Cryo-EM structures of the active NLRP3 inflammasome disc.

Inflammasomes are cytosolic innate immune complexes that activate caspase-1 following detection of pathogenic and endogenous dangers1-5, and NACHT-, leucine-rich repeat (LRR)- and pyrin domain (PYD)-containing protein 3 (NLRP3) is an inflammasome sensor of membrane damage highly important in regard to the induction of inflammation2,6,7. Here we report cryogenic electron microscopy structures of disc-shaped active NLRP3 oligomers in complex with adenosine 5'-O-(3-thio)triphosphate, the centrosomal NIMA-related kinase 7 (NEK7) and the adaptor protein ASC, which recruits caspase-1. In these NLRP3-NEK7-ASC complexes, the central NACHT domain of NLRP3 assumes an ATP-bound conformation in which two of its subdomains rotate by about 85° relative to the ADP-bound inactive conformation8-12. The fish-specific NACHT-associated domain conserved in NLRP3 but absent in most NLRPs13 becomes ordered in its key regions to stabilize the active NACHT conformation and mediate most interactions in the disc. Mutations on these interactions compromise NLRP3-mediated caspase-1 activation. The N-terminal PYDs from all NLRP3 subunits combine to form a PYD filament that recruits ASC PYD to elicit downstream signalling. Surprisingly, the C-terminal LRR domain and the LRR-bound NEK7 do not participate in disc interfaces. Together with previous structures of an inactive NLRP3 cage in which LRR-LRR interactions play an important role8-11, we propose that the role of NEK7 is to break the inactive cage to transform NLRP3 into the active NLRP3 inflammasome disc.

Probing the effect of NEK7 and cofactor interactions on dynamics of NLRP3 monomer using molecular simulation.

The NLRP3 inflammasome is a cytoplasmic complex that regulates the activation of inflammatory cytokines and, given its implication in a range of diseases, is an important therapeutic target. The cofactor ATP and the centrosomal kinase NEK7 are important for NLRP3 activation. Here we have constructed and simulated computational models of full-length monomeric NLRP3 to shed light on the importance of NEK7 and cofactor interactions for its conformation and dynamics in aqueous solution. We find that molecular dynamics simulation reproduces well the features of the recently published cryo-EM structure of the ADP-bound NLRP3-NEK7 complex; on the removal of NEK7, the NLRP3 molecule adopts a more compact closed form during simulations. Replacement of ADP by ATP promotes a rearrangement of hydrogen-bonding interactions, domain interfaces, and a degree of opening of the NLRP3 conformation. We also examine the dynamics of an acidic loop of the LRR domain of NLRP3, which samples in a region observed in the NEK7-bound cryo-EM structure but not in an oligomeric form of inactive NLRP3. During the molecular dynamics simulations of NLRP3, we find some plasticity in its topology that suggests access routes for ATP to the cofactor pocket not immediately evident from the existing NEK7-bound cryo-EM structure. These computed dynamical trajectories of NLRP3 provide insight into coordinates of deformation that may be key for cofactor binding and inflammasome activation.

Inhibitory Role of Berberine, an Isoquinoline Alkaloid, on NLRP3 Inflammasome Activation for the Treatment of Inflammatory Diseases.

The pyrin domain-containing multiprotein complex NLRP3 inflammasome, consisting of the NLRP3 protein, ASC adaptor, and procaspase-1, plays a vital role in the pathophysiology of several inflammatory disorders, including neurological and metabolic disorders, chronic inflammatory diseases, and cancer. Several phytochemicals act as promising anti-inflammatory agents and are usually regarded to have potential applications as complementary or alternative therapeutic agents against chronic inflammatory disorders. Various in vitro and in vivo studies have reported the anti-inflammatory role of berberine (BRB), an organic heteropentacyclic phytochemical and natural isoquinoline, in inhibiting NLRP3 inflammasome-dependent inflammation against many disorders. This review summarizes the mechanism and regulation of NLRP3 inflammasome activation and its involvement in inflammatory diseases, and discusses the current scientific evidence on the repressive role of BRB on NLRP3 inflammasome pathways along with the possible mechanism(s) and their potential in counteracting various inflammatory diseases.

Gasdermin D pore structure reveals preferential release of mature interleukin-1.

As organelles of the innate immune system, inflammasomes activate caspase-1 and other inflammatory caspases that cleave gasdermin D (GSDMD). Caspase-1 also cleaves inactive precursors of the interleukin (IL)-1 family to generate mature cytokines such as IL-1ß and IL-18. Cleaved GSDMD forms transmembrane pores to enable the release of IL-1 and to drive cell lysis through pyroptosis1-9. Here we report cryo-electron microscopy structures of the pore and the prepore of GSDMD. These structures reveal the different conformations of the two states, as well as extensive membrane-binding elements including a hydrophobic anchor and three positively charged patches. The GSDMD pore conduit is predominantly negatively charged. By contrast, IL-1 precursors have an acidic domain that is proteolytically removed by caspase-110. When permeabilized by GSDMD pores, unlysed liposomes release positively charged and neutral cargoes faster than negatively charged cargoes of similar sizes, and the pores favour the passage of IL-1ß and IL-18 over that of their precursors. Consistent with these findings, living-but not pyroptotic-macrophages preferentially release mature IL-1ß upon perforation by GSDMD. Mutation of the acidic residues of GSDMD compromises this preference, hindering intracellular retention of the precursor and secretion of the mature cytokine. The GSDMD pore therefore mediates IL-1 release by electrostatic filtering, which suggests the importance of charge in addition to size in the transport of cargoes across this large channel.

Structure, Activation and Regulation of NLRP3 and AIM2 Inflammasomes.

The inflammasome is a three-component (sensor, adaptor, and effector) filamentous signaling platform that shields from multiple pathogenic infections by stimulating the proteolytical maturation of proinflammatory cytokines and pyroptotic cell death. The signaling process initiates with the detection of endogenous and/or external danger signals by specific sensors, followed by the nucleation and polymerization from sensor to downstream adaptor and then to the effector, caspase-1. Aberrant activation of inflammasomes promotes autoinflammatory diseases, cancer, neurodegeneration, and cardiometabolic disorders. Therefore, an equitable level of regulation is required to maintain the equilibrium between inflammasome activation and inhibition. Recent advancement in the structural and mechanistic understanding of inflammasome assembly potentiates the emergence of novel therapeutics against inflammasome-regulated diseases. In this review, we have comprehensively discussed the recent and updated insights into the structure of inflammasome components, their activation, interaction, mechanism of regulation, and finally, the formation of densely packed filamentous inflammasome complex that exists as micron-sized punctum in the cells and mediates the immune responses.

Structural basis for distinct inflammasome complex assembly by human NLRP1 and CARD8.

Nod-like receptor (NLR) proteins activate pyroptotic cell death and IL-1 driven inflammation by assembling and activating the inflammasome complex. Closely related sensor proteins NLRP1 and CARD8 undergo unique auto-proteolysis-dependent activation and are implicated in auto-inflammatory diseases; however, their mechanisms of activation are not understood. Here we report the structural basis of how the activating domains (FIINDUPA-CARD) of NLRP1 and CARD8 self-oligomerize to assemble distinct inflammasome complexes. Recombinant FIINDUPA-CARD of NLRP1 forms a two-layered filament, with an inner core of oligomerized CARD surrounded by an outer ring of FIINDUPA. Biochemically, self-assembled NLRP1-CARD filaments are sufficient to drive ASC speck formation in cultured human cells-a process that is greatly enhanced by NLRP1-FIINDUPA which forms oligomers in vitro. The cryo-EM structures of NLRP1-CARD and CARD8-CARD filaments, solved here at 3.7 Å, uncover unique structural features that enable NLRP1 and CARD8 to discriminate between ASC and pro-caspase-1. In summary, our findings provide structural insight into the mechanisms of activation for human NLRP1 and CARD8 and reveal how highly specific signaling can be achieved by heterotypic CARD interactions within the inflammasome complexes.

Mechanism of filament formation in UPA-promoted CARD8 and NLRP1 inflammasomes.

NLRP1 and CARD8 are related cytosolic sensors that upon activation form supramolecular signalling complexes known as canonical inflammasomes, resulting in caspase-1 activation, cytokine maturation and/or pyroptotic cell death. NLRP1 and CARD8 use their C-terminal (CT) fragments containing a caspase recruitment domain (CARD) and the UPA (conserved in UNC5, PIDD, and ankyrins) subdomain for self-oligomerization, which in turn form the platform to recruit the inflammasome adaptor ASC (apoptosis-associated speck-like protein containing a CARD) or caspase-1, respectively. Here, we report cryo-EM structures of NLRP1-CT and CARD8-CT assemblies, in which the respective CARDs form central helical filaments that are promoted by oligomerized, but flexibly linked, UPAs surrounding the filaments. Through biochemical and cellular approaches, we demonstrate that the UPA itself reduces the threshold needed for NLRP1-CT and CARD8-CT filament formation and signalling. Structural analyses provide insights on the mode of ASC recruitment by NLRP1-CT and the contrasting direct recruitment of caspase-1 by CARD8-CT. We also discover that subunits in the central NLRP1CARD filament dimerize with additional exterior CARDs, which roughly doubles its thickness and is unique among all known CARD filaments. Finally, we engineer and determine the structure of an ASCCARD-caspase-1CARD octamer, which suggests that ASC uses opposing surfaces for NLRP1, versus caspase-1, recruitment. Together these structures capture the architecture and specificity of the active NLRP1 and CARD8 inflammasomes in addition to key heteromeric CARD-CARD interactions governing inflammasome signalling.

NLRP3 Sensing of Diverse Inflammatory Stimuli Requires Distinct Structural Features.

The NLRP3 inflammasome is central to host defense and implicated in various inflammatory diseases and conditions. While the favored paradigm of NLRP3 inflammasome activation stipulates a unifying signal intermediate that de-represses NLRP3, this view has not been tested. Further, structures within NLRP3 required for inflammasome activation are poorly defined. Here we demonstrate that while the NLRP3 LRRs are not auto-repressive and are not required for inflammasome activation by all agonists, distinct sequences within the NLRP3 LRRs positively and negatively modulate inflammasome activation by specific ligands. In addition, elements within the HD1/HD2 "hinge" of NLRP3 and the nucleotide-binding domain have contrasting functions depending upon the specific agonists. Further, while NLRP3 1-432 is minimally sufficient for inflammasome activation by all agonists tested, the pyrin, and linker domains (1-134) function cooperatively and are sufficient for inflammasome activation by certain agonists. Conserved cysteines 8 and 108 appear important for inflammasome activation by sterile, but not infectious insults. Our results define common and agonist-specific regions of NLRP3 that likely mediate ligand-specific responses, discount the hypothesis that NLRP3 inflammasome activation has a unified mechanism, and implicate NLRP3 as an integrator of agonist-specific, inflammasome activating signals.

Alternative splicing regulates stochastic NLRP3 activity.

Leucine-rich repeat (LRR) domains are evolutionarily conserved in proteins that function in development and immunity. Here we report strict exonic modularity of LRR domains of several human gene families, which is a precondition for alternative splicing (AS). We provide evidence for AS of LRR domain within several Nod-like receptors, most prominently the inflammasome sensor NLRP3. Human NLRP3, but not mouse NLRP3, is expressed as two major isoforms, the full-length variant and a variant lacking exon 5. Moreover, NLRP3 AS is stochastically regulated, with NLRP3 ∆ exon 5 lacking the interaction surface for NEK7 and hence loss of activity. Our data thus reveals unexpected regulatory roles of AS through differential utilization of LRRs modules in vertebrate innate immunity.

Targeting NLRP3 Inflammasome in Inflammatory Bowel Disease: Putting out the Fire of Inflammation.

Inflammatory bowel disease (IBD) is a group of inflammatory conditions of the colon and small intestine, comprised of ulcerative colitis and Crohn's disease. Among the complicated pathogenic factors of IBD, the overaction of inflammatory and immune reaction serves as an important factor. Inflammasome is a form of innate immunity as well as inflammation. Among all kinds of inflammasomes, the NOD-like receptor family, pyrin domain-containing 3 (NLRP3) inflammasome is the most studied one, and has been revealed to be involved in the pathogenesis and progression of IBD. Here, in this review, the association between the NLRP3 inflammasome and IBD will be discussed. Furthermore, several NLRP3 inflammasome inhibitors which have been demonstrated to be effective in the alleviation of IBD will be described in this review.

Sinomenine Attenuates Cartilage Degeneration by Regulating miR-223-3p/NLRP3 Inflammasome Signaling.

Sinomenine (SIN) has been shown to protect against IL-1ß-induced chondrocyte apoptosis in vitro. However, the role of SIN in the anterior cruciate ligament transection (ACLT)-induced osteoarthritis (OA) mouse model and its underlying molecular mechanisms remain unclear. In the present study, the protective effect of SIN on ACLT-induced articular cartilage degeneration and IL-1ß-induced chondrocyte apoptosis miR-223-3p/NLRP3 signaling regulation was investigated. Safranin O staining was performed to evaluate the pathological changes of articular cartilage. Chondrocyte apoptosis was measured with Annexin V-fluorescein isothiocyanate/polyimide (annexin V-FITC/PI) staining using flow cytometry. Gene and protein expression were detected by RT-qPCR and Western blotting, respectively. SIN administration markedly improved articular cartilage degradation in mice undergoing ACLT surgery. In addition, SIN treatment downregulated the levels of inflammatory cytokines and the protein expression of NLRP3 inflammasome components and upregulated the expression of miR-223-3p in OA mice and IL-1ß-stimulated chondrocytes. In vitro, we found that NLRP3 was a direct target of miR-223-3p, and overexpression of miR-223-3p blocked IL-1ß-induced apoptosis and the inflammatory response in chondrocytes. These findings indicate that miR-223-3p/NLRP3 signaling could be used as a potential target of SIN for the treatment of OA.

NLRP3 lacking the leucine-rich repeat domain can be fully activated via the canonical inflammasome pathway.

NLRP3 is a cytosolic sensor triggered by different pathogen- and self-derived signals that plays a central role in a variety of pathological conditions, including sterile inflammation. The leucine-rich repeat domain is present in several innate immune receptors, where it is frequently responsible for sensing danger signals and regulation of activation. Here we show by reconstitution of truncated and chimeric variants into Nlrp3-/- macrophages that the leucine-rich repeat domain is dispensable for activation and self-regulation of NLRP3 by several different triggers. The pyrin domain on the other hand is required to maintain NLRP3 in the inactive conformation. A fully responsive minimal NLRP3 truncation variant reconstitutes peritonitis in Nlrp3-/- mice. We demonstrate that in contrast to pathogen-activated NLRC4, the constitutively active NLRP3 molecule cannot engage wild-type NLRP3 molecules in a self-catalytic oligomerization. This lack of signal amplification is likely a protective mechanism to decrease sensitivity to endogenous triggers to impede autoinflammation.

Oridonin is a covalent NLRP3 inhibitor with strong anti-inflammasome activity.

Oridonin (Ori) is the major active ingredient of the traditional Chinese medicinal herb Rabdosia rubescens and has anti-inflammatory activity, but the target of Ori remains unknown. NLRP3 is a central component of NLRP3 inflammasome and has been involved in a wide variety of chronic inflammation-driven human diseases. Here, we show that Ori is a specific and covalent inhibitor for NLRP3 inflammasome. Ori forms a covalent bond with the cysteine 279 of NLRP3 in NACHT domain to block the interaction between NLRP3 and NEK7, thereby inhibiting NLRP3 inflammasome assembly and activation. Importantly, Ori has both preventive or therapeutic effects on mouse models of peritonitis, gouty arthritis and type 2 diabetes, via inhibition of NLRP3 activation. Our results thus identify NLRP3 as the direct target of Ori for mediating Ori's anti-inflammatory activity. Ori could serve as a lead for developing new therapeutics against NLRP3-driven diseases.

Regulatory Roles of Flavonoids on Inflammasome Activation during Inflammatory Responses.

Inflammation is an innate immune response to noxious stimuli to protect the body from pathogens. Inflammatory responses consist of two main steps: priming and triggering. In priming, inflammatory cells increase expressions of inflammatory molecules, while in triggering, inflammasomes are activated, resulting in cell death and pro-inflammatory cytokine secretion. Inflammasomes are protein complexes comprising intracellular pattern recognition receptors (PRRs) (e.g., nucleotide-binding oligomerization domain-like receptors (NLRs), absent in melanoma 2 (AIM2), and caspases-4/5/11) and pro-caspase-1 with or without a bipartite adaptor molecule ASC. Inflammasome activation induces pyroptosis, inflammatory cell death, and stimulates caspase-1-mediated secretion of interleukin (IL)-1b and IL-18. Flavonoids are secondary metabolites found in various plants and are considered as critical ingredients promoting health and ameliorating various disease symptoms. Anti-inflammatory activity of flavonoids and underlying mechanisms have been widely studied. This review introduces current knowledge on different types of inflammasomes and their activation during inflammatory responses and discusses recent studies regarding anti-inflammatory roles of flavonoids as suppressors of inflammasomes in inflammatory conditions. Understanding the regulatory effects of flavonoids on inflammasome activation will increase our knowledge of flavonoid-mediated anti-inflammatory activity and provide new insights into the development of flavonoid preparations to prevent and treat human inflammatory diseases.

Inflammasome Activation Characterizes Lesional Skin of Folliculitis Decalvans.

Folliculitis decalvans (FD) is a chronic inflammatory disease leading to scarring alopecia with poorly defined pathogenesis. The aim of this study was to investigate the expression of markers associated with the activation of innate immune signals, such as inflammasome (NALP1 and NALP3), interleukin (IL)-1ß and IL-8 and type I interferon (MxA). A retrospective monocentric study was conducted and included 17 patients with FD with available biopsies. Disease activity (stable vs. active) was defined clinically and histologically. Immunostaining was performed using antibodies directed against NALP1, NALP3, IL-1ß, IL-8, and MxA on FD skin biopsies. Results were compared with normal controls and lichen planopilaris. Eleven patients had active disease and 6 had stable disease. NALP1, NALP3, and IL-1ß expression were significantly increased in hair follicles in FD compared with controls and lichen planopilaris. This study highlights the predominant immune signal associated with inflammasome activation in FD, suggesting the use of IL-1ß blockade in FD.

AIM2 inflammasome activation and regulation: A structural perspective.

Absent in melanoma 2 (AIM2) inflammasome is a multi-protein platform that recognizes aberrant cytoplasmic dsDNA and induces cytokine maturation, release and pyroptosis. It is composed of AIM2, apoptosis-associated speck-like protein containing a CARD (ASC), and caspase-1. Recent X-ray crystallographic and high resolution cryo-electron microscopic (cryo-EM) studies have revealed a series of structures in AIM2 inflammasome activation and regulation. One prominent feature common in multiple steps is the assembly of high-order structures, especially helical filaments nucleated by upstream molecules, rather than stoichiometric complexes. In this review, we track the AIM2 inflammasome activation process step by step, using high-resolution structures to illustrate the overall architecture of AIM2 inflammasome and its assembly and regulatory mechanisms.

Nlrp9b inflammasome restricts rotavirus infection in intestinal epithelial cells.

Rotavirus, a leading cause of severe gastroenteritis and diarrhoea in young children, accounts for around 215,000 deaths annually worldwide. Rotavirus specifically infects the intestinal epithelial cells in the host small intestine and has evolved strategies to antagonize interferon and NF-κB signalling, raising the question as to whether other host factors participate in antiviral responses in intestinal mucosa. The mechanism by which enteric viruses are sensed and restricted in vivo, especially by NOD-like receptor (NLR) inflammasomes, is largely unknown. Here we uncover and mechanistically characterize the NLR Nlrp9b that is specifically expressed in intestinal epithelial cells and restricts rotavirus infection. Our data show that, via RNA helicase Dhx9, Nlrp9b recognizes short double-stranded RNA stretches and forms inflammasome complexes with the adaptor proteins Asc and caspase-1 to promote the maturation of interleukin (Il)-18 and gasdermin D (Gsdmd)-induced pyroptosis. Conditional depletion of Nlrp9b or other inflammasome components in the intestine in vivo resulted in enhanced susceptibility of mice to rotavirus replication. Our study highlights an important innate immune signalling pathway that functions in intestinal epithelial cells and may present useful targets in the modulation of host defences against viral pathogens.

Inhibition of NLRP3 Inflammasome Prevents LPS-Induced Inflammatory Hyperalgesia in Mice: Contribution of NF-κB, Caspase-1/11, ASC, NOX, and NOS Isoforms.

The nucleotide-binding domain and leucine-rich repeat protein 3 (NLRP3), an intracellular signaling molecule that senses many environmental- and pathogen/host-derived factors, has been implicated in the pathogenesis of several diseases associated with inflammation. It has been suggested that NLRP3 inflammasome inhibitors may have a therapeutic potential in the treatment of NLRP3-related inflammatory diseases. The aim of this study was to determine whether inhibition of NLRP3 inflammasome prevents inflammatory hyperalgesia induced by lipopolysaccharide (LPS) in mice as well as changes in expression/activity of nuclear factor κB (NF-κB), caspase-1/11, nicotinamide adenine dinucleotide phosphate oxidase (NOX), and endothelial/neuronal/inducible nitric oxide synthase (eNOS/nNOS/iNOS) that may regulate NLRP3/apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC)/pro-caspase-1 inflammasome formation and activity by using a selective NLRP3 inflammasome inhibitor, MCC950. Male mice received saline (10 ml/kg; i.p.), LPS (10 mg/kg; i.p.), and/or MCC950 (3 mg/kg; i.p.). Reaction time to thermal stimuli within 1 min was evaluated after 6 h. The mice were killed and the brains, hearts, and lungs were collected for measurement of NF-κB, caspase-1, caspase-11, NLRP3, ASC, NOX subunits (gp91phox; NOX2), and p47phox; NOXO2), nitrotyrosine, eNOS, nNOS, iNOS, and ß-actin protein expression, NOS activity, and interleukin (IL)-1ß levels. LPS-induced hyperalgesia was associated with a decrease in eNOS, nNOS, and iNOS protein expression and activity as well as an increase in expression of NF-κB p65, caspase-1 p20, caspase-11 p20, NLRP3, ASC, gp91phox, p47phox, and nitrotyrosine proteins in addition to elevated IL-1ß levels. The LPS-induced changes were prevented by MCC950. The results suggest that inhibition of NLRP3/ASC/pro-caspase-1 inflammasome formation and activity prevents inflammatory hyperalgesia induced by LPS in mice as well as changes in NF-κB, caspase-11, NOX2, NOXO2, and eNOS/nNOS/iNOS expression/activity.

Inflammasome-Derived Exosomes Activate NF-κB Signaling in Macrophages.

Exosomes are secreted small vesicles that mediate various biological processes, such as tumorigenesis and immune response. However, whether the inflammasome signaling leads to the change of constituent of exosomes and its roles in immune response remains to be determined. We isolated the exosomes from macrophages with treatment of mock, endotoxin, or endotoxin/nigericin. A label-free quantification method by MS/MS was used to identify the components of exosomes. In total, 2331 proteins were identified and 513 proteins were exclusively detected in exosomes with endotoxin and nigericin treatment. The differentially expressed proteins were classified by Gene Ontology and KEGG pathways. The immune response-related proteins and signaling pathways were specifically enriched in inflammasome-derived exosomes. Moreover, we treated macrophages with the exosomes from different stimulation. We found that inflammasome-derived exosomes directly activate NF-κB signaling pathway, while the control or endotoxin-derived exosomes have no effect. The inflammatory signaling was amplified in neighbor cells in an exosome-dependent way. The inflammasome-derived exosomes might be used to augment the immune response in disease treatment, and preventing the transfer of these exosomes might ameliorate autoimmune diseases.

Role of NLRP3 Inflammasome in Eosinophilic and Non-eosinophilic Chronic Rhinosinusitis with Nasal Polyps.

The pathophysiologic mechanisms of human chronic rhinosinusitis with nasal polyps (CRSwNP) remain unclear. We aimed to elucidate expression and biologic role of NLRP3 inflammasome in CRSwNP. Immunohistochemistry (IHC) was conducted to assess NLRP3 immunolabeling, real-time polymerase chain reaction (PCR) was used for IL-9 and NLRP3, and caspase-1 level quantitation in CRSwNP and control subjects. In addition, enzyme-linked immunosorbent assay (ELISA) was employed for analyzing concentrations of IL-1ß and IL-18 in the homogenates prepared from tissue specimens. Moreover, human nasal epithelial cells (HNECs) were used to evaluate the effects of lipopolysaccharide (LPS) and glyburide on NLRP3 inflammasome signaling pathway. Results showed that NLRP3 and caspase-1 were overexpressed in CRSwNP, especially in eosinophilic CRSwNP (ECRSwNP). Interestingly, NLRP3 expression had close correlation to that of caspase-1. Concentrations of IL-1ß and IL-18 were elevated. NLRP3 inflammasome signaling pathway was augmented by LPS but suppressed by glyburide. In conclusion, NLRP3 inflammasome signaling pathway played a pro-inflammatory role in the pathogenesis of CRSwNP, especially in ECRSwNP. NLRP3 inflammasome signaling pathway was augmented by LPS, but suppressed by glyburide.