Upon ligand binding, RIPK1 is recruited to tumor necrosis factor receptor superfamily (TNFRSF) and Toll-like receptor (TLR) complexes promoting prosurvival and inflammatory signaling. RIPK1 also directly regulates caspase-8-mediated apoptosis or, if caspase-8 activity is blocked, RIPK3-MLKL-dependent necroptosis. We show that C57BL/6 Ripk1(-/-) mice die at birth of systemic inflammation that was not transferable by the hematopoietic compartment. However, Ripk1(-/-) progenitors failed to engraft lethally irradiated hosts properly. Blocking TNF reversed this defect in emergency hematopoiesis but, surprisingly, Tnfr1 deficiency did not prevent inflammation in Ripk1(-/-) neonates. Deletion of Ripk3 or Mlkl, but not Casp8, prevented extracellular release of the necroptotic DAMP, IL-33, and reduced Myd88-dependent inflammation. Reduced inflammation in the Ripk1(-/-)Ripk3(-/-), Ripk1(-/-)Mlkl(-/-), and Ripk1(-/-)Myd88(-/-) mice prevented neonatal lethality, but only Ripk1(-/-)Ripk3(-/-)Casp8(-/-) mice survived past weaning. These results reveal a key function for RIPK1 in inhibiting necroptosis and, thereby, a role in limiting, not only promoting, inflammation.

RIPK1 is involved in signaling from TNF and TLR family receptors. After receptor ligation, RIPK1 not only modulates activation of both canonical and NIK-dependent NF-B, but also regulates caspase-8 activation and cell death. Although overexpression of RIPK1 can cause caspase-8-dependent cell death, when RIPK1؊/؊ cells are exposed to TNF and low doses of cycloheximide, they die more readily than wild-type cells, indicating RIPK1 has pro-survival as well as pro-apoptotic activities (1, 2). To determine how RIPK1 promotes cell survival, we compared wild-type and RIPK1 ؊/؊ cells treated with TNF.Although TRAF2 levels remained constant in TNF-treated wildtype cells, TNF stimulation of RIPK1 ؊/؊ cells caused TRAF2 and cIAP1 to be rapidly degraded by the proteasome, which led to an increase in NIK levels. This resulted in processing of p100 NF-B2 to p52, a decrease in levels of cFLIP L , and activation of caspase-8, culminating in cell death. Therefore, the pro-survival effect of RIPK1 is mediated by stabilization of TRAF2 and cIAP1.In mouse embryonic fibroblasts (MEFs), 2 TNFR1 signaling is mediated by binding of the adaptor protein TRADD to its cytoplasmic domains. TRADD then recruits TRAF2 and RIPK1. TRAF2 binds to cIAP1 and cIAP2, which mediate ubiquitylation of RIPK1 (3-5). A series of ubiquitylation and phosphorylation events follow, leading to activation of TAK1, p65/RelA NF-B, and JNK (6 -8). When pro-survival signals mediated by NF-B are blocked, for example by deletion of genes for p65/ RelA, or addition of the translation inhibitor cycloheximide, ligation of TNFR1 results in recruitment of FADD, binding and activation of caspase-8, and apoptosis.Ligation-induced recruitment of RIPK1 to TNFR1 was thought to be necessary for activation of the IKK complex that phosphorylates IkB prior to its ubiquitylation by SCF BTrCP . In this way, RIPK1 was proposed to promote cell survival by allowing activation of the canonical NF-B. Consistent with this model, gene deletion of RIPK1 sensitizes cells to killing by TNF in combination with low doses of cycloheximide (2). However, we have recently shown that RIPK1 is not essential for activation of canonical p65/RelA NF-B in response to TNF, but plays a redundant role (1). This suggests that the survival effect of RIPK1 in response to TNF is mediated by some other mechanism.Like RIPK1 Ϫ/Ϫ cells, those deficient for TRAF2 are also more likely to die than wild-type MEFs when treated with low doses of cycloheximide and TNF. This sensitization was shown to be due to loss of function of the RING domain of TRAF2 (9). The fact that RIPK1 has been shown to interact directly with TRAF2 raised the possibility that they act in concert to inhibit cell death in response to TNF, but to date this has not been confirmed, and the mechanism has not been elucidated (10).We reasoned that the ability of TRAF2 and RIPK1 to promote survival of TNF-treated cells might be related. To test this hypothesis we compared TRAF2 Ϫ/Ϫ and RIPK1 Ϫ/Ϫ genedeleted cells. Here we show that RIPK1 prevents activatio...

There were several errors in this article. PAGE 13284:The figure legend of with 100 ng/ml Fc-TNF for 0, 5, and 15 min. Cells were then lysed in DISC lysis buffer and split into soluble and insoluble fractions. 25 g of protein from both fractions were run on SDS-PAGE and analyzed for levels of indicated proteins by Western blotting analysis." An asterisk denotes a nonspecific band. PAGE 13285:In the figure legend for Fig. 2A, the text "Keratinocytes were isolated from day old RIPK Ϫ/Ϫ 1-day-old pups" does not refer to panelA of the figure and should be removed. The corrected text for the description of Fig. 2A should read, "E19 embryos were taken, and thymus was harvested. Thymi were dispersed into single cell suspensions, and half of each was left untreated, whereas the other half was treated with 100 ng/ml Fc-TNF for 1 h. Cells were lysed in DISC lysis buffer and analyzed for TRAF2 and cIAP1 by Western blotting analysis. Genotypes were confirmed by Western blotting analysis and PCR." PAGE 13286:At the end of the figure legend for Fig. 4, the text "To control for NEC-1 activity, L929 cells" does not refer to this figure and should be removed. PAGE 13287:The following sentence should be added to the end of the legend to Fig. 6. "Error bars show S.E. of at least three independent experiments." PAGE 13288:In Fig. 7A, the caspase-8 long exposure immunoblot was incorrectly cropped and the molecular mass of the ␤-actin immunoblot was incorrectly labeled. These errors have been corrected so that there are now six lanes in the caspase-8 (long exposure), and the molecular weight of actin reflects the actual size.The following sentence should be added to the figure legend to supplemental Fig. 2. "Error bars show S.E. of at least three independent experiments."These errors do not affect the results or conclusions of the work.

Introduction Cell death can be triggered by many stimuli leading to apoptosis, pyroptosis (Caspase-1-dependent cell death) or necroptosis (Receptor-interacting serine/threonine-protein kinase (RIPK)-1/RIPK3-dependent cell death). RIPK1 is engaged by TNFR or Fas/CD95 ligation, and can induce NF-κB activation and cell death. FADD and Caspase-8 modulate RIPK1 and RIPK3 activity to prevent inappropriate induction of necroptosis (Oberst et al., Nature 2011, 471: 363-367; Zhang et al., Nature 2011, 471: 373-376). Modulation of necroptosis by small molecule inhibitors of RIPK1 has emerged as an exciting approach to intervene in inflammatory disease, ischemia reperfusion injury, pancreatitis and in mouse models of sepsis (He et al., Cell 2009, 137: 1100-1111; McNeal et al., Shock 2011, 35: 499-505; Oerlemans et al., Basic Res Cardiol 2012, 107: 270; Lukens et al., Nature 2013, 498: 224-227). However, RIPK1-deficient neonates die at birth and exhibit inflammatory disease and anemia, suggesting that inhibitors of RIPK1 may alter hematopoiesis. We have therefore investigated the hematological consequences of RIPK1 deficiency. Methods Fetal liver chimeras and competitive transplants were generated using E13.5 Ripk1-/-, Ripk3-/-and Ripk1-/-Ripk3-/- fetal liver cells. Serial transplants were established using 106 fetal liver cells for primary transplants and 0.2-5 x 106 bone marrow cells for secondary transplants. The survival of recipient mice and frequency of donor, competitor and recipient cells was assessed by flow cytometry up to 6 months post transplantation. The frequency of hematopoietic progenitor cells was assessed using in vitro clonal culture assays of E13.5-E18.5 fetal liver cells stimulated with SCF+IL-3+Epo in the presence or absence of TNFα or FasL. The contribution of TNFα and FasL to hematopoiesis was examined using TNFα neutralizing antibody in lethally-irradiated recipients of Ripk1-/- cells or by engrafting Ripk1-/- cells into lethally-irradiated Tnfa-/-Faslgld/gldrecipient mice. Results Ripk1 -/- fetal liver cells fail to engraft in lethally-irradiated recipients, with defects evident in lymphoid and myeloid lineages in the bone marrow, peripheral blood and spleen between 4 and 26 weeks post-transplant. In competitive fetal liver transplant experiments, Ripk1-/- hematopoietic stem and progenitor cells failed to compete with wild-type counterparts, indicating a cell-intrinsic defect in hematopoietic progenitor cells that cannot be attributed to the inflammatory disease evident in Ripk1-/- embryos. Ripk1-/- myeloid progenitor cells were sensitive to death induced by TNFα or FasL stimulation. Only minor abnormalities in hematopoiesis were detected when Ripk1-/- fetal liver cells were transplanted into lethally-irradiated Tnfa-/-Faslgld/gld recipient mice, or when lethally-irradiated wild-type recipient mice receiving Ripk1-/- fetal liver cells were treated with a TNFα neutralizing antibody, indicating key roles for TNFα and FasL during engraftment. A compound deficiency in RIPK3 rescued the reconstitution defects seen in Ripk1-/- cells suggesting that RIPK1-deficient hematopoietic cells undergo RIPK3-dependent necroptotic death. A residual defect in Ripk1-/-Ripk3-/-T lymphopoiesis suggests that RIPK1 deficiency induces other forms of cell death or that RIPK1 is required for other essential signaling pathways such as NF-κB signaling. Conclusion These data demonstrate essential roles for RIPK1 in hematopoiesis at steady state. Our results indicate that small molecule RIPK1 inhibitors should be used with caution in the clinic to avoid activation of RIPK3-dependent cell death pathways leading to cytopenia, immunosuppression and bone marrow failure. Finally, this work highlights that studies using RIPK1-deficient cells to study the roles for RIPK1 in inflammatory disease must draw conclusions with care considering the critical role of RIPK1 in hematopoiesis. Disclosures: No relevant conflicts of interest to declare.