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.

Neutrophil Extracellular Traps and Microcrystals

“…In a recent study performed by Sil et al, we found that PMNs need to attempt to phagocytose MSU crystals in order to perform subsequent NET release and to form aggNETs [23]. PMNs do not really phagocytose MSU crystals since most of the crystals are far longer than PMNs themselves [23]. Our data indicated that only a small fraction of PMNs engaged in attempting MSU crystal phagocytosis but NET-releasing PMNs were all associated with MSU crystals [23].…”

“…PMNs do not really phagocytose MSU crystals since most of the crystals are far longer than PMNs themselves [23]. Our data indicated that only a small fraction of PMNs engaged in attempting MSU crystal phagocytosis but NET-releasing PMNs were all associated with MSU crystals [23]. This let us conclude that MSU crystal phagocytosis is a prerequisite for NET formation [23].…”

“…Our data indicated that only a small fraction of PMNs engaged in attempting MSU crystal phagocytosis but NET-releasing PMNs were all associated with MSU crystals [23]. This let us conclude that MSU crystal phagocytosis is a prerequisite for NET formation [23]. We proposed the involvement of the purinergic P2Y6 receptor in this mechanism based on a strong reduction of MSU crystal-induced NET release by general purinergic receptor inhibitors and the P2Y6-specific inhibitor MRS2578 [23].…”

“…MSU crystals are negatively birefringent, needle-shaped, and generally 5–25  μ m (sometimes 100  μ m) in length [22, 23]. Uric acid is a degradation product of nucleic acid metabolism and crystallizes in the joints of gout patients in the form of needle-shaped crystals [21].…”

“…Thus, the relationship between NET formation and PMN necroptosis remains to be studied in more detail. In a recent study performed by Sil et al, we found that PMNs need to attempt to phagocytose MSU crystals in order to perform subsequent NET release and to form aggNETs [23]. PMNs do not really phagocytose MSU crystals since most of the crystals are far longer than PMNs themselves [23].…”