Hematopoietic RIPK1 deficiency results in bone marrow failure caused by apoptosis and RIPK3-mediated necroptosis

Abstract

Receptor-interacting serine/threonine-protein kinase 1 (RIPK1) is recruited to the TNF receptor 1 to mediate proinflammatory signaling and to regulate TNF-induced cell death. RIPK1 deficiency results in postnatal lethality, but precisely why Ripk1 −/− mice die remains unclear. To identify the lineages and cell types that depend on RIPK1 for survival, we generated conditional Ripk1 mice. Tamoxifen administration to adult RosaCreER T2 Ripk1fl/fl mice results in lethality caused by cell death in the intestinal and hematopoietic lineages. Similarly, Ripk1 deletion in cells of the hematopoietic lineage stimulates proinflammatory cytokine and chemokine production and hematopoietic cell death, resulting in bone marrow failure. The cell death reflected cell-intrinsic survival roles for RIPK1 in hematopoietic stem and progenitor cells, because Ripk3−/− progenitors remain TNF sensitive in vitro and fail to repopulate irradiated mice. These genetic studies reveal that hematopoietic RIPK1 deficiency triggers both apoptotic and necroptotic death that is partially prevented by RIPK3 deficiency. Therefore, RIPK1 regulates hematopoiesis and prevents inflammation by suppressing RIPK3 activation.tumor necrosis factor | programmed necrosis | hematopoietic failure T he proinflammatory cytokine TNF stimulates receptorinteracting serine/threonine-protein kinase 1 (RIPK1) ubiquitination, NFκB and MAPK activation, and induction of apoptosis or necroptosis (1, 2). TNF signaling via TNF receptor 1 (TNFR1) is highly regulated and results in the recruitment of several adapter proteins including TNFR1-associated death domain (TRADD) protein, the E3 ubiquitin ligases cellular inhibitor of apoptosis protein-1 and -2 (cIAP1/2), and TNFRassociated factor 2 (TRAF2) or 5, and the serine threonine death domain-containing kinase RIPK1 (complex I) (1). We have demonstrated that the kinase activity of RIPK1 is not required for NFκB activation (3); rather, RIPK1 is modified by the addition of Lys63-linked and linear polyubiquitin chains (3-6). Polyubiquitinated RIPK1 then recruits NEMO/IκB kinase-γ (IKKγ) to mediate IKK activation and TAK1/TAB2/3 to mediate MAPK activation, resulting in antiapoptotic and proinflammatory gene expression (7,8). Deubiquitination of RIPK1 by cylindromatosis (CYLD) results in the formation of a cytosolic complex containing TRADD, Fas-associated death domain protein (FADD), caspase-8, and RIPK1 (complex IIa) (2). Caspase-8 cleaves and inactivates RIPK1 and CYLD and stimulates apoptosis (9-11). In the absence of caspase-8 or the presence of caspase inhibitors, TNF family members and potentially other ligands stimulate the kinase activity of RIPK1 to induce necroptosis (9, 11-16). RIPK1 also is recruited to the Toll-like receptor adapter TRIF via the Rip homotypic interaction motif (RHIM) to mediate NFκB activation (17) and, under conditions of caspase-8 inhibition, initiates necroptosis (14, 16). Necrostatin-1 (Nec-1), an allosteric RIPK1 inhibitor, inhibits necroptosis induced by TNF or the TLR3 ligand poly I:C and abolish...

K45A mutation of RIPK1 results in poor necroptosis and cytokine signaling in macrophages, which impacts inflammatory responses in vivo

“…5 Besides the kinase activity, RIPK1 appears to have a scaffolding function, which may influence immune homeostasis. 6,7 Depending on the interacting partners and posttranslational modifications, RIPK1 has a multifaceted role in cell signaling and cell survival. 8 Following TNF-R1 signaling, RIPK1 transitions between pro-survival and pro-cell death signaling complexes.…”

“…9 RIPK1-deficient mice die within the first week of birth 5 as a result of uncontrolled activation of necrosis and apoptosis mediated by RIPK3 and caspase 8,6,30 suggesting that RIPK1, RIPK3 and caspase 8 regulate the overactivation of each other. As RIPK3-deficient mice are viable, 31 this suggests that RIPK1 has additional roles besides necroptosis.…”

“…5 Besides the kinase activity, RIPK1 appears to have a scaffolding function, which may influence immune homeostasis. 6,7 Depending on the interacting partners and posttranslational modifications, RIPK1 has a multifaceted role in cell signaling and cell survival.8 Following TNF-R1 signaling, RIPK1 transitions between pro-survival and pro-cell death signaling complexes. 9,10 Necroptosis is a form of regulated necrosis of cells that operates in the absence of caspase activity 9 and is initiated by the engagement of various TLRs or cytokine receptors.…”

PolyI:C–Induced, TLR3/RIP3-Dependent Necroptosis Backs Up Immune Effector–Mediated Tumor Elimination In Vivo

“…Although RIP1 and RIP3 are required to initiate necroptotic signaling, necroptosis is induced in the absence of RIP1 (40,41), which has been reported to protect some tissue/organ cells from necroptosis, reflecting the complex arrays of necroptosis. Similar to L929 cells, CT26 cells can be sensitized by polyI:C to induce necroptosis in the presence of zVAD (24).…”

Necroptosis in spontaneously-mutated hematopoietic cells induces autoimmune bone marrow failure in mice

“…Importantly, although necroptosis has been implicated in BMF in hematopoietic-specific Tak1 and receptor interacting protein kinase-1 (Rip1)-knockout mice, these studies suggested that both Tak1 and Rip1 are required for the survival of HSPCs by repressing apoptosis and necroptosis. 25,30,31 However, due to the rapid death of the knockout mice, none of these studies was able to detect the activation of immune cells. In addition, although it has been proposed that necroptotic cells can be immunogenic, 23,24,32 to our knowledge, we are the first to experimentally demonstrate that necroptotic cells actually do initiate autoimmune diseases.…”