Active Caspase-1 Induces Plasma Membrane Pores That Precede Pyroptotic Lysis and Are Blocked by Lanthanides

Abstract

Canonical inflammasome activation induces a caspase-1/gasdermin D (Gsdmd) dependent lytic cell death called pyroptosis which promotes anti-microbial host defense but may contribute to sepsis. The nature of the caspase-1-dependent change in plasma membrane (PM) permeability during pyroptotic progression remains incompletely defined. We assayed propidium2+ (Pro2+) influx kinetics during NLRP3 or Pyrin inflammasome activation in murine bone marrow-derived macrophages (BMDM) as an indicator of this PM permeabilization. BMDM were characterized by rapid Pro2+ influx after initiation of NLRP3 or Pyrin inflammasomes by nigericin or C. difficile toxin B (TcdB), respectively. No Pro2+ uptake in response to nigericin or TcdB was observed in Caspase-1−/− or ASC−/− BMDM. The cytoprotectant glycine profoundly suppressed nigericin and TcdB-induced lysis but not Pro2+ influx. The absence of Gsdmd expression resulted in suppression of nigericin-stimulated Pro2+ influx and pyroptotic lysis. Extracellular La3+ and Gd3+ rapidly and reversibly blocked the induced Pro2+ influx and markedly delayed pyroptotic lysis without limiting upstream inflammasome assembly and caspase-1 activation. Thus, caspase-1 driven pyroptosis requires induction of initial pre-lytic pores in the PM that are dependent on Gsdmd expression. These PM pores also facilitated the efflux of cytosolic ATP and influx of extracellular Ca2+. Although lanthanides and Gsdmd deletion both suppressed PM pore activity and pyroptotic lysis, robust IL-1β release was observed in lanthanide-treated BMDM but not in Gsdmd-deficient cells. This suggests roles for Gsdmd in both passive IL-1β release secondary to pyroptotic lysis and in non-lytic/non-classical IL-1β export.

Active MLKL triggers the NLRP3 inflammasome in a cell-intrinsic manner

“…As such, we could not conclusively determine if IL-1β secretion was an active or passive process during necroptosis. This question of whether IL-1β can be actively secreted from cells after caspase-1 activation remains controversial (20,23,(35)(36)(37). These experiments nonetheless suggested that NLRP3 activation could be cell-intrinsic, because glycine treatment had no effect on mature IL-1β secretion following MLKL activation.…”

“…Caspase-1 subsequently cleaves and activates the potent proinflammatory cytokines IL-1β and IL-18 and induces their secretion. Although it has been suggested that cell lysis is required for IL-1β release, it has been reported by a number of groups that mature IL-1β secretion is an active cellular process and can occur in the absence of cell death (20)(21)(22)(23).…”

Gasdermins: Effectors of Pyroptosis

“…Thus, in a cell undergoing pyroptosis without sufficient compensatory mechanisms, the membrane rupture likely occurs before significant amounts of propidium can be detected inside the cell. However, caution should be used since propidium is very small and, thus, passes freely through the gasdermin pore[28]. It is therefore formally possible that a cell can repair its membrane to remove the gasdermin pore, as was recently shown during necroptosis[24].…”

Roles of Caspases in Necrotic Cell Death

“…However, since the release of proinflammatory cytokines is also blocked by GSDMD deficiency (Shi et al, 2015), and mice deficient for IL-1R type I, the receptor for both IL-1α and IL-1β are highly resistant to LPS (Joosten et al, 2010), a major role of pyropotosis in sepsis in vivo might still be related to the release of proinflammatory cytokines. The cleavage of GSDMD by caspase-1 may play a role in the release of IL-1β by forming an ion-permeable conduit that can be inhibited by broadly acting channel inhibitors such as lanthanides (La 3+ and Gd 3+ ), before pyroptotic cell death (Russo et al, 2016). Caspase-11 mediated cleavage of pannexin-1 can also trigger the release of intracellular ATP through this non-selective large-pore channel, and the subsequent activation of the purinergic receptor P2X ligand-gated ion channel (P2X7), both critical for caspase-11-mediated pyroptosis (Yang et al, 2015).…”