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AIM2 forms a complex with pyrin and ZBP1 to drive PANoptosis and host defence

Abstract

Inflammasomes are important sentinels of innate immune defence, sensing pathogens and inducing cell death in infected cells1. There are several inflammasome sensors that each detect and respond to a specific pathogen- or damage-associated molecular pattern (PAMP or DAMP, respectively)1. During infection, live pathogens can induce the release of multiple PAMPs and DAMPs, which can simultaneously engage multiple inflammasome sensors2,3,4,5. Here we found that AIM2 regulates the innate immune sensors pyrin and ZBP1 to drive inflammatory signalling and a form of inflammatory cell death known as PANoptosis, and provide host protection during infections with herpes simplex virus 1 and Francisella novicida. We also observed that AIM2, pyrin and ZBP1 were members of a large multi-protein complex along with ASC, caspase-1, caspase-8, RIPK3, RIPK1 and FADD, that drove inflammatory cell death (PANoptosis). Collectively, our findings define a previously unknown regulatory and molecular interaction between AIM2, pyrin and ZBP1 that drives assembly of an AIM2-mediated multi-protein complex that we term the AIM2 PANoptosome and comprising multiple inflammasome sensors and cell death regulators. These results advance the understanding of the functions of these molecules in innate immunity and inflammatory cell death, suggesting new therapeutic targets for AIM2-, ZBP1- and pyrin-mediated diseases.

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Fig. 1: HSV1 induces AIM2-, pyrin- and ZBP1-mediated caspase-1 activation, cytokine release and cell death.
Fig. 2: ZBP1 cooperates with pyrin to drive AIM2-mediated caspase-1 activation, cytokine release and cell death.
Fig. 3: AIM2, pyrin and ZBP1 promote inflammatory cell death in response to HSV1 and F. novicida infections.
Fig. 4: AIM2-mediated signalling acts as an upstream regulator of pyrin and ZBP1, which are required to form the AIM2 PANoptosome.

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.

References

  1. 1.

    Man, S. M., Karki, R. & Kanneganti, T. D. Molecular mechanisms and functions of pyroptosis, inflammatory caspases and inflammasomes in infectious diseases. Immunol. Rev. 277, 61–75 (2017).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  2. 2.

    Karki, R. et al. Concerted activation of the AIM2 and NLRP3 inflammasomes orchestrates host protection against Aspergillus infection. Cell Host Microbe 17, 357–368 (2015).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  3. 3.

    Broz, P. et al. Redundant roles for inflammasome receptors NLRP3 and NLRC4 in host defense against Salmonella. J. Exp. Med. 207, 1745–1755 (2010).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  4. 4.

    Man, S. M. et al. Inflammasome activation causes dual recruitment of NLRC4 and NLRP3 to the same macromolecular complex. Proc. Natl Acad. Sci. USA 111, 7403–7408 (2014).

    ADS  CAS  PubMed  PubMed Central  Article  Google Scholar 

  5. 5.

    Kalantari, P. et al. Dual engagement of the NLRP3 and AIM2 inflammasomes by plasmodium-derived hemozoin and DNA during malaria. Cell Rep. 6, 196–210 (2014).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  6. 6.

    Man, S. M. et al. The transcription factor IRF1 and guanylate-binding proteins target activation of the AIM2 inflammasome by Francisella infection. Nat. Immunol. 16, 467–475 (2015).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  7. 7.

    Sharma, B. R., Karki, R. & Kanneganti, T. D. Role of AIM2 inflammasome in inflammatory diseases, cancer and infection. Eur. J. Immunol. 49, 1998–2011 (2019).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  8. 8.

    Lammert, C. R. et al. AIM2 inflammasome surveillance of DNA damage shapes neurodevelopment. Nature 580, 647–652 (2020).

    ADS  CAS  PubMed  PubMed Central  Article  Google Scholar 

  9. 9.

    Hornung, V. et al. AIM2 recognizes cytosolic dsDNA and forms a caspase-1-activating inflammasome with ASC. Nature 458, 514–518 (2009).

    ADS  CAS  PubMed  PubMed Central  Article  Google Scholar 

  10. 10.

    Rathinam, V. A. et al. The AIM2 inflammasome is essential for host defense against cytosolic bacteria and DNA viruses. Nat. Immunol. 11, 395–402 (2010).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  11. 11.

    Fernandes-Alnemri, T. et al. The AIM2 inflammasome is critical for innate immunity to Francisella tularensis. Nat. Immunol. 11, 385–393 (2010).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  12. 12.

    Zhu, Q., Man, S. M., Karki, R., Malireddi, R. K. S. & Kanneganti, T. D. Detrimental type i interferon signaling dominates protective AIM2 inflammasome responses during Francisella novicida infection. Cell Rep. 22, 3168–3174 (2018).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  13. 13.

    Maruzuru, Y. et al. Herpes simplex virus 1 VP22 inhibits AIM2-dependent inflammasome activation to enable efficient viral replication. Cell Host Microbe 23, 254–265.e257 (2018).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  14. 14.

    Kuriakose, T. et al. ZBP1/DAI is an innate sensor of influenza virus triggering the NLRP3 inflammasome and programmed cell death pathways. Sci. Immunol. 1, aag2045 (2016).

    PubMed  PubMed Central  Article  Google Scholar 

  15. 15.

    Kesavardhana, S. et al. The Zα2 domain of ZBP1 is a molecular switch regulating influenza-induced PANoptosis and perinatal lethality during development. J. Biol. Chem. 295, 8325–8330 (2020).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  16. 16.

    Newton, K. et al. RIPK1 inhibits ZBP1-driven necroptosis during development. Nature 540, 129–133 (2016).

    ADS  CAS  PubMed  Article  PubMed Central  Google Scholar 

  17. 17.

    Park, Y. H., Wood, G., Kastner, D. L. & Chae, J. J. Pyrin inflammasome activation and RhoA signaling in the autoinflammatory diseases FMF and HIDS. Nat. Immunol. 17, 914–921 (2016).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  18. 18.

    Gao, W., Yang, J., Liu, W., Wang, Y. & Shao, F. Site-specific phosphorylation and microtubule dynamics control Pyrin inflammasome activation. Proc. Natl Acad. Sci. USA 113, E4857–E4866 (2016).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  19. 19.

    Xu, H. et al. Innate immune sensing of bacterial modifications of Rho GTPases by the Pyrin inflammasome. Nature 513, 237–241 (2014).

    ADS  CAS  PubMed  Article  PubMed Central  Google Scholar 

  20. 20.

    Masters, S. L. et al. Familial autoinflammation with neutrophilic dermatosis reveals a regulatory mechanism of pyrin activation. Sci. Transl. Med. 8, 332ra345 (2016).

    Article  CAS  Google Scholar 

  21. 21.

    Van Gorp, H. et al. Familial Mediterranean fever mutations lift the obligatory requirement for microtubules in Pyrin inflammasome activation. Proc. Natl Acad. Sci. USA 113, 14384–14389 (2016).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  22. 22.

    Kesavardhana, S. et al. ZBP1/DAI ubiquitination and sensing of influenza vRNPs activate programmed cell death. J. Exp. Med. 214, 2217–2229 (2017).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  23. 23.

    Place, D. E., Lee, S. & Kanneganti, T. D. PANoptosis in microbial infection. Curr. Opin. Microbiol. 59, 42–49 (2021).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  24. 24.

    Malireddi, R. K., Ippagunta, S., Lamkanfi, M. & Kanneganti, T. D. Cutting edge: proteolytic inactivation of poly(ADP-ribose) polymerase 1 by the Nlrp3 and Nlrc4 inflammasomes. J. Immunol 185, 3127–3130 (2010).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  25. 25.

    Lamkanfi, M. et al. Targeted peptidecentric proteomics reveals caspase-7 as a substrate of the caspase-1 inflammasomes. Mol. Cell Proteomics 7, 2350–2363 (2008).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  26. 26.

    Lukens, J. R. et al. Dietary modulation of the microbiome affects autoinflammatory disease. Nature 516, 246–249 (2014).

    ADS  CAS  PubMed  PubMed Central  Article  Google Scholar 

  27. 27.

    Gurung, P. et al. FADD and caspase-8 mediate priming and activation of the canonical and noncanonical Nlrp3 inflammasomes. J. Immunol. 192, 1835–1846 (2014).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  28. 28.

    Karki, R. et al. Synergism of TNF-α and IFN-γ triggers inflammatory cell death, tissue damage, and mortality in SARS-CoV-2 infection and cytokine shock syndromes. Cell 184, 149–168.e117 (2021).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  29. 29.

    Malireddi, R. K. S. et al. Innate immune priming in the absence of TAK1 drives RIPK1 kinase activity-independent pyroptosis, apoptosis, necroptosis, and inflammatory disease. J. Exp. Med. 217, e20191644 (2020).

    PubMed  Article  CAS  PubMed Central  Google Scholar 

  30. 30.

    Malireddi, R. K. S. et al. TAK1 restricts spontaneous NLRP3 activation and cell death to control myeloid proliferation. J. Exp. Med. 215, 1023–1034 (2018).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  31. 31.

    Zheng, M., Karki, R., Vogel, P. & Kanneganti, T. D. Caspase-6 is a key regulator of innate immunity, inflammasome activation, and host defense. Cell 181, 674–687.e613 (2020).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  32. 32.

    Banerjee, I. et al. Gasdermin D restrains type I interferon response to cytosolic DNA by disrupting ionic homeostasis. Immunity 49, 413–426 e415 (2018).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  33. 33.

    Christgen, S. et al. Identification of the PANoptosome: a molecular platform triggering pyroptosis, apoptosis, and necroptosis (PANoptosis). Front. Cell Infect. Microbiol. 10, 237 (2020).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  34. 34.

    Sharma, D., Malik, A., Guy, C., Vogel, P. & Kanneganti, T. D. TNF/TNFR axis promotes pyrin inflammasome activation and distinctly modulates pyrin inflammasomopathy. J. Clin. Invest. 129, 150–162 (2019).

    PubMed  Article  PubMed Central  Google Scholar 

  35. 35.

    Jones, J. W. et al. Absent in melanoma 2 is required for innate immune recognition of Francisella tularensis. Proc. Natl Acad. Sci. USA 107, 9771–9776 (2010).

    ADS  CAS  PubMed  PubMed Central  Article  Google Scholar 

  36. 36.

    Henry, T., Brotcke, A., Weiss, D. S., Thompson, L. J. & Monack, D. M. Type I interferon signaling is required for activation of the inflammasome during Francisella infection. J. Exp. Med. 204, 987–994 (2007).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  37. 37.

    Kanneganti, T. D. et al. Bacterial RNA and small antiviral compounds activate caspase-1 through cryopyrin/Nalp3. Nature 440, 233–236 (2006).

    ADS  CAS  PubMed  Article  PubMed Central  Google Scholar 

  38. 38.

    Mariathasan, S. et al. Differential activation of the inflammasome by caspase-1 adaptors ASC and Ipaf. Nature 430, 213–218 (2004).

    ADS  CAS  PubMed  Article  PubMed Central  Google Scholar 

  39. 39.

    Ishii, K. J. et al. TANK-binding kinase-1 delineates innate and adaptive immune responses to DNA vaccines. Nature 451, 725–729 (2008).

    ADS  CAS  PubMed  Article  PubMed Central  Google Scholar 

  40. 40.

    Alexopoulou, L., Holt, A. C., Medzhitov, R. & Flavell, R. A. Recognition of double-stranded RNA and activation of NF-κB by Toll-like receptor 3. Nature 413, 732–738 (2001).

    ADS  CAS  PubMed  Article  PubMed Central  Google Scholar 

  41. 41.

    Yamamoto, M. et al. Role of adaptor TRIF in the MyD88-independent toll-like receptor signaling pathway. Science 301, 640–643 (2003).

    ADS  CAS  PubMed  Article  PubMed Central  Google Scholar 

  42. 42.

    Gitlin, L. et al. Essential role of mda-5 in type I IFN responses to polyriboinosinic:polyribocytidylic acid and encephalomyocarditis picornavirus. Proc. Natl Acad. Sci. USA 103, 8459–8464 (2006).

    ADS  CAS  PubMed  PubMed Central  Article  Google Scholar 

  43. 43.

    Kumar, H. et al. Essential role of IPS-1 in innate immune responses against RNA viruses. J. Exp. Med. 203, 1795–1803 (2006).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  44. 44.

    Chen, G. Y., Liu, M., Wang, F., Bertin, J. & Núñez, G. A functional role for Nlrp6 in intestinal inflammation and tumorigenesis. J. Immunol. 186, 7187–7194 (2011).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  45. 45.

    Zaki, M. H. et al. The NOD-like receptor NLRP12 attenuates colon inflammation and tumorigenesis. Cancer Cell 20, 649–660 (2011).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  46. 46.

    Ozören, N. et al. Distinct roles of TLR2 and the adaptor ASC in IL-1β/IL-18 secretion in response to Listeria monocytogenes. J. Immunol. 176, 4337–4342 (2006).

    PubMed  Article  PubMed Central  Google Scholar 

  47. 47.

    Man, S. M. et al. IRGB10 liberates bacterial ligands for sensing by the AIM2 and Caspase-11-NLRP3 inflammasomes. Cell 167, 382–396.e317 (2016).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  48. 48.

    Van Opdenbosch, N. et al. Caspase-1 engagement and TLR-induced c-FLIP expression suppress ASC/Caspase-8-dependent apoptosis by inflammasome sensors NLRP1b and NLRC4. Cell Rep. 21, 3427–3444 (2017).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  49. 49.

    Karki, R. et al. IRF8 regulates transcription of Naips for NLRC4 inflammasome activation. Cell 173, 920–933.e913 (2018).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  50. 50.

    Tummers, B. et al. Caspase-8-dependent inflammatory responses are controlled by its adaptor, FADD, and necroptosis. Immunity 52, 994–1006.e1008 (2020).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  51. 51.

    Lakhani, S. A. et al. Caspases 3 and 7: key mediators of mitochondrial events of apoptosis. Science 311, 847–851 (2006).

    ADS  CAS  PubMed  PubMed Central  Article  Google Scholar 

  52. 52.

    Zheng, T. S. et al. Deficiency in caspase-9 or caspase-3 induces compensatory caspase activation. Nat. Med. 6, 1241–1247 (2000).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  53. 53.

    Newton, K., Sun, X. & Dixit, V. M. Kinase RIP3 is dispensable for normal NF-κBs, signaling by the B-cell and T-cell receptors, tumor necrosis factor receptor 1, and Toll-like receptors 2 and 4. Mol. Cell. Biol. 24, 1464–1469 (2004).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  54. 54.

    Oberst, A. et al. Catalytic activity of the caspase-8FLIPL complex inhibits RIPK3-dependent necrosis. Nature 471, 363–367 (2011).

    ADS  CAS  PubMed  PubMed Central  Article  Google Scholar 

  55. 55.

    Dillon, C. P. et al. Survival function of the FADD–CASPASE-8–cFLIPL complex. Cell Rep. 1, 401–407 (2012).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  56. 56.

    Kuehne, S. A. et al. Importance of toxin A, toxin B, and CDT in virulence of an epidemic Clostridium difficile strain. J. Infect. Dis. 209, 83–86 (2014).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  57. 57.

    Tweedell, R. E., Malireddi, R. K. S. & Kanneganti, T. D. A comprehensive guide to studying inflammasome activation and cell death. Nat. Protoc. 15, 3284–33339 (2020).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  58. 58.

    Lee, S. et al. Influenza restriction factor MxA functions as inflammasome sensor in the respiratory epithelium. Sci. Immunol. 4, eaau4643 (2019).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  59. 59.

    Lee, S., Hirohama, M., Noguchi, M., Nagata, K. & Kawaguchi, A. Influenza A virus infection triggers pyroptosis and apoptosis of respiratory epithelial cells through the type I interferon signaling pathway in a mutually exclusive manner. J. Virol. 92, e00396–18 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

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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.

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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 to Thirumala-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). df, 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. gi, 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. jl, 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. mo, 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). df, 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. pr, 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). eh, 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.

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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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Supplementary information

Supplementary Information

This file contains uncropped blots with molecular weight and size markers and an indication of how the images were cropped.

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Supplementary Table 1

Exact P values for Figs. 1–4 and Extended Data Figs. 1–10.

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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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