AIM2 forms a complex with pyrin and ZBP1 to drive PANoptosis and host defence (original) (raw)

Data availability

The datasets generated and analysed during the current study are contained within the manuscript and the accompanying extended data figures. Source data are provided with this paper.

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Acknowledgements

We thank members of the Kanneganti laboratory for their comments and suggestions and R. Tweedell for scientific editing and writing support. _Mavs_−/− mutant mice were kindly provided by M. Gale. We thank M. Yamamoto for the _Trif_−/− mutant mouse strain. T.-D.K. is supported by NIH grants AI101935, AI124346, AI160179, AR056296 and CA253095 and by the American Lebanese Syrian Associated Charities. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Author information

Authors and Affiliations

1. Department of Immunology, St Jude Children’s Research Hospital, Memphis, TN, USA
   SangJoon Lee, Rajendra Karki, Yaqiu Wang, Lam Nhat Nguyen & Thirumala-Devi Kanneganti
2. Department of Structural Biology, St Jude Children’s Research Hospital, Memphis, TN, USA
   Ravi C. Kalathur

Authors

1. SangJoon Lee
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2. Rajendra Karki
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3. Yaqiu Wang
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4. Lam Nhat Nguyen
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5. Ravi C. Kalathur
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6. Thirumala-Devi Kanneganti
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Contributions

S.L., R.K. and T.-D.K. conceptualized the study; S.L. and R.K. designed the methodology; S.L., R.K., Y.W., L.N.N. and R.C.K. performed the experiments; S.L., R.K., Y.W. and L.N.N. conducted the analysis; S.L., R.K. and T.-D.K. wrote the manuscript. T.-D.K. acquired the funding and provided overall supervision.

Corresponding author

Correspondence toThirumala-Devi Kanneganti.

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The authors declare no competing interests.

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Extended data figures and tables

Extended Data Fig. 1 F. novicida induces AIM2-, Pyrin-, ZBP1-mediated caspase-1 activation, cytokine release and cell death.

a, Immunoblot analysis of pro–caspase-1 (CASP1; P45) and cleaved CASP1 (P20) in _F. novicida_-infected or poly(dA:dT)-transfected wild type (WT) or Aim2 −/− bone marrow-derived macrophages (BMDMs). b, Cell death in BMDMs after F. novicida infection for 16 h. Red indicates dead cells. c, Quantification of the cell death in (b). d–f, Immunoblot analysis of CASP1 (d), cell death images at 16 h post-infection (e), and cell death quantification (f) from WT or Nlrp3 −/− BMDMs after F. novicida infection or LPS plus nigericin (LPS + Ni) treatment. g–i, Immunoblot analysis of CASP1 (g), cell death images at 16 h post-infection (h), and cell death quantification (i) from WT or Nlrc4 −/− BMDMs after F. novicida or Salmonella Typhimurium infection. j–l, Immunoblot analysis of CASP1 (j), cell death images at 16 h post-infection (k), and cell death quantification (l) from WT or Mefv −/− BMDMs after F. novicida infection or C. difficile Toxin AB+ supernatant treatment. m–o, Immunoblot analysis of CASP1 (m), cell death images at 16 h post-infection (n), and cell death quantification (o) from WT or Zbp1 −/− BMDMs after F. novicida or influenza A virus (IAV) infection. a, d, g, j, m, Data are representative of at least three independent experiments. b, e, h, k, n, Images are representative of at least three independent experiments. Scale bar, 50 μm. c, f, i, l, o, Data are mean ± s.e.m. ns, not significant; ****P < 0.0001 (two-tailed t-test; n = 8 from 4 biologically independent samples). Exact P values are presented in Supplementary Table 1. For gel source data, see Supplementary Figure 1.

Source data

Extended Data Fig. 2 Innate immune sensors TLR3, MDA5, NLRP6 and NLRP12 and adaptors Trif and MAVS are not required for caspase-1 activation and cell death after HSV1 and F. novicida infections.

a, Immunoblot analysis of pro–caspase-1 (CASP1; P45) and cleaved CASP1 (P20) in HSV1-infected wild type (WT), Tlr3 −/− , Trif −/− or Asc −/− bone marrow-derived macrophages (BMDMs). b, Cell death in BMDMs after HSV1 infection for 16 h. Red indicates dead cells. c, Quantification of the cell death in (b). d–f, Immunoblot analysis of CASP1 (d), cell death images at 16 h post-infection (e), and cell death quantification (f) from WT, Mda5 −/− or Mavs −/− BMDMs after HSV1 infection. g–i, Immunoblot analysis of CASP1 (g), cell death images at 16 h post-infection (h), and cell death quantification (i) from WT, Nlrp6 −/− or Nlrp12 −/− BMDMs after HSV1 infection. j–l, Immunoblot analysis of CASP1 (j), cell death images at 16 h post-infection (k), and cell death quantification (l) from WT, Tlr3 −/− or Trif −/− BMDMs after F. novicida infection. m–o, Immunoblot analysis of CASP1 (m), cell death images (n), and cell death quantification (o) from WT, Mda5 −/− or Mavs −/− BMDMs after F. novicida infection. p–r, Immunoblot analysis of CASP1 (p), cell death images at 16 h post-infection (q), and cell death quantification (r) from WT, Nlrp6 −/− or Nlrp12 −/− BMDMs after F. novicida infection. a, d, g, j, m, p, Data are representative of at least three independent experiments. b, e, h, k, n, q, Images are representative of at least three independent experiments. Scale bar, 50 μm. c, f, i, l, o, r, Data are mean ± s.e.m. ns, not significant (one-way ANOVA with Dunnett’s multiple comparisons test; n = 8 from 4 biologically independent samples). Exact P values are presented in Supplementary Table 1. For gel source data, see Supplementary Figure 1.

Source data

Extended Data Fig. 3 ZBP1 cooperates with Pyrin to drive AIM2-mediated cell death and cytokine release.

a, Cell death in bone marrow-derived macrophages (BMDMs) after HSV1 infection with or without colchicine (Col). Red indicates dead cells. Data are representative of at least three independent experiments. Scale bar, 50 μm. b, Quantification of the cell death from (a). Data are mean ± s.e.m. ns, not significant; ****P < 0.0001 (one-way ANOVA with Dunnett’s multiple comparisons test; n = 12 from 3 biologically independent samples). c, Cell death in BMDMs after F. novicida infection with or without Col. Red indicates dead cells. Data are representative of at least three independent experiments. Scale bar, 50 μm. d, Quantification of the cell death from (c). Data are mean ± s.e.m. ns, not significant; ****P < 0.0001 (one-way ANOVA with Dunnett’s multiple comparisons test; n = 9 from 3 biologically independent samples). e–h, Release of IL-1β (e, g) or IL-18 (f, h) following HSV1 (e, f) or F. novicida (g, h) infections with or without Col. Data are mean ± s.e.m. ns, not significant; ****P < 0.0001 (one-way ANOVA with Dunnett’s multiple comparisons test; n = 6 from 3 biologically independent samples). Exact P values are presented in Supplementary Table 1.

Source data

Extended Data Fig. 4 Pyrin and ZBP1 are required for AIM2-mediated cell death following HSV1 infection, but not in response to poly(dA:dT).

a, b, Quantification of cell death in wild type (WT), Aim2 −/− , Mefv −/−, Zbp1 −/− or Mefv −/− Zbp1 −/− bone marrow-derived macrophages (BMDMs) over time during HSV1 (a) and F. novicida (b) infections. Data are mean ± s.e.m. *P < 0.05; ***P < 0.001; ****P < 0.0001 (one-way ANOVA with Dunnett’s multiple comparisons test; n = 4). Data are representative of at least three independent experiments. c, Cell death in THP-1 macrophages treated with control siRNA (Control) or siRNA targeted to AIM2 (AIM2 KD), MEFV (MEFV KD) and/or ZBP1 (ZBP1 KD) after HSV1 infection. Red indicates dead cells. Images are representative of at least three independent experiments. Scale bar, 50 μm. d, Quantification of the cell death from (c). Data are mean ± s.e.m. ****P < 0.0001 (one-way ANOVA with Dunnett’s multiple comparisons test; n = 4). Data are representative of at least three independent experiments. e, Immunoblot analysis of caspase-1 (CASP1) activation and AIM2, Pyrin and ZBP1 expression in the indicated THP-1 cells. Data are representative of two independent experiments. f, Cell death in WT, Aim2 −/− , Mefv −/−, Zbp1 −/− or Mefv −/− Zbp1 −/− BMDMs after poly(dA:dT) transfection. Red indicates dead cells. Images are representative of at least three independent experiments. Scale bar, 50 μm. g, Quantification of the cell death from (f). Data are mean ± s.e.m. ns, not significant; ****P < 0.0001 (one-way ANOVA with Dunnett’s multiple comparisons test; n = 9 from 3 biologically independent samples). Exact P values are presented in Supplementary Table 1. h, Immunoblot analysis of CASP1 in the indicated BMDMs after poly(dA:dT) transfection. Data are representative of at least three independent experiments. For gel source data, see Supplementary Figure 1.

Source data

Extended Data Fig. 5 AIM2 acts as an upstream regulator of RhoA modifications, and the ZBP1 Zα2 domain is required for cell death.

a, RhoA-GTP activity in wild type (WT), Aim2 −/−, Mefv −/− or Zbp1 −/− bone marrow-derived macrophages (BMDMs) infected with HSV1 or treated with TcdB for 12 h. Activity was normalized to total RhoA levels. Data are mean ± s.e.m. from three independent experiments. ns, not significant; ***P < 0.001; ****P < 0.0001 (one-way ANOVA with Dunnett’s multiple comparisons test; n = 3, 6, 7 or 9). b, c, Activated RhoA (RhoA-GTP) assessed using a pull-down assay with Rhotekin-RBD beads from WT, Aim2 −/− , Asc −/− or Casp1 −/− BMDMs infected with HSV1 (b) or F. novicida (c). Data are representative of at least three independent experiments. d, RhoA-GTP activity in WT BMDMs infected with HSV1 or transfected with poly(dA:dT) for 12 h. Data are mean ± s.e.m. from three independent experiments. ns, not significant; **P < 0.01 (one-way ANOVA with Dunnett’s multiple comparisons test; n = 3). e, Activated RhoA (RhoA-GTP) assessed using a pull-down assay with Rhotekin-RBD beads from WT, Aim2 −/− , Asc −/− or Casp1 −/− BMDMs transfected with poly(dA:dT). Data are representative of at least three independent experiments. f, g Cell death in WT, Zbp1 −/− , Zbp1 ∆Za2/∆Za2 or Ripk3 −/− BMDMs after HSV1 (f) or F. novicida (g) infections. Red indicates dead cells. Images are representative of at least three independent experiments. Scale bar, 50 μm. h, i, Quantification of the cell death from f (h) or g (i). Data are mean ± s.e.m. ****P < 0.0001 (one-way ANOVA with Dunnett’s multiple comparisons test; n = 9 from 3 biologically independent samples). Exact P values are presented in Supplementary Table 1. For gel source data, see Supplementary Figure 1.

Source data

Extended Data Fig. 6 The expression of Pyrin and ZBP1 is not regulated by GSDMD or caspase-8, -7, -3 or -6 during HSV1 or F. novicida infections.

a, Immunoblot analysis of caspase-1 (CASP1) activation and ZBP1 and Pyrin expression in wild type (WT) or Gsdmd −/− bone marrow-derived macrophages (BMDMs) after HSV1 or F. novicida infection. Data are representative of at least three independent experiments. b, c, Immunoblot analysis of ZBP1, Pyrin and AIM2 expression in the indicated BMDMs after HSV1 (b) or F. novicida (c) infections. Data are representative of at least three independent experiments. d, Immunoblot analysis of ZBP1, Pyrin and AIM2 expression in the indicated BMDMs after influenza A virus (IAV) infection. Data are representative of at least three independent experiments. For gel source data, see Supplementary Figure 1.

Extended Data Fig. 7 The expression of Pyrin and ZBP1 is regulated by AIM2 during HSV1 and F. novicida infections.

a–d, Relative expression of Zbp1 (a, b) and Mefv (c, d) in wild type (WT), Aim2 −/− , Asc −/− or Casp1 −/− bone marrow-derived macrophages (BMDMs) after HSV1 (a, c) or F. novicida (b, d) infections. Expression presented relative to that of the control gene Gapdh. Data are mean ± s.e.m. from three independent experiments. ****P < 0.0001 (one-way ANOVA with Dunnett’s multiple comparisons test, n = 6). e, f, Release of IFN-β following HSV1 (e) or F. novicida (f) infections. Data are mean ± s.e.m. ****P < 0.0001 (one-way ANOVA with Dunnett’s multiple comparisons test; n = 6 from 3 biologically independent samples). Exact P values are presented in Supplementary Table 1. g, h, Immunoblot analysis of ZBP1 and Pyrin expression in the indicated BMDMs after HSV1 (g) or F. novicida (h) infections with or without IFN-β treatment. Data are representative of at least three independent experiments. For gel source data, see Supplementary Figure 1.

Source data

Extended Data Fig. 8 Loss of AIM2 or combined loss of Pyrin and ZBP1 prevents the formation of the AIM2 complex during HSV1 and F. novicida infections.

a, b, Immunoprecipitation (IP) in wild type (WT), Aim2 −/− , Mefv −/− , Zbp1 −/− or Mefv −/− Zbp1 −/− bone marrow-derived macrophages (BMDMs) with either IgG control antibodies or anti-ASC antibodies after HSV1 (a) or F. novicida (b) infection. Data are representative of three independent experiments. c, d, IP in WT, Ripk3 −/− , Ripk3 −/− Casp8 −/− or Ripk3 −/− Fadd −/− BMDMs with either IgG control antibodies or anti-ASC antibodies after HSV1 (c) or F. novicida (d) infection. Data are representative of three independent experiments. For gel source data, see Supplementary Figure 1.

Extended Data Fig. 9 ASC speck colocalizes with AIM2, Pyrin and ZBP1, caspase-8 and RIPK3 in the same cell during HSV1 and F. novicida infections, and formation of this complex drives cell death.

a, Immunofluorescence images of wild type (WT) bone marrow-derived macrophages (BMDMs) at 12 h after F. novicida infection. Scale bars, 5 μm. Arrowheads indicate the ASC speck. Images are representative of three independent experiments. b, Quantification of the percentage of cells with ASC+AIM2+Pyrin+ZBP1+specks among the ASC speck+ cells. Data are mean ± s.e.m. ****P < 0.0001 (one-way ANOVA with Dunnett’s multiple comparisons test; n = 6 from 3 biologically independent samples). c, Immunofluorescence images of WT BMDMs at 12 h after HSV1 infection. Scale bars, 5 μm. Arrowheads indicate the ASC speck. Images are representative of three independent experiments. d, Quantification of the percentage of cells with ASC+RIPK3+CASP8+ specks among the ASC speck+ cells. Data are mean ± s.e.m. ****P < 0.0001 (one-way ANOVA with Dunnett’s multiple comparisons test; n = 6 from 3 biologically independent samples). e, Immunofluorescence images of WT BMDMs at 12 h after F. novicida infection. Scale bars, 5 μm. Arrowheads indicate the ASC speck. Images are representative of three independent experiments. f, Quantification of the percentage of cells with ASC+RIPK3+CASP8+ specks among the ASC speck+ cells. Data are mean ± s.e.m. ****P < 0.0001 (one-way ANOVA with Dunnett’s multiple comparisons test; n = 6 from 3 biologically independent samples). g, Cell death in WT, Ripk3 −/− Casp8 −/− or Ripk3 −/− Fadd −/− BMDMs at 16 h post-infection with HSV1 or F. novicida. Red indicates dead cells. Data are representative of at least three independent experiments. Scale bar, 50 μm. h, Quantification of the cell death from (g). Data are mean ± s.e.m. ****P < 0.0001 (one-way ANOVA with Dunnett’s multiple comparisons test; n = 9 from 3 biologically independent samples). Exact P values are presented in Supplementary Table 1.

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Extended Data Fig. 10 AIM2 regulates Pyrin and ZBP1 expression in vivo, and AIM2 provides host protection against HSV1 and F. novicida.

a, b, Immunoblot analysis of pro- (P45) and activated (P20) caspase-1 (CASP1), pro- (P53) and activated (P30) gasdermin D (GSDMD), pro- (P55) and cleaved (P18) caspase-8 (CASP8), pro- (P35) and cleaved (P17/P19) caspase-3 (CASP3), pro- (P35) and cleaved (P20) caspase-7 (CASP7), phosphorylated MLKL (pMLKL), total MLKL (tMLKL), ZBP1, Pyrin and AIM2 in lung from uninfected animals (PBS) or wild type (WT) or Aim2 −/− mice 3 days after HSV1 (a) or F. novicida (b) infection. Each lane indicates independent biological replicates. c, Viral quantification in WT, Aim2 −/− , Mefv −/− , Zbp1 −/− or Mefv −/− Zbp1 −/− BMDMs at 16 h post-infection with HSV1. Data are mean ± s.e.m. ****P < 0.0001 (one-way ANOVA with Dunnett’s multiple comparisons test; n = 6 from 3 biologically independent samples). d, Bacterial quantification in WT, Aim2 −/− , Mefv −/− , Zbp1 −/− or Mefv −/− Zbp1 −/− BMDMs after F. novicida infection. Data are mean ± s.e.m. *P < 0.05 and ****P < 0.0001 (one-way ANOVA with Dunnett’s multiple comparisons test; n = 3 from 3 biologically independent samples). e, In vivo bacterial quantification in lung, liver or spleen from WT or Aim2 −/− mice 2 days after F. novicida infection (n = 5). Each symbol represents one mouse. Data are pooled from two independent experiments. Data are mean ± s.e.m. **P < 0.01 and ***P < 0.001 (two-tailed t-test). f, Survival of WT, Aim2 −/−, Mefv −/− and Zbp1 −/− mice infected subcutaneously with 5 × 105 CFU of F. novicida in 200 μl PBS. Survival data are pooled from three independent experiments. **P < 0.01; ****P < 0.0001 (log-rank (Mantel-Cox) test). Exact P values are presented in Supplementary Table 1. For gel source data, see Supplementary Figure 1.

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Lee, S., Karki, R., Wang, Y. et al. AIM2 forms a complex with pyrin and ZBP1 to drive PANoptosis and host defence.Nature 597, 415–419 (2021). https://doi.org/10.1038/s41586-021-03875-8

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• Received: 17 March 2021
• Accepted: 04 August 2021
• Published: 01 September 2021
• Issue Date: 16 September 2021
• DOI: https://doi.org/10.1038/s41586-021-03875-8