NLRP3 inflammasome activation and cell death

The NLRP3 inflammasome is a cytosolic multiprotein complex composed of the innate immune receptor protein NLRP3, adapter protein ASC, and inflammatory protease caspase-1 that responds to microbial infection, endogenous danger signals, and environmental stimuli. The assembled NLRP3 inflammasome can activate the protease caspase‐1 to induce gasdermin D-dependent pyroptosis and facilitate the release of IL-1β and IL-18, which contribute to innate immune defense and homeostatic maintenance. However, aberrant activation of the NLRP3 inflammasome is associated with the pathogenesis of various inflammatory diseases, such as diabetes, cancer, and Alzheimer’s disease. Recent studies have revealed that NLRP3 inflammasome activation contributes to not only pyroptosis but also other types of cell death, including apoptosis, necroptosis, and ferroptosis. In addition, various effectors of cell death have been reported to regulate NLRP3 inflammasome activation, suggesting that cell death is closely related to NLRP3 inflammasome activation. In this review, we summarize the inextricable link between NLRP3 inflammasome activation and cell death and discuss potential therapeutics that target cell death effectors in NLRP3 inflammasome-associated diseases.     

Inflammasome activation in multiple sclerosis and experimental autoimmune encephalomyelitis (EAE)

The aptly named inflammasomes are powerful signaling complexes that sense inflammatory signals under a myriad of conditions, including those from infections and endogenous sources. The inflammasomes promote inflammation by maturation and release of the pro‐inflammatory cytokines, IL‐1β and IL‐18. Several inflammasomes have been identified so far, but this review focuses mainly on the NLRP3 inflammasome. By still ill‐defined activation mechanisms, a sensor molecule, NLRP3 (NACHT, LRR and PYD domains‐containing protein 3), responds to danger signals and rapidly recruits ASC (apoptosis‐associated speck‐like protein containing a CARD) and pro‐caspase‐1 to form a large oligomeric signaling platform—the inflammasome. Involvement of the NLRP3 inflammasome in infections, metabolic disorders, autoinflammation, and autoimmunity, underscores its position as a central player in sensing microbial and damage signals and coordinating pro‐inflammatory immune responses. Indeed, evidence in patients with multiple sclerosis (MS) suggests inflammasome activation occurs during disease. Experiments with the mouse model of MS, experimental autoimmune encephalomyelitis (EAE), specifically describe the NLRP3 inflammasome as critical and necessary to disease development. This review discusses recent studies in EAE and MS which describe associations of inflammasome activation with promotion of T cell pathogenicity, infiltration of cells into the central nervous system (CNS) and direct neurodegeneration during EAE and MS.     

Caspase‐11 activates a canonical NLRP3 inflammasome by promoting K+ efflux

Recognition of microbe‐associated molecular patterns or endogenous danger signals by a subset of cytosolic PRRs results in the assembly of multiprotein signaling complexes, the so‐called inflammasomes. Canonical inflammasomes are assembled by NOD‐like receptor (NLR) or PYHIN family members and activate caspase‐1, which promotes the induction of pyroptosis and the release of mature interleukin‐1β/‐18. Recently, a noncanonical inflammasome pathway was discovered that results in caspase‐11 activation in response to bacterial lipopolysaccharide (LPS) in the cytosol. Interestingly, caspase‐11 induces pyroptosis by itself, but requires NLRP3, the inflammasome adapter ASC, and caspase‐1 to promote cytokine secretion. Here, we have studied the mechanism by which caspase‐11 controls IL‐1β secretion. Investigating NLRP3/ASC complex formation, we find that caspase‐11 functions upstream of a canonical NLRP3 inflammasome. The activation of NLRP3 by caspase‐11 during LPS transfection is a cell‐intrinsic process and is independent of the release of danger signals. Furthermore, we show that active caspase‐11 leads to a drop of intracellular potassium levels, which is necessary to activate NLRP3. Our study, therefore, sheds new light on the mechanism of noncanonical inflammasome signaling.     

Ticagrelor inhibits the NLRP3 inflammasome to protect against inflammatory disease independent of the P2Y12 signaling pathway

Ticagrelor is the first reversibly binding oral P2Y₁₂ receptor antagonist to inhibit platelet activation and has been approved by the Food and Drug Administration for the treatment of coronary artery disease. At present, the other pharmacological functions of ticagrelor remain poorly understood. The NOD-, LRR-, and pyrin domain-containing protein 3 (NLRP3) inflammasome plays a critical role in the innate immune system, but its excessive activation also contributes to the pathogenesis of complex diseases. In this study, we systematically examined the effects of ticagrelor on the NLRP3 inflammasome and found that ticagrelor inhibits NLRP3 inflammasome activation in macrophages independent of its classic inhibitory effect on the P2Y₁₂ signaling pathway. Further mechanistic studies demonstrate that ticagrelor attenuates the oligomerization of apoptosis-associated speck-like protein containing a CARD (ASC) by blocking chloride efflux, an effect achieved through the degradation of chloride intracellular channel proteins (CLICs) and blockade of the translocation of CLICs to the plasma membrane. Moreover, experiments on lipopolysaccharide-induced sepsis and alum-induced peritonitis in mice confirmed that ticagrelor mitigates the severity of systemic inflammation independent of P2Y₁₂ receptor antagonism. Importantly, oral administration of ticagrelor rapidly and strongly inhibited NLRP3 inflammasome activation in peripheral blood mononuclear cells from patients with acute coronary syndrome. Overall, our study reveals a novel pharmacological function of ticagrelor in addition to its classic antiplatelet properties, which suggests that ticagrelor may serve as a potential therapeutic agent for use in NLRP3-associated diseases.     

Trichothecene mycotoxins activate NLRP3 inflammasome through a P2X7 receptor and Src tyrosine kinase dependent pathway

Inflammasome is an intracellular molecular platform of the innate immunity that is a key mediator of inflammation. The inflammasome complex detects pathogens and different danger signals, and triggers cysteine protease caspase-1-dependent processing of pro-inflammatory cytokines IL-1β, and IL-18 in dendritic cells and macrophages. Previously, we have shown that water-damaged building associated trichothecene mycotoxins, including roridin A, trigger IL-1β and IL-18 secretion in human macrophages. However, the molecular basis as well as mechanism behind this trichothecene-induced cytokine secretion has remained uncharacterized. Here, we show that the trichothecene-induced IL-1β secretion is dependent on NLRP3 inflammasome in human primary macrophages. Pharmacological inhibition and small interfering RNA approach showed that the trichothecene-induced NLRP3 inflammasome activation is mediated through ATP-gated P2X₇ receptor. Moreover, we show that trichothecene-triggered NLRP3 inflammasome activation is dependent on Src tyrosine kinase activity. In addition, gene silencing of c-Cbl, a negative autophagy-related regulator of c-Src, resulted in enhanced secretion of IL-1β and IL-18 in response to trichothecene mycotoxin stimulation in human macrophages. In conclusion, our results suggest that roridin A, a fungal trichothecene mycotoxin, acts as microbial danger signals that trigger activation of NLRP3 inflammasome through P2X₇R and Src tyrosine kinase signaling dependent pathway in human primary macrophages.     

Initiation and perpetuation of NLRP3 inflammasome activation and assembly

The NLRP3 (NOD-like receptor family, pyrin domain containing 3) inflammasome is a multiprotein complex that orchestrates innate immune responses to infection and cell stress through activation of caspase-1 and maturation of inflammatory cytokines pro-interleukin-1β (pro-IL-1β) and pro-IL-18. Activation of the inflammasome during infection can be protective, but unregulated NLRP3 inflammasome activation in response to non-pathogenic endogenous or exogenous stimuli can lead to unintended pathology. NLRP3 associates with mitochondria and mitochondrial molecules, and activation of the NLRP3 inflammasome in response to diverse stimuli requires cation flux, mitochondrial Ca²⁺ uptake, and mitochondrial reactive oxygen species accumulation. It remains uncertain whether NLRP3 surveys mitochondrial integrity and senses mitochondrial damage, or whether mitochondria simply serve as a physical platform for inflammasome assembly. The structure of the active, caspase-1-processing NLRP3 inflammasome also requires further clarification, but recent studies describing the prion-like properties of ASC have advanced the understanding of how inflammasome assembly and caspase-1 activation occur while raising new questions regarding the propagation and resolution of NLRP3 inflammasome activation. Here, we review the mechanisms and pathways regulating NLRP3 inflammasome activation, discuss emerging concepts in NLRP3 complex organization, and expose the knowledge gaps hindering a comprehensive understanding of NLRP3 activation.     

NLRP3 Inflammasome and Host Protection against Bacterial Infection

The inflammasome is a multi-protein complex that induces maturation of inflammatory cytokines interleukin (IL)-1β and IL-18 through activation of caspase-1. Several nucleotide binding oligomerization domain-like receptor family members, including NLRP3, recognize unique microbial and danger components and play a central role in inflammasome activation. The NLRP3 inflammasome is critical for maintenance of homeostasis against pathogenic infections. However, inflammasome activation acts as a double-edged sword for various bacterial infections. When the IL-1 family of cytokines is secreted excessively, they cause tissue damage and extensive inflammatory responses that are potentially hazardous for the host. Emerging evidence has shown that diverse bacterial pathogens or their components negatively regulate inflammasome activation to escape the immune response. In this review, we discuss the current knowledge of the roles and regulation of the NLRP3 inflammasome during bacterial infections. Activation and regulation of the NLRP3 inflammasome should be tightly controlled to prevent virulence and pathology during infections. Understanding the roles and regulatory mechanisms of the NLRP3 inflammasome is essential for developing potential treatment approaches against pathogenic infections.     

Extracellular ADP augments microglial inflammasome and NF-κB activation via the P2Y12 receptor

The NLRP3 inflammasome is a molecular complex that translates signals from pathogens and tissue damage into inflammatory responses, and plays crucial roles in numerous neurological diseases. Activation of the NLRP3 inflammasome leads to caspase-1 dependent cleavage of pro-IL-1β to form mature IL-1β. By acting on the P2X7 purinergic receptor, extracellular ATP is one of the major stimuli that activates the NLRP3 inflammasome. Although microglia express multiple purinergic receptors, their roles in inflammasome-mediated inflammation are largely unknown. We studied the role of the P2Y12 receptor, a metabotropic P2Y receptor enriched in microglia, on inflammation in vitro. Inhibition of the microglial P2Y12 receptor by PSB0739 or siRNA knockdown suppressed IL-1β release. P2Y12 receptor-deficient microglia displayed markedly attenuated IL-1β mRNA expression and release. P2Y12 receptor blockade also suppressed IL-6 production. Both IL-1β and IL-6 responses were augmented by extracellular ADP or ADP-βS and were abrogated by PSB0739. Mechanistically, ADP-βS potentiated NF-κB activation. In addition, ADP altered mitochondrial membrane potential in combination with ATP and increased the number of caspase-1 positive cells through the P2Y12 receptor. These results elucidate a novel inflammatory mechanism by which extracellular ADP acts on the P2Y12 receptor to activate NF-κB and the NLRP3 inflammasome to enhance microglial inflammation.     

Flavonoid VI-16 protects against DSS-induced colitis by inhibiting Txnip-dependent NLRP3 inflammasome activation in macrophages via reducing oxidative stress

Emerging evidence suggests that NLRP3 inflammasome was associated with various kinds of immunological diseases including colitis. However, there are few drugs targeting inflammasomes in the treatment of colitis. Several flavonoids have been found to affect the inflammasome pathway, but the mechanism is still confusing. Here we report that VI-16, a synthetic flavonoid compound, exerts potent anti-inflammatory effects on macrophages in DSS-induced colitis mice, which intervened in the activation of NLRP3 inflammasome without affecting intestinal epithelial cells. However, the protection of VI-16 against DSS-induced colitis was dependent on NLRP3 expression in hematopoietic cells. Furthermore, this inhibitory effect of VI-16 was found to be at least partially achieved by decreasing the mitochondrial ROS generation without affecting autophagy. Further studies confirm that VI-16 inhibits the binding of Txnip to NLRP3 by reducing oxidative stress and ultimately inhibits NLRP3 inflammasome. This demonstrates the ability of VI-16 to inhibit the NLRP3 inflammasome activation and its potential use in the treatment of inflammatory bowel disease.     

Mitochondria: Sovereign of inflammation?

NLRP3 inflammasome-dependent inflammatory responses are triggered by a variety of signals of host danger, including infection, tissue damage and metabolic dysregulation. How these diverse activators cause inflammasome activation is poorly understood. Recent data suggest that the mitochondria integrate these distinct signals and relay this information to the NLRP3 inflammasome. Dysfunctional mitochondria generate ROS, which is required for inflammasome activation. On the contrary, the NLRP3 inflammasome is negatively regulated by autophagy, which is a catabolic process that removes damaged or otherwise dysfunctional organelles, including mitochondria. In addition to the processing and secretion of pro-inflammatory cytokines such as IL-1β, NLRP3 inflammasome activation also influences cellular metabolic pathways such as glycolysis and lipogenesis. Mapping the connections between mitochondria, metabolism and inflammation is of great interest, as malfunctioning of this network is associated with many chronic inflammatory diseases.     

The purinergic 2X7 receptor participates in renal inflammation and injury induced by high‐fat diet: possible role of NLRP3 inflammasome activation

Renal disease associated with type 2 diabetes and the metabolic syndrome is characterized by a distinct inflammatory phenotype. The purinergic 2X₇ receptor (P2X₇R) and the nucleotide‐binding and oligomerization domain‐like receptor containing a pyrin domain 3 (NLRP3) inflammasome have been separately shown to play a role in two models of non‐metabolic chronic kidney disease. Moreover, the NLRP3 inflammasome has been implicated in chronic low‐grade sterile inflammation characterizing metabolic disorders, though the mechanism(s) involved in inflammasome activation under these conditions are still unknown. We investigated the role of P2X₇R (through activation of the NLRP3 inflammasome) in renal inflammation and injury induced by a high‐fat diet, an established model of the metabolic syndrome. On a high‐fat diet, mice lacking P2X₇R developed attenuated renal functional and structural alterations as well as reduced inflammation, fibrosis, and oxidative/carbonyl stress, as compared with wild‐type animals, in the absence of significant differences in metabolic parameters. This was associated with blunted up‐regulation of the NLRP3 inflammasome components NLRP3, apoptosis‐associated speck‐like protein containing a caspase recruitment domain (ASC), pro‐caspase 1, pro‐interleukin (IL)‐1β, and pro‐IL‐18, as well as reduced inflammasome activation, as evidenced by decreased formation of mature caspase 1, whereas mature IL‐1β and IL‐18 were not detected. Up‐regulated expression of NLRP3 and pro‐caspase 1, post‐translational processing of pro‐caspase‐1, and release of IL‐18 in response to lipopolysaccharide + 2′(3′)‐O‐(4‐benzoylbenzoyl)ATP were attenuated by P2X₇R silencing in cultured mouse podocytes. Protein and mRNA expression of P2X₇R, NLRP3, and ASC were also increased in kidneys from subjects with type 2 diabetes and the metabolic syndrome, showing histologically documented renal disease. These data provide evidence of a major role for the purinergic system, at least in part through activation of the NLRP3 inflammasome, in the process driving ‘metabolic’ renal inflammation and injury and identify P2X₇R and NLRP3 as novel therapeutic targets. Copyright © 2013 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Aggregatibacter actinomycetemcomitans Cytolethal Distending Toxin Activates the NLRP3 Inflammasome in Human Macrophages,Leading to the Release of Proinflammatory Cytokines

The cytolethal distending toxin (Cdt) is produced from a number of bacteria capable of causing infection and inflammatory disease. Our previous studies with Actinobacillus actinomycetemcomitans Cdt demonstrate not only that the active toxin subunit functions as a phosphatidylinositol-3,4,5-triphosphate (PIP3) phosphatase but also that macrophages exposed to the toxin were stimulated to produce proinflammatory cytokines. We now demonstrate that the Cdt-induced proinflammatory response involves the activation of the NLRP3 inflammasome. Specific inhibitors and short hairpin RNA (shRNA) were employed to demonstrate requirements for NLRP3 and ASC as well as caspase-1. Furthermore, Cdt-mediated inflammasome activation is dependent upon upstream signals, including reactive oxygen species (ROS) generation and Cdt-induced increases in extracellular ATP levels. Increases in extracellular ATP levels contribute to the activation of the P2X₇ purinergic receptor, leading to K⁺ efflux. The relationship between the abilities of the active toxin subunit CdtB to function as a lipid phosphatase, activate the NLRP3 inflammasome, and induce a proinflammatory cytokine response is discussed. These studies provide new insight into the virulence potential of Cdt in mediating the pathogenesis of disease caused by Cdt-producing organisms such as Aggregatibacter actinomycetemcomitans.     

NLRP3: A promising therapeutic target for autoimmune diseases

NLRP3, a member of nucleotide-binding domain-(NOD) like receptor family, can be found in large varieties of immune and non-immune cells. Upon activation, the NLRP3, apoptosis-associated speck-like protein (ASC) and pro-caspase-1 would assemble into a multimeric protein, called the NLRP3 inflammasome. Then the inflammasome promotes inflammation (through specific cleavage and production of bioactive IL-1β and IL-18) and pyroptotic cell death. Previous studies have indicated the importance of NLRP3 in regulating innate immunity. Recently, numerous studies have revealed their significance in autoimmune diseases, such as systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), systemic sclerosis (SSc) and inflammatory bowel disease (IBD). In this review, we will briefly discuss the biological features of NLRP3 and summarize the recent progression of the involvement of NLRP3 in the development and pathogenesis of autoimmune diseases, as well as its clinical implications and therapeutic potential.     

Sensing and reacting to microbes through the inflammasomes

Inflammasomes are multiprotein complexes that activate caspase-1, which leads to maturation of the proinflammatory cytokines interleukin 1β (IL-1β) and IL-18 and the induction of pyroptosis. Members of the Nod-like receptor (NLR) family, including NLRP1, NLRP3 and NLRC4, and the cytosolic receptor AIM2 are critical components of inflammasomes and link microbial and endogenous danger signals to the activation of caspase-1. In response to microbial infection, activation of the inflammasomes contributes to host protection by inducing immune responses that limit microbial invasion, but deregulated activation of inflammasomes is associated with autoinflammatory syndromes and other pathologies. Thus, understanding inflammasome pathways may provide insight into the mechanisms of host defense against microbes and the development of inflammatory disorders.     

The inhibitor effect of RKIP on inflammasome activation and inflammasome-dependent diseases

Aberrant inflammasome activation contributes to the pathogenesis of various human diseases, including atherosclerosis, gout, and metabolic disorders. Elucidation of the underlying mechanism involved in the negative regulation of the inflammasome is important for developing new therapeutic targets for these diseases. Here, we showed that Raf kinase inhibitor protein (RKIP) negatively regulates the activation of the NLRP1, NLRP3, and NLRC4 inflammasomes. RKIP deficiency enhanced caspase-1 activation and IL-1β secretion via NLRP1, NLRP3, and NLRC4 inflammasome activation in primary macrophages. The overexpression of RKIP in THP-1 cells inhibited NLRP1, NLRP3, and NLRC4 inflammasome activation. RKIP-deficient mice showed increased sensitivity to Alum-induced peritonitis and Salmonella typhimurium-induced inflammation, indicating that RKIP inhibits NLRP3 and NLRC4 inflammasome activation in vivo. Mechanistically, RKIP directly binds to apoptosis-associated speck-like protein containing a caspase-recruitment domain (ASC) and competes with NLRP1, NLRP3, or NLRC4 to interact with ASC, thus interrupting inflammasome assembly and activation. The depletion of RKIP aggravated inflammasome-related diseases such as monosodium urate (MSU)-induced gouty arthritis and high-fat diet (HFD)-induced metabolic disorders. Furthermore, the expression of RKIP was substantially downregulated in patients with gouty arthritis or type 2 diabetes (T2D) compared to healthy controls. Collectively, our findings suggest that RKIP negatively regulates NLRP1, NLRP3, and NLRC4 inflammasome activation and is a potential therapeutic target for the treatment of inflammasome-related diseases.     

Posttranslational modifications of NLRP3 and their regulatory roles in inflammasome activation

The NACHT, LRR, and PYD domains-containing protein 3 (NLRP3) inflammasome is a multimolecular complex that plays a fundamental role in inflammation. Optimal activation of NLRP3 inflammasome is crucial for host defense against pathogens and the maintenance of immune homeostasis. Aberrant NLRP3 inflammasome activity has been implicated in various inflammatory diseases. Posttranslational modifications (PTMs) of NLRP3, a key inflammasome sensor, play critical roles in directing inflammasome activation and controlling the severity of inflammation and inflammatory diseases, such as arthritis, peritonitis, inflammatory bowel disease, atherosclerosis, and Parkinson's disease. Various NLRP3 PTMs, including phosphorylation, ubiquitination, and SUMOylation, could direct inflammasome activation and control inflammation severity by affecting the protein stability, ATPase activity, subcellular localization, and oligomerization of NLRP3 as well as the association between NLRP3 and other inflammasome components. Here, we provide an overview of the PTMs of NLRP3 and their roles in controlling inflammation and summarize potential anti-inflammatory drugs targeting NLRP3 PTMs.     

Endothelial NLRP3 Inflammasome Activation and Enhanced Neointima Formation in Mice by Adipokine Visfatin

Inflammasomes serve as an intracellular machinery to initiate inflammatory response to various danger signals. The present study tested whether an inflammasome centered on nucleotide oligomerization domain-like receptor protein 3 (NLRP3) triggers endothelial inflammatory response to adipokine visfatin, a major injurious adipokine during obesity. NLRP3 inflammasome components were abundantly expressed in cultured mouse microvascular endothelial cells, including NLRP3, apoptosis-associated speck-like protein, and caspase-1. These NLRP3 inflammasome molecules could be aggregated to form an inflammasome complex on stimulation of visfatin, as shown by fluorescence confocal microscopy and size exclusion chromatography. Correspondingly, visfatin significantly increased caspase-1 activity and IL-1β release in microvascular endothelial cells, indicating an activation of NLRP3 inflammasomes. In animal experiments, direct infusion of visfatin in mice with partially ligated left carotid artery were found to have significantly increased neointimal formation, which was correlated with increased NLRP3 inflammasome formation and IL-1β production in the intima. Further, visfatin-induced neointimal formation, endothelial inflammasome formation, and IL-1β production in mouse partially ligated left carotid artery were abolished by caspase-1 inhibition, local delivery of apoptosis-associated speck-like protein shRNA or deletion of the ASC gene. In conclusion, the formation and activation of NLRP3 inflammasomes by adipokine visfatin may be an important initiating mechanism to turn on the endothelial inflammatory response leading to arterial inflammation and endothelial dysfunction in mice during early stage obesity.Obesity is a major risk factor for cardiovascular disease and has been strongly associated with endothelial dysfunction and coronary atherosclerosis. Obese patients have significantly elevated morbidity and mortality due to coronary artery disease.¹ However, weight loss can decrease cardiovascular risk, improve endothelial function, and protect coronary arteries from atherosclerotic injury. However, mechanisms underlying obesity-associated coronary atherosclerotic injury and endothelial dysfunction are not fully understood. Numerous studies have reported a critical role of vascular inflammation in the development of coronary atherosclerosis, which has been characterized as an inflammatory disease.^2–6 To date, the precise mechanism that mediates the early inflammatory responses of endothelial cells (ECs) during obesity remains unknown.Recently, the inflammasome as an intracellular inflammatory machinery has been reported to switch on the inflammatory response of tissues or organs to various danger signals.^7,8 Among different types of inflammasomes, the nucleotide oligomerization domain (Nod)-like receptor family pyrin domain containing 3 (NLRP3) inflammasome is well characterized in a variety of mammalian cells, especially as a receptor for endogenous danger signals such as ATP, cholesterol crystal, β-amyloid, and monosodium urate.^2,9–14 The NLRP3 inflammasome is characteristic of a proteolytic complex mainly composed of NLRP3, the adaptor protein apoptosis-associated speck-like protein (ASC), and caspase-1. On stimulation, NLRP3 inflammasomes oligomerize to form large multimolecular complexes that control the caspase-1 activity and subsequent bioactive IL-1β production.^10,15–18 More recently, NLRP3 inflammasomes have been implicated in the development of obesity and insulin resistance.¹¹ For example, the consumption of a high-fat diet (HFD) has been considered as critical contributor to type 2 diabetes, and NLRP3 inflammasome might be an important pathway of HFD mediating insulin resistance leading to inflammation.¹⁹ These findings led us to wonder whether activation of NLRP3 inflammasome is an initiating mechanism for obesity-induced endothelial inflammatory responses.Adipose tissue as an active metabolic tissue secretes multiple metabolically important proteins known as ‘adipokines.''^20,21 Visfatin is a newly identified adipokine and a major injurious factor during obesity-associated diseases, including diabetes,²² carotid and coronary atherosclerosis,^23,24 and chronic kidney disease.^25,26 Visfatin has also been considered as a pro-inflammatory adipokine to promote endothelial inflammation and injury.^27,28 The present study was designed to test the hypothesis that activation of NLRP3 inflammasomes is one of the important mechanisms that mediate endothelial inflammatory response to visfatin during early-stage obesity. We used a series of molecular and physiological approaches both in vitro and in vivo to test this hypothesis.