Adaptor Protein ASC Research Articles

Potential Impact of Bioactive Compounds as NLRP3 Inflammasome Inhibitors: An Update.

The inflammasome NLRP3 comprises a caspase recruitment domain, a pyrin domain containing receptor 3, an apoptosis-linked protein like a speck containing a procaspase-1, and an attached nucleotide domain leucine abundant repeat. There are a wide variety of stimuli that can activate the inflammasome NLRP3. When activated, the protein NLRP3 appoints the adapter protein ASC. Adapter ASC protein then recruits the procaspase-1 protein, which causes the procaspase- 1 protein to be cleaved and activated, which induces cytokines. At the same time, abnormal activation of inflammasome NLRP3 is associated with many diseases, such as diabetes, atherosclerosis, metabolic syndrome, cardiovascular and neurodegenerative diseases. As a result, a significant amount of effort has been put into comprehending the mechanisms behind its activation and looking for their specific inhibitors. In this review, we primarily focused on phytochemicals that inhibit the inflammasome NLRP3, as well as discuss the defects caused by NLRP3 signaling. We conducted an in-depth research review by searching for relevant articles in the Scopus, Google Scholar, and PubMed databases. By gathering information on phytochemical inhibitors that block NLRP3 inflammasome activation, a complicated balance between inflammasome activation or inhibition with NLRP3 as a key role was revealed in NLRP3-driven clinical situations.

Short-term respiratory cadmium exposure partially activates pulmonary NLRP3 inflammasome by inducing ferroptosis in mice

Cadmium (Cd) is a common environmental metal. Previous studies indicated that long-term respiratory Cd exposure caused lung injury and airway inflammation. The purpose of this study was to evaluate whether short-term respiratory Cd exposure induces pulmonary ferroptosis and NLRP3 inflammasome activation. Adult C57BL/6J mice were exposed to Cd by inhaling CdCl2 aerosol (0, 10, or 100 ppm) for 5 days. Serum and lung Fe2+ contents were elevated in Cd-exposed mice. Oxidized AA metabolites, the major oxidized lipids during ferroptosis, were upregulated in Cd-exposed mouse lungs. Pulmonary MDA content and 4-HNE-positive cells were increased in Cd-exposed mice. ACSL4 and COX-2, two lipoxygenases, were upregulated in Cd-exposed mouse lungs. Further analyses found that phosphorylated NF-kB p65 was elevated in Cd-exposed mouse lungs. Innate immune receptor protein NLRP3 and adapter protein ASC were upregulated in Cd-exposed mouse lungs. Caspase-1 was activated and IL-1β and IL-18 were upregulated in Cd-exposed mouse lungs. Fer-1, a specific inhibitor of ferroptosis, attenuated Cd-induced elevation of pulmonary NLRP3 and ASC, caspase-1 activation, and IL-1β and IL-18 upregulation. Finally, mitoquinone (MitoQ), a mitochondria-target antioxidant, suppressed Cd-caused ferroptosis and NLRP3 inflammasome activation. Our results demonstrate that ferroptosis might partially mediate Cd-evoked activation of NLRP3 inflammasome in the lungs.

Discovery of natural product derivative triptolidiol as a direct NLRP3 inhibitor by reducing K63-specific ubiquitination.

NLRP3 is up-regulated in inflammatory and autoimmune diseases. The development of NLRP3 inhibitors is challenged by the identification of compounds with distinct mechanisms of action avoiding side effects and toxicity. Triptolide is a natural product with multiple anti-inflammatory activities, but a narrow therapeutic window. Natural product triptolide derivatives were screened for NLRP3 inhibitors in human THP-1 and mouse bone marrow-derived macrophages. The efficacy of potent NLRP3 inhibitors was evaluated in LPS-induced acute lung injury and septic shock models. Triptolidiol was identified as a selective inhibitor of NLRP3 with high potency. Triptolidiol inactivated the NLRP3 inflammasome in human THP-1 and mouse primary macrophages primed with LPS. Triptolidiol specifically inhibited pro-caspase 1 cleavage downstream of NLRP3, but not AIM2 or NLRC4 inflammasomes. Based on the structure-activity relationship study, the C8-β-OH group was critical for its binding to NLRP3. Triptolidiol exhibited a submicromolar KD for NLRP3, binding to residue C280. This binding prevented the interaction of NLRP3 with NEK7, the key regulator of NLRP3 inflammasome oligomerization and assembly, but not with the inflammasome adaptor protein ASC. Triptolidiol decreased the K63-specific ubiquitination of NLRP3, leading NLRP3 to a "closed" inactive conformation. Intraperitoneal administration of triptolidiol significantly attenuated LPS-induced acute lung injury and lethal septic shock. Triptolidiol is a novel NLRP3 inhibitor that regulates inflammasome assembly and activation by decreasing K63-linked ubiquitination. Triptolidiol has novel structural features that make it distinct from reported NLRP3 inhibitors and represents a viable therapeutic lead for inflammatory diseases.

The Inflammasome Adaptor Protein ASC in Plasma as a Biomarker of Early Cognitive Changes.

Dementia is a group of symptoms including memory loss, language difficulties, and other types of cognitive and functional impairments that affects 57 million people worldwide, with the incidence expected to double by 2040. Therefore, there is an unmet need to develop reliable biomarkers to diagnose early brain impairments so that emerging interventions can be applied before brain degeneration. Here, we performed biomarker analyses for apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), neurofilament light chain (NfL), glial fibrillary acidic protein (GFAP), and amyloid-β 42/40 (Aβ42/40) ratio in the plasma of older adults. Participants had blood drawn at baseline and underwent two annual clinical and cognitive evaluations. The groups tested either cognitively normal on both evaluations (NN), cognitively normal year 1 but cognitively impaired year 2 (NI), or cognitively impaired on both evaluations (II). ASC was elevated in the plasma of the NI group compared to the NN and II groups. Additionally, Aβ42 was increased in the plasma in the NI and II groups compared to the NN group. Importantly, the area under the curve (AUC) for ASC in participants older than 70 years old in NN vs. NI groups was 0.81, indicating that ASC is a promising plasma biomarker for early detection of cognitive decline.

Staphylococcus aureus–specific skin resident memory T cells protect against bacteria colonization but exacerbate atopic dermatitis–like flares in mice

BackgroundThe contribution of Staphylococcus aureus (S. aureus) to the exacerbation of atopic dermatitis (AD) is widely documented, but its role as a primary trigger of AD skin symptoms remains poorly explored. ObjectiveTo reappraise the main bacterial factors and underlying immune mechanisms by which S. aureus triggers AD-like inflammation. MethodsWe capitalized on a pre-clinical model, in which different clinical isolates were applied in the absence of any prior experimental skin injury. ResultsWe report that the development of S. aureus-induced dermatitis depended on the nature of the S. aureus strain, its viability, the concentration of the applied bacterial suspension, the production of secreted and non-secreted factors, as well as the activation of accessory gene regulatory quorum sensing system. In addition, the rising dermatitis, which exhibited the well-documented AD cytokine signature, was significantly inhibited in inflammasome adaptor protein ASC- and monocyte/macrophage-deficient animals, but not in T- and B-cell-deficient mice, suggesting a major role for the innate response in the induction of skin inflammation. However, bacterial exposure generated a robust adaptive immune response against S. aureus, and an accumulation of S. aureus-specific γδ and CD4+ tissue resident memory T (Trm) cells at the site of previous dermatitis. The latter both contributed to worsen the flares of AD-like dermatitis upon new bacteria exposures, but also, protected the mice from persistent bacterial colonization. ConclusionThese data highlight the induction of unique AD-like inflammation, with the generation of pro-inflammatory but protective Trm cells in a context of natural exposure to pathogenic S. aureus strains.

NLRP12 interacts with NLRP3 to block the activation of the human NLRP3 inflammasome.

Inflammasomes are multiprotein complexes that drive inflammation and contribute to protective immunity against pathogens and immune pathology in autoinflammatory diseases. Inflammasomes assemble when an inflammasome scaffold protein senses an activating signal and forms a signaling platform with the inflammasome adaptor protein ASC. The NLRP subfamily of NOD-like receptors (NLRs) includes inflammasome nucleators (such as NLRP3) and also NLRP12, which is genetically linked to familial autoinflammatory disorders that resemble diseases caused by gain-of-function NLRP3 mutants that generate a hyperactive NLRP3 inflammasome. We performed a screen to identify ASC inflammasome-nucleating proteins among NLRs that have the canonical pyrin-NACHT-LRR domain structure. Only NLRP3 and NLRP6 could initiate ASC polymerization to form "specks," and NLRP12 failed to nucleate ASC polymerization. However, wild-type NLRP12 inhibited ASC inflammasome assembly induced by wild-type and gain-of-function mutant NLRP3, an effect not seen with disease-associated NLRP12 mutants. The capacity of NLRP12 to suppress NLRP3 inflammasome assembly was limited to human NLRP3 and was not observed for wild-type murine NLRP3. Furthermore, peripheral blood mononuclear cells from patients with an NLRP12 mutant-associated inflammatory disorder produced increased amounts of the inflammatory cytokine IL-1β in response to NLRP3 stimulation. Thus, our findings provide insights into NLRP12 biology and suggest that NLRP3 inhibitors in clinical trials for NLRP3-driven diseases may also be effective in treating NLRP12-associated autoinflammatory diseases.

Eriodictyol inhibits the motility, angiogenesis and tumor growth of hepatocellular carcinoma via NLRP3 inflammasome inactivation

NLRP3 involves in the development of hepatocellular carcinoma (HCC). Eriodictyol has shown its inhibitory effect on HCC cell proliferation. However, the underlying mechanism of eriodictyol in HCC is still unclear. This study aimed to explore the effect of and mechanism of eriodictyol on HCC. In this study, compared with eriodictyol (0 μM) group, eriodictyol significantly suppressed HepG2 cells (eriodictyol of 25, 50 and 100 μM) and Huh-7 cells (eriodictyol of 50 and 100 μM) viability, invasion, tube formation, metastasis-related genes MMP3, MMP16 and angiogenesis regulator VEGFA expressions with IC50 of 45.63 μM and 78.26 μM in vitro, respectively. Besides, eriodictyol significantly repressed NLRP3 expression, and reduced the protein levels of NLRP3 inflammasome-related proteins, adapter protein ASC, caspase-1, interleukin (IL)-18, and IL-1β in HepG2 (eriodictyol of 25, 50 and 100 μM) and Huh-7 cells (eriodictyol of 50 and 100 μM), respectively. Meanwhile, compared with control group, NLRP3 overexpression reversed the anti-metastatic effects of 100 μM eriodictyol on HCC cells invasion, tube formation, and metastasis-related genes MMP3, MMP16 and angiogenesis regulator VEGFA expressions, whereas NLRP3 knockdown enhanced the anti-metastatic effects of 100 μM eriodictyol on HCC cells. In vivo, compared with control group, eriodictyol significantly reduced the tumor growth, liver damage, inhibited the activation of NLRP3 inflammasome, and improved liver function, whereas NLRP3 overexpressing neutralized the anti-tumor effects of eriodictyol and degraded liver function. Hence, eriodictyol inhibited HCC cell viability, motility, angiogenesis and tumor growth via NLRP3 inflammasome inactivation both in vitro and in vivo.

Inflammasome Activation and IL-1β Release Triggered by Nanosecond Pulsed Electric Fields in Murine Innate Immune Cells and Skin.

Although electric field-induced cell membrane permeabilization (electroporation) is used in a wide range of clinical applications from cancer therapy to cardiac ablation, the cellular- and molecular-level details of the processes that determine the success or failure of these treatments are poorly understood. Nanosecond pulsed electric field (nsPEF)-based tumor therapies are known to have an immune component, but whether and how immune cells sense the electroporative damage and respond to it have not been demonstrated. Damage- and pathogen-associated stresses drive inflammation via activation of cytosolic multiprotein platforms known as inflammasomes. The assembly of inflammasome complexes triggers caspase-1-dependent secretion of IL-1β and in many settings a form of cell death called pyroptosis. In this study we tested the hypothesis that the nsPEF damage is sensed intracellularly by the NLRP3 inflammasome. We found that 200-ns PEFs induced aggregation of the inflammasome adaptor protein ASC, activation of caspase-1, and triggered IL-1β release in multiple innate immune cell types (J774A.1 macrophages, bone marrow-derived macrophages, and dendritic cells) and invivo in mouse skin. Efflux of potassium from the permeabilized cell plasma membrane was partially responsible for nsPEF-induced inflammasome activation. Based on results from experiments using both the NRLP3-specific inhibitor MCC950 and NLRP3 knockout cells, we propose that the damage created by nsPEFs generates a set of stimuli for the inflammasome and that more than one sensor can drive IL-1β release in response to electrical pulse stimulation. This study shows, to our knowledge, for the first time, that PEFs activate the inflammasome, suggesting that this pathway alarms the immune system after treatment.

Nanoscale organization of the endogenous ASC speck

The NLRP3 inflammasome is a central component of the innate immune system. Its activation leads to formation of the ASC speck, a supramolecular assembly of the inflammasome adaptor protein ASC. Different models, based on ASC overexpression, have been proposed for the structure of the ASC speck. Using dual-color 3D super-resolution imaging (dSTORM and DNA-PAINT), we visualized the ASC speck structure following NLRP3 inflammasome activation using endogenous ASC expression. A complete structure was only obtainable by labeling with both anti-ASC antibodies and nanobodies. The complex varies in diameter between ∼800 and 1000nm, and is composed of a dense core with emerging filaments. Dual-color confocal fluorescence microscopy indicated that the ASC speck does not colocalize with the microtubule-organizing center at late time points after Nigericin stimulation. From super-resolution images of whole cells, the ASC specks were sorted into a pseudo-time sequence indicating that they become denser but not larger during formation.

IL11 activates the placental inflammasome to drive preeclampsia.

Preeclampsia is a life-threatening disorder of pregnancy unique to humans. Interleukin (IL)11 is elevated in serum from pregnancies that subsequently develop early-onset preeclampsia and pharmacological elevation of IL11 in pregnant mice causes the development of early-onset preeclampsia-like features (hypertension, proteinuria, and fetal growth restriction). However, the mechanism by which IL11 drives preeclampsia is unknown. Pregnant mice were administered PEGylated (PEG)IL11 or control (PEG) from embryonic day (E)10-16 and the effect on inflammasome activation, systolic blood pressure (during gestation and at 50/90 days post-natal), placental development, and fetal/post-natal pup growth measured. RNAseq analysis was performed on E13 placenta. Human 1st trimester placental villi were treated with IL11 and the effect on inflammasome activation and pyroptosis identified by immunohistochemistry and ELISA. PEGIL11 activated the placental inflammasome causing inflammation, fibrosis, and acute and chronic hypertension in wild-type mice. Global and placental-specific loss of the inflammasome adaptor protein Asc and global loss of the Nlrp3 sensor protein prevented PEGIL11-induced fibrosis and hypertension in mice but did not prevent PEGIL11-induced fetal growth restriction or stillbirths. RNA-sequencing and histology identified that PEGIL11 inhibited trophoblast differentiation towards spongiotrophoblast and syncytiotrophoblast lineages in mice and extravillous trophoblast lineages in human placental villi. Inhibition of ASC/NLRP3 inflammasome activity could prevent IL11-induced inflammation and fibrosis in various disease states including preeclampsia.

C4b-binding protein inhibits particulate- and crystalline-induced NLRP3 inflammasome activation.

Dysregulated NLRP3 inflammasome activation drives a wide variety of diseases, while endogenous inhibition of this pathway is poorly characterised. The serum protein C4b-binding protein (C4BP) is a well-established inhibitor of complement with emerging functions as an endogenously expressed inhibitor of the NLRP3 inflammasome signalling pathway. Here, we identified that C4BP purified from human plasma is an inhibitor of crystalline- (monosodium urate, MSU) and particulate-induced (silica) NLRP3 inflammasome activation. Using a C4BP mutant panel, we identified that C4BP bound these particles via specific protein domains located on the C4BP α-chain. Plasma-purified C4BP was internalised into MSU- or silica-stimulated human primary macrophages, and inhibited MSU- or silica-induced inflammasome complex assembly and IL-1β cytokine secretion. While internalised C4BP in MSU or silica-stimulated human macrophages was in close proximity to the inflammasome adaptor protein ASC, C4BP had no direct effect on ASC polymerisation in in vitro assays. C4BP was also protective against MSU- and silica-induced lysosomal membrane damage. We further provide evidence for an anti-inflammatory function for C4BP in vivo, as C4bp-/- mice showed an elevated pro-inflammatory state following intraperitoneal delivery of MSU. Therefore, internalised C4BP is an inhibitor of crystal- or particle-induced inflammasome responses in human primary macrophages, while murine C4BP protects against an enhanced inflammatory state in vivo. Our data suggests C4BP has important functions in retaining tissue homeostasis in both human and mice as an endogenous serum inhibitor of particulate-stimulated inflammasome activation.

Lyososome Membrane Permeabilization Induces Plasma Membrane Macropores to Activate K +-Efflux-Dependent NLRP3 Inflammasomes in Murine Dendritic Cells

Abstract NLRP3 is a cytosolic sensor protein in myeloid leukocytes that recruits the adaptor protein ASC and activates caspase-1 in functional NLRP3 inflammasomes. Caspase-1 coordinately cleaves pro-IL-1β to bioactive IL-1β and gasdermin D (GSDMD) to induce N-GSDMD macropores in the plasma membrane (PM). The latter mediate efflux of mature IL-1β and initiate progression to pyroptosis, a lytic cell death. NLRP3 activation is triggered by perturbed cellular homeostasis with decreased cytosolic [K +] being the best-characterized signal. The required K +efflux is typically induced by the ionophore nigericin or extracellular ATP as an agonist for P2X7 receptor channels. K +efflux-dependent activation of NLRP3 is also triggered by soluble or particulate agents which accumulate in lysosomes to induce lysosomal membrane permeabilization (LMP) and release of hydrolytic enzymes into the cytosol; particulates include alum widely used as a vaccine adjuvant. The mechanisms by which LMP rapidly triggers changes in plasma membrane permeability remain undefined. We used a model of murine bone marrow derived dendritic cells (BMDC) treated with the soluble LMP agent Leu-Leu-O-methyl ester (LLME) to elicit K+ efflux and NLRP3 activation. Notably, this K+ efflux was coincident with a rapidly induced influx of propidiumiodide (PI), a 668 Da organic salt routinely used to track assembly of plasma membrane macropores such as GSDMD. Equivalent LLME-induced PI influx was observed in wildtype and Gsdmd−/−BMDC and was unaffected by inhibition of NLRP3 or caspase-1. These observations indicate that LMP triggers rapid activation of an endogenous pore-forming protein distinct from GSDMD. Current studies are evaluating gasdermin E or pannexin-1 as candidates. Supported by grants from the NIH (P01-AI141350 and R01-EY014362)

Porphyromonas gingivalis Evades Immune Clearance by Regulating Lysosome Efflux

Porphyromonas gingivalis, a major periodontal pathogen, invades autophagosomes of cells, including gingival epithelial cells, endothelial cells, gingival fibroblasts, macrophages, and dendritic cells, to escape antimicrobial autophagy and lysosome fusion. However, it is not known how P. gingivalis resists autophagic immunity, survives within cells, and induces inflammation. Thus, we investigated whether P. gingivalis could escape antimicrobial autophagy by promoting lysosome efflux to block autophagic maturation, leading to intracellular survival, and whether the growth of P. gingivalis within cells results in cellular oxidative stress, causing mitochondrial damage and inflammatory responses. P. gingivalis invaded human immortalized oral epithelial cells in vitro and mouse oral epithelial cells of gingival tissues in vivo. The production of reactive oxygen species (ROS) increased upon bacterial invasion, as well as mitochondrial dysfunction-related parameters with downregulated mitochondrial membrane potential and intracellular adenosine triphosphate (ATP), upregulated mitochondrial membrane permeability, intracellular Ca2+ influx, mitochondrial DNA expression, and extracellular ATP. Lysosome excretion was elevated, the number of intracellular lysosomes was diminished, and lysosomal-associated membrane protein 2 was downregulated. Expression of autophagy-related proteins, microtubule-associated protein light chain 3, sequestosome-1, the NLRP3 inflammasome, and interleukin-1β increased with P. gingivalis infection. P. gingivalis may survive in vivo by promoting lysosome efflux, blocking autophagosome-lysosome fusion, and destroying autophagic flux. As a result, ROS and damaged mitochondria accumulated and activated the NLRP3 inflammasome, which recruited the adaptor protein ASC and caspase 1, leading to the production of proinflammatory factor interleukin-1β and inflammation.

Inflammasome activation by Gram-positive bacteria: Mechanisms of activation and regulation.

The inflammasomes are intracellular multimeric protein complexes consisting of an innate immune sensor, the adapter protein ASC and the inflammatory caspases-1 and/or -11 and are important for the host defense against pathogens. Activaton of the receptor leads to formation of the inflammasomes and subsequent processing and activation of caspase-1 that cleaves the proinflammatory cytokines IL-1β and IL-18. Active caspase-1, and in some instances caspase-11, cleaves gasdermin D that translocates to the cell membrane where it forms pores resulting in the cell death program called pyroptosis. Inflammasomes can detect a range of microbial ligands through direct interaction or indirectly through diverse cellular processes including changes in ion fluxes, production of reactive oxygen species and disruption of various host cell functions. In this review, we will focus on the NLRP3, NLRP6, NLRC4 and AIM2 inflammasomes and how they are activated and regulated during infections with Gram-positive bacteria, including Staphylococcus spp., Streptococcus spp. and Listeria monocytogenes.

Methods to Activate the NLRP3 Inflammasome.

The inflammasome-nucleating cytoplasmic sensor protein NLRP3 (NACHT-, LRR, and PYD domains-containing protein 3, also known as NOD-like receptor pyrin domain-containing 3, NALP3, or cryopyrin) is triggered by a broad spectrum of sterile endogenous danger signals and environmental irritants. Upon activation, NLRP3 engages the adapter protein ASC that in turn recruits the third inflammasome component, the protease caspase-1. Subsequent caspase-1 activation leads to its auto-processing and maturation of the leaderless IL-1 family cytokines IL-1β and IL-18 as well as cleavage of the pore-forming protein Gasdermin D (GSDMD). GSDMD plasma membrane pores, formed by its N-terminus, facilitate IL-1 release and, typically, subsequent cell lysis (pyroptosis). This protocol explains standard methods, which are routinely used in our laboratory to study NLRP3 inflammasome biology in vitro. It includes experimental approaches using primary murine bone marrow-derived macrophages (BMDMs) and bone marrow-derived dendritic cells (BMDCs), human peripheral blood mononuclear cells (PBMCs), as well as inflammasome-competent cell lines (HoxB8 and THP-1 cells). The protocol covers the use of a broad spectrum of established NLRP3 activators and outlines the use of common inhibitors blocking NLRP3 itself or its upstream triggering events. We also provide guidelines for experimental set-up and crucial experimental controls to investigate NLRP3 inflammasome signaling or study new activators and inhibitors.

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.

Spirodalesol analog 8A inhibits NLRP3 inflammasome activation and attenuates inflammatory disease by directly targeting adaptor protein ASC

Pharmacological inhibition of the Nod-like receptor family protein 3 (NLRP3) inflammasome contributes to the treatment of numerous inflammation-related diseases, making it a desirable drug target. Spirodalesol, derived from the ascomycete fungus Daldinia eschscholzii, has been reported to inhibit NLRP3 inflammasome activation. Based on the structure of spirodalesol, we synthesized and screened a series of analogs to find a more potent inhibitor. Analog compound 8A was identified as the most potent selective inhibitor for NLRP3 inflammasome assembly, but 8A did not inhibit the priming phase of the inflammasome. Specifically, while 8A did not reduce NLRP3 oligomerization, we found that it inhibited the oligomerization of adaptor protein apoptosis-associated speck-like protein containing a caspase activation and recruitment domain (ASC), as ASC speck formation was significantly reduced. Also, 8A interrupted the assembly of the NLRP3 inflammasome complex and inhibited the activation of caspase-1. Subsequently, we used a cellular thermal shift assay and microscale thermophoresis assay to demonstrate that 8A interacts directly with ASC, both invitro and exvivo. Further, 8A alleviated lipopolysaccharide-induced endotoxemia, as well as monosodium urate-induced peritonitis and gouty arthritis in mice by suppressing NLRP3 inflammasome activation. Thus, 8A was identified as a promising ASC inhibitor to treat inflammasome-driven diseases.

Endothelial Acid Sphingomyelinase Promotes NLRP3 Inflammasome and Neointima Formation During Hypercholesterolemia

The NOD-like receptor pyrin domain 3 (NLRP3) inflammasome is activated during atherogenesis, but how this occurs is unclear. Here, we explored the mechanisms activating and regulating NLRP3 inflammasomes via the acid sphingomyelinase (ASM)-ceramide signaling pathway. As a neointima formation model, partial left carotid ligations were performed on endothelial cell (EC)-specific ASM transgene mice (Smpd1trg/ECcre) and their control littermates (Smpd1trg/WT and WT/WT) fed on the Western diet (WD). We found neointima formation remarkably increased in Smpd1trg/ECcre mice over their control littermates. Next, we observed enhanced colocalization of NLRP3 versus adaptor protein ASC (the adaptor molecule apoptosis-associated speck-like protein containing a CARD) or caspase-1 in the carotid ECs of WD-treated Smpd1trg/ECcre mice but not in their control littermates. In addition, we used membrane raft (MR) marker flotillin-1 and found more aggregation of ASM and ceramide in the intima of Smpd1trg/ECcre mice than their control littermates. Moreover, we demonstrated by in situ dihydroethidium staining, carotid intimal superoxide levels were much higher in WD-treated Smpd1trg/ECcre mice than in their control littermates. Using ECs from Smpd1trg/ECcre and WT/WT mice, we showed ASM overexpression markedly enhanced 7-ketocholesterol (7-Ket)-induced increases in NLRP3 inflammasome formation, accompanied by enhanced caspase-1 activity and elevated interleukin-1β levels. These 7-Ket-induced increases were significantly attenuated by ASM inhibitor amitriptyline. Furthermore, we determined that increased MR clustering with NADPH oxidase subunits to produce superoxide contributes to 7-Ket-induced NLRP3 inflammasome activation via a thioredoxin-interacting protein-mediated controlling mechanism. We conclude that ceramide from ASM plays a critical role in NLRP3 inflammasome activation during hypercholesterolemia via MR redox signaling platforms to produce superoxide, which leads to TXNIP dissociation.

Single cell analysis of PANoptosome cell death complexes through an expansion microscopy method.

In response to infection or sterile insults, inflammatory programmed cell death is an essential component of the innate immune response to remove infected or damaged cells. PANoptosis is a unique innate immune inflammatorycell death pathway regulated by multifaceted macromolecular complexes called PANoptosomes, which integrate components from other cell death pathways. Growing evidence shows that PANoptosis can be triggered in many physiological conditions, including viral and bacterial infections, cytokine storms, and cancers. However, PANoptosomes at the single cell level have not yet been fully characterized. Initial investigations have suggested that key pyroptotic, apoptotic, and necroptotic molecules including the inflammasome adaptor protein ASC, apoptotic caspase-8 (CASP8), and necroptotic RIPK3 are conserved components of PANoptosomes. Here, we optimized an immunofluorescence procedure to probe the highly dynamic multiprotein PANoptosome complexes across various innate immune cell death-inducing conditions. We first identified and validated antibodies to stain endogenous mouse ASC, CASP8, and RIPK3, without residual staining in the respective knockout cells. We then assessed the formation of PANoptosomes across innate immune cell death-inducing conditions by monitoring the colocalization of ASC with CASP8 and/or RIPK3. Finally, we established an expansion microscopy procedure using these validated antibodies to image the organization of ASC, CASP8, and RIPK3 within the PANoptosome. This optimized protocol, which can be easily adapted to study other multiprotein complexes and other cell death triggers, provides confirmation of PANoptosome assembly in individual cells and forms the foundation for a deeper molecular understanding of the PANoptosome complex and PANoptosis to facilitate therapeutic targeting.

POP1 inhibits MSU-induced inflammasome activation and ameliorates gout.

Canonical inflammasomes are innate immune protein scaffolds that enable the activation of inflammatory caspase-1, and subsequently the processing and release of interleukin (IL)-1β, IL-18, and danger signals, as well as the induction of pyroptotic cell death. Inflammasome assembly and activation occurs in response to sensing of infectious, sterile and self-derived molecular patterns by cytosolic pattern recognition receptors, including the Nod-like receptor NLRP3. While these responses are essential for host defense, excessive and uncontrolled NLRP3 inflammasome responses cause and contribute to a wide spectrum of inflammatory diseases, including gout. A key step in NLRP3 inflammasome assembly is the sequentially nucleated polymerization of Pyrin domain (PYD)- and caspase recruitment domain (CARD)-containing inflammasome components. NLRP3 triggers polymerization of the adaptor protein ASC through PYD-PYD interactions, but ASC polymerization then proceeds in a self-perpetuating manner and represents a point of no return, which culminates in the activation of caspase-1 by induced proximity. In humans, small PYD-only proteins (POPs) lacking an effector domain regulate this key process through competitive binding, but limited information exists on their physiological role during health and disease. Here we demonstrate that POP1 expression in macrophages is sufficient to dampen MSU crystal-mediated inflammatory responses in animal models of gout. Whether MSU crystals are administered into a subcutaneous airpouch or into the ankle joint, the presence of POP1 significantly reduces neutrophil infiltration. Also, airpouch exudates have much reduced IL-1β and ASC, which are typical pro-inflammatory indicators that can also be detected in synovial fluids of gout patients. Exogenous expression of POP1 in mouse and human macrophages also blocks MSU crystal-induced NLRP3 inflammasome assembly, resulting in reduced IL-1β and IL-18 secretion. Conversely, reduced POP1 expression in human macrophages enhances IL-1β secretion. We further determined that the mechanism for the POP1-mediated inhibition of NLRP3 inflammasome activation is through its interference with the crucial NLRP3 and ASC interaction within the inflammasome complex. Strikingly, administration of an engineered cell permeable version of POP1 was able to ameliorate MSU crystal-mediated inflammation in vivo, as measured by neutrophil infiltration. Overall, we demonstrate that POP1 may play a crucial role in regulating inflammatory responses in gout.