On the Road to Discovering the Elusive Executioner of Pyroptosis

Programmed cell death is a regulated process that multicellular organisms use to dispose of cells that are no longer needed, injured, or infected. The first regulated cell death pathway was called apoptosis, from the Greek phrase for “falling off.” After the realization that cells undergo cell death in a controlled manner, many pathways have been identified and characterized by their distinct morphological changes and the unique molecular pathways that control them. These distinct forms of cell death include intrinsic and extrinsic apoptosis, mitochondrial permeability transition-driven necrosis, necroptosis, ferroptosis, immunogenic cell death, autophagy-dependent cell death, lysosome-dependent cell death, parthanatosis, entosis, NETosis, and pyroptosis (1). Because of the importance of cell death in organismal development and the number of diseases resulting from dysregulation and mutation of the pathways involved in regulating cell death, understanding how the process works is critical for developing treatments.

Pyroptotic cell death in particular is important in regulating the immune response to pathogens. Similar to apoptosis, the term pyroptosis derives from the Greek “pyro,” meaning fire, because this form of cell death drives a proinflammatory immune response by the release of cytokines, including IL-1β and IL-18 (2). Since the discovery of this distinct form of cell death, researchers have identified multiple proteins that regulate the initiation and execution of pyroptosis. In this Pillars of Immunology article by S.L. Fink and B.T. Cookson, a critical step of pyroptosis was described (3). Prior to this discovery, researchers knew that bacterial pathogens could induce a unique form of caspase-1–dependent cell death and that this was mediated by a complex called the inflammasome, consisting of a sensor protein, an adaptor, and caspase-1 (4). Others had observed that extracellular ATP stimulation led to membrane permeability, which was attributed to the formation of pores via P2X7 receptors (5, 6). The study by Fink and Cookson showed that formation of membrane pores was dependent upon the activity of caspase-1.

By using a Salmonella infection model to study pyroptosis, Fink and Cookson discovered that the formation of 1–2 nm pores in macrophages led to rapid cell swelling and osmotic lysis. At the time, it was clear that sensor proteins such as NLRP3 or NLRC4 were required for different stimuli to drive inflammasome-dependent caspase-1 activation and cell death (7–9). By using an inhibitory peptide (Ac-YVAD-cmk) to block caspase-1 activity and glycine as an osmoprotectant in the extracellular media, the authors were able to demonstrate that the caspase-1–dependent membrane pores led to the eventual cell swelling and lysis following infection. This finding contributed to our understanding of how pyroptosis is regulated in a stepwise manner and raised new questions about what substrate of caspase-1 was responsible for generating lytic pores.

In 2010, gasdermin D (GSDMD) was first identified as a substrate cleaved by caspase-1, but its significance was not realized until 2015, when GSDMD cleavage was found to be required for cell death by pyroptosis (10–13). Mechanistically, cleavage of GSDMD releases its N-terminal domain from its autoinhibitory C-terminal domain, leading to oligomerization and insertion into the plasma membrane. The membrane pores formed by GSDMD facilitate the release of the inflammasome-dependent cytokines IL-1β and IL-18 and mediate the lytic cell death (14, 15). Later studies found that GSDMD formed pores 10–16 nm in diameter, much larger than 1–2 nm pores originally suggested by Fink and Cookson. Importantly, GSDMD is required for both canonical and noncanonical, caspase-11/4/5–dependent, inflammasome activation. These findings led to the realization that other gasdermin family members, which contain a similar N-terminal domain, could also regulate cell death and explain the significance of gain of function mutations in these proteins in diseases.

More recently, gasdermin family members have been implicated in regulating cell death in surprising contexts. The protein GSDME (previously DFNA5), was found to be cleaved by caspase-3 to mediate a unique lytic form of cell death in cancer cells treated with chemotherapy agents (16). In contrast to the observations from initial studies, caspase-8 has been found to cleave GSDMD (17, 18). The conserved N-terminal fragment of GSDMD is thought to bind to phosphatidylinositol, phosphatidic acid, phosphatidylserine in the plasma membrane, and additional phospholipids that are enriched in intracellular organelles (19). Other gasdermin family members exhibit different lipid-binding preferences that warrant further studies (19). Differences in cell type and tissue distribution of the gasdermin family members and caspases will likely reveal distinct enzyme/substrate combinations that are important for driving pathogenesis in many diseases.

Similar to pyroptosis, lytic cell death is also observed during necroptosis. Rather than relying on caspases and gasdermin family members, the protein MLKL was found to generate ion channels following phosphorylation by the kinase RIPK3 (20, 21). Similar to GSDMD, MLKL seems to bind phosphatidylinositol and cardiolipin, and ion channel formation by both these proteins can activate the NLRP3 inflammasome, highlighting the capability of these proteins in facilitating cross-talk between different cell death pathways (22, 23). The similarity between these different pathways also suggests the possibility of many common regulators of necroptosis and pyroptosis. Recent studies have also shown that the pores formed by both MLKL and GSDMD can be repaired prior to cell death by recruitment of the ESCRT complex (24, 25). Future studies may further evaluate the importance of this repair mechanism in infections or other disease models.

Overall, the study by Fink and Cookson in 2006 indicated that active caspase-1 led to pore formation to drive the characteristic lytic cell death of pyroptosis. This study, together with the discoveries of caspase-1 inflammasomes at the same time, spurred research into discovering the molecular mechanisms that drive this form of cell death. Emerging studies on this pathway further highlight the importance of this Pillars of Immunology article and will continue to lead to new research into how cell death is regulated and how its dysregulation leads to a multitude of diseases.