Deletion of the Casp8 gene in epithelial tissues of mice results in severe inflammatory pathologies. Its ubiquitous deletion, or its specific deletion in endothelial cells, results in intrauterine death associated with capillary damage. These pathologies are all preventable by co-deletion of Casp8 and the genes encoding either the RIPK1 or the RIPK3 protein kinase. Since activation of RIPK3 in Caspase-8-deficient cells can trigger necroptotic cell death, and since RIPK1 can activate RIPK3, it is widely assumed that the inflammatory states resulting from Caspase-8 deficiency occur as a consequence of RIPK3-induced necroptosis. Here, we report that although on a Ripk3-null background Casp8 deletion in mice does not result in outright pathological changes, it triggers enhanced expression of a variety of inflammatory genes in utero, which gradually subsides after birth. Deletion of Ripk1, or even of only one of its two alleles, obliterates this activation. Resembling the embryonic pathology observed in RIPK3-expressing cells, the activation of inflammatory genes observed on a Ripk3-null background seems to be initiated in endothelial cells. Analysis of endothelial cells isolated from livers of Caspase-8-deficient embryos revealed neither an increase in the amount of RIPK1 in these cells after Casp8 deletion, nor triggering of RIPK1 phosphorylation. These findings indicate that the triggering of inflammation by Casp8 deletion in mice occurs, in part, independently of necroptosis or other functions of RIPK3, and rather reflects enhanced RIPK1-dependent signaling for activation of inflammatory genes.

Phosphorylation of the pseudokinase mixed lineage kinase domain-like protein (MLKL) by the protein kinase RIPK3 targets MLKL to the cell membrane, where it triggers necroptotic cell death. We report that conjugation of K63-linked polyubiquitin chains to distinct lysine residues in the N-terminal HeLo domain of phosphorylated MLKL (facilitated by the ubiquitin ligase ITCH that binds MLKL via a WW domain) targets MLKL instead to endosomes. This results in the release of phosphorylated MLKL within extracellular vesicles. It also prompts enhanced endosomal trafficking of intracellular bacteria such as Listeria monocytogenes and Yersinia enterocolitica to the lysosomes, resulting in decreased bacterial yield. Thus, MLKL can be directed by specific covalent modifications to differing subcellular sites, whence it signals either for cell death or for non-deadly defense mechanisms.