Nina Van Opdenbosch

The Nlrp3 inflammasome is critical for host immunity, but the mechanisms controlling its activation are enigmatic. In this study, we show that loss of FADD or caspase-8 in a RIP3-deficient background, but not RIP3 deficiency alone, hampered transcriptional priming and posttranslational activation of the canonical and noncanonical Nlrp3 inflammasome. Deletion of caspase-8 in the presence or absence of RIP3 inhibited caspase-1 and caspase-11 activation by Nlrp3 stimuli but not the Nlrc4 inflammasome. In addition, FADD deletion prevented caspase-8 maturation, positioning FADD upstream of caspase-8. Consequently, FADD- and caspase-8-deficient mice had impaired IL-1β production when challenged with LPS or infected with the enteropathogen Citrobacter rodentium. Thus, our results reveal FADD and caspase-8 as apical mediators of canonical and noncanonical Nlrp3 inflammasome priming and activation.

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.

Despite its clinical importance in infection and autoimmunity, the activation mechanisms of the NLRP1b inflammasome remain enigmatic. Here we show that deletion of the inflammasome adaptor ASC in BALB/c mice and in C57BL/6 macrophages expressing a functional NLRP1b prevents anthrax lethal toxin (LeTx)-induced caspase-1 autoproteolysis and speck formation. However, ASC−/− macrophages undergo normal LeTx-induced pyroptosis and secrete significant amounts of interleukin (IL)-1β. In contrast, ASC is critical for caspase-1 autoproteolysis and IL-1β secretion by the NLRC4, NLRP3 and AIM2 inflammasomes. Notably, LeTx-induced inflammasome activation is associated with caspase-1 ubiquitination, which is unaffected in ASC-deficient cells. In vivo, ASC-deficient mice challenged with LeTx produce significant levels of IL-1β, IL-18 and HMGB1 in circulation, although caspase-1 autoproteolysis is abolished. As a result, ASC−/− mice are sensitive to rapid LeTx-induced lethality. Together, these results demonstrate that ASC-driven caspase-1 autoprocessing and speck formation are dispensable for the activation of caspase-1 and the NLRP1b inflammasome. The NLRP1b inflammasome activation may lead to pyroptosis and secretion of the inflammatory cytokines IL-1ß and IL-18 but the mechanisms behind these processes are not fully understood. Here, the authors show that they can occur independently of the inflammasome adaptor ASC and without caspase-1 autoprocessing.