PLK4 deubiquitination by Spata2-CYLD suppresses NEK7-mediated NLRP3 inflammasome activation at the centrosome

Abstract

The innate immune sensor NLRP3 assembles an inflammasome complex with NEK7 and ASC to activate caspase-1 and drive the maturation of proinflammatory cytokines IL-1β and IL-18. NLRP3 inflammasome activity must be tightly controlled, as its over-activation is involved in the pathogenesis of inflammatory diseases. Here, we show that NLRP3 inflammasome activation is suppressed by a centrosomal protein Spata2. Spata2 deficiency enhances NLRP3 inflammasome activity both in the macrophages and in an animal model of peritonitis. Mechanistically, Spata2 recruits the deubiquitinase CYLD to the centrosome for deubiquitination of polo-like kinase 4 (PLK4), the master regulator of centrosome duplication. Deubiquitination of PLK4 facilitates its binding to and phosphorylation of NEK7 at Ser204. NEK7 phosphorylation in turn attenuates NEK7 and NLRP3 interaction, which is required for NLRP3 inflammasome activation. Pharmacological or shRNA-mediated inhibition of PLK4, or mutation of the NEK7 Ser204 phosphorylation site, augments NEK7 interaction with NLRP3 and causes increased NLRP3 inflammasome activation. Our study unravels a novel centrosomal regulatory pathway of inflammasome activation and may provide new therapeutic targets for the treatment of NLRP3-associated inflammatory diseases.

Keywords: CYLD; NEK7; NLRP3; Spata2; deubiquitination.

Figure 1. Spata2 is a negative regulator of inflammation

1. Survival of age‐ and sex‐matched (8–10 week old) Spata2 ^+/+ (WT) and Spata2 ^−/− (KO) mice injected intraperitoneally (i.p.) with LPS (20 mg/kg bodyweight). Data pooled from three independent experiments are shown. Statistical analysis was performed using long‐rank test.

2. ELISA of serum IL‐1β, IL‐18, TNFα, and IL‐6 at 12 h after injection of WT or Spata2‐KO mice with 20 mg/kg LPS. Circles and triangles represent individual mice. Horizontal bars indicate mean values. **P < 0.01; ****P < 0.0001. Statistical analysis was performed using unpaired two‐tailed Student's t‐test.

3. Immunoblot analysis of the indicated phosphorylated (P) and total proteins in lysates of LPS‐stimulated BMDMs derived from WT or Spata2‐KO mice. Molecular weights in kDa are indicated to the right.

4. qRT–PCR analysis of the indicated mRNAs in untreated or LPS‐stimulated WT or Spata2‐KO BMDMs. Bars and error bars represent the mean ± SD of triplicate experiments. Statistical analysis was performed using unpaired two‐tailed Student's t‐test.

5. ELISA of TNFα and IL‐6 in the supernatants from untreated or LPS‐stimulated WT or Spata2‐KO BMDMs. Bars and error bars represent the mean ± SD of triplicate experiments. Statistical analysis was performed using unpaired two‐tailed Student's t‐test.

Source data are available online for this figure.

Figure 2. Spata2 suppresses NLRP3 inflammasome activation in vitro and in vivo

1. A, B

   ELISA of IL‐1β secretion by WT or Spata2‐KO BMDMs that were either untreated (−) or LPS‐primed and then treated with the indicated NLRP3 inducers (A) or stimulated with the NLRC4 inducer (Salmonella infection) or AIM2 inducer (Poly(dA:dT) transfection) (B). Bars and error bars represent the mean ± SD of triplicate experiments. *P < 0.05; **P < 0.01; ***P < 0.001. Statistical analysis was performed using unpaired two‐tailed Student's t‐test.

2. C

   Immunoblot analysis of the mature IL‐1β p17 and the active caspase‐1 p20 in cell supernatants (Sup) and the indicated proteins in the cell extracts (Cell ext) of WT or Spata2‐KO BMDMs stimulated as in (A).

3. D

   WT and Spata2 CRISPR knockout (KO) iBMDMs stably expressing GFP‐ASC were primed with LPS and stimulated with Nigericin for 1 h. GFP‐ASC specks were imaged by confocal microscope, and percentage of ASC speck‐positive cells was quantified. Scale bar, 10 μm. Bars and error bars represent the mean ± SD of triplicate experiments. ****P < 0.0001. Statistical analysis was performed using unpaired two‐tailed Student's t‐test.

4. E

   BMDMs from Spata2 ^+/+ (WT) and Spata2 ^−/− (KO) mice were pretreated with caspase‐1 inhibitor, Z‐YVAD‐FMK, for 30 min, primed with LPS, and then stimulated with Nigericin for 1 h. ASC specks were assessed by immunofluorescence with antibody against ASC and imaged by confocal microscope, and percentage of ASC speck‐positive cells was quantified. Scale bar, 10 μm. Bars and error bars represent the mean ± SD of triplicate experiments. *P < 0.05. Statistical analysis was performed using unpaired two‐tailed Student's t‐test.

5. F

   Immunoblot analysis of ASC in soluble cell lysate and DSS cross‐linked insoluble fraction of WT or Spata2‐KO BMDMs primed with LPS and stimulated without or with nigericin (Nig) for 1 h. Relative levels of ASC in cross‐linked samples from LPS+ Nig treated cells were quantitated and shown below.

6. G‐I

   Spata2 ^+/+ (WT) and Spata2 ^−/− (KO) mice were injected intraperitoneally (i.p.) with alum (20 mg/kg bodyweight) and sacrificed 6 h latter to collect peritoneal lavages. Absolute numbers of neutrophils recruited to the peritoneum were counted by FACS (G); levels of IL‐1β (H) and IL‐6 (I) in the peritoneal fluids were measured by ELISA. Circles and triangles represent individual mice. Horizontal bars indicate mean values. *P < 0.05. Statistical analysis was performed using unpaired two‐tailed Student's t‐test.

Data information: For all Immunoblotting data, molecular weights in kDa are indicated to the left. Source data are available online for this figure.

Figure 3. CYLD suppresses NLRP3 inflammasome activation in a manner dependent on Spata2‐CYLD interaction

1. ELISA of IL‐1β secretion by WT or CYLD KO BMDMs that were either untreated (−) or LPS‐primed and then treated with the indicated NLRP3 inducers. Bars and error bars represent the mean ± SD of triplicate experiments. *P < 0.05; **P < 0.01; ***P < 0.001. Statistical analysis was performed using unpaired two‐tailed Student's t‐test. ND, not detected.

2. Immunoblot analysis of IL‐1β and active caspase‐1 p20 in cell supernatants (Sup) and the indicated proteins in cell extracts (Cell ext) from WT and CYLD KO BMDMs, stimulated as in (A).

3. BMDMs from WT and CYLD KO mice were pretreated with caspase‐1 inhibitor, Z‐YVAD‐FMK, for 30 min, primed with LPS, and then stimulated with Nigericin for 30 min. ASC specks were assessed by immunofluorescence with antibody against ASC, and the percentage of ASC speck‐positive cells was quantified. Bars and error bars represent the mean ± SD of triplicate experiments. *P < 0.05. Statistical analysis was performed using unpaired two‐tailed Student's t‐test.

4. LDH release from WT and CYLD KO BMDMs primed with LPS and stimulated as indicated. Bars and error bars represent the mean ± SD of triplicate experiments. **P < 0.01. Statistical analysis was performed using unpaired two‐tailed Student's t‐test.

5. Immunoblotting analysis of active caspase‐1 p20 and IL‐1β in cell supernatants (Sup) and the indicated proteins in cell extracts (Cell ext) of WT iBMDMs and Spata2 KO iBMDMs reconstituted with WT Spata2 or a Spata2 mutant (F108A) defective in CYLD binding that were primed with LPS and stimulated with Nigericin for 1 h.

Data information: For all Immunoblotting data, molecular weights in kDa are indicated to the left.Source data are available online for this figure.

Figure 4. Centrosomal localization of Spata2 is critical for recruiting CYLD to the centrosome and suppressing NLRP3 inflammasome activation

1. Confocal immunofluorescence analysis of Spata2 centrosome localization in HEK293 cells stably expressing control shRNA or shRNA against Spata2, stained with Spata2 and γ‐tubulin antibodies and the nuclear dye DAPI. Scale bar, 10 μm.

2. Confocal immunofluorescence analysis of Spata2 localization in RAW264.7 cells stably expressing GFP‐Spata2, stained with γ‐tubulin antibody and DAPI. Scale bar, 10 μm.

3. Confocal microscopy of HEK293 cells stably expressing GFP‐tagged Spata2 or Spata2 mutants (ΔPUB or Δ51–81) that were stained with γ‐tubulin antibody and DAPI.

4. Confocal microscopy of HEK293 cells stably expressing GFP‐tagged CYLD along with an empty vector or vectors encoding Flag‐tagged Spata2 or Spata2 mutants (ΔPUB or Δ51–81) stained as in (C). Scale bar, 10 μm.

5. Co‐immunoprecipitation analysis of CYLD physical interaction with Spata2 or Spata2 mutants (ΔPUB or Δ51–81) in HEK293 cells transfected with the indicated expression vectors. CYLD and Spata2 proteins were detected by immunoblot using anti‐HA and anti‐Flag antibodies, respectively.

6. Immunoblotting analysis of the active caspase‐1 p20 in cell supernatants (Sup) and pro‐caspase‐1 and Flag‐Spata2 proteins in cell extracts (Cell ext) from WT iBMDMs and Spata2 KO iBMDMs reconstituted with WT Spata2 or Spata2 mutant defective in centrosome localization (ΔPUB or Δ51–81). Cells were primed with LPS and stimulated with Nigericin for 1 h.

Data information: For all immunoblotting data, molecular weights in kDa are indicated to the left.Source data are available online for this figure.

Figure 5. Centrosomal kinase PLK4 suppresses NLRP3 inflammasome activation in a kinase activity‐dependent manner

1. A

   WT and Spata2 KO iBMDMs stably expressing an empty vector or a vector encoding Flag‐NEK7 were untreated (NT), primed with LPS for 4 h, or primed with LPS for 4 h followed by stimulation with Nigericin for 30 min. Flag‐NEK7 was immunoprecipitated, and the association of endogenous NLRP3 was assessed by immunoblotting. Relative levels of NLRP3 in immunoprecipitates were normalized to Flag‐NEK7 and shown below.

2. B

   Co‐immunoprecipitation analysis of PLK4 interaction with endogenous Spata2 and CYLD in parental HEK293 cells and HEK293 cells stably expressing GFP‐PLK4.

3. C

   iBMDMs stably expressing GFP‐PLK4 and parental iBMDMs were unstimulated or stimulated with 0.5 μg/ml LPS for 4 h. The PLK4 interaction with endogenous CYLD was analyzed by co‐immunoprecipitation with anti‐GFP antibody.

4. D

   Confocal microscopy analysis of ASC specks in iBMDMs stably expressing GFP‐ASC, pretreated for 2 days with DMSO and two different PLK4 inhibitors, Centrinone (150 nM) and Centrinone‐B(500 nM), primed with LPS for 4 h, and stimulated with Nigericin for 30 min. GFP‐ASC specks were imaged and quantified (> 500 cells counted). Scale bar, 10 μm. Bars and error bars represent the mean ± SD of triplicate experiments. *P < 0.05; ***P < 0.001. Statistical analysis was performed using unpaired two‐tailed Student's t‐test.

5. E, F

   Immunoblot analysis of the indicated proteins in cell supernatants (Sup) and cell extracts (Cell ext) of LPS‐primed and Nigericin‐stimulated BMDMs that were pretreated for 2 days with PLK4 inhibitors, Centrinone (150 nM) and Centrinone‐B (500 nM) (E), or of LPS‐primed and Nigericin‐stimulated iBMDMs stably expressing a control shRNA or three different PLK4‐specific shRNAs (F).

6. G

   Immunoblot analysis of active caspase‐1 (p20) generation in HEK293 cells that were transfected with inflammasome components (Flag‐tagged NLRP3, NEK7, ASC, and caspase‐1) along with EGFP, GFP‐tagged PLK4, PLK4 kinase dead mutant (D154A), or PLK1 and stimulated with 10 μM Nigericin for 1 h 24 h post‐transfection.

Data information: For all Immunoblotting data, molecular weights in kDa are indicated to the left.Source data are available online for this figure.

Figure 6. PLK4 inhibits NEK7‐NLRP3 interaction by mediating NEK7 phosphorylation at Ser204

1. WT and Spata2 CRISPR knockout (KO) iBMDMs stably expressing GFP‐ASC were pretreated with DMSO or PLK4 inhibitor Centrinone at a concentration of 150 nM for 2 days, primed with LPS, and stimulated with Nigericin for 1 h. GFP‐ASC specks were imaged by confocal microscope, and percentage of ASC speck‐positive cells was quantified. Bars and error bars represent the mean ± SD of triplicate experiments. *P < 0.05; **P < 0.01. Statistical analysis was performed using unpaired two‐tailed Student's t‐test. See Appendix Fig S9 for images of ASC specks and levels of IL‐1β and LDH.

2. Co‐immunoprecipitation analysis of NEK7/NLRP3 interaction in iBMDMs stably expressing Flag‐NEK7 that were transduced with control or PLK4‐specific shRNA. The cells were untreated, primed with LPS, or primed with LPS and stimulated with Nigericin. Flag‐NEK7 was immunoprecipitated, and the association of endogenous NLRP3 was assessed by immunoblot analysis. Relative levels of NLRP3 in immunoprecipitates were normalized to Flag‐NEK7 and shown below.

3. Co‐immunoprecipitation analysis of NEK7/NLRP3 interaction in HEK293 cells transfected with HA‐NEK7 and Flag‐NLRP3 along with (+) or without (−) GFP‐PLK4. The cells were either treated (+) or not treated (−) with the PLK4 inhibitor Centrinone.

4. iBMDMs stably expressing Flag‐tagged NEK7 or NEK7 mutant S204A were treated with LPS. Anti‐Flag immunoprecipitates were prepared for immunoblot analysis for the interaction between NEK7 and endogenous NLRP3.

5. Immunoblot analysis of active caspase‐1 (p20) in supernatants (Sup) and the indicated proteins in cell extracts (Cell ext) of iBMDMs stably expressing WT NEK7 or NEK7 mutant (S204A), primed with LPS and stimulated with nigericin.

6. iBMDMs stably expressing Flag‐tagged WT NEK7 or NEK7 mutant S204A were left untreated or stimulated with LPS for indicated time. Anti‐Flag immunoprecipitates were prepared for immunoblot analysis for NEK7 phosphorylation at Ser204 (P‐NEK7).

7. iBMDMs stably expressing Flag‐tagged WT NEK7 were left untreated or pretreated with PLK4 inhibitor Centrinone, and stimulated without or with LPS for 4 h. Anti‐Flag immunoprecipitates were prepared for immunoblot analysis for NEK7 phosphorylation at Ser204.

8. iBMDMs stably expressing GFP‐tagged PLK4 and parental iBMDMs were left untreated or stimulated with LPS for 4 h. Anti‐GFP immunoprecipitates were prepared for immunoblot analysis for the interaction between GFP‐PLK4 and endogenous NEK7.

Data information: For all Immunoblotting data, molecular weights in kDa are indicated to the left.Source data are available online for this figure.

Figure 7. Spata2 and CYLD regulate PLK4 ubiquitination and function in NLRP3 inflammasome inhibition

1. Co‐immunoprecipitation analysis of NEK7/NLRP3 interaction in HEK293 cells transfected with the indicated expression vectors, treated with either DMSO or 150 nM PLK4 inhibitor Centrinone.

2. WT and Spata2 KO iBMDMs stably expressing Flag‐tagged WT NEK7 were left untreated or pretreated with the PLK4 inhibitor Centrinone, and stimulated without or with LPS for 4 h. Anti‐Flag immunoprecipitates were prepared for immunoblot analysis for NEK7 phosphorylation at Ser204.

3. HEK293 cells stably expressing GFP‐PLK4Δ24 were transfected with a control siRNA or siRNAs targeting Spata2 or CYLD. GFP‐PLK4Δ24 was immunoprecipitated under denaturing conditions and immunoblotted for K63‐linked, K48‐linked, and linear ubiquitination. Relative levels of PLK4 ubiquitination were normalized to GFP‐PLK4 and shown below.

4. Co‐immunoprecipitation analysis of the PLK4‐NEK7 interaction in HEK293 cells co‐transfected with Flag‐NEK7 and HA‐PLK4 in the presence (+) or absence (−) of GFP, GFP‐Spata2, GFP‐CYLD, or GFP‐CYLD C601S, a catalytically inactive mutant.

5. A model of NLRP3 inflammasome regulation by PLK4 and Spata2/CYLD. Polyubiquitination of PLK4 suppresses PLK4 binding to and phosphorylation of NEK7. This polyubiquitination‐mediated suppression can be reversed by Spata2 and CYLD‐mediated deubiquitination of PLK4, thereby allowing for PLK4 phosphorylation of NEK7, which attenuates NEK7 binding to NLRP3 and NLRP3 inflammasome activation. Spata2 or CYLD deficiency would cause accumulation of PLK4 polyubiquitination, reduction in NEK7 phosphorylation, increase in NEK7 binding to NLRP3, and eventually hyperactivation of NLRP3 inflammasome.

Data information: For all Immunoblotting data, molecular weights in kDa are indicated to the left.Source data are available online for this figure.