A Dominant Autoinflammatory Disease Caused By Non-cleavable Variants Of RIPK1

The concept of autoinflammatory disease arose from the recognition of monogenic disorders,including familial Mediterranean fever,with seemingly unprovoked inflammation without the high-titer autoantibodies or antigen-specific T cells seen in classic autoimmune diseases.The main clinical manifestations include recurrent fever,rash,elevated CRP,lymphadenopathy,heptospenomegaly,arthritis,IBD,vasculitis,CNS involvement,interstitial lung disease,etc.Identifying pathogenic genetic variants and studying the molecular mechanisms are of great significance for targeted therapy of patients and functional studies of genes.RIPK1 plays a crucial role in mediating signals triggered by DRs and PRRs.It enables the NF-κB-mediated pro-survival signaling as well as caspase-8-dependent apoptotic or RIPK3/MLKL-dependent necroptotic cell death.When the cell is stimulated by cell death ligand,such as tumor necrosis factor(TNF),TNF receptor 1(TNFR1)on cell surface will trimerize and recruit TNFR1-associated death domain(TRADD)and RIPK1 to form complex I,which mediates NF-κB cascade activation.In this process,ubiquitination and phosphorylation limit the activation of RIPK1.When the ubiquitination or phosphorylation of RIPK1 are dysregulated,RIPK1 will dimerize and trans-phosphorylate itself to promote further activation.Activated RIPK1 interacts with FAS-associated death domain(FADD)and caspase-8 to form complex IIa and activate downstream RIPK1 dependent extrinsic apoptotic events.When caspase-8activation is blocked,RIPK1 co-aggregates with RIPK3 and MLKL forms the complex IIb to cause necroptosis by disrupting the plasma membrane.Fadd knockout leads to excessive cell necroptosis and embryonic lethality,which can be rescued by knocking out Ripk1.Casp8 knockout also causes embryonic lethality,which can be rescued by knocking out Ripk3 or Mlkl.This suggest that the activation of FADD-caspase-8 can inhibit RIPK1/RIPK3/MLKL-dependent necroptosis to ensure mouse embryo development,but the molecular mechanisms and its physiological significance in human diseases are still unclear.Here we show that the heterozygous missense mutations D324 V and D324 H prevent caspase cleavage of RIPK1 in humans and lead to distinct symptoms of recurrent fevers and lymphadenopathy.The patients showed strong RIPK1-dependent activation of inflammatory signalling pathways and overproduction of inflammatory cytokines and chemokines,such as IL-6.The expression of the RIPK1 mutants D325 V or D325 H in mouse embryonic fibroblasts confers not only increased sensitivity to RIPK1 activation-mediated apoptosis and necroptosis,but also induction of proinflammatory cytokines.Furthermore,RIPK1 D324 variants sensitized patients’ peripheral blood mononuclear cells to RIPK1 activation,apoptosis and necroptosis induced by TNF.By contrast,patient-derived fibroblasts showed reduced expression of RIPK1 and TNFR1 and downregulated production of reactive oxygen species,resulting in resistance to necroptosis and ferroptosis.Supporting a pathogenic role of excessive IL-6 production,a patient experienced clinical improvement and the PBMCs displayed normalized expression of inflammatory mediators after treatment with tocilizumab(monoclonal antibody against IL-6R).Together,these data suggest that activated caspase-8 can cleave RIPK1 at residue D324 to attenuate RIPK1 activity.Human non-cleavable RIPK1 variants promote activation of RIPK1,and lead to a new autoinflammatory disease characterized by hypersensitivity to apoptosis and necroptosis and increased inflammatory response in peripheral blood mononuclear cells,as well as a compensatory mechanism to protect against several pro-death stimuli in fibroblasts.