Pyroptosis is a type of programmed, lytic, and inflammatory cell death pathway that occurs following the assembly and activation of inflammasomes. As an innate immune mechanism, pyroptosis can enhance the host’s ability to resist pathogens (4). Inflammasomes are multimeric protein complexes, which usually consist of sensors, adaptor proteins, and the caspase family (26). The sensor is a pattern recognition receptor (PRR), such as nucleotide-binding domain-like receptors (NLRs), AIM2, and pyrin (27). Recognition of inflammatory ligands leads to sensor activation, oligomerization, and subsequent recruitment of adapters (28). Besides the caspase recruitment domain (CARD), the adaptor protein ASC also has a death-fold domain: the pyrin domain (PYD) (28). The structural characteristics of ASC provide support for the binding of CASP1 to the sensor, and thus the generation of CASP1 with catalytic activity, which induces pyroptosis and promotes the release of IL-1β and IL-18. It has been confirmed currently that PYD-containing NLRP3, AIM2, and pyrin recruit ASC to form inflammasomes, while CARD-containing NLRP1 and NLRC4 assemble inflammasomes via ASC-independent pathway, that is, directly recruiting CASP1 (27, 29, 30). Bacterial type III secretion system (T3SS) components, flagellin, abnormal or foreign DNA, dsRNA and other kinds of PAMPs and DAMPs can be specifically recognized by these sensors, and activate CASP1 (31–33). CASP1 with enzyme activity cleaves gasdermin D (GSDMD), pro-IL-1β and pro-IL-18, and promotes pyroptosis and inflammation (27, 31, 34). In addition, inflammasomes can also activate CASP8, the regulator of pyroptosis, apoptosis and necroptosis. CASP8 with enzyme activity can cleave GSDMD and GSDME (35), while CASP8 without enzyme activity can induce ASC aggregation and then activate CASP1, thus inducing pyroptosis (36).

Apoptosis is a highly regulated process of cell death that elicits no inflammatory responses. Such stimuli as pathogen infection, DNA damage, hypoxia, and the expression of certain cancer proteins can induce apoptosis (37, 38). Apoptosis can be divided into the endogenous pathway and the exogenous pathway. The former is caused by DNA damage or endoplasmic reticulum stress, while the latter is mediated by the activation of death receptor family members (39). Apoptosis is regulated by several components, including signaling molecules, receptors, enzymes, and gene regulatory proteins, the most important of which are the BCL-2 protein family, caspase family, and tumor necrosis factor (TNF) receptor superfamily (40, 41). The Bcl-2 protein family regulates the permeability of the mitochondrial membrane, the release of substances such as cytochrome c, and the activity of caspases (42–44). CASP2/8/9 acts as an activator, while CASP3/6/7 acts as an executioner (40). As an activator of caspases, apoptotic protease-activating factor-1 (APAF-1) is closely related to endogenous apoptosis (45, 46). Cytochrome c and dATP in the cytoplasm initiate the assembly of APAF-1, and procaspase-9 is activated after binding to APAF-1 through CARD, thus activating downstream CASP3/7, and finally leading to the occurrence of apoptosis (47, 48). The TNF receptor superfamily is a key membrane receptor involved in apoptosis and an important participant in caspase-mediated apoptosis (49). Simply put, TNF receptor superfamily members recruit adaptor proteins after interacting with ligands to transmit death signals and promote apoptosis by activating CASP8 or CASP9 (49).

Necroptosis is primarily mediated by RIPK1, RIPK3, and mixed lineage kinase domain-like protein (MLKL), which is a form of lytic cell death independent of caspases (50, 51). TNF is the main regulator of cell survival, apoptosis, and necroptosis, and the study of TNF signaling is an important pathway for recognizing necroptosis because it plays an important role in the inflammatory response induced by infection or injury (52, 53). When the activity of CASP8 is blocked, the stimulation of receptors such as TNFR1 and TLR can induce necroptosis, which involves the phosphorylation and activation of RIPK1. Some studies have shown that the catalytic activity of RIPK3 may determine whether the death pathway is apoptosis or necrotic apoptosis (53). The formation of the RIPK1-RIPK3 complex can be observed during virus infection (54). When caspase defects or inhibitors exist, the apoptosis induced by TNF and RIPK1 is transformed into necroptosis. In this process, RIPK3 regulates the phosphorylation of RIPK1 and forms a necrosome with it, and regulates the production of reactive oxygen species induced by TNF (54–56). MLKL is phosphorylated and activated by necrosome, leading to plasma membrane destruction and necroptosis (57, 58). In addition, RIPK1, RIPK3, and MLKL are also involved in the activation of inflammasomes, which is beneficial to the maturation and secretion of IL-1β and IL-18 (59, 60). In addition to TNFR, Fas, TRAILR, TLR3, and TLR4 stimulation can also induce necroptosis in the absence of CASP-8 activity (53).

Several decades have passed since programmed cell death (PCD) was identified. Among all the proposed forms of PCD, pyroptosis, apoptosis and necroptosis were the most well-defined in terms of the molecular machinery responsible for the initiation, transduction, and execution of cell death (61). Studies have found that there is a complex dynamic molecular network between these programs, and the processes of cell death caused by the three are not absolutely independent. Apoptosis was initially thought to be an immune-silent form, but in some cell types, it is also inflammatory (62, 63). Pyroptosis and apoptosis both involve the activation of caspase family members, indicating that the two forms may have a common evolutionary origin. As mentioned, activation of CASP1 triggers pyroptosis, a process mediated by GSDMD. Studies have found that CASP1 induces apoptosis in GSDMD-deficient cells via the Bid-CASP9-CASP3 axis (Bid is a member of the BCL-2 family) or other pathways (64). CASP8, as the initiator of apoptosis, can inhibit the necroptosis mediated by RIPK3 and MLKL. Notably, CASP8 scaffolding function is involved in the activation of NLRP3 inflammasome induced by dsRNA (59), while the expression of enzymatically inactive CASP8 can trigger the formation of ASC specks, the activation of CASP1, and the secretion of inflammatory cytokines (65). Both pyroptosis and necroptosis belong to the lytic cell death pathway, which means that there is likely to be a crossover between them. RIPK3 and MLKL are essential for the occurrence of necroptosis. The latest study found that in addition to the activation of MLKL, the activation of RIPK3 was also accompanied by the activation of NLRP3 inflammasome and CASP1 (66). In addition, active MLKL can also activate the NLRP3 inflammasome (67). These shreds of evidence suggest that necroptotic stimuli can trigger pyroptosis. Cell death signals are constantly evolving and diversifying to cope with pathogen infection and other risk factors. Multiple PCD pathways ensure that when particular proteins are suppressed, the body can fulfill its defensive goal via other means. The extensive cross-talk between pyroptosis, apoptosis, and necroptosis led to the conceptualization of “PANoptosis” (‘P’, Pyroptosis; ‘A’, Apoptosis; ‘N’, Necroptosis; and ‘optosis’, a form of programmed cell death). The phenomenon when pyroptosis, apoptosis, and necroptosis are regulated at the same time was named PANoptosis by the team of Professor Kanneganti in their studies in 2019 (11, 68, 69). PANoptosis was originally defined as “combines the main features of pyroptosis, apoptosis, and necroptosis”.

A variety of proteins can form multi-protein complexes that regulate PCD, which is based on the interaction between various protein domains that can be divided into three categories: sensing domain, assembly domain, and catalytic domain (9). For example, assembly domains include CARD, DEATH, death effector domain (DED), PYD, and receptor-interacting protein homotypic interaction motif (RHIM), among which the first four are also known as death fold domains due to their importance to the assembly of PCD-executing complexes. A variety of protein molecules, including sensors, adapters, and CASP1, are assembled into inflammasomes through PYD-PYD or CARD-CARD interactions to mediate pyroptosis (26). Apoptosis is regulated by the complex apoptosome, and a typical example is that APAF-1 senses mitochondrial-derived cytochrome C and interacts with CASP9 through the CARD domain (70). Under the inhibition of CASP8 activity, RIPK3 and RIPK1 formed a necrosome that mediated necroptosis through RHIM-RHIM interaction (71). It was concluded that the death-inducing complexes inflammasome, apoptosome, and necrosome have many core members such as NLRP3, FADD, and RIPK1 (65, 72–74). Taken together, it is not difficult to speculate that multiple proteins interacting through key domains may also be an important molecular basis for PANoptosome formation. Then in 2020, PANoptosome, a cell death-inducing complex with the molecular characteristics of pyroptosis, apoptosis, and necroptosis, was identified to induce and regulate PANoptosis (75). The PANoptosome was initially shown to contain RIPK1, ASC, NLRP3, and CASP8 (76). The subsequent study determined that RIPK3, CASP6, ZBP1, and CASP1 are also components of the PANoptosome in response to IAV infection (77). ZBP1 initiates ZBP1-PANoptosome assembly to drive inflammasome activation and cell death, and CASP6 interacts with RIPK3 to enhance the interaction between RIPK3 and ZBP1 to promote PANoptosome assembly (78). The studies of Malireddi et al. connected CASP1 (the inflammasome sensors) and CASP11 (components of pyroptosis) with CASP8, CASP7, PARP (components of apoptosis), RIPK1, and RIPK3 (components of necroptosis) (79). To sum up, these studies indicated that the PANoptosome contains molecules crucial for activating pyroptosis, apoptosis, and necroptosis to execute pro-inflammatory cell death. As a result, PANoptosis was defined as “a programmed cell death pathway induced by the PANoptosome, which provides a molecular scaffold that allows for interactions and activation of the machinery required for pyroptosis, apoptosis, and necroptosis”. However, studies focused on thoroughly elucidating the role and regulation of PANoptosis were lagging behind (79).

Recent studies highlighted the crosstalk and redundancies among pyroptosis, apoptosis, and necroptosis. The viral sensor ZBP1 regulates NLRP3 inflammasome activation and pro-inflammatory cytokine production during IAV infection through the RIPK1-RIPK3-CASP8 axis, triggering the co-occurrence of pyroptosis, apoptosis, and necroptosis (PANoptosis) in infected cells (10). Activation of CASP1, CASP8, CASP3, and phosphorylation of MLKL in macrophages infected with IAV suggest that IAV infection triggers PANoptosis in host cells. ZBP1 deletion inhibited PANoptosis, but blockade of either pyroptosis, apoptosis, or necroptosis alone failed to prevent cell death (10, 77). Studies have shown that CASP8 is a molecular converter of pyroptosis, apoptosis, and necroptosis (65). CASP8 can regulate pyroptosis through different pathways, mediating apoptosis induced by the death receptor in the exogenous pathway, and inhibiting necroptosis mediated by RIPK3 and MLKL (65, 80). In recent years, more and more molecules involved in PANoptosis have been discovered, and their roles and mechanisms are gradually clear. For example, CASP6 promotes the activation of pyroptosis, apoptosis, and necroptosis (77), and RIPK1 plays a key role in the regulation of Yersinia-induced PANoptosis (12). In addition to experimental evidence, the use of the STRING database enables visual analysis of the physical interactions among the molecular components of pyroptosis, apoptosis, and necroptosis, providing strong evidence for crosstalk between these three PCD pathways and also supporting the study of PANoptosis (81, 82). The results of STRING analysis indicate that CASP8 is the hub of pyroptosis, apoptosis, and necroptosis (81), which is consistent with experimental evidence showing the function of CASP8 in the three types of PCD pathways. What’s more, the role and regulation of PANoptosis in different physiological and pathological scenarios [like infections (83, 84), and cancers (85–87)] are elucidated more clearly. So, PANoptosis was described as “a unique innate immune inflammatory cell death pathway regulated by PANoptosomes, complexes that integrate molecules from other cell death pathways” (88), and the totality of biological effects in PANoptosis cannot be individually accounted for by pyroptosis, apoptosis, or necroptosis alone”.

To conclude, PANoptosis is a changing and dynamic field. As the studies go further, there is no doubt that PANoptosis will continue to get further conceptualized. It has recently been discovered that a variety of pathogens such as viruses, bacteria, fungi, and even parasites, as well as other non-infectious factors such as cytokines in tumors, may trigger PANoptosis in host cells (Table 1).

The concept of PANoptosome was proposed and revealed by Christian et al. in 2020, who found that molecules from pyroptotic, apoptotic, and necroptotic cell death can interact to form a single molecular complex (75). After transient overexpression of molecules known to be essential to PANoptosis in HEK293T cells, it was found that NLRP3 could co-precipitate with ZBP1, RIPK3, and RIPK1 respectively through protein interaction experiments, and CASP8, ASC, RIPK1, NLRP3, and ZBP1 could form protein complexes regardless of the presence or absence of RIPK3 (75). This method confirms the direct interaction between PANoptotic molecules, but whether these molecules can also assemble into complexes in individual cells still needs to be determined. Microscopy is a powerful and informative tool for assessing complex formations and dynamics. Wang et al. developed a method to validate and analyze PANoptosome complexes in single cells by expansion microscopy (99). This protocol allows the formation of multi-protein complexes containing ASC, CASP8, and RIPK3 to be visualized in individual cells under a variety of innate immune cell death-inducing conditions, providing evidence of PANoptosome assembly in individual cells. Up to now, different species of PANoptosomes have been identified, such as the ZBP1 PANoptosome, the AIM2 PANoptosome, and the RIPK1 PANoptosome (8, 12, 100).

The proteins that constitute PANoptosome can be divided into three types: 1) PAMPs or DAMPs sensors such as ZBP1, AIM2, and NLRP3; 2) adaptors such as ASC and FADD; 3) catalytic effectors such as RIPK1, RIPK3, CASP1 and CASP8 (9). The classification of these proteins is not absolute, for example, RIPK1 with kinase activity is required for necroptosis, where RIPK1 acts as a catalytic effector molecule; whereas the scaffold function of RIPK1 without kinase activity is required for NLRP3 inflammasome activation and cell death in TAK1-deficient cells, suggesting that RIPK1 may also act as an adaptor (76).

With the deepening of research, it has been found that a variety of proteins participate in or coordinate the assembly of PANoptosome, and the composition of PANoptosomes induced by different factors tends to vary as well (Figure 2A). During C. albicans or A. fumigatus infection, ZBP1 senses special components and mediates the formation of the ZBP1/RIPK3/RIPK1/FADD/CASP8 complex (11). CASP8 and RIPK3-deficient BMDMs are largely protected from fungus-induced PANoptosis (11). TNF-α and IFN-γ produced by SARS-CoV-2 infection can promote the formation of RIPK1/RIPK3/FADD/CASP8 PANoptosome through signal transduction (91), and ZBP1 is highly likely to be involved in this process (89). Recently, it has been found that CASP6 is required for pyroptosis, apoptosis, and necroptosis but its caspase activity is not necessary during IAV infection. This is because the interaction of CASP6 with RIPK3 can promote the binding of RIPK3 and ZBP1, which in turn promotes the formation of ZBP1 PANoptosome (77). Notably, Yersinia induced a ZBP1-independent PANoptosome composed of RIPK1/RIPK3/CASP8, and the formation of this complex was associated with TNFR/TLR4 signaling activation and MAPKs and NF-κB signaling inhibition (12).

PANoptosis is closely related to the inflammasome, and in addition to the classical NLRP3 inflammasome, the NLRC4 inflammasome also has an important role in PANoptosis. Upon detection of the T3SS protein, NAIP binds to NLRC4 to induce assembly and activation of the NAIP/NLRC4 inflammasome, which triggers PANoptosis in infected cells via CASP1-GSDMD and CASP8/3/7 as well as MLKL pathways (101). The components of the PANoptosome protein complex are very complicated and interrelated. The composition of PANoptosome induced by various factors may be different, but they all can induce the co-occurrence of pyroptosis, apoptosis, and necroptosis.

With further research, a variety of PANoptosomes have been identified, among which ZBP1 PANoptosome is representative (Figure 2B). The structural characteristics of ZBP1 are that its N-terminus contains two Z-nucleic acid binding domains (Zα domains), and the middle of the protein sequence contains two RHIM domains (100). The presence of Zα domains allows ZBP1 to act as a key innate immune sensor for Z-RNA produced by DNA and RNA viruses. Kuriakose et al. also identified ZBP1 as a sensor for nucleoprotein and polymerase subunit PB1 of IAV (10). Further studies confirmed that the C-terminal domain of ZBP1 is essential for the interaction with IAV protein, and Zα domains are also required for the interaction with PB1 (10). The structure of ZBP1 confers the ability to regulate PANoptosome formation and trigger PANoptosis in IAV infection, which has been verified: deletion of the ZBP1 Zα2 domain eliminated IAV infection-induced PANoptosis and inflammation (11, 102).

The existence of RHIM domains enables ZBP1 to interact with other RHIM-containing proteins, such as RIPK1 and RIPK3. The ZBP1-RIPK1 complex formed during IAV infection can transduce NF-κB signaling and regulate the production of IL-6 and TNF (10). The formation of the ZBP1-RIPK3 complex is conducive to recruiting RIPK1, and further forming the ZBP1 PANoptosome mainly composed of ZBP1/RIPK3/RIPK1/FADD/CASP8 (10, 77, 103). ZBP1 PANoptosome induces pyroptosis, apoptosis, and necroptosis by activating NLRP3/ASC/CASP1-GSDMD and CASP3/6/7 and MLKL pathways respectively, which ultimately triggers PANoptosis (77).

Pyroptosis, as one of the arms of PANoptosis in IAV infection, mainly relies on the NLRP3 inflammasome. Studies have found that activation of NLRP3 inflammasomes is essential in host defense against IAV and that CASP1-deficient mice have higher mortality after IAV infection (104, 105). Interestingly, inhibition of NLRP3 at late stages of infection reduced IAV infection severity (106). Therefore, the resistance to IAV depended to some extent on the activation of NLRP3 inflammasome in the early stage of infection, which was completely eliminated by the deletion of ZBP1 (10, 100, 107). It should be noted that NLRP3 inflammasome activation in other pathogen infections may not depend on ZBP1, such as the vesicular stomatitis virus (10). Previous studies have found that RIPK1 and RIPK3 are required for NLRP3 inflammasome activation induced by RNA viruses, including IAV (108). Mechanistically, infection triggers the formation of the RIPK1-RIPK3 complex, which promotes phosphorylation of dynamin-related protein 1 (DRP1) and translocation to mitochondria, thereby driving mitochondrial damage and NLRP3 inflammasome activation (108). The formation of the ZBP1-RIPK3 complex is conducive to the recruitment of RIPK1 and other proteins. Therefore, the formation of ZBP1 PANoptosome during IAV infection is very important for the activation of NLRP3 inflammasome. CASP8, which is involved in constituting ZBP1 PANoptosome, also has a regulatory effect on NLRP3 inflammasome activation. Compared with the deletion of RIPK3, the deletion of RIPK3 and CASP8 is more unfavorable to inflammasome activation, consistent with the effect of ZBP1 loss during IAV infection (10). Structurally, CASP8 has no domain that can interact with ZBP1, so it needs to rely on RIPK1 for recruitment through FAS-associated protein with dead domains (FADD). GSDMD cleavage was impaired in IAV-infected Ripk3^-/-and Ripk3^-/-Casp8^-/-BMDMs compared to WT BMDMs, suggesting that RIPK3 and CASP8 are the main components of ZBP1 PANoptosome that mediate pyroptosis (77). MLKL is a substrate of RIPK3 and simultaneously an executor of necroptosis. However, experiments have shown that the deaths of WT and Mlkl^-/- BMDMs infected with IAV are almost the same (10), indicating that MLKL is not necessary for cell death caused by ZBP1 and RIPK3-mediated IAV infection, which provides evidence for the existence of other forms of cell death. Compared with the wild type, the activation of CASP8/3/7 in Zbp1^-/- BMDMs infected with IAV was abrogated, confirming that ZBP1 indeed mediates apoptosis in IAV infection (10).

It was found that fibroblasts and BMDMs lacking RIPK3 did not die due to IAV infection (10). This strongly demonstrates the importance of RIPK3 in ZBP1 PANoptosome, which plays a dominant role in the activation of the NLRP3 inflammasome, the phosphorylation of MLKL, and the activation of CASP8/3/7. Furthermore, it was confirmed that concurrent use of CASP8 inhibitors and MLKL inhibitors prevented pyroptosis, apoptosis, and necroptosis of WT BMDMs caused by IAV infection (109). We hypothesized that this may be due to the involvement of CASP8 in the regulation of both pyroptosis and apoptosis, together with the inhibition of MLKL preventing necroptosis from occurring. In short, ZBP1, RIPK3, RIPK1, FADD, and CASP8 constitute ZBP1 PANoptosome, which together coordinate pyroptosis, apoptosis, and necroptosis with parallel contribution as well as inflammatory response in IAV infection, and infected cells do not die unless three pathways are blocked at the same time.

It is known that AIM2 can detect dsDNA released during the pathological attack and cellular perturbation, and its recognition of dsDNA leads to the assembly of AIM2 inflammasome and the occurrence of pyroptosis, which plays an important role in infectious diseases, inflammatory diseases, and cancer (110, 111). Herpes simplex virus 1(HSV1) and Francisella novicida are known pathogens that can activate the AIM2 inflammasome (8, 112). Recently, it has been found that the function of AIM2 is not limited to activating inflammasome and triggering pyroptosis. For example, AIM2 and NLRP3 can form a dual cytoplasmic surveillance system, which has a stronger protective effect on A. fumigatus infection than either of the two alone (113).

The role of AIM2 in PANoptosis has been identified as regulating pyrin and ZBP1 to drive PANoptosis and inflammatory responses in HSV1 or F. novicida infection (8). It was found that the expressions of pyrin and ZBP1 in Aim2^–/–, Asc^–/–, Casp1^–/– BMDMs infected with HSV1 or F. novicida were decreased, but not in im2^–/–, Asc^–/–, Casp1^–/–BMDMs infected with IAV (8). Interferon (IFN)-β production was noted in HSV1 or F. novicida infected WT BMDMS (8), and IFN signaling has previously been found to promote the expression of pyrin and ZBP1 (10, 75). IFN-β was not found in Aim2^–/–, Asc^–/–, Casp1^–/–BMDMs, and the expression of pyrin and ZBP1 recovered after IFN-β supplementation (8), which suggested that the production of IFN-β mediated by AIM2/ASC/CASP1 pathway is necessary for the expression of pyrin and ZBP1 in HSV1 or F. novicida infection. Infection with HSV1 or F. novicida can induce AIM2-dependent and NLRP3/NLRC4-independent activation of CASP1, followed by the release of IL-1β and IL-18 and cell death (8). However, under the same condition, the inflammasome activation and cell death were decreased in Mefv^–/– BMDMs, suggesting that the function of AIM2 is driven by pyrin to some extent (8). In addition, the absence of ZBP1 also attenuated the effect of AIM2. Compared with WT BMDMs, the activation of CASP1/GSDMD/GSDME, CASP8/3/7, and RIPK3/MLKL in Mefv^–/– and Zbp1^–/– BMDMs decreased after infection with HSV1 or F. novicida, and completely inactive in Aim2^–/– and Mefv^–/–Zbp1^–/– BMDMs (8). These results suggested that ZBP1 and pyrin synergistically drive AIM2-mediated PANoptosis in HSV1 or F. novicida infection. HSV1 or F. novicida infection activates AIM2 and promotes the formation of PANoptosome consisting of AIM2, pyrin, and ZBP1 as well as molecules such as ASC, RIPK3, RIPK1, FADD, CASP1, and CASP8, which is necessary for cell death (PANoptosis). The limited assembly of AIM2 PANoptosome, such as the lack of AIM2, pyrin, or ZBP1, will lead to the death of infected mice (8).

Notably, HSV1 and F. novicida, while activating AIM2, also attract the ZBP1 Zα domain and inhibit Rho-GTP activity, thus enabling the assembly of PANoptosome containing a variety of sensors (8). Similarly, other pathogens that can bind to multiple sensors may also form PANoptosomes with shared central components.