Cleavage of RIPK1 by caspase-8 is crucial for limiting apoptosis and necroptosis

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Abstract

The aspartate-specific cysteine protease caspase-8 suppresses necroptotic cell death mediated by RIPK3 and MLKL. Indeed, mice that lack caspase-8 die in a RIPK3- and MLKL-dependent manner during embryogenesis1,2,3. In humans, caspase-8 deficiency is associated with immunodeficiency4 or very early onset inflammatory bowel disease5. The substrates that are cleaved by caspase-8 to prevent necroptosis in vivo have not been defined. Here we show that knock-in mice that express catalytically inactive caspase-8(C362A) die as embryos owing to MLKL-dependent necroptosis, similar to caspase-8-deficient mice. Thus, caspase-8 must cleave itself, other proteins or both to inhibit necroptosis. Mice that express caspase-8(D212A/D218A/D225A/D387A), which cannot cleave itself, were viable, as were mice that express c-FLIP or CYLD proteins that had been mutated to prevent cleavage by caspase-8. By contrast, mice that express RIPK1(D325A), in which the caspase-8 cleavage site Asp325 had been mutated, died mid-gestation. Embryonic lethality was prevented by inactivation of RIPK1, loss of TNFR1, or loss of both MLKL and the caspase-8 adaptor FADD, but not by loss of MLKL alone. Thus, RIPK1(D325A) appears to trigger cell death mediated by TNF, the kinase activity of RIPK1 and FADD–caspase-8. Accordingly, dying endothelial cells that contain cleaved caspase-3 were abnormally abundant in yolk sacs of Ripk1D325A/D325A embryos. Heterozygous Ripk1D325A/+ cells and mice were viable, but were also more susceptible to TNF-induced cell death than were wild-type cells or mice. Our data show that Asp325 of RIPK1 is essential for limiting aberrant cell death in response to TNF, consistent with the idea that cleavage of RIPK1 by caspase-8 is a mechanism for dismantling death-inducing complexes.

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Fig. 1: Catalytic activity of caspase-8 prevents necroptosis during mouse embryogenesis.
Fig. 2: RIPK1(D325A) triggers FADD-dependent cell death.
Fig. 3: RIPK1(D325A) heterozygosity sensitizes cells to TNF toxicity.
Fig. 4: Ripk1D325A/+ mice require RIPK1 enzymatic activity and FADD for TNF sensitivity.

Data availability

The datasets generated during and/or analysed during the current study are available from the corresponding authors on reasonable request.

References

  1. 1.

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

  2. 2.

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

  3. 3.

    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).

  4. 4.

    Chun, H. J. et al. Pleiotropic defects in lymphocyte activation caused by caspase-8 mutations lead to human immunodeficiency. Nature 419, 395–399 (2002).

  5. 5.

    Lehle, A. S. et al. Intestinal inflammation and dysregulated immunity in patients with inherited caspase-8 deficiency. Gastroenterology 156, 275–278 (2019).

  6. 6.

    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).

  7. 7.

    Ch’en, I. L., Tsau, J. S., Molkentin, J. D., Komatsu, M. & Hedrick, S. M. Mechanisms of necroptosis in T cells. J. Exp. Med. 208, 633–641 (2011).

  8. 8.

    Kaiser, W. J. et al. Toll-like receptor 3-mediated necrosis via TRIF, RIP3, and MLKL. J. Biol. Chem. 288, 31268–31279 (2013).

  9. 9.

    Kang, T. B. et al. Caspase-8 serves both apoptotic and nonapoptotic roles. J. Immunol. 173, 2976–2984 (2004).

  10. 10.

    Webster, J. D., Solon, M., Haller, S. & Newton, K. Detection of necroptosis by phospho-RIPK3 immunohistochemical labeling. Methods Mol. Biol. 1857, 153–160 (2018).

  11. 11.

    Medema, J. P. et al. FLICE is activated by association with the CD95 death-inducing signaling complex (DISC). EMBO J. 16, 2794–2804 (1997).

  12. 12.

    Pop, C. et al. FLIPL induces caspase 8 activity in the absence of interdomain caspase 8 cleavage and alters substrate specificity. Biochem. J. 433, 447–457 (2011).

  13. 13.

    O’Donnell, M. A. et al. Caspase 8 inhibits programmed necrosis by processing CYLD. Nat. Cell Biol. 13, 1437–1442 (2011).

  14. 14.

    Lin, Y., Devin, A., Rodriguez, Y. & Liu, Z. G. Cleavage of the death domain kinase RIP by caspase-8 prompts TNF-induced apoptosis. Genes Dev. 13, 2514–2526 (1999).

  15. 15.

    Feng, S. et al. Cleavage of RIP3 inactivates its caspase-independent apoptosis pathway by removal of kinase domain. Cell. Signal. 19, 2056–2067 (2007).

  16. 16.

    Kang, T. B. et al. Mutation of a self-processing site in caspase-8 compromises its apoptotic but not its nonapoptotic functions in bacterial artificial chromosome-transgenic mice. J. Immunol. 181, 2522–2532 (2008).

  17. 17.

    Watanabe-Fukunaga, R., Brannan, C. I., Copeland, N. G., Jenkins, N. A. & Nagata, S. Lymphoproliferation disorder in mice explained by defects in Fas antigen that mediates apoptosis. Nature 356, 314–317 (1992).

  18. 18.

    Zhang, X., Dowling, J. P. & Zhang, J. RIPK1 can mediate apoptosis in addition to necroptosis during embryonic development. Cell Death Dis. 10, 245 (2019).

  19. 19.

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

  20. 20.

    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).

  21. 21.

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

  22. 22.

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

  23. 23.

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

  24. 24.

    Gong, Y. N. et al. ESCRT-III acts downstream of MLKL to regulate necroptotic cell death and its consequences. Cell 169, 286–300.e16 (2017).

  25. 25.

    Mandal, P. et al. RIP3 induces apoptosis independent of pronecrotic kinase activity. Mol. Cell 56, 481–495 (2014).

  26. 26.

    Dondelinger, Y. et al. RIPK3 contributes to TNFR1-mediated RIPK1 kinase-dependent apoptosis in conditions of cIAP1/2 depletion or TAK1 kinase inhibition. Cell Death Differ. 20, 1381–1392 (2013).

  27. 27.

    Holler, N. et al. Fas triggers an alternative, caspase-8-independent cell death pathway using the kinase RIP as effector molecule. Nat. Immunol. 1, 489–495 (2000).

  28. 28.

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

  29. 29.

    Peschon, J. J. et al. TNF receptor-deficient mice reveal divergent roles for p55 and p75 in several models of inflammation. J. Immunol. 160, 943–952 (1998).

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Acknowledgements

We thank M. Long, M. Dempsey, A. Verducci, T. Scholl, T. Alcantar, and E. Lu for mouse husbandry, A. Strasser, D. Siler, and the Genentech genetic analysis and histology laboratories for technical assistance, G. Salvesen and D. Vucic for discussions, and W. Alexander (WEHI, Australia) for Mlkl−/− mice.

Author information

M.R.-G. generated the Casp81×DA/+, Casp84×DA/+, Cflar2×DA/+, and Ripk1D325A/+ mice; K.N., A.M., D.L.D., and K.E.W. designed and performed experiments; L.K. performed confocal microscopy; M.D. performed immunohistochemistry; J.D.W. analysed histological data; 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.

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Competing interests

All authors were employees of Genentech.

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

Extended data figures and tables

Extended Data Fig. 1 Catalytic activity of caspase-8 is required to prevent necroptosis, but autoprocessing of caspase-8 is not required.

a, Schematic showing the organization of the different Casp8 alleles and of pro-caspase-8 protein. Non-coding and coding Casp8 exons are represented by grey and black boxes, respectively. b, Graph indicates cleavage of a proluminogenic caspase-8 substrate in primary MEF lysates at 6 h after treatment (−, no treatment; C, cycloheximide; T, TNF). RLU, relative luminescence unit. Circles, cells from different embryos (Mlkl−/−, n = 3; Casp8C362A/C362A Mlkl−/−, n = 3; Casp8−/− Mlkl−/−, n = 3). Bars, mean ± s.e.m. Unpaired two-tailed t-test. c, Graph indicates the percentage of primary MEFs that were viable 16 h after treatment (F, FasL; Tr, TRAIL). Circles, cells from different embryos (Mlkl−/−, n = 3; Casp8C362A/C362A Mlkl−/−, n = 2; Casp8−/− Mlkl−/−, n = 3). Bars, mean ± s.e.m. d, Western blots of primary MEFs. Each lane represents cells from a different embryo (Mlkl−/−, n = 3; Casp8C362A/C362A Mlkl−/−, n = 2; Casp8−/− Mlkl−/−, n = 3). β-Actin loading control performed after caspase-8. e, Body weights of E18.5 embryos. Circles, different embryos (Mlkl−/−, n = 11; Casp8C362A/C362A Mlkl−/−, n = 11). Lines, mean ± s.e.m. P = 0.7; unpaired two-tailed t-test. f, Kaplan–Meier survival curve of wild-type (n = 25) and Casp81×DA/1×DA (n = 29) littermates. P = 0.9; two-sided log-rank test. g, Western blots of thymocytes. The pan-caspase inhibitor emricasan revealed caspase-dependent cleavage events. Asterisk, non-specific band detected by the caspase-8 antibody. β-Actin loading control performed after cleaved caspase-8. Results representative of two independent experiments. h, Kaplan–Meier survival curve of Casp83×DA/3×DA (n = 40) and Casp84×DA/4×DA (n = 43) littermates. P = 0.2; two-sided log-rank test. i, Graph indicates the percentage of viable thymocytes before treatment (−), and after culture in either medium alone (Med.) or FasL for 24 h. Circles, cells from different mice (wild-type, n = 3; Casp81×DA/1×DA, n = 3; Casp83×DA/3×DA, n = 3; Casp84×DA/4×DA, n = 3). Bars, mean ± s.e.m. Unpaired two-tailed t-test. j, Graph indicates cleavage of a proluminogenic caspase-8 substrate in thymocyte lysates. Circles, cells from different mice (wild-type, n = 3; Casp84×DA/4×DA, n = 3). Bars, mean ± s.e.m. Unpaired two-tailed t-test. k, Graph indicates leukocyte numbers in spleen and lymph nodes (axillary, brachial, inguinal, and mesenteric) of mice aged 12–16 months (spleen, n = 3 per genotype; lymph nodes, n = 4 wild-type, n = 5 Casp81×DA/1×DA). Circles, cells from different mice. Lines, mean ± s.e.m. l, Flow cytometric analysis of lymph node cells from 1 wild-type and 2 Casp81×DA/1×DA mice aged 16 months, and as a control, one Casp8−/− Mlkl−/− mouse aged 3 months (this strain develops lymphadenopathy due to impaired Fas-induced apoptosis3). m, Graph indicates leukocyte numbers in spleen and mesenteric lymph node of mice aged 4–5 months (n = 3 mice per genotype). Circles, cells from different mice. Lines, mean ± s.e.m. n, Flow cytometric analysis of mesenteric lymph node cells from the mice in m. Percentages represent the mean ± s.e.m. The three gates in l were also applied to these samples. For gel source data, see Supplementary Fig. 1. Source data

Extended Data Fig. 2 Characterization of mice expressing c-FLIP(D371A/D377A) or CYLD(D215A).

a, Western blots of thymocytes that were freshly isolated (−), or cultured in either medium alone or FasL for 2.5 h. β-Actin loading control performed after caspase-8. Results representative of two independent experiments. b, Kaplan–Meier survival curve of Cflar+/+ (n = 10) and Cflar2×DA/2×DA littermates (n = 14). P = 0.3, two-sided log-rank test. c, Graph shows the percentage of thymocytes that were viable 24 h after treatment. Circles, cells from different mice (Cflar+/+, n = 3; Cflar2×DA/2×DA, n = 3). Bars, mean ± s.e.m. Unpaired two-tailed t-test. d, Flow cytometric analysis of mesenteric lymph node cells of wild-type (n = 2) and Cflar2×DA/2×DA (n = 2) littermates, and as controls, Fadd−/− Mlkl−/− mice (n = 2). Numbers below each genotype indicate total lymph node cellularity. Analyses used the gating strategy shown in Extended Data Fig. 11. e, Schematic showing the organization of the CyldD215A allele. f, Western blots of thymocytes. Arrowheads highlight caspase-dependent CYLD cleavage products. β-Actin loading control performed after cleaved caspase-8. Results representative of two independent experiments. g, Littermate males aged 18 days. Results representative of 29 wild-type and 23 CyldD215A/D215A mice. h, Kaplan–Meier survival curve of Cyld+/+ (n = 29) and CyldD215A/D215A (n = 23) littermates. P = 0.9, two-sided log-rank test. For gel source data, see Supplementary Fig. 1. Source data

Extended Data Fig. 3 Ripk1D325A/D325A Mlkl−/− Fadd−/− mice develop splenomegaly and lymphadenopathy similar to Mlkl−/− Fadd−/− mice.

a, Western blots of 293T cells overexpressing mouse caspase-8 and mouse RIPK1 (left), primary MEFs (middle), or BMDMs (right). Results representative of two independent experiments. The N-terminal RIPK1 cleavage product appears larger in the 293T cells owing to the N-terminal Flag tag. T, TNF; C, cycloheximide. β-Actin loading control performed after MYC (left) or caspase-8 (middle, right). For gel source data, see Supplementary Fig. 1. b, E12.5 littermates representative of six Ripk1D325A/+ Ripk3−/− and four Ripk1D325A/D325A Ripk3−/− embryos. c, E10.5 embryo sections immunolabelled for either RIPK1 or caspase-8 (brown). Results representative of five wild-type and six Casp8−/− embryos. Scale bar, 100 μm. Asterisk shows maternally derived Casp8+/− decidua in the placenta. d, Schematic showing the organization of the Fadd knockout allele. e, Leukocyte numbers in mice aged 10–12 weeks (wild-type, n = 2; Mlkl−/−, n = 3; Mlkl−/− Fadd−/−, n = 4; Ripk1D325A/D325A Mlkl−/− Fadd−/−, n = 4). Circles, different mice. Lines, mean ± s.e.m. f, Flow cytometric analysis of mesenteric lymph node cells from the mice in e. Mean ± s.e.m. Source data

Extended Data Fig. 4 Ripk1D325A/D325A Tnfr1−/− and Ripk1D138N, D325A/D138N, D325A mice survive until after birth.

aRipk1D325A/+ Tnfr1−/− female with her 9-day-old male pups. Results representative of two Ripk1D325A/D325A Tnfr1−/− mice. b, Body weights of Ripk1D138N,D325A/D138N,D325A pups (n = 3 males, 2 females) and their littermates (n = 1 male, 4 females) aged 14–16 days. Circles, individual mice. Lines, mean ± s.e.m. P = 3 × 10−5; unpaired two-sided t-test with Welch’s correction. c, Tissues from seven-day-old littermates stained with haematoxylin and eosin. Results representative of two Ripk1D325A/D325A Tnfr1−/− and three Tnfr1−/− mice. Scale bar, 50 μm. d, Tissues from 17-day-old Ripk1D138N,D325A/D138N,D325A mice. Asterisks, granulocytic infiltrates. Results representative of two mice. Scale bar, 100 μm. e, P4–P7 offspring from intercrossing Casp8+/− mice. f, E15.5 littermates. Results representative of two mice of each genotype. g, E13.5 embryo sections immunolabelled for p-RIPK3(T231/S232) (brown). Results representative of three Casp8−/− Ripk1D138N/D138N and two Ripk1D138N/D138N embryos. Scale bar, 200 μm. h, E12.5 littermates. Results representative of four Casp8−/− Ripk1D325A/D325A and three Ripk1D325A/+ control embryos. i, Western blots of primary MEFs. β-Actin loading control performed after IκBα. For gel source data, see Supplementary Fig. 1. Results representative of two independent experiments. Source data

Extended Data Fig. 5 The RIPK3 scaffold contributes to TNF-induced apoptosis in Ripk1D325A/D325A MEFs.

a, ce, Graphs show the percentage of primary MEFs (a, c) or BMDMs (d, e) that were viable at 24 h (a), 8 h (c, e), or 5 h (d) after treatment (−, untreated; T, TNF; N, Nec-1; i, TAK1 inhibitor 5Z-7-oxozeaenol; G, RIPK3 inhibitor GSK′843). Circles, cells from different mice (Ripk1+/+, n = 4 (a), 2 (c), and 3 (d, e); Ripk1D325A/D325A, n = 4 (a); Ripk1D325A/D325A Ripk3−/−, n = 3 (a); Ripk3−/−, n = 4 (a); Ripk1D325A/+, n = 2 (c) and 3 (d, e)). Bars, mean ± s.e.m. Unpaired two-sided t-test. not determined. b, f, Western blots of primary MEFs after 4 h treatment (b) or of BMDMs (f). β-Actin loading control performed after c-FLIP (b) or p-RIPK1 (f). For gel source data, see Supplementary Fig. 1. Results representative of two independent experiments. Source data

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