Activity of caspase-8 determines plasticity between cell death pathways

Article metrics

Abstract

Caspase-8 is a protease with both pro-death and pro-survival functions: it mediates apoptosis induced by death receptors such as TNFR11, and suppresses necroptosis mediated by the kinase RIPK3 and the pseudokinase MLKL2,3,4. Mice that lack caspase-8 display MLKL-dependent embryonic lethality4, as do mice that express catalytically inactive CASP8(C362A)5. Casp8C362A/C362AMlkl−/− mice die during the perinatal period5, whereas Casp8−/−Mlkl−/− mice are viable4, which indicates that inactive caspase-8 also has a pro-death scaffolding function. Here we show that mutant CASP8(C362A) induces the formation of ASC (also known as PYCARD) specks, and caspase-1-dependent cleavage of GSDMD and caspases 3 and 7 in MLKL-deficient mouse intestines around embryonic day 18. Caspase-1 and its adaptor ASC contributed to the perinatal lethal phenotype because a number of Casp8C362A/C362AMlkl−/−Casp1−/− and Casp8C362A/C362AMlkl−/−Asc−/− mice survived beyond weaning. Transfection studies suggest that inactive caspase-8 adopts a distinct conformation to active caspase-8, enabling its prodomain to engage ASC. Upregulation of the lipopolysaccharide sensor caspase-11 in the intestines of both Casp8C362A/C362AMlkl−/− and Casp8C362A/C362AMlkl−/−Casp1−/− mice also contributed to lethality because Casp8C362A/C362AMlkl−/−Casp1−/−Casp11−/− (Casp11 is also known as Casp4) neonates survived more often than Casp8C362A/C362AMlkl−/−Casp1−/− neonates. Finally, Casp8C362A/C362ARipk3−/−Casp1−/−Casp11−/− mice survived longer than Casp8C362A/C362AMlkl−/−Casp1−/−Casp11−/− mice, indicating that a necroptosis-independent function of RIPK3 also contributes to lethality. Thus, unanticipated plasticity in death pathways is revealed when caspase-8-dependent apoptosis and MLKL-dependent necroptosis are inhibited.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Fig. 1: CASP8(C362A) causes MLKL-independent inflammation.
Fig. 2: Caspase-1, caspase-11 and RIPK3 promote lethality in Casp8C362A/C362AMlkl−/− mice.
Fig. 3: CASP8(C362A) promotes the formation of ASC specks.
Fig. 4: Expression of CASP8(C362A) in necroptosis-deficient intestinal epithelial cells triggers cleavage of caspase-1 substrates.

Data availability

Bulk and single-cell RNA-sequencing data are available in full through the GEO database (accession GSE132134). Source Data for Figs. 13 and Extended Data Figs. 14, 68 are provided with the paper. Other datasets generated during and/or analysed during the current study are available from the corresponding authors on reasonable request.

References

  1. 1.

    Varfolomeev, E. E. et al. Targeted disruption of the mouse Caspase 8 gene ablates cell death induction by the TNF receptors, Fas/Apo1, and DR3 and is lethal prenatally. Immunity 9, 267–276 (1998).

  2. 2.

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

  3. 3.

    Kaiser, W. J. et al. RIP3 mediates the embryonic lethality of caspase-8-deficient mice. Nature 471, 368–372 (2011).

  4. 4.

    Alvarez-Diaz, S. et al. The pseudokinase MLKL and the kinase RIPK3 have distinct roles in autoimmune disease caused by loss of death-receptor-induced apoptosis. Immunity 45, 513–526 (2016).

  5. 5.

    Newton, K. et al. Cleavage of RIPK1 by caspase-8 is crucial for limiting apoptosis and necroptosis. Nature 574, 428–431 (2019).

  6. 6.

    Henry, C. M. & Martin, S. J. Caspase-8 acts in a non-enzymatic role as a scaffold for assembly of a pro-inflammatory “FADDosome” complex upon TRAIL stimulation. Mol. Cell 65, 715–729 (2017).

  7. 7.

    Hartwig, T. et al. The TRAIL-induced cancer secretome promotes a tumor-supportive immune microenvironment via CCR2. Mol. Cell 65, 730–742 (2017).

  8. 8.

    Kang, S. et al. Caspase-8 scaffolding function and MLKL regulate NLRP3 inflammasome activation downstream of TLR3. Nat. Commun. 6, 7515 (2015).

  9. 9.

    Kang, T. B., Jeong, J. S., Yang, S. H., Kovalenko, A. & Wallach, D. Caspase-8 deficiency in mouse embryos triggers chronic RIPK1-dependent activation of inflammatory genes, independently of RIPK3. Cell Death Differ. 25, 1107–1117 (2018).

  10. 10.

    Rickard, J. A. et al. RIPK1 regulates RIPK3–MLKL-driven systemic inflammation and emergency hematopoiesis. Cell 157, 1175–1188 (2014).

  11. 11.

    Dillon, C. P. et al. RIPK1 blocks early postnatal lethality mediated by caspase-8 and RIPK3. Cell 157, 1189–1202 (2014).

  12. 12.

    Kaiser, W. J. et al. RIP1 suppresses innate immune necrotic as well as apoptotic cell death during mammalian parturition. Proc. Natl Acad. Sci. USA 111, 7753–7758 (2014).

  13. 13.

    Afonina, I. S., Müller, C., Martin, S. J. & Beyaert, R. Proteolytic processing of interleukin-1 family cytokines: variations on a common theme. Immunity 42, 991–1004 (2015).

  14. 14.

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

  15. 15.

    Schauvliege, R., Vanrobaeys, J., Schotte, P. & Beyaert, R. Caspase-11 gene expression in response to lipopolysaccharide and interferon-γ requires nuclear factor-κB and signal transducer and activator of transcription (STAT) 1. J. Biol. Chem. 277, 41624–41630 (2002).

  16. 16.

    Aglietti, R. A. & Dueber, E. C. Recent insights into the molecular mechanisms underlying pyroptosis and gasdermin family functions. Trends Immunol. 38, 261–271 (2017).

  17. 17.

    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).

  18. 18.

    Pierini, R. et al. AIM2/ASC triggers caspase-8-dependent apoptosis in Francisella-infected caspase-1-deficient macrophages. Cell Death Differ. 19, 1709–1721 (2012).

  19. 19.

    Man, S. M. et al. Salmonella infection induces recruitment of caspase-8 to the inflammasome to modulate IL-1β production. J. Immunol. 191, 5239–5246 (2013).

  20. 20.

    Stutz, A., Horvath, G. L., Monks, B. G. & Latz, E. ASC speck formation as a readout for inflammasome activation. Methods Mol. Biol. 1040, 91–101 (2013).

  21. 21.

    Vajjhala, P. R. et al. The inflammasome adaptor ASC induces procaspase-8 death effector domain filaments. J. Biol. Chem. 290, 29217–29230 (2015).

  22. 22.

    Georgiades, P. et al. Vav cre transgenic mice: a tool for mutagenesis in hematopoietic and endothelial lineages. Genesis 34, 251–256 (2002).

  23. 23.

    Madison, B. B. et al. cis elements of the Villin gene control expression in restricted domains of the vertical (crypt) and horizontal (duodenum, cecum) axes of the intestine. J. Biol. Chem. 277, 33275–33283 (2002).

  24. 24.

    Weng, D. et al. Caspase-8 and RIP kinases regulate bacteria-induced innate immune responses and cell death. Proc. Natl Acad. Sci. USA 111, 7391–7396 (2014).

  25. 25.

    Philip, N. H. et al. Caspase-8 mediates caspase-1 processing and innate immune defense in response to bacterial blockade of NF-κB and MAPK signaling. Proc. Natl Acad. Sci. USA 111, 7385–7390 (2014).

  26. 26.

    Nailwal, H. & Chan, F. K. Necroptosis in anti-viral inflammation. Cell Death Differ. 26, 4–13 (2019).

  27. 27.

    Chan, F. K. et al. A role for tumor necrosis factor receptor-2 and receptor-interacting protein in programmed necrosis and antiviral responses. J. Biol. Chem. 278, 51613–51621 (2003).

  28. 28.

    Thome, M. et al. Viral FLICE-inhibitory proteins (FLIPs) prevent apoptosis induced by death receptors. Nature 386, 517–521 (1997).

  29. 29.

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

  30. 30.

    Mariathasan, S. et al. Cryopyrin activates the inflammasome in response to toxins and ATP. Nature 440, 228–232 (2006).

  31. 31.

    Kayagaki, N. et al. Non-canonical inflammasome activation targets caspase-11. Nature 479, 117–121 (2011).

  32. 32.

    Kayagaki, N. et al. Caspase-11 cleaves gasdermin D for non-canonical inflammasome signalling. Nature 526, 666–671 (2015).

  33. 33.

    Newton, K. et al. Activity of protein kinase RIPK3 determines whether cells die by necroptosis or apoptosis. Science 343, 1357–1360 (2014).

  34. 34.

    Murphy, J. M. et al. The pseudokinase MLKL mediates necroptosis via a molecular switch mechanism. Immunity 39, 443–453 (2013).

  35. 35.

    Wu, T. D. & Nacu, S. Fast and SNP-tolerant detection of complex variants and splicing in short reads. Bioinformatics 26, 873–881 (2010).

  36. 36.

    Ritchie, M. E. et al. limma powers differential expression analyses for RNA-sequencing and microarray studies. Nucleic Acids Res. 43, e47 (2015).

  37. 37.

    Butler, A., Hoffman, P., Smibert, P., Papalexi, E. & Satija, R. Integrating single-cell transcriptomic data across different conditions, technologies, and species. Nat. Biotechnol. 36, 411–420 (2018).

  38. 38.

    Haber, A. L. et al. A single-cell survey of the small intestinal epithelium. Nature 551, 333–339 (2017).

Download references

Acknowledgements

We thank M. Dempsey, T. Scholl, F. Gallardo and B. Torres for animal husbandry, J. Zhang, K.-H. Sun, S. Haller and members of the Genentech genetic analysis laboratory for technical assistance, and C. Print and W. Alexander for Vavcre and Mlkl−/− mice, respectively.

Author information

K.N., K.E.W., A.M., D.L.D., M.S.S. and Z.M. designed and performed experiments, M.R.-G. generated Casp8cC362A/+ mice, Y.Z. and R.R. analysed RNA-sequencing data, J.D.W. analysed histological data and V.M.D. contributed to experimental design. K.N. wrote the paper with input from all authors.

Correspondence to Kim Newton or Vishva M. Dixit.

Ethics declarations

Competing interests

All authors are employees of Genentech.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Peer review information Nature thanks Igor E. Brodsky, William Kaiser and Seamus J. Martin for their contribution to the peer review of this work.

Extended data figures and tables

Extended Data Fig. 1 Characterization of Casp8C362AMlkl−/− mice.

ac, Serum concentrations of cytokines and chemokines at E17.5 (a) or E18.5 (b, c). Circles, individual mice. Data are mean ± s.e.m. P values are shown if P < 0.05; unpaired, two-tailed t-test. The concentration of IL-18 was below the limit of detection in c. d, Concentrations of cytokines and chemokines in the intestine at E17.5. Circles, individual mice. e, E17.5 intestines. Results representative of five Mlkl−/− mice (left) and five Casp8C362A/C362AMlkl−/− mice (middle and right), two of which had intestinal atrophy (middle) and the other three showed a normal intestinal morphology. Scale bars, 100 μm. f, Western blots of E17.5 intestine, E18.5 skin and E18.5 liver. Each lane is for a different mouse (n = 2 per genotype for intestine, 3 per genotype for skin and liver). g, Western blots of E16.5 intestine before (input) and after immunoprecipitation (IP) with anti-caspase-8 antibody. Results are representative of five independent experiments. For gel source data, see Supplementary Fig. 1. Source data

Extended Data Fig. 2 Characterization of Casp8C362A/C362ARipk3−/−, Casp8C362A/C362AMlkl−/−Casp1−/−, Casp8C362A/C362AMlkl−/−Casp1−/−Casp11−/− and Casp8C362A/C362ARipk3−/−Casp1−/−Casp11−/− mice.

a, Mouse body weights. Circles, individual mice. Data are mean ± s.e.m. P values were calculated by unpaired, two-tailed t-test. b, Red blood cells (RBCs) and haemoglobin in mouse peripheral blood at 5–14 weeks. Circles, individual mice. Data are mean ± s.e.m. P values were calculated by unpaired, two-tailed t-test with Welch’s correction. c, Spleen weight as a percentage of body weight for mice aged 6–43 weeks. Circles, individual mice. Data are mean ± s.e.m. P values by unpaired, two-tailed t-test with Welch’s correction. d, Sections of spleen, liver, lungs and small intestine. Results are representative of two wild-type, four Casp8C362A/C362ARipk3−/−, three Mlkl−/−Casp1−/− and three Casp8C362A/C362AMlkl−/−Casp1−/− mice aged 3–8 weeks. Scale bars, 200 μm (spleen), 100 μm (intestine) or 50 μm (liver and lungs). Source data

Extended Data Fig. 3 Flow cytometry analysis of Casp8C362A/C362ARipk3−/−, Casp8C362A/C362AMlkl−/−Casp1−/−, Casp8C362A/C362AMlkl−/−Casp1−/−Casp11−/− and Casp8C362A/C362ARipk3−/−Casp1−/−Casp11−/− splenocytes.

a, Representative dot plots of splenocytes from mice aged 3–15 weeks (Casp8C362A/C362ARipk3−/− and Ripk3−/−) or 14–38 weeks (Casp8C362A/C362AMlkl−/−Casp1−/−, Mlkl−/−Casp1−/−, Casp8C362A/C362AMlkl−/−Casp1−/−Casp11−/−, Mlkl−/−Casp1−/−Casp11−/−, Casp8C362A/C362ARipk3−/−Casp1−/−Casp11−/− and Ripk3−/−Casp1−/−Casp11−/−). Percentages are mean ± s.e.m. SSC, side scatter. b, Splenic leukocyte subsets. Circles, individual mice. Data are mean ± s.e.m. P values are shown if P < 0.05; unpaired, two-sided t-test. Source data

Extended Data Fig. 4 Flow cytometry analysis of Casp8C362A/C362ARipk3−/−, Casp8C362A/C362AMlkl−/−Casp1−/−, Casp8C362A/C362AMlkl−/−Casp1−/−Casp11−/− and Casp8C362A/C362ARipk3−/−Casp1−/−Casp11−/− mesenteric lymph node or bone marrow cells.

a, b, Representative dot plots of mouse mesenteric lymph node (a) and bone marrow (b) cells. The initial three gates in Extended Data Fig. 3 were also applied to these samples. Percentages are mean ± s.e.m. c, Bone marrow leukocyte subsets. Circles, individual mice. Data are mean ± s.e.m. P values are shown if P < 0.05; unpaired, two-sided t-test. Mice in ac were aged 3–15 weeks (Casp8C362A/C362ARipk3−/− and Ripk3−/−) or 14–38 weeks (Casp8C362A/C362AMlkl−/−Casp1−/−, Mlkl−/−Casp1−/−, Casp8C362A/C362AMlkl−/−Casp1−/−Casp11−/−, Mlkl−/−Casp1−/−Casp11−/−, Casp8C362A/C362ARipk3−/−Casp1−/−Casp11−/− and Ripk3−/−Casp1−/−Casp11−/−). Source data

Extended Data Fig. 5 Single-cell RNA sequencing of E18.5 intestines.

a, UMAP plots of E18.5 intestines analysed by single-cell RNA sequencing (Mlkl−/−, n = 1,328 cells; Casp8C362A/C362AMlkl−/−, n = 874 cells). b, Density plots indicate expression of Casp11 mRNA by cells in the eight clusters shown in a. Density corresponds to smoothed values for cell number. UMI, unique molecular identifier.

Extended Data Fig. 6 The pro-domain of caspase-8 is necessary and sufficient for enhanced aggregation of ASC.

a, Serum LPS for 7-day-old mice. Circles, individual mice (n = 4 per genotype). Data are mean ± s.e.m. P = 0.89; unpaired, two-tailed t-test with Welch’s correction. b, FITC-labelled dextran in the serum of 3–4-week-old mice at 5 h after gavage dosing with FITC–dextran. Circles, individual mice (n = 3, Casp8C362A/C362AMlkl−/−Casp1−/−; n = 4, Casp8C362A/+Mlkl−/−Casp1−/−). Data are mean ± s.e.m. P = 0.83; unpaired, two-tailed t-test with Welch’s correction. c, Western blots of HEK293T cells transfected with caspase-8 and/or ASC. Results are representative of two independent experiments. Blotting of the loading control β-actin was performed after Flag. d, HEK293T cells labelled for transfected ASC (red), transfected caspase-8 (wild-type caspase-8 or mutant CASP8(C362A); green) and DNA (blue). The table indicates the proportion of cells co-expressing ASC and caspase-8 that contained speck-like structures (indicated by arrows in the image). Scale bars, 10 μm. Results representative of two independent experiments. e, Western blots of HEK293T cells transfected with ASC and different caspase-8 mutants and then lysed with 1% Triton X-100. Results are representative of two independent experiments. Blotting of the loading control β-actin was performed after Flag (soluble fraction) or Myc (insoluble fraction). f, Western blots of E18.5 intestines. Lanes, different embryos (n = 2 per genotype). Blotting of the loading control β-actin was performed after phosphorylated (p-)RIPK1. For gel source data, see Supplementary Fig. 1. Source data

Extended Data Fig. 7 Characterization of mice expressing CASP8(C362A) only in haematopoietic and endothelial cells.

a, Schematic showing the organization of the Casp8cC362A conditional allele. b, Body weights of female littermates (Casp8cC362A/+Mlkl−/−Vavcre, n = 2; Casp8cC362A/cC362AMlkl−/−Vavcre, n = 3). c, Spleen weight as a percentage of body weight for mice aged 4–6 months. Circles, individual mice (Mlkl−/−Vavcre, n = 3; Casp8cC362A/cC362AMlkl−/−Vavcre, n = 4). Lines, mean ± s.e.m. P = 0.09, unpaired, two-tailed t-test with Welch’s correction. d, Leukocyte numbers in mice aged 2–7 months. Bone marrow was from two femurs. Circles, individual mice (Mlkl−/−Vavcre, n = 2 males, 1 female; Casp8cC362A/cC362AMlkl−/−Vavcre, n = 1 male, 3 females). Data are mean ± s.e.m. P values were calculated by unpaired, two-tailed t-test. eg, Flow cytometry analysis of bone marrow (e), spleen (f) and mesenteric lymph node (g) cells from the mice in d. Samples analysed using the gating strategy shown in Extended Data Fig. 3. Source data

Extended Data Fig. 8 Characterization of mice expressing CASP8(C362A) only in intestinal epithelial cells.

a, Mouse body weights. Circles, individual mice (Villincre, n = 3 females, 4 males; Casp8cC362A/cC362AMlkl−/−Villincre, n = 8 females, 8 males at 3 weeks, or 8 females, 4 males at 5 weeks). Data are mean ± s.e.m. P values by unpaired, two-tailed t-test. b, Small and large intestines of mice aged 19 weeks. Results representative of five Casp8cC362A/cC362AVillincre mice aged 3–19 weeks. Scale bars, 100 μm. c, Kaplan–Meier survival curves of Villincre (n = 13) and Casp8cC362A/cC362AVillincre (n = 19) littermates. P = 0.06, two-sided Gehan–Breslow–Wilcoxon test. d, Body weights of littermates (n = 2 per genotype). e, Littermate females aged 11 days. Results representative of two mice of each genotype. f, Small and large intestines of mice aged 4 weeks. Scale bars, 100 μm (small intestine) or 50 μm (large intestine). Results are representative of two mice of each genotype. g, Small intestine from 1-day-old littermates. Scale bars, 100 μm. Similar histological changes were observed in two out of three newborn (P0) Casp8cC362A/cC362AMlkl−/−Villincre pups. Source data

Supplementary information

Supplementary Information

This file contains the uncropped blots used in the main figures and extended data figures.

Reporting Summary

Source data

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Newton, K., Wickliffe, K.E., Maltzman, A. et al. Activity of caspase-8 determines plasticity between cell death pathways. Nature 575, 679–682 (2019) doi:10.1038/s41586-019-1752-8

Download citation

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.