Inflammasomes are important cytoplasmic signaling complexes of the innate immune system that sense infection, cellular injury, and metabolic abnormalities. They activate inflammatory Caspases, promote the maturation of IL-1β and IL-18, and drive Gasdermin-mediated pyroptosis. Inflammasomes such as NLRP3, AIM2, and NLRC4 have distinct upstream sensing mechanisms. Among them, NLRP3 responds to diverse danger signals including ionic-homeostasis disturbances, crystal deposition, and organelle injury and is therefore a major target in current immunoinflammatory research.

Keywords: inflammasome; NLRP3; Caspase-1; GSDMD; pyroptosis; IL-1β; AIM2; innate immunity

1 Composition and Major Types of Inflammasomes

1.1 Basic Composition

Canonical inflammasomes are composed of a cytoplasmic pattern-recognition receptor, the adaptor protein ASC, and an inflammatory Caspase. After activation, the sensor undergoes conformational changes and oligomerization and recruits ASC and pro-Caspase-1 through PYD or CARD domains to form a higher-order signaling complex. Mature Caspase-1 cleaves pro-IL-1β, pro-IL-18, and GSDMD, thereby converting danger-signal recognition into inflammatory-cytokine release and pyroptotic cell death.

1.2 Priming and Activation

(1) Priming

Receptors such as TLRs, TNFR, and IL-1R can increase NLRP3 and pro-IL-1β expression through NF-κB, establishing the molecular basis for subsequent inflammasome assembly. Increased NLRP3 protein mainly reflects enhanced priming and cannot alone demonstrate that the inflammasome has been activated.

(2) Activation

Stimuli such as ATP, Nigericin, crystalline particles, cytoplasmic DNA, and bacterial structures can further promote sensor activation, ASC aggregation, and inflammatory-Caspase maturation. Inflammasome activation should generally be evaluated by combining ASC speck formation, Caspase-1 maturation, IL-1β release, and GSDMD cleavage.

1.3 Major Inflammasomes

Table 1 Major Inflammasomes and Their Functional Positioning

2 Activation Mechanisms of the NLRP3 Inflammasome

Figure 1. Two-signal activation of the NLRP3 inflammasome and downstream inflammatory effector responses

2.1 Priming Signals

Stimuli such as LPS can increase NLRP3 and pro-IL-1β expression through the TLR4-NF-κB pathway. TNF-α- and IL-1-related signals can also generate priming through NF-κB. This stage mainly changes the intracellular abundance of inflammatory proteins and the cellular state, thereby providing the conditions required for a second signal to trigger inflammasome assembly.

2.2 Activation Signals

(1) K⁺ Efflux

Reduced intracellular K⁺ is an important process shared by many NLRP3 stimuli. Extracellular ATP can promote K⁺ efflux through P2X7-associated changes in ion permeability, whereas Nigericin rapidly disrupts the K⁺ gradient through ion exchange. Both are therefore commonly used to establish NLRP3 activation models.

(2) Lysosomal Injury

After phagocytosis, particles such as Monosodium Urate crystals, Cholesterol crystals, and Silica can damage lysosomal membranes, allowing Cathepsins and other danger signals to enter the cytoplasm. These stimuli are suitable for models of gout, atherosclerosis, and particulate inflammation.

(3) Mitochondrial Stress

Abnormal mitochondrial membrane potential, increased ROS, mtDNA release, and impaired mitochondrial quality control can lower the threshold for NLRP3 activation. These changes often coexist with altered ionic homeostasis and metabolic reprogramming, so increased ROS alone is insufficient to attribute a response to NLRP3.

2.3 NLRP3-ASC-Caspase-1 Assembly

Activated NLRP3 oligomerizes and recruits ASC. ASC then forms higher-order aggregates that bring pro-Caspase-1 molecules into close proximity and promote their activation. ASC specks, ASC oligomers, and NLRP3-ASC interactions can be used to evaluate inflammasome assembly, whereas Caspase-1 maturation indicates that the complex has entered the functional-output stage.

2.4 GSDMD and Pyroptosis

Caspase-1 cleavage of GSDMD releases GSDMD-N, which binds the plasma membrane and forms pores, disrupting ionic gradients, increasing membrane permeability, and facilitating IL-1β and IL-18 release. When pore formation exceeds the capacity of membrane-repair mechanisms, cellular swelling and terminal membrane rupture may occur. GSDMD cleavage is therefore a key node linking inflammasome activation to pyroptosis.

3 Other Major Inflammasome Pathways

3.1 AIM2 Inflammasome

AIM2 recognizes cytoplasmic double-stranded DNA through its HIN domain and recruits ASC through PYD-PYD interactions. ASC subsequently recruits pro-Caspase-1 through CARD-CARD interactions and promotes its activation. Bacterial or DNA-virus infection, abnormal leakage of nuclear DNA, and mitochondrial DNA release can all establish conditions for AIM2 activation. Because cytoplasmic DNA can also activate cGAS-STING, Caspase-1/GSDMD and TBK1/IRF3 signaling can be analyzed together to distinguish the two pathways.

3.2 NLRC4 Inflammasome

NAIP proteins recognize bacterial Flagellin or components of type III secretion systems and promote NLRC4 oligomerization. NLRC4 contains a CARD domain and can directly recruit Caspase-1, while ASC can further enhance cytokine maturation. This pathway is mainly used to study intracellular bacterial infections such as Salmonella and Legionella.

3.3 Noncanonical Inflammasome

Human Caspase-4 and Caspase-5 and mouse Caspase-11 respond to cytoplasmic LPS and directly cleave GSDMD. K⁺ efflux caused by GSDMD pore formation can subsequently activate NLRP3-Caspase-1, allowing the noncanonical pathway to connect cytoplasmic LPS sensing, pyroptosis, and IL-1β/IL-18 maturation.

4 Major Disease-Research Directions for Inflammasomes

4.1 Infectious Inflammation

NLRP3, NLRC4, AIM2, and noncanonical inflammasomes respond to infection-associated cellular injury, bacterial structures, cytoplasmic DNA, and cytoplasmic LPS, respectively. Inflammasome activation can promote pathogen clearance and immune-cell recruitment, but excessive Caspase-1/GSDMD signaling may also amplify tissue injury. Infection models should therefore evaluate both pathogen control and inflammatory damage.

4.2 Metabolic and Sterile Inflammation

Monosodium Urate crystals, Cholesterol crystals, lipid accumulation, and mitochondrial abnormalities can provide persistent NLRP3 stimulation, allowing IL-1β-associated inflammation to contribute to gout, atherosclerosis, and certain metabolic diseases. The central objective in such studies is to demonstrate whether the disease phenotype truly depends on the NLRP3-Caspase-1-GSDMD axis rather than merely observing increased inflammatory factors.

4.3 Neuroinflammation

Microglia can respond to ATP, protein aggregates, mitochondrial injury, and neuron-derived danger signals and generate NLRP3-associated inflammatory responses. Nervous-system models can combine analysis of NLRP3 assembly, IL-1β release, GSDMD cleavage, microglial activation, and neuronal injury to determine the mechanistic level at which inflammasomes contribute to neurodegenerative changes.

4.4 Ischemia and Tissue Injury

Ischemia-reperfusion can cause ATP release, increased ROS, mitochondrial injury, and release of necrotic-cell components, providing multiple stimuli for NLRP3 activation. Cardiac, cerebral, and renal ischemia models can all be used to investigate inflammasome-mediated sterile inflammation and secondary tissue injury.

5 Experimental Applications in Inflammasome Research

5.1 Classical NLRP3 Models

(1) LPS + ATP

LPS provides priming, whereas ATP promotes K⁺ efflux through P2X7-associated signaling. This model is suitable for evaluating NLRP3 assembly, Caspase-1 maturation, and IL-1β release.

(2) LPS + Nigericin

Nigericin rapidly alters the K⁺ gradient and generally produces robust NLRP3 activation, making it suitable for mechanistic studies and inhibitor screening. Excessive stimulation can cause nonspecific membrane injury, so treatment concentration and duration should be controlled.

(3) Crystal Models

Particulate stimuli such as Monosodium Urate or Cholesterol crystals activate NLRP3 through phagocytosis, lysosomal injury, and organelle stress and more closely reproduce sterile inflammatory environments such as gout and atherosclerosis.

5.2 Detection of Inflammasome Activation

(1) Complex Assembly

ASC speck immunofluorescence, ASC oligomerization, and NLRP3-ASC protein interactions can be used to analyze inflammasome assembly. Measurement of total NLRP3 protein alone cannot replace these functional indicators.

(2) Caspase-1 and Inflammatory Cytokines

Western blotting or activity probes can detect Caspase-1 maturation, whereas ELISA and Western blotting can detect IL-1β and IL-18. Intracellular pro-IL-1β should be distinguished from extracellular mature IL-1β to differentiate priming from complete inflammasome activation.

(3) Execution of Pyroptosis

GSDMD cleavage, PI/SYTOX uptake, LDH release, and real-time cell imaging can be used to evaluate pyroptosis. Changes in LDH release and membrane permeability are not specific to pyroptosis and should be interpreted together with GSDMD and inflammatory-Caspase indicators.

Table 2 Common Detection Indicators in Inflammasome Research

5.3 Inflammasome Inhibitor Research

(1) NLRP3 Inhibition

Candidate NLRP3 inhibitors can be cross-validated in ATP-, Nigericin-, and crystal-based models, while AIM2- or NLRC4-stimulation systems can be used to evaluate pathway selectivity. If a compound reduces both NLRP3 and pro-IL-1β expression, its effects on NF-κB-mediated priming should be excluded.

(2) Caspase-1 Inhibition

Caspase-1 inhibition generally reduces mature IL-1β and GSDMD cleavage, while upstream NLRP3 and ASC assembly may remain intact. This feature can help distinguish inhibition of inflammasome assembly from inhibition of downstream enzyme activity.

(3) GSDMD Inhibition

GSDMD inhibition mainly affects membrane pore formation and terminal pyroptosis, whereas upstream ASC aggregation and Caspase-1 activation may remain present. Layered analysis of upstream assembly, Caspase activity, and membrane permeability helps determine the specific site of action of candidate compounds.

6 Frequently Asked Questions

6.1 Does Increased NLRP3 Expression Indicate Inflammasome Activation?

No. Increased NLRP3 expression mainly reflects priming. Complete activation should additionally be evaluated through ASC aggregation, Caspase-1 maturation, IL-1β release, and GSDMD cleavage.

6.2 Can Increased IL-1β Prove NLRP3 Involvement?

No. AIM2, NLRC4, NLRP1, Pyrin, and other inflammasomes can also promote IL-1β maturation, while NF-κB can increase pro-IL-1β expression. Selective pharmacological or genetic methods are therefore required for pathway attribution.

6.3 Can LPS Alone Activate the Classical NLRP3 Inflammasome?

In classical two-signal models, LPS mainly provides priming. ATP, Nigericin, or another second signal is generally required to obtain robust Caspase-1 maturation, IL-1β release, and GSDMD cleavage.

Not necessarily. GSDMD cleavage indicates that the pore-forming program has been initiated, but cells may temporarily maintain membrane integrity through repair mechanisms. Terminal pyroptosis should be evaluated using membrane permeability, LDH release, and cellular morphology.

6.5 Can ATP and Nigericin Be Used Interchangeably?

No. Both can induce K⁺ efflux, but ATP depends on P2X7-associated membrane signaling, whereas Nigericin mainly alters ionic gradients directly. The stimulus should be selected according to the specific upstream mechanism being investigated.

6.6 Why Can Cell Death Still Occur After Caspase-1 Inhibition?

Cells may lose membrane integrity through other pathways involving Caspase-4/5/11, Caspase-8, GSDME, or RIPK3-MLKL. Complex inflammatory models therefore require combined analysis of different cell-death execution systems.

6.7 Is Inflammasome Research Limited to Macrophages?

No. Monocytes and macrophages are commonly used models, but microglia, neutrophils, dendritic cells, and certain epithelial cells can also form inflammasomes. Priming requirements and the extent of pyroptosis differ among cell types.

7 Products for Inflammasome Research

7.1 Products for Inflammasome Activation and Model Construction

7.2 NLRP3 and Inflammatory-Signaling Inhibitors

7.3 Caspase-1, GSDMD, and Related Pathway Research Products

7.4 P2X7-, Lysosome-, and Oxidative-Stress-Related Products

Inflammasomes connect danger-signal recognition with inflammatory Caspase activation, IL-1β/IL-18 maturation, and GSDMD-mediated pyroptosis. Experimental research should clearly define the inflammasome subtype under investigation and distinguish among priming, complex assembly, Caspase activation, and membrane pore formation to avoid attributing pathway activation solely on the basis of a single inflammatory cytokine or cell-death indicator.

References

[1] Man SM, et al. Regulation of inflammasome activation. Immunol Rev. 2015;265(1):6-21.

[2] Jiang Q, et al. Inflammasome and Its Therapeutic Targeting in Rheumatoid Arthritis. Front Immunol. 2022;12:816839.

[3] Bergsbaken T, et al. Pyroptosis: host cell death and inflammation. Nat Rev Microbiol. 2009;7(2):99-109.

[4] Paerewijck O, et al. The human inflammasomes. Mol Aspects Med. 2022;88:101100.

[5] Chen C, et al. Activation and Pharmacological Regulation of Inflammasomes. Biomolecules. 2022;12(7):1005.

[6] Zhang C, et al. A small molecule inhibitor MCC950 ameliorates kidney injury in diabetic nephropathy by inhibiting NLRP3 inflammasome activation. Diabetes Metab Syndr Obes. 2019;12:1297-1309.

For more related articles, please see below:

[1] Activation and monitoring of inflammatory vesicles

[2] How to Map the NF-κB Pathway and Choose Inhibitors: Bringing Inflammatory Transcriptional Output into a “Controllable Range” (Tables A–F)