Single-cell analysis of pyroptosis dynamics reveals conserved GSDMD-mediated subcellular events that precede plasma membrane rupture

TLDR

Pyroptosis is an inflammasome‑dependent, caspase‑activated form of necrosis that releases IL‑1β and IL‑18, with gasdermin D pore formation, yet the subcellular events preceding cell lysis remain poorly defined. This study aims to trigger primary macrophages to undergo pyroptosis via three inflammasome types and record their dynamics, thereby establishing a dynamic framework and chronological sequence of GSDMD‑mediated subcellular events. The authors used high‑resolution live‑cell spinning‑disk confocal laser microscopy to monitor primary macrophages undergoing pyroptosis from the three inflammasome types. Quantitative single‑cell analysis revealed that pyroptotic disintegration begins with opening of GSDMD‑dependent ion channels or pores more restrictive than previously proposed, followed by osmotic swelling, organelle commitment, and then sudden plasma membrane rupture with full permeability to intracellular proteins.

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.