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.
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Bulk and single-cell RNA-sequencing data are available in full through the GEO database (accession GSE132134). Source Data for Figs. 1–3 and Extended Data Figs. 1–4, 6–8 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.
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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.
All authors are employees of Genentech.
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
a–c, 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 a–c 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
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. e–g, 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
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Newton, K., Wickliffe, K.E., Maltzman, A. et al. Activity of caspase-8 determines plasticity between cell death pathways. Nature 575, 679–682 (2019). https://doi.org/10.1038/s41586-019-1752-8
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