Lysosomal Membrane Permeabilization Triggers GSDMD-mediated Pyroptosis through Cytosolic Release of Cathepsin B and L

Abstract

Lysosomal membrane permeabilization (LMP) in macrophages by detergents, crystals and infectious agents triggers an inflammatory cell death, called pyroptosis, which is caused by cleavage and activation of a pore-forming protein, called gasdermin D (GSDMD), that disrupts mitochondrial and cell membranes and serves as a conduit that releases inflammatory cytokines. LMP-mediated cell death has been shown to play an important role in atherosclerosis caused by cholesterol crystals, gout caused by uric acid crystals, silicosis caused by silica crystals and asbestosis caused by asbestos fibers. Pyroptosis in macrophages is often caused by activation of cytosolic sensors of invasive infection or signs of intracellular damage, called inflammasomes, that recruit a family of inflammatory death-inducing proteases called caspases to cleave gasdermin D. However, the mechanism responsible for LMP-induced inflammatory cell death is not well studied. Here, we show that LMP in mouse and human macrophages triggers gasdermin D-dependent pyroptosis that is unexpectedly independent of the caspases and the inflammasome NLRP3 previously thought to cause pyroptosis in response to LMP. Instead, we show that LMP caused by crystals and lysosomal detergents releases the lysosomal proteases, cathepsins B and L, into the cytosol, where they activate gasdermin D to induce pyroptosis. Pharmacological inhibition of cathepsin B and L suppresses LMP-induced pyroptosis. Knockout of either cathepsin B or L on its own does not inhibit LMP-induced pyroptosis, but knockout of both cathepsins strongly blunts pyroptosis, indicating that either cathepsin can activate gasdermin D. Genetic deficiency of both cathepsins also reduces the release of the inflammatory cytokine interleukin-1beta (IL-1beta) ) from LMP-induced pyroptotic macrophages. However, unlike cell death, IL-1beta release also requires NLRP3 activation of caspase-1 to cleave pro-IL-1beta to its mature form, since it is blocked by the NLRP3 inhibitor MCC950, indicating that pro-IL-1beta is not a substrate of the cathepsins. Thus, lysosomal rupture triggered by macrophage phagocytosis of disease-causing crystals leads to release of cathepsins B and L into the cytosol, where they activate gasdermin D to form membrane pores, triggering caspase-independent pyroptosis.Graduate Educatio