title: P2Y6 Receptor Antagonist MRS2578 Inhibits Neutrophil Activation and Aggregated Neutrophil Extracellular Trap Formation Induced by Gout-Associated Monosodium Urate Crystals.
abstract: Human neutrophils (polymorphonuclear leukocytes [PMNs]) generate inflammatory responses within the joints of gout patients upon encountering monosodium urate (MSU) crystals. Neutrophil extracellular traps (NETs) are found abundantly in the synovial fluid of gout patients. The detailed mechanism of MSU crystal-induced NET formation remains unknown. Our goal was to shed light on possible roles of purinergic signaling and neutrophil migration in mediating NET formation induced by MSU crystals. Interaction of human neutrophils with MSU crystals was evaluated by high-throughput live imaging using confocal microscopy. We quantitated NET levels in gout synovial fluid supernatants and detected enzymatically active neutrophil primary granule enzymes, myeloperoxidase, and human neutrophil elastase. Suramin and PPADS, general P2Y receptor blockers, and MRS2578, an inhibitor of the purinergic P2Y6 receptor, blocked NET formation triggered by MSU crystals. AR-C25118925XX (P2Y2 antagonist) did not inhibit MSU crystal-stimulated NET release. Live imaging of PMNs showed that MRS2578 represses neutrophil migration and blocked characteristic formation of MSU crystal-NET aggregates called aggregated NETs. Interestingly, the store-operated calcium entry channel inhibitor (SK&F96365) also reduced MSU crystal-induced NET release. Our results indicate that the P2Y6/store-operated calcium entry/IL-8 axis is involved in MSU crystal-induced aggregated NET formation, but MRS2578 could have additional effects affecting PMN migration. The work presented in the present study could lead to a better understanding of gouty joint inflammation and help improve the treatment and care of gout patients.
authors.affiliation: Department of Infectious Diseases, College of Veterinary Medicine, University of Georgia, Athens, GA 30602.
authors.forename: Payel
authors.initials: P
authors.lastname: Sil
authors.affiliation: Department of Infectious Diseases, College of Veterinary Medicine, University of Georgia, Athens, GA 30602.
authors.forename: Craig P
authors.initials: CP
authors.lastname: Hayes
authors.affiliation: Department of Infectious Diseases, College of Veterinary Medicine, University of Georgia, Athens, GA 30602.
authors.forename: Barbara J
authors.initials: BJ
authors.lastname: Reaves
authors.affiliation: Institute of Bioinformatics, University of Georgia, Athens, GA 30602.
authors.forename: Patrick
authors.identifier: 0000-0002-3510-6281
authors.initials: P
authors.lastname: Breen
authors.affiliation: Department of Computer Science, Franklin College of Arts and Sciences, University of Georgia, Athens, 30602 GA.
authors.forename: Shannon
authors.identifier: 0000-0002-8916-6335
authors.initials: S
authors.lastname: Quinn
authors.affiliation: Stanford University School of Medicine, Stanford, CA 94305; and.|Internal Medicine and Rheumatology, VA Palo Alto Health Care System, Palo Alto, CA 94034.
authors.forename: Jeremy
authors.initials: J
authors.lastname: Sokolove
authors.affiliation: Department of Infectious Diseases, College of Veterinary Medicine, University of Georgia, Athens, GA 30602; radab@uga.edu.
authors.forename: Balázs
authors.initials: B
authors.lastname: Rada
mesh_terms: D002634:Chemotaxis, Leukocyte / Q000187:drug effects; D004797:Enzyme-Linked Immunosorbent Assay; D065206:Extracellular Traps / Q000187:drug effects* / Q000276:immunology; D005455:Fluorescent Antibody Technique; D006073:Gout / Q000276:immunology / Q000378:metabolism / Q000473:pathology; D057166:High-Throughput Screening Assays; D006801:Humans; D066298:In Vitro Techniques; D017879:Isothiocyanates / Q000494:pharmacology*; D018613:Microscopy, Confocal; D018375:Neutrophil Activation / Q000187:drug effects* / Q000276:immunology; D009504:Neutrophils / Q000187:drug effects / Q000276:immunology; D018048:Receptors, Purinergic P2 / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects / Q000276:immunology; D013582:Synovial Fluid / Q000276:immunology / Q000378:metabolism; D013890:Thiourea / Q000031:analogs & derivatives* / Q000494:pharmacology; D014527:Uric Acid / Q000276:immunology*
chemical_list: D017879:Isothiocyanates; C541384:N,N''-1,4-butanediylbis(N'-(3-isothiocyanatophenyl))thiourea; D018048:Receptors, Purinergic P2; C099845:purinoceptor P2Y6; D014527:Uric Acid; D013890:Thiourea