Abstract
Caspase-8 is the initiator caspase of extrinsic apoptosis1,2 and inhibits necroptosis mediated by RIPK3 and MLKL. Accordingly, caspase-8 deficiency in mice causes embryonic lethality3, which can be rescued by deletion of either Ripk3 or Mlkl4,5,6. Here we show that the expression of enzymatically inactive CASP8(C362S) causes embryonic lethality in mice by inducing necroptosis and pyroptosis. Similar to Casp8−/− mice3,7, Casp8C362S/C362S mouse embryos died after endothelial cell necroptosis leading to cardiovascular defects. MLKL deficiency rescued the cardiovascular phenotype but unexpectedly caused perinatal lethality in Casp8C362S/C362S mice, indicating that CASP8(C362S) causes necroptosis-independent death at later stages of embryonic development. Specific loss of the catalytic activity of caspase-8 in intestinal epithelial cells induced intestinal inflammation similar to intestinal epithelial cell-specific Casp8 knockout mice8. Inhibition of necroptosis by additional deletion of Mlkl severely aggravated intestinal inflammation and caused premature lethality in Mlkl knockout mice with specific loss of caspase-8 catalytic activity in intestinal epithelial cells. Expression of CASP8(C362S) triggered the formation of ASC specks, activation of caspase-1 and secretion of IL-1β. Both embryonic lethality and premature death were completely rescued in Casp8C362S/C362SMlkl−/−Asc−/− or Casp8C362S/C362SMlkl−/−Casp1−/− mice, indicating that the activation of the inflammasome promotes CASP8(C362S)-mediated tissue pathology when necroptosis is blocked. Therefore, caspase-8 represents the molecular switch that controls apoptosis, necroptosis and pyroptosis, and prevents tissue damage during embryonic development and adulthood.
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Acknowledgements
We thank M. Menning, A. Manav, T. Roth and R. Hoppe for technical assistance; the CECAD in vivo Research Facility and their Transgenic Core Unit for mouse care and the generation of transgenic mice (B. Zevnik); Imaging Facilities of the CECAD and the Collaborative Research Center 670 (SFB670, Z2); T. Wunderlich for the HTNCre-expressing bacterial strain; G. Malchau for supporting blood analyses; S. Hedrick for the Casp8fl/fl mouse and J. Tschopp for the Asc−/− mouse. This work was supported by the Deutsche Forschungsgemeinschaft (DFG) CRC670, CRC1218, CRU286 and The German Cancer Aid to H.K. and by the ERC (grant agreements 323040 and 787826) to M.P.
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Contributions
M.F. generated the Casp8C362S mice, performed genetic crosses and carried out most of the experimental work. S.D.G. performed microscopy analysis, BMDM experiments, mouse preparations and designed the figures. R.S. was involved in designing the knock-in strategy for Casp8C362S mice and carried out the histopathology analysis of intestines. M.-C.A. performed immunoprecipitation experiments, endothelial cell experiments and mouse preparations. F.S. carried out overexpression studies, generated knockout cell lines and performed BMDM experiments. J.P.W. performed BMDM experiments and statistical analysis of intestinal inflammation and pyroptosis. L.M.S. and N.S. isolated and carried out endothelial cell work. H.S. evaluated knockout cell lines. J.M.S. supported mouse work. M.K. provided essential reagents. M.L. provided essential mouse lines. M.P. provided essential mouse lines and was involved in the design of the study. H.K. designed and supervised the study. All authors analysed the data, discussed the results and commented on the manuscript.
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Extended data figures and tables
Extended Data Fig. 1 The enzymatic activity of caspase-8 is required to inhibit necroptosis.
a, Top, schematic illustration of the Casp8 gene with the domain structure and the position of catalytic cysteine (star). Bottom, targeted genomic sequence of Casp8 and representative sequence analysis of embryos at E11.5 with respective genotypes. b, c, Expected and observed numbers of mice per genotype obtained from the indicated crossings. d, Representative images of Casp8WT/flTie2cre (n = 2) and Casp8fl/flTie2cre (n = 5) mouse embryos at E11.5 (top). Whole-mount yolk sacs stained with anti-CD31 antibody as endothelial marker (bottom). Scale bars, 100 µm. e, Representative images of 9-day-old Casp8WT/flK14cre (n = 5) and Casp8fl/flK14cre (n = 4) mice (top left) and skin sections stained with H&E (top right and bottom). Scale bars, 100 µm (magnification, top) and 300 µm (bottom).
Extended Data Fig. 2 The enzymatic activity of caspase-8 is required to inhibit necroptosis.
a, Representative images of ileal sections from 10-week-old Casp8WT/flVillincre (n = 4) and Casp8fl/flVillincre (n = 3) mice stained with H&E (top), immunostained for lysozyme (Paneth cells, middle) and PAS (bottom). Scale bars, 100 µm. b, Representative images of ileal sections from 10-week-old Casp8WT/flVillincre (n = 4), Casp8fl/flVillincre (n = 3), Casp8C362S/fl (n = 3) and Casp8C362S/flVillincre (n = 4) mice stained with PAS. Arrows, dead cells. Scale bars, 50 µm.
Extended Data Fig. 3 CASP8(C362S) induces necroptosis-independent tissue destruction.
a, Genotyping PCR of respective endothelial cells (ECs) after treatment with cell-permeable recombinant HTNCre protein. Results are representative of two individual experiments. b, Analysis of caspase-8 and caspase-3 processing by western blot after treatment with TNF (10 ng ml−1), CHX (2.5 µg ml−1) or both (TNF and CHX). Results are representative of two individual experiments. c, Top, viability of endothelial cells after treatment with TNF (10 ng ml−1), Nec-1 (30 µM) or both (TNF and Nec-1) for 6 h (top) in biologically independent replicates. Dots represent individual biological replicates (n = 3). Data are mean ± s.e.m. One-way ANOVA followed by Sidak’s post-analysis. n = 3, representative of two individual experiments. Bottom, western blot analysis of cell lysates examining phosphorylated MLKL (P-MLKL) and β-actin. Results are representative of two individual experiments. d, Expected, observed and weaned numbers of mice per genotype obtained from the indicated crossings. e, Spleen weight and spleen:body weight ratios of 8- and 15-week-old mice of the indicated genotypes. Dots and circles, individual mice. Data are mean ± s.e.m. One-way ANOVA followed by Sidak’s post-analysis compared to the corresponding Casp8WT/WT values. f, Representative images of spleen, axial and inguinal lymph nodes (LN) and mesenteric lymph nodes (left) as well as splenic sections from Casp8WT/WTRipk3−/− (n = 3), Casp8C362S/WTRipk3−/− (n = 3) and Casp8C362S/C362SRipk3−/− mice (n = 3) stained with H&E (right) from 15-week-old mice. Scale bars, 300 µm. g, Cardiac blood was analysed for cell numbers, and haematocrit and haemoglobin concentrations from 8–15-week-old mice. Dots and circles, individual mice. Data are mean ± s.e.m. One-way ANOVA followed by Dunnett’s post-analysis compared to the corresponding Casp8WT/WTRipk3−/− values. Exact P values (from left to right): erythrocytes, P = 0.9774, P = 0.0607; haematocrit: P = 0.9961, P = 0.0462; lymphocytes, P = 0.5812, P = 0.2426; monocytes, P = 0.9944, P = 0.0693; neutrophils, P = 0.4614, P = 0.4349; haemoglobin, P = 0.8326, P = 0.0025. h, Expected, observed and weaned numbers of embryos (left) and mice (right) per genotype obtained from the indicated crossings.
Extended Data Fig. 4 CASP8(C362S) induces necroptosis-independent tissue destruction.
a, Representative images of Casp8WT/WTMlkl−/− (n = 8), Casp8C362S/WTMlkl−/− (n = 7) and Casp8C362S/C362SMlkl−/− (n = 3) mouse embryos at E13.5 (top). Whole-mount yolk sacs stained with anti-CD31 antibody as endothelial marker (bottom). Scale bars, 100 µm. b, Representative images of spleen, inguinal and mesenteric lymph nodes from Casp8C362S/flTie2creMlkl−/− (n = 2) and Casp8fl/flMlkl−/− (n = 2) mice. c, Representative images of 9-day-old mice (top left) and skin sections stained with H&E (top right, bottom). Scale bars, 100 µm (magnification, top) and 300 µm (bottom). d, Expected, observed and weaned numbers of mice per genotype obtained from the indicated crossings.
Extended Data Fig. 5 CASP8(C362S) activates the ASC inflammasome in IECs.
a, Count of Paneth cells (per crypt per mouse, n = 3). Dots, individual mice. Data are mean ± s.e.m. One-way ANOVA followed by Sidak’s post-analysis. b, Cytokine array (AYOXXA Lunaris) to detect the indicated cytokines in ileal lysates derived from 5-week-old mice (left) or P1 neonates (right). Dots and circles, individual mice. Data are mean ± s.e.m. One-way ANOVA followed by Turkey’s post-analysis. c, d, Ponceau staining of ileal lysates from 5-week-old mice (n = 2) (c) and P1 neonates (n = 1) (d) (Fig. 3b, c). Lanes, individual mice. e, Western blot analysis of CASP8WT/WT and CASP8−/− HCT-116 and HEK293T CRISPR–Cas9 cell clones. Results are representative of one individual experiment. f, Western blot analysis of the soluble and insoluble fraction of CASP8−/− HCT-116 clone 2 with overexpression of either human wild-type caspase-8 or CASP8(C360S) after treatment with IDN-6556 (20 µM), Nec-1 (10 µM) or both (IDN-6556 and Nec-1) for 14 h. Results are representative of two individual experiments.
Extended Data Fig. 6 CASP8(C362S) activates the ASC inflammasome in IECs.
a, Immunofluorescence confocal images of CASP8−/− HCT-116 clone 2 overexpressing either human wild-type caspase-8 or CASP8(C360S) together with DsRed–ASC untreated or treated with IDN-6556 (20 µM) and stained for caspase-8 after 24 h. Scale bar, 20 µm. Results are representative of two individual experiments. b, Immunoprecipitation of CASP8−/− HEK293T clone 1 lysates overexpressing either human wild-type caspase-8, CASP8(C360S) or empty vector (−) together with DsRed–ASC untreated or treated with IDN-6556 (20 µM) as indicated. Results are representative of two individual experiments. c, Immunofluorescence confocal images of BMDMs derived from Casp8fl/fl or Casp8C362S/fl mice treated with HTNCre for 24 h and stained with an anti-ASC antibody (top) or measurement of IL-1β levels in the supernatant of BMDMs (bottom; n = 3 biologically independent replicates). Scale bar, 20 µm. Dots and circles represent individual biological replicates. Data are mean ± s.e.m. One-way ANOVA followed by Sidak’s post-analysis compared to the corresponding untreated value. Results are representative of two individual experiments. d, ASC-speck-positive BMDMs (top; n = 100 of one representative experiment), measurements of IL-1β levels (middle; n = 3 biologically independent replicates) and LDH release (bottom; n = 3 biologically independent replicates) in the supernatants of BMDMs after treatment with LPS (200 ng ml−1), IDN-6556 (20 µM) or both (LPS and IDN-6556) for 24 h. Dots and circles represent individual biological replicates. Data are mean ± s.e.m. One-way ANOVA followed by Sidak’s post-analysis compared to the corresponding untreated value and shown for P > 0.1. Results are representative of two individual experiments.
Extended Data Fig. 7 ASC or caspase-1 deficiency rescues embryonic lethality of mice expressing CASP8(C362S).
a, Expected, observed and weaned numbers of mice per genotype obtained from the indicated crossings. b, Representative images of 8-week-old mice (top) and spleen, axial and inguinal and mesenteric lymph nodes (middle) as well as splenic sections stained with H&E (bottom) from Casp8C362S/WTMlkl−/−Asc−/− (n = 3), Casp8C362S/C362SMlkl−/−Asc−/− (n = 3), Casp8C362S/WTMlkl−/−Casp1−/− (n = 3) and Casp8C362S/C362SMlkl−/−Casp1−/− (n = 3) 8-week-old mice. Scale bars, 300 µm. c, Immunofluorescence confocal images of endothelial cells treated with HTNCre and stained with an anti-ASC antibody after 24 h (top) Scale bar, 20 µm. Measurement of IL-1β levels in supernatants of endothelial cells after 24-h HTNCre treatment (bottom; n = 3 biologically independent replicates). Dots and circles represent individual biological replicates. Data are mean ± s.e.m. One-way ANOVA followed by Sidak’s post-analysis. Results are representative of two individual experiments. d, Ponceau staining of ileal lysates from 5-week-old mice (n = 2) (Fig. 4d). Lanes, individual mice.
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Fritsch, M., Günther, S.D., Schwarzer, R. et al. Caspase-8 is the molecular switch for apoptosis, necroptosis and pyroptosis. Nature 575, 683–687 (2019). https://doi.org/10.1038/s41586-019-1770-6
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DOI: https://doi.org/10.1038/s41586-019-1770-6
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