Identification of the PANoptosome: A Molecular Platform Triggering Pyroptosis, Apoptosis, and Necroptosis (PANoptosis) - PubMed (original) (raw)

doi: 10.3389/fcimb.2020.00237. eCollection 2020.

Min Zheng 1, Sannula Kesavardhana 1, Rajendra Karki 1, R K Subbarao Malireddi 1, Balaji Banoth 1, David E Place 1, Benoit Briard 1, Bhesh Raj Sharma 1, Shraddha Tuladhar 1, Parimal Samir 1, Amanda Burton 1, Thirumala-Devi Kanneganti 1

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Identification of the PANoptosome: A Molecular Platform Triggering Pyroptosis, Apoptosis, and Necroptosis (PANoptosis)

Shelbi Christgen et al. Front Cell Infect Microbiol. 2020.

Abstract

Programmed cell death plays crucial roles in organismal development and host defense. Recent studies have highlighted mechanistic overlaps and extensive, multifaceted crosstalk between pyroptosis, apoptosis, and necroptosis, three programmed cell death pathways traditionally considered autonomous. The growing body of evidence, in conjunction with the identification of molecules controlling the concomitant activation of all three pathways by pathological triggers, has led to the development of the concept of PANoptosis. During PANoptosis, inflammatory cell death occurs through the collective activation of pyroptosis, apoptosis, and necroptosis, which can circumvent pathogen-mediated inhibition of individual death pathways. Many of the molecular details of this emerging pathway are unclear. Here, we describe the activation of PANoptosis by bacterial and viral triggers and report protein interactions that reveal the formation of a PANoptosome complex. Infection of macrophages with influenza A virus, vesicular stomatitis virus, Listeria monocytogenes, or Salmonella enterica serovar Typhimurium resulted in robust cell death and the hallmarks of PANoptosis activation. Combined deletion of the PANoptotic components caspase-1 (CASP1), CASP11, receptor-interacting serine/threonine-protein kinase 3 (RIPK3), and CASP8 largely protected macrophages from cell death induced by these pathogens, while deletion of individual components provided reduced or no protection. Further, molecules from the pyroptotic, apoptotic, and necroptotic cell death pathways interacted to form a single molecular complex that we have termed the PANoptosome. Overall, our study identifies pathogens capable of activating PANoptosis and the formation of a PANoptosome complex.

Keywords: ASC; NLRP3; PANoptosis; PANoptosome; RIPK1; RIPK3; caspase-1; caspase-8.

Copyright © 2020 Christgen, Zheng, Kesavardhana, Karki, Malireddi, Banoth, Place, Briard, Sharma, Tuladhar, Samir, Burton and Kanneganti.

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Figures

Figure 1

Figure 1

Loss of PANoptotic molecules prevents infection-induced cell death. Cell death analysis of BMDMs lacking different components of pyroptosis, apoptosis, or necroptosis. (A) Representative cell death images with the red mask indicating dead cells and (B) quantification of cell death over time in BMDMs after S. Typhimurium, L. monocytogenes, IAV, and VSV infection.

Figure 2

Figure 2

Bacterial and viral infections activate PANoptosis in vitro. Western blot analysis of PANoptosis activation markers after (A) S. Typhimurium, (B) L. monocytogenes, (C) IAV, and (D) VSV infection. Activation of pyroptosis was measured by immunoblotting of cleaved CASP1 (p20) and activated GSDMD (p20/p30). Activation of apoptosis was measured by immunoblotting of active CASP8 (p18), active CASP7 (p20), and active CASP3 (p17/19). Activation of necroptosis was measured by phosphorylation of MLKL (pMLKL). Red asterisks denote a non-specific band. (E) Confocal imaging of PANoptosis activation in wild-type BMDMs following S. Typhimurium infection. ASC speck formation (white triangle), cleavage of CASP3 (red triangle), and pMLKL localization to the membrane (purple triangle) were used as readouts of PANoptosis. Scale bar, 10 μm.

Figure 3

Figure 3

PANoptotic molecules directly interact. (A) Co-immunoprecipitation of NLRP3 expressed in HEK293T cells with ZBP1, RIPK3, and RIPK1 individually. (B) Co-immunoprecipitation of the PANoptosome complex in HEK293T cells expressing CASP8, ASC, RIPK1, NLRP3, and ZBP1 with or without RIPK3. Red asterisks denote a non-specific band.

Figure 4

Figure 4

Inhibition of TAK1 promotes PANoptosis and PANoptosome formation. (A) Representative cell death images of BMDMs lacking different components of pyroptosis, apoptosis, or necroptosis after LPS priming and inhibition of TAK1. (B) Western blot analysis of PANoptosis activation after LPS priming and TAK1 inhibition. (C) Co-immunoprecipitation of PANoptosome components from primary BMDMs after TAK1 and caspase inhibition. Red asterisks denote a non-specific band.

Figure 5

Figure 5

Graphical representation of a PANoptosome. Schematic summary of a representative PANoptosome formed after IAV infection. Domains hypothesized to be crucial for mediating PANoptosome formation are highlighted.

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