Pyroptosis is a proinflammatory form of regulated cell death mediated by pore formation by Gasdermin family proteins. This process is generally initiated by inflammasome assembly and inflammatory caspase activation, leading to increased plasma membrane permeability, cellular swelling and rupture, and the release of IL-1β, IL-18, and damage-associated molecules. Pyroptosis is an important host-defense mechanism for eliminating intracellular pathogens, but excessive activation can aggravate tissue injury, chronic inflammation, and disease progression.

Keywords: pyroptosis; inflammasome; Gasdermin D; Gasdermin E; caspase-1; caspase-4; caspase-5; caspase-11; IL-1β; IL-18

1 Basic Characteristics and Biological Significance of Pyroptosis

1.1 Concept of Pyroptosis

Pyroptosis is a form of regulated cell death (RCD) whose core event is proteolytic cleavage of Gasdermin family proteins, releasing pore-forming N-terminal domains. Gasdermin N-terminal fragments oligomerize in the plasma membrane and form transmembrane pores, causing disruption of ion gradients, water influx, cellular swelling, and eventual membrane rupture. Unlike apoptosis, in which plasma membrane integrity is generally maintained during the early stages, pyroptosis has prominent proinflammatory characteristics.

1.2 Morphological Characteristics of Pyroptosis

Pyroptotic cells generally exhibit increased cell volume, bubble-like protrusions of the plasma membrane, increased membrane permeability, and cell lysis. The nucleus may show chromatin condensation and DNA damage, but regular apoptotic bodies are generally not formed as in typical apoptosis. After Gasdermin pores are formed, membrane-impermeable nucleic acid dyes such as propidium iodide and 7-AAD can enter cells, while lactate dehydrogenase and cytoplasmic proteins gradually increase in the culture supernatant.

1.3 Immune Functions of Pyroptosis

Pyroptosis eliminates cells infected with intracellular pathogens and promotes immune-cell recruitment through the release of pathogen-associated molecules, inflammatory cytokines, and damage-associated molecular patterns. Moderate pyroptosis helps restrict pathogen replication and dissemination, whereas excessive or persistent pyroptosis amplifies inflammatory responses and can cause increased vascular permeability, tissue injury, and organ dysfunction.

2 Inflammasomes and Gasdermin Proteins

2.1 Basic Composition of Inflammasomes

An inflammasome is a cytoplasmic multiprotein signaling complex generally composed of an upstream sensor protein, the adaptor protein ASC, and the effector protease caspase-1. The sensor recognizes pathogen-associated molecular patterns, damage-associated molecular patterns, or disturbances in cellular homeostasis. ASC connects the upstream sensor with pro-caspase-1 through its PYD and CARD domains, promoting pro-caspase-1 clustering, autocleavage, and activation.

2.2 NLRP3 Inflammasome

NLRP3 responds to extracellular ATP, potassium efflux, mitochondrial damage, lysosomal rupture, crystalline particles, and various microbial stimuli. NLRP3 inflammasome activation generally involves two stages: transcriptional priming and complex assembly. During priming, expression of NLRP3, pro-IL-1β, and related molecules is increased. A second signal then promotes NLRP3 oligomerization and recruitment of ASC and caspase-1.

2.3 NLRC4 Inflammasome

NLRC4 primarily responds to bacterial flagellin and components associated with type III secretion systems. NLRC4 contains a CARD domain and can directly recruit caspase-1, while ASC can further enhance caspase-1 activation and inflammatory cytokine processing. NLRC4-associated pyroptosis is commonly studied in intracellular bacterial infection models involving Salmonella, Shigella, and Legionella.

2.4 AIM2 Inflammasome

AIM2 recognizes double-stranded DNA in the cytoplasm and binds ASC through its PYD domain, subsequently recruiting and activating caspase-1. This pathway participates in inflammatory responses caused by DNA-virus infection, intracellular bacterial infection, and abnormal accumulation of self-DNA.

2.5 NLRP1 Inflammasome

NLRP1 can sense specific microbial proteases, cellular stress, and disturbances in protein homeostasis. NLRP1 protein structure and activation mechanisms differ substantially among species. Studies of NLRP1-associated pyroptosis should therefore distinguish between human and mouse models and evaluate responses within the relevant cell type.

2.6 Gasdermin D

Gasdermin D (GSDMD) is the principal executioner protein in canonical and noncanonical pyroptosis pathways. Full-length GSDMD consists of a pore-forming N-terminal domain and an autoinhibitory C-terminal domain. Cleavage of the linker region by caspase-1, caspase-4, caspase-5, or caspase-11 releases the GSDMD N-terminal fragment. This fragment binds phospholipids on the inner leaflet of the plasma membrane, oligomerizes, and forms pores that permit the passage of ions and small molecules.

2.7 Gasdermin E

Gasdermin E (GSDME) can be cleaved by activated caspase-3 to generate a pore-forming GSDME N-terminal fragment. In cells with high GSDME expression, caspase-3 signaling that would otherwise primarily induce apoptosis may instead produce pyroptosis characterized by cellular swelling, membrane rupture, and inflammatory release. GSDME expression can therefore influence the final cell-death phenotype induced by chemotherapeutic agents and mitochondrial apoptotic stimuli.

Figure 1 Canonical and non-canonical pyroptosis pathways mediated by Gasdermin pore formation

3 Caspase-1-Dependent Canonical Pyroptosis

3.1 Inflammasome Priming

Inflammasomes such as NLRP3 generally require a first signal for transcriptional priming. Lipopolysaccharide, cytokines, or other pattern-recognition receptor ligands can activate NF-κB signaling and increase expression of NLRP3, pro-IL-1β, and other inflammation-associated proteins. Priming mainly provides the molecular basis for subsequent inflammasome assembly and is generally insufficient by itself to induce complete pyroptosis.

3.2 Inflammasome Assembly

Second signals such as ATP, nigericin, crystalline particles, and organelle damage can induce potassium efflux, mitochondrial dysfunction, increased reactive oxygen species, or lysosomal rupture, thereby promoting oligomerization of inflammasome sensors. After the sensor, ASC, and pro-caspase-1 form a large molecular complex, proximity-induced autocleavage converts pro-caspase-1 into catalytically active caspase-1.

3.3 GSDMD Cleavage and Pore Formation

Activated caspase-1 directly cleaves GSDMD and releases the GSDMD N-terminal fragment. GSDMD-N forms pores in the plasma membrane, causing potassium efflux and sodium and water influx, eventually resulting in cellular swelling and plasma membrane rupture. As membrane integrity declines, lactate dehydrogenase, HMGB1, and other cytoplasmic components can be released into the extracellular environment.

3.4 Maturation of IL-1β and IL-18

Caspase-1 also processes pro-IL-1β and pro-IL-18 into mature IL-1β and IL-18. These mature cytokines can be released through GSDMD pores or after cell lysis, promoting inflammatory-cell recruitment, interferon production, and amplification of local inflammatory signaling. Cytokine maturation, pore formation, and complete cellular rupture may occur sequentially and do not necessarily reach their maximum levels at the same time.

4 Noncanonical Pyroptosis and Other Gasdermin Pathways

4.1 Caspase-4, Caspase-5, and Caspase-11 Pathways

Human caspase-4 and caspase-5 and mouse caspase-11 can directly sense Gram-negative bacterial lipopolysaccharide that has entered the cytoplasm. Activated inflammatory caspases directly cleave GSDMD and induce membrane-pore formation but do not efficiently process pro-IL-1β and pro-IL-18. Potassium efflux caused by GSDMD pores can subsequently activate the NLRP3 inflammasome and caspase-1, thereby promoting maturation of IL-1β and IL-18.

4.2 Caspase-3/GSDME Pathway

Death-receptor signaling, mitochondrial apoptosis, or certain chemotherapeutic agents can activate caspase-3. In cells with high GSDME expression, the N-terminal fragment generated by caspase-3-mediated GSDME cleavage forms pores in the plasma membrane, shifting the cellular phenotype from apoptosis-like changes toward pyroptosis-like membrane rupture. This pathway illustrates mechanistic crosstalk between apoptosis and pyroptosis.

4.3 Caspase-8-Associated Gasdermin Activation

Under specific conditions involving infection, death-receptor activation, or disruption of caspase signaling, caspase-8 can participate in cleavage of GSDMD or other Gasdermin family proteins. Caspase-8 occupies a central position at the intersection of apoptosis, necroptosis, and inflammatory signaling. Its function depends on the cell type, stimulus, and activity states of other caspases and RIPK proteins.

4.4 Crosstalk Among Pyroptosis Pathways

Extensive crosstalk exists among inflammasome signaling, apoptosis, and necroptosis. When one cell-death pathway is inhibited, cells may switch to another death program. Changes in a single caspase or inflammatory cytokine are insufficient to establish pyroptosis. Gasdermin cleavage, membrane integrity, inflammatory cytokine release, and genetic intervention should be evaluated together.

5 Differences Between Pyroptosis and Other Forms of Regulated Cell Death

Autophagy is generally a protective process that maintains cellular homeostasis. Autophagy-dependent cell death should not be diagnosed solely on the basis of increased LC3-II, increased numbers of autophagosomes, or changes in lysosome abundance. This designation is appropriate only when inhibition of core autophagy genes or autophagic flux significantly prevents cell death.

6 Experimental Detection and Interpretation of Pyroptosis

6.1 Cell Morphology and Membrane Integrity

Microscopic observation can reveal cellular swelling, bubble-like membrane protrusions, and eventual lysis in pyroptotic cells. Entry of propidium iodide or 7-AAD reflects increased plasma membrane permeability, whereas increased lactate dehydrogenase in the culture supernatant indicates membrane rupture. These indicators can also occur during necroptosis, secondary necrosis, and nonspecific cell injury and therefore cannot independently serve as pyroptosis-specific evidence.

6.2 Gasdermin Protein Cleavage

Western blotting can detect full-length GSDMD and its N-terminal cleavage fragment and is an important method for evaluating canonical and noncanonical pyroptosis. Studies of caspase-3-associated pyroptosis should detect both full-length GSDME and the GSDME N-terminal fragment. Different antibodies may recognize different epitopes, so it is necessary to confirm whether an antibody detects the target cleavage fragment and to include positive, negative, and inhibitor controls.

6.3 Inflammatory Caspase Activation

Canonical pyroptosis can be evaluated by detecting cleaved caspase-1, caspase-1 enzymatic activity, ASC-speck formation, or inflammasome-complex assembly. Noncanonical pathways require detection of caspase-4, caspase-5, or caspase-11 according to the species. Increased pro-caspase expression indicates only a change in protein abundance and does not demonstrate catalytic activation of the protease.

6.4 IL-1β and IL-18 Release

ELISA, immunoblotting, and other protein-quantification methods can detect mature IL-1β and IL-18 in culture supernatants. Data interpretation should distinguish among transcriptional expression of precursor proteins, caspase-mediated protein processing, and release of mature cytokines. Increased cytokine expression should not be directly interpreted as evidence of pyroptosis.

6.5 Cell Viability and Mitochondrial Function

Metabolic assays such as CCK-8 can evaluate the overall effect of stimuli on cell viability, whereas JC-1 or JC-10 can detect changes in mitochondrial membrane potential. Both are auxiliary measurements and cannot distinguish pyroptosis from apoptosis, ferroptosis, or other forms of cell death. They should be combined with Gasdermin cleavage, membrane-permeability measurements, and inflammatory caspase activation.

6.6 Integrated Criteria for Pyroptosis

Reliable evidence of pyroptosis should include Gasdermin-specific cleavage, increased plasma membrane permeability, activation of the corresponding inflammatory caspase or inflammasome, and attenuation of the phenotype after pathway intervention. Cross-validation using gene knockdown, gene knockout, targeted inhibitors, or inhibitors of multiple cell-death pathways can reduce the risk of misclassifying nonspecific cell lysis as pyroptosis.

7 Pyroptosis and Disease

7.1 Infectious Diseases

Bacterial, viral, fungal, and parasitic infections can induce pyroptosis. Intracellular pathogens such as Salmonella, Shigella flexneri, Listeria monocytogenes, and Legionella pneumophila can activate different inflammasomes or noncanonical LPS-sensing pathways. Pyroptosis can eliminate intracellular sites of pathogen replication and recruit immune cells, but excessive activation can also contribute to sepsis, vascular-barrier disruption, and multiorgan injury.

7.2 Cardiovascular Diseases

The NLRP3 inflammasome and Gasdermin-mediated membrane injury are associated with atherosclerosis, myocardial infarction, ischemia-reperfusion injury, diabetic cardiomyopathy, and myocarditis. Endothelial-cell pyroptosis can increase vascular inflammation and monocyte adhesion, macrophage pyroptosis can promote formation of the necrotic core of atherosclerotic plaques, and cardiomyocyte pyroptosis may expand ischemic and inflammation-associated tissue injury.

7.3 Neurological Diseases

Abnormal inflammasome activation in neurons, microglia, and astrocytes is associated with cerebral ischemia, Alzheimer's disease, Parkinson's disease, epilepsy, and neural injury. Abnormal protein aggregation, extracellular ATP, disruption of ionic homeostasis, and mitochondrial damage can promote inflammasome activation, resulting in IL-1β release and amplification of neuroinflammation.

7.4 Metabolic Diseases

High glucose, free fatty acids, urate crystals, cholesterol crystals, and oxidative stress can promote NLRP3 inflammasome activation. Persistent pyroptosis in adipose tissue, pancreatic islet cells, renal cells, and vascular cells may contribute to insulin resistance, diabetic complications, metabolic dysfunction-associated steatotic liver disease, and chronic low-grade inflammation.

7.5 Cancer

Pyroptosis has bidirectional effects in cancer. Pyroptosis of tumor cells can release tumor antigens and inflammatory mediators, promoting dendritic-cell activation and antitumor immunity. However, persistent inflammation may also promote tumor growth, angiogenesis, tissue remodeling, and immune suppression. Expression of GSDMD, GSDME, and inflammasome proteins can influence the form of cell death induced by chemotherapeutic agents and reshape the tumor immune microenvironment.

8 Pyroptosis Models, Intervention Strategies, and Related Products

8.1 Establishment of Pyroptosis Models

Canonical NLRP3 pyroptosis models generally use LPS or another inflammatory stimulus for transcriptional priming, followed by ATP, nigericin, crystalline particles, or another second signal. Intracellular delivery of LPS can be used to study noncanonical pyroptosis mediated by human caspase-4/5 or mouse caspase-11. Certain chemotherapeutic agents, death-receptor agonists, and mitochondrial-damage conditions can be used to investigate the caspase-3/GSDME pathway. Model conditions should be optimized according to species, cell type, Gasdermin expression, and inflammasome composition.

8.2 Control Design and Pathway Validation

Pyroptosis experiments should include vehicle controls, model-stimulation controls, positive cell-death controls, and pathway-inhibition controls. NLRP3 inhibitors, caspase-1 inhibitors, and compounds associated with inhibition of GSDMD pore formation can be used to validate canonical pyroptosis signaling. Necrostatin-1, Ferrostatin-1, Erastin, and related products can be used to compare necroptosis and ferroptosis. Pharmacological findings should, where possible, be confirmed by gene knockdown, gene knockout, and protein-cleavage analysis.

8.3 Products for Pyroptosis and Related Cell-Death Research

Pyroptosis should be identified using a complete evidence chain encompassing inflammasome or related protease activation, Gasdermin cleavage, membrane-pore formation, and inflammatory mediator release. Combining morphological observation, membrane-integrity measurements, biochemical indicators, pharmacological inhibition, and genetic validation can reduce the risk of misclassifying nonspecific cell lysis as pyroptosis and provide a reliable experimental basis for research on infectious, inflammatory, metabolic, and neoplastic diseases.

References

[1] Fang Y, et al. Pyroptosis: A New Frontier in Cancer. Biomed Pharmacother. 2020;121:109595.

[2] Fuchs Y, et al. Live to Die Another Way: Modes of Programmed Cell Death and the Signals Emanating From Dying Cells. Nat Rev Mol Cell Biol. 2015;16(6):329-344.

[3] Bergsbaken T, et al. Pyroptosis: Host Cell Death and Inflammation. Nat Rev Microbiol. 2009;7(2):99-109.

[4] Jha S, et al. Inflammasome-Associated Nucleotide-Binding Domain, Leucine-Rich Repeat Proteins and Inflammatory Diseases. J Immunol. 2009;183(12):7623-7629.

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