. Scale bar, 2 μm. b FRAP of HeLa cells stably expressing mNG-hNLRP3, treated with nigericin for the indicated time. The white boxes indicate bleached sites. Scale bar, 1 μm. The normalized FRAP rate was shown on the right. s, second; n = 3. c HeLa cells stably expressing APEX-hNLRP3 -EGFP or APEX-hNLRP3-3K/A-EGFP (hN3-3K/A) were stimulated with 8 μM nigericin for 1 h before imaging by the transmission electron microscopy. The white boxes (a, b) indicate magnified regions of right images. Scale bars, as indicated. d In vitro NLRP3 droplet formation after diluting mNG-hNLRP3 protein to the indicated concentrations into a phase-separation buffer at room temperature (RT). e NLRP3 (1 μM) LLPS in low salt (60 mM KCl), high salt (250 mM KCl) or high salt with 1.75% PEG8000. Scale bar, 10 μm. Coomassie blue staining of purified mNG-hNLRP3 protein was shown on the right. f NLRP3 droplets formed as in e. Images of hNLRP3 aggregates before and after photobleaching. Scale bar, 10 μm. g Quantification of FRAP data in f. s, second; n = 5. h Live cell images (left) or percentage of cells with NLRP3 aggregates (right) in HeLa cells stably expressing mNG-hNLRP3 with indicated mutants after 40 μg/mL imiquimod treatment for 1 h. Scale bar, 10 μm. i Palmitoylation of the WT, Δ140–152, or C^130/261S hNLRP3 in HEK293T cells with ZDHHC7 expression detected by the ABE assay. j NLRP3 activation in NLRP3^–/– THP-1 cells reconstituted with the indicated hNLRP3 mutants. Cells were pretreated with 1 μg/mL LPS for 3 h, followed by 4 μM nigericin or 30 μg/mL imiquimod for another 1 h. k Live cell images (left) or percentage of cells with NLRP3 aggregates (right) in HeLa cells stably expressing mNG-hNLRP3 or mNG-hNLRP3-VFI/TYT. Cells were treated with 40 μg/mL imiquimod for 1 h. l NLRP3 activation in NLRP3^–/– THP-1 cells reconstituted with hNLRP3 or VFI/TYT. Cells were pretreated with 1 μg/mL LPS for 3 h, followed by 4 μM nigericin for another 1 h. m Phase-separation diagram of hNLRP3, KCl and NaCl at the indicated concentrations at RT after 1 min in phase-separation buffer.

Fig. 6. K⁺ efflux, imiquimod, cardiolipin or palmitate induces NLRP3 phase separation.

a NLRP3 activation in THP-1 cells in control DMEM (145 mM NaCl/5 mM KCl) or K⁺ free DMEM (150 mM NaCl) for 2 h. b, c Live cell images (b) or percentage of cells with NLRP3 aggregates (c) in HeLa cells stably expressing mNG-hNLRP3 as in a for 5 h. d Live cell images (left) or percentage of cells with NLRP3 aggregates (right) in HeLa cells stably expressing mNG-hNLRP3 treated with 40 μg/mL R848, 40 μg/mL imiquimod or 40 μg/mL CL097 for 1 h. e The structure of R848, imiquimod, and CL097. f Condensation of purified mNG-hNLRP3 (1 μM) in phase-separation buffer with 60 mM KCl in the presence of 100 μg/mL R848, 100 μg/mL imiquimod, or 100 μg/mL CL097. g Thermostability of NLRP3 at the indicated temperatures in the presence of 1% DMSO (Con), 100 μg/mL R848, 100 μg/mL imiquimod (up), or 100 μg/mL CL097 (bottom). h Live cell images of HeLa cells stably expressing mNG-hNLRP3 or Δ94–134 treated with 40 μg/mL imiquimod or CL097 for 1 h. Scale bar, 10 μm. i Thermostability of NLRP3 or NLRP3 Δ94–134 at the indicated temperatures in the presence of 1% DMSO (Con), 100 μg/mL imiquimod (IMQ, top), or 100 μg/mL CL097 (bottom). j, k Live cell images (left) or percentage of cells with NLRP3 vesicles or aggregates (right) in HeLa cells stably expressing mNG-hNLRP3-WT treated with 8 μM nigericin or 40 μg/mL imiquimod for 1 h (j) or cells with mNG-hNLRP3-3K/A treated with 8 μM nigericin (k) in the absence or presence of 20 μM MCC950. l In vitro NLRP3 (1 μM) LLPS in a phase-separation buffer in the presence of 1 mM MCC950. m Immunoblotting analysis of NLRP3 activation in THP-1 cells pretreated with 2 μM MCC950, 10 μM parthenolide (PTL), 10 μM Bay11-7082, 10 μM MNS or 20 μM CY-09 for 30 min, and then treated with 1 μg/mL LPS for 3 h, followed by 4 μM nigericin treatment for another 1 h. n Live cell images of HeLa cells stably expressing mNG-hNLRP3 (top) or mNG-hNLRP3 -3K/A (bottom) untreated (Control), or pretreated with DMSO, 2 μM MCC950, 10 μM PTL, 10 μM Bay11-7082, 10 μM MNS or 20 μM CY-09 for 30 min, followed by 40 μg/mL imiquimod (top) or 8 μM nigericin treatment (bottom) for 1 h. Scale bar, 10 μm. o In vitro NLRP3 LLPS as in l with the indicated concentrations of KCl and cardiolipin. p Images (left) or percentage of cells with NLRP3 aggregates (right) in HeLa cells stably expressing mNG-hNLRP3 treated with 120 μM cardiolipin for 5 h before live cell imaging. q Thermostability of NLRP3 at the indicated temperatures in the presence of 5% methanol (Con), 20 μM or 50 μM cardiolipin. r In vitro NLRP3 LLPS as in l with indicated concentrations of KCl and palmitate acid. s Images (left) or percentage of cells with NLRP3 aggregates (right) in HeLa cells stably expressing mNG-hNLRP3 treated with 100 μM palmitate for 3 h before live cell imaging. t Thermostability of NLRP3 at the indicated temperatures in the presence of 3% methanol and 1% chloroform (Con), 200 μM or 500 μM palmitate acid.

Fig. 7. Amphiphilic molecules induce NLRP3 phase separation and activation.

a In vitro NLRP3 LLPS as in Fig. 6l with the indicated amphiphilic molecules at different concentrations (7.5% or 10%). Scale bar, 10 μm. b The solubility of NLRP3 in whole cell lysates of HEK293T cells expressing Flag-hNLRP3 in the presence of indicated concentration of amphiphilic di-alcohols including 1,5-PD and 1,2-PD. Precipitated NLRP3 (Pre. NLRP3) was detected by immunoblotting. c The solubility of NLRP3 in cell lysates of WT and ZDHHC7^−/− THP-1 cells in the presence of the indicated concentrations of ammonium sulfate (g/100 mL whole cell lysate) was analyzed. Experiment was conducted as in (b). d NLRP3 activation in WT or NLRP3^–/– THP-1 cells treated with 1 μg/mL LPS for 3 h, followed by 4 μM nigericin for another 1 h, or cells pretreated with 1 μg/mL LPS for 30 min, followed by 1,5-PD (1.2%, 2.5%) or 1,2-PD (0.6%, 1.2%) treatment for another 3 h. e NLRP3 activation in THP-1 cells treated by 4 μM nigericin, 2.5% 1,5-PD or 1.2% 1,2-PD in the presence of different concentrations of KCl (5, 10, 20 mM). f NLRP3 activation in the WT or ZDHHC7^–/– THP-1 cells treated by nigericin (4 μM), 1,5-PD (2.5%) or 1,2-PD (1.2%) as in (d). g NLRP3 activation in THP-1 cells treated by nigericin in the presence of 1,5-PD, 1,2-PD or not. Cells were pretreated with 1 μg/mL LPS for 2 h, followed by 0.6% 1,5-PD or 0.3% 1,2-PD treatment for 1 h, and 2 μM nigericin treatment for another 1 h. h Live cell images (left) or percentage of cells with NLRP3 aggregates (right) in HeLa cells stably expressing mNG-hNLRP3 with the indicated treatment, 0.5% 1,2-PD or 0.5% 1,5-PD for 4 h, 0.1% HFIP or 20 μM doxorubicin for 3 h, or 5 μM paclitaxel for 4 h. i NLRP3 activation in WT, ZDHHC7^–/–, or NLRP3^–/– THP-1 cells pretreated with 0.2 μg/mL LPS for 3 h and 2 μM nigericin for another 1 h, or pretreated with 0.2 μg/mL LPS for 30 min and doxorubicin (DOX, 2.5 μM, 5 μM) for another 8 h. j NLRP3 solubility in cell lysate from HEK293T cells expressing Flag-hNLRP3 in the presence of indicated concentration of doxorubicin. Precipitated NLRP3 (Pre. NLRP3) was detected by immunoblotting.