Gasdermin D Mediates a Mitochondrial Cell Death Pathway That Contributes to Pyroptosis.

Abstract

Pyroptosis is an inflammatory cell death program triggered in innate immune cells by intracellular infections and sterile danger signals. The presence of intracellular DAMPs and PAMPs leads to inflammasome formation, inflammatory caspase activation and the cleavage of gasdermin D (GSDMD) into N-terminal (NT) and C-terminal fragments. GSDMD-NT forms pores on the plasma membrane, which release pro-inflammatory cytokines and cause cell death. We previously identified specific phospholipids to which GSDMD-NT binds, suggesting where pores may form during pyroptosis. Surprisingly, GSDMD-NT binds to and permeabilizes membranes containing cardiolipin, a phospholipid predominantly found on the inner mitochondrial membrane (IMM) and bacterial membranes. Studies suggest that cardiolipin can translocate to the outer mitochondrial membrane (OMM) through poorly defined mechanisms including mitochondrial scramblase PLS3, potentially becoming accessible to cytosolic GSDMD-NT. However, it remains unclear whether GSDMD-NT pores form on mitochondria, which may contribute to pyroptosis. Here, we demonstrate that GSDMD-NT permeabilizes mitochondria, leading to the release of mitochondrial proteins and DNA. Furthermore, GSDMD-NT permeabilization increases mitochondrial ROS production and reduces mitochondrial trans-membrane potential (MMP). In THP-1 cells undergoing NLRP3-mediated canonical pyroptosis GSDMD-NT localizes to mitochondria and releases mitochondrial proteins into the cytosol prior to permeabilization of the plasma membrane. Inhibition of cardiolipin synthesis and IMM-to-OMM translocation by siRNA knockdown of cardiolipin synthase (CLS) and PLS3, respectively, reduced pyroptosis. Based on these findings, we propose that GSDMD-NT mediates a mitochondrial pathway in pyroptosis which promotes cell death.