title: IL-1β production is dependent on the activation of purinergic receptors and NLRP3 pathway in human macrophages.
abstract: The Nod-like receptor family protein 3 (NLRP3)-inflammasome pathway is known to be activated by danger signals such as monosodium urate (MSU). We investigated the role of P2 purinergic receptors in the activation of NLRP3-inflammasome pathway after MSU treatment of primary human monocyte-derived macrophages (MDMs). After initial stimulation with a low concentration of LPS (0.1 µg/ml), a 6 h treatment with MSU crystals (250, 500, and 1000 µg/ml) induced the MDMs to release IL-1β, IL-1α, and IL-6 in a dose-dependent manner. Moreover, the caspase 1 inhibitor Z-YVAD-FMK and the cathepsin B inhibitor CA-074Me reduced production of IL-1β in a dose-dependent manner after LPS + MSU treatment. We used real-time reverse transcription-quantitative PCR to show that treatment with LPS and MSU (500 µg/ml) induced significantly greater expression of NLRP3 and IL-1β than after treatment with LPS. We also found that MSU treatment induced P2X purinergic receptor 7 (P2X7R) mRNA and protein expression. Furthermore, addition of the P2X7 purinergic receptor antagonist A-740003 significantly impeded IL-1β production and pro-IL-1β cleavage after treatment with LPS + MSU. Remarkably, RNA silencing of P2X7R (but not P2X4R) inhibited the release of IL-1β and other M1 macrophage cytokines (such as IL-1α, IL-6, and TNF-α) from MDMs stimulated with LPS + MSU. Taken as a whole, our results show that P2 purinergic receptors and the NLRP3 inflammasome pathway are involved in the secretion of IL-1β from MSU-stimulated human macrophages. This pathway may constitute a novel therapeutic target for controlling the inflammatory process in several associated pathologies.
authors.affiliation: *Unité Mixte de Recherche 991, Institut National de la Santé et de la Recherche Médicale, Université de Rennes 1, Rennes, France; Laboratoire de Toxicologie Biologique et Médico-Légale, Centre Hospitalier Universitaire de Rennes, Rennes, France; and Experimental and Molecular Immunology and Neurogenetics, Centre National de la Recherche Scientifique, Unité Mixte de Recherche 7355, Université d'Orleans, Orleans, France thomas.gicquel@univ-rennes1.fr.
authors.forename: Thomas
authors.initials: T
authors.lastname: Gicquel
authors.affiliation: *Unité Mixte de Recherche 991, Institut National de la Santé et de la Recherche Médicale, Université de Rennes 1, Rennes, France; Laboratoire de Toxicologie Biologique et Médico-Légale, Centre Hospitalier Universitaire de Rennes, Rennes, France; and Experimental and Molecular Immunology and Neurogenetics, Centre National de la Recherche Scientifique, Unité Mixte de Recherche 7355, Université d'Orleans, Orleans, France.
authors.forename: Sacha
authors.initials: S
authors.lastname: Robert
authors.affiliation: *Unité Mixte de Recherche 991, Institut National de la Santé et de la Recherche Médicale, Université de Rennes 1, Rennes, France; Laboratoire de Toxicologie Biologique et Médico-Légale, Centre Hospitalier Universitaire de Rennes, Rennes, France; and Experimental and Molecular Immunology and Neurogenetics, Centre National de la Recherche Scientifique, Unité Mixte de Recherche 7355, Université d'Orleans, Orleans, France.
authors.forename: Pascal
authors.initials: P
authors.lastname: Loyer
authors.affiliation: *Unité Mixte de Recherche 991, Institut National de la Santé et de la Recherche Médicale, Université de Rennes 1, Rennes, France; Laboratoire de Toxicologie Biologique et Médico-Légale, Centre Hospitalier Universitaire de Rennes, Rennes, France; and Experimental and Molecular Immunology and Neurogenetics, Centre National de la Recherche Scientifique, Unité Mixte de Recherche 7355, Université d'Orleans, Orleans, France.
authors.forename: Tatiana
authors.initials: T
authors.lastname: Victoni
authors.affiliation: *Unité Mixte de Recherche 991, Institut National de la Santé et de la Recherche Médicale, Université de Rennes 1, Rennes, France; Laboratoire de Toxicologie Biologique et Médico-Légale, Centre Hospitalier Universitaire de Rennes, Rennes, France; and Experimental and Molecular Immunology and Neurogenetics, Centre National de la Recherche Scientifique, Unité Mixte de Recherche 7355, Université d'Orleans, Orleans, France.
authors.forename: Aude
authors.initials: A
authors.lastname: Bodin
authors.affiliation: *Unité Mixte de Recherche 991, Institut National de la Santé et de la Recherche Médicale, Université de Rennes 1, Rennes, France; Laboratoire de Toxicologie Biologique et Médico-Légale, Centre Hospitalier Universitaire de Rennes, Rennes, France; and Experimental and Molecular Immunology and Neurogenetics, Centre National de la Recherche Scientifique, Unité Mixte de Recherche 7355, Université d'Orleans, Orleans, France.
authors.forename: Catherine
authors.initials: C
authors.lastname: Ribault
authors.affiliation: *Unité Mixte de Recherche 991, Institut National de la Santé et de la Recherche Médicale, Université de Rennes 1, Rennes, France; Laboratoire de Toxicologie Biologique et Médico-Légale, Centre Hospitalier Universitaire de Rennes, Rennes, France; and Experimental and Molecular Immunology and Neurogenetics, Centre National de la Recherche Scientifique, Unité Mixte de Recherche 7355, Université d'Orleans, Orleans, France.
authors.forename: Florence
authors.initials: F
authors.lastname: Gleonnec
authors.affiliation: *Unité Mixte de Recherche 991, Institut National de la Santé et de la Recherche Médicale, Université de Rennes 1, Rennes, France; Laboratoire de Toxicologie Biologique et Médico-Légale, Centre Hospitalier Universitaire de Rennes, Rennes, France; and Experimental and Molecular Immunology and Neurogenetics, Centre National de la Recherche Scientifique, Unité Mixte de Recherche 7355, Université d'Orleans, Orleans, France.
authors.forename: Isabelle
authors.initials: I
authors.lastname: Couillin
authors.affiliation: *Unité Mixte de Recherche 991, Institut National de la Santé et de la Recherche Médicale, Université de Rennes 1, Rennes, France; Laboratoire de Toxicologie Biologique et Médico-Légale, Centre Hospitalier Universitaire de Rennes, Rennes, France; and Experimental and Molecular Immunology and Neurogenetics, Centre National de la Recherche Scientifique, Unité Mixte de Recherche 7355, Université d'Orleans, Orleans, France.
authors.forename: Elisabeth
authors.initials: E
authors.lastname: Boichot
authors.affiliation: *Unité Mixte de Recherche 991, Institut National de la Santé et de la Recherche Médicale, Université de Rennes 1, Rennes, France; Laboratoire de Toxicologie Biologique et Médico-Légale, Centre Hospitalier Universitaire de Rennes, Rennes, France; and Experimental and Molecular Immunology and Neurogenetics, Centre National de la Recherche Scientifique, Unité Mixte de Recherche 7355, Université d'Orleans, Orleans, France.
authors.forename: Vincent
authors.initials: V
authors.lastname: Lagente
mesh_terms: D000081:Acetamides / Q000494:pharmacology; D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D045744:Cell Line, Tumor; D002478:Cells, Cultured; D004151:Dipeptides / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D005434:Flow Cytometry; D015870:Gene Expression / Q000187:drug effects; D006801:Humans; D015151:Immunoblotting; D053582:Interleukin-1alpha / Q000235:genetics / Q000378:metabolism; D053583:Interleukin-1beta / Q000235:genetics / Q000378:metabolism*; D015850:Interleukin-6 / Q000235:genetics / Q000378:metabolism; D008070:Lipopolysaccharides / Q000494:pharmacology; D008264:Macrophages / Q000187:drug effects / Q000378:metabolism*; D000071199:NLR Family, Pyrin Domain-Containing 3 Protein; D011804:Quinolines / Q000494:pharmacology; D058486:Receptors, Purinergic P2X7 / Q000235:genetics / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction; D014527:Uric Acid / Q000494:pharmacology
chemical_list: C515928:(N-(1-(((cyanoimino)(5-quinolinylamino) methyl) amino)-2,2-dimethylpropyl)-2-(3,4-dimethoxyphenyl)acetamide); D000081:Acetamides; D000590:Amino Acid Chloromethyl Ketones; C400541:CA 074 methyl ester; D002352:Carrier Proteins; D004151:Dipeptides; D053582:Interleukin-1alpha; D053583:Interleukin-1beta; D015850:Interleukin-6; D008070:Lipopolysaccharides; D000071199:NLR Family, Pyrin Domain-Containing 3 Protein; C453881:NLRP3 protein, human; D011804:Quinolines; D058486:Receptors, Purinergic P2X7; C460579:benzyloxycarbonyltyrosyl-valyl-alanyl-aspartic acid fluoromethyl ketone; D014527:Uric Acid
keywords: NLRP3 inflammasome; P2X7 receptor; cytokines; uric acid