Neutrophil extracellular trap (NET) formation contributes to gout, autoimmune vasculitis, thrombosis, and atherosclerosis. The outside-in signaling pathway triggering NET formation is unknown. Here, we show that the receptor-interacting protein kinase (RIPK)-1-stabilizers necrostatin-1 or necrostatin-1s and the mixed lineage kinase domain-like (MLKL)-inhibitor necrosulfonamide prevent monosodium urate (MSU) crystal-or PMAinduced NET formation in human and mouse neutrophils. These compounds do not affect PMA-or urate crystal-induced production of ROS. Moreover, neutrophils of chronic granulomatous disease patients are shown to lack PMA-induced MLKL phosphorylation. Genetic deficiency of RIPK3 in mice prevents MSU crystal-induced NET formation in vitro and in vivo. Thus, neutrophil death and NET formation may involve the signaling pathway defining necroptosis downstream of ROS production. These data imply that RIPK1, RIPK3, and MLKL could represent molecular targets in gout or other crystallopathies. Additional supporting information may be found in the online version of this article at the publisher's web-site
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IntroductionNeutrophil extracellular trap (NET) formation was first described to enhance bacterial killing via the release of histones, ROS, and proteases [1,2], but also contributes to autoinflammatory Correspondence: Prof. Hans-Joachim Anders e-mail: hjanders@med.uni-muenchen.de and autoimmune disorders [3][4][5][6][7]. For example, monosodium urate (MSU) crystals trigger NET formation, which first drives massive inflammation and subsequently fosters the resolution of inflammation, explaining both gouty arthritis and tophusrelated immune energy [8]. MSU crystals, cytokines, and bacterial * These authors contributed equally to this work.
Results and discussionPMA triggered death and NET formation of human neutrophils release around 50% of total DNA after 2 h (Supporting Information Fig. 1A). During these processes neutrophils release IL-1β (but not TNF-α) in time-dependent manner into the supernatant. However, pretreatment with IL-1R antagonist anakinra, TNF blocker etanercept or anti-TLR4 as well as lack of Fas (Fas lpr mice) had no effect on DNA release upon PMA or MSU stimulation as compared to their respective controls, which excludes their role in this process (Supporting Information Fig. 1B-F).In addition, neither zVAD-FMK nor ferrostatin-1 (Fer-1) had an effect (Fig. 1A), excluding caspase-mediated extrinsic apoptosis or pyroptosis, ferroptosis [12], respectively. In contrast, firstgeneration necrostatin (Nec)-1, Nec-1s, and the MLKL inhibitor necrosulfonamide (NSA) decreased overall cell death and NET formation as assessed by nuclear Sytox uptake, the release of DNA using PICO green dye, and chromatin release to induce NET structures. Nec-1 inhibits necroptosis via modulating RIPK1 and preventing RIPK3 and MLKL phosphorylation and necrosome formation [13][14][15][16]. Indeed, MSU-, LPS-, and PMA-induced cell death and NET formation of human neutrophils-induced RIPK3 expression and phospho...
