GSDMD breaches both mitochondrial membranes before the plasma membrane: mitochondrial damage accelerates and amplifies pyroptosis

Pyroptosis is known to be accompanied by mitochondrial damage, but is that damage a consequence or a cause? The conclusion of this paper is that the N-terminal pore-forming fragment of GSDMD (GSDMD-NT) permeabilizes both the outer and inner mitochondrial membranes before causing plasma membrane rupture. Mitochondrial contents, including mtDNA and intermembrane-space proteins, are released into the cytosol and, in turn, accelerate and amplify pyroptosis, IL-1β release, and inflammation. All of this depends on cardiolipin on the outer mitochondrial membrane—a phospholipid found mainly in the inner mitochondrial membrane and bacterial membranes—and is independent of the BAX/BAK-mediated mitochondrial permeabilization that occurs during apoptosis.

Step 1: Mitochondria are damaged before the plasma membrane during pyroptosis (Figures 1–2). The authors activate canonical (LPS + nigericin) or noncanonical (cytosolic LPS) inflammasomes in immortalized mouse macrophages (iBMDMs). They find that as soon as GSDMD is cleaved, mitochondria show loss of membrane potential, increased reactive oxygen species, decreased oxidative phosphorylation, reduced numbers, and mitophagy, all before plasma membrane damage. Fluorescently tagged GSDMD-NT translocates to mitochondria early in pyroptosis. These results indicate that mitochondrial damage is an early event in pyroptosis, rather than a secondary phenomenon following cell rupture.

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Step 2: Ruling out apoptotic mitochondrial permeabilization (Figure 3). Using BAX/BAK double-knockout cells and treatment with inhibitors of mPTP (the mitochondrial permeability transition pore), the authors find that pyroptotic mitochondrial damage is unaffected. Moreover, whereas apoptosis permeabilizes only the outer membrane (MOMP), pyroptosis permeabilizes both the outer and inner mitochondrial membranes, allowing matrix contents to leak out as well. These results indicate that pyroptotic mitochondrial damage is caused directly by GSDMD-NT and does not proceed through the apoptotic BAX/BAK pathway.

Step 3: Cardiolipin anchors GSDMD-NT-mediated mitochondrial damage (Figures 4–6). GSDMD-NT has a higher affinity for cardiolipin than for plasma membrane phospholipids. The authors incubate recombinant GSDMD-NT with isolated mitochondria in vitro and directly observe permeabilization of both membranes. In cells, knocking out the cardiolipin synthase CRLS1 or the transporter PLSCR3, which flips cardiolipin from the inner to the outer membrane, almost completely eliminates mitochondrial damage. These results indicate that GSDMD-NT permeabilizes mitochondria by binding cardiolipin on the outer membrane; the presence and localization of cardiolipin determine whether damage can occur.

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Step 4: The functional consequences of mitochondrial damage (Figures 5–7). In Crls1- or Plscr3-knockout macrophages, both pyroptosis and IL-1β release induced by canonical and noncanonical inflammasomes are markedly inhibited. One mechanism is that permeabilized mitochondria release the intermembrane-space exoribonuclease PNPT1 into the cytosol, causing global mRNA degradation and amplifying pyroptosis. In vivo, the authors use Plscr3-knockout J774 mouse macrophage tumors in a transplantation model and find impaired antitumor immunity induced by tumor-cell pyroptosis and faster tumor growth. This dependence also extends to GSDMA- and GSDME-mediated pyroptosis. These results indicate that mitochondrial damage is required for pyroptosis to proceed fully and for inflammatory amplification, and affects antitumor immunity in vivo.

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1. Mitochondrial readouts during pyroptosis are not merely bystander indicators: membrane potential, reactive oxygen species, and mtDNA release in turn determine the intensity of pyroptosis. Monitoring them is equivalent to monitoring an upstream amplifier of pyroptosis.
2. BAX/BAK double knockout cannot be used to show that “mitochondria do not participate in pyroptosis”: pyroptotic mitochondrial damage proceeds through the GSDMD-NT–cardiolipin pathway, in which neither BAX/BAK nor mPTP participates. The two mechanisms need to be distinguished using different genetic tools.
3. CRLS1/PLSCR3 are genetic handles for modulating the intensity of pyroptosis: when a control is needed that “weakens only the mitochondrial amplification loop without affecting GSDMD cleavage,” these two knockouts provide a cleaner approach than global inhibitors.

• Further reading | Gasdermin permeabilizes mitochondria to amplify caspase-3 (Rogers 2019, Nat Commun): Rogers 2019 demonstrates that gasdermin-N permeabilizes mitochondria and amplifies caspase-3 through the apoptosome during apoptosis. This paper demonstrates that GSDMD-NT permeabilizes both mitochondrial membranes and amplifies pyroptosis itself in a cardiolipin-dependent manner—the mechanism of “gasdermin attacking mitochondria” serves two cell-death pathways.
• Further reading | Sublytic GSDME-mediated IL-1β release (Zhou & Abbott 2021, Cell Rep): Together, the two papers refine our understanding of “how gasdermin pores quantitatively control IL-1β release.” This paper demonstrates that GSDMD pores amplify release through mitochondrial damage, whereas Zhou & Abbott demonstrate that GSDME pores can permit IL-1β release at a sublytic level without lysing the cell.

(The cross-paper comparisons above are a synthesized interpretation, rather than direct conclusions of either original paper.)