Pyroptosis

Pyroptosis is a form of programmed cell death that is inflammatory and lytic in nature, triggered by the detection of pathogens or endogenous danger signals within the cytosol, leading to cell membrane permeabilization, swelling, rupture, and the release of pro-inflammatory cytokines such as interleukin-1β (IL-1β) and IL-18.^[1] This process is mediated primarily by gasdermin family proteins, particularly gasdermin D (GSDMD), which, upon cleavage by inflammatory caspases (e.g., caspase-1, -4, -5, or -11), oligomerize to form pores in the plasma membrane, disrupting ion balance and facilitating the extrusion of danger-associated molecular patterns (DAMPs) and alarmins.^[2] Unlike apoptosis, which is immunologically silent, pyroptosis amplifies innate immune responses but can contribute to tissue damage if uncontrolled.^[1] The mechanisms of pyroptosis involve two main pathways: the canonical pathway, where sensor proteins like NLRP3 form inflammasomes in response to stimuli such as ATP or bacterial toxins, activating caspase-1 to process GSDMD and pro-IL-1β; and the non-canonical pathway, activated directly by lipopolysaccharide (LPS) from Gram-negative bacteria via caspase-4/5 (in humans) or caspase-11 (in mice).^[2] Additional executors include other gasdermins like GSDME, which can be cleaved by caspase-3 during apoptosis to induce pyroptotic features, and ninjurin-1 (NINJ1), which aids in the final membrane rupture.^[1] These pores, approximately 10–20 nm in diameter, allow the passive release of cellular contents while maintaining initial membrane integrity for cytokine secretion.^[2] First described in the early 1990s as a distinct form of macrophage death induced by bacterial pathogens like Shigella flexneri and Bacillus anthracis, pyroptosis was formally named in 2001 to highlight its fever-inducing inflammatory properties, derived from the Greek words "pyro" (fire) and "ptosis" (falling).^[2] The identification of gasdermins as the central effectors in 2015 marked a breakthrough, linking pyroptosis to broader gasdermin-mediated pyroptotic pathways.^[1] In host defense, pyroptosis eliminates intracellular pathogens and signals danger to neighboring cells, enhancing antimicrobial immunity against infections like salmonellosis or yersiniosis.^[2] However, excessive pyroptosis drives pathology in conditions such as sepsis, where it exacerbates cytokine storms; autoinflammatory diseases like cryopyrin-associated periodic syndromes (CAPS) and familial Mediterranean fever (FMF); and chronic disorders including cardiovascular disease, neurodegeneration, and cancer, where it may suppress tumors by boosting immunity or promote progression via inflammation.^[1] Therapeutic strategies targeting pyroptosis, such as GSDMD inhibitors, are under investigation to balance its protective and detrimental effects.^[2]

Overview

Definition and Key Features

Pyroptosis is a lytic and pro-inflammatory form of programmed cell death that is dependent on caspases and mediated by gasdermin proteins, resulting in plasma membrane rupture and the release of intracellular contents without the formation of apoptotic bodies.^[3] Unlike non-lytic cell death pathways, pyroptosis leads to the extrusion of pro-inflammatory mediators such as interleukin-1β (IL-1β) and IL-18, which amplify immune responses.^[2] The term "pyroptosis" was coined in 2001 by Cookson and Brennan to describe a caspase-1-dependent death observed in macrophages infected with Salmonella, derived from the Greek words "pyro" (fire or fever) and "ptosis" (falling), emphasizing its fever-inducing inflammatory nature.^[4] Key features of pyroptosis include rapid cell swelling due to osmotic imbalance, formation of pores in the plasma membrane by the N-terminal fragments of gasdermins—primarily gasdermin D (GSDMD)—and subsequent cell lysis that generates pyroptotic bodies.^[5] GSDMD, a member of the gasdermin family, is cleaved by inflammatory caspases (such as caspase-1, -4, -5, or -11) to release its pore-forming N-terminal domain, which oligomerizes to create ~20 nm diameter pores that permeabilize the membrane.^[3] This process is typically triggered by inflammasome activation in response to microbial or endogenous danger signals, distinguishing pyroptosis as an innate immune defense mechanism.^[2] The primary outcomes of pyroptosis involve the release of damage-associated molecular patterns (DAMPs), such as high-mobility group box 1 (HMGB1), alongside cytokines, which propagate systemic inflammation and recruit immune cells to infection sites or damaged tissues.^[3] This contrasts with non-lytic forms of cell death by actively promoting a hyperinflammatory state rather than silent clearance, thereby enhancing host defense but potentially contributing to pathology if dysregulated.^[2]

Distinction from Other Cell Death Pathways

Pyroptosis differs fundamentally from apoptosis in its pro-inflammatory nature and lytic execution. While apoptosis is an anti-inflammatory process characterized by non-lytic cell shrinkage, chromatin condensation, membrane blebbing, and formation of apoptotic bodies that prevent content leakage, pyroptosis involves rapid cell swelling, membrane rupture via gasdermin D (GSDMD) pores, and release of intracellular contents, including pro-inflammatory cytokines like IL-1β and IL-18.^[2] Apoptosis relies on caspase-3 and caspase-7 activation without GSDMD involvement or inflammasome priming, serving to maintain tissue homeostasis without alerting the immune system, whereas pyroptosis lacks caspase-3/7 activation and is driven by caspase-1 or caspase-11 to amplify innate immune responses.^[2] In contrast to necroptosis, another lytic form of cell death, pyroptosis is specifically dependent on inflammasome activation and caspases-1/11, leading to targeted cytokine release and GSDMD-mediated pore formation. Necroptosis, triggered by death receptor signaling such as TNF-α, involves the RIPK1/RIPK3/MLKL pathway, where phosphorylated MLKL oligomerizes to disrupt the plasma membrane, but it does not require inflammasome priming or result in IL-1 family cytokine maturation. Both pathways cause membrane permeabilization and pro-inflammatory damage-associated molecular pattern (DAMP) release, yet pyroptosis's reliance on pathogen-associated molecular patterns (PAMPs) or DAMPs distinguishes it as a primary defense against microbial infection, while necroptosis acts as a backup mechanism when apoptosis is inhibited. Pyroptosis also contrasts with ferroptosis, which features iron-dependent lipid peroxidation as its core mechanism without significant involvement of inflammasomes or cytokine release. Ferroptosis leads to lytic cell death through accumulation of lipid hydroperoxides and glutathione depletion, often resulting in mitochondrial shrinkage but eventual membrane rupture, and it engages pro-inflammatory pathways through DAMP exposure like HMGB1. In pyroptosis, lipid peroxidation plays a negligible role, with execution centered on GSDMD pores and caspase-dependent processing, emphasizing its role in innate immunity rather than ferroptosis's association with oxidative stress responses in conditions like ischemia or cancer.^[2] The following table summarizes key distinctions across these pathways:

Aspect	Pyroptosis	Apoptosis	Necroptosis	Ferroptosis
Triggers	PAMPs/DAMPs via inflammasomes	Death ligands (e.g., TNF), DNA damage	TNF-α, viral infection, TLRs	Iron overload, lipid peroxidation inducers
Executioners	GSDMD pores (caspase-1/11-cleaved)	Caspases-3/7/9	MLKL (RIPK3-phosphorylated)	Lipid peroxidation (no specific protein)
Morphology	Lytic: swelling, membrane rupture	Non-lytic: shrinkage, apoptotic bodies	Lytic: swelling, membrane rupture	Lytic: mitochondrial shrinkage, membrane rupture
Inflammatory Outcome	Pro-inflammatory (IL-1β/IL-18 release)	Anti-inflammatory (no content leakage)	Pro-inflammatory (DAMP release)	Pro-inflammatory (HMGB1/ATP)
Physiological Role	Innate immunity, pathogen clearance	Tissue homeostasis, development	Backup death, inflammation	Tumor suppression, oxidative defense

History

Initial Discovery

The initial observation of what would later be recognized as pyroptosis occurred in 1992, when Zychlinsky and colleagues reported that infection of macrophages with the Gram-negative bacterium Shigella flexneri triggered a form of programmed cell death resembling apoptosis, characterized by DNA fragmentation and cell lysis. This death was distinct from bacterial replication in non-phagocytic cells and was proposed to contribute to the inflammatory response in shigellosis by limiting intracellular pathogen growth. Subsequent work in 1994 by the same group demonstrated that Shigella-infected macrophages released mature interleukin-1β (IL-1β), linking the cell death process to pro-inflammatory cytokine production during infection.^[6] Parallel early observations in the 1990s included macrophage death induced by Bacillus anthracis lethal toxin, which was later shown to activate caspase-1 and promote inflammatory cell lysis similar to Shigella-induced death.^[7] Further characterization in 1997 revealed that this macrophage death was dependent on the interleukin-1β-converting enzyme (ICE), now known as caspase-1, as inhibition of ICE with the peptide YVAD-CHO blocked both cell death and IL-1β processing in Shigella-infected murine macrophages. This finding highlighted caspase-1's role in the pathway but also contributed to initial confusion with classical apoptosis, given the shared involvement of caspases and morphological similarities like nuclear condensation. Experiments using caspase-1 knockout mice, reported in 1998, confirmed the enzyme's necessity, as macrophages from these animals resisted Shigella-induced death while remaining susceptible to other apoptotic stimuli.^[8] In 2000, Brennan and Cookson extended these observations to Salmonella infections, showing that the bacterium induced rapid, caspase-1-dependent macrophage death accompanied by IL-1β secretion but lacking typical apoptotic features such as caspase-3 activation or phosphatidylserine exposure. This process limited Salmonella replication by promoting bacterial expulsion from dying cells, providing early evidence of its protective role in host defense. The following year, Cookson and Brennan coined the term "pyroptosis" to describe this distinct, pro-inflammatory form of cell death, emphasizing its rapid kinetics, inflammatory cytokine release, and divergence from non-inflammatory apoptosis.^[9] Key in vivo validation came from studies in caspase-1-deficient mice, which exhibited markedly reduced acute inflammation and impaired clearance of Shigella flexneri following oral infection, underscoring pyroptosis's contribution to orchestrating immune responses against intracellular bacteria.^[10]

Major Milestones and Recent Advances

The identification of gasdermin D (GSDMD) as the key executor of pyroptosis marked a pivotal breakthrough in 2015, when independent studies by the Shi, Kayagaki, and Ding groups demonstrated that GSDMD is cleaved by inflammatory caspases (caspase-1 in the canonical pathway and caspase-11/4/5 in the non-canonical pathway) to release its N-terminal domain, which forms plasma membrane pores leading to cell lysis and interleukin-1β release. In 2016, the Ding group further elucidated the pore-forming mechanism of the GSDMD N-terminal fragment, confirming its oligomerization into 10- to 20-subunit pores approximately 16-24 nm in diameter, which disrupts osmotic balance and triggers pyroptotic cell death.^[11] Building on this, the non-canonical inflammasome pathway was more precisely characterized in 2014, revealing that mouse caspase-11 (human orthologs caspase-4/5) directly senses cytosolic lipopolysaccharide from Gram-negative bacteria and cleaves GSDMD to induce pyroptosis independent of canonical inflammasome sensors like NLRP3. This pathway highlighted pyroptosis's role beyond inflammasome activation, emphasizing direct pathogen detection as a rapid innate immune response.^[12] Research in 2017 expanded pyroptosis beyond GSDMD dependence, with studies showing that gasdermin E (GSDME) mediates an alternative form of pyroptosis in cancer cells through caspase-3 cleavage, particularly in response to chemotherapeutic agents, thereby enhancing antitumor immunity by releasing damage-associated molecular patterns.^[13] A 2024 study uncovered pyroptosis's physiological role in neural development, where AIM2 inflammasome-driven GSDMD activation clears DNA-damaged neural progenitor cells during cortical proliferation, preventing neurodevelopmental disorders like anxiety-like behaviors.^[14] As of 2025, key advances include the discovery that S-palmitoylation of NLRP3 at multiple cysteine residues, catalyzed by ZDHHC enzymes, sequentially regulates inflammasome assembly and activation, offering new targets for modulating pyroptotic inflammation in diseases like atherosclerosis.^[15] Plant-derived compounds such as curcumin and kaempferol have emerged as selective pyroptosis inducers in drug-resistant tumors, activating GSDME-mediated death while sparing healthy cells and synergizing with immunotherapy.^[16] Additionally, small-molecule GSDMD inhibitors like AI-screened pore blockers (e.g., SK56) and repurposed drugs targeting N-terminal oligomerization have advanced to preclinical trials, demonstrating efficacy in attenuating excessive pyroptosis in sepsis and autoinflammatory conditions without compromising pathogen clearance.^[17] Overall, these milestones reflect a paradigm shift in pyroptosis research from its initial focus on infection defense to its broader implications in cancer therapy, neurodevelopment, and chronic inflammation, with therapeutic strategies increasingly targeting gasdermin pores for precision modulation.^[2]

Cellular Characteristics

Morphological Changes

Pyroptosis begins with the formation of pores in the plasma membrane mediated by gasdermin D (GSDMD), which initiates a cascade of structural alterations in the affected cell.^[11] In the early phase, these pores, measuring 10-20 nm in diameter, permit non-selective ion fluxes, including initial potassium (K⁺) efflux followed by sodium (Na⁺) and chloride (Cl⁻) influx, leading to osmotic imbalance and rapid cell swelling. This swelling is observable through live-cell microscopy and can result in up to a 1.5-fold increase in cell volume prior to further progression.^[18] As pyroptosis advances, the accumulating osmotic pressure causes the plasma membrane to rupture, releasing intracellular contents such as lactate dehydrogenase (LDH), which serves as a quantifiable marker of membrane integrity loss.^[18] Electron microscopy visualizations confirm the presence of these GSDMD-induced pores on the membrane surface, contributing to the lytic nature of the process.^[11] In the late phase, cells form balloon-like protrusions or pyroptotic bodies, characterized by membrane blebbing and eventual fragmentation into corpse-like structures, distinct from the apoptotic shrinkage.^[18] Nuclear changes during pyroptosis include DNA condensation without the internucleosomal fragmentation typical of apoptosis, as observed via live-cell imaging techniques that track these morphological dynamics in real time.^[11] These alterations culminate in the complete lysis of the cell, facilitating the release of proinflammatory mediators while preserving key subcellular components within the resulting pyroptotic remnants.^[18]

Biochemical and Molecular Markers

Pyroptosis is characterized by specific biochemical and molecular signatures that distinguish it from other forms of cell death, primarily involving the activation of inflammatory caspases and the pore-forming protein gasdermin D (GSDMD). The hallmark marker is the cleavage of GSDMD into its N-terminal fragment (GSDMD-NT), which oligomerizes to form plasma membrane pores, leading to cell lysis and release of intracellular contents. This cleavage occurs at aspartate residue 275 in humans (D275) or 276 in mice (D276) by active caspase-1, caspase-11 (in mice), or caspase-4/5 (in humans), producing the ~30 kDa GSDMD-NT fragment detectable by Western blot analysis of cell lysates or supernatants.^[3] Another key indicator is the processing of pro-caspase-1 into its active p20 subunit (along with p10), which drives GSDMD cleavage and is similarly identified via Western blot, confirming inflammasome activation.^[3] Additionally, the maturation and extracellular release of pro-inflammatory cytokines interleukin-1β (IL-1β) and IL-18, processed by caspase-1, serve as soluble markers enriched in culture supernatants or serum, reflecting the lytic nature of pyroptosis.^[3]^[19] Detection of these markers relies on established assays tailored to pyroptotic events. Western blotting remains the gold standard for visualizing cleaved GSDMD-NT and the p20 subunit of caspase-1/11, often using antibodies specific to the N-terminal domain or neo-epitopes generated post-cleavage, allowing quantification of pyroptosis induction in response to stimuli like lipopolysaccharide (LPS).^[3]^[19] Cytokine release is quantified via enzyme-linked immunosorbent assay (ELISA) for mature IL-1β and IL-18 in supernatants, providing a sensitive measure of inflammasome-driven pyroptosis without requiring cell lysis.^[3]^[20] Flow cytometry distinguishes pyroptotic cells by their Annexin V-negative/propidium iodide (PI)-positive profile, indicating membrane permeabilization and loss of integrity prior to full lysis, in contrast to apoptotic cells which are Annexin V-positive/PI-negative; this method can be combined with antibodies against cleaved GSDMD for enhanced specificity.^[20]^[19] In vivo, pyroptosis manifests through systemic indicators of cell damage and inflammasome activity. Elevated serum lactate dehydrogenase (LDH) levels, released due to GSDMD pore-mediated membrane rupture, serve as a non-specific but quantitative proxy for pyroptotic cell death in tissues or circulation, measurable by enzymatic assays in animal models of infection or sterile inflammation.^[20]^[21] ASC specks—oligomeric complexes of apoptosis-associated speck-like protein containing a CARD (ASC)—are released extracellularly following pyroptosis and can be detected in tissues via immunohistochemistry using anti-ASC antibodies, highlighting persistent inflammasome foci that amplify inflammation.^[3]^[22] To differentiate pyroptosis from apoptosis or other necrotic pathways, functional validation assays are essential. Genetic knockout of GSDMD abolishes pore formation and IL-1β release while preserving caspase-1 activation and cytokine processing, confirming GSDMD dependency and ruling out alternative death modes.^[2] Pharmacological inhibitors like disulfiram, which covalently modifies GSDMD at cysteine 191/192 to block NT oligomerization, selectively prevent pyroptotic lysis and LDH release without interfering with apoptotic caspase-3 activation, enabling mechanistic dissection in experimental settings.^[23]^[24] As of 2025, advances in understanding NLRP3 regulation include its S-palmitoylation at cysteine residues (e.g., Cys126) by ZDHHC enzymes such as ZDHHC7, which promotes inflammasome assembly and serves as an early marker of pyroptotic priming; this modification is detected using indirect methods like acyl-biotin exchange (ABE) assays in immunoblotting and immunofluorescence.^[25]^[26]

Molecular Mechanisms

Canonical Inflammasome Pathway

The canonical inflammasome pathway is the primary mechanism by which pyroptosis is induced in response to microbial and sterile stimuli, involving the assembly of pattern recognition receptors such as NLRP3 or AIM2 with the adaptor protein ASC and pro-caspase-1 to form an active inflammasome complex.^[2] This pathway culminates in the activation of caspase-1, which processes pro-inflammatory cytokines and executes cell lysis through gasdermin D (GSDMD) pore formation.^[5] Unlike other cell death modalities, this process is tightly regulated to balance innate immune defense with tissue integrity.^[27] The pathway requires a two-step process for inflammasome activation. In the priming phase (signal 1), pathogen-associated molecular patterns (PAMPs) or damage-associated molecular patterns (DAMPs) are recognized by Toll-like receptors (TLRs), leading to NF-κB translocation and transcriptional upregulation of NLRP3, pro-IL-1β, and pro-IL-18.^[27] This step prepares the cell by increasing the availability of inflammasome components without immediate cytotoxicity.^[28] The activation phase (signal 2) is triggered by diverse stimuli, including potassium (K⁺) efflux, lysosomal membrane destabilization, or mitochondrial reactive oxygen species (mtROS) production, which promote NLRP3 oligomerization and recruitment of ASC via PYRIN domains. For AIM2, double-stranded DNA from pathogens or damaged cells directly binds and activates the sensor, bypassing some priming requirements but often integrating with NF-κB signals.^[2] Inflammasome assembly results in the recruitment and autocleavage of pro-caspase-1 into its active form, a p20/p10 heterotetramer, which then cleaves substrates.^[5] This active caspase-1 processes pro-IL-1β and pro-IL-18 into their mature, secreted forms, amplifying inflammation.^[27] Critically, caspase-1 also cleaves GSDMD at Asp275 (in humans) or Asp276 (in mice), separating the N-terminal fragment (GSDMD-NT, residues 1-275/276) from the inhibitory C-terminal domain (GSDMD-CT).^[5] The GSDMD-NT domain oligomerizes and inserts into the plasma membrane, forming non-selective pores approximately 10-20 nm in diameter that permit the efflux of ions, water influx, and cytokine release, ultimately causing osmotic cell swelling and lysis characteristic of pyroptosis.^[2] This pore formation is essential for pyroptotic execution, as GSDMD-deficient cells resist lysis despite inflammasome activation.^[5] Representative triggers illustrate the pathway's versatility. The bacterial toxin nigericin induces K⁺ efflux from cells, a key signal for NLRP3 activation in primed macrophages. Similarly, monosodium urate (MSU) crystals, associated with gout, destabilize lysosomes to release cathepsin B, promoting NLRP3 assembly and downstream pyroptosis.^[27] These examples highlight how environmental and microbial cues converge on common intracellular signals to drive the canonical response.^[2]

Non-Canonical Inflammasome Pathway

The non-canonical inflammasome pathway represents a distinct mechanism of pyroptosis activation in response to intracellular lipopolysaccharide (LPS) from Gram-negative bacteria, primarily mediated by caspase-11 in mice and caspases-4 and -5 in humans. Unlike the canonical pathway, this route operates independently of inflammasome sensors such as NLRP3 or NLRC4 and does not require the adaptor protein ASC or prior transcriptional priming signals. It enables rapid detection of cytosolic LPS, triggering inflammatory caspase activation and subsequent cell lysis to restrict pathogen replication. The activation process begins with direct binding of LPS to the catalytic p20 subunit of pro-caspase-11/4/5 in the cytosol, facilitated by an exosite on the caspase that recognizes the lipid A moiety of LPS. This interaction induces oligomerization of the zymogen into dimers or higher-order structures, promoting autocleavage at specific aspartate residues (e.g., Asp289 in mouse caspase-11) to generate the active heterotetramer. Autocleavage is essential for protease activity but occurs without the need for external processing, distinguishing this pathway as a single-step response to cytosolic bacterial components. The process is particularly prominent during Gram-negative bacterial infections where LPS is released into the host cytoplasm, such as upon vacuole lysis.^[29] Upon activation, caspase-11/4/5 directly cleaves gasdermin D (GSDMD) at Asp276 (mouse) or Asp275 (human), liberating the N-terminal GSDMD fragment that oligomerizes to form plasma membrane pores. These pores cause osmotic cell swelling, membrane rupture, and pyroptotic cell death, releasing intracellular contents including alarmins. Indirectly, GSDMD pore formation leads to potassium efflux, which can secondarily activate the canonical NLRP3 inflammasome and caspase-1, enabling maturation and secretion of interleukin-1β (IL-1β); however, IL-1β release is limited compared to canonical activation. Key differences from the canonical pathway include the absence of potassium efflux as a primary trigger, reliance on direct LPS sensing rather than two-step pattern recognition, and a stronger emphasis on pyroptosis over cytokine processing in Gram-negative contexts.^[30] Experimental evidence for this pathway emerged from studies in the early 2010s demonstrating that caspase-11-deficient mice are highly resistant to lethal endotoxin shock induced by high-dose LPS, even in the absence of Toll-like receptor 4 (TLR4) signaling, which handles extracellular LPS detection. Transfection of synthetic LPS into macrophages from wild-type but not caspase-11 knockout cells induced pyroptosis, confirming direct cytosolic sensing. Similarly, human monocytes and macrophages respond to cytosolic LPS via caspases-4 and -5, with knockdown protecting against pyroptotic responses to Gram-negative bacteria like Escherichia coli. These findings established caspase-11/4/5 as critical sensors for intracellular bacterial threats.^[31] Recent advances highlight cross-talk with guanylate-binding proteins (GBPs), interferon-inducible effectors that target pathogen-containing vacuoles and facilitate LPS exposure to caspases-11/4/5. GBPs, such as GBP1 and GBP2, disrupt bacterial outer membranes or endosomal compartments, releasing LPS into the cytosol to enhance non-canonical activation during infections like Salmonella or Legionella. This GBP-mediated targeting amplifies host defense by promoting pyroptosis specifically against cytosolic invaders, with disruptions in GBP function impairing caspase-11 responses in vivo. Studies as recent as 2023 underscore GBPs' role in integrating non-canonical signaling with broader innate immunity, including autophagy modulation, to fine-tune inflammatory outcomes.

Caspase-3-Dependent Pathway

The caspase-3-dependent pathway of pyroptosis represents a non-canonical mechanism that intersects with apoptosis, where executioner caspase-3 cleaves gasdermin E (GSDME) to trigger lytic cell death instead of the typical apoptotic fragmentation. This pathway was first identified in 2017 by Wang et al., who showed that in gastric cancer cells expressing high levels of GSDME, chemotherapeutic agents like cisplatin activate the apoptotic cascade, leading caspase-3 to cleave GSDME and induce pyroptotic features such as membrane rupture and inflammatory cytokine release. Independently, Zhang et al. in 2018 demonstrated that cleaved GSDME functions as the key effector downstream of caspase-3 in various cancer cell lines treated with chemotherapy, confirming the switch from apoptosis to pyroptosis in GSDME-expressing cells.^[32] Mechanistically, stimuli such as chemotherapeutic drugs or other apoptosis inducers (e.g., staurosporine) engage the extrinsic pathway via caspase-8 or the intrinsic pathway via caspase-9, culminating in caspase-3 activation from its pro-form. Active caspase-3 then specifically cleaves GSDME at the Asp270 residue, liberating the N-terminal domain (GSDME-N) that assembles into oligomeric pores on the plasma membrane, disrupting osmotic balance and causing cell swelling and lysis. This process can be represented as:

\text{pro-caspase-3} \to \text{active caspase-3} \to \text{GSDME-NT} \to \text{membrane pores}

Unlike GSDMD-mediated pyroptosis in the canonical inflammasome pathway, GSDME cleavage by caspase-3 does not directly mature IL-1β or IL-18, but the pore formation enables passive release of these cytokines along with other intracellular contents. Cell-type specificity is a hallmark of this pathway, as it predominates in tissues or cells with elevated GSDME expression, such as gastric and breast cancer cells, where it converts intended apoptotic responses into pyroptotic ones, enhancing antitumor immunity through inflammation. In neurons, which also express high GSDME, this mechanism contributes to pyroptotic cell death under stress conditions, shifting from quiet apoptosis to immunogenic lysis. In contrast, many tumor cells with low or silenced GSDME undergo standard apoptosis without pore formation, underscoring GSDME's role as a molecular switch.^[32]^[33] The outcomes of caspase-3/GSDME-mediated pyroptosis include rapid cell lysis followed by secondary necrosis-like inflammation, driven by the efflux of damage-associated molecular patterns (DAMPs) and alarmins that recruit immune cells and amplify local responses. While direct IL-1β processing is absent, indirect release occurs through cellular damage signals, promoting broader inflammatory cascades without the need for inflammasome assembly. Recent research has extended this pathway's relevance to neurodegeneration, showing that in HIV-infected brains, convergent caspase-1 and -3 activation cleaves GSDME to induce neuronal pyroptosis, contributing to cognitive decline in HIV-associated neurocognitive disorders.^[33]

Emerging Gasdermin-Centric Pathways

Recent discoveries have revealed gasdermin-centric pathways in pyroptosis that operate independently of the classical caspase-1/11 or caspase-3 activations, highlighting diverse proteolytic and post-translational mechanisms that fine-tune inflammatory cell death. These emerging routes often involve alternative proteases or integrated cell death programs, expanding the scope of pyroptosis beyond canonical inflammasomes to include immune cell interactions and stress responses. Such pathways underscore the adaptability of gasdermins in host defense and pathology, with implications for targeted therapies. One prominent GSDMD-independent mechanism involves cleavage of gasdermin B (GSDMB) by granzyme A secreted from natural killer (NK) cells and cytotoxic T lymphocytes during immune synapse formation. Granzyme A proteolytically activates GSDMB by cleaving at specific lysine residues (e.g., K229 or K244), releasing the N-terminal fragment that oligomerizes to form plasma membrane pores, thereby inducing pyroptosis in target cells such as tumor cells. This process enhances anti-tumor immunity by promoting the release of inflammatory contents from lysed cells, as demonstrated in esophageal carcinoma models where GSDMB expression correlates with NK cell-mediated cytotoxicity. Similarly, granzyme B can cleave GSDMB at distinct sites, further diversifying pyroptotic execution in immune responses against infected or malignant cells.^[34] In neutrophils, GSDMD activation occurs independently of caspases through cleavage by neutrophil elastase released during lysosomal membrane permeabilization in aging or activated cells. This protease targets GSDMD at non-canonical sites, facilitating pore formation and pyroptosis that contributes to inflammatory resolution or exacerbation in conditions like sepsis. Such elastase-mediated processing allows neutrophils to undergo lytic death without inflammasome involvement, releasing antimicrobial contents while amplifying local inflammation at immune synapses with pathogens or other immune cells. A key integration of pyroptosis with other cell death modalities occurs in PANoptosis, where ZBP1 senses viral or sterile triggers to assemble a PANoptosome complex incorporating RIPK3 and caspase-8, concurrently driving pyroptosis, apoptosis, and necroptosis. In influenza A virus infection, ZBP1 recruits RIPK3 to phosphorylate MLKL for necroptosis while enabling GSDMD cleavage for pyroptotic pores, amplifying inflammatory signaling without exclusive reliance on inflammasomes. This unified pathway, observed in diverse infections, ensures robust host defense by lytic cell death but can exacerbate tissue damage if dysregulated. Post-translational modifications have emerged as critical regulators of gasdermin function, with recent studies (2024–2025) elucidating their roles in pore stability and inhibition. Palmitoylation of the GSDMD N-terminal fragment (GSDMD-NT) at conserved cysteine residues (e.g., Cys191/192) promotes its membrane translocation and oligomerization, stabilizing pores essential for pyroptosis execution in response to lipopolysaccharide or reactive oxygen species. This lipid modification, enhanced by mitochondrial ROS, is indispensable for GSDMD's insertion into lipid bilayers, as mutants lacking palmitoylation fail to induce cell lysis. Conversely, ubiquitination of GSDMD-NT modulates its activity; for example, K48-linked ubiquitination by E3 ligases like TRIM21 can inhibit oligomerization by altering membrane targeting, thereby suppressing excessive pyroptosis in inflammatory settings. These modifications provide dynamic control points, with 2025 research highlighting ubiquitination's role in directing GSDMD degradation or relocation to prevent uncontrolled inflammation. Beyond GSDMD, GSDMB in gastrointestinal epithelia undergoes activation via caspase-3 or caspase-8 cleavage during apoptosis-pyroptosis crosstalk, or by bacterial proteases like those from Vibrio cholerae, leading to epithelial pyroptosis and barrier disruption. In the gut, caspase-8 processes GSDMB at apoptotic sites to release its pore-forming domain, exacerbating inflammation in conditions like colitis, while bacterial enzymes exploit this for pathogen escape. This dual activation underscores GSDMB's context-specific role in mucosal immunity. Alternative cleavage sites on GSDMD itself, such as those targeted by caspase-8 in tumor necrosis factor (TNF)-induced death, represent another emerging pathway. Caspase-8 cleaves GSDMD at the canonical Asp275 (humans)/Asp276 (mice) site, generating the pore-forming N-terminal fragment and promoting lethality in TNF-challenged cells, independent of caspase-1. This mechanism integrates extrinsic apoptosis signals with pyroptosis, as evidenced in genetic models where caspase-8/GSDMD double deficiency protects against TNF shock, highlighting its relevance in systemic inflammation.^[35]

Regulation

Activators and Triggers

Pyroptosis is triggered by a diverse array of upstream signals that alert the innate immune system to microbial invasion, cellular damage, or environmental perturbations, leading to the activation of inflammasomes or related pathways. These activators encompass pathogen-associated molecular patterns (PAMPs), damage-associated molecular patterns (DAMPs), and synthetic or environmental stimuli, which converge on sensors such as NLRP3, AIM2, NLRC4, or non-canonical caspases to initiate the process.^[2] Microbial triggers primarily involve PAMPs recognized by cytosolic sensors. Lipopolysaccharide (LPS) from Gram-negative bacteria directly binds and activates human caspase-4/5 or murine caspase-11 in the non-canonical inflammasome pathway, independent of TLR4 priming, thereby sensing cytosolic bacterial presence. Flagellin, a component of bacterial flagella, is detected by neuronal apoptosis inhibitory proteins (NAIPs), which recruit and activate NLRC4 to form an inflammasome complex, as demonstrated in responses to Salmonella infection. Similarly, double-stranded DNA (dsDNA) or RNA from viruses and bacteria engages the AIM2 sensor, which oligomerizes upon binding to initiate caspase-1 activation, a mechanism pivotal in defending against intracellular pathogens like Francisella tularensis. Endogenous activators arise from host-derived danger signals released during cellular stress or injury. Mitochondrial DNA (mtDNA), extruded into the cytosol following mitochondrial damage, acts as a DAMP to stimulate the NLRP3 inflammasome, contributing to sterile inflammation in conditions like non-alcoholic fatty liver disease. Extracellular ATP, often liberated from dying cells, binds the P2X7 receptor to induce potassium efflux and NLRP3 activation, amplifying inflammatory responses.^[2] Cholesterol crystals, prevalent in atherosclerotic plaques, are phagocytosed by macrophages, causing lysosomal destabilization and NLRP3 inflammasome engagement, which drives IL-1β secretion and promotes atherogenesis.^[36] In sterile inflammatory diseases such as gout, monosodium urate crystals similarly serve as DAMPs to trigger NLRP3-dependent pyroptosis.^[2] Environmental and pharmacological factors can mimic pathogenic signals to provoke pyroptosis. Nigericin, a potassium ionophore derived from Streptomyces hygroscopicus, depletes intracellular K⁺, serving as a canonical second signal for NLRP3 inflammasome activation in primed macrophages, widely used to model bacterial toxin effects. Lysosomal destabilizers, such as L-leucyl-L-leucine methyl ester (LLOMe), rupture lysosomes to release cathepsins, indirectly activating NLRP3 and inducing pyroptosis in immune cells.^[2] Cell-specific mechanisms fine-tune trigger responsiveness. For NLRP3, priming via NF-κB signaling—often induced by Toll-like receptor ligands like LPS—upregulates NLRP3 and pro-IL-1β expression, sensitizing cells to subsequent activators such as ATP or nigericin. Guanylate-binding proteins (GBPs), interferon-inducible effectors, facilitate LPS delivery to caspase-11 by targeting bacterial outer membranes and promoting lipopolysaccharide aggregation, essential for non-canonical inflammasome sensing of Gram-negative invaders. Recent advances highlight viral contributions to pyroptosis triggers. SARS-CoV-2 infection induces ZBP1-dependent PANoptosis in bystander cells via secreted inflammatory factors such as 2′3′-cGAMP, TNF-α, and IFN-β, exacerbating COVID-19 pathology, as reported in 2025 studies.^[37]

Inhibitors and Modulatory Mechanisms

Pyroptosis is tightly regulated by endogenous inhibitors that prevent unwarranted activation of its core components. The C-terminal domain (GSDMD-CT) of gasdermin D (GSDMD) exerts autoinhibition by binding to and masking the pore-forming N-terminal domain (GSDMD-NT), thereby suppressing oligomerization and membrane perforation until proteolytic cleavage occurs.^[38] Similarly, post-translational modifications of NLRP3, such as phosphorylation at Ser803 within its leucine-rich repeat domain, disrupt the interaction with NEK7, a kinase essential for NLRP3 oligomerization and inflammasome assembly, thus inhibiting downstream caspase-1 activation and pyroptosis.^[39] Ubiquitination of NLRP3 by E3 ligases like MARCH5 on mitochondrial surfaces promotes NLRP3-NEK7 complex formation under basal conditions but can be reversed by deubiquitinases to dissociate NEK7 and attenuate inflammasome signaling.^[40] Pharmacological agents target key steps in pyroptosis to modulate its execution. Disulfiram, an FDA-approved alcohol dehydrogenase inhibitor, covalently modifies Cys191 (human) or Cys192 (mouse) in GSDMD-NT, blocking pore formation and thereby preventing IL-1β release and cell lysis without affecting upstream inflammasome activation. Necrosulfonamide (NSA), a small-molecule inhibitor, similarly targets a reactive cysteine in GSDMD to inhibit its oligomerization into plasma membrane pores, suppressing pyroptosis in inflammatory models. MCC950, a potent NLRP3-specific inhibitor, binds the NACHT domain's Walker B motif, blocking NLRP3's ATPase activity and preventing ASC oligomerization, which halts caspase-1-mediated GSDMD cleavage and pyroptotic cell death. Post-translational modifications further fine-tune pyroptosis by stabilizing inactive protein conformations. O-GlcNAcylation of GSDMD at specific serine residues, such as Ser338, hinders its cleavage by inflammatory caspases, reducing GSDMD-NT release and subsequent pore formation in endothelial cells during sepsis.^[41] Recent advances have identified palmitoylation of GSDMD-NT at Cys191 as a critical checkpoint for membrane targeting; inhibitors like NU6300, which covalently react with this residue, stabilize inactive GSDMD forms, suppress pyroptosis in macrophages, and improve outcomes in septic models as demonstrated in 2024 studies.^[42] Negative feedback loops provide additional layers of control to limit pyroptosis and prevent excessive inflammation. IL-1 receptor (IL-1R) signaling, triggered by pyroptosis-derived IL-1β, induces expression of suppressors of cytokine signaling (SOCS) proteins, particularly SOCS1 and SOCS3, which inhibit further NLRP3 inflammasome activation by promoting its ubiquitination and degradation, thereby downregulating the pyroptotic cascade.^[43] Autophagy serves as a degradative mechanism that selectively targets inflammasome components, such as NLRP3 and ASC specks, for lysosomal breakdown, thereby preventing caspase-1 activation and GSDMD-mediated pyroptosis in infected macrophages. Certain physiological triggers exhibit dual roles in pyroptosis regulation depending on concentration. Reactive oxygen species (ROS), while typically promoting NLRP3 activation at moderate levels, inhibit pyroptosis at high concentrations by oxidizing critical cysteines in caspase-1, such as Cys285, which impairs its autocatalytic activity and blocks GSDMD cleavage.^[44]

Physiological Roles

Role in Host Defense Against Pathogens

Pyroptosis serves as a critical component of innate immunity by enabling the lytic death of infected cells, which disrupts intracellular pathogen replication and amplifies inflammatory signals to orchestrate broader host defense. In bacterial infections, pyroptosis effectively restricts pathogens such as Salmonella typhimurium and Listeria monocytogenes through caspase-1-mediated cleavage of gasdermin D, leading to plasma membrane rupture that ejects bacteria into the extracellular space for subsequent elimination by neutrophils via reactive oxygen species.^[45] This process is particularly prominent in the spleen, where pyroptosis acts as the primary clearance mechanism against Gram-negative bacteria like Chromobacterium violaceum, reducing bacterial burden and preventing systemic dissemination.^[45] Concurrently, the release of interleukin-1β (IL-1β) and IL-18 from pyroptotic cells recruits and activates neutrophils and natural killer cells, enhancing local inflammation and pathogen containment without excessive tissue damage in neutrophils, which resist pyroptosis themselves.^[46] In viral infections, ZBP1 (Z-DNA binding protein 1) emerges as a key sensor that initiates pyroptosis to counter threats like influenza A virus (IAV) and herpes simplex virus (HSV-1). ZBP1 detects viral nucleic acids, such as IAV nucleoprotein and polymerase basic 1, or HSV-1 DNA, activating the NLRP3 inflammasome and caspase-1 to drive gasdermin D pore formation, thereby inducing inflammatory cell death that limits viral propagation.^[47] This response promotes IL-1β and IL-18 secretion, bolstering antiviral immunity, though it represents a double-edged sword as viruses employ various evasion tactics to block ZBP1 signaling and prevent inflammatory cell death.^[48] For fungal and parasitic pathogens, the AIM2 inflammasome detects cytosolic double-stranded DNA from microbes, including fungal elements and parasitic invaders, triggering caspase-1 activation that culminates in pyroptosis to curtail pathogen spread.^[49] This DNA-sensing mechanism restricts replication of DNA pathogens, such as certain fungal DNA mimics or parasites like Toxoplasma gondii, by eliminating infected host cells and releasing alarmins that amplify innate responses.^[28] The protective role of pyroptosis extends across evolutionary lineages, with gasdermin-mediated pore formation conserved from fish to mammals, underscoring its ancient origins in antimicrobial defense.^[50] In teleosts like zebrafish, inflammasome components and gasdermins enable similar lytic responses to pathogens, highlighting functional preservation over 450 million years of divergence.^[51] Beyond immediate innate effects, pyroptosis enhances adaptive immunity by liberating pathogen-associated antigens from ruptured cells, which are captured by dendritic cells to prime T-cell responses and generate long-term memory.^[52] Recent 2025 analyses further illuminate pyroptosis's context-dependent benefits in viral infections like COVID-19, where early activation eliminates SARS-CoV-2-infected cells via NLRP3-gasdermin D pathways, releasing IL-1β and IL-18 to recruit antiviral effectors and control initial replication. As of 2025, studies highlight pyroptosis's role in defending against emerging zoonotic viruses via ZBP1-PANoptosis integration.^[53]^[54] However, unchecked pyroptosis can escalate to hyperinflammation, contributing to cytokine storms in severe cases, emphasizing the need for balanced regulation.^[53]

Involvement in Tissue Development and Homeostasis

Pyroptosis plays a pivotal role in embryonic development by facilitating the clearance of damaged neural progenitor cells in the cortex, thereby preventing the accumulation of DNA damage and ensuring proper neurogenesis. During cortical development, high levels of replicative stress induce DNA damage in neural progenitors, triggering gasdermin D (GSDMD)-mediated pyroptosis via the AIM2 inflammasome pathway. This process eliminates defective cells in the ventricular and subventricular zones, promoting balanced proliferation and differentiation of healthy progenitors. Disruption of this mechanism leads to excessive progenitor accumulation, impaired neuronal maturation, and long-term neurodevelopmental abnormalities, such as autism-like behaviors in mice.^[55] In tissue homeostasis, pyroptosis contributes to maintaining epithelial barrier integrity in the gut by selectively removing stressed or senescent enterocytes without compromising overall tissue function. Controlled activation of pyroptosis in intestinal epithelial cells helps regulate microbial interactions and prevents barrier leakage, supporting mucosal homeostasis through the timely clearance of damaged cells.^[56] This balanced inflammatory response is essential for tissue repair in various organs.^[57] Pyroptosis also regulates stem cell populations by pruning excess progenitors, particularly in hematopoiesis, where it maintains hematopoietic stem cell (HSC) balance under stress conditions. In HSCs, GSDME-mediated pyroptosis eliminates overproliferating or damaged cells in response to genotoxic agents like cisplatin, preventing clonal expansion and preserving long-term repopulation capacity. Similar pruning mechanisms operate in skin stem cell niches, where pyroptosis of keratinocytes removes aberrant cells to sustain epidermal renewal and barrier function without disrupting tissue architecture.^[58]^[59] In non-inflammatory contexts, low-level GSDMD activation supports tissue remodeling by forming transient pores that enable cytokine release and cellular content extrusion without inducing full lytic cell death or a cytokine storm. This sub-lytic pore formation facilitates coordinated cell extrusion in epithelial sheets, aiding in wound healing and developmental morphogenesis while preserving tissue integrity. Such regulated activity contrasts with robust pyroptosis, allowing pyroptosis to contribute to homeostasis through subtle adjustments rather than overt inflammation.^[60] Animal models underscore these roles, with GSDMD knockout mice exhibiting developmental defects in the brain, including disrupted cortical layering and progenitor overaccumulation due to failed DNA damage clearance. In the gut, GSDMD deficiency impairs epithelial turnover, leading to barrier dysfunction and altered microbial homeostasis, highlighting pyroptosis's necessity for normal tissue maturation. These phenotypes demonstrate that GSDMD-mediated pyroptosis is indispensable for preventing developmental anomalies and sustaining adult tissue equilibrium.^[55]^[61]

Pathological Implications

In Infectious Diseases

In bacterial infections, excessive activation of the NLRP3 inflammasome leads to pyroptosis, contributing to severe pathology in conditions like sepsis. For instance, lipopolysaccharide (LPS) from Escherichia coli triggers NLRP3-dependent pyroptosis in endothelial cells, driving endotoxic shock through gasdermin D (GSDMD) pore formation and release of pro-inflammatory cytokines.^[62] This process amplifies systemic inflammation, as miR-21 enhances NLRP3 inflammasome activity, promoting pyroptosis and septic shock via NF-κB pathway dysregulation.^[63] In tuberculosis caused by Mycobacterium tuberculosis, pyroptosis initially aids host defense by lysing infected macrophages and releasing IL-1β to recruit immune cells, but in chronic phases, excessive NLRP3 and AIM2 inflammasome activation causes tissue damage and facilitates bacterial dissemination.^[47]^[64] Viral infections also exploit or dysregulate pyroptosis for pathogenesis. In HIV-1 infection, pyroptosis of CD4+ T cells, mediated by caspase-1 cleavage of GSDMD, drives immune depletion and viral persistence by reducing antiviral responses, with HIV-1 proteins like Vpu contributing to neurotoxicity through caspase-3/GSDME pathways in the brain.^[65]^[66] Similarly, SARS-CoV-2 induces ZBP1-dependent PANoptosis—a coordinated pyroptotic, apoptotic, and necroptotic cell death—in lung epithelial cells, leading to cytokine storm with elevated IL-1β and TNF-α levels that exacerbate acute respiratory distress.^[67]^[68] In parasitic infections such as malaria, AIM2 inflammasome activation by Plasmodium DNA triggers pyroptosis in infected cells, releasing IL-1β and IL-18 that intensify neuroinflammation and exacerbate cerebral damage. Dual engagement of AIM2 and NLRP3 by hemozoin and parasite DNA further promotes systemic inflammation, contributing to blood-brain barrier disruption and neuronal injury in cerebral malaria.^[69]^[70] As of 2025, pyroptosis exhibits a double-edged role in antibiotic-resistant infections, where it enhances clearance of resistant Gram-negative bacteria like Enterobacter species by restricting intracellular replication, but excessive activation worsens tissue damage in persistent infections. Plant-derived compounds, such as polyphenols and flavonoids from ginseng and food sources, act as inducers of targeted pyroptosis in macrophages, boosting bacterial clearance while minimizing hyperinflammation in models of resistant infections.^[71]^[47]^[72] Dysregulated pyroptosis in these infections culminates in septic shock and IL-1β-mediated tissue damage, with elevated cytokines causing vascular leakage and organ failure. Animal models demonstrate that caspase-1 inhibitors, such as AC-YVAD-CMK, reduce mortality in LPS-induced sepsis by blocking GSDMD cleavage and IL-1β release, alleviating kidney and lung injury.^[73]^[74]^[75]

In Neurovascular and Cardiovascular Diseases

Pyroptosis plays a critical role in neurovascular diseases, particularly ischemic stroke, where the NLRP3 inflammasome activates gasdermin D (GSDMD) to mediate inflammatory cell death in neurons, microglia, and endothelial cells. This process leads to blood-brain barrier (BBB) breakdown through caspase-1-dependent cleavage of GSDMD, forming membrane pores that release pro-inflammatory cytokines such as IL-1β and IL-18, thereby amplifying ischemic injury and promoting neuronal death.^[76] In mouse models of middle cerebral artery occlusion/reperfusion (MCAO/R), NLRP3 activation via ROS/TXNIP signaling exacerbates BBB permeability and infarct expansion, while inhibition of this pathway with compounds like astragaloside IV reduces pyroptosis and decreases infarct volume, and improves neurological function.^[76] Similarly, GSDMD knockout in mice subjected to MCAO diminishes neutrophil infiltration, lowers cytokine levels (e.g., IL-1β, IL-6, TNF-α), and reduces infarct size by approximately 30-50% at day 3 post-stroke, alongside enhanced sensory and motor recovery.^[77] In neurodegenerative conditions like Alzheimer's disease (AD), amyloid-β (Aβ) peptides activate the AIM2 inflammasome in microglia, triggering GSDMD-mediated pyroptosis that releases damage-associated molecular patterns (DAMPs) and cytokines, which in turn amplify tau hyperphosphorylation and aggregation. This creates a feed-forward loop of neuroinflammation, where pyroptotic microglia exacerbate plaque formation and neuronal loss, contributing to cognitive decline.^[78] Studies in APP/PS1 mouse models demonstrate that AIM2 or NLRP3 inhibition attenuates Aβ-induced microglial pyroptosis, reduces tau pathology, and lowers Aβ deposition, highlighting the pathway's role in disease progression.^[78] Emerging evidence also indicates sex differences, with female AD models showing elevated NLRP1 inflammasome signaling and greater pyroptotic susceptibility compared to males, potentially linked to hormonal influences on microglial activation.^[79] In cardiovascular diseases, GSDMD-driven pyroptosis contributes to atherosclerosis by promoting macrophage death within plaques, leading to instability and rupture; macrophage-derived GSDMD perforates mitochondria, releasing mtDNA that activates the STING-IRF3/NF-κB axis and sustains inflammation.^[80] In ApoE^{-/-} mice on a high-fat diet, GSDMD knockout reduces plaque lesion area by over 50%, decreases macrophage content from 56% to 21%, and lowers IL-1β/IL-18 levels, confirming its role in plaque progression.^[80] For myocardial infarction (MI), mitochondrial reactive oxygen species (mtROS) initiate NLRP3 inflammasome assembly in cardiomyocytes, culminating in GSDMD pore formation and cell lysis during ischemia/reperfusion.^[81] Recent 2025 analyses underscore GSDMD's effector dominance in MI, where its conditional knockout in cardiomyocytes shrinks infarct size and preserves cardiac function in murine models.^[82] Across these conditions, DAMPs such as HMGB1 and ATP, released from initial pyroptotic cells, bind pattern recognition receptors to propagate NLRP3/AIM2 activation, establishing self-amplifying inflammatory cycles that worsen tissue damage.^[83] In both stroke and MI animal models, GSDMD ablation interrupts this cycle, mitigating global inflammation and cell loss while improving outcomes like reduced cognitive impairment in AD or heart failure post-MI.^[84]^[85]

In Cancer

Pyroptosis exhibits a dual role in cancer, acting as both a tumor suppressor and promoter depending on the cellular context and triggering pathway. In its suppressive capacity, gasdermin E (GSDME)-mediated pyroptosis in chemotherapy-sensitive tumors releases damage-associated molecular patterns (DAMPs), such as HMGB1 and ATP, which activate dendritic cells and enhance CD8+ T-cell infiltration into the tumor microenvironment, thereby amplifying antitumor immune responses.^[86] This mechanism is particularly evident in tumors where chemotherapeutic agents like cisplatin cleave GSDME via caspase-3, converting apoptosis to immunogenic pyroptosis and improving therapeutic efficacy.^[87] Conversely, in promotive scenarios, NLRP3 inflammasome activation leading to pyroptosis in tumor-associated macrophages (TAMs) releases IL-1β and IL-18, which polarize additional macrophages toward an M2-like immunosuppressive phenotype, fostering tumor progression and immune evasion.^[88] High NLRP3 expression in TAMs correlates with poorer outcomes in cancers such as head and neck squamous cell carcinoma.^[89] Cell-type specificity further modulates pyroptosis's impact, with elevated gasdermin B (GSDMB) expression observed in epithelial cancers, including breast and lung, where it drives non-canonical pyroptosis via caspase-4/5 or granzyme A, potentially contributing to tumor cell death but also inflammation that supports metastasis in certain contexts.^[90] Low GSDME expression, frequently downregulated in gastric, colorectal, and breast cancers through promoter hypermethylation, is associated with aggressive disease and poor prognosis, as it impairs pyroptotic responses to therapies and reduces immunogenic cell death.^[87] In hepatocellular carcinoma, for instance, diminished GSDME levels predict worse survival and radioresistance.^[91] Recent advances as of 2025 highlight plant-derived inducers, such as flavonoids from sources like quercetin and apigenin, which activate NLRP3 or caspase-3/GSDME pathways to trigger pyroptosis in drug-resistant cancers, including multidrug-resistant ovarian and lung tumors, thereby restoring sensitivity and eliciting antitumor immunity with minimal toxicity.^[16] Targeting pyroptosis in immunotherapy contexts has shown promise in overcoming PD-1 resistance; for example, inducing GSDME-dependent pyroptosis enhances T-cell effector functions and reverses immunosuppressive barriers in colorectal cancer models resistant to checkpoint blockade.^[92] Clinically, pyroptosis-related gene signatures, incorporating markers like GSDMD and NLRP3, predict favorable responses to CAR-T cell therapy in hematologic malignancies by indicating robust inflammasome activity that boosts cytokine release and T-cell persistence.^[88] Mouse models further demonstrate that engineered pyroptosis induction, such as via cytokine-armed GSDME activation, significantly enhances anti-tumor immunity, leading to tumor regression in solid tumors like melanoma and promoting long-term T-cell memory.^[93]

In Metabolic and Autoimmune Disorders

Pyroptosis plays a significant role in metabolic disorders such as obesity and type 2 diabetes, where activation of the NLRP3 inflammasome in adipose tissue macrophages promotes inflammation and insulin resistance. In obese individuals, NLRP3-driven pyroptosis in visceral adipose tissue exacerbates the release of pro-inflammatory cytokines like IL-1β, contributing to systemic insulin desensitization and metabolic dysfunction. Recent studies have highlighted the IL-9–NLRP3 axis as a modulator in this process, with reduced IL-9 levels correlating with enhanced NLRP3 activity and worsened insulin resistance in type 2 diabetes patients.^[94] Similarly, hyperglycemia in diabetes induces caspase-1 activation in pancreatic β-cells, triggering GSDMD-mediated pyroptosis and β-cell dysfunction, which impairs insulin secretion and accelerates disease progression.^[95] This mechanism is evidenced by high-glucose conditions promoting NLRP3/GSDMD signaling, leading to inflammatory cell death in β-cells. In gout, monosodium urate (MSU) crystals, formed from hyperuricemia, directly induce pyroptosis in macrophages and neutrophils, perpetuating a cycle of inflammation and uric acid accumulation in joints. MSU crystals activate the NLRP3 inflammasome, resulting in caspase-1 cleavage of GSDMD and subsequent pore formation, which amplifies IL-1β release and sustains acute flares. This pyroptotic response in synovial macrophages is critical for the pathological progression of gouty arthritis, as inhibiting GSDMD reduces crystal-induced cell death and inflammation. Autoimmune disorders, particularly cryopyrin-associated periodic syndromes (CAPS), arise from gain-of-function mutations in NLRP3, leading to constitutive inflammasome activation, excessive IL-1β production, and pyroptosis in affected tissues. These mutations cause uncontrolled caspase-1 activity, resulting in skin rashes, joint inflammation, and systemic autoinflammation characteristic of CAPS. Pathogenic NLRP3 variants enhance spontaneous inflammasome assembly, driving pyroptotic cell death and IL-1β-mediated flares without external triggers. Recent advancements as of 2025 underscore the involvement of gasdermin D (GSDMD) in non-alcoholic fatty liver disease (NAFLD) progression, where GSDMD-mediated pyroptosis in hepatocytes exacerbates steatosis and fibrosis through NLRP3-caspase-1 signaling. Additionally, AIM2 inflammasome activation has been linked to pyroptosis in autoimmune encephalitis models, such as experimental autoimmune encephalomyelitis (EAE), where AIM2 regulates microglial inflammation and neuronal pyroptosis, contributing to central nervous system autoimmunity. Organelle stresses, including mitochondrial dysfunction and endoplasmic reticulum stress, amplify pyroptosis in these metabolic contexts by potentiating NLRP3 activation and cytokine release, forming a vicious cycle of inflammation. Therapeutically, IL-1 blockers like anakinra, a recombinant IL-1 receptor antagonist, effectively mitigate pyroptosis-driven flares in autoimmune conditions such as CAPS by neutralizing IL-1β and reducing inflammasome-mediated cell death. Anakinra rapidly alleviates symptoms in NLRP3-related autoinflammatory diseases, highlighting its role in targeting downstream pyroptotic outcomes.

Therapeutic Targeting

Pharmacological Inhibitors

Pharmacological inhibitors of pyroptosis target key components of the inflammasome pathway, including NLRP3, gasdermin D (GSDMD), and caspases, to mitigate excessive inflammation in various diseases. These agents primarily act by blocking inflammasome assembly, GSDMD cleavage and pore formation, or caspase activation, thereby preventing the release of pro-inflammatory cytokines like IL-1β and IL-18, as well as lytic cell death. Small molecules and biologics in this class have shown promise in preclinical models, though clinical translation remains limited by specificity and delivery hurdles.^[96]^[97] NLRP3 inhibitors represent a major focus due to the central role of the NLRP3 inflammasome in pyroptosis initiation. MCC950, a sulfonylurea-based small molecule, selectively inhibits NLRP3 by binding to its NACHT domain and blocking ATPase activity, thereby preventing ASC oligomerization and downstream caspase-1 activation without affecting other inflammasomes like AIM2 or NLRC4. In preclinical studies, MCC950 has demonstrated efficacy in models of cryopyrin-associated periodic syndromes (CAPS) by reducing IL-1β production and pyroptosis in patient-derived cells, and in gout models where it attenuates monosodium urate crystal-induced inflammation and neutrophil death. Similarly, CY-09 inhibits NLRP3 inflammasome activation by disrupting ASC oligomerization, showing neuroprotective effects in models of cerebral ischemia by limiting pyroptosis and cognitive deficits. Both compounds remain in preclinical stages for CAPS and gout, with ongoing efforts to optimize MCC950 analogs for better potency and reduced toxicity. As of 2025, NLRP3 inhibitors like dapansutrile have advanced to Phase III trials for acute myocardial infarction, showing reduced inflammation without affecting apoptosis.^[98]^[99]^[43]^[100] Direct targeting of GSDMD, the executor of pyroptosis, offers a downstream approach to block pore formation and cell lysis. Disulfiram, an FDA-approved alcohol dehydrogenase inhibitor repurposed for pyroptosis, covalently modifies cysteine residues in the GSDMD N-terminal fragment (GSDMD-NT), preventing pore assembly on the plasma membrane and reducing IL-1β release in inflammatory models. Ac-FLTD-CMK, a peptide-based caspase inhibitor derived from the GSDMD cleavage site, specifically blocks caspase-1, -4, -5, and -11-mediated GSDMD processing (IC₅₀ of 46.7 nM for caspase-1), thereby inhibiting pyroptosis in macrophages and improving outcomes in sepsis and liver injury models. Recent advancements include 2025 developments in small-molecule GSDMD inhibitors, such as necrosulfonamide (NSA) derivatives and novel compounds like GI-Y1, which target GSDMD pores or cleavage to attenuate myocardial injury in sepsis, though these are still preclinical.^[101]^[102]^[103] Broad-spectrum caspase inhibitors, such as VX-765, indirectly suppress pyroptosis by inhibiting caspase-1 activity. VX-765, an oral prodrug of the selective caspase-1 inhibitor VRT-043198, reduces IL-1β maturation, GSDMD cleavage, and pyroptotic cell death in models of acute liver failure, atherosclerosis, and epilepsy, with demonstrated reductions in inflammation via PPARα upregulation. However, selectivity challenges arise, as VX-765 can partially inhibit caspase-3 and -7 at higher doses, potentially interfering with apoptosis and complicating therapeutic use in tissues where both cell death pathways coexist.^[104]^[105]^[106] Clinically, IL-1 pathway blockers like anakinra (IL-1 receptor antagonist) and canakinumab (anti-IL-1β monoclonal antibody) indirectly dampen pyroptosis by neutralizing downstream cytokines, reducing inflammasome-driven inflammation without directly targeting upstream components. These biologics have been evaluated in Phase II trials for acute ischemic stroke as of 2025, showing safety, reduced systemic inflammation, and potential neuroprotection in small randomized controlled trials, though larger efficacy studies are needed.^[107]^[100] Key challenges in developing pyroptosis inhibitors include off-target effects on apoptosis, particularly with caspase inhibitors that may promote unintended cell survival in tumors or chronic inflammation, and poor cytosolic delivery for intracellular targets like NLRP3 and GSDMD, limiting efficacy in vivo. Ongoing research emphasizes structure-based design to enhance specificity and nanoparticle formulations for targeted delivery.^[108]^[97]

Inducers and Immunotherapeutic Strategies

Pyroptosis can be induced through various endogenous and exogenous stimuli that activate inflammasomes or caspases, leading to the cleavage and oligomerization of gasdermin family proteins such as GSDMD and GSDME. Canonical pathways involve caspase-1 activation by NLRP3 or AIM2 inflammasomes in response to damage-associated molecular patterns (DAMPs) such as ATP or pathogen-associated molecular patterns (PAMPs) like double-stranded DNA (for AIM2), resulting in GSDMD pore formation and release of IL-1β and IL-18.^[109] Non-canonical induction occurs via caspase-4/5/11 sensing cytosolic lipopolysaccharide (LPS), while alternative routes include caspase-3-mediated GSDME cleavage triggered by apoptotic signals or granzyme B from cytotoxic lymphocytes.^[110] Pharmacological inducers encompass chemotherapeutic agents like cisplatin and paclitaxel, which activate caspase-3/GSDME in tumor cells, and natural compounds such as docosahexaenoic acid (DHA), which stimulates NLRP3 inflammasomes.^[111] Emerging nanomedicines, including gold nanoparticles delivering GSDMA3 N-terminal domains or mRNA-lipid nanoparticles encoding GSDMD N-terminal fragments, precisely target pyroptosis to enhance therapeutic efficacy.^[112] Reactive oxygen species (ROS) generators, such as photosensitizers (e.g., NIR-II AIEgens), and ion-overloading agents like Fe²⁺-loaded nanoparticles further promote gasdermin activation by disrupting cellular homeostasis.^[113] Immunotherapeutic strategies leverage pyroptosis induction to transform immunosuppressive tumor microenvironments into immunogenic ones by releasing tumor antigens, DAMPs (e.g., HMGB1), and cytokines that recruit and activate dendritic cells, T cells, and natural killer cells. In cancer settings, combining pyroptosis inducers with immune checkpoint inhibitors (ICIs) like anti-PD-1/PD-L1 antibodies has shown synergistic effects; for instance, cisplatin-induced GSDME pyroptosis in small-cell lung cancer upregulates IL-12 signaling, enhancing ICI responsiveness and improving survival in preclinical models.^[114] CAR-T cell therapies exploit granzyme B-mediated GSDME cleavage to trigger pyroptosis in target tumors, amplifying cytokine release and T-cell infiltration while mitigating exhaustion, as demonstrated in lymphoma models where GSDME overexpression boosted antitumor activity.^[115] Oncolytic viruses, such as parapoxvirus ovis, induce pyroptosis via caspase-8/GSDMD activation, acting as in situ vaccines that prime adaptive immunity and synergize with ICIs in melanoma.^[116] Nanocarrier-based approaches, including CRISPR scaffolds or ZIF-8 nanoparticles co-delivering GSDMD inducers and PD-1 blockers, have eradicated "cold" tumors in mouse models by sustaining pyroptotic signaling and preventing immune escape.^[117] Clinical trials, such as NCT03349710 evaluating nivolumab with cisplatin in advanced cancers, underscore the translational potential of these strategies to overcome resistance to monotherapy.^[118]