Activity of caspase-8 determines plasticity between cell death pathways

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 MLKL2–4. 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. Alternative cell death pathways are revealed in the absence of caspase-8-dependent apoptosis and MLKL-dependent necroptosis.