Apoptotic cell death in disease-Current understanding of the NCCD 2023.

Apoptosis is a form of regulated cell death (RCD) that involves proteases of the caspase family. Pharmacological and genetic strategies that experimentally inhibit or delay apoptosis in mammalian systems have elucidated the key contribution of this process not only to (post-)embryonic development and adult tissue homeostasis, but also to the etiology of multiple human disorders. Consistent with this notion, while defects in the molecular machinery for apoptotic cell death impair organismal development and promote oncogenesis, the unwarranted activation of apoptosis promotes cell loss and tissue damage in the context of various neurological, cardiovascular, renal, hepatic, infectious, neoplastic and inflammatory conditions. Here, the Nomenclature Committee on Cell Death (NCCD) gathered to critically summarize an abundant pre-clinical literature mechanistically linking the core apoptotic apparatus to organismal homeostasis in the context of disease.

Raptinal Induces Gasdermin E-Dependent Pyroptosis in Naïve and Therapy-Resistant Melanoma.

Lack of response and acquired resistance continue to be limitations of targeted and immune-based therapies. Pyroptosis is an inflammatory form of cell death characterized by the release of inflammatory damage-associated molecular patterns (DAMP) and cytokines via gasdermin (GSDM) protein pores in the plasma membrane. Induction of pyroptosis has implications for treatment strategies in both therapy-responsive, as well as resistance forms of melanoma. We show that the caspase-3 activator, raptinal, induces pyroptosis in both human and mouse melanoma cell line models and delays tumor growth in vivo. Release of DAMPs and inflammatory cytokines was dependent on caspase activity and GSDME expression. Furthermore, raptinal stimulated pyroptosis in melanoma models that have acquired resistance to BRAF and MEK inhibitor therapy. These findings add support to efforts to induce pyroptosis in both the treatment-naïve and resistant settings. IMPLICATIONS: Raptinal can rapidly induce pyroptosis in naïve and BRAFi plus MEKi-resistant melanoma, which may be beneficial for patients who have developed acquired resistance to targeted therapies.

Multiple Autonomous Cell Death Suppression Strategies Ensure Cytomegalovirus Fitness.

Programmed cell death pathways eliminate infected cells and regulate infection-associated inflammation during pathogen invasion. Cytomegaloviruses encode several distinct suppressors that block intrinsic apoptosis, extrinsic apoptosis, and necroptosis, pathways that impact pathogenesis of this ubiquitous herpesvirus. Here, we expanded the understanding of three cell autonomous suppression mechanisms on which murine cytomegalovirus relies: (i) M38.5-encoded viral mitochondrial inhibitor of apoptosis (vMIA), a BAX suppressor that functions in concert with M41.1-encoded viral inhibitor of BAK oligomerization (vIBO), (ii) M36-encoded viral inhibitor of caspase-8 activation (vICA), and (iii) M45-encoded viral inhibitor of RIP/RHIM activation (vIRA). Following infection of bone marrow-derived macrophages, the virus initially deflected receptor-interacting protein kinase (RIPK)3-dependent necroptosis, the most potent of the three cell death pathways. This process remained independent of caspase-8, although suppression of this apoptotic protease enhances necroptosis in most cell types. Second, the virus deflected TNF-mediated extrinsic apoptosis, a pathway dependent on autocrine TNF production by macrophages that proceeds independently of mitochondrial death machinery or RIPK3. Third, cytomegalovirus deflected BCL-2 family protein-dependent mitochondrial cell death through combined TNF-dependent and -independent signaling even in the absence of RIPK1, RIPK3, and caspase-8. Furthermore, each of these cell death pathways dictated a distinct pattern of cytokine and chemokine activation. Therefore, cytomegalovirus employs sequential, non-redundant suppression strategies to specifically modulate the timing and execution of necroptosis, extrinsic apoptosis, and intrinsic apoptosis within infected cells to orchestrate virus control and infection-dependent inflammation. Virus-encoded death suppressors together hold control over an intricate network that upends host defense and supports pathogenesis in the intact mammalian host.

Chloride sensing by WNK1 regulates NLRP3 inflammasome activation and pyroptosis.

The NLRP3 inflammasome mediates the production of proinflammatory cytokines and initiates inflammatory cell death. Although NLRP3 is essential for innate immunity, aberrant NLRP3 inflammasome activation contributes to a wide variety of inflammatory diseases. Understanding the pathways that control NLRP3 activation will help develop strategies to treat these diseases. Here we identify WNK1 as a negative regulator of the NLRP3 inflammasome. Macrophages deficient in WNK1 protein or kinase activity have increased NLRP3 activation and pyroptosis compared with control macrophages. Mice with conditional knockout of WNK1 in macrophages have increased IL-1ß production in response to NLRP3 stimulation compared with control mice. Mechanistically, WNK1 tempers NLRP3 activation by balancing intracellular Cl- and K+ concentrations during NLRP3 activation. Collectively, this work shows that the WNK1 pathway has a critical function in suppressing NLRP3 activation and suggests that pharmacological inhibition of this pathway to treat hypertension might have negative clinical implications.

Tristetraprolin regulates necroptosis during tonic Toll-like receptor 4 (TLR4) signaling in murine macrophages.

The necrosome is a protein complex required for signaling in cells that results in necroptosis, which is also dependent on tumor necrosis factor receptor (TNF-R) signaling. TNFα promotes necroptosis, and its expression is facilitated by mitogen-activated protein (MAP) kinase-activated protein kinase 2 (MK2) but is inhibited by the RNA-binding protein tristetraprolin (TTP, encoded by the Zfp36 gene). We have stimulated murine macrophages from WT, MyD88-/-, Trif-/-, MyD88-/-Trif-/-, MK2-/-, and Zfp36-/- mice with graded doses of lipopolysaccharide (LPS) and various inhibitors to evaluate the role of various genes in Toll-like receptor 4 (TLR4)-induced necroptosis. Necrosome signaling, cytokine production, and cell death were evaluated by immunoblotting, ELISA, and cell death assays, respectively. We observed that during TLR4 signaling, necrosome activation is mediated through the adaptor proteins MyD88 and TRIF, and this is inhibited by MK2. In the absence of MK2-mediated necrosome activation, lipopolysaccharide-induced TNFα expression was drastically reduced, but MK2-deficient cells became highly sensitive to necroptosis even at low TNFα levels. In contrast, during tonic TLR4 signaling, WT cells did not undergo necroptosis, even when MK2 was disabled. Of note, necroptosis occurred only in the absence of TTP and was mediated by the expression of TNFα and activation of JUN N-terminal kinase (JNK). These results reveal that TTP plays an important role in inhibiting TNFα/JNK-induced necrosome signaling and resultant cytotoxicity.

Mutant BRAF and MEK Inhibitors Regulate the Tumor Immune Microenvironment via Pyroptosis.

Combinations of BRAF inhibitors and MEK inhibitors (BRAFi + MEKi) are FDA-approved to treat BRAF V600E/K-mutant melanoma. Efficacy of BRAFi + MEKi associates with cancer cell death and alterations in the tumor immune microenvironment; however, the links are poorly understood. We show that BRAFi + MEKi caused durable melanoma regression in an immune-mediated manner. BRAFi + MEKi treatment promoted cleavage of gasdermin E (GSDME) and release of HMGB1, markers of pyroptotic cell death. GSDME-deficient melanoma showed defective HMGB1 release, reduced tumor-associated T cell and activated dendritic cell infiltrates in response to BRAFi + MEKi, and more frequent tumor regrowth after drug removal. Importantly, BRAFi + MEKi-resistant disease lacked pyroptosis markers and showed decreased intratumoral T-cell infiltration but was sensitive to pyroptosis-inducing chemotherapy. These data implicate BRAFi + MEKi-induced pyroptosis in antitumor immune responses and highlight new therapeutic strategies for resistant melanoma. SIGNIFICANCE: Targeted inhibitors and immune checkpoint agents have advanced the care of patients with melanoma; however, detailed knowledge of the intersection between these two research areas is lacking. We describe a molecular mechanism of targeted inhibitor regulation of an immune-stimulatory form of cell death and provide a proof-of-principle salvage therapy concept for inhibitor-resistant melanoma.See related commentary by Smalley, p. 176.This article is highlighted in the In This Issue feature, p. 161.

Gasdermins in Apoptosis: New players in an Old Game.

Apoptosis is a form of programmed cell death (PCD) that plays critical physiological roles in removing superfluous or dangerous cell populations that are unneeded or threatening to the health of the host organism. Although the molecular pathways leading to activation of the apoptotic program have been extensively studied and characterized starting in the 1970s, new evidence suggests that members of the gasdermin superfamily are novel pore-forming proteins that augment apoptosis by permeabilizing the mitochondria and participate in the final stages of the apoptotic program by inducing secondary necrosis/pyroptosis. These findings may explain outstanding questions in the field such as why certain gasdermin members sensitize cells to apoptosis, and why some apoptotic cells also show morphological features of necrosis. Furthermore, the interplay between the gasdermins and apoptosis may also explain why genetic and epigenetic alterations in these genes cause diseases and disorders like cancer and hearing loss. This review focuses on our current understanding of the function of several gasdermin superfamily members, their role in apoptosis, and how they may contribute to pathophysiological conditions.

Gasdermins: novel mitochondrial pore-forming proteins.

Gasdermin proteins have been extensively characterized for their ability to form necrotic pores in the plasma membrane, however, their interactions with other organelles have yet to be described. We recently demonstrated that some gasdermin proteins can also permeabilize the mitochondria to augment apoptotic signaling which may be important in the context of cancer and hearing loss.

Gasdermin pores permeabilize mitochondria to augment caspase-3 activation during apoptosis and inflammasome activation.

Gasdermin E (GSDME/DFNA5) cleavage by caspase-3 liberates the GSDME-N domain, which mediates pyroptosis by forming pores in the plasma membrane. Here we show that GSDME-N also permeabilizes the mitochondrial membrane, releasing cytochrome c and activating the apoptosome. Cytochrome c release and caspase-3 activation in response to intrinsic and extrinsic apoptotic stimuli are significantly reduced in GSDME-deficient cells comparing with wild type cells. GSDME deficiency also accelerates cell growth in culture and in a mouse model of melanoma. Phosphomimetic mutation of the highly conserved phosphorylatable Thr6 residue of GSDME, inhibits its pore-forming activity, thus uncovering a potential mechanism by which GSDME might be regulated. Like GSDME-N, inflammasome-generated gasdermin D-N (GSDMD-N), can also permeabilize the mitochondria linking inflammasome activation to downstream activation of the apoptosome. Collectively, our results point to a role of gasdermin proteins in targeting the mitochondria to promote cytochrome c release to augment the mitochondrial apoptotic pathway.

The NLRP6 Inflammasome Recognizes Lipoteichoic Acid and Regulates Gram-Positive Pathogen Infection.

The activator and composition of the NLRP6 inflammasome remain poorly understood. We find that lipoteichoic acid (LTA), a molecule produced by Gram-positive bacteria, binds and activates NLRP6. In response to cytosolic LTA or infection with Listeria monocytogenes, NLRP6 recruited caspase-11 and caspase-1 via the adaptor ASC. NLRP6 activation by LTA induced processing of caspase-11, which promoted caspase-1 activation and interleukin-1ß (IL-1ß)/IL-18 maturation in macrophages. Nlrp6-/- and Casp11-/- mice were less susceptible to L. monocytogenes infection, which was associated with reduced pathogen loads and impaired IL-18 production. Administration of IL-18 to Nlrp6-/- or Casp11-/- mice restored the susceptibility of mutant mice to L. monocytogenes infection. These results reveal a previously unrecognized innate immunity pathway triggered by cytosolic LTA that is sensed by NLRP6 and exacerbates systemic Gram-positive pathogen infection via the production of IL-18.

The mitochondrial protease HtrA2 restricts the NLRP3 and AIM2 inflammasomes.

Activation of the inflammasome pathway is crucial for effective intracellular host defense. The mitochondrial network plays an important role in inflammasome regulation but the mechanisms linking mitochondrial homeostasis to attenuation of inflammasome activation are not fully understood. Here, we report that the Parkinson's disease-associated mitochondrial serine protease HtrA2 restricts the activation of ASC-dependent NLRP3 and AIM2 inflammasomes, in a protease activity-dependent manner. Consistently, disruption of the protease activity of HtrA2 results in exacerbated NLRP3 and AIM2 inflammasome responses in macrophages ex vivo and systemically in vivo. Mechanistically, we show that the HtrA2 protease activity regulates autophagy and controls the magnitude and duration of inflammasome signaling by preventing prolonged accumulation of the inflammasome adaptor ASC. Our findings identify HtrA2 as a non-redundant mitochondrial quality control effector that keeps NLRP3 and AIM2 inflammasomes in check.

Molecular mechanisms of cell death: recommendations of the Nomenclature Committee on Cell Death 2018.

Over the past decade, the Nomenclature Committee on Cell Death (NCCD) has formulated guidelines for the definition and interpretation of cell death from morphological, biochemical, and functional perspectives. Since the field continues to expand and novel mechanisms that orchestrate multiple cell death pathways are unveiled, we propose an updated classification of cell death subroutines focusing on mechanistic and essential (as opposed to correlative and dispensable) aspects of the process. As we provide molecularly oriented definitions of terms including intrinsic apoptosis, extrinsic apoptosis, mitochondrial permeability transition (MPT)-driven necrosis, necroptosis, ferroptosis, pyroptosis, parthanatos, entotic cell death, NETotic cell death, lysosome-dependent cell death, autophagy-dependent cell death, immunogenic cell death, cellular senescence, and mitotic catastrophe, we discuss the utility of neologisms that refer to highly specialized instances of these processes. The mission of the NCCD is to provide a widely accepted nomenclature on cell death in support of the continued development of the field.

Cleavage of DFNA5 by caspase-3 during apoptosis mediates progression to secondary necrotic/pyroptotic cell death.

Apoptosis is a genetically regulated cell suicide programme mediated by activation of the effector caspases 3, 6 and 7. If apoptotic cells are not scavenged, they progress to a lytic and inflammatory phase called secondary necrosis. The mechanism by which this occurs is unknown. Here we show that caspase-3 cleaves the GSDMD-related protein DFNA5 after Asp270 to generate a necrotic DFNA5-N fragment that targets the plasma membrane to induce secondary necrosis/pyroptosis. Cells that express DFNA5 progress to secondary necrosis, when stimulated with apoptotic triggers such as etoposide or vesicular stomatitis virus infection, but disassemble into small apoptotic bodies when DFNA5 is deleted. Our findings identify DFNA5 as a central molecule that regulates apoptotic cell disassembly and progression to secondary necrosis, and provide a molecular mechanism for secondary necrosis. Because DFNA5-induced secondary necrosis and GSDMD-induced pyroptosis are dependent on caspase activation, we propose that they are forms of programmed necrosis.

The Yersinia pestis Effector YopM Inhibits Pyrin Inflammasome Activation.

Type III secretion systems (T3SS) are central virulence factors for many pathogenic Gram-negative bacteria, and secreted T3SS effectors can block key aspects of host cell signaling. To counter this, innate immune responses can also sense some T3SS components to initiate anti-bacterial mechanisms. The Yersinia pestis T3SS is particularly effective and sophisticated in manipulating the production of pro-inflammatory cytokines IL-1ß and IL-18, which are typically processed into their mature forms by active caspase-1 following inflammasome formation. Some effectors, like Y. pestis YopM, may block inflammasome activation. Here we show that YopM prevents Y. pestis induced activation of the Pyrin inflammasome induced by the RhoA-inhibiting effector YopE, which is a GTPase activating protein. YopM blocks YopE-induced Pyrin-mediated caspase-1 dependent IL-1ß/IL-18 production and cell death. We also detected YopM in a complex with Pyrin and kinases RSK1 and PKN1, putative negative regulators of Pyrin. In contrast to wild-type mice, Pyrin deficient mice were also highly susceptible to an attenuated Y. pestis strain lacking YopM, emphasizing the importance of inhibition of Pyrin in vivo. A complex interplay between the Y. pestis T3SS and IL-1ß/IL-18 production is evident, involving at least four inflammasome pathways. The secreted effector YopJ triggers caspase-8- dependent IL-1ß activation, even when YopM is present. Additionally, the presence of the T3SS needle/translocon activates NLRP3 and NLRC4-dependent IL-1ß generation, which is blocked by YopK, but not by YopM. Taken together, the data suggest YopM specificity for obstructing the Pyrin pathway, as the effector does not appear to block Y. pestis-induced NLRP3, NLRC4 or caspase-8 dependent caspase-1 processing. Thus, we identify Y. pestis YopM as a microbial inhibitor of the Pyrin inflammasome. The fact that so many of the Y. pestis T3SS components are participating in regulation of IL-1ß/IL-18 release suggests that these effects are essential for maximal control of innate immunity during plague.

Caspase-8 scaffolding function and MLKL regulate NLRP3 inflammasome activation downstream of TLR3.

TLR2 promotes NLRP3 inflammasome activation via an early MyD88-IRAK1-dependent pathway that provides a priming signal (signal 1) necessary for activation of the inflammasome by a second potassium-depleting signal (signal 2). Here we show that TLR3 binding to dsRNA promotes post-translational inflammasome activation through intermediate and late TRIF/RIPK1/FADD-dependent pathways. Both pathways require the scaffolding but not the catalytic function of caspase-8 or RIPK1. Only the late pathway requires kinase competent RIPK3 and MLKL function. Mechanistically, FADD/caspase-8 scaffolding function provides a post-translational signal 1 in the intermediate pathway, whereas in the late pathway it helps the oligomerization of RIPK3, which together with MLKL provides both signal 1 and 2 for inflammasome assembly. Cytoplasmic dsRNA activates NLRP3 independent of TRIF, RIPK1, RIPK3 or mitochondrial DRP1, but requires FADD/caspase-8 in wildtype macrophages to remove RIPK3 inhibition. Our study provides a comprehensive analysis of pathways that lead to NLRP3 inflammasome activation in response to dsRNA.

Non-transcriptional regulation of NLRP3 inflammasome signaling by IL-4.

Th2 cytokine IL-4 has been previously shown to suppress the production of proinflammatory cytokines in monocytes. However, the underlying molecular mechanism by which IL-4 signaling antagonizes proinflammatory responses is poorly characterized. In particular, whether IL-4 can modulate inflammasome signaling remains unknown. Here, we provide evidence that IL-4 suppresses NLRP3-dependent caspase-1 activation and the subsequent IL-1ß secretion but does not inhibit absent in melanoma 2 (AIM2)- or NLRC4 (NOD-like receptor family, CARD domain-containing 4)-dependent caspase-1 activation in THP-1 and mouse bone marrow-derived macrophages. Upon lipopolysaccharide (LPS) or LPS/ATP stimulation, IL-4 markedly inhibited the assembly of NLRP3 inflammasome, including NLRP3-dependent ASC (apoptosis-associated speck-like protein containing a caspase recruitment domain) oligomerization, NLRP3-ASC interaction and NLRP3 speck-like oligomeric structure formation. The negative regulation of NLRP3 inflammasome by IL-4 was not due to the impaired mRNA or protein production of NLRP3 and proinflammatory cytokines. Supporting this observation, IL-4 attenuated NLRP3 inflammasome activation even in reconstituted NLRP3-expressing macrophages in which NLRP3 expression is not transcriptionally regulated by TLR-NF-κB signaling. Furthermore, the IL-4-mediated suppression of NLRP3 inflammasome was independent of STAT6-dependent transcription and mitochondrial reactive oxygen species (ROS). Instead, IL-4 inhibited subcellular redistribution of NLRP3 into mitochondria and microtubule polymerization upon NLRP3-activating stimulation. Our results collectively suggest that IL-4 could suppress NLRP3 inflammasome activation in a transcription-independent manner, thus providing an endogenous regulatory machinery to prevent excessive inflammasome activation.

IKKα negatively regulates ASC-dependent inflammasome activation.

The inflammasomes are multiprotein complexes that activate caspase-1 in response to infections and stress, resulting in the secretion of pro-inflammatory cytokines. Here we report that IκB kinase α (IKKα) is a critical negative regulator of apoptosis-associated specklike protein containing a C-terminal caspase-activation-andrecruitment (CARD) domain (ASC)-dependent inflammasomes. IKKα controls the inflammasome at the level of the adaptor ASC, which interacts with IKKα in the nucleus of resting macrophages in an IKKα kinase-dependent manner. Loss of IKKα kinase activity results in inflammasome hyperactivation. Mechanistically, the downstream nuclear effector IKK-related kinase (IKKi) facilitates translocation of ASC from the nucleus to the perinuclear area during inflammasome activation. ASC remains under the control of IKKα in the perinuclear area following translocation of the ASC/IKKα complex. Signal 2 of NLRP3 activation leads to inhibition of IKKα kinase activity through the recruitment of PP2A, allowing ASC to participate in NLRP3 inflammasome assembly. Taken together, these findings reveal a IKKi-IKKα-ASC axis that serves as a common regulatory mechanism for ASC-dependent inflammasomes.

Inactivation of Omi/HtrA2 protease leads to the deregulation of mitochondrial Mulan E3 ubiquitin ligase and increased mitophagy.

Omi/HtrA2 is a nuclear encoded mitochondrial serine protease with dual and opposite functions that depend entirely on its subcellular localization. During apoptosis, Omi/HtrA2 is released into the cytoplasm where it participates in cell death. While confined in the inter-membrane space of the mitochondria, Omi/HtrA2 has a pro-survival function that may involve the regulation of protein quality control (PQC) and mitochondrial homeostasis. Loss of Omi/HtrA2's protease activity causes the neuromuscular disorder of the mnd2 (motor neuron degeneration 2) mutant mice. These mice develop multiple defects including neurodegeneration with parkinsonian features. Loss of Omi/HtrA2 in non-neuronal tissues has also been shown to cause premature aging. The normal function of Omi/HtrA2 in the mitochondria and how its deregulation causes neurodegeneration or premature aging are unknown. Here we report that the mitochondrial Mulan E3 ubiquitin ligase is a specific substrate of Omi/HtrA2. During exposure to H(2)O(2), Omi/HtrA2 degrades Mulan, and this regulation is lost in cells that carry the inactive protease. Furthermore, we show accumulation of Mulan protein in various tissues of mnd2 mice as well as in Omi/HtrA2(-/-) mouse embryonic fibroblasts (MEFs). This causes a significant decrease of mitofusin 2 (Mfn2) protein, and increased mitophagy. Our work describes a new stress-signaling pathway that is initiated in the mitochondria and involves the regulation of Mulan by Omi/HtrA2 protease. Deregulation of this pathway, as it occurs in mnd2 mutant mice, causes mitochondrial dysfunction and mitophagy, and could be responsible for the motor neuron disease and the premature aging phenotype observed in these animals.

Inflammasome priming by lipopolysaccharide is dependent upon ERK signaling and proteasome function.

Caspase-1 activation is a central event in innate immune responses to many pathogenic infections and tissue damage. The NLRP3 inflammasome, a multiprotein scaffolding complex that assembles in response to two distinct steps, priming and activation, is required for caspase-1 activation. However, the detailed mechanisms of these steps remain poorly characterized. To investigate the process of LPS-mediated NLRP3 inflammasome priming, we used constitutively present pro-IL-18 as the caspase-1-specific substrate to allow study of the early events. We analyzed human monocyte caspase-1 activity in response to LPS priming, followed by activation with ATP. Within minutes of endotoxin priming, the NLRP3 inflammasome is licensed for ATP-induced release of processed IL-18, apoptosis-associated speck-forming complex containing CARD, and active caspase-1, independent of new mRNA or protein synthesis. Moreover, extracellular signal-regulated kinase 1 (ERK1) phosphorylation is central to the priming process. ERK inhibition and small interfering RNA-mediated ERK1 knockdown profoundly impair priming. In addition, proteasome inhibition prevents ERK phosphorylation and blocks priming. Scavenging reactive oxygen species with diphenylene iodonium also blocks both priming and ERK phosphorylation. These findings suggest that ERK1-mediated posttranslational modifications license the NLRP3 inflammasome to respond to the second signal ATP by inducing posttranslational events that are independent of new production of pro-IL-1ß and NOD-like receptor components.