PANoptosis is an inflammatory PCD pathway activated by specific triggers and regulated by the PANoptosome complex, which integrates key features of pyroptosis, apoptosis, and/or necroptosis. This is also the source of the "P," "A," and "N" in the term PANoptosis, but it cannot be solely represented by any one of these death modalities^[1].

In 2019, American scholar Malireddi named this novel form of cell death, characterized by features of pyroptosis, apoptosis, and necroptosis, as PANoptosis, proposing that the innate immune sensors ZBP1 and the TAK1 kinase play crucial roles in the regulation of PANoptosome complex assembly^[2].

Cell death is crucial for normal organismal development and resistance to pathogen invasion, serving as a host defense mechanism against pathogenic infections. Studies have found that PANoptosis occurs following influenza A virus (IAV) infection and the loss of TAK1 activity. Additionally, various pathogens such as viruses, bacteria, fungi, and even parasites, as well as other non-infectious factors like cytokines in tumors, can trigger PANoptosis in host cells (Table 1)^[3].

Multiple proteins can form multiprotein complexes that regulate programmed cell death (PCD), which can be classified into three categories based on interactions among various protein domains: sensing domains, assembly domains, and catalytic domains^[2].

PANoptosome

PANoptosis is regulated by a cascade of upstream receptors and molecular signals, which assemble into a polymeric complex known as the PANoptosome. The PANoptosome and its upstream receptors not only serve as an activation platform for downstream molecules but also act as the "master switch" for initiating the three PCD pathways^[4][5]. The PANoptosome functions as a molecular scaffold, allowing key molecules involved in pyroptosis, apoptosis, and/or necroptosis to couple and interact^[1]. Upon sensing pathogen components, sensor proteins mediate the assembly of RIPK3, RIPK1, CASP8, FADD, and other proteins into the PANoptosome complex, thereby inducing PANoptosis^[2][6].

The proteins that constitute the PANoptosome can generally be divided into three categories: (1) ZBP1 and NLRP3 as putative PAMP and DAMP sensors, (2) ASC and FADD as adaptors, and (3) RIPK1, RIPK3, CASP1, and CASP8 as catalytic effectors^[2].

How do upstream receptors specifically recognize pathogenic microbial infections and how do these components interact? The specific mechanisms remain unknown. To date, three upstream molecules have been identified to play a clear role in PANoptosis: ZBP1, RIPK1, and AIM2. These molecules can sense specific stimuli and trigger the assembly of the PANoptosome, forming three types of PANoptosomes with different sensors and regulatory factors: ZBP1-PANoptosomes, AIM2-PANoptosomes, and RIPK1-PANoptosomes. Furthermore, the activation of the PANoptosome can also be inhibited by TAK1, PSTPIP2, SHARPIN, HOIP, HOIL-1, and A20^[6].

Detection Methods and Indicators for PANoptosis

1.Observation of Cell Morphology: Pyroptosis causes cytoplasmic swelling and membrane rupture; key morphological features of apoptosis include chromatin condensation, DNA fragmentation, membrane blebbing, cell shrinkage, and the formation of apoptotic bodies.

2.Detection of Key Proteins in Different PCD Pathways: Pyroptosis-related: Caspase-1, Caspase-3, Gasdermins, AIM2/Pyrin/NLRP3, etc; Apoptosis-related: Caspase-3, Caspase-7, Caspase-8, PARP, Bax/Bcl, etc；Necroptosis-related: MLKL, RIPK1, RIPK3, ZBP1, etc.

3.Other Indicator Tests: Annexin V-FITC and PI double staining; TUNEL assay; JC-1 detection; ELISA for measuring the release of inflammatory factors; Techniques such as Western blotting and flow cytometry to assess the expression of the inflammasome NLRP3 and the activation of Caspase-1.

Literature case (IF=39.3 )

Recently, Jin-Fei Lin and colleagues discovered that phosphorylated NFS1 can weaken the sensitivity of colorectal cancer to oxaliplatin by preventing PANoptosis^[11].

The authors used a CRISPR-Cas9 library based on metabolic enzyme genes and found that the loss of NFS1 significantly enhanced CRC cell sensitivity to oxaliplatin. In vitro and in vivo results indicated that NFS1 deficiency cooperates with oxaliplatin to induce PANoptosis by increasing intracellular reactive oxygen species (ROS) levels.

To investigate the type of cell death occurring, various inhibitors of common cell death pathways were used in conjunction with microscopy to observe cell morphology, YP1/PI staining, and flow cytometry to detect cell death. The results showed that the pyroptosis (GSDME) inhibitor disulfiram and the autophagy inhibitor 3-methyladenine had no significant effect, while the apoptosis inhibitor Z-VAD-FMK , necroptosis inhibitor necrostatin-1 , ferroptosis inhibitor ferrostatin-1 , and pyroptosis (GSDME) inhibitor Ac-DMPD/DMLD-CMK partially reversed (but did not completely restore) the decrease in cell viability and increased cytotoxicity caused by NFS1 deficiency under oxaliplatin treatment (Figure 5).

To further confirm the occurrence of PANoptosis, the authors found that NFS1 knockdown combined with oxaliplatintreatment significantly increased the number of dead cells, including YP1-positive cells indicative of apoptosis or necroptosis, and PI-positive cells indicative of necroptosis, pyroptosis, or ferroptosis (Figure 6: left panel, a-b and right panel a-b).

Analysis of HCT116 cells also showed that the number of large bubbles emerging from the plasma membrane in the combination group was significantly higher than that in the negative control group, NFS1 knockdown group, and oxaliplatin treatment group, indicating the occurrence of pyroptosis (Figure 6: left panel, a). Furthermore, oxaliplatin treatment after NFS1 depletion quantitatively increased the number of early/late apoptotic cells and significantly reduced the number of viable cells (Figure 6: left panel, c-d and right panel c-d). Additionally, the levels of lipid ROS detected in the NFS1 knockdown and oxaliplatin treatment groups were higher than in the control group. This increase was more pronounced in the combination group, indicating the occurrence of ferroptosis (Figure 6: left panel, e and right panel e-f). Therefore, these data suggest that NFS1 deficiency combined with oxaliplatin contributes to the activation of PANoptosis.

Moreover, the authors evaluated the specific mechanism by which NFS1 deficiency induces PANoptosis under oxaliplatin treatment. In summary, oxaliplatin-mediated oxidative stress can enhance the serine phosphorylation level of NFS1, and NFS1 prevents the activation of PANoptosis under oxaliplatin treatment in a phosphorylation-dependent manner at S293.