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. Pyrodeath, apoptosis and necrosis are three programmed cell death pathways, which have been widely studied and have good characteristics. The work of Kanneganti's group at St. Jude Children's Research Hospital has shown that these three pathways do not always operate in isolation from each other and that their work describes a new inflammatory programmed cell death pathway, PAN apoptosis. PAN apoptosis is called PAN apoptosis because it involves a series of collective behaviors of cell death, apoptosis, and programmed necrosis.
PANoptosis is Regulated by the PANoptosome
The main regulator of PAN cell apoptosis is a polymeric somatomeric protein complex called PAN apoptosome, which consists of proteins involved in death, apoptosis and programmed necrosis. 1,4 It may include NOD-, LRR-, and pyridine-containing domain protein 3 (NLRP3), apoptosis-associated spot-like protein (ASC) containing CARD, and apoptotic proteases, proteins that act in death and inflammatory bodies (FIG. 1). Apoptotic proteins caspase 8 antigen and necrotic protein receptor interaction protein kinase 1 (RIPK1) and RIPK3 can also be incorporated into PAN apoptotic bodies. Other protein components include cysteamine protease protein, which acts as a scaffold, Z-DNA binding protein 1 (ZBP1), which acts as an innate immune sensor, and fatty acid synthase associated death domain (FADD), which acts as an adapter. Downstream effector gasdermin D (GSDMD), caspase -3 and -7, and mixed lineage kinase domain-like (MLKL) are activated through the signaling pathway of PAN apoptotic bodies to perform cell death, apoptosis, and programmed necrosis, respectively.
Figure 1. Activation of the ZBP1-dependent PANoptosome triggers pyroptosis, apoptosis, and necroptosis in response to IAV infection.
Natural immune sensor ZBP1 is a positive regulator of the formation and activation of PAN apoptotic bodies. 1,4,5 During IAV infection, ZBP1 recognizes viral ribonucleoproteins and induces the formation of ZBP1-dependent ubiquitones. The ZBP1-dependent panthenosome consists of ZBP1(sensor), RIPK3 and RIPK1(necrotic protein), NLRP3, ASC, apoptotic protease-1 (inflammasome/necrotic protein), apoptotic protease-8 (apoptotic protein), and scaffold apoptotic protease-6 (FIG. 1). The formation of this PAN apoptotic body leads to the activation of RIPK3, apoptotic protease-8 and NLRP3 inflammasome, leading to PAN apoptosis.
Tgf-β-activated kinase 1 (TAK1) was a negative regulator of PAN cell apoptosis. Inhibition of TAK1 is coupled to signals from Toll-like receptors (TLRs) or death receptors such as TNF receptor 1 (TNFR1), Fas, TRAIL-R, or DR3, promoting the formation of RIPK1-dependent PAN apoptotic bodies (Figure 2) A bacterium that induces panpanism in this way is Yersinia. Pathogenic strains of Yersinia can secrete effector protein YopJ into macrophages to inhibit TAK1 and NF-κB kinase inhibitors (IKK). 2,4 This leads to activation of PAN apoptosis, clearance of intracellular pathogens, and release of pro-inflammatory cytokines including IL-1β and IL-18.
Figure 2. Pathogenic Yersinia strains trigger PANoptosis through inhibition of TAK1, which leads to PANoptosome activation.
As noted above, IAV, VSV, L. monocytogenes, and enterostreptococcal serotype typhimurium all induce PAN cell death in macrophages. Inhibition of death, apoptosis, or necrosis alone is not sufficient to protect macrophages from pathogen induced cell death. Only by inhibiting all three pathways simultaneously, for example by deleting genes encoding apoptotic proteinase-1 and apoptotic proteinase-11, apoptotic proteinase-8, and the necrosis protein RIPK3, can macrophages be protected from PAN cell death induced by these pathogens. This suggests that all three branches of PAN apoptosis are involved by cells and mediate cell death in response to these pathogens.
Coronaviruses Activate Programmed Cell Death Pathways
Japanese coronavirus-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)
Figure 3. Coronaviruses activate multiple programmed cell death pathways
SARS-CoV, SARs-CoV-2, and MERS-CoV all cause death and apoptosis In the case of SARS-CoV and SARS-CoV-2, these pathogene-induced death is accompanied by secretion of the pro-inflammatory cytokine IL-1β and activation of the NLRP3 inflammasome. HCoV-OC43 is a coronavirus that causes the common cold and can induce nerve cell necrosis in humans. More research is needed to determine whether SARS-CoV, SARS-CoV-2 and MERS-CoV can also induce necrosis. However, mouse coronavirus MHV has been shown to activate all three photogenic cell death pathways in mouse macrophages, and this photogenic 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 research by Karki et al. suggests that PAN apoptosis may play a role in the inflammatory response in patients with severe COVID-19 19.3. In mice, the combination of pro-inflammatory cytokines TNF-α and IFN-γ resulted in increased mortality and reflected various phenotypes observed in patients with severe COVID-19, including elevated serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), and ferritin levels. There are also thrombocytopenia and an increased ratio of neutrophils to lymphocytes. In cells, TNF-α and IFN-γ induced global apoptosis and cell death. Mice lacking the PAN apoptotic body components RIPK3 and apoptotic protease-8 were protected from TNF-α- and IFN-γ- induced death. Macrophages from these mice were also protected from TNF-α- and IFN-γ- induced global vision and death. This suggests that PAN apoptosis is induced by a combination of TNF-α and IFN-γ, and its pathology is similar to that of severe COVID-19.
The authors then attempted to elucidate the TNF-α and IFN-gam.3 induced panpanism signaling pathways and found that the JAK/STAT1/IRF1 signaling pathway is important for regulation. In this pathway, Janus kinase 2 (JAK2) phosphorylates JAK1 and then activates the transcription factor STAT1, inducing transcription of genes including IFN regulatory factor 1 (Irf1) (Figure 4). JAK2 and Irf1 genes are upregulated in TNF-α and IFN-γ treated mouse macrophages, as well as in severe COVID-19 patients. Disruption of this signaling pathway by deletion of Irf1 or Stat1 protects mouse macrophages from TNF-α- and IFN-γ- induced cell death, as well as induced holopsia deletion in the absence of Irf1. Similarly, Stat1-/- mice were protected from TNF- and IFN-γ- induced death.
Figure 4. TNF-α and IFN-γ induce JAK/STAT1/IRF1 signaling and PANoptosis
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 PAN cell death (Figure 4). 3 The expression of inducible nitric oxide synthase (iNOS) and its encoding gene Nos2 can be decreased in IRF1 -/- mouse macrophages. Consistent with this, both Irf1-/- and Stat1-/- cells reduced NO production when stimulated by TNF-α and IFN-γ compared with wild-type cells. Interfering with NO production by deleting Nos2 or using the no-production inhibitors L-NAME or 1400W protects cells from TNF-α- and IFN-γ- induced cell death.
Given that combined treatment with TNF-α and IFN-γ induces symptoms similar to those seen in patients with COVID-19, we explored whether blocking the TNF-α and IFN-γ signaling pathways prevents SARS-CoV-2-induced death in a mouse model of infection. They found that using a combination of neutralizing antibodies against these two cytokines to block TNF-α and IFN-γ protected mice from death caused by SARS-CoV-2. Neutralizing antibodies against TNF-α and IFN-γ also protected mice from death induced by the LPS combination after lethal doses of LPS or poly I:C initiation, which mimicked the severe systemic inflammatory syndrome hemophagocytohistiocytosis (HLH). These results suggest that TNF-α- and INF-γ- mediated pandemic-loss is involved in the pathology of many inflammatory diseases, including COVID-19, and provide new avenues for the treatment of these diseases.
Aladdin provides a variety of tools to study PAN apoptosis and other programmed cell death pathways, including small molecule inhibitors of PAN apoptotic body components and regulators, other cell death pathways, JAK/STAT signaling, and induced nitric oxide synthase production.
Inhibitors of PANoptosome Components and Regulators | |||
PANoptosome Component/Regulator | Inhibitors | PANoptosome Component/Regulator | Inhibitors |
NLRP3 | NLRP3i | RIPK3/RIP3 Kinase | GSK872 |
MCC950 | RIPK1/RIP1 Kinase | (±)-Necrostatin-2 | |
Dapansutrile | Necrostatin-1 | ||
INF39 | Necrostatin-5 | ||
Caspase-1 | Ac-YVAD-CMK | GSK2982772 | |
Ac-YVAD-CHO | GSK481 | ||
VX-765 | TAK1 | (5Z)-7-Oxozeaenol | |
Caspase-8 | Ac-IETD-CHO (trifluoroacetate salt) | Takinib | |
Caspase-6 | Ac-VEID-CHO (trifluoroacetate salt) |
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Additional Inhibitors of Pyroptosis, Apoptosis, and Necroptosis | |||
Target | Inhibitors | Target | Inhibitors |
Pan-caspase | Z-VAD(OMe)-FMK | Caspase-3 | Z-DEVD-FMK |
Q-VD-OPH | Ac-DEVD-CMK | ||
Z-VAD(OH)-FMK | Caspase-3 Inhibitor VII | ||
Emricasan | Caspase-1/3 | Z-YVAD-CMK (trifluoroacetate salt) | |
Boc-D-FMK | MLKL | Necrosulfonamide | |
Z-Asp-CH2-DCB | Necroptosis | Necrostatin-2 | |
Caspase-3/7 | Ac-DEVD-CHO | Necrostatin-7 | |
Caspase-3/7 Inhibitor I |
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JAK Inhibitors | iNOS Inhibitors |
Ruxolitinib | L-NAME (hydrochloride) |
Baricitinib | 1400W (hydrochloride) |
Filgotinib | L-NIL (hydrochloride) |
TG101348 (Fedratinib) | Diphenyleneiodonium (chloride) |
See all JAK inhibitors | AMT (hydrochloride) |
| Aminoguanidine (hydrochloride) |