炎症小体是由传感蛋白、炎症性半胱天冬酶以及在某些情况下连接这两者的适配蛋白组成的细胞质多蛋白复合体。它们能够响应多种内源性和外源性刺激，激活经典的半胱天冬酶-1、非经典的半胱天冬酶-11（或人类中的半胱天冬酶-4和半胱天冬酶-5）或半胱天冬酶-8。这一过程导致IL-1β和IL-18的分泌以及凋亡和焦亡的细胞死亡。
炎症小体的适当激活对于宿主抵抗外来病原体或组织损伤至关重要。然而，炎症小体的异常激活可能导致无法控制的组织反应，这可能会促进各种疾病的发展，包括自身炎性疾病、心脏代谢性疾病、癌症和神经退行性疾病。因此，必须精确调节炎症小体的激活和抑制，以维持炎症小体组装和效应器功能之间的细致平衡。
最近的研究聚焦于阐明炎症小体信号调节的结构和分子机制，尝试解析在感知多种刺激后炎症小体组装和激活的顺序性以及这些过程的严格调节。除此之外，当前的研究也在探索将炎症小体研究转化为治疗手段，以改变炎症小体调节的人类疾病。
总的来说，炎症小体在宿主防御、组织损伤响应及疾病发展中发挥关键作用，对其进行合理的调节和治疗干预有望成为治疗相关炎症和免疫性疾病的新策略。
Abstract
- Inflammasomes are cytoplasmic multiprotein complexes comprising a sensor protein, inflammatory caspases, and in some but not all cases an adapter protein connecting the two.
- They can be activated by a repertoire of endogenous and exogenous stimuli, leading to enzymatic activation of canonical caspase-1, noncanonical caspase-11 (or the equivalent caspase-4 and caspase-5 in humans) or caspase-8, resulting in secretion of IL-1β and IL-18, as well as apoptotic and pyroptotic cell death.
- Appropriate inflammasome activation is vital for the host to cope with foreign pathogens or tissue damage, while aberrant inflammasome activation can cause uncontrolled tissue responses that may contribute to various diseases, including autoinflammatory disorders, cardiometabolic diseases, cancer and neurodegenerative diseases.
- Therefore, it is imperative to maintain a fine balance between inflammasome activation and inhibition, which requires a fine-tuned regulation of inflammasome assembly and effector function.
- Recently, a growing body of studies have been focusing on delineating the structural and molecular mechanisms underlying the regulation of inflammasome signaling.
- In the present review, we summarize the most recent advances and remaining challenges in understanding the ordered inflammasome assembly and activation upon sensing of diverse stimuli, as well as the tight regulations of these processes.
- Furthermore, we review recent progress and challenges in translating inflammasome research into therapeutic tools, aimed at modifying inflammasome-regulated human diseases.
Introduction
- Inflammation is a vital physiological response triggered by noxious agents in all metazoan organisms.
- Virtually any challenge to the body’s homeostasis may elicit an inflammatory response at the local or systemic levels1.
- The innate immune system’s task is to generate a protective response against signals of danger including both pathogenic microorganisms and sterile incursions such as trauma, cancer, ischemia, and metabolic perturbations.
- Technically, factors eliciting an innate inflammatory response can be classified as pathogen-associated molecular patterns (PAMPs), conserved compounds of infectious agents, and damage-associated molecular patterns (DAMPs), which are signals of host cellular distress.
- PAMPs and DAMPs are sensed by an increasingly appreciated variety of pattern recognition receptors (PRRs) and cells of innate and adaptive immunity 2,3.
- Upon encounter of a pathogenic agent or tissue injury, the innate immune system is challenged to integrate a wealth of signals to initiate a proper response.
- In addition to Toll-like receptors (TLR)4, Lectin receptors5, RIG-I-like receptors6, and oligoadenylate synthase (OAS)-like receptor7, inflammasomes emerged in the last decade to constitute fundamental processing units contributing to PAMP and DAMP sensing, which actively participate in integration of their downstream signaling3,8.
- The term “inflammasome” has originally been coined by Martinon et al.8 in a seminal report in 2002 that described the assembly of these supramolecular structures in the cytoplasm of activated immune cells, thereby leading to proteolytic activation of proinflammatory caspases, which drives subsequent systemic immune responses and inflammation.
- Importantly, while inflammasome signaling has been shown to be critical to host defense, the elicited immune response needs to be tightly regulated in order to limit collateral damage to the host.
- This implies that appropriate regulation of inflammasomes is intrinsic to the control circuit of the associated inflammatory processes.
- Several cytoplasmic PRRs are able to assemble into an inflammasome complex and are classified by their protein domain structures.
- For example, the NBD leucine-rich repeat-containing receptor (NLR) family implicates sub-families distinguishable by their N-terminal effector domains.
- There are four recognizable NLR N-terminal domains: the acidic transactivation domain, pyrin domain, caspase recruitment domain (CARD), and baculoviral inhibitory repeat (BIR)-like domains9.
- Another class of inflammasome assembling PRRs is represented by PYHIN protein family members, such as absent in melanoma 2 (AIM2), which contain HIN200 and pyrin domains10.
- Inflammasome-assembling PRRs are expressed in many cell types, including macrophages, dendritic cells (DCs), neutrophils, and epithelial cells11 .
- The final common pathway of inflammasome signaling is inflammatory caspase-activation.
- This task is achieved by the assembly of a hetero-oligomeric complex based on a scaffold protein, such as NOD-like receptor-pyrin-containing proteins (NLRP) or AIM2 protein, and in some inflammasomes the additional recruitment of adapter and effector partners, such as apoptosis-associated speck-like protein containing a CARD (ASC)12.
- The activation of caspases results in the proteolytic activation of the proinflammatory cytokines interleukin-1β (IL-1β) and/or interleukin-18 (IL-18).
- In particular, IL-1β is considered a gatekeeper cytokine which is critically involved in many events related to activation and regulation of inflammation13.
- Considering the potency of the inflammasome-dependent immune responses, it is not surprising that dysregulated inflammasome activity is associated with a number of inflammatory disorders or of multi-factorial diseases involving an inflammatory component, including autoinflammatory disorders, cardiometabolic diseases, infection, cancer and neurological disorders12,14–16 .
- Therefore, regulation of inflammasome activity and therapeutic interventions targeting structures related to inflammasome signaling constitute promising areas of basic and translational research.
- As it is impossible for a single review to fully cover the large and rapidly growing body of high-quality research in inflammasome biology, we aim to provide an introduction to key concepts and an update on recent evidence highlighting new aspects of inflammasome signaling regulation and its implications in health and disease, while referring to reviews dedicated to more specific features of inflammasome research throughout the text.
Inflammasome activation and assembly
- As an important arm of innate immunity, one of the most outstanding functions of inflammasomes is to detect and sense a variety of endogenous or exogenous, sterile or infectious stimuli that are encountered within the cell, and to induce cellular responses and effector mechanisms.
- The assembly of the inflammasome platform is a critical and well-organized process involving several core parts: the upstream sensors recognizing activating signals, the adapters and the downstream effectors (Fig. 1).Pathogen-derived activating signals
- A plethora of PAMPs play key roles in initiating activation of various inflammasomes, among which the most well-studied are bacteria-associated signals.
- Pathogenic activators of the NLRC4 inflammasome are mainly derived from Gram-negative bacteria namely Salmonella, Legionella, Shigella, and Pseudomonas spp.
- These bacteria possess flagellin, or a type III (T3SS) or type IV (T4SS) secretion system rod proteins that are recognized by the NAIP proteins, constituting unique binding partners of NLRC417.
- The murine NLRP1b inflammasome detects Bacillus anthracis lethal toxin in the cytoplasm18.
- The NLRP3 inflammasome can be activated by the pore-forming activity of a wide array of Gram-positive and Gram-negative bacteria, including Staphylococcus aureus, Streptococcus pneumoniae, enterohemorrhagic Escherichia coli and others, mainly through triggering potassium (K ) eflux19–21.
- NLRP6 inflammasome can sense lipoteichoic acid derived from Gram-positive pathogens like Listeria monocytogenes22.
- In human macrophages, the NLRP7 inflammasome recognizes acylated lipopeptides, a microbial cell wall components23.
- Free cytosolic DNA released from a variety of bacteria species, including but not limited to Francisella novicida, is required to activate the inflammasome-forming DNA sensor AIM224.
- Mod-ification and inactivation of the Rho GTPases by bacterial toxins, for example the Clostridium difficile cytotoxin TcdB, and Clostridium botulinum ADP-ribosylating C3 toxin, are important to activate the Pyrin inflammasome, dependent on their enzymatic activities 25.
- In addition, intracellular lipopolysaccharide (LPS) from Gram-negative bacteria is known to be recognized by the mouse caspase-11, or human caspase-4/5, in activating non-canonical inflammasomes26.
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Notably, although the inflammasome activation facilitates host defense against intracellular pathogenic infections, some bacteria have developed effective strategies to dampen or evade inflammasome activation, which has been recently reviewed in detail elsewhere27. For example, bacteria such as Streptococcus spp. can restrain inflammasome activation by producing hydrogen peroxide, which in turn dampens bacterial clearance from host cells28.
Moreover, specific factors from viruses, fungi and parasites have been recently shown to also activate inflammasomes. For example, the NLRP3 inflammasome in macrophages can be activated by a multitude of viruses and viral proteins, such as the hepatitis C virus core protein29, severe acute respiratory syndrome coronavirus (SARS-CoV) viroporin30, Influenza virus M231, and Encephalomyocarditis virus viroporin 2B 32. NLRP9b, mainly expressed in intestinal epithelial cells (IECs), is capable of recognizing the short double-stranded RNA of rotavirus through host RNA helicase Dhx933. Major fun- gal PAMPs such as β-glucan upon Aspergillus fumigatus infection34, fungal CPG35, Candidalysin secreted by Candida albicans36,37 are direct inducers of inflamma- some assembly, mainly for the NLRP3 inflammasome. Moreover, activation of both canonical and noncanonical inflammasomes (mainly involving mouse caspase-11 or human caspase-4/5) by diverse parasitic stimuli, such as Leishmania and its lipophosphoglycan38,39 and Fasciola hepatica-derived molecule cathepsin L340 have recently been discovered as an important strategy for the restric- tion and control of parasitic invasion. The mammalian body is inhabited by a complex com- munity of microorganisms, collectively termed the microbiome. Selective commensal bacteria can not only induce IL-18 secretion in IECs, which is mediated by NLRP6 inflammasome41, but also induce IL-1β maturation mediated by NLRP3 inflammasome signaling in intestinal monocytes, which subsequently promotes intestinal inflammation42. NLRP3 inflammasome activation induced +by commensal bacteria is K efflux-dependent, but does not depend on bacterial viability or phagocytosis43. Sen- sing of commensal gut fungi through the Card9–Syk sig- naling axis promotes inflammasome activation and maturation of IL-18, which plays a protective role in colitis and colitis-associated carcinogenesis44 . Host-derived activating signals In addition to PAMPs, endogenous DAMPs (or host danger signals), which are released upon tissue injury, emerged as another major source for inflammasome activation. For example, NLRP3 inflammasome which is activated by a diverse numbers and structures of exo- genous stimuli ranging from particulate matter (such as silica) to bacterial-derived toxins45 can also sense other host-derived signals downstream of all these exogenous stimuli. Some of these endogenous signals, including ion efflux, mitochondrial dysfunction and reactive oxygen species (ROS) are well-characterized (see below), while others remain uncharacterized to date. An elegant example of a new host-related activator was demonstrated in a recent study showing that diverse NLRP3 stimuli can trigger the disassembly of trans-Golgi network (TGN), and the dispersed TGN acts, in turn, as a scaffold for NLRP3 recruitment through ionic bonding and sub- sequent inflammasome assembly and activation46. +Cytosolic K efflux is a common trigger involved in both canonical NLRP3 inflammasome activation21 and caspase-11-mediated noncanonical activation47. NEK7, a mitosis-related serine-threonine kinase, is essential to regulate NLRP3 oligomerization and activation down- stream of K+ efflux48,49. Intracellular chloride efflux, +another event downstream of K efflux, is a critical upstream event for NLRP3 activation50. The role of cal- cium (Ca2+) signaling in activating NLRP3 inflammasome remains debatable. Although some studies showed that inflammasome activation essentially requires elevation of intracellular and extracellular Ca2+51,52, other studies argued that Ca2+ is not strictly required for initiation of NLRP3 signaling53,54. The roles and mechanisms of other upstream ion flux disturbances, including Na+ and Zn2+ in orchestrating NLRP3 inflammasome activation have been extensively reviewed elsewhere55. Although it remains to be answered whether these ions directly bind to NLRP3 and trigger downstream processes, such advances provide potential ion-associated targets for modulating NLRP3-driven disorders. Mitochondrial dysfunction is another emerging elicitor of NLRP3 activation, operating via mitochondrial DNA (mtDNA) release, mitophagy and apoptosis. For example, in aging hematopoietic stem cells, increased mitochon- drial stress leads to aberrant NLRP3 inflammasome acti- vation and contributes to characteristic aging-associated defects56. Recently, it was found that mtDNA synthesis and oxidized mtDNA release into the cytosol driven by TLR signaling is crucial to prime NLRP3 activation, while inhibition of mtDNA synthesis via TFAM or IRF1 abla- tion prevents NLRP3 inflammasome activation 57. How- ever, other studies show that TFAM depletion leads to increased rather than decreased cytosolic mtDNA to activate antiviral immune responses through the cGAS–STING pathway58, and that IRF1 is dispensable for the NLRP3 inflammasome activation59. Furthermore, cholesterol-dependent mtDNA accumulation in macro- phages results in AIM2, but not necessarily NLRP3 inflammasome activation60. Cytosolic mtDNA activates the NLRP3 inflammasome and promotes the release of IL- 1β and IL-18, while the translocation of mtDNA into cytosol, in turn, requires NLRP3, indicating that NLRP3 might act both upstream and downstream of mtDNA release61. On the other hand, damaged mitochondria, stimulated by NLRP3 activators, initiate an intrinsic “NF-κB-p62-mitophagy” pathway through which NF-κB limits NLRP3 inflammasome activation62. These results highlight a regulatory loop existing between the NLRP3 inflammasome and mitochondria. In addition, oxidized mtDNA released into the cytosol during apoptosis binds to the NLRP3 inflammasome, highlighting a link between apoptosis and inflammasome activation63. However, this conclusion has been challenged by another study showing that genetic deletion of important executioners of mito- chondrial apoptosis does not affect the activation of NLRP3 inflammasome64. Indeed, besides mtDNA, intrinsic apoptosis resulting from mitochondrial mem- brane damage in macrophages activates Caspase-3 and -7 to drive IL-1β secretion downstream of both NLRP3 inflammasome and caspase-8 signaling65. Emerging evidence suggests that ROS constitute another central signaling hub among a diversity of NLRP3-related stimuli45. ROS induce the binding of thioredoxin-interacting protein to NLRP3, which is essential for NLRP3 inflammasome activation66. Although some studies suggest that NLRP3 inflammasome activa- tion may be triggered by ROS generated by an NADPH oxidase67, other studies show that activation of the NLRP3 inflammasome is independent upon NADPH oxidase- generating ROS68 or its oxidative activity69, challenging the proposed indispensable role of ROS in NLRP3 inflammasome activation. Of note, ROS generation is frequently accompanied by mitochondrial dysfunction and ion efflux. It would be worth investigating the inter- play among these triggers in contributing to inflamma- some activation. Growing evidence suggests that dysregulated metabolic factors may constitute novel stimuli of inflammasome activation. These include, as an example, alterations in sterol biosynthesis70 and glycolysis metabolism71 . Cho- lesterol overload leads to activation of the AIM2 inflam- masome through impairing mitochondrial metabolism and eliciting mtDNA release60. Increased choline uptake and metabolism in macrophages keeps the NLRP3 inflammasome in an active state by maintaining func- tional mitochondria72. In the central nervous system, cholesterol accumulation in myelin debris in aged mice results in NLRP3 inflammasome activation, which ham- pers remyelination and nerve repair73. Collectively, the discovery of multiple metabolic activators of inflamma- somes necessitates the investigation of the underlying mechanisms by which metabolic dysfunction impacts on inflammasome biology. Assembly of inflammasomes Upon activation, assembly of inflammasomes requires interactions between the inflammasome sensor and inflammatory caspase-1 or noncanonical caspase-11, with or without co-binding of the adapter protein ASC. With the help of advanced techniques such as cryo-electron microscopy, rapidly growing evidence begins to disen- tangle the structural mechanisms of inflammasome assembly. NLRC4 inflammasome The interaction between the nucleotide-binding domain (NBD) and winged-helix domain (WHD) is essential to keep NLRC4 in an auto-inhibited state74. Studies using a purified PrgJ–NAIP2–NLRC4 inflammasome revealed that one single PrgJ-bound NAIP2 molecule with a cata- lytic surface is sufficient to activate NLRC4 by matching its oligomerization surface, which comprises a large por- tion of NBD and a small part of LRR. Activated NLRC4 undergoes substantial structural reorganization, interacts, and activates another quiescent NLRC4 molecule in a self-propagating approach to ultimately form a 10- to 12- spoke wheel- or disk-like architecture75,76. Similarly, stu- dies on the assembled flagellin–NAIP5–NLRC4 inflam- masome showed that the conserved regions of the flagellin ligand recognize multiple domains of the NAIP5 molecule, resulting in NAIP5 activation77 and mediating progressive NLRC4 oligomerization78. More importantly, the extreme C-terminal side of different bacterial fla- gellins forms a key structural epitope for their specific detection by NAIP5, potentially explaining why different bacteria possess different potency in inducing the NLRC4 inflammasome79. How different NAIPs precisely interact with their respective ligands provides an intriguing question for further investigations. NLRP3 inflammasome Unlike NLRC4, the assembly of the NLRP3 inflamma- some requires the presence of NEK7, a serine and threonine kinase that is critically involved in mitotic cell cycle progression48,80. NEK7 directly binds to the LRR domain of NLRP3 to promote inflammasome assembly during cell interphase49. A recent cryo-electron micro- scopic study focusing on the human NLRP3-NEK7 com- plex found that NEK7 is indispensable to bridge the gaps between adjacent NLRP3 subunits and mediate NLRP3 oligomerization, thereby clarifying the structural basis of NEK7-mediated NLRP3 inflammasome activation81. Despite these advances, it remains to be investigated whether NEK7 serves as a common sensor for multiple stimuli-induced NLRP3 activation states, and whether NEK7 is sufficient to trigger a nucleated oligomerization. Other NLRP inflammasomes In vitro reconstitution of the human NLRP1 inflam- masome by purified recombinant proteins has character- ized that NLRP1 oligomerization can directly recruit caspase-1 via its CARD domain, and can be further enhanced by binding to ASC via the pyrin domain (PYD)82. Crystal structure analysis suggests that interac- tion between human NLRP1 and procaspase-1 CARDs is potentially mediated by electrostatic attractions 83. The role of enzymatic cleavage of murine NLRP1b in its activation is discussed in detail below. Another NLRP subfamily member, NLRP6, is capable of forming fila- mentous structures though the PYD by self-assembly. Remarkable conformational changes following this step, enable subsequent recruitment of the ASC adapter through PYD–PYD interaction, while the NBD of NLRP6 features a synergistic role in enhancing the assembly process84. NLRP7 can self-associate to form an oligomer through NACHT domain interaction upon activation85, which essentially requires the ATP-binding and hydrolysis activities of the NLRP7 NBD86. In a recent study, a pre- viously uncharacterized member of the NLR family, NLRP9b, has been shown to assemble an inflammasome in the intestine upon murine enteric rotavirus infection33. The authors demonstrated that NLRP9b recognizes short dsRNA of rotavirus to form inflammasome complexes with ASC and caspase-1 to promote maturation of IL-18 and GSDMA-induced cell death33. AIM2 and IFI16 inflammasomes Assemblies of the AIM2 and IFI16 inflammasomes are considered different from the aforementioned NLR- dependent inflammasomes, since both the AIM2 and IFI16 sensors specifically recognize cytosolic DNA through their hematopoietic interferon-inducible nuclear (HIN) domain, while lacking the NOD for self- oligomerization. Electrostatic binding of double-stranded DNA backbone to the positively charged HIN domain liberates AIM2 autoinhibition87,88. Following this event, the helical assemblies of AIM2PYD generate a poly- merization platform to nucleate downstream ASCPYD filaments, underpinning the assembly of an inflamma- some89,90. Unlike AIM2, the isolated IFI16 HIN domain possesses relatively weaker DNA-binding affinity87, which can be strengthened by the presence of a full length IFI16 protein91. Furthermore, the non-DNA-binding PYD of IFI16 is necessary for the cooperative assembly of IFI16 filaments on dsDNA91. Recruitment of ASC and caspase-1 In CARD-absent NLRs including AIM2, IFI16, NLRP3, NLRP6, and NLRP7, PYD–PYD interactions between NLRs and ASC nucleate the PYD filaments of ASC, which in turn nucleates CARD filaments of caspase-1 through CARD–CARD interactions92 . This brings monomers of pro-caspase-1 into close proximity and initiates caspase-1 self-cleavage and activation. Another innate immune sensor encoded by MEFV gene, pyrin, also forms an inflammasome complex in an ASC-dependent approach93. Multiple binding sites on both PYDs of pyrin and ASC identified by spectrometry provide a molecular basis for their interactions94. In NLRC4, which contains a CARD, the nucleation and activation of downstream caspase-1 can occur through the homotypic CARD–CARD binding, and this process can be inde- pendent of ASC95. The orientation of the CARD of NLRC4 and the ability to integrate ASC into the structure is currently unknown and warrants further studies. Noncanonical inflammasome activation While the final common pathway of canonical inflam- masome activation involves recruitment of caspase-1 in response to multiple microbial or danger signals, the newly discovered “noncanonical inflammasome” signals in a caspase-1-independent manner through direct recognition of the cytosolic LPS by the CARDs of caspase-4 and caspase-5 (in humans) and caspase-11 (in mice). This elicits caspase-dimerization and activation, resulting in cleavage of GSDMD96,97 . Activation and regulation of non-canonical inflammasomes is described in detail below. Inflammasome effector functions * Inflammasome-mediated maturation of IL-1 family cytokines IL-1β and IL-18 Secretion of the proinflammatory cytokines IL-1β and IL-18, together with induction of pyroptotic cell death (see below), represent the main outcomes of inflamma- some activation upon caspase-1 activation98. Akin to caspase-1, IL-1β, and IL-18 accumulate in the cytoplasm as inactive precursors (pro-IL-1β and pro-IL-18, respec- tively). Caspase-1 cleaves pro-IL-1β into a 17 kDa mature 99fragment and proIL-18 into a 17.2 kDa mature pro- tein100. IL-1β stimulates the release of other cytokines such as IL-6, tumor necrosis factor (TNF)-α and IL-1α, as well as other crucial factors responsible for growth and differentiation of immune cells13. Both IL-1α and IL-1β bind to IL-1R1, thereby enabling recruitment of its co- receptor IL-1RAc. Similarly, upon binding of IL-18 to IL- 18Rα, the latter heterodimerizes with IL-18Rβ. Approx- imation of the intracellular TIR domains of the IL-1R or IL-18R complex results in recruitment of MyD88 followed by a cascade of downstream events, which ultimately results in the activation of important signaling proteins and transcription factors, such as NF-κB, regulating inflammation101. * Pyroptosis Both canonical inflammasome signaling engaging caspase-1 and noncanonical inflammasome activation recruiting caspase-4, caspase-5 (in humans), and caspase- 11 (in mice) elicit an inflammatory type of cell death termed “pyroptosis”102. Pyroptosis is a lytic form of pro- grammed cell death in response to sensing of pathogens or host-derived danger signals. It is morphologically * Apoptosis Caspase-8-driven apoptosis, also regarded as “secondary pyroptosis”, is increasingly studied as another effector function of inflammasome activation. Studies have con- firmed the existence of a caspase-8 dependent apoptotic death pathway activated by AIM2, NLRP3, and NAIP–NLRC4 inflammasomes, which is parallel to, but distinct from the canonical caspase-1 dependent pyr- optosis, and is present in various cell types109–111. In the absence of caspase-1, these inflammasomes are capable of triggering caspase-8-dependent apoptosis upon sensing of diverse stimuli112,113. Importantly the presence of caspase-1 protease activity suppresses activation and induction of caspase-8-mediated apoptosis by the sensor NLRP1b and NLRC4114. Procaspase-8 binds to the ASC through the adapter protein FADD, and co-localizes to ASC inflammasome specks, adding another layer of complexity to the inflammasome structure109,114. Inter- estingly, a recent study shows that apoptosis induced by inflammasome stimuli can be initiated by caspase-1 itself, in the absence of GSDMD, through the Bid–caspase- 9–caspase-3 axis115. The molecular mechanisms regulat- ing inflammasome-induced apoptosis, and the interaction between apoptosis and pyroptosis, remain to be further investigated.