Abstract
BackgroundPyroptosis is a type of proinflammatory regulated cell death (RCD) in which caspase-1 proteolytically cleaves gasdermin D (GSDMD) to yield a cytotoxic pore-forming protein. Recent studies have suggested that additional cell death pathways may interact with GSDMD under certain circumstances to execute pyroptosis. Microglia/macrophages in the central nervous system (CNS) undergo GSDMD-associated pyroptosis in multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE) but the contribution of other cell death pathways to this phenomenon is unknown. Herein, we tested the hypothesis that multiple RCD pathways underlie microglial pyroptosis in the context of neuroinflammation.MethodsA siRNA screen of genes with known RCD functions was performed in primary human microglia to evaluate their role in nigericin-induced pyroptosis using supernatant lactate dehydrogenase activity as a read-out of cell lysis. Activation of apoptotic executioner proteins and their contribution to pyroptosis was assessed using semi-quantitative confocal microscopy, high-sensitivity ELISA, immunoblot, cell lysis assays, and activity-based fluorescent probes. Quantification of pyroptosis-related protein expression was performed in CNS lesions from patients with progressive MS and mice with MOG35-55-induced EAE, and in matched controls.ResultsAmong progressive MS patients, activated caspase-3 was detected in GSDMD immunopositive pyroptotic microglia/macrophages within demyelinating lesions. In the siRNA screen, suppression of caspase-3/7, caspase-1, or GSDMD expression prevented plasma membrane rupture during pyroptosis. Upon exposure to pyroptotic stimuli (ATP or nigericin), human microglia displayed caspase-3/7 activation and cleavage of caspase-3/7-specific substrates (e.g., DFF45, ROCK1, and PARP), with accompanying features of pyroptosis including GSDMD immunopositive pyroptotic bodies, IL-1β release, and membrane rupture. Pyroptosis-associated nuclear condensation and pyroptotic body formation were suppressed by caspase-3/7 inhibition. Pharmacological and siRNA-mediated inhibition of caspase-1 diminished caspase-3/7 activation during pyroptosis. In mice with EAE-associated neurological deficits, activated caspase-3 colocalized with GSDMD immunopositivity in lesion-associated macrophages/microglia.ConclusionsActivation of executioner caspases-3/7, widely considered key mediators of apoptosis, contributed to GSDMD-associated microglial pyroptosis under neuroinflammatory conditions. Collectively, these observations highlight the convergence of different cell death pathways during neuroinflammation and offer new therapeutic opportunities in neuroinflammatory disease.
Highlights
Pyroptosis is a type of proinflammatory regulated cell death (RCD) in which caspase-1 proteolytically cleaves gasdermin D (GSDMD) to yield a cytotoxic pore-forming protein
Pyroptosis relies upon caspase activation as an initiating event in the cell death program; while apoptosis is initiated by caspases-8 and -9 and executed by caspases-3 and -7, pyroptosis is initiated by the caspase1 family members and executed by the pore-forming protein, gasdermin D (GSDMD), or in certain circumstances, gasdermin E (GSDME) [7]
Cleaved GSDMD-immunopositive macrophages/microglia are present in multiple sclerosis (MS) lesions Earlier studies demonstrated GSDMD immunoreactivity localized to macrophages/microglia in normal-appearing white matter (NAWM) of progressive MS patients, representing the first evidence of GSDMD-mediated pyroptosis in the central nervous system (CNS) [9]
Summary
Pyroptosis is a type of proinflammatory regulated cell death (RCD) in which caspase-1 proteolytically cleaves gasdermin D (GSDMD) to yield a cytotoxic pore-forming protein. Pyroptosis relies upon caspase activation as an initiating event in the cell death program; while apoptosis is initiated by caspases-8 and -9 and executed by caspases-3 and -7, pyroptosis is initiated by the caspase family members (caspases-1 and -11 in mice, caspases and -4 in humans) and executed by the pore-forming protein, gasdermin D (GSDMD), or in certain circumstances, gasdermin E (GSDME) [7]. Due to local changes in osmotic pressure, pyroptotic bodies form along the membrane, which swell and eventually rupture catastrophically to cause cell lysis [10,11,12,13,14] This process releases intracellular alarmins (e.g., heat shock proteins), soluble cytoplasmic proteins (e.g., LDH), and inflammatory mediators (e.g., IL-1β and IL-18) into the extracellular milieu, propagating local inflammation. To categorize cells as immunopositive for proteins of interest, the threshold MFI for “immunopositive” was established as triple the background MFI for each channel and positive cells manually counted in Volocity 6.3 analysis software as previously described [9]
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