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TNF is a master proinflammatory cytokine whose pathogenic role in inflammatory disorders can, in certain conditions, be attributed to RIPK1 kinase-dependent cell death. Survival, however, is the default response of most cells to TNF stimulation, indicating that cell demise is normally actively repressed and that specific checkpoints must be turned off for cell death to proceed. We identified RIPK1 as a direct substrate of MK2 in the TNFR1 signalling pathway. Phosphorylation of RIPK1 by MK2 limits cytosolic activation of RIPK1 and the subsequent assembly of the death complex that drives RIPK1 kinase-dependent apoptosis and necroptosis. In line with these in vitro findings, MK2 inactivation greatly sensitizes mice to the cytotoxic effects of TNF in an acute model of sterile shock caused by RIPK1-dependent cell death. In conclusion, we identified MK2-mediated RIPK1 phosphorylation as an important molecular mechanism limiting the sensitivity of the cells to the cytotoxic effects of TNF. Dondelinger et al. and Menon et al. show that MAPKAP kinase-2 (MK2) phosphorylates RIPK1 to regulate TNF-mediated cell death as well as RIPK1 signalling in inflammation and bacterial infection.

Necroptosis is a form of programmed cell death that depends on the activation of receptor interacting protein kinase-1 (RIPK1) and RIPK3 by receptors such as tumor necrosis factor (TNF) receptor-1. Structural studies indicate that activation of RIPK3 by RIPK1 involves the formation of oligomers via interactions of the RIP homotypic interaction motif (RHIM) domains shared by both proteins; however, the molecular mechanisms by which this occurs are not fully understood. To gain insight into this process, we constructed versions of RIPK3 that could be induced to dimerize or oligomerize in response to a synthetic drug. Using this system, we find that although the formation of RIPK3 dimers is itself insufficient to trigger cell death, this dimerization seeds a RHIM-dependent complex, the propagation and stability of which is controlled by caspase-8 and RIPK1. Consistent with this idea, we find that chemically enforced oligomerization of RIPK3 is sufficient to induce necroptosis, independent of the presence of the RHIM domain, TNF stimulation or RIPK1 activity. Further, although RIPK1 contributes to TNF-mediated RIPK3 activation, we find that RIPK1 intrinsically suppresses spontaneous RIPK3 activation in the cytosol by controlling RIPK3 oligomerization. Cells lacking RIPK1 undergo increased spontaneous RIPK3-dependent death on accumulation of the RIPK3 protein, while cells containing a chemically inhibited or catalytically inactive form of RIPK1 are protected from this form of death. Together, these data indicate that RIPK1 can activate RIPK3 in response to receptor signaling, but also acts as a negative regulator of spontaneous RIPK3 activation in the cytosol.

NEK7, a member of the NIMA-related kinase family, is identified as a regulator of NLRP3 inflammasome oligomerization and activation; NEK7 functions downstream of potassium efflux in a manner that is independent of its kinase activity. NEK7 mediates NLRP3 inflammasome activation The NLRP3 inflammasome, a critical component of the innate immune system, has been linked to multiple acquired and inherited diseases. However, the molecular mechanism that leads to NLRP3 oligomerization and activation remains elusive. Here Gabriel Núñez and colleagues identify a member of the family of NIM related kinases (NEK7) as a regulator of NLRP3 inflammasome oligomerization and activation. NEK7 functions downstream of potassium efflux in a manner that is independent of its kinase activity. Inflammasomes are intracellular protein complexes that drive the activation of inflammatory caspases 1 . So far, four inflammasomes involving NLRP1, NLRP3, NLRC4 and AIM2 have been described that recruit the common adaptor protein ASC to activate caspase-1, leading to the secretion of mature IL-1β and IL-18 proteins 2 , 3 . The NLRP3 inflammasome has been implicated in the pathogenesis of several acquired inflammatory diseases 4 , 5 as well as cryopyrin-associated periodic fever syndromes (CAPS) caused by inherited NLRP3 mutations 6 , 7 . Potassium efflux is a common step that is essential for NLRP3 inflammasome activation induced by many stimuli 8 , 9 . Despite extensive investigation, the molecular mechanism leading to NLRP3 activation in response to potassium efflux remains unknown. Here we report the identification of NEK7, a member of the family of mammalian NIMA-related kinases (NEK proteins) 10 , as an NLRP3-binding protein that acts downstream of potassium efflux to regulate NLRP3 oligomerization and activation. In the absence of NEK7, caspase-1 activation and IL-1β release were abrogated in response to signals that activate NLRP3, but not NLRC4 or AIM2 inflammasomes. NLRP3-activating stimuli promoted the NLRP3–NEK7 interaction in a process that was dependent on potassium efflux. NLRP3 associated with the catalytic domain of NEK7, but the catalytic activity of NEK7 was shown to be dispensable for activation of the NLRP3 inflammasome. Activated macrophages formed a high-molecular-mass NLRP3–NEK7 complex, which, along with ASC oligomerization and ASC speck formation, was abrogated in the absence of NEK7. NEK7 was required for macrophages containing the CAPS-associated NLRP3(R258W) activating mutation to activate caspase-1. Mouse chimaeras reconstituted with wild-type, Nek7 −/− or Nlrp3 −/− haematopoietic cells showed that NEK7 was required for NLRP3 inflammasome activation in vivo . These studies demonstrate that NEK7 is an essential protein that acts downstream of potassium efflux to mediate NLRP3 inflammasome assembly and activation.

The enzyme RIPK1 functions through its RHIM domain to prevent ZBP1-mediated activation of RIPK3–MLKL-dependent necroptosis, thus preventing perinatal lethality and skin inflammation in adult mice. RIPK1 inhibition of inflammation Manolis Pasparakis and colleagues report that receptor-interacting protein kinase 1 (RIPK1) functions via its RIP homotypic interaction motif (RHIM) to prevent skin inflammation in mice by inhibiting activation of RIPK3–MLKL-dependent necroptosis mediated by Z-DNA binding protein 1 (ZBP1; also known as DAI). The finding that ZBP1 is a critical mediator of inflammation beyond its previously known role in antiviral defence suggests that ZBP1 might be involved in the pathogenesis of necroptosis-associated inflammatory diseases. Receptor-interacting protein kinase 1 (RIPK1) regulates cell death and inflammation through kinase-dependent and -independent functions 1 , 2 , 3 , 4 , 5 , 6 , 7 . RIPK1 kinase activity induces caspase-8-dependent apoptosis and RIPK3 and mixed lineage kinase like (MLKL)-dependent necroptosis 8 , 9 , 10 , 11 , 12 , 13 . In addition, RIPK1 inhibits apoptosis and necroptosis through kinase-independent functions, which are important for late embryonic development and the prevention of inflammation in epithelial barriers 14 , 15 , 16 , 17 , 18 . The mechanism by which RIPK1 counteracts RIPK3–MLKL-mediated necroptosis has remained unknown. Here we show that RIPK1 prevents skin inflammation by inhibiting activation of RIPK3–MLKL-dependent necroptosis mediated by Z-DNA binding protein 1 (ZBP1, also known as DAI or DLM1). ZBP1 deficiency inhibited keratinocyte necroptosis and skin inflammation in mice with epidermis-specific RIPK1 knockout. Moreover, mutation of the conserved RIP homotypic interaction motif (RHIM) of endogenous mouse RIPK1 (RIPK1 mRHIM ) caused perinatal lethality that was prevented by RIPK3, MLKL or ZBP1 deficiency. Furthermore, mice expressing only RIPK1 mRHIM in keratinocytes developed skin inflammation that was abrogated by MLKL or ZBP1 deficiency. Mechanistically, ZBP1 interacted strongly with phosphorylated RIPK3 in cells expressing RIPK1 mRHIM , suggesting that the RIPK1 RHIM prevents ZBP1 from binding and activating RIPK3. Collectively, these results show that RIPK1 prevents perinatal death as well as skin inflammation in adult mice by inhibiting ZBP1-induced necroptosis. Furthermore, these findings identify ZBP1 as a critical mediator of inflammation beyond its previously known role in antiviral defence and suggest that ZBP1 might be implicated in the pathogenesis of necroptosis-associated inflammatory diseases.

Haematopoietic expression of the adaptor protein Trib1 is shown to be required for the presence of adipose-tissue-resident macrophages with an M2-like phenotype; Trib1 deficiency leads to aberrant expression of C/EBPα and impaired adipose tissue function. Trib1 protein role in macrophage function Macrophages are classified loosely into two types: M1 cells are immune cells active against microbial infection, and M2 cells have a broad spectrum of activities involving tissue repair, helminth infection, tumour progression and various metabolic disorders. This paper demonstrates that Tribbles homolog 1 (Trib1), an adaptor protein involved in protein degradation through interaction with COP1 ubiquitin ligase, is essential for the development of adipose-tissue-resident macrophages with an M2-like phenotype. Trib1 deficiency leads to aberrant expression of the transcription factor C/EBPα and impaired adipose tissue function. TRIB1 mutations have been implicated in metabolic disorders including atherosclerosis and hyperlipidaemia, and this work points to possible explanation of the relations between TRIB1 and metabolic disorders in humans. Macrophages consist of at least two subgroups, M1 and M2 (refs 1 , 2 , 3 ). Whereas M1 macrophages are proinflammatory and have a central role in host defence against bacterial and viral infections 4 , 5 , M2 macrophages are associated with responses to anti-inflammatory reactions, helminth infection, tissue remodelling, fibrosis and tumour progression 6 . Trib1 is an adaptor protein involved in protein degradation by interacting with COP1 ubiquitin ligase 7 . Genome-wide association studies in humans have implicated TRIB1 in lipid metabolism 8 , 9 , 10 . Here we show that Trib1 is critical for the differentiation of F4/80 + MR + tissue-resident macrophages—that share characteristics with M2 macrophages (which we term M2-like macrophages)—and eosinophils but not for the differentiation of M1 myeloid cells. Trib1 deficiency results in a severe reduction of M2-like macrophages in various organs, including bone marrow, spleen, lung and adipose tissues. Aberrant expression of C/EBPα in Trib1-deficient bone marrow cells is responsible for the defects in macrophage differentiation. Unexpectedly, mice lacking Trib1 in haematopoietic cells show diminished adipose tissue mass accompanied by evidence of increased lipolysis, even when fed a normal diet. Supplementation of M2-like macrophages rescues the pathophysiology, indicating that a lack of these macrophages is the cause of lipolysis. In response to a high-fat diet, mice lacking Trib1 in haematopoietic cells develop hypertriglyceridaemia and insulin resistance, together with increased proinflammatory cytokine gene induction. Collectively, these results demonstrate that Trib1 is critical for adipose tissue maintenance and suppression of metabolic disorders by controlling the differentiation of tissue-resident M2-like macrophages.

In the absence of RIPK1, ZBP1 engages RIPK3 in a RHIM-dependent manner and acts as a critical activator of RIPK3/MLKL-dependent necroptosis. RIPK1 regulation of necroptosis Receptor-interacting protein kinase 1 (RIPK1) is known to act as a brake on apoptosis and necroptosis during development. Here Kim Newton et al . report that, in the absence of RIPK1, the adapter protein ZBP1 (also known as DAI) engages RIPK3 in a RIP homotypic interaction motif (RHIM)-dependent manner and acts as a critical activator of RIPK3–MLKL-dependent necroptosis. This finding, together with related analyses, suggests an unexpected role for ZBP1 in triggering necroptosis in the perinatal period. Receptor-interacting protein kinase 1 (RIPK1) promotes cell survival—mice lacking RIPK1 die perinatally, exhibiting aberrant caspase-8-dependent apoptosis and mixed lineage kinase-like (MLKL)-dependent necroptosis 1 , 2 , 3 . However, mice expressing catalytically inactive RIPK1 are viable 2 , 4 , 5 , and an ill-defined pro-survival function for the RIPK1 scaffold has therefore been proposed. Here we show that the RIP homotypic interaction motif (RHIM) in RIPK1 prevents the RHIM-containing adaptor protein ZBP1 (Z-DNA binding protein 1; also known as DAI or DLM1) from activating RIPK3 upstream of MLKL. Ripk1 RHIM/RHIM mice that expressed mutant RIPK1 with critical RHIM residues IQIG mutated to AAAA died around birth and exhibited RIPK3 autophosphorylation on Thr231 and Ser232, which is a hallmark of necroptosis 6 , in the skin and thymus. Blocking necroptosis with catalytically inactive RIPK3(D161N), RHIM mutant RIPK3, RIPK3 deficiency, or MLKL deficiency prevented lethality in Ripk1 RHIM/RHIM mice. Loss of ZBP1, which engages RIPK3 in response to certain viruses 7 , 8 but previously had no defined role in development, also prevented perinatal lethality in Ripk1 RHIM/RHIM mice. Consistent with the RHIM of RIPK1 functioning as a brake that prevents ZBP1 from engaging the RIPK3 RHIM, ZBP1 interacted with RIPK3 in Ripk1 RHIM/RHIM Mlkl −/− macrophages, but not in wild-type, Mlkl −/− or Ripk1 RHIM/RHIM Ripk3 RHIM/RHIM macrophages. Collectively, these findings indicate that the RHIM of RIPK1 is critical for preventing ZBP1/RIPK3/MLKL-dependent necroptosis during development.

Stimulation of TNFR1 by TNFα can promote three distinct alternative mechanisms of cell death: necroptosis, RIPK1-independent and -dependent apoptosis. How cells decide which way to die is unclear. Here, we report that TNFα-induced phosphorylation of RIPK1 in the intermediate domain by TAK1 plays a key role in regulating this critical decision. Using phospho-Ser321 as a marker, we show that the transient phosphorylation of RIPK1 intermediate domain induced by TNFα leads to RIPK1-independent apoptosis when NF-κB activation is inhibited by cycloheximide. On the other hand, blocking Ser321 phosphorylation promotes RIPK1 activation and its interaction with FADD to mediate RIPK1-dependent apoptosis (RDA). Finally, sustained phosphorylation of RIPK1 intermediate domain at multiple sites by TAK1 promotes its interaction with RIPK3 and necroptosis. Thus, absent, transient and sustained levels of TAK1-mediated RIPK1 phosphorylation may represent distinct states in TNF-RSC to dictate the activation of three alternative cell death mechanisms, RDA, RIPK1-independent apoptosis and necroptosis. TNFα can promote three distinct mechanisms of cell death: necroptosis, RIPK1-independent and dependent apoptosis. Here the authors show that TNFα-induced phosphorylation of RIPK1 in the intermediate domain by TAK1 plays a key role in regulating this decision.

Deletion of the Hippo pathway component Salvador in mouse hearts with established ischaemic heart failure after myocardial infarction induces a reparative genetic program with increased scar border vascularity, reduced fibrosis, and recovery of pumping function. Salvador deletion reverses heart failure Previous work has shown that interfering with Hippo signalling during myocardial injury improves heart function in mice. Clinical outcomes of acute myocardial infarction in humans have improved as a result of better emergency care, but chronic heart failure, whereby the heart tissue undergoes pathological remodelling, remains a leading cause of death. James Martin and colleagues now show that the failing heart has a previously unrecognized capacity for repair. They show that blocking Hippo signalling can rescue established heart failure in mice. Deletion of the Hippo pathway component Salvador (Salv) or virus-mediated delivery of Salv short hairpin RNA when ischaemic heart failure is established can improve heart function in mice. The authors attribute the effect to the induction of a reparative genetic program, including increased expression of stress response genes and proliferative genes and preservation of mitochondrial quality control. Mammalian organs vary widely in regenerative capacity. Poorly regenerative organs, such as the heart are particularly vulnerable to organ failure. Once established, heart failure commonly results in mortality 1 . The Hippo pathway, a kinase cascade that prevents adult cardiomyocyte proliferation and regeneration 2 , is upregulated in human heart failure. Here we show that deletion of the Hippo pathway component Salvador (Salv) in mouse hearts with established ischaemic heart failure after myocardial infarction induces a reparative genetic program with increased scar border vascularity, reduced fibrosis, and recovery of pumping function compared with controls. Using translating ribosomal affinity purification, we isolate cardiomyocyte-specific translating messenger RNA. Hippo-deficient cardiomyocytes have increased expression of proliferative genes and stress response genes, such as the mitochondrial quality control gene, Park2 . Genetic studies indicate that Park2 is essential for heart repair, suggesting a requirement for mitochondrial quality control in regenerating myocardium. Gene therapy with a virus encoding Salv short hairpin RNA improves heart function when delivered at the time of infarct or after ischaemic heart failure following myocardial infarction was established. Our findings indicate that the failing heart has a previously unrecognized reparative capacity involving more than cardiomyocyte renewal.

Necroptosis is a type of programmed cell death with necrotic morphology, occurring in a variety of biological processes, including inflammation, immune response, embryonic development and metabolic abnormalities. The current nomenclature defines necroptosis as cell death mediated by signal transduction from receptor-interacting serine/threonine kinase (RIP) 1 to RIP3 (hereafter called RIP1/RIP3). However, RIP3-dependent cell death would be a more precise definition of necroptosis. RIP3 is indispensable for necroptosis, while RIP1 is not consistently involved in the signal transduction. Notably, deletion of RIP1 even promotes RIP3-mediated necroptosis under certain conditions. Necroptosis was previously thought as an alternate process of cell death in case of apoptosis inhibition. Currently, necroptosis is recognized to serve a pivotal role in regulating various physiological processes. Of note, it mediates a variety of human diseases, such as ischemic brain injury, immune system disorders and cancer. Targeting and inhibiting necroptosis, therefore, has the potential to be used for therapeutic purposes. To date, research has elucidated the suppression of RIP1/RIP3 via effective inhibitors and highlighted their potential application in disease therapy. The present review focused on the molecular mechanisms of RIP1/RIP3-mediated necroptosis, explored the functions of RIP1/RIP3 in necroptosis, discussed their potential as a novel therapeutic target for disease therapy, and provided valuable suggestions for further study in this field.

Necroptosis is a caspase-independent form of cell death that is triggered by activation of the receptor interacting serine/threonine kinase 3 (RIPK3) and phosphorylation of its pseudokinase substrate mixed lineage kinase-like (MLKL), which then translocates to membranes and promotes cell lysis. Activation of RIPK3 is regulated by the kinase RIPK1. Here we analyze the contribution of RIPK1, RIPK3, or MLKL to several mouse disease models. Loss of RIPK3 had no effect on lipopolysaccharide-induced sepsis, dextran sodium sulfate-induced colitis, cerulein-induced pancreatitis, hypoxia-induced cerebral edema, or the major cerebral artery occlusion stroke model. However, kidney ischemia–reperfusion injury, myocardial infarction, and systemic inflammation associated with A20 deficiency or high-dose tumor necrosis factor (TNF) were ameliorated by RIPK3 deficiency. Catalytically inactive RIPK1 was also beneficial in the kidney ischemia–reperfusion injury model, the high-dose TNF model, and in A20 −/− mice. Interestingly, MLKL deficiency offered less protection in the kidney ischemia–reperfusion injury model and no benefit in A20 −/− mice, consistent with necroptosis-independent functions for RIPK1 and RIPK3. Combined loss of RIPK3 (or MLKL) and caspase-8 largely prevented the cytokine storm, hypothermia, and morbidity induced by TNF, suggesting that the triggering event in this model is a combination of apoptosis and necroptosis. Tissue-specific RIPK3 deletion identified intestinal epithelial cells as the major target organ. Together these data emphasize that MLKL deficiency rather than RIPK1 inactivation or RIPK3 deficiency must be examined to implicate a role for necroptosis in disease.