Updated insights into the molecular networks for NLRP3 inflammasome activation

Abstract

Over the past decade, significant advances have been made in our understanding of how NACHT-, leucine-rich-repeat-, and pyrin domain-containing protein 3 (NLRP3) inflammasomes are activated. These findings provide detailed insights into the transcriptional and posttranslational regulatory processes, the structural-functional relationship of the activation processes, and the spatiotemporal dynamics of NLRP3 activation. Notably, the multifaceted mechanisms underlying the licensing of NLRP3 inflammasome activation constitute a focal point of intense research. Extensive research has revealed the interactions of NLRP3 and its inflammasome components with partner molecules in terms of positive and negative regulation. In this Review, we provide the current understanding of the complex molecular networks that play pivotal roles in regulating NLRP3 inflammasome priming, licensing and assembly. In addition, we highlight the intricate and interconnected mechanisms involved in the activation of the NLRP3 inflammasome and the associated regulatory pathways. Furthermore, we discuss recent advances in the development of therapeutic strategies targeting the NLRP3 inflammasome to identify potential therapeutics for NLRP3-associated inflammatory diseases. As research continues to uncover the intricacies of the molecular networks governing NLRP3 activation, novel approaches for therapeutic interventions against NLRP3-related pathologies are emerging.

Keywords: Inflammatory disease; Licensing; NLRP3 inflammasome; Post-translational modification (PTM); Pyroptosis; Spatiotemporal.

Fig. 1

Transcriptional regulation of NLRP3 expression. The activation of TLRs by DAMPs, PAMPs, and pro-inflammatory cytokines such as TNF and IL-1β triggers NF-κB activation, which subsequently induces the transcription of the NLRP3 gene. This NF-κB-dependent transcription is mediated by FADD and caspase-8. During IAV infection, NLRP3 transcription is also upregulated via the ERK/c-Jun/AP-1 signaling pathway. In response to α-synuclein, Fyn kinase regulates PKCδ-mediated NF-κB activation, thereby promoting NLRP3 expression. Furthermore, atheroprone flow activates the NLRP3 inflammasome through SREBP2 activation. Under hypertonic stress, NFAT5 functions as a transcription factor to enhance NLRP3 expression. In endothelial cells exposed to high glucose conditions, ELF3 interacts with SET8 to regulate NLRP3 transcription. Conversely, during TCDD exposure, AhR suppresses NLRP3 expression. In models of LPS-induced septic shock and DSS-induced colitis, GSNOR modulates NLRP3 expression by reducing S-nitrosylated MAPK14 levels. Under hypoxic conditions, the accumulation of HIF-1α inhibits NLRP3 transcription by downregulating mTOR signaling and promoting autophagy, particularly in the context of DSS-induced colitis. AhR aryl hydrocarbon receptor, DAMP damage-associated molecular pattern, DSS dextran sodium sulfate, ELF3 transcription factor E74-like factor 3, FADD fas-associated death domain, GSNOR S-nitrosoglutathione reductase, HIF-1α hypoxia-inducible factor 1-alpha, IAV influenza A virus, LPS lipopolysaccharide, MAPK14 mitogen-activated protein kinase 14, mTOR mammalian target of rapamycin, NFAT5 nuclear factor of activated T cells 5, PAMP pattern-associated molecular pattern, PKCδ protein kinase Cδ, RNS reactive nitrogen species, SNO S-nitrosation, SREBP2 sterol regulatory element-binding protein2, TCDD 2,3,7,8-tetrachlorodibenzo-p-dioxin, TLR toll-like receptor, TNF tumor necrosis factor, TNFR tumor necrosis factor receptor

Fig. 2

Second signal of NLRP3 inflammasome activation. A Perturbation of ion homeostasis perturbation. Membrane damage induced by Mycobacterium tuberculosis and TLR4 activation triggered by ornithine lead to K⁺ efflux. In addition, Ca²⁺ influx through the mechanosensitive channel PIEZO is converted into K⁺ efflux via KCNN4, thereby promoting NLRP3 inflammasome activation following stimulation with LPS and Yoda1. K⁺ efflux is further facilitated by various ion channels, including Kv1.3, KCa3.1, and TREK-1, ultimately contributing to NLRP3 activation. HBV activates the NLRP3 inflammasome through both K⁺ efflux and Na⁺ influx. Moreover, extracellular histones induce Ca²⁺ influx and recruit TWIK2 to the plasma membrane, resulting in K⁺ efflux through the TWIK2 channel. Extracellular ATP, released via pannexin-1 channels, binds to P2X7 receptors, leading to both Ca²⁺ influx and K⁺ efflux. Subsequently, K⁺ efflux promotes Cl⁻ efflux through CLIC1 and CLIC4, further contributing to NLRP3 inflammasome activation. Additionally, Ca²⁺ influx via TRPM2 induces mtROS production, providing an additional activation signal. VRAC activation, ATP release, and subsequent P2YR activation also participate in this process. Together, these ionic fluxes converge to promote NLRP3 inflammasome activation. B Perturbation of mitochondrial homeostasis. Extracellular mtDNA, SFTSV, forchlorfenuron, and aristolochic acid I have been shown to induce mitochondrial dysfunction. Gasdermin processing is essential for the release of mtDNA into the cytosol. Imiquimod inhibits mitochondrial complex I of the ETC and NQO2, leading to the production of mtROS. In contrast, PCr, generated through ETC activity, helps maintain intracellular ATP levels. ROS generated via FADDosome induction, along with ox-mtDNA produced through the TLR4–CMPK2 signaling axis, translocate to the cytosol through the mPTP and VDAC, where they trigger NLRP3 inflammasome activation. Furthermore, cardiolipin interacts with NLRP3 to promote inflammasome assembly. The recruitment of pro-IL-1α to mitochondrial cardiolipin impairs mitophagy and further enhances NLRP3 inflammasome activation during LPS stimulation. C Perturbation of lysosomal homeostasis. Various stimuli—including imatinib, masitinib, LL-37, LLME, LPC, Candida albicans, H-ferritin, nicotine, particulate matter and crystals, carbon nanotubes, and lecithinase from Clostridium perfringens—induce lysosomal damage. This damage leads to the release of cathepsins into the cytosol, which subsequently triggers K⁺ efflux and activates the NLRP3 inflammasome. ATP adenosine triphosphate, Casp8 caspase-8, CLIC chloride intracellular channels, CMPK2 cytidine monophosphate kinase 2, ER endoplasmic reticulum, ETC electron transport chain, FADD fas-associated death domain, GSDMD gasdermin D, GSDME gasdermin E, HBV hepatitis B virus, H-ferritin, heavy chain-ferritin, IP3R2 inositol 1,4,5-trisphosphate receptor type 2, KCNN4 potassium–calcium-activated channel subfamily N member 4, LLME Leu-Leu-O-methyl ester, LPC lysophosphatidylcholine, LPS lipopolysaccharide, mPTP mitochondrial permeability transition pore, mtDNA mitochondrial DNA, N- N terminal, NQO2 quinone oxidoreductase 2, ox-mtDNA oxidized-mitochondrial DNA, PCr phosphocreatine, ROS reactive oxygen species, RTK receptor tyrosine kinase, SFTSV severe fever with thrombocytopenia syndrome virus, TLR4 toll-like receptor 4, TRPM2 transient receptor potential melastatin 2, VDAC voltage-dependent anion channel, VRAC volume-regulated anion channel

Fig. 3

Schematic diagram depicting the phosphorylation or dephosphorylation of the NLRP3 inflammasome. NLRP3 consists of three domains, including the PYD, NACHT, and LRR domains. AKT phosphorylates S5(h)/3(m), hindering TRIM31-associated ubiquitination and degradation as well as NLRP3 oligomerization. PP2A dephosphorylates S5(h)/3(m), facilitating NLRP3 assembly. PTEN dephosphorylates Y32(h)/30(m), triggering NLRP3 inflammasome activation. BTK phosphorylates Y136(h)/132(m), Y140(h)/136(m), Y143(h)/145(m), and Y168(h)/ 164(m), subsequently activating the NLRP3 inflammasome. BTK also suppresses the activation of PP2A, resulting in the inhibition of S5(h)/3(m) dephosphorylation under TLR2/4 priming conditions. EphA2 phosphorylates Y136(h)/132(m), thereby inhibiting inflammasome activation. JNK1 phosphorylates S198(h)/194(m), facilitating the deubiquitination of NLRP3 by BRCC3. The phosphorylation of S295(h)/291(m) by PKA inhibits the NLRP3 inflammasome through ubiquitination. PKD promotes S295(h)/293(m) phosphorylation, releasing NLRP3 for ASC assembly. Pak1 phosphorylates T659 (h/m), leading to the NLRP3–NEK7 interaction, IL-1β maturation, and bacterial clearance. The phosphorylation of NLRP3 at S728(h)/725(m) by MINK1 is required for the priming and activation of the NLRP3 inflammasome. CK1α phosphorylates S806(h)/803(m) and then recruits NEK7 to NLRP3, thus activating the NLRP3 inflammasome. The dephosphorylation of NLRP3 at Y861(h)/859(m) by PTPN22 suppresses inflammasome activation. Lyn kinase phosphorylates Y918(m), resulting in the ubiquitination and subsequent degradation of NLRP3. ASC apoptosis-associated speck-like protein containing a caspase-recruitment domain, BRCC3 BRCA1/BRCA2-containing complex subunit 3, BTK Bruton’s tyrosine kinase, CK1α casein kinase 1 alpha, EphA2 ephrin type-A receptor 2, h human, JNK1 c-Jun N-terminal kinase 1, LRR leucine-rich repeat domain, m mouse, MINK1 misshapen/Nck-interacting kinase-related kinase 1, NEK7 NIMA-related kinase 7, NLRP3 NACHT- leucine-rich-repeat- and pyrin domain-containing protein 3, P phosphorylation, Pak1 p21-activated kinase 1, PKA protein kinase A, PKD protein kinase D, PP2A protein phosphatase 2A, PTEN phosphatase and tensin homolog, PTPN22 protein tyrosine phosphatase nonreceptor 22, PYD pyrin domain, TRIM31 tripartite motif-containing protein 31, TLR toll-like receptor, Ub ubiquitination

Fig. 4

Posttranslational modification of NLRP3 inflammasome components via ubiquitination and deubiquitination. Ubiquitination is a reversible PTM in which Ub is covalently attached to lysine (K) residues of target proteins, particularly involving K48-, K63-, and K27/K29-linked ubiquitination. Pellino-1 promotes NLRP3 inflammasome activation by attaching K63-linked ubiquitin chains to K55 of ASC, whereas USP50 counteracts this process by removing K63-linked chains from ASC, thereby inhibiting activation. HUWE1 and MARCH5 catalyze K27-linked polyubiquitination of NLRP3 at unknown sites within the PYD domain and at K324 and K430 in the NACHT domain, respectively. Ubc13 facilitates K63-linked polyubiquitination of NLRP3 at K565 and K687, while OTUD6A removes K48-linked ubiquitin chains from K430 and K689. Additionally, ABRO1 enhances NLRP3 inflammasome activation by competing with WWP2 for binding to BRCC3. TET2 deficiency promotes JNK1 activation and BRCC3-mediated deubiquitination, further contributing to NLRP3 activation. TRIM50 inhibits NLRP3 ubiquitination, thereby inducing inflammasome activation. Similarly, TRIM62 and RNF31 enhance the K63-linked ubiquitination of NLRP3, promoting its activation. HSPA8 facilitates the degradation of SKP2, which in turn decreases NLRP3 ubiquitination and leads to inflammasome activation. Conversely, STING negatively regulates NLRP3 activation by reducing both K48- and K63-linked ubiquitination of NLRP3. Several deubiquitinating enzymes (DUBs), including USP30, USP9X, USP14, UCHL5, USP47, USP7, and ZNF70, promote NLRP3 inflammasome activation by removing ubiquitin chains from NLRP3. Specifically, USP47 also enhances activation by suppressing miR-138-5p through a ZNF883-dependent mechanism. In contrast, TRIM31 mediates K48-linked polyubiquitination of NLRP3 at K496, targeting it for proteasomal degradation. YAP facilitates NLRP3 degradation via β-TRCP1-mediated K27-linked ubiquitination at K380. TRIM65 induces both K48- and K63-linked ubiquitination of NLRP3, disrupting the NEK7–NLRP3 interaction. TNFAIP3 promotes K48-linked ubiquitination of NEK7 at K189 and K293, leading to its proteasomal degradation, while METTL3 enhances the degradation of TNFAIP3 transcripts. Parkin and CHIP mediate K48-linked polyubiquitination of NLRP3, resulting in its proteasomal degradation in microglia and other contexts, respectively. Furthermore, TRIM24, TRIM40, and TRIM59 promote NLRP3 ubiquitination. The E3 ligase gp78/Insig-1 mediates K48-/K63-linked ubiquitination, thereby inhibiting NLRP3 oligomerization. STAMBP restrains inflammasome activity by removing K63-linked polyubiquitin chains from NLRP3. SLC25A3 promotes NLRP3 ubiquitination, disrupting the NLRP3–NEK7 interaction. Finally, TIMP2 enhances NLRP3 ubiquitination and facilitates its degradation via the autophagy-lysosome pathway. ABRO1 Abraxas brother protein 1, ASC apoptosis-associated speck-like protein containing a CARD, BRCC3 BRCA1/BRCA2-containing complex subunit 3, CARD caspase recruitment domain, β-TrCP1 β-transducin repeat-containing E3 ubiquitin protein ligase 1, Drp1 dynamin-related protein 1, DUB, deubiquitinating enzyme; HSPA8 heat shock protein (HSP) family A member 8, HUWE1 HECT, UBA, and WWE domain-containing E3 ubiquitin protein ligase 1, Insig1 insulin induced gene 1, JNK1 c-Jun N-terminal kinase 1, LRR leucine-rich repeat, MARCH5 membrane-associated ring-CH-type finger 5, METTL3 methyltransferase-like 3, NACHT central NAIP, CIITA, HET-E, and TP1, NEK7 NIMA-related kinase 7,NLRP3 NOD-like receptor family, pyrin domain containing 3, OTUD6A OTU deubiquitinase 6A, PGAM5 phosphoglycerate mutase 5, PYD pyrin domain, RNF31 RING finger protein 31, SERTAD1 SERTA domain-containing 1, SKP2 S-phase kinase-associated protein 2, SLC25A3 solute carrier family 25 member 3, STAMBP STAM-binding protein, STING stimulator of interferon genes, TET2, Tet methylcytosine dioxygenase 2, TNFAIP3 tumor necrosis factor, alpha-induced protein 3 TRIM tripartite motif-containing, Ubc13 ubiquitin-conjugating enzyme E2 13, UCHL5 ubiquitin c-terminal hydrolase L5, USP ubiquitin-specific peptidase, WWP WW domain-containing E3, ZNF70 zinc finger protein 70

Fig. 5

Activation of NLRP3 inflammasome complex through acetylation or deacetylation. Posttranslational modifications via acetylation and deacetylation critically regulate NLRP3 inflammasome activity across various pathological conditions. A The acetyltransferase KAT5 directly acetylates NLRP3 at K24. B Rheumatoid arthritis, acute respiratory distress syndrome, and colorectal cancer. In rheumatoid arthritis, KAT2A promotes NLRP3 inflammasome activation by suppressing NRF2 transcriptional activity and downregulating antioxidant signaling. In acute respiratory distress syndrome, increased SPHK2 enhances S1P production, which in turn increases p53 acetylation, contributing to inflammasome activation. In colorectal cancer patients with poor prognosis, the upregulated deacetylase HDAC2 represses H3K27 acetylation, thereby modulating NLRP3 transcription through the BRD4/p-p65 complex. C Viral infections. During respiratory syncytial virus infection, histone hyperacetylation drives the upregulation of ORMDL3 expression, which in turn regulates NLRP3 expression levels. D Microglia in neurodegeneration, inflammatory cells, and endothelial cells. In microglia with elevated tau levels, tau protein acetylates the PYD domain of NLRP3 at K21, K22, and K24 residues. Additionally, PHGDH-mediated serine biosynthesis regulates the acetylation status of both NLRP3 and ASC via NAD+-dependent modulation of SIRT1 and SIRT3 in inflammatory macrophages. In septic macrophages, upregulated GITR competitively binds to MARCH7 instead of NLRP3, leading to the degradation of the deacetylase SIRT2 and resulting in increased NLRP3 acetylation. In endothelial cells, SIRT6 suppresses ASC acetylation, thereby inhibiting inflammasome assembly. E Myocardial fibrosis. In myocardial fibrotic cells, acetylation of HADHa at K255 promotes NLRP3 inflammasome activation. ASC adaptor apoptosis-associated speck-like protein containing a caspase-recruitment domain, BRD4 Bromodomain-containing protein 4, GITR glucocorticoid-induced TNFR-related, HADHa hydroxyacyl-CoA dehydrogenase, HDACs histone deacetylases, HDAC2 histone deacetylase 2, KAT2A lysine acetyltransferase 2A, KAT5 lysine acetyltransferase 5, MARCH7 membrane-associated ring-CH-type finger 7, NRF2 nuclear factor erythroid 2-related factor 2, ORMDL3 orosomucoid-like protein 3, PYD pyrin domain, S1P sphingosine-1-phosphate, SIRT2 sirtuin 2, SIRT6 sirtuin 6, SPHK2 sphingosine kinase 2

Fig. 6

Posttranslational modification of NLRP3 inflammasome components by SUMOylation and ISGylation. A The E3 ligase MAPL mediates SUMOylation of NLRP3 at K689 in a UBC9-dependent manner, generating a polymeric SUMO2/3 chain rather than a single SUMO1 moiety, thereby impairing NLRP3 activation. However, the deSUMOylases SENP6 and SENP7 counteract this modification, enhancing NLRP3 inflammasome activation. Additionally, NLRP3 interacts with UBC9, which promotes SUMO1-mediated SUMOylation of NLRP3 at residue K204, resulting in inflammasome activation, whereas SENP3 deSUMOylates NLRP3 to inhibit its activity. Ursolic acid inhibits SUMO1-mediated SUMOylation of NLRP3, thereby preventing its activation and limiting excessive inflammation. Furthermore, TRIM28 interacts with NLRP3 to facilitate SUMOylation at an as-yet unidentified site, which inhibits K48-linked ubiquitination and subsequent proteasomal degradation of NLRP3. B ISGylation is another PTM in which ISG15 is conjugated to target proteins through an enzymatic cascade involving E1 (UBE1L), E2 (UBCH8) and E3 (HERCs) ligases. TLR activation and SARS-CoV-2 infection upregulate HERC expression (HERC5 in humans and HERC6 in mice), allowing ISG15 to conjugate to NLRP3 at K799. This inhibits K48-linked ubiquitination, which leads to increased NLRP3 inflammasome activation. HERC HECT domain- and RCC1-like domain-containing proteins, IGF15 interferon-stimulated gene 15, LRR leucine-rich repeat, MAPL mitochondrial-anchored protein ligase, NACHT central NAIP, CIITA, HET-E, and TP1, PYD pyrin domain, SENP Sentrin/SUMO-specific protease, SUMO small ubiquitin-like modifier, TLRs toll-like receptors, TRIM28 tripartite motif-containing protein 28, UBC9 ubiquitin-conjugating enzyme E2 9

Fig. 7

Palmitoylation of NLRP3 regulates inflammasome activation. A Palmitoylation at C130(h)/C126(m), C898(m), C958(h)/C955(m), and the teleost-specific sites C946(Lc NLRP3)/C1037(Dr NLRP3) facilitates NLRP3 trafficking to PI4P-enriched, dTGN membranes, promoting inflammasome activation. Among these, palmitoylation at C958(h)/C955(m) stabilizes the double-ring, cage-like oligomer during the priming phase. Palmitoylation at C419(h) and C837/838(h) enhances NEK7 interaction, with C837/838(h) further promoting ASC recruitment. These modifications are catalyzed by the palmitoyltransferases zDHHC1, zDHHC5, zDHHC7, zDHHC17, and zDHHC18. FASN supports palmitoylation at C898(m), a process inhibited by cerulenin, while ABHD17A depalmitoylates C837/838(h). Palmitoylation inhibitors such as 2-BP (a general and non-selective palmitoylation inhibitor), MY-D4 (a zDHHC-targeting tool compound), and disulfiram (a thiol-reactive compound that blocks palmitoylation) suppress NLRP3 inflammasome activation. B Additionally, palmitoylation at C6(m)/C8(h) protects NLRP3 from lysosome-mediated autophagic degradation, stabilizing the intracellular NLRP3 protein pool and indirectly promoting inflammasome readiness. In diabetic wound healing, the depalmitoylase PPT1 antagonizes this stabilization, whereas phenylpyruvate inhibits PPT1 activity, thereby enhancing NLRP3 persistence. C Conversely, palmitoylation at C844(h)/C841(m), mediated by zDHHC12, facilitates HSC70 binding and directs NLRP3 to LAMP2A-dependent chaperone-mediated autophagy, thereby restraining excessive inflammasome activation. 2-BP 2-bromopalmitate, ABHD17A alpha/beta hydrolase domain containing 17A, Dr Danio rerio, dTGN dispersed trans-Golgi network, FASN fatty acid synthase, HSP70 heat shock protein 70, LAMP2A lysosome-associated membrane protein 2A, Lc, Larimichthys crocea, MTOC microtubule-organizing center, PI4P phosphatidylinositol-4-phosphate, PPT1 palmitoyl-protein thioesterase 1, zDHHC zinc finger DHHC (Asp-His-His-Cys motif-containing) domain-containing palmitoyltransferase

Fig. 8

NLRP3 inflammasome activation is tightly regulated by subcellular localization dynamics. MAMs serve as key signaling hubs for NLRP3 assembly by promoting ER-mitochondria tethering and local calcium flux. Mitochondrial ASC binds to NLRP3 on the ER through acetylated α-tubulin- and dynein-dependent mitochondrial transport, facilitating the formation of MAMs. ER-mitochondria contacts mediated by ORMDL3 and Fis1, as well as Ca²⁺ flux via VDAC oligomerization (triggered by HK2 dissociation), further promote NLRP3 assembly. NEK7 and PLK1, located at the MTOC, are also involved in NLRP3 inflammasome activation. NLRP3 recruited to the dTGN can be transported to the MTOC by HDAC6 and MARK4. Furthermore, NLRP3 interacts with SREBP2 and SCAP to form a ternary complex, which translocates to the Golgi apparatus near the mitochondria, facilitating optimal inflammasome assembly and activation of the NLRP3 inflammasome. PKD, a key effector of DAG, induces MAMs to localize near Golgi membranes, facilitating NLRP3 oligomerization and activation. Activated GSK3β phosphorylates PI4K2A, enhancing PI4P production at the TGN, which facilitates NLRP3 localization and oligomerization. HDAC6 enhances the binding of NLRP3 to Lamtor1 located on the lysosome, leading to NLRP3 inflammasome activation. Ac acetylated, Ca calcium, DAG diacylglycerol, dTGN dispersed trans-Golgi network, ER endoplasmic reticulum, Fis1 fission 1, GSK3β glycogen synthase kinase 3β, HDAC6 dynein adaptor histone deacetylase 6, HK2 hexokinase 2, MAM mitochondria-associated membrane, MARK4 microtubule-affinity regulating kinase 4, MTOC microtubule-organizing center, NEK7 NIMA-related kinase 7, ORMDL3 orosomucoid-like protein 3, PI4K2A phosphatidylinositol 4-kinase type 2 alpha, PKD protein kinase D, PLK1 polo-like kinase 1, SCAP SREBP cleavage-activating protein, SREBP sterol regulatory element binding protein, VDAC voltage-dependent anion channel