Inhibitory Effects of Chloride on the Activation of Caspase-1, IL-1β Secretion, and Cytolysis by the P2X7 Receptor

Abstract Muscle tissue damage is one of the local effects described in bothropic envenomations. Bothropstoxin-I (BthTX-I), from Bothrops jararacussu venom, is a K49-phospholipase A2 (PLA2) that induces a massive muscle tissue injury, and, consequently, local inflammatory reaction. The NLRP3 inflammasome is a sensor that triggers inflammation by activating caspase 1 and releasing interleukin (IL)-1β and/or inducing pyroptotic cell death in response to tissue damage. We, therefore, aimed to address activation of NLRP3 inflammasome by BthTX-I-associated injury and the mechanism involved in this process. Intramuscular injection of BthTX-I results in infiltration of neutrophils and macrophages in gastrocnemius muscle, which is reduced in NLRP3- and Caspase-1-deficient mice. The in vitro IL-1β production induced by BthTX-I in peritoneal macrophages (PMs) requires caspase 1/11, ASC and NLRP3 and is dependent on adenosine 5′-triphosphate (ATP)-induced K+ efflux and P2X7 receptor (P2X7R). BthTX-I induces a dramatic release of ATP from C2C12 myotubes, therefore representing the major mechanism for P2X7R-dependent inflammasome activation in macrophages. A similar result was obtained when human monocyte-derived macrophages (HMDMs) were treated with BthTX-I. These findings demonstrated the inflammatory effect of BthTX-I on muscle tissue, pointing out a role for the ATP released by damaged cells for the NLRP3 activation on macrophages, contributing to the understanding of the microenvironment of the tissue damage of the Bothrops envenomation.

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P2X7 Receptor-Stimulated Secretion of MHC Class II-Containing Exosomes Requires the ASC/NLRP3 Inflammasome but Is Independent of Caspase-1

The Journal of Immunology ◽

10.4049/jimmunol.0802968 ◽

2009 ◽

Vol 182 (8) ◽

pp. 5052-5062 ◽

Cited By ~ 92

Author(s):

Yan Qu ◽

Lakshmi Ramachandra ◽

Susanne Mohr ◽

Luigi Franchi ◽

Clifford V. Harding ◽

...

Keyword(s):

Mhc Class Ii ◽

Nlrp3 Inflammasome ◽

P2x7 Receptor ◽

Class Ii ◽

Caspase 1

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DOP21 Role of P2X7 in intestinal fibrosis and inflammasome activation: Relevance in Crohn′s Disease

Journal of Crohn s and Colitis ◽

10.1093/ecco-jcc/jjab073.060 ◽

2021 ◽

Vol 15 (Supplement_1) ◽

pp. S061-S061

Author(s):

L Lis-López ◽

C Bauset ◽

D Ortiz-Masia ◽

L Gisbert-Ferrándiz ◽

S Coll ◽

...

Keyword(s):

Protein Expression ◽

P2x7 Receptor ◽

Inflammasome Activation ◽

Chronic Colitis ◽

Intestinal Fibrosis ◽

Caspase 1 ◽

Ht29 Cells ◽

Gene And Protein Expression ◽

Collagen Layer

Abstract Background Crohn’s disease (CD) is a chronic inflammatory disorder of the gastrointestinal tract whose etiology is unknown. CD is associated with complications such as fibrosis or fistula, which cannot be pharmacologically reversed, requiring repeated surgery. Although a profibrotic effect of the P2X7 receptor has been described in some scenarios such as lung, heart and liver, its role in intestinal fibrosis has not been analysed yet. Given the crosstalk between fibrosis and inflammasome, we aim to analyze the relevance of P2X7 in intestinal fibrosis and inflammasome activation. Methods Surgical intestinal resections of CD patients and healthy ileum of carcinoma patients were obtained. Murine chronic colitis was induced by 4 cycles of DSS in wild-type (WT) or P2X7-/- (KO) mice. HT29 cells were treated 24 hours with an inflammasome activator cocktail (LPS, TNF-α and IFN-γ) and the P2X7 antagonist A-80. Gene and protein expression of P2X7, inflammasome markers (NLRP3, ASC, CASPASE1, IL1β and IL18) and alternative inflammasome pathways (APIs) (NLRP1, NLRC4 and AIM2) were analysed by qPCR and Western Blot. The collagen layer was analysed by Sirius Red Staining. Results are expressed by mean±SEM. Statistical analysis was performed with one-way ANOVA and correlations were analysed with Spearman coefficient. Results In CD patients, the expression of P2X7 (2.97±0.50), Nlrp3 (2.53±0.41), Asc (5.61±0.76), Caspase1 (6.90±1.41), IL18 (4.17±0.89) and APIs Nlrp1 (3.07±0.40), Nlrc4 (6.99±1.19) is significantly increased vs non-IBD patients. Moreover, P2X7 expression positively and significantly correlates with the expression of the inflammasome markers NLRP3 (r=0.51), ASC (r=0.38), CASPASE1 (r=0.46), IL18 (r=0.36) and API such as NLRP1 (r=0.73), NRLC4 (r=0.67) and AIM2 (r=0.51) in CD patients (n≥45). The chronic murine model of DSS revealed that: a) KO-DSS showed more aggravated colitis with lower survival and greater weight loss compared with WT-DSS; b) the expression of NLRP3, IL18, IL1β and NLRP1 were significantly increased in KO-DSS (101.00±16.33, 3.28±1.49, 327.50±113.90, 4.92±1.00 respectively) vs WT-DSS; c) the thickness of the collagen layer in KO-DSS was increased vs WT-DSS. As expected, HT29 cells treated with the inflammasome cocktail increased protein expression of caspase-1 and the treatment with the P2X7 antagonist A-80 impaired the inflammasome activation since it significantly reduced the protein expression of caspase-1. Conclusion An increased expression of P2X7 receptor, the inflammasome and its APIs is detected in CD patients. Lack of P2X7 worsens chronic colitis associated with an increased activation of the inflammasome. Additional studies are needed in order to elucidate this dual role of P2X7 in intestinal fibrosis.

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A Crucial Role for Caspase 1 in Thrombo-Inflammatory Microparticle Release

Blood ◽

10.1182/blood.v124.21.2806.2806 ◽

2014 ◽

Vol 124 (21) ◽

pp. 2806-2806

Author(s):

Andrea S Rothmeier ◽

Patrizia Marchese ◽

Christian Furlan-Freguia ◽

Brian G. Petrich ◽

Mark H. Ginsberg ◽

...

Keyword(s):

Cardiovascular Diseases ◽

Cell Surface ◽

P2x7 Receptor ◽

Receptor Activation ◽

Common Mechanism ◽

Inflammasome Activation ◽

Disulfide Exchange ◽

Phalloidin Staining ◽

Cytoskeletal Remodeling ◽

Caspase 1

Abstract Macrophages are central orchestrators in the detrimental cycle of inflammation and coagulation in cardiovascular diseases. Cell injury signals trigger the macrophages P2X7 receptor and thereby induce the release both proinflammatory IL-1β and prothrombotic MP. Prothrombotic MP carry tissue factor (TF) and high content of phosphatidylserine (PS), and can induce thrombosis causing major clinical complications in patients. We previously identified the P2X7 receptor as a crucial component of thrombosis in mice, but the mechanistic details of macrophage MP release in this thrombo-inflammatory pathway remain incompletely understood. The the generation of these MP requires thiol-disulfide exchange-dependent activation of the inflammasome and is accompanied by the release of various soluble proteins into the extracellular space. We hypothesized that the released proteome presents regulators and structural components of the MP generation pathway and employed proteomics to unveil their identity. Amongst the most abundant proteins were γ-actin and actin cytoskeleton associated proteins, including PS-binding proteins annexin 1 and annexin 5. Cytoskeletal remodeling processes leading to formation of filopodia downstream of P2X7 receptor activation were crucial for the biogenesis of thrombo-inflammatory MP, since pharmacological inhibitors of inflammasome activation, cytoskeletal remodeling and the thioredoxin system attenuated both, filopodia formation and the release of highly procoagulant MP. Confocal microscopy demonstrated raft dependent translocation of TF onto filopodia that was prevented by the same inhibitory strategies. Surprisingly, phalloidin-staining of non-permeabilized macrophages revealed that F-actin is exposed to the cell surface decorating the base of filopodia. Positive phalloidin-staining of thrombo-inflammatory MP further demonstrated that F-actin remained associated with the MP surface. Strikingly, blocking surface actin by incubation with high concentration of phalloidin prevented the release of PS-rich MP, demonstrating that exposure of F-actin during filopodia formation is functionally linked to the biogenesis of thrombo-inflammatory MP. As the underlying common mechanism for thiol-disulfide exchange-dependent cell surface actin exposure and MP release, we showed that blockade of the cysteine protease caspase 1, which mediates processing and release of IL-1β downstream of inflammasome activation is also required for the release of thrombo-inflammatory MP. Although caspase 1-mediated activation of caplain was required for the release of filamin A localized TF to the cell cortex, calpain was not involved in the release of thrombo-inflammatory MP release. The N-terminus of γ-actin harbors a recognition and cleavage motif for caspase 1. Residual γ-actin released from caspase 1-blocked macrophages showed decreased electrophoretic mobility, indicating prior cleavage of actin that becomes exposed on the cell surface. We show here that the proteome released during thrombo-inflammatory activation of macrophages includes critical players in the biogenesis of MP and may provide diagnostic fingerprints in complex biological samples. Our data demonstrate an entirely unexpected role for caspase 1 and surface exposure of polymerized actin in the severing of prothrombotic MP from filopodia and thus position this protease upstream of both IL-1β processing and thrombo-inflammatory MP in cardiovascular diseases. Disclosures No relevant conflicts of interest to declare.

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Juglone Suppresses LPS-induced Inflammatory Responses and NLRP3 Activation in Macrophages

Molecules ◽

10.3390/molecules25133104 ◽

2020 ◽

Vol 25 (13) ◽

pp. 3104 ◽

Cited By ~ 3

Author(s):

Nam-Hun Kim ◽

Hong-Ki Kim ◽

Ji-Hak Lee ◽

Seung-Il Jo ◽

Hye-Min Won ◽

...

Keyword(s):

Nlrp3 Inflammasome ◽

Inflammatory Responses ◽

Inflammasome Activation ◽

No Production ◽

Dependent Manner ◽

Inhibitory Effects ◽

Concentration Dependent Manner ◽

Inflammatory Cytokine Production ◽

Caspase 1 ◽

Nlrp3 Inflammasome Activation

The NLRP3 (NACHT, LRR and PYD domains-containing protein 3) inflammasome has been implicated in a variety of diseases, including atherosclerosis, neurodegenerative diseases, and infectious diseases. Thus, inhibitors of NLRP3 inflammasome have emerged as promising approaches to treat inflammation-related diseases. The aim of this study was to explore the effects of juglone (5-hydroxyl-1,4-naphthoquinone) on NLRP3 inflammasome activation. The inhibitory effects of juglone on nitric oxide (NO) production were assessed in lipopolysaccharide (LPS)-stimulated J774.1 cells by Griess assay, while its effects on reactive oxygen species (ROS) and NLRP3 ATPase activity were assessed. The expression levels of NLRP3, caspase-1, and pro-inflammatory cytokines (IL-1β, IL-18) and cytotoxicity of juglone in J774.1 cells were also determined. Juglone was non-toxic in J774.1 cells when used at 10 μM (p < 0.01). Juglone treatment inhibited the production of ROS and NO. The levels of NLRP3 and cleaved caspase-1, as well as the secretion of IL-1β and IL-18, were decreased by treatment with juglone in a concentration-dependent manner. Juglone also inhibited the ATPase activities of NLRP3 in LPS/ATP-stimulated J774.1 macrophages. Our results suggested that juglone could inhibit inflammatory cytokine production and NLRP3 inflammasome activation in macrophages, and should be considered as a therapeutic strategy for inflammation-related diseases.

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