PANoptosis: An Inflammatory Programmed Cell Death Pathway

Programmed cell death pathways are activated by the innate immune system in response to microbial infections and other cellular stressors. Pyroptosis, apoptosis, and necroptosis are three such programmed cell death pathways that have been extensively studied and are very well-characterized. Work from the Kanneganti group at St. Jude Children’s Research Hospital has indicated that these three pathways do not always operate in isolation from one another.1 Their work describes a novel inflammatory programmed cell death pathway, PANoptosis, so-called because it involves the collective activation of pyroptosis, apoptosis, and necroptosis.

PANoptotic cell death can be induced by a variety of bacterial and viral pathogens, including L. monocytogenesS. enterica serovar Typhimurium, vesicular stomatitis virus (VSV), and influenza A virus (IAV).2 PANoptosis may also play a role in inflammation in patients with severe COVID-19, which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).3 In this article, we review the mechanism and regulation of this newly described programmed cell death pathway, how coronaviruses trigger programmed cell death, and discuss recent research suggesting a role for PANoptosis in COVID-19-associated inflammation, providing possible opportunities for therapeutic intervention through modulation of this cell death pathway.

PANoptosis is Regulated by the PANoptosome

The master regulator of PANoptosis is a multimeric cytoplasmic protein complex called the PANoptosome that consists of proteins involved in pyroptosis, apoptosis, and necroptosis.1,4 It can include NOD-, LRR-, and pyrin domain-containing protein 3 (NLRP3), apoptosis-associated speck-like protein containing a CARD (ASC), and caspase-1, proteins that function in pyroptosis and the inflammasome (Figure 1). The apoptotic protein caspase-8 and the necroptotic proteins receptor-interacting protein kinase 1 (RIPK1) and RIPK3 can also be incorporated into the PANoptosome. Other protein components can include caspase-6, which acts as a scaffold, Z-DNA binding protein 1 (ZBP1), which acts as an innate immune sensor, and Fas-associated death domain (FADD), which acts as an adaptor. Signaling through the PANoptosome activates the downstream effectors gasdermin D (GSDMD), caspases-3 and -7, and mixed lineage kinase domain-like (MLKL), which execute pyroptosis, apoptosis, and necroptosis, respectively.2-4

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Figure 1. Activation of the ZBP1-dependent PANoptosome triggers pyroptosis, apoptosis, and necroptosis in response to IAV infection. Image adapted from Place, et al., 2021.4

The innate immune sensor ZBP1 is a positive regulator of PANoptosome formation and activation.1,4,5 During IAV infection, ZBP1 recognizes viral ribonucleoproteins and induces formation of the ZBP1-dependent PANoptosome. The ZBP1-dependent PANoptosome consists of ZBP1 (the sensor), RIPK3 and RIPK1 (necroptotic proteins), NLRP3, ASC, and caspase-1 (inflammasome/pyroptotic proteins), caspase-8 (an apoptotic protein), and the scaffold caspase-6 (Figure 1). Formation of this PANoptosome leads to activation of RIPK3, caspase-8, and the NLRP3 inflammasome, leading to PANoptosis.

TGF-β-activated kinase 1 (TAK1), on the other hand, is a negative regulator of PANoptosis. Inhibition of TAK1 coupled with signaling through toll-like receptors (TLRs) or death receptors such as TNF receptor 1 (TNFR1), Fas, TRAIL-R, or DR3 promotes formation of a RIPK1-dependent PANoptosome (Figure 2).4 One bacterium that induces PANoptosis in this way is Yersinia. Pathogenic strains of Yersinia can secrete the effector protein YopJ into macrophages to inhibit TAK1 and inhibitor of NF-κB kinase (IKK).2,4 This leads to activation of PANoptosis, intracellular pathogen clearance, and the release of pro-inflammatory cytokines including IL-1β and IL-18.

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Figure 2. Pathogenic Yersinia strains trigger PANoptosis through inhibition of TAK1, which leads to PANoptosome activation. Image adapted from Place, et al., 2021.4

As mentioned above, IAV, VSV, L. monocytogenes, and S. enterica serovar Typhimurium all induce PANoptotic cell death in macrophages.2 Inhibiting pyroptosis, apoptosis, or necroptosis alone is not sufficient to protect macrophages from pathogen-induced cell death. Only by simultaneously inhibiting all three pathways, such as through deletion of the genes encoding the pyroptotic proteins caspase-1 and caspase-11, the apoptotic protein caspase-8, and the necroptotic protein RIPK3, can macrophages be protected from PANoptotic cell death induced by these pathogens. This demonstrates that all three arms of PANoptosis are engaged by the cell and mediate cell death in response to these pathogens.

Coronaviruses Activate Programmed Cell Death Pathways

The betacoronaviruses SARS-CoV, SARS-CoV-2, Middle East respiratory syndrome coronavirus (MERS-CoV), and murine hepatitis virus (MHV) have been shown to activate programmed cell death pathways (Figure 3).6,7

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Figure 3. Coronaviruses activate multiple programmed cell death pathways. Image adapted from Lee, et al., 2020.6

SARS-CoV, SARS-CoV-2, and MERS-CoV can induce both pyroptosis and apoptosis.6 Pyroptosis induced by these pathogens is accompanied by secretion of the pro-inflammatory cytokine IL-1β, as well as activation of the NLRP3 inflammasome in the case of SARS-CoV and SARS-CoV-2. HCoV-OC43, a coronavirus that can cause the common cold, induces necroptosis in human neural cells. More research is needed to determine whether SARS-CoV, SARS-CoV-2, and MERS-CoV can also induce necroptosis. The mouse coronavirus MHV, however, has been shown to activate all three PANoptotic cell death pathways in murine macrophages, and this PANoptotic cell death is accompanied by the release of the pro-inflammatory cytokines IL-1β, IL-18, IL-6, and TNF.7

TNF-α and IFN-γ Induce PANoptotic Cell Death and Inflammation Resembling COVID-19

Recent work from Karki et al. suggests that PANoptosis may play a role in the inflammatory response in patients with severe COVID-19.3 In mice, co-administration of the pro-inflammatory cytokines TNF-α and IFN-γ causes an increase in mortality, as well as a variety of phenotypes that mirror observations in patients with severe COVID-19, including elevated serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), and ferritin, as well as thrombocytopenia and an increased neutrophil-to-lymphocyte ratio. In cells, TNF-α and IFN-γ induce PANoptosis and cell death. Mice lacking the PANoptosome components RIPK3 and caspase-8 are protected from TNF-α- and IFN-γ-induced mortality. Macrophages from these mice are also protected from TNF-α- and IFN-γ-induced PANoptosis and death. This suggests that PANoptosis is induced by the combination of TNF-α and IFN-γ, the pathology of which has similarities with severe COVID-19.

The authors next sought to elucidate the signaling pathway involved in the induction of PANoptosis by TNF-α and IFN-γ.3 They identified the JAK/STAT1/IRF1 signaling pathway as important for regulation. In this pathway, Janus kinase 2 (JAK2) phosphorylates JAK1, which then activates the transcription factor STAT1 to induce transcription of genes including IFN regulatory factor 1 (Irf1) (Figure 4). The genes Jak2 and Irf1 are upregulated in mouse macrophages treated with TNF-α and IFN-γ, as well as in patients with severe COVID-19. Disrupting this signaling pathway via deletion of Irf1 or Stat1 protects mouse macrophages against TNF-α- and IFN-γ-induced cell death, as well as induction of PANoptosis in the case of Irf1 deletion. Similarly, Stat1-/- mice are protected from TNF-α- and IFN-γ-induced mortality.

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Figure 4. TNF-α and IFN-γ induce JAK/STAT1/IRF1 signaling and PANoptosis. Image adapted from Karki, et al., 2020.3

Karki et al. also demonstrated that nitric oxide (NO) production induced by the JAK/STAT1/IRF1 pathway contributes to TNF-α- and IFN-γ-induced PANoptotic cell death (Figure 4).3Irf1-/- mouse macrophages have reduced expression of inducible nitric oxide synthase (iNOS) and the gene encoding it, Nos2. Consistent with this, both Irf1-/- and Stat1-/- cells have decreased NO production compared with wild-type cells when stimulated by TNF-α and IFN-γ. Disrupting NO production via deletion of Nos2 or treatment with the NO production inhibitors L-NAME or 1400W protects cells against TNF-α- and IFN-γ-induced cell death.

Given that treating mice with a combination of TNF-α and IFN-γ induces symptoms similar to those observed in patients with COVID-19, the authors explored whether blocking TNF-α and IFN-γ signaling would protect against SARS-CoV-2-induced mortality in a mouse model of infection. They found that blocking TNF-α and IFN-γ using a combination of neutralizing antibodies against the two cytokines protects mice against mortality induced by SARS-CoV-2. Neutralizing antibodies against TNF-α and IFN-γ also protect mice against mortality induced by a lethal dose of LPS or a combination of poly I:C priming followed by LPS, which models the severe systemic inflammatory syndrome hemophagocytic lymphohistiocytosis (HLH). These results suggest that TNF-α- and INF-γ-mediated PANoptosis is involved in the pathology of a variety of inflammatory conditions, including COVID-19, and presents new avenues to explore in the treatment of these conditions.

Cayman offers a variety of tools to study of PANoptosis and other programmed cell death pathways, including small molecule inhibitors of PANoptosome components and regulators, other cell death pathways, JAK/STAT signaling, and NO production.

Inhibitors of PANoptosome Components and Regulators​

 

PANoptosome Component/Regulator

Inhibitors

PANoptosome Component/Regulator

Inhibitors

NLRP3 ​ NLRP3iRIPK3/RIP3 KinaseGSK872
MCC950RIPK1/RIP1 Kinase​ ​ (±)-Necrostatin-2
DapansutrileNecrostatin-1
INF39Necrostatin-5
Caspase-1​ Ac-YVAD-CMKGSK2982772
Ac-YVAD-CHOGSK481
VX-765TAK1​(5Z)-7-Oxozeaenol
Caspase-8Ac-IETD-CHO (trifluoroacetate salt)Takinib
Caspase-6Ac-VEID-CHO (trifluoroacetate salt)  

Additional Inhibitors of Pyroptosis, Apoptosis, and Necroptosis​

Target

Inhibitors

Target

Inhibitors

Pan-caspase​ ​ ​ ​ Z-VAD(OMe)-FMKCaspase-3​ Z-DEVD-FMK
Q-VD-OPHAc-DEVD-CMK
Z-VAD(OH)-FMKCaspase-3 Inhibitor VII
EmricasanCaspase-1/3Z-YVAD-CMK (trifluoroacetate salt)
Boc-D-FMKMLKLNecrosulfonamide
Z-Asp-CH2-DCBNecroptosis​Necrostatin-2
Caspase-3/7Ac-DEVD-CHONecrostatin-7
Caspase-3/7 Inhibitor I  

JAK Inhibitors

iNOS Inhibitors

RuxolitinibL-NAME (hydrochloride)
Baricitinib1400W (hydrochloride)
FilgotinibL-NIL (hydrochloride)
TG101348 (Fedratinib)Diphenyleneiodonium (chloride)
See all JAK inhibitorsAMT (hydrochloride)
 Aminoguanidine (hydrochloride)

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