Pyroptosis: Mechanisms mediating cell death and pro-inflammatory cytokine release

Fri, 06/18/2021 - 15:49

Immunohistochemical staining of formalin-fixed tissue of adenocarcinoma of the rectum using Caspase-1 antibody, peroxidase conjugate, and DAB chromogen.

By Victoria Osinski

Pyroptosis is an inflammatory form of programmed cell death characterized by the release of pro-inflammatory cytokines IL-1β and IL-18.1,10 It is a process distinct from apoptosis and necrosis (Table 1) and has been observed in the setting of multiple diseases including, but not limited to, microbial infection10, atherosclerosis and other cardiovascular diseases6, sepsis4, and liver disease5. Of note, cell death is defined as “programmed” when a cell relies on its own intrinsic cellular signaling to initiate the process.2 The canonical form of pyroptosis is defined as caspase-1-dependent, though multiple studies have identified caspase-1-independent pyroptosis pathways. Pyroptosis and its molecular mechanisms are a growing area of interest because they serve as potential novel therapeutic targets for a number of diseases.9

Immunohistochemical analysis of paraffin-embedded human tonsil tissue probed with anti-IL-1 beta/IL-1F2 polyclonal antibody, followed by HRP polymer antibody, stained with DAB, and counterstained with hematoxylin.



Immunohistochemical analysis from paraffin-embedded sections of human tonsil showing IL‑1 beta /IL‑1F2 detected by using Goat Anti-Human IL‑1 beta /IL‑1F2 Polyclonal Antibody (AF-201-NA) followed by incubation with IgG VisUCyte™ HRP Polymer Antibody (VC004). Tissue was then stained with DAB (brown) and counterstained using hematoxylin (blue). Specific staining was localized to cytoplasm in lymphocytes.



Table 1: Differences between apoptosis, necrosis, and pyroptosis.

  Apoptosis Necrosis Pyroptosis





Membrane maintained?




Components of cellular process

Cytoplasmic and nuclear condensation, DNA damage, Formation of apoptotic bodies, Exposure of surface molecules that recruit phagocytosis

Organelle swelling, DNA damage (sometimes)

Caspase-1 activation, DNA damage, Cytokine release

Inflammatory cellular components release?




Caspases involved

Caspase-2, -3, -6, -7, -8, -9 (mouse), -10 (human)

Caspase-1, -3, -11 (mouse); -4, -5 (human)

Based on findings summarized in References 2, 3, 10

Western blot of lysates from monocyte-derived macrophages (MDMs) either untreated or infected with P. gingivalis (Pg), Heat-Inactivated Pg (HI-Pg), outer membrane vesicles (OMVs), or HI-OMVs, and probed from inflammasome components using anti-caspase-1 antibody and anti-IL-1 beta antibody, showing differential induction

Western blot highlighting that P. gingivalis and its outer membrane vesicles (OMVs) differentially induce inflammasome signaling and pyroptosis in human macrophages. Monocyte-derived macrophages (MDM) were infected with viable P. gingivalis (Pg), heat-killed-Pg (HK-Pg), OMVs, or heat-inactivated-OMVs (HI-OMVs) and the activation of inflammasome components in the lysates was measured after 24 h by Western blot using Mouse Anti-Caspase-1 Monoclonal Antibody (NB100-56565) and Goat Anti-IL-1 beta Polyclonal Antibody (AF-401-NA); beta-actin serves as a loading control. Image collected and cropped by CiteAb from the following publication (// .2017.00351/full), licensed under a CC-BY license.

Caspase-1-dependent pyroptosis:

Caspase 1 is induced by the pattern recognition receptors Nucleotide-binding oligomerization domain and leucine-rich repeat receptors (NLRs), including NLRP3, NLRP1b, and NLRC4/CARD12.1 These receptors can recognize and respond to PAMPs and DAMPs originating from bacterial, viral, and host cell components. Following NLR-Caspase 1 activation, the inflammasome is formed. The inflammasome is a multi-protein structure formed by sensors (such as NLRP3), inflammatory caspases, and an inflammasome adaptor protein such as apoptosis-associated speck-like protein containing a carboxy-terminal CARD (ASC) (Table 2).8,9,10

In addition to forming the inflammasome, inflammatory Caspases-1, -4/5, and -11 also activate Gasdermin D, which forms pores in the plasma membrane to release pro-inflammatory cytokines.7,10 This component of pyroptosis, the active release of pro-inflammatory cytokines, is what make it particularly unique relative to other forms of cell death. Pro-inflammatory cytokines IL-1β and IL-18 are first translated into non-active forms, which are then cleaved and activated by Caspase-1.3,7,10 These cytokines can then be released via Gasdermin D-formed pores.

Learn more about PAMPs and DAMPs

Caspase-1-independent pyroptosis:

Gasdermin D can also be activated by Caspase-4,-5, and -11 in a Caspase-1-independent form of pyroptosis. Though, it should be noted that activation of pro-inflammatory IL-1β and IL-18 still rely on Caspase-1 activity in this context.10 Caspase-3 has also been shown to activate Gasdermin E to form a pore for release of DAMPs and cytokines.3

New information concerning the mechanisms driving pyroptosis continues to emerge and it is evident that this is a complex problem. These advances in our understanding of pyroptosis also reveal its importance in human health and provide opportunities to identify new therapeutic targets.

Table 2: Inflammasome components

Sensor molecule Adaptor protein Caspases

NLRP1, -3, -4, -6, -7, -12; NLRC4; AIM2; pyrin; IFI16; RIG-1

ASC, Pycard

Caspase-1, -4/5*, -11*

*Not necessary for inflammasome assembly, but can contribute
Based on findings summarized in References 1, 8, 9

Immunocytochemical analysis of THP-1 human acute monocytic leukemia cells either untreated (bottom) or treated with PMA and LPS (top) and probed with anti-NLRP3/NALP3 antibody followed by NorthernLights 557-conjugated secondary antibody (red).




Immunocytochemical staining of immersion fixed THP‑1 human acute monocytic leukemia cells either treated with PMA and LPS (upper panel) or untreated (lower panel) and probed with Anti-NLRP3/NALP3 Polyclonal Antibody (AF6789), followed by staining with NorthernLights™ 557-conjugated Secondary Antibody (NL010) (red) and counterstained with DAPI (blue). Specific staining was localized to cytoplasm.

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Victoria OsinskiVictoria Osinski, Doctoral Candidate   
University of Virginia
Victoria studies cellular mechanisms regulating vascular growth during peripheral artery disease and obesity.


  1. Bergsbaken, T., Fink, S. L., & Cookson, B. T. (2009). Pyroptosis: host cell death and inflammationNature reviews. Microbiology.
  2. Fink, S. L., & Cookson, B. T. (2005). Apoptosis, pyroptosis, and necrosis: mechanistic description of dead and dying eukaryotic cellsInfection and immunity.
  3. Frank, D., & Vince, J. E. (2019). Pyroptosis versus necroptosis: similarities, differences, and crosstalkCell death and differentiation.
  4. Gao, Y. L., Zhai, J. H., & Chai, Y. F. (2018). Recent Advances in the Molecular Mechanisms Underlying Pyroptosis in SepsisMediators of inflammation.
  5. Guo, H., Xie, M., Zhou, C., & Zheng, M. (2019). The relevance of pyroptosis in the pathogenesis of liver diseasesLife sciences.
  6. Jia, C., Chen, H., Zhang, J., Zhou, K., Zhuge, Y., Niu, C., Qiu, J., Rong, X., Shi, Z., Xiao, J., Shi, Y., & Chu, M. (2019). Role of pyroptosis in cardiovascular diseases. International immunopharmacology.
  7. Kesavardhana, S., & Kanneganti, T. D. (2017). Mechanisms governing inflammasome activation, assembly and pyroptosis inductionInternational immunology.
  8. Latz, E., Xiao, T. S., & Stutz, A. (2013). Activation and regulation of the inflammasomesNature reviews. Immunology.
  9. Malik, A., & Kanneganti, T. D. (2017). Inflammasome activation and assembly at a glanceJournal of cell science.
  10. Man, S. M., Karki, R., & Kanneganti, T. D. (2017). Molecular mechanisms and functions of pyroptosis, inflammatory caspases and inflammasomes in infectious diseasesImmunological reviews.
  11. Swanson, K. V., Deng, M., & Ting, J. P. (2019). The NLRP3 inflammasome: molecular activation and regulation to therapeuticsNature reviews. Immunology.

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