Endothelial cell expression of a STING gain-of-function mutation initiates pulmonary lymphocytic infiltration.

Patients afflicted with Stimulator of interferon gene (STING) gain-of-function mutations frequently present with debilitating interstitial lung disease (ILD) that is recapitulated in mice expressing the STINGV154M mutation (VM). Prior radiation chimera studies revealed an unexpected and critical role for non-hematopoietic cells in initiating ILD. To identify STING-expressing non-hematopoietic cell types required for the development of ILD, we use a conditional knockin (CKI) model and direct expression of the VM allele to hematopoietic cells, fibroblasts, epithelial cells, or endothelial cells. Only endothelial cell-targeted VM expression results in enhanced recruitment of immune cells to the lung associated with elevated chemokine expression and the formation of bronchus-associated lymphoid tissue, as seen in the parental VM strain. These findings reveal the importance of endothelial cells as instigators of STING-driven lung disease and suggest that therapeutic targeting of STING inhibitors to endothelial cells could potentially mitigate inflammation in the lungs of STING-associated vasculopathy with onset in infancy (SAVI) patients or patients afflicted with other ILD-related disorders.

Gene coexpression networks reveal a broad role for lncRNAs in inflammatory bowel disease.

The role of long noncoding RNAs (lncRNAs) in disease is incompletely understood, but their regulation of inflammation is increasingly appreciated. We addressed the extent of lncRNA involvement in inflammatory bowel disease (IBD) using biopsy-derived RNA-sequencing data from a large cohort of deeply phenotyped patients with IBD. Weighted gene correlation network analysis revealed gene modules of lncRNAs coexpressed with protein-coding genes enriched for biological pathways, correlated with epithelial and immune cell signatures, or correlated with distal colon expression. Correlation of modules with clinical features uncovered a module correlated with disease severity, with an enriched interferon response signature containing the hub lncRNA IRF1-AS1. Connecting genes to IBD-associated single nucleotide polymorphisms (SNPs) revealed an enrichment of SNP-adjacent lncRNAs in biologically relevant modules. Ulcerative colitis-specific SNPs were enriched in distal colon-related modules, suggesting that disease-specific mechanisms may result from altered lncRNA expression. The function of the IBD-associated SNP-adjacent lncRNA IRF1-AS1 was explored in human myeloid cells, and our results suggested IRF1-AS1 promoted optimal production of TNF-α, IL-6, and IL-23. A CRISPR/Cas9-mediated activation screen in THP-1 cells revealed several lncRNAs that modulated LPS-induced TNF-α responses. Overall, this study uncovered the expression patterns of lncRNAs in IBD that identify functional, disease-relevant lncRNAs.

Nanoparticle delivery of innate immune agonists combines with senescence-inducing agents to mediate T cell control of pancreatic cancer.

Pancreatic ductal adenocarcinoma has quickly risen to become the 3rd leading cause of cancer-related death. This is in part due to its fibrotic tumor microenvironment (TME) that contributes to poor vascularization and immune infiltration and subsequent chemo- and immunotherapy failure. Here we investigated an innovative immunotherapy approach combining local delivery of STING and TLR4 innate immune agonists via lipid-based nanoparticles (NPs) co-encapsulation with senescence-inducing RAS-targeted therapies that can remodel the immune suppressive PDAC TME through the senescence-associated secretory phenotype. Treatment of transplanted and autochthonous PDAC mouse models with these regimens led to enhanced uptake of NPs by multiple cell types in the PDAC TME, induction of type I interferon and other pro-inflammatory signaling, increased antigen presentation by tumor cells and antigen presenting cells, and subsequent activation of both innate and adaptive immune responses. This two-pronged approach produced potent T cell-driven and Type I interferon-dependent tumor regressions and long-term survival in preclinical PDAC models. STING and TLR4-mediated Type I interferon signaling were also associated with enhanced NK and CD8+ T cell immunity in human PDAC. Thus, combining localized immune agonist delivery with systemic tumor-targeted therapy can synergize to orchestrate a coordinated innate and adaptive immune assault to overcome immune suppression and activate durable anti-tumor T cell responses against PDAC.

Expression of a STING Gain-of-function Mutation in Endothelial Cells Initiates Lymphocytic Infiltration of the Lungs.

Patients afflicted with STING gain-of-function mutations frequently present with debilitating interstitial lung disease ( ILD ) that is recapitulated in mice expressing the STING V154M mutation ( VM ). Prior radiation chimera studies revealed an unexpected and critical role for non-hematopoietic cells in the initiation of ILD. To identify STING-expressing non-hematopoietic cell types relevant to ILD, we generated a conditional knock-in ( CKI ) model in which expression of the VM allele was directed to hematopoietic cells, fibroblasts, epithelial cells, or endothelial cells. Only endothelial cell-targeted expression of the mutant allele resulted in the recruitment of immune cells to the lung and the formation of bronchus-associated lymphoid tissue, as seen in the parental VM strain. These findings reveal the importance of endothelial cells as instigators of STING-driven lung disease and suggest that therapeutic targeting of STING inhibitors to endothelial cells could potentially mitigate inflammation in the lungs of SAVI patients or patients afflicted with other ILD-related disorders. Summary: Patients with STING gain-of-function (GOF) mutations develop life-threatening lung autoinflammation. In this study, Gao et al. utilize a mouse model of conditional STING GOF to demonstrate a role for endothelial STING GOF in initiating immune cell recruitment into lung tissues of SAVI mice.

STING-dependent interferon signatures restrict osteoclast differentiation and bone loss in mice.

Stimulator of interferon genes (STING) is a key mediator of type-I interferon (IFN-I) signaling in response to a variety of stimuli, but the contribution of STING to homeostatic processes is not fully characterized. Previous studies showed that ligand activation of STING limits osteoclast differentiation in vitro through the induction of IFNß and IFN-I interferon-stimulated genes (ISGs). In a disease model (SAVI) driven by the V154M gain-of-function mutation in STING, fewer osteoclasts form from SAVI precursors in response to receptor activator of NF-kappaB ligand (RANKL) in an IFN-I-dependent manner. Due to the described role of STING-mediated regulation of osteoclastogenesis in activation settings, we sought to determine whether basal STING signaling contributes to bone homeostasis, an unexplored area. Using whole-body and myeloid-specific deficiency, we show that STING signaling prevents trabecular bone loss in mice over time and that myeloid-restricted STING activity is sufficient for this effect. STING-deficient osteoclast precursors differentiate with greater efficiency than wild types. RNA sequencing of wild-type and STING-deficient osteoclast precursor cells and differentiating osteoclasts reveals unique clusters of ISGs including a previously undescribed ISG set expressed in RANKL naïve precursors (tonic expression) and down-regulated during differentiation. We identify a 50 gene tonic ISG signature that is STING dependent and shapes osteoclast differentiation. From this list, we identify interferon-stimulated gene 15 (ISG15) as a tonic STING-regulated ISG that limits osteoclast formation. Thus, STING is an important upstream regulator of tonic IFN-I signatures shaping the commitment to osteoclast fates, providing evidence for a nuanced and unique role for this pathway in bone homeostasis.

TBK1 inhibition unleashes RIPK1, resensitizing tumors to immunotherapy.

Resistance mechanisms have curbed the potential of immune checkpoint blockade (ICB) therapies. Understanding mechanisms that contribute to this resistance should reveal new targets for combinatorial therapy. Tank-binding kinase 1 (TBK1) represents such a target. In recent work by Sun et al., inhibition of TBK1 restored the efficacy of such treatments by sensitizing tumors to RIPK1 kinase-dependent inflammatory cell death.

Type-1 interferon-dependent and -independent mechanisms in cyclic GMP-AMP synthase-stimulator of interferon genes-driven auto-inflammation.

The cyclic cyclic gaunosine monophosphate adenosine monophosphate (GMP-AMP) synthase-stimulator of interferon genes (cGAS-STING) pathway senses cytosolic dsDNA and initiates immune responses against pathogens. It is also implicated in several auto-inflammatory diseases known as monogenic interferonopathies, specifically Three prime repair exonuclease 1 (Trex1) loss-of-function (LOF), Dnase2 LOF, and stimulator of interferon genes-associated-vasculopathy-with-onset-in-infancy (SAVI). Although monogenic interferonopathies have diverse clinical presentations, they are distinguished by the elevation of type-1 interferons (T1IFNs). However, animal models have demonstrated that T1IFNs contribute to only some disease outcomes and that cGAS-STING activation also promotes T1IFN-independent pathology. For example, while T1IFNs drive the immunopathology associated with Trex1 LOF, disease in Dnase2 LOF is partially independent of T1IFNs, while disease in SAVI appears to occur entirely independent of T1IFNs. Additionally, while the cGAS-STING pathway is well characterized in hematopoietic cells, these animal models point to important roles for STING activity in nonhematopoietic cells in disease. Together, these models illustrate the complex role that cGAS-STING-driven responses play in the pathogenesis of inflammatory diseases across tissues.

Activation of the NLRP1 inflammasome in human keratinocytes by the dsDNA mimetic poly(dA:dT).

The accrual of cytosolic DNA leads to transcription of type I IFNs, proteolytic maturation of the IL-1 family of cytokines, and pyroptotic cell death. Caspase-1 cleaves pro-IL1ß to generate mature bioactive cytokine and gasdermin D which facilitates IL-1 release and pyroptotic cell death. Absent in melanoma-2 (AIM2) is a sensor of dsDNA leading to caspase-1 activation, although in human monocytes, cGAS-STING acting upstream of NLRP3 mediates the dsDNA-activated inflammasome response. In healthy human keratinocytes, AIM2 is not expressed yet caspase-1 is activated by the synthetic dsDNA mimetic poly(dA:dT). Here, we show that this response is not mediated by either AIM2 or the cGAS-STING-NLRP3 pathway and is instead dependent on NLRP1. Poly(dA:dT) is unique in its ability to activate NLRP1, as conventional linear dsDNAs fail to elicit NLRP1 activation. DsRNA was recently shown to activate NLRP1 and prior work has shown that poly(dA:dT) is transcribed into an RNA intermediate that stimulates the RNA sensor RIG-I. However, poly(dA:dT)-dependent RNA intermediates are insufficient to activate NLRP1. Instead, poly(dA:dT) results in oxidative nucleic acid damage and cellular stress, events which activate MAP3 kinases including ZAKα that converge on p38 to activate NLRP1. Collectively, this work defines a new activator of NLRP1, broadening our understanding of sensors that recognize poly(dA:dT) and advances the understanding of the immunostimulatory potential of this potent adjuvant.

Regulation of Synovial γδ T Cell Ligand Expression by Mitochondrial Reactive Oxygen Species and Gasdermin-D.

γδ T cells reside at mucosal and epithelial barriers, and they often accumulate at sites of inflammation, both infectious and autoimmune, as well as in certain tumors. However, progress in understanding their function is considerably hampered by a lack of full understanding of the ligands recognized by TCR-γδ and how expression of these ligands is regulated. We recently developed a soluble human TCR-γδ (Vγ9Vδ1) tetramer from a synovial γδ T cell clone of a Lyme arthritis patient and observed that it stains monocytes activated by Borrelia burgdorferi. Those findings are extended in the current study to further examine the physiological regulation of ligand expression on monocytes. The TCR-γδ ligand is induced by a variety of TLR agonists and requires NF-κB activation. Of particular interest is that ligand expression also requires caspase activation of the inflammasome and is dependent on active metabolism, mitochondrial reactive oxygen species, and activation of gasdermin-D. Consistent with these observations, the TCR-γδ ligand is expressed by a subset of metabolically active CD14+CD16+ monocytes and colocalizes intracellularly with mitochondria. The findings suggest a model in which synovial γδ T cell ligand is a self-antigen whose surface expression is increased by inflammatory conditions and mitochondrial stress.

Dual Inhibitors of Main Protease (MPro) and Cathepsin L as Potent Antivirals against SARS-CoV2.

Given the current impact of SARS-CoV2 and COVID-19 on human health and the global economy, the development of direct acting antivirals is of paramount importance. Main protease (MPro), a cysteine protease that cleaves the viral polyprotein, is essential for viral replication. Therefore, MPro is a novel therapeutic target. We identified two novel MPro inhibitors, D-FFRCMKyne and D-FFCitCMKyne, that covalently modify the active site cysteine (C145) and determined cocrystal structures. Medicinal chemistry efforts led to SM141 and SM142, which adopt a unique binding mode within the MPro active site. Notably, these inhibitors do not inhibit the other cysteine protease, papain-like protease (PLPro), involved in the life cycle of SARS-CoV2. SM141 and SM142 block SARS-CoV2 replication in hACE2 expressing A549 cells with IC50 values of 8.2 and 14.7 nM. Detailed studies indicate that these compounds also inhibit cathepsin L (CatL), which cleaves the viral S protein to promote viral entry into host cells. Detailed biochemical, proteomic, and knockdown studies indicate that the antiviral activity of SM141 and SM142 results from the dual inhibition of MPro and CatL. Notably, intranasal and intraperitoneal administration of SM141 and SM142 lead to reduced viral replication, viral loads in the lung, and enhanced survival in SARS-CoV2 infected K18-ACE2 transgenic mice. In total, these data indicate that SM141 and SM142 represent promising scaffolds on which to develop antiviral drugs against SARS-CoV2.

Lymphocyte crosstalk is required for monocyte-intrinsic trained immunity to Plasmodium falciparum.

Plasmodium falciparum (P. falciparum) induces trained innate immune responses in vitro, where initial stimulation of adherent PBMCs with P. falciparum-infected RBCs (iRBCs) results in hyperresponsiveness to subsequent ligation of TLR2. This response correlates with the presence of T and B lymphocytes in adherent PBMCs, suggesting that innate immune training is partially due to adaptive immunity. We found that T cell-depleted PBMCs and purified monocytes alone did not elicit hyperproduction of IL-6 and TNF-α under training conditions. Analysis of P. falciparum-trained PBMCs showed that DCs did not develop under control conditions, and IL-6 and TNF-α were primarily produced by monocytes and DCs. Transwell experiments isolating purified monocytes from either PBMCs or purified CD4+ T cells, but allowing diffusion of secreted proteins, enabled monocytes trained with iRBCs to hyperproduce IL-6 and TNF-α after TLR restimulation. Purified monocytes stimulated with IFN-γ hyperproduced IL-6 and TNF-α, whereas blockade of IFN-γ in P. falciparum-trained PBMCs inhibited trained responses. Assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-Seq) on monocytes from patients with malaria showed persistently open chromatin at genes that appeared to be trained in vitro. Together, these findings indicate that the trained immune response of monocytes to P. falciparum is not completely cell intrinsic but depends on soluble signals from lymphocytes.

REL and BHLHE40 Variants Are Associated with IL-12 and IL-10 Responses and Tuberculosis Risk.

The major human genes regulating Mycobacterium tuberculosis-induced immune responses and tuberculosis (TB) susceptibility are poorly understood. Although IL-12 and IL-10 are critical for TB pathogenesis, the genetic factors that regulate their expression in humans are unknown. CNBP, REL, and BHLHE40 are master regulators of IL-12 and IL-10 signaling. We hypothesized that common variants in CNBP, REL, and BHLHE40 were associated with IL-12 and IL-10 production from dendritic cells, and that these variants also influence adaptive immune responses to bacillus Calmette-Guérin (BCG) vaccination and TB susceptibility. We characterized the association between common variants in CNBP, REL, and BHLHE40, innate immune responses in dendritic cells and monocyte-derived macrophages, BCG-specific T cell responses, and susceptibility to pediatric and adult TB in human populations. BHLHE40 single-nucleotide polymorphism (SNP) rs4496464 was associated with increased BHLHE40 expression in monocyte-derived macrophages and increased IL-10 from peripheral blood dendritic cells and monocyte-derived macrophages after LPS and TB whole-cell lysate stimulation. SNP BHLHE40 rs11130215, in linkage disequilibrium with rs4496464, was associated with increased BCG-specific IL-2+CD4+ T cell responses and decreased risk for pediatric TB in South Africa. SNPs REL rs842634 and rs842618 were associated with increased IL-12 production from dendritic cells, and SNP REL rs842618 was associated with increased risk for TB meningitis. In summary, we found that genetic variations in REL and BHLHE40 are associated with IL-12 and IL-10 cytokine responses and TB clinical outcomes. Common human genetic regulation of well-defined intermediate cellular traits provides insights into mechanisms of TB pathogenesis.

A genetic screen in macrophages identifies new regulators of IFNγ-inducible MHCII that contribute to T cell activation.

Cytokine-mediated activation of host immunity is central to the control of pathogens. Interferon-gamma (IFNγ) is a key cytokine in protective immunity that induces major histocompatibility complex class II molecules (MHCII) to amplify CD4+ T cell activation and effector function. Despite its central role, the dynamic regulation of IFNγ-induced MHCII is not well understood. Using a genome-wide CRISPR-Cas9 screen in murine macrophages, we identified genes that control MHCII surface expression. Mechanistic studies uncovered two parallel pathways of IFNγ-mediated MHCII control that require the multifunctional glycogen synthase kinase three beta (GSK3ß) or the mediator complex subunit 16 (MED16). Both pathways control distinct aspects of the IFNγ response and are necessary for IFNγ-mediated induction of the MHCII transactivator Ciita, MHCII expression, and CD4+ T cell activation. Our results define previously unappreciated regulation of MHCII expression that is required to control CD4+ T cell responses.

UMP-CMP kinase 2 gene expression in macrophages is dependent on the IRF3-IFNAR signaling axis.

Toll-like receptors (TLRs) are highly-conserved pattern recognition receptors that mediate innate immune responses to invading pathogens and endogenous danger signals released from damaged and dying cells. Activation of TLRs trigger downstream signaling cascades, that culminate in the activation of interferon regulatory factors (IRFs), which subsequently leads to type I interferon (IFN) response. In the current study, we sought to expand the scope of gene expression changes in THP1-derived macrophages upon TLR4 activation and to identify interferon-stimulated genes. RNA-seq analysis led to the identification of several known and novel differentially expressed genes, including CMPK2, particularly in association with type I IFN signaling. We performed an in-depth characterization of CMPK2 expression, a nucleoside monophosphate kinase that supplies intracellular UTP/CTP for nucleic acid synthesis in response to type I IFN signaling in macrophages. CMPK2 was significantly induced at both RNA and protein levels upon stimulation with TLR4 ligand-LPS and TLR3 ligand-Poly (I:C). Confocal microscopy and subcellular fractionation indicated CMPK2 localization in both cytoplasm and mitochondria of THP-1 macrophages. Furthermore, neutralizing antibody-based inhibition of IFNAR receptor in THP-1 cells and BMDMs derived from IFNAR KO and IRF3 KO knockout mice further revealed that CMPK2 expression is dependent on LPS/Poly (I:C) mediated IRF3- type I interferon signaling. In summary, our findings suggest that CMPK2 is a potential interferon-stimulated gene in THP-1 macrophages and that CMPK2 may facilitate IRF3- type I IFN-dependent anti-bacterial and anti-viral roles.

AIM2 regulates anti-tumor immunity and is a viable therapeutic target for melanoma.

The STING and absent in melanoma 2 (AIM2) pathways are activated by the presence of cytosolic DNA, and STING agonists enhance immunotherapeutic responses. Here, we show that dendritic cell (DC) expression of AIM2 within human melanoma correlates with poor prognosis and, in contrast to STING, AIM2 exerts an immunosuppressive effect within the melanoma microenvironment. Vaccination with AIM2-deficient DCs improves the efficacy of both adoptive T cell therapy and anti-PD-1 immunotherapy for "cold tumors," which exhibit poor therapeutic responses. This effect did not depend on prolonged survival of vaccinated DCs, but on tumor-derived DNA that activates STING-dependent type I IFN secretion and subsequent production of CXCL10 to recruit CD8+ T cells. Additionally, loss of AIM2-dependent IL-1ß and IL-18 processing enhanced the treatment response further by limiting the recruitment of regulatory T cells. Finally, AIM2 siRNA-treated mouse DCs in vivo and human DCs in vitro enhanced similar anti-tumor immune responses. Thus, targeting AIM2 in tumor-infiltrating DCs is a promising new treatment strategy for melanoma.

cGAS-STING Pathway Does Not Promote Autoimmunity in Murine Models of SLE.

Detection of DNA is an important determinant of host-defense but also a driver of autoinflammatory and autoimmune diseases. Failure to degrade self-DNA in DNAseII or III(TREX1)-deficient mice results in activation of the cGAS-STING pathway. Deficiency of cGAS or STING in these models ameliorates disease manifestations. However, the contribution of the cGAS-STING pathway, relative to endosomal TLRs, in systemic lupus erythematosus (SLE) is controversial. In fact, STING deficiency failed to rescue, and actually exacerbated, disease manifestations in Fas-deficient SLE-prone mice. We have now extended these observations to a chronic model of SLE induced by the i.p. injection of TMPD (pristane). We found that both cGAS- and STING-deficiency not only failed to rescue mice from TMPD-induced SLE, but resulted in increased autoantibody production and higher proteinuria levels compared to cGAS STING sufficient mice. Further, we generated cGASKOFaslpr mice on a pure MRL/Faslpr background using Crispr/Cas9 and found slightly exacerbated, and not attenuated, disease. We hypothesized that the cGAS-STING pathway constrains TLR activation, and thereby limits autoimmune manifestations in these two models. Consistent with this premise, mice lacking cGAS and Unc93B1 or STING and Unc93B1 developed minimal systemic autoimmunity as compared to cGAS or STING single knock out animals. Nevertheless, TMPD-driven lupus in B6 mice was abrogated upon AAV-delivery of DNAse I, implicating a DNA trigger. Overall, this study demonstrated that the cGAS-STING pathway does not promote systemic autoimmunity in murine models of SLE. These data have important implications for cGAS-STING-directed therapies being developed for the treatment of systemic autoimmunity.

Overcoming innate immune barriers that impede AAV gene therapy vectors.

The field of gene therapy has made considerable progress over the past several years. Adeno-associated virus (AAV) vectors have emerged as promising and attractive tools for in vivo gene therapy. Despite the recent clinical successes achieved with recombinant AAVs (rAAVs) for therapeutics, host immune responses against the vector and transgene product have been observed in numerous preclinical and clinical studies. These outcomes have hampered the advancement of AAV gene therapies, preventing them from becoming fully viable and safe medicines. The human immune system is multidimensional and complex. Both the innate and adaptive arms of the immune system seem to play a concerted role in the response against rAAVs. While most efforts have been focused on the role of adaptive immunity and developing ways to overcome it, the innate immune system has also been found to have a critical function. Innate immunity not only mediates the initial response to the vector, but also primes the adaptive immune system to launch a more deleterious attack against the foreign vector. This Review highlights what is known about innate immune responses against rAAVs and discusses potential strategies to circumvent these pathways.

HDAC6 mediates an aggresome-like mechanism for NLRP3 and pyrin inflammasome activation.

Inflammasomes are supramolecular complexes that play key roles in immune surveillance. This is accomplished by the activation of inflammatory caspases, which leads to the proteolytic maturation of interleukin 1ß (IL-1ß) and pyroptosis. Here, we show that nucleotide-binding domain, leucine-rich repeat, and pyrin domain-containing protein 3 (NLRP3)- and pyrin-mediated inflammasome assembly, caspase activation, and IL-1ß conversion occur at the microtubule-organizing center (MTOC). Furthermore, the dynein adapter histone deacetylase 6 (HDAC6) is indispensable for the microtubule transport and assembly of these inflammasomes both in vitro and in mice. Because HDAC6 can transport ubiquitinated pathological aggregates to the MTOC for aggresome formation and autophagosomal degradation, its role in NLRP3 and pyrin inflammasome activation also provides an inherent mechanism for the down-regulation of these inflammasomes by autophagy. This work suggests an unexpected parallel between the formation of physiological and pathological aggregates.

Succination inactivates gasdermin D and blocks pyroptosis.

Activated macrophages undergo a metabolic switch to aerobic glycolysis, accumulating Krebs' cycle intermediates that alter transcription of immune response genes. We extended these observations by defining fumarate as an inhibitor of pyroptotic cell death. We found that dimethyl fumarate (DMF) delivered to cells or endogenous fumarate reacts with gasdermin D (GSDMD) at critical cysteine residues to form S-(2-succinyl)-cysteine. GSDMD succination prevents its interaction with caspases, limiting its processing, oligomerization, and capacity to induce cell death. In mice, the administration of DMF protects against lipopolysaccharide shock and alleviates familial Mediterranean fever and experimental autoimmune encephalitis by targeting GSDMD. Collectively, these findings identify GSDMD as a target of fumarate and reveal a mechanism of action for fumarate-based therapeutics that include DMF, for the treatment of multiple sclerosis.

Direct Binding to NLRP3 Pyrin Domain as a Novel Strategy to Prevent NLRP3-Driven Inflammation and Gouty Arthritis.

OBJECTIVE: The NLRP3 inflammasome is closely linked to the pathophysiology of a wide range of inflammatory diseases. This study was undertaken to identify small molecules that directly bind to NLRP3 in order to develop pharmacologic interventions for NLRP3-related diseases. METHODS: A structure-based virtual screening analysis was performed with ~62,800 compounds to select efficient NLRP3 inhibitors. The production of caspase 1-p10 and interleukin-1ß (IL-1ß) was measured by immunoblotting and enzyme-linked immunosorbent assay to examine NLRP3 inflammasome activation. Two gouty arthritis models and an air pouch inflammation model induced by monosodium urate monohydrate (MSU) crystal injection were used for in vivo experiments. Primary synovial fluid cells from gout patients were used to determine the relevance of NLRP3 inflammasome inhibition in human gout. RESULTS: Beta-carotene (provitamin A) suppressed the NLRP3 inflammasome activation induced by various activators, including MSU crystals, in mouse bone marrow-derived primary macrophages (P < 0.05). Surface plasmon resonance analysis demonstrated the direct binding of ß-carotene to the pyrin domain (PYD) of NLRP3 (KD = 3.41 × 10-6 ). Molecular modeling and mutation assays revealed the interaction mode between ß-carotene and the NLRP3 PYD. Inflammatory symptoms induced by MSU crystals were attenuated by oral administration of ß-carotene in gouty arthritis mouse models (P < 0.05), correlating with its suppressive effects on the NLRP3 inflammasome in inflamed tissues. Furthermore, ß-carotene reduced IL-1ß secretion from human synovial fluid cells isolated from gout patients (P < 0.05), showing its inhibitory efficacy in human gout. CONCLUSION: Our results present ß-carotene as a selective and direct inhibitor of NLRP3, and the binding of ß-carotene to NLRP3 PYD as a novel pharmacologic strategy to combat NLRP3 inflammasome-driven diseases, including gouty arthritis.