Aernoud Fiolet

273 Colchicine reduces extracellular vesicle NLRP3 inflammasome protein levels Visualization was done with a Super Signal enhanced luminol-based chemiluminescent kit (Thermoscientific) and image capture and analysis was done on a Biorad Chemidoc MP. NLRP3 and interleukin-1β measurements NLRP3 was quantified using a Human NLRP3 Enzyme-Linked Immunosorbent Assay (ELISA) kit (cat No. EH4202, Fine Test, Fine Biological Technology, Wuhan, China) according to the manufacturer’s instruction. The kit had a sensitivity of 0.47 ng/mL and a range of 0.78 to 50.0 ng/mL. NLRP3 was measured in EVs and in a random selection (2/3) of patients in serum without EV isolation. In vitro, IL-1 β was measured using a research IL-1 β kit (Human IL-1 beta/IL-1F2 Quantikine HS ELISA Kit, R&D Systems, Minneapolis, MN, USA). This assay had a sensitivity of 0.063 pg/mL and an assay range of 0.1 to 8.0 pg/mL. High sensitivity C-reactive protein and Interleukin-6 measurements Hs-CRPwas measured using a research ELISA kit (Hycult Biotech #HK369, Uden, the Netherlands). The lower detection limit of this assaywas 0.4 ng/L and the inter- and intra-assay coefficients of variation were <6.9% and <6.3%, respectively. IL-6 levels were measured by highly sensitive human IL-6 immunoassay (R&D Systems #D6050, Minneapolis, MN, USA). This assay had a sensitivity of 0.7 pg/mL and the intra-assay and inter-assay coefficients of variation ranged from 4.2% to 6.4%. This assay had a sensitivity of 0.7 pg/mL. Statistical analysis Continuous data are displayed using median values and 25th and 75th percentile when non-normally distributed. Differences in continuous data were tested using unpaired t-tests when normally distributed or Mann-Whitney U tests when non- normally distributed data. A Hodges–Lehmann estimator was used to provide a confidence interval (CI) for the estimated differences between non-parametric distributions. The linear relationship of two continuous parameters was assessed using the Spearman's rank correlation coefficient after data were log transformed. All statistical analyses were performed using IBM SPSS Version 25.0 (IBM Corp, Armonk, NY, USA).