PANoptosis: A Cell Death Characterized by Pyroptosis, Apoptosis, and Necroptosis

Introduction

Regulated cell death plays an important role in host defense. Pyroptosis, apoptosis and necroptosis are three types of regulated cell death. Recent studies have found that necroptosis, apoptosis and pyroptosis are superimposed among each other, thus a concept of total cell death called PANoptosis (P, pyroptosis; A, apoptosis; N, necroptosis) has been proposed.¹ Studies have reported that PANoptosis is involved in many diseases, including infectious diseases and tumor diseases.^2,3 Therefore, it is important to understand the mechanism of PANoptosis and provide treatment for human diseases. PANoptosis is regulated by a cascade of upstream receptor and molecular signals, which assemble into polymeric complexes known as PANoptosome.⁴ PANoptosis is a new cell death proposed by Malireddi et al¹ in 2019, which is characterized by necroptosis, apoptosis and pyroptosis, but cannot be explained by any of them alone.^5,6 In the following text, we provide a brief review of pyroptosis, apoptosis, and necroptosis to help understand the fundamental mechanism of PANoptosis.

Pyroptosis involves classical signaling pathway mediated by cysteinyl aspartate specific proteinase-1 (caspase-1) and non-classical signaling pathway mediated by caspase-4, 5, 11 (human caspase-11 is a murine ortholog of caspase-4/5).⁷ Activation of caspase-1 is mediated by inflammasome, which binds to caspase-1 and cleaves caspase-1 precursor into catalytically active caspase-1. Caspase-1 activates downstream molecules, forming pores in cell envelope and destroys the osmotic pressure balance, resulting in ion imbalance on the envelope, leading to cell swelling, lysis and death.⁸ In addition, activated caspase-1 can also cleave interleukin-1β (IL-1β) and IL-18 precursor, promoting IL-1β and IL-18 release, mediating pyroptosis.⁹ The non-classical pyroptosis inflammasome pathway is also known as the non-caspase-1-dependent cell death, which is mainly mediated by caspase-4, 5, 11. After activation of caspase-4, 5, 11, the cleavage of gasdermin D (GSDMD) causes pyroptosis, or the Pannexin-1 channel is opened under stimulation, the release of adenosine triphosphate (ATP) leads to ion imbalance, and then the pyroptosis process is initiated.¹⁰

There are three ways of apoptosis: endogenous apoptosis, exogenous apoptosis and endoplasmic reticulum stress. Endogenous apoptosis pathway is also known as mitochondrial apoptosis pathway. After stimulation, the outer membrane of mitochondria is permeated, which promotes the release of cytochrome C (cyto-C). Cyto-C activates caspase-9, and then, caspase-3, 6, 7 are activated, leading to cell apoptosis.¹¹ The initiation of mitochondrial apoptosis is regulated by B-cell lymphoma-2 (Bcl-2) family proteins.¹² Exogenous apoptosis pathway is mainly mediated by the interaction of receptors and ligands to transmit apoptosis signals. Receptors bind to caspase-8 precursor through their Fas-associating protein with a novel death domain (FADD) or tumor necrosis factor (TNF) receptor 1 associated via death domain (TRADD), and then activate caspase-8, which further activates caspase-3, 6, 7 to induce apoptosis.^13,14 Endoplasmic reticulum stress apoptosis pathway is a relatively new apoptosis regulatory pathway. When the body experiences endoplasmic reticulum stress due to various factors, the system will respond to endoplasmic reticulum stress through unfolded protein response. The unfolded protein response is activated by three endoplasmic reticulum stress integrated proteins: inositol-requiring enzyme 1 (IRE1), protein kinase R-like endoplasmic reticulum kinase (PERK) and activating transcription factor 6 (ATF6).¹⁵ The interaction between IRE1 and adaptor protein TNF receptor associated factor 2 (TRAF2) leads to the activation of Jun n-terminal kinase (JNK), which promotes the expression of pro-apoptosis protein Bcl-2 associated death promoter (Bad), then inducing cell apoptosis.¹⁶ PERK promotes the phosphorylation of eukaryotic translation initiation factor 2α (eIF2α) and activates the translation of ATF4, which can promote C/EBP-homologous protein (CHOP) expression, an endoplasmic reticulum stress protein.¹⁷ CHOP promotes apoptosis by upregulating pro-apoptosis proteins Bcl-2 associated X protein (Bax) and Bcl-2 antagonist/killer (Bak).

Necroptosis is regulated by receptor-interacting serine/threonine-protein kinase 1 (RIP1), RIP3 and mixed lineage kinase domain-like (MLKL). RIP1 and RIP3 combine to form a complex called necrosome, which can induce RIP3 auto phosphorylation. And then RIP3 phosphorylates MLKL protein, induces the formation of MLKL oligomers and translocation to cell membrane, resulting in rupture and eventually cell destruction.¹⁸

In this review, from the perspective of PANoptosis, we focus on the relationship between necroptosis, apoptosis and pyroptosis, the key molecules in the process of PANoptosis and the formation of PANoptosome, as well as the role of PANoptosis in diseases. Studies have reported that PANoptosis is involved in many diseases, including infectious diseases and tumor diseases.^2,3 We aim to understand the mechanism of PANoptosis and provide a basis for targeted PANoptosis to treat human diseases.

The Interaction Between Pyroptosis, Apoptosis and Necroptosis

For a long time, it has been considered that cell death pathways operate in parallel, without overlap. However, it is clear that pyroptosis, apoptosis and necroptosis are closely linked and can regulate each other.^19,20 In the rat sepsis associated encephalopathy model, treatment of apoptosis inhibitor could suppress pyroptosis and activate necroptosis. Pyroptosis inhibitor could suppress pyroptosis and inhibit apoptosis, but activate necroptosis.²¹ However, both apoptosis and pyroptosis were activated when necroptosis was inhibited.²¹ The differences between pyroptosis, apoptosis and necroptosis, including the mechanisms of membrane damage and subcellular localization, have been widely reported and no more repeat. In the following, we focused on the relationship and regulation among pyroptosis, apoptosis and necroptosis.

One of the first identified bridges between different cell death types is caspase-8, which is an important regulator in necroptosis, apoptosis and pyroptosis.²² Caspases can be divided into inflammatory caspases (caspase-1, 4, 5 and 11) and apoptosis caspases (caspase-3, 6, 7, 8, 9 and −10).^23,24 However, caspase-3 and caspase-8 were found to mediate pyroptosis.^22,25 Caspase-8 is considered to be a molecular switch of cell death, which can promote apoptosis, pyroptosis or necroptosis, respectively.^26,27

GSDMD is also an important molecular regulating pyroptosis, apoptosis and necroptosis. Activation of caspase-3 and caspase-7 inactivates GSDMD by processing GSDMD to produce p20 fragment, which is a form of inactivated GSDMD.²⁸ In the absence of GSDMD, caspase-1 fails to trigger pyroptosis and instead redirects cell fate to apoptosis pathway.²⁹ Studies on macrophage apoptosis have reported that in the absence of GSDMD, caspase-1 activates caspase-3 and caspase-7 to induce apoptosis; conversely, during apoptosis, caspase-3 and caspase-7 specifically prevented pyroptosis by processing GSDMD, indicating a close interaction between pyroptosis and apoptosis.²⁸

RIP3 protein is reported to be the key molecule to regulate cell apoptosis or cell necroptosis. If RIP3 expression is high, cells will go to the necroptosis pathway, while RIP3 expression is low, cells will go to the apoptosis pathway.³⁰ RIP3 can also promote IL-1β release mediated by NLRP3-caspase-1 axis.³¹ These findings indicate that RIP3 regulates the balance of pyroptosis, apoptosis and necroptosis.

MLKL, an important molecule in necroptosis, has also been reported to activate NLRP3 inflammasome.³¹ MLKL inhibitor necrosulfonamide can suppress GSDMD, a key protein of pyroptosis.³² These suggest a cross between pyroptosis and necroptosis. In addition, loss of nod-like receptor (NLR) family CARD domain containing 4 (NLRC4) inflammasome resulted in enhanced MLKL phosphorylation and necroptosis. During P. aeruginosa challenge, in the absence of NLRC4, alternative cell-death molecules such as RIP1 and MLKL were activated, whereas activation of caspase-1, 3, 7, 8 was reduced.³³ Therefore, there are inseparable interaction relationships among necroptosis, apoptosis and pyroptosis. The relationship among necroptosis, apoptosis and pyroptosis is summarized in Figure 1.

Figure 1 The interaction among pyroptosis, apoptosis and necroptosis. FADD mediates the exogenous apoptosis pathway. Bax mediates the endogenous apoptosis pathway. Caspase-7, caspase-9 and caspase-3 are the hubs of the two pathways. Caspase-3 can also mediate pyroptosis through GSDME. RIP1, RIP3 and ZBP1 mediate necroptosis. Caspase-8 is the hub of apoptosis and necroptosis. ZBP1 can also activate inflammasome, which can activate caspase-1/4/5 and induce pyroptosis.

Key Molecules During PANoptosis and the Formation of PANoptosome

Z-DNA-Binding Protein 1 (ZBP1)

ZBP1 is a nucleic acid innate immune sensor that regulates host defense response.³⁴ Activation of ZBP1 triggers necroptosis, apoptosis and pyroptosis (PANoptosis) through activating RIP3, caspase-8 and NLR thermal protein domain associated protein 3 (NLRP3).^35,36 ZBP1 is considered to be a key component of PANoptosome. PANoptosome is mainly composed of receptors (ZBP1, inflammasome), adapters (apoptosis-associated speck-like protein containing a CARD (ASC) and FADD) and catalytic effectors (caspase-1, RIP3, RIP1, and caspase-8).⁵ ZBP1 and its Zα2 domain are required for the activation of NLRP3 and PANoptosis. Loss of ZBP1 and Zα2 domains leads to decreased activation of NLRP3 and pyroptosis, decreased cleavage of caspase-3, caspase-8 and caspase-7 (apoptosis), and decreased MLKL phosphorylation (necroptosis), which means decreased PANoptosis. These results indicate that ZBP1 and its Zα2 domain play a key role in PANoptosis. Moreover, the Zα2 domain of ZBP1 is required to promote the interaction between ZBP1 and RIP3. In the absence of Zα2 domain, the interaction between RIP3 and ZBP1 disappeared and PANoptosis was blocked.^37,38 Caspase-6 is a factor regulating the interaction between RIP3 and ZBP1, and caspase-6 promotes the interaction between RIP3 and ZBP1 by binding to RIP3.^39,40 The role of ZBP1 and its Zα2 domain during influenza A virus and β-coronavirus infection has also been described. The Zα2 domain of ZBP1 can regulate influenza-induced PANoptosis, and deficiency of Zα2 domain abrogated influenza A virus-induced activation of PANoptosis. Moreover, loss of Zα2 domain rescued mice from death caused by ZBP1-driven inflammation.³⁵ Besides, ZBP1 is upregulated in severe patients with corona virus disease-19 (COVID-19) during β-coronavirus infection. ZBP1 drives cytokine storm and death in mice infected with β-coronavirus, and its Zα2 domain is essential for β-coronavirus-induced cell death (PANoptosis). Further studies demonstrated that ZBP1-mediated activation of RIP3-dependent regulated cell death was responsible for β-coronavirus-induced cell death. ZBP1-dependent inflammatory cell death, regulated cell death, and cytokine storm can disrupt the efficacy of interferon (IFN) treatment.⁴¹

RIP1

RIP1 regulation on PANoptosis is required for cell death and inflammatory response.^42,43 Loss of RIP1 can activate RIP3-regulated PANoptosis through caspase-8 and FADD, leading to cell death.^44,45 In disease, deletion of RIP1 abolished Yersinia-induced inflammasome activation/pyroptosis and apoptosis, but enhanced necroptosis. Yersinia induced the assembly of PANoptosome complex (RIP1, caspase-8, FADD, NLRP3, ASC, RIP3), which could regulate all three pathways of PANoptosis.² Transforming growth factor-β (TGF-β)-activated kinase 1 (TAK1) was discovered to be the regulatory switch of PANoptosome. Under the condition of TAK1 deficiency, RIP1 recruited NLRP3 and ASC to form a cell death complex that activates inflammasome, caspase-8 and GSDMD to drive pyroptosis and apoptosis.^1,46 Further studies found that the lack of TAK1 could also induce the activation of RIP1 non-dependent PANoptosis pathway, which was mainly mediated by RIP3-MLKL. Inactivation of TAK1 led to myelodysplasia and severe sepsis-like syndrome driven by the RIP3‑caspase-8 signaling axis, without triggering high expression of RIP1.⁴⁷ RIP1 is also involved in the FADD/caspase-8 axis driving TNF-α and IFN-γ-induced PANoptosis. However, knockdown of RIP3 and caspase-8 or RIP3 and FADD protected cells from death.⁴⁸ Therefore, RIP1 plays a key role in PANoptosis and host defense.

Inflammasome

Inflammasome is one of the important components of PANoptosome, and the activation of inflammasome is crucial for pyroptosis and PANoptosis.⁴⁹ There are five identified inflammasome sensors, including NLRP1, NLRP3, absent in melanoma 2 (AIM2), NLRC4 and Pyrin. The last four have been found to be involved in PANoptosis. NLRP3 activation and GSDMD-mediated pyroptosis play key roles in apoptosis and necroptosis. After NLRP3 inflammasome activation, inflammatory cells die through PANoptosis. Knockdown of NLRP3 or GSDMD led to a decrease in cell death at an early stage, but the incidence of inflammatory cell death increased substantially over time, which was mediated by caspase-8 and RIP3, suggesting that in the absence of NLRP3 or pyroptosis, increased cell death is dependent on caspase-8 and RIP3.⁵⁰ After P. aeruginosa infection, in the absence of NLRC4, alternative cell death molecules such as RIP1 and MLKL are activated, while the activation of caspase-1, 3, 7, 8 is reduced. This suggests the important effect of the NLRC4 in PANoptosis.³³ However, recent studies have reported that caspase-1 cleavage, IL-1β and IL-18 release, and cell death induced by herpes simplex virus 1 (HSV1) infection are AIM2-dependent, not NLRP3 and NLRC4 dependent. AIM2 was found to regulate pyrin and ZBP1 to drive PANoptosome formation, PANoptosis, and provide protection against HSV1 infection. The composition of this multiprotein complex mediated by AIM2 includes AIM2, ZBP1, RIP1, RIP3, caspase-1, ASC, caspase-8, pyrin and FADD.⁵¹ The process of PANoptosis is summarized in Figure 2.

Figure 2 The process of PANoptosis and the formation of PANoptosome. After stimulated, ZBP1, RIP1, RIP3, caspase-1, ASC, NLRP3, FADD and caspase-8 form PANoptosome, mediating PANoptosis. Caspase-6 is a factor regulating the interaction between RIP3 and ZBP1.

PANoptosis and Diseases

PANoptosis in Infectious Diseases

The role of PANoptosis in infectious diseases has been reported in several articles, including bacterial, viral and fungal infections.^52–55 It has been reported that Yersinia can induce assembly of the PANoptosome complex, and RIP1 knockdown can eliminate Yersinia-induced PANoptosis.² Furthermore, macrophages lacking the molecules required for PANoptosis were resistant to cell death induced by B. thailandensis. In contrast to wild-type or pyroptosis deficient macrophages, PANoptosis deficient macrophages failed to restrict cell–cell fusion induced by B. thailandensis.⁵⁶ In addition, when enterococcus faecalis infected macrophage, expression of caspase-1, GSDMD, caspase-3, MLKL, RIP3, NLRP3, IL-1β, IL-18 and caspase-3 and p-MIKL were upregulated in macrophage, which induced different degrees of cell death.⁵⁷ In sepsis associated encephalopathy rat model, PANoptosis occured in rat cortical nerve cells. The upstream mechanism may be that toll-like receptor 9 (TLR9) activates PANoptosis through mitogen-activated protein kinase (MAPK) signaling pathway. Inhibition of TLR9 resulted in suppression of MAPK pathway, which induced suppression of PANoptosis, improved survival rate in sepsis associated encephalopathy rats.²¹

In addition to bacterial infections, PANoptosis is also an effective defense against viral infections.^58,59 HSV1 induced caspase-1 cleavage in an AIM2-dependent manner, driving the formation of PANoptosome, causing PANoptosis, and providing protection against HSV-1 infection.⁵¹ When the host was infected with influenza A virus, ZBP1 could mediate the activation of NLRP3 inflammasome, and combine with RIP3 to induce PANoptosis.³⁵ The role of PANoptosis in coronaviruses is also of wide interest to researchers.^60,61 SARS-CoV-2 virus can cause multiple organ failure, and the failure symptoms may be related to cytokine storm. The strong release of cytokines during COVID-19 may be related to PANoptosis.⁶² During coronavirus infection, ZBP1 is upregulated in critical patients with COVID-19. ZBP1 drives cell death and mice death in β-coronavirus infected mice, and the Zα2 domain of ZBP1 is essential for β-coronavirus induced cell death. ZBP1-dependent cell death can disrupt the efficacy of IFN treatment. Therefore, the combination strategy of blocking ZBP1 and providing IFN therapy may be beneficial to the prognosis of patients.⁴¹ In addition, murine coronavirus mouse hepatitis virus (MHV) infection can activate NLRP3 and PANoptosis.⁵⁰ Together, these data suggest that PANoptosis plays a key role in virus-induced cell damage.

Studies of PANoptosis in fungal infections have also been reported.⁶³ Candida albicans and Aspergillus fumigatus have been reported to activate caspase-1, GSDMD, caspase-8, 3, 7, phosphorylate MLKL, and knock down PANoptosis-related molecules (such as caspase-8, RIP3, caspase-1) can reduce PANoptosis caused by Candida albicans and Aspergillus fumigatus.³⁷ Besides, P. aeruginosa has been reported to induce PANoptosis in murine macrophage. Only the combined deletion of caspase-1, 11, 8 and RIP3 protected mice macrophage from cell death.³³

PANoptosis in Non-Infectious Diseases

The role of PANoptosis in tumors, especially colorectal cancer, has been reported.^3,64–66 According to the results of gene analysis, the expression of PANoptosis-related genes was different between colon cancer samples and normal samples. PANoptosis-related genes were mutated, among which NLRP3 had the highest mutation frequency, and ZBP1, AIM2, NLRP3 and GSDMD had the highest copy number variation. NLRP3, caspase-7, RIP1 and RIP3 were down-regulated in tumor samples. Molecular clustering and prognostic features based on PANoptosis can predict the survival and immune status of colon cancer patients.⁴ Multivariate Cox analysis showed that PANoptosis-related lncRNA SNHG7 was significantly associated with the prognosis of colon adenocarcinoma. Subsequent analysis showed that its expression was related to lymph node metastasis and tumor stage. In addition, susceptibility analysis showed that lncRNA SNHG7 was involved in the drug resistance of colon adenocarcinoma.⁶⁷ Since the occurrence of PANoptosis is reduced in colorectal cancer, activation of PANoptosis may be a strategy to kill cancer cells. Interferon regulatory factor 1 can prevent colorectal cancer by regulating PANoptosis.⁶⁸ Besides, the synergistic effect of cysteine desulfurase nitrogen fixation 1 (NFS1) deletion and oxaliplatin induced PANoptosis by increasing intracellular reactive oxygen species (ROS), which improved the antitumor efficacy of platinum-based chemotherapy in colorectal cancer treatment.⁶⁹ It was also found that the combination of TNF-α and IFN-γ stimulation in multiple colon cancer cell lines could cause the activation of PANoptosis, and the inhibition of Janus kinase (JAK) signaling pathway significantly reduced the cell death caused by TNF-α and IFN-γ, indicating that the cell death induced by TNF-α and IFN-γ is an important mechanism to inhibit tumor growth and kill cancer cells.⁷⁰ The role of PANoptosis in other tumors has also been reported. Most PANoptosis genes are considered as favorable genes for the prognosis of gastric cancer, and the expression level of PANoptosis genes is positively correlated with the survival ability of patients, that is, the lower expression level, the worse survival ability.⁷¹ In addition, the activation of pyroptosis, apoptosis and necroptosis (PANoptosis) promotes cancer cell death, thus preventing adrenocortical carcinoma and melanoma development.^72,73

In addition to tumors, PANoptosis also occurs in the process of acute respiratory distress syndrome (ARDS) and acute lung injury, which is manifested by the formation of ASC-caspase-8-RIP3 complex accompanied by caspase-3, GSDMD, phosphorylated MLKL, ZBP1 increased.^74,75 A study showed that genes associated with PANoptosis were differentially expressed in mice middle cerebral artery ischemia-reperfusion injury and control brain tissue samples.⁷⁶ The occurrence of PANoptosis has also been found in cerebral ischemia or ischemia-reperfusion injury,⁷⁷ indicating that PANoptosis plays an important role in ischemia-reperfusion injury.^78,79 In addition, an in vivo and in vitro experiment found that PANoptosis occurred in retinal neurons under ischemia-reperfusion injury from morphological characteristics to gene level, specifically manifested in significantly increased cleaved caspase-3, NLRP3, cleaved caspase-1 (p20), GSDMD, phosphorylated RIP3 and MLKL, and high expression of pro-apoptotic protein BAX and proinflammatory cytokines IL-1β (p17), IL-18 (p22). However, the expression of these molecules decreased after inhibition of GSDMD or administration of necroptosis inhibitors. When the three pathways of death were intervened simultaneously, the cell survival rate was significantly improved compared with intervention with one death alone.⁸⁰ PANoptosis is also involved in retinal ganglion cell (RGC) death and diabetic retinopathy caused by acute ocular hypertension. Melatonin and Dickkopf-1 can inhibit cell death by inhibiting PANoptosis.^81,82 These studies show that PANoptosis is widely present in the pathological process of various diseases. A deeper understanding of the pathogenesis of PANoptosis will not only provide preliminary experimental clues for the future study of this novel regulatory cell death, but also develop a promising therapeutic target for the treatment of the disease.