MLKL, a key component downstream of RIPK3, is suggested to be a terminal executor of necroptosis. Genetic studies have revealed that Ripk3 ablation rescues embryonic lethality in Fadd- or Caspase-8-deficient mice. Given that RIPK3 has also been implicated in non-necroptotic pathways including apoptosis and inflammatory signaling, it remains unclear whether the lethality in Fadd(-/-) mice is indeed caused by necropotosis. Here, we show that genetic deletion of Mlkl rescues the developmental defect in Fadd-deficient mice and that Fadd(-/-)Mlkl(-/-) mice are viable and fertile. Mlkl(-/-)Fadd(-/-) mice display significantly accelerated lymphoproliferative disease characterized by lymphadenopathy and splenomegaly when compared to Ripk3(-/-)Fadd(-/-) mice. Mlkl(-/-)Fadd(-/-) bone-marrow-derived macrophages and dendritic cells have impaired NLRP3 inflammasome activation associated with defects in ASC speck formation and NF-κB-dependent NLRP3 transcription. Our findings reveal that MLKL and FADD play critical roles in preventing lymphoproliferative disease and activating the NLRP3 inflammasome.

RIP1 is an essential regulator of TNF-induced signaling complexes mediating NF-κB activation, apoptosis and necroptosis. Loss of Rip1 rescues the embryonic lethality of Fadd or Caspase-8-deficient mice, even though the double knockout mice die shortly after birth like Rip1-deficient mice. Recent studies demonstrated that mice expressing RIP1 kinase-dead mutants developed normally and resisted necroptotic stimuli in vitro and in vivo. However, the impact of RIP1 kinase activity on Fadd embryonic development remains unknown. Here, we engineered two RIP1 kinase inactive mutant mouse lines, a Rip1 mouse line as previously reported and a novel Rip1 mouse line with an altered P-loop in the kinase domain. While RIP1 could not rescue the embryonic lethality of Fadd-deficient mice at E11.5, RIP1 rescued lethality of Fadd mice at E11.5 and FaddRip1 mice eventually died at E16.5 due to excessive death of fetal liver cells and unregulated inflammation. Under necropotosis-inducing conditions, comparing to Rip1 cells, Rip1cells displayed reduced phosphorylation and oligomerization of RIP3 and MLKL, which lead to increased cell viability. Thus, our study provides genetic evidence that different kinase inactive mutations have distinct impacts on the embryogenesis of Fadd-deficient mice, which might attribute to their extents of protection on necroptosis signaling.

In mammalian cells, signaling pathways triggered by TNF can be switched from NF-κB activation to apoptosis and/or necroptosis. The in vivo mechanisms underlying the mutual regulation of these three signaling pathways are poorly understood. In this article, we report that the embryonic lethality of -deficient mice is partially prevented by the deletion of or but it is fully rescued by the combined ablation of and or or by blocking RIP1 kinase activity (RIP1). triple-knockout (TKO) and mice displayed bacterial pneumonia leading to death ∼2 wk after birth. Moreover, mice, but not TKO mice, developed severe inflammation associated with inflammatory skin lesion. Antibiotic treatment improved bacterial pneumonia, extended the lifespan of TKO and mice, and alleviated skin inflammation in mice. These results show the mechanisms underlying the in vivo mutual regulation between NF-κB activation and the cell death pathway and provide new insights into this interplay in embryonic development and host immune homeostasis.