Mature IL-1β Research Articles

Structural Mechanisms of NLRP3 Inflammasome Assembly and Activation.

As an important sensor in the innate immune system, NLRP3 detects exogenous pathogenic invasions and endogenous cellular damage and responds by forming the NLRP3 inflammasome, a supramolecular complex that activates caspase-1. The three major components of the NLRP3 inflammasome are NLRP3, which captures the danger signals and recruits downstream molecules; caspase-1, which elicits maturation of the cytokines IL-1β and IL-18 and processing of gasdermin D to mediate cytokine release and pyroptosis; and ASC (apoptosis-associated speck-like protein containing a caspase recruitment domain), which functions as a bridge connecting NLRP3 and caspase-1. In this article, we review the structural information that has been obtained on the NLRP3 inflammasome and its components or subcomplexes, with special focus on the inactive NLRP3 cage, the active NLRP3-NEK7 (NIMA-related kinase 7)-ASC inflammasome disk, and the PYD-PYD and CARD-CARD homotypic filamentous scaffolds of the inflammasome. We further implicate structure-derived mechanisms for the assembly and activation of the NLRP3 inflammasome.

Ring finger protein 10 improves pirarubicin-induced cardiac inflammation by regulating the AP-1/Meox2 signaling pathway

ObjectivesPirarubicin (THP) is widely used in clinical antitumor therapy, but its cardiotoxicity seriously affects the therapeutic effect in patients. In the study, we investigated the role of ring finger protein 10 (RNF10) in cardiotoxicity induced by THP. Materials and methodsA cardiac toxicity model in Sprague-Dawley (SD) rats induced by THP was established. Changes in diet, weight, electrocardiogram (ECG), and echocardiography were observed. Serum levels of brain natriuretic peptide (BNP), creatine kinase MB (CK-MB), cardiac troponin T (cTnT), and lactate dehydrogenase (LDH) were measured. The expression of RNF10 in myocardium was observed by immunohistochemistry. The expressions of RNF10, activator protein-1 (AP-1), mesenchyme homeobox 2 (Meox2), total nuclear factor (NF)-κB p65 (T-P65), phosphorylated NF-κB p65 (PP65), monocyte chemoattractant protein-1 (MCP-1), tumor necrosis factor (TNF)-α, interleukin (IL)-6, and mature IL-1β were detected by Western blot. A THP-induced H9c2 myocardial cell injury model was established. RNF10 was downregulated or overexpressed by RNF10 siRNA and a RNF10 lentiviral vector, respectively. Then, cell viability was measured. The expression of RNF10 in H9c2 cells was observed by immunofluorescence. All of the above signaling pathways were verified by Western blots. FindingsTHP caused a series of cardiotoxic manifestations in SD rats. Our studies suggested that THP caused cardiac inflammation by inhibiting the expression of RNF10, while overexpression of RNF10 antagonized the cardiotoxicity induced by THP. SignificanceOur study showed RNF10 improved THP-induced cardiac inflammation by regulating the AP-1/Meox2 signaling pathway. RNF10 may be a new target to treat THP-induced cardiotoxicity.

Dimethyl fumarate ameliorates fungal keratitis by limiting fungal growth and inhibiting pyroptosis.

We aimed to investigate the therapeutic role of dimethyl fumarate (DMF) in fungal keratitis. Human corneal epithelial cells (HCECs) and mouse models of fungal keratitis were used in this study. The antifungal effect of DMF on Aspergillus fumigatus (A. fumigatus) was confirmed by examining the minimum inhibitory concentration (MIC), biofilm formation, conidial adherence and corneal fungal loads. Slit-lamp photography, haematoxylin and eosin staining and immunostaining were used to assess the severity of corneal impairment. RT-PCR, western blot, ELISA, immunohistochemistry and immunostaining were performed to examine the effects of DMF on the expression of the inflammatory mediators during fungal infection. In vitro, DMF limited A. fumigatus growth, biofilm formation, and conidial adherence and reduced the mRNA levels of AldA, GlkA, GAPDH, HxkA, PgkA, Sdh2, GelA and ChsF in A. fumigatus. In vivo, DMF effectively decreased corneal fungal loads. DMF attenuated corneal inflammatory impairment by suppressing inflammatory cell accumulation and downregulating cytokine expression. DMF notably downregulated the high expression of NLRP3, cleaved GSDMD, cleaved caspase-1, mature IL-1β and mature IL-18 induced by fungi. The production of Nrf2 and HO-1 could be further increased by DMF in infected HCECs. Nrf2 siRNA pretreatment counteracted DMF-mediated downregulation of the expression of the active forms of IL-18, IL-1β, caspase-1 and GSDMD. DMF limits fungal growth by suppressing biofilm formation, conidial adherence and respiratory metabolism. It also exerts an anti-inflammatory effect on fungal keratitis by inhibiting pyroptosis, which could be regulated by Nrf2. Our results suggest that DMF plays a therapeutic role in fungal keratitis.

EuHD1 protects against inflammatory injury driven by NLRP3 inflammasome.

Non-steroidal anti-inflammatory drugs (NSAIDs) possessing anti-inflammatory, analgesic and antipyretic activities, are widely used in the treatment of osteoarthritis, rheumatism and rheumatoid arthritis. However, its long-term or large use will cause serious gastrointestinal injury or cardiovascular adverse reactions, which limits its clinical application. We have synthesized a new class of NSAIDs, EuHD1, which can release hydrogen sulfide and have better gastrointestinal safety. However, the anti-inflammatory molecular mechanism of the drug is still unclear. In this paper, we explored the mechanism of EuHD1 on NLRP3 inflammasome and its effects on acute lung injury and acute liver injury in mice. In vitro results demonstrated that EuHD1 inhibited macrophage pyroptosis and LDH release induced by LPS combined with ATP. In addition, EuHD1 blocked NLRP3 inflammasome activation and suppressed following Caspase-1 activation and secretion of mature IL-1β. EuHD1 restrained intracellular ROS production and the formation of ASC oligomers, which inhibited the assembly and activation of NLRP3 inflammasome. In vivo results further showed that EuHD1 alleviated LPS-induced acute lung injury in mice, and inhibited the production of mature IL-1β and Caspase-1 (p20). Besides, EuHD1 improved D-GalN/LPS-induced acute liver injury, and inhibited SOD/MDA levels and oxidative stress injury, and blocked the activation of NLRP3 inflammasome. In summary, we found that EuHD1 inhibits the assembly and activation of NLRP3 inflammasome through restraining the production of ROS and the formation of ASC oligomers, and has therapeutic effects on acute lung injury and liver injury in mice, indicating that EuHD1 has the potential to treat NLRP3 inflammasome-related diseases.

Inhibition of sphingosine-1-phosphate receptor 3 suppresses ATP-induced NLRP3 inflammasome activation in macrophages via TWIK2-mediated potassium efflux.

Inhibition of sphingosine kinase 1 (SphK1), which catalyzes bioactive lipid sphingosine-1-phosphate (S1P), attenuates NLRP3 inflammasome activation. S1P exerts most of its function by binding to S1P receptors (S1PR1-5). The roles of S1P receptors in NLRP3 inflammasome activation remain unclear. The mRNA expressions of S1PRs in bone marrow-derived macrophages (BMDMs) were measured by real-time quantitative polymerase chain reaction (qPCR) assays. BMDMs were primed with LPS and stimulated with NLRP3 activators, including ATP, nigericin, and imiquimod. Interleukin-1β (IL-1β) in the cell culture supernatant was detected by enzyme-linked immunosorbent assay (ELISA). Intracellular potassium was labeled with a potassium indicator and was measured by confocal microscopy. Protein expression in whole-cell or plasma membrane fraction was measured by Western blot. Cecal ligation and puncture (CLP) was induced in C57BL/6J mice. Mortality, lung wet/dry ratio, NLRP3 activation, and bacterial loads were measured. Macrophages expressed all five S1PRs in the resting state. The mRNA expression of S1PR3 was upregulated after lipopolysaccharide (LPS) stimulation. Inhibition of S1PR3 suppressed NLRP3 and pro-IL-1β in macrophages primed with LPS. Inhibition of S1PR3 attenuated ATP-induced NLRP3 inflammasome activation, enhanced nigericin-induced NLRP3 activation, and did not affect imiquimod-induced NLRP3 inflammasome activation. In addition, inhibition of S1PR3 suppressed ATP-induced intracellular potassium efflux. Inhibition of S1PR3 did not affect the mRNA or protein expression of TWIK2 in LPS-primed BMDMs. ATP stimulation induced TWIK2 expression in the plasma membrane of LPS-primed BMDMs, and inhibition of S1PR3 impeded the membrane expression of TWIK2 induced by ATP. Compared with CLP mice treated with vehicle, CLP mice treated with the S1PR3 antagonist, TY52156, had aggravated pulmonary edema, increased bacterial loads in the lung, liver, spleen, and blood, and a higher seven-day mortality rate. Inhibition of S1PR3 suppresses the expression of NLRP3 and pro-IL-1β during LPS priming, and attenuates ATP-induced NLRP3 inflammasome activation by impeding membrane trafficking of TWIK2 and potassium efflux. Although inhibition of S1PR3 decreases IL-1β maturation in the lungs, it leads to higher bacterial loads and mortality in CLP mice.

A Staphylococcal Glucosaminidase Drives Inflammatory Responses by Processing Peptidoglycan Chains to Physiological Lengths.

The peptidoglycan of Staphylococcus aureus is a critical cell envelope constituent and virulence factor that subverts host immune defenses and provides protection against environmental stressors. Peptidoglycan chains of the S. aureus cell wall are processed to characteristically short lengths by the glucosaminidase SagB. It is well established that peptidoglycan is an important pathogen-associated molecular pattern (PAMP) that is recognized by the host innate immune system and promotes production of proinflammatory cytokines, including interleukin-1β (IL-1β). However, how bacterial processing of peptidoglycan drives IL-1β production is comparatively unexplored. Here, we tested the involvement of staphylococcal glucosaminidases in shaping innate immune responses and identified SagB as a mediator of IL-1β production. A ΔsagB mutant fails to promote IL-1β production by macrophages and dendritic cells, and processing of peptidoglycan by SagB is essential for this response. SagB-dependent IL-1β production by macrophages is independent of canonical pattern recognition receptor engagement and NLRP3 inflammasome-mediated caspase activity. Instead, treatment of macrophages with heat-killed cells from a ΔsagB mutant leads to reduced caspase-independent cleavage of pro-IL-1β, resulting in accumulation of the pro form in the macrophage cytosol. Furthermore, SagB is required for virulence in systemic infection and promotes IL-1β production in a skin and soft tissue infection model. Taken together, our results suggest that the length of S. aureus cell wall glycan chains can drive IL-1β production by innate immune cells through a previously undescribed mechanism related to IL-1β maturation.

Ac-YVAD-cmk ameliorated sevoflurane-induced cognitive dysfunction and revised mitophagy impairment.

It is common for elderly patients to develop postoperative cognitive dysfunction (POCD), but the pathophysiological mechanisms have not yet been fully explored. NLRP3 inflammasome activation and mitophagy impairment was involved in neurodegenerative disease. This study investigated the interaction of NLRP3 inflammasome and mitophagy in sevoflurane-induced cognitive dysfunction. We found that sevoflurane induced cleaved caspase-1 level, IL-1β and IL-18 maturation, and activated NLRP3 inflammasome in aged mice and the primary hippocampus neuron. The cleaved caspase-1 was demonstrated in microglia of hippocampus. Ac-YVAD-cmk, a selected caspase-1 inhibitor, reduced the expression of cleaved caspase-1, IL-1β, IL-18 and NLRP3 inflammasome activation induced by sevoflurane. Ac-YVAD-cmk ameliorated learning ability impairment in aged mice induced by sevoflurane using Morris water maze. Moreover, Ac-YVAD-cmk reversed the mitophagy flux dysfunction induced by sevoflurane in aged mice by western blotting, immunostaining and mt-Keima reporters. For the first time, we found caspase-1 inhibitor mitigated mitochondria dysfunction and revised mitophagy impairment induced by sevoflurane.

TRIM72 Alleviates Muscle Inflammation in mdx Mice via Promoting Mitophagy-Mediated NLRP3 Inflammasome Inactivation.

Chronic muscle inflammation exacerbates the pathogenesis of Duchenne muscular dystrophy (DMD), which is characterized by progressive muscle degeneration and weakness. NLRP3 (nucleotide-binding domain and leucine-rich repeat pyrin domain containing 3) inflammasome plays a key role in the inflammatory process, and its abnormal activation leads to a variety of inflammatory or immune diseases. TRIM72 (MG53) is a protective myokine for tissue repair and regeneration. However, little is known about the potential impact of TRIM72 in the crosstalk between mitophagy and inflammatory process of DMD. Here, 10-week-old male mdx mice were injected intramuscularly with adeno-associated virus (AAV-TRIM72) to overexpress TRIM72 protein for 6 weeks. Then, skeletal muscle samples were collected, and relevant parameters were measured by histopathological analysis and molecular biology techniques. C2C12 cell line was transfected with lentivirus (LV-TRIM72) to overexpress or siRNA (si-TRIM72) to suppress the TRIM72 expression for the following experiment. Our data firstly showed that the TRIM72 expression was decreased in skeletal muscles of mdx mice. Then, we observed the increased NLRP3 inflammasome and impaired mitophagy in mdx mice compared with wild type mice. In mdx mice, administration of AAV-TRIM72 alleviated the accumulation of NLRP3 inflammasome and the consequent IL-18 and IL1β maturation by inducing autophagy, while this protective effect was reversed by chloroquine. Mitochondrial reactive oxygen species (mtROS), as a recognized activator for NLRP3 inflammasome, was attenuated by TRIM72 through the induction of mitophagy in C2C12 cells. Additionally, we proposed that the TRIM72 overexpression might promote mitophagy through both the early stage by PI3K-AKT pathway and the late stage by autolysosome fusion. In conclusion, the current study suggests that TRIM72 prevents DMD inflammation via decreasing NLRP3 inflammasomes and enhancing mitophagy. Collectively, our study provides insight into TRIM72 as a promising target for therapeutic intervention for DMD.

Caspase-8-driven apoptotic and pyroptotic crosstalk causes cell death and IL-1β release in X-linked inhibitor of apoptosis (XIAP) deficiency.

Genetic lesions in X-linked inhibitor of apoptosis (XIAP) pre-dispose humans to cell death-associated inflammatory diseases, although the underlying mechanisms remain unclear. Here, we report that two patients with XIAP deficiency-associated inflammatory bowel disease display increased inflammatory IL-1β maturation as well as cell death-associated caspase-8 and Gasdermin D (GSDMD) processing in diseased tissue, which is reduced upon patient treatment. Loss of XIAP leads to caspase-8-driven cell death and bioactive IL-1β release that is only abrogated by combined deletion of the apoptotic and pyroptotic cell death machinery. Namely, extrinsic apoptotic caspase-8 promotes pyroptotic GSDMD processing that kills macrophages lacking both inflammasome and apoptosis signalling components (caspase-1, -3, -7, -11 and BID), while caspase-8 can still cause cell death in the absence of both GSDMD and GSDME when caspase-3 and caspase-7 are present. Neither caspase-3 and caspase-7-mediated activation of the pannexin-1 channel, or GSDMD loss, prevented NLRP3 inflammasome assembly and consequent caspase-1 and IL-1β maturation downstream of XIAP inhibition and caspase-8 activation, even though the pannexin-1 channel was required for NLRP3 triggering upon mitochondrial apoptosis. These findings uncouple the mechanisms of cell death and NLRP3 activation resulting from extrinsic and intrinsic apoptosis signalling, revealhow XIAP loss can co-opt dual cell death programs, and uncover strategies for targeting the celldeath and inflammatory pathways that result from XIAP deficiency.

The neuropeptide CGRP enters the macrophage cytosol to suppress the NLRP3 inflammasome during pulmonary infection.

The NLRP3 inflammasome plays an essential role in resistance to bacterial infection. The nervous system secretes multiple neuropeptides affecting the nervous system as well as immune cells. The precise impact of the neuropeptide CGRP on NLRP3 inflammasome activation is still unclear. Here, we show that CGRP negatively regulates the antibacterial process of host cells. CGRP prevents NLRP3 inflammasome activation and reduces mature IL-1β secretion. Following NLRP3 inflammasome stimulation that triggers endosome leakage, CGRP internalized to endosomal compartments is released into the cell cytosol. Cytosolic CGRP binds directly to NLRP3 and dismantles the NLRP3-NEK7 complex, which is crucial for NLRP3 inflammasome activation. CGRP administration exacerbates bacterial infection, while thetreatment with a CGRP antagonist has the opposite effect. Our study uncovers a unique role of CGRP in inhibiting inflammasome activation during infections, which might shed new light on antibacterial therapies in the future.

Assessment of NLRP3 Inflammasome Activation and NLRP3-NEK7 Complex Assembly.

The NLRP3 inflammasome is a critical component of innate immunity that activates caspase-1 to induce inflammation in response to a wide spectrum of endogenous and exogenous stimuli. NLRP3 inflammasome activation has been shown by assays for the cleavage of caspase-1 and gasdermin D, the maturation of IL-1β and IL-18, and ASC speck formation in innate immune cells such as macrophages and monocytes. Recently, NEK7 has been revealed as an essential regulator for NLRP3 inflammasome activation by forming high-molecular-weight complexes with NLRP3. Blue native polyacrylamide gel electrophoresis (BN-PAGE) has been used to study multi-protein complexes in many experimental systems. Here, we provide a detailed protocol to detect NLRP3 inflammasome activation and NLRP3-NEK7 complex assembly in mouse macrophages by Western blot and BN-PAGE.

Inflammasome Activation in Human Macrophages: IL-1β Cleavage Detection by Fully Automated Capillary-Based Immunoassay.

Interleukin (IL)-1β is a key mediator of inflammation and activates via pattern recognition receptors (PRR) of the inflammasome family by proteolytic maturation. Proteolysis is driven by proteases such as caspase-1 (also known as IL-1 converting enzyme, ICE) and converts the intact pro-IL-1β ~31kDa pro-peptide into a mature, ~17kDa form that can exit cells through nanomolecular pores or via microvesicles. Whereas pro-IL-1β fails to trigger IL-1 receptor (IL-1R) activation, mature IL-1β, upon release from the cell, triggers pleiotropic downstream effects, establishing an inflammatory state. Hence, being able to detect IL-1β conversion is physiologically relevant for measuring inflammation, but it cannot be easily accomplished by conventional ELISA or flow cytometry as most commercially available antibodies do not discriminate mature and pro-form. Furthermore, unlike for other cytokines, the mere induction and translation of IL1B mRNA cannot serve as a proxy of inflammasome PRR activation. Rather the cleavage of IL-1β needs to be verified. Hence, conventional immunoblotting has emerged as the gold standard for demonstrating inflammasome activation as the difference in molecular weight between pro- and mature form can easily be detected. However, conventional immunoblotting suffers from poor standardization, quantification, and reproducibility, may require sample concentration, and is also not suitable for medium to high throughput. Some of these shortcomings are prohibitive for analysis of human primary samples but can be overcome by fully automated capillary-based immunoassay as we outline here. We here provide a practical guide to quantify pro- vs mature IL-1β directly from unconcentrated supernatants of human monocyte-derived macrophages. The assay may be useful for more standardized and medium-throughput analysis in these cells or other biospecimen.

Pro-Inflammatory of PRDM1/SIRT2/NLRP3 Axis in Monosodium Urate-Induced Acute Gouty Arthritis

PR domain-containing 1 with zinc finger domain (PRDM1) has been reported as a promoter of inflammation, which is a critical process involved in the pathogenesis of acute gouty arthritis. Herein, we sought to ascertain the function of PRDM1 in the development of acute gouty arthritis and related mechanisms. At first, peripheral blood-derived monocytes from patients with acute gouty arthritis and healthy individuals were collected as experimental samples. Then, macrophages were induced from monocytes using phorbol myristate acetate (PMA). The expression patterns of PRDM1, sirtuin 2 (SIRT2), and NLR family, pyrin domain-containing 3 (NLRP3) were characterized by RT-qPCR and Western blot assay. PMA-induced macrophages were stimulated by monosodium urate (MSU) for in vitro experimentation. Meanwhile, a murine model of MSU-induced acute gouty arthritis was established for in vivo validation. PRDM1 was highly expressed while SIRT2 poorly expressed in patients with acute gouty arthritis. Loss of PRDM1 could reduce NLRP3 inflammasome and mature IL-1β levels and downregulate inflammatory cytokines in macrophages, which contributed to protection against acute gouty arthritis. Furthermore, results showed that PRDM1 could inhibit SIRT2 expression via binding to the deacetylase SIRT2 promoter. Finally, the in vivo experiments demonstrated that PRDM1 increased NLRP3 inflammasome and mature IL-1β through transcriptional inhibition of SIRT2, whereby aggravating MSU-induced acute gouty arthritis. To sum up, PRDM1 increased NLRP3 inflammasome through inhibiting SIRT2, consequently aggravating MSU-induced acute gouty arthritis.

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.

Monitoring Calcium Fluxes and Lysosome Exocytosis During Pyroptosis.

Inflammasome-mediated activation of inflammatory caspases (caspase-1, caspase-4, caspase-5, caspase-11) initiates a cascade of cellular events that lead to proinflammatory cell death, or pyroptosis. Proteolytic cleavage of gasdermin D results in the formation of transmembrane pores that allow the release of mature cytokines IL-1β and IL-18. Gasdermin pores also allow calcium influx through the plasma membrane, triggering the fusion of lysosomal compartments with the cell surface and release of their contents into the extracellular milieu in a process termed lysosome exocytosis. This chapter outlines methods for measuring calcium flux, lysosome exocytosis, and membrane disruption after inflammatory caspase activation.

In Vitro Study of TLR4-NLRP3-Inflammasome Activation in Innate Immune Response.

Toll-like receptors (TLRs) are pivotal players in mediating immune responses. TLR4 is the main receptor for LPS, a strong activator of immune cells. LPS/TLR4-dependent pathway, by inducing NF-κB activation, is responsible for the release of several mediators, including IL-1β, one of the most powerful cytokines deeply involved in inflammatory and immune responses. The same pathway is also involved in NLRP3-inflammasome activation, essential for IL-1β maturation. NLRP3 is a major component of innate immune responses, being a crucial player of host immune defense against virus, bacterial, or fungal infections. NLRP3-inflammasome and IL-1β hyperactivation have been associated to the pathogenesis of a wide range of disorders and represent therapeutic targets for the development of new treatments of inflammasome-driven inflammatory and autoimmune diseases.Here, we describe an in vitro protocol to induce LPS/TLR4-dependent NLRP3-inflammasome/IL-1β activation in immune cells, in order to provide a useful assay to study the efficacy of different anti-inflammatory/immune-modulatory agents.

IL-1β, an important cytokine affecting Helicobacter pylori-mediated gastric carcinogenesis.

Infection with Helicobacter pylori (H. pylori) is prevalent around the world and responsible for gastric cancer (GC). The development of GC from gastritis is closely associated with the bacterial virulence and the body's immune response ability. In this process, interleukin-1β (IL-1β) plays an important role. Under H. pylori infection, IL-1β is highly expressed that result in gastric acid inhibition, GC-related gene methylations and disfunctions, angiogenesis. Nod-like receptor pyrin domain containing 3 (NLRP3) inflammasome mediates IL-1β maturation in cells such as macrophages, neutrophils and dendritic cells. But how does IL-1β get released across the cell membrane still unclear. In this review, we focus on the secretion mechanism of IL-1β across the membrane, and to explore the role of IL-1β in the progression of GC.

Resting time after phorbol 12-myristate 13-acetate in THP-1 derived macrophages provides a non-biased model for the study of NLRP3 inflammasome.

The activation of NLRP3 inflammasome in macrophages has been proven to play a crucial role in the development of cardiovascular diseases. THP-1 monocytes can be differentiated to macrophages by incubation with phorbol-12-myristate 13-acetate (PMA), providing a suitable model for in vitro studies. However, PMA has been shown to have effects on the levels of IL-1β, the main mediator of NLRP3 inflammasome, while the effects on the other mediators of the inflammasome have not been reported before. THP-1 monocytes were incubated without (THP-1), with 5ng/ml PMA for 48h (PMA48h) or with 5ng/ml PMA for 48h plus 24h in fresh medium (PMArest). Morphological changes and the expression of macrophage surface markers (CD14, CD11b, CD36 and CD204) were evaluated by flow cytometry. Changes in intracellular levels of inflammasome components (NLRP3, ASC, pro-caspase-1, pro-IL1β) were analyzed by western blot and release of mature IL-1β in cell supernatant was analyzed by ELISA. ASC speck formation was determined by immunofluorescence. After 48h incubation with PMA or subsequent rest in fresh medium, cells became adherent, and the differential expression of CD36, CD11b, CD14 and CD204 compared to THP-1 cells confirmed that PMArest resemble macrophages from a molecular point of view. Changes in the levels were detected in PMA48h group for all the NLRP3-related proteins, with increase of NLRP3 and pro-IL-1β and secretion of mature IL-1β. In PMArest, no pro-IL-1β and lower amounts of mature IL-1β were detected. No ASC speck was found in PMA treated groups, but the addition of a second stimulus to PMArest resulted in ASC speck formation, together with IL-1β production, confirming the responsiveness of the model. Differentiation of THP-1 with 5ng/ml PMA followed by 24h resting period provides a model that morphologically and molecularly resembles macrophages. However, even at low concentrations, PMA induces production of IL-1β. The 24h rest period provides for down-regulation of pro-IL-1β in PMArest group, without affecting its ability to respond to a second stimulus through activation of inflammasome.

Effects and mechanisms of SGLT2 inhibitors on the NLRP3 inflammasome, with a focus on atherosclerosis.

Atherosclerosis is a lipid-driven chronic inflammatory disease that is widespread in the walls of large and medium-sized arteries. Its pathogenesis is not fully understood. The currently known pathogenesis includes activation of pro-inflammatory signaling pathways in the body, increased oxidative stress, and increased expression of cytokines/chemokines. In the innate immune response, inflammatory vesicles are an important component with the ability to promote the expression and maturation of inflammatory factors, release large amounts of inflammatory cytokines, trigger a cascade of inflammatory responses, and clear pathogens and damaged cells. Studies in the last few years have demonstrated that NLRP3 inflammatory vesicles play a crucial role in the development of atherosclerosis as well as its complications. Several studies have shown that NLRP3 binding to ligands promotes inflammasome formation, activates caspase-1, and ultimately promotes its maturation and the maturation and production of IL-1β and IL-18. IL-1β and IL-18 are considered to be the two most prominent inflammatory cytokines in the inflammasome that promote the development of atherosclerosis. SGLT2 inhibitors are novel hypoglycemic agents that also have significant antiatherosclerotic effects. However, their exact mechanism is not yet clear. This article is a review of the literature on the effects and mechanisms of SGLT2 inhibitors on the NLRP3 inflammasome, focusing on their role in antiatherosclerosis.

Scoparone suppresses mitophagy-mediated NLRP3 inflammasome activation in inflammatory diseases.

Recent evidence shows that targeting NLRP3 inflammasome activation is an important means to treat inflammasome-driven diseases. Scoparone, a natural compound isolated from the Chinese herb Artemisia capillaris Thunb, has anti-inflammatory activity. In this study we investigated the effect of scoparone on NLRP3 inflammasome activation in inflammatory diseases. In LPS-primed, ATP or nigericin-stimulated mouse macrophage J774A.1 cells and bone marrow-derived macrophages (BMDMs), pretreatment with scoparone (50 μM) markedly restrained canonical and noncanonical NLRP3 inflammasome activation, evidenced by suppressed caspase-1 cleavage, GSDMD-mediated pyroptosis, mature IL-1β secretion and the formation of ASC specks. We then conducted a transcriptome analysis in scoparone-pretreated BMDMs, and found that the differentially expressed genes were significantly enriched in mitochondrial reactive oxygen species (ROS) metabolic process, mitochondrial translation and assembly process, as well as in inflammatory response. We demonstrated in J774A.1 cells and BMDMs that scoparone promoted mitophagy, a well-characterized mechanism to control mitochondrial quality and reduce ROS production and subsequent NLRP3 inflammasome activation. Mitophagy blockade by 3-methyladenine (3-MA, 5 mM) reversed the protective effects of scoparone on mitochondrial damage and inflammation in the murine macrophages. Moreover, administration of scoparone (50 mg/kg) exerted significant preventive effects via inhibition of NLRP3 activation in mouse models of bacterial enteritis and septic shock. Collectively, scoparone displays potent anti-inflammatory effects via blocking NLRP3 inflammasome activation through enhancing mitophagy, highlighting a potential action mechanism in treating inflammasome-related diseases for further clinical investigation.