Unraveling Pyroptosis: Mechanisms and Detection Methods

The Fundamental Process of Pyroptosis

Pyroptosis is a form of PCD triggered by the activation of inflammasomes. Its primary function is to induce a robust inflammatory response, thereby protecting the host against microbial infections. Pyroptosis is initiated by inflammatory caspases with cleavage activity (Caspase-1, -4, -5, -11), and by cleaving the execution protein Gasdermin-D (GSDMD), it releases its N-terminal fragment on the cell membrane to form a pore, ultimately causing the cell to undergo lysis and death^[1].

The process of pyroptosis can be broadly divided into four sequential steps: initiation by danger signals and inflammasome assembly, caspase activation, Gasdermin-mediated membrane perforation, and amplification of inflammation leading to cell lysis.

1. Signal triggering and inflammasome assembly: Danger signals released upon pathogen invasion (e.g., bacterial toxins) or cellular damage (e.g., ATP, DNA) activate intracellular pattern recognition receptors (PRRs) such as NLRP3, AIM2. These receptors recruit adaptor proteins (e.g., ASC) and pro-caspase precursors (e.g., pro-caspase-1/4/5/11), forming a multi-protein complex known as the "inflammasome".

2. Caspase activation: Upon inflammasome assembly, caspases (e.g., caspase-1/4/5/11) are activated through self-cleavage. These activated caspases serve as the key "molecular switches" regulating pyroptosis.

3. Gasdermin cleavage and membrane perforation: Activated caspases specifically cleave Gasdermin family proteins (e.g., GSDMD), releasing their N-terminal domains. These domains oligomerize on the cell membrane to form pores, causing osmotic imbalance and the leakage of cellular contents.

4. Inflammation amplification and cell death: Following membrane perforation, intracellular pro-inflammatory factors (e.g., IL-1β, IL-18) are released into the extracellular space along with other cellular contents, recruiting immune cells and amplifying the inflammatory response. The cell eventually swells and ruptures, completing the pyroptosis process.

To highlight the unique features of pyroptosis, we compared it with other forms of PCD. Table 1 summarizes the morphological, biochemical, and molecular characteristics of major PCD types, illustrating how pyroptosis is distinct yet interconnected with other cell death pathways.

Different Mechanisms of Pyroptosis

Although the fundamental process of pyroptosis involves inflammasome assembly, caspase activity, and Gasdermin-mediated membrane perforation, different stimuli or cell types can engage distinct caspases or granzymes, resulting in multiple specific execution pathways.

Cell pyroptosis mainly occurs via four pathways: the canonical pathway mediated by Caspase-1, the non-canonical pathway mediated by Caspase-4/5/11, the cross-talk pyroptosis pathway mediated by Caspase-3/8, and the granzyme-mediated pathway mediated by GzmA/GzmB. Among them, Caspase-1 must be activated through cleavage by the inflammasome before it can cleave GSDMD^[7].

In the canonical pathway, stimuli such as viruses, bacteria, toxins, ATP or reactive oxygen species (ROS) activate the inflammasome, which in turn activates caspase-1. Active caspase-1 cleaves the precursors of IL-1β and IL-18, as well as GSDMD, releasing the N-terminal domain (GSDMD-NT). GSDMD-NT forms membrane pores, leading to inflammatory responses and pyroptosis. Canonical inflammasome-mediated pyroptosis primarily occurs in immune cells and serves as a host defend mechanism against pathogen infections^[1][8].

The non-canonical pathway is initially independent of inflammasomes. Upon stimulation by lipopolysaccharide (LPS), activated Caspase-4/5/11 cleaves the GSDMD protein, causing GSDMD-N to punch holes in the cell membrane and inducing pyroptosis. Additionally, the N-terminal fragment of GSDMD activates the NLRP3 inflammasome, further promoting Caspase-1 activation and maturation of IL-1β and IL-18^[1][8].

Caspase-3, a classical apoptotic marker, specifically cleaves GSDME, leading to mitochondrial damage, apoptosis, and pyroptosis^[9].

Caspase-8 mediates apoptosis and necroptosis via RIPK1/MLKL. However, when the Yersinia effector YopJ of Yersinia and its mimic 5z7 inhibit RIPK1 and activate TLR4, Caspase-8 mediates pyroptosis instead. TLR4 recruits the RIPK1/Caspase-8/FADD complex to Rag-Ragulator, activating Caspase-8 and cleaving GSDMD. Additionally, α-ketoglutarate-induced ROS can recruit Caspase-8 via the death receptor DR6, leading to GSDMC activation and pyroptosis^[10].

Granzyme A (GZMA) is the most abundant serine protease in cytotoxic lymphocytes and is a key mediator of cell death. In this pathway, GZMA or GZMB derived from cytotoxic lymphocytes can respectively cleave GSDMB or GSDME, forming membrane pores and inducing pyroptosis. GzmB also rapidly activates caspase-3 in target cells, subsequently activating GSDME and promoting extensive pyroptosis. Both GzmA and GzmB enter target cells via perforin: GzmA hydrolyzes GSDMB, while GzmB directly activates GSDME^[7][11].

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Detection Methods for Pyroptosis

Understanding the diverse molecular pathways of pyroptosis provides a foundation for monitoring this form of cell death. To study pyroptosis in vitro and in vivo, researchers use various methods to detect and quantify pyroptotic cell death, typically focusing on two aspects: cell phenotype and biological function.

TEM, transmission electron microscopy; SEM, scanning electron microscope; GSDM, gasdermin; AFM, atomic force microscopy; GzmA, granzyme A; GzmB, granzyme B.

To capture pyroptosis at the cellular level, researchers first examine characteristic morphological changes using microscopy and staining techniques. Figure 4 illustrates representative phenotypic features observed during pyroptosis.

(A-B) Cellular morphological features during pyroptosis, apoptosis, and necroptosis;(C) Representative microscopic images of SCC7 cells after Cisplatin (DDP) (HY-17394) treatment;(D) Representative transmission electron microscopy images of SCC7 cells treated with DDP.

During the early stage, apoptotic cells swell and expand, forming bubble-like protrusions on the membrane. In the later stage, pores appear in the cell membrane, compromising its integrity, and the cells collapse and rupture. At this point, the nucleus remains centrally located. As morphology changes, the nucleus shrinks and the DNA fragmentation occurs^[12].

Morphological changes can be observed using optical microscopy or scanning/transmission electron microscopy. Membrane integrity can also be assessed with fluorescence staining techniques: the plasma membrane of pyroptotic cells becomes more permeable, allowing nucleic acid dyes that normally cannot cross the cell membrane to enter the cell and stain the cell nucleus, emitting corresponding fluorescence.

GSDM proteins also induce mitochondria damage during pyroptosis. Activated GSDM binds to mitochondrial cardiolipin, forming mitochondrial pores that disrupt the double-membrane structure, leading to ROS production, altered oxidative phosphorylation (OXPHOS), release of cytotoxic mediators, and mitochondrial autophagy. Notably, mitochondrial damage precedes plasma membrane rupture, allowing the early stage of pyroptosis to be detected through mitochondrial phenotypic analysis^[13].

Phenotypic changes in cells provide early visual cues of pyroptosis, while functional assays capture the underlying molecular events.

(A and B) Relative mRNA levels of NLRP3, caspase-1, GSDMD, GSDMD-C, IL-1β, IL-18 were measured by qRT-PCR; (C and D) Protein levels of NLRP3, caspase-1 p45/p20, GSDMD, GSDMD-C, IL-1β p31/ p17, and IL-18 was measured by Western blot.

The GSDM protein and inflammatory factors in pyroptotic cells are activated and cleaved, leading to increased mRNA expression and elevated levels of their active protein forms. when pyroptosis is inhibited, these indicators correspondingly decrease.

• Common indicators: GSDM-B/C/D/E (N-GSDM, C-GSDM), Caspase-1/3/4/5/8/11, NLRP3 inflammasome, and increased levels of mature IL-1β (p17) and IL-18.

Activated Caspases proteins cleave GSDM family members and the precursors of IL-1β/IL-18, generating the pore-forming GSDM-N fragment and the mature form of IL-1β/IL-18.

• Common indicators: Detection of N-GSDM and C-GSDM fragments, as well as active/mature IL-1β/IL-18.

During pyroptosis, rupture of the plasma membrane causes intracellular components to leak into the extracellular environment. For instance, lactate dehydrogenase (LDH) is released upon membrane disruption. Measuring soluble extracellular biomarkers such as LDH in culture supernatants or plasma helps determine whether pyroptosis has occurred.

• Common indicators: Increased extracellular levels of HMGB1, ATP, LDH, ROS, and mature IL-1β/IL-18.

Pyroptosis is a form of programmed cell death that relies on immune processes involved in antimicrobial defense and inflammation. It can be executed through canonical and non-canonical pathways mediated by caspases. It can be detected by both phenotypic changes, such as cell swelling and membrane perforation, and functional markers, including caspase activation and release of inflammatory mediators. Understanding pyroptosis provides valuable insights for research in cancer, inflammatory diseases, and immunotherapy. Further development of precise strategies to modulate pyroptosis and deeper exploration of its conserved mechanisms across species will be essential for advancing translational applications^[15].

Note: MCE can provide products for research use only. We do not sell to patients.

Note: MCE can provide products for research use only. We do not sell to patients.