NET formation induced by gout-associated monosodium urate crystals requires IL-8-mediated neutrophil migration and is inhibited by the P2Y6 receptor antagonist MRS2578.

Abstract Human neutrophils generate inflammatory responses within the joints of gout patients upon encountering Monosodium Urate (MSU) crystals. Studies (including ours) show that neutrophil extracellular traps (NETs) are found abundantly in the synovial fluid (SF) of gout patients and are crucial for the onset of the autoinflammatory cascade. Our goal is to shed light on possible roles of purinergic signaling and neutrophil migration in mediating in vitro NET formation induced by MSU crystals. Our data show that gout SF supernatants have significantly higher levels of the neutrophil granule markers, myeloperoxidase (MPO) and human neutrophil elastase (HNE), and NETs than SF specimens from MSU-negative osteoarthritis patients. MSU crystal-triggered NETs in human neutrophils contain active DNA-bound MPO and HNE. MRS2578, a purinergic P2Y6 receptor antagonist, reduces the release of extracellular DNA, MPO and HNE in MSU crystal-stimulated neutrophils. Releases of MPO, HNE and NETs were measured by commercial ELISA and our proprietary NET-DNA ELISA assays. We found that MRS2578 reduces MSU crystal-induced IL-8 release, neutrophil chemotaxis and characteristic formation of DNA aggregates. The store operated calcium entry (SOCE) channel inhibitor (SK96365) blocked MSU crystal-induced NET release. Our results indicating that the P2Y6-SOCE-IL-8 axis is involved in MSU crystal-induced NET formation provide a novel mechanism potentially relevant in gout inflammation. Our work on the role of the P2Y6-SOCE-IL-8 axis in MSU crystal-triggered NET formation could potentially lead to a better understanding of the mechanism of gout joint inflammation and, thereby, help in improving the care of gout patients.