Aernoud Fiolet

279 Colchicine reduces extracellular vesicle NLRP3 inflammasome protein levels Therefore, EV NLPR3 protein levels may provide a more specific indicator of NLRP3 inflammasome activity. Unfortunately, we were not able to investigate the effects of colchicine on in vitro EV NLRP3 protein levels since colchicine in combination with LPS and ATP stimulation significantly reduced cell viability. NLRP3 inflammasome in atherosclerosis The involvement of theNLRP3 inflammasome and its downstreamcytokines in the development and progression of atherosclerosis was described over a decade ago. 28 Since then, multiple signals (e.g. cholesterol crystals, oxidized LDL-cholesterol, calcium phosphate crystals), present in the atherosclerotic lesions, have shown the ability to activate the NLRP3 inflammasome and incite a vicious circle of NLRP3 inflammasome activation and infiltration of leukocytes that leads to chronic vascular inflammation that drives detrimental atherosclerotic progression. 8,9 NLRP3 components and extracellular vesicles The NLRP3 inflammasome consists of three components; 1) NLRP3, 2) apoptosis-associated speck-like protein containing a caspase activation and recruitment domain (ASC), and 3) caspase-1 [10]. Several in vitro studies have shown components of the NLRP3 inflammasome in secreted EVs. 16,29 Stimulated macrophages and dendritic cells release vesicles containing NLRP3 protein and caspase-1. 16,17,30 Furthermore, EVs were associated with the release of both the pro- and mature form of IL-1 β and ATP is known to activate rapid vesicle-mediated protein secretion from human macrophages, including caspase-1 and ASC. 31–34 The NLRP3 inflammasome is expressed in human inflammatory cells. Since NLRP3 protein is a NLRP3 inflammasome component, this indicates that EV NLRP3 protein levels reflect the status of the NLRP3 inflammasome in circulating immune cells and other cells and regions (atherosclerotic plaques, endothelial cells) where NLRP3 inflammasome mediated signaling is present. Along with immune cells, platelets also express the NLRP3 inflammasome. 35 Several recent in vitro studies showed relevant effects of colchicine on platelets and serum samples also contain a large proportion of EVs derived from platelets. 36–38 Therefore one could hypothesize that platelet-derived EVs could also be a source of EV NLRP3 protein. In the current study, however, we did not observe EV NLRP3 protein release following platelet activation and full aggregation. Based on these results, we therefore consider it unlikely that platelets are a source of EV NLRP3 protein. We were not able to investigate if EV NLRP3 protein has relevant physiological effects on other cells or if it solely reflects completed NLRP3 inflammasome