Left, TNF binding to TNFR1 triggers the formation of complex I, and subsequent ubiquitylation and phosphorylation of RIPK1. These post-translational modifications (PTMs) inhibit the cytotoxic activity of RIPK1. Complex I formation activates NF-κB- and MAPK-dependent survival genes such as CFLAR, which encodes cFLIP. Subsequently, a cytosolic complex II containing FADD, caspase-8, RIPK1 and cFLIP is formed. In this complex, cFLIP inhibits caspase-8 activity so that a restricted number of substrates (such as RIPK1) are cleaved, but others (such as pro-caspase-3) are not. Cleavage of RIPK1 dismantles complex II. Activation of the NF-κB and MAPK signalling pathways PTM of RIPK1 prevent TNF from inducing cell death, resulting in cell survival (top left). Inhibition of the NF-κB or MAPK signalling pathways reduces levels of cFLIP and accelerates formation of complex II, resulting in cell death via apoptosis (middle left). When NF-κB or MAPK signalling is disrupted in caspase-8-deficient conditions, RIPK1 is not cleaved and autophosphorylates, which triggers the recruitment of RIPK3 and its autophosphorylation. RIPK3 phosphorylates MLKL and necroptosis occurs (bottom left). Right, according to this model, lack of RIPK1 cleavage could result in several distinct outcomes, as follows. (1) RIPK1 accumulation could stabilize complex II, and the presence of cFLIP and inhibitory PTMs to RIPK1 may prevent caspase-8 from killing, resulting in cell survival. (2) The accumulation of ‘uncleavable’ RIPK1 to complex II could override the inhibitory RIPK1 PTMs, resulting in autophosphorylation of RIPK1 and recruitment of RIPK3, leading to necroptosis. (3) RIPK1 accumulation could result in activated caspase-8 that cleaves RIPK3, resulting in cell survival. (4) Stabilization of complex II could result in recruitment and activation of caspase-8 that induces apoptosis and possibly prevents necroptosis by cleaving RIPK3. (5) Finally, the accumulation of RIPK1 could result in activation of both RIPK3 and caspase-8 and therefore induce both apoptotic and necroptotic cell death. In terms of how these potential outcomes match with our data, in homozygote Ripk1D325A cells, both caspase-8 and RIPK3 are activated after TNF signalling, which suggests that apoptosis and necroptosis occur at the same time (Figs. 2d, 3a, b). However, according to these models, loss of RIPK3 limits caspase-8 activation (Fig. 3a, b). This suggests that the recruitment of RIPK3 to complex II increases the recruitment and activation of caspase-8. A precedent for this observation comes from experiments in which RIPK3 inhibitors promoted RIPK1-dependent caspase-8 activation42,43, in a manner we term ‘reverse activation’. In our experiments, however, RIPK3 activation occurs downstream of TNF signalling, which suggests that reverse activation might represent a physiological amplification loop that increases caspase-8 activation. Yet, this requirement for RIPK3 is not present in all cells, as the embryonic lethality of the RIPK1-cleavage mutant is only partially rescued by loss of Ripk3. In the heterozygote Ripk1D325A cells, caspase-8 cleaves wild-type RIPK1, thus limiting TNF-induced cell death as compared to homozygote cells. However, reduction of cFLIP and/or RIPK1 PTMs by treatment with IAP, TAK1, IKK or translational inhibitors decreases the threshold of TNF sensitivity (Extended Data Fig. 4). This may cause the hyper-inflammatory response observed in patients with CRIA syndrome (Fig. 1).