GSE132133
Single cell RNA-seq of intestine from E18.5 Mlkl -/- and Casp8 C362A/C362A Mlkl -/- embryos
Caspase-8 is a protease with both pro-death and pro-survival functions: it is required  for apoptosis induced by death receptors such as TNFR1 (tumour necrosis factor   receptor 1), and it has a critical role in suppressing necroptosis mediated by the   kinase RIPK3 (receptor interacting protein kinase 3) and the pseudokinase MLKL  (mixed lineage kinase-like). Mice lacking caspase-8 display MLKL-dependent   embryonic lethality, as do mice expressing catalytically inactive caspase-8 mutant   C362A. However, Casp8C362A/C362A Mlkl-/- mice die in the perinatal period, whereas   Casp8-/- Mlkl-/- mice are viable, indicating that inactive caspase-8 also has a pro-death   scaffolding function. Here we show that caspase-8 C362A triggers ASC speck   formation and caspase-1-dependent pyroptosis in MLKL-deficient intestinal epithelial   cells (IECs) around embryonic day 18. Pyroptosis contributed to the perinatal lethal   phenotype because a number of Casp8 C362A/C362A Mlkl-/- Casp1-/- mice survived beyond   weaning. Transfection studies suggested inactive caspase-8 adopts a distinct   conformation to wild-type caspase-8, enabling it to engage the caspase-1 adaptor ASC.   Wild-type caspase-8 was found in the Triton X-100 soluble fraction, whereas wild-type   caspase-8 inhibited with the pan-caspase inhibitor emricasan, or inactive caspase-8   mutant C362A, were detected in the insoluble fraction. Moreover, inhibited or   inactive caspase-8 shifted ASC into the insoluble fraction. Perinatal lethality was   recapitulated when expression of caspase-8 C362A was restricted to IECs, but   intriguingly, only in the absence of MLKL. Hence, unanticipated plasticity in death   pathways is revealed such that IECs can undergo caspase-1-dependent death when   caspase-8-dependent apoptosis and MLKL-dependent necroptosis are inhibited. Overall design: Whole intestines from E18.5 embryos (One E18.5 Mlkl -/- and one Casp8 C362A/C362A Mlkl -/- ) were dissociated into single cells and sequenced.