Abstract
Pyroptosis is rapidly emerging as a mechanism of anti-microbial host defense, and of extracellular release of the inflammasome-dependent cytokines interleukin (IL)-1β and IL-18, which contributes to autoinflammatory pathology. Caspases 1, 4, 5 and 11 trigger this regulated form of necrosis by cleaving the pyroptosis effector gasdermin D (GSDMD), causing its pore-forming amino-terminal domain to oligomerize and perforate the plasma membrane. However, the subcellular events that precede pyroptotic cell lysis are ill defined. In this study, we triggered primary macrophages to undergo pyroptosis from three inflammasome types and recorded their dynamics and morphology using high-resolution live-cell spinning disk confocal laser microscopy. Based on quantitative analysis of single-cell subcellular events, we propose a model of pyroptotic cell disintegration that is initiated by opening of GSDMD-dependent ion channels or pores that are more restrictive than recently proposed GSDMD pores, followed by osmotic cell swelling, commitment of mitochondria and other membrane-bound organelles prior to sudden rupture of the plasma membrane and full permeability to intracellular proteins. This study provides a dynamic framework for understanding cellular changes that occur during pyroptosis, and charts a chronological sequence of GSDMD-mediated subcellular events that define pyroptotic cell death at the single-cell level.
Highlights
Pyroptosis is a lytic form of regulated cell death that is induced by inflammatory caspases 1, 4, 5 and 11 [1, 2]
C57BL/6J mouse bone marrow-derived macrophages (BMDMs) that express a functional NLRP1b allele (B6Nlrp1b+) can be induced to undergo caspase-1-dependent pyroptosis when stimulated with Bacillus anthracis lethal toxin (LeTx) [13]
Our singlecell analysis of pyroptosis kinetics revealed that pyroptosis is characterized by ionic fluxing and cell swelling that is accompanied by mitochondrial depolarization and lysosome leakage well before cells lost their plasma membrane integrity concomitant with evidence of late-stage nuclear condensation
Summary
Pyroptosis is a lytic form of regulated cell death that is induced by inflammatory caspases 1, 4, 5 and 11 [1, 2]. VIB Bioimaging Core, VIB, Ghent B-9000, Belgium 4 Present address: Janssen Immunosciences, World Without Disease. Unlike for IL-1β and IL-18, each of the aforementioned inflammatory caspases can induce pyroptosis directly by cleaving gasdermin D (GSDMD) at the central linker peptide, which separates the pore-forming amino-terminal domain (GSDMDN) from the inhibitory carboxy-terminal (GSDMDC) domain [5,6,7,8]. This cleavage event causes GSDMDN to oligomerize and insert in the plasma membrane, giving rise to rapid cell lysis
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