RIPK3 promotes cell death and NLRP3 inflammasome activation in the absence of MLKLRIPK3 and its substrate MLKL are essential for necroptosis, a lytic cell death proposed to cause inflammation via the release of intracellular molecules. Whether and how RIPK3 might drive inflammation in a manner independent of MLKL and cell lysis remains unclear. Here we show that following LPS treatment, or LPS-induced necroptosis, the TLR adaptor protein TRIF and inhibitor of apoptosis proteins (IAPs: X-linked IAP, cellular IAP1 and IAP2) regulate RIPK3 and MLKL ubiquitylation. Hence, when IAPs are absent, LPS triggers RIPK3 to activate caspase-8, promoting apoptosis and NLRP3-caspase-1 activation, independent of RIPK3 kinase activity and MLKL. In contrast, in the absence of both IAPs and caspase-8, RIPK3 kinase activity and MLKL are essential for TLR-induced NLRP3 activation. Consistent with in vitro experiments, interleukin-1 (IL-1)-dependent autoantibody-mediated arthritis is exacerbated in mice lacking IAPs, and is reduced by deletion of RIPK3, but not MLKL. Therefore RIPK3 can promote NLRP3 inflammasome and IL-1β inflammatory responses independent of MLKL and necroptotic cell death.
RIPK1 Regulates RIPK3-MLKL-Driven Systemic Inflammation and Emergency HematopoiesisMutations that prevent caspase cleavage of RIPK1 cause autoinflammatory diseaseTNFR1-dependent cell death drives inflammation in Sharpin-deficient miceSHARPIN regulates immune signaling and contributes to full transcriptional activity and prevention of cell death in response to TNF in vitro. The inactivating mouse Sharpin cpdm mutation causes TNF-dependent multi-organ inflammation, characterized by dermatitis, liver inflammation, splenomegaly, and loss of Peyer's patches. TNF-dependent cell death has been proposed to cause the inflammatory phenotype and consistent with this we show Tnfr1, but not Tnfr2, deficiency suppresses the phenotype (and it does so more efficiently than Il1r1 loss). TNFR1-induced apoptosis can proceed through caspase-8 and BID, but reduction in or loss of these players generally did not suppress inflammation, although Casp8 heterozygosity significantly delayed dermatitis. Ripk3 or Mlkl deficiency partially ameliorated the multi-organ phenotype, and combined Ripk3 deletion and Casp8 heterozygosity almost completely suppressed it, even restoring Peyer's patches. Unexpectedly, Sharpin, Ripk3 and Casp8 triple deficiency caused perinatal lethality. These results provide unexpected insights into the developmental importance of SHARPIN.
The Mitochondrial Apoptotic Effectors BAX/BAK Activate Caspase-3 and -7 to Trigger NLRP3 Inflammasome and Caspase-8 Driven IL-1β ActivationIntrinsic apoptosis resulting from BAX/BAK-mediated mitochondrial membrane damage is regarded as immunologically silent. We show here that in macrophages, BAX/BAK activation results in inhibitor of apoptosis (IAP) protein degradation to promote caspase-8-mediated activation of IL-1β. Furthermore, BAX/BAK signaling induces a parallel pathway to NLRP3 inflammasome-mediated caspase-1-dependent IL-1β maturation that requires potassium efflux. Remarkably, following BAX/BAK activation, the apoptotic executioner caspases, caspase-3 and -7, act upstream of both caspase-8 and NLRP3-induced IL-1β maturation and secretion. Conversely, the pyroptotic cell death effectors gasdermin D and gasdermin E are not essential for BAX/BAK-induced IL-1β release. These findings highlight that innate immune cells undergoing BAX/BAK-mediated apoptosis have the capacity to generate pro-inflammatory signals and provide an explanation as to why IL-1β activation is often associated with cellular stress, such as during chemotherapy.