SUMMARY Activated caspases are a hallmark of apoptosis induced by the intrinsic pathway, but they are dispensable for cell death and the apoptotic clearance of cells in vivo. This has led to the suggestion that caspases are activated not just to kill but to prevent dying cells from triggering a host immune response. Here, we show that the caspase cascade suppresses type I interferon (IFN) production by cells undergoing Bak/Bax-mediated apoptosis. Bak and Bax trigger the release of mitochondrial DNA. This is recognized by the cGAS/STING-dependent DNA sensing pathway, which initiates IFN production. Activated caspases attenuate this response. Pharmacological caspase inhibition or genetic deletion of caspase-9, Apaf-1, or caspase-3/7 causes dying cells to secrete IFN-β. In vivo, this precipitates an elevation in IFN-β levels and consequent hematopoietic stem cell dysfunction, which is corrected by loss of Bak and Bax. Thus, the apoptotic caspase cascade functions to render mitochondrial apoptosis immunologically silent.

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A current paradigm proposes that mitochondrial damage is a critical determinant of NLRP3 inflammasome activation. Here, we genetically assess whether mitochondrial signalling represents a unified mechanism to explain how NLRP3 is activated by divergent stimuli. Neither co-deletion of the essential executioners of mitochondrial apoptosis BAK and BAX, nor removal of the mitochondrial permeability transition pore component cyclophilin D, nor loss of the mitophagy regulator Parkin, nor deficiency in MAVS affects NLRP3 inflammasome function. In contrast, caspase-8, a caspase essential for death-receptor-mediated apoptosis, is required for efficient Toll-like-receptor-induced inflammasome priming and cytokine production. Collectively, these results demonstrate that mitochondrial apoptosis is not required for NLRP3 activation, and highlight an important non-apoptotic role for caspase-8 in regulating inflammasome activation and proinflammatory cytokine levels.

Deletion of Bak and Bax, the effectors of mitochondrial apoptosis, does not affect platelet production, however, loss of prosurvival Bcl-xL results in megakaryocyte apoptosis and failure of platelet shedding.

BH3 mimetics are a new class of proapoptotic anticancer agents that have shown considerable promise in preclinical animal models and early-stage human trials. These agents act by inhibiting the prosurvival function of one or more Bcl-2-related proteins. Agents that inhibit Bcl-x L induce rapid platelet death that leads to thrombocytopenia; however, their impact on the function of residual circulating platelets remains unclear. In this study, we demonstrate that the BH3 mimetics, ABT-737 or ABT-263, induce a time-and dose-dependent decrease in platelet adhesive function that correlates with ectodomain shedding of the major platelet adhesion receptors, glycoprotein Ib␣ and glycoprotein VI, and functional downregulation of integrin ␣ IIb ␤ 3 . Analysis of platelets from mice treated with higher doses of BH3 mimetics revealed the presence of a subpopulation of circulating platelets undergoing cell death that have impaired activation responses to soluble agonists. Functional analysis of platelets by intravital microscopy revealed a timedependent defect in platelet aggregation at sites of vascular injury that correlated with an increase in tail bleeding time.Overall, these studies demonstrate that Bcl-x L -inhibitory BH3 mimetics not only induce thrombocytopenia but also a transient thrombocytopathy that can undermine the hemostatic function of platelets. (Blood. 2011;118(6):1663-1674) IntroductionApoptosis is an evolutionarily conserved process important for mammalian development, tissue homeostasis, and immune tolerance. Apoptosis also acts as an important barrier against malignant transformation. Resistance to apoptosis is a hallmark feature of cancer, and several pathways regulating apoptosis are commonly altered in human malignancies. 1,2 For example, Ͼ 50% of all human cancers contain mutations in the p53 tumor suppressor gene, with nearly all of these mutations preventing p53 from triggering apoptosis. 3 Furthermore, the overexpression or enhanced function of certain prosurvival proteins, such as Bcl-2, 2 Bcl-x L , 2 PI3K/Akt/mTOR, 4 and nuclear factor-B, 5 is found in many types of human cancers. As a consequence, strategies targeting specific prosurvival pathways have gained prominence as novel anticancer therapies.Progress unraveling the complex pathways underlying apoptosis and cell survival, and elucidation of their roles in human cancers, has led to the development of several potential anticancer drugs, such as inhibitors of the Bcl-2 protein family, 6 inhibitors of apoptosis (IAPs), 7 MDM2, 8 PI3K/Akt/mTOR, 9 as well as TNFrelated apoptosis-inducing ligand. 10 A particularly promising approach is the ability to inhibit tumor cell survival through the use of agents that mimic the proapoptotic Bcl-2 homology 3 (BH3) domain proteins. BH3 proteins play an important role in inducing apoptosis by antagonizing the function of prosurvival Bcl-2 family proteins. Preclinical studies with the BH3 mimetic ABT-737, which selectively antagonizes Bcl-2, Bcl-x L , and Bcl-w, have demonstrated potent cytotoxic activity a...

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During immune responses, neutrophils must integrate survival and death signals from multiple sources to regulate their lifespan. Signals that activate either the Bcl-2- or death receptor-regulated apoptosis pathways can provide powerful stimuli for neutrophils to undergo cell death, but whether they act cooperatively in parallel or directly cross-talk in neutrophils is not known. Previous studies suggested that Bcl-2 family proteins are not required for Fas-induced cell death in neutrophils, but did not examine whether they could modulate its rapid onset. By monitoring the rate of change in neutrophil viability associated with activation of the Fas-triggered death receptor pathway using real-time cell imaging, we show that the Bcl-2-related proteins Bid, Bax, and Bak accelerate neutrophil apoptosis but are not essential for cell death. Increased Bcl-2 or Mcl-1 expression prevents efficient induction of apoptosis by Fas stimulation indicating that the Bcl-2-regulated apoptosis pathway can directly interfere with Fas-triggered apoptosis. Fas has been shown to initiate NFκB activation and gene transcription in cell lines, however gene transcription is not altered in Fas-activated Bid −/− neutrophils, indicating that apoptosis occurs independently of gene transcription in neutrophils. The specification of kinetics of neutrophil apoptosis by Bid impacts on the magnitude of neutrophil IL-1β production, implicating a functional role for the Bcl-2-regulated pathway in controlling neutrophil responses to FasL. These data demonstrate that the intrinsic apoptosis pathway directly controls the kinetics of Fas-triggered apoptosis in neutrophils.