RIPK3 cleavage is dispensable for necroptosis inhibition but restricts NLRP3 inflammasome activation

Caspase-8 activity is required to inhibit Necroptosis during embryogenesis in mice. In vitro studies have suggested that Caspase-8 directly cleaves RIPK1, CYLD and the key necroptotic effector kinase RIPK3 to repress Necroptosis. However, recent studies have shown that mice expressing uncleavable RIPK1 die during embryogenesis due to excessive Apoptosis, while uncleavable CYLD mice are viable. Therefore, these results raise important questions about the role of RIPK3 cleavage. To evaluate the physiological significance of RIPK3 cleavage, we generated Ripk3^D333A/D333A mice harbouring a point mutation in the conserved Caspase-8 cleavage site. These mice are viable, demonstrating that RIPK3 cleavage is not essential for blocking Necroptosis during development. Furthermore, unlike RIPK1 cleavage-resistant cells, Ripk3^D333A/D333A cells were not significantly more sensitive to necroptotic stimuli. Instead, we found that the cleavage of RIPK3 by Caspase-8 restricts NLRP3 inflammasome activation-dependent Pyroptosis and IL-1β secretion when Inhibitors of Apoptosis (IAP) are limited. These results demonstrate that Caspase-8 does not inhibit Necroptosis by directly cleaving RIPK3 and further underscore a role for RIPK3 in regulating the NLRP3 inflammasome.