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. 2021 Aug 3;11:15676. doi: 10.1038/s41598-021-95028-0

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© The Author(s) 2021

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

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Increased levels of NETs was induced by sera of active AOSD patients is mitochondrial ROS-dependent. (a) Increased oxidative stress (8-OHdG) levels in active AOSD patients. (b-d) Normal human neutrophils were stimulated with PMA, or the serum of active AOSD (aAOSD) patient in the presence or absence of DPI (25 μM) or MitoTEMPO (10 μM). (b) The dynamic of NETs formation was analyzed by using Sytox Green with plate reader. The fluorescence readout obtained from cells lysed with 0.5% (vol/vol) Triton X-100 was considered as 100% DNA release, and the relative NETs formation was calculated at each time point. (c) The levels of NETs DNA release in the supernatant of cells at 4 h post-treatment was quantified using PicoGreen fluorescence dye with plate reader. (d) The NETs formation was detected by confocal microscopy. (e) Neutrophils were stimulated with serum of active AOSD (n = 8) or inactive AOSD (n = 8) patients in the presence of the indicated ROS inhibitor and the levels NETs DNA release were quantified. (f, g) The levels of cytosolic ROS were analyzed using DHR123 with plate reader. (h) The levels of mitochondrial ROS were analyzed using MitoSOX Red dye with flow cytometry. All experiments were performed in triplicate, and data are presented as the mean ± SD. **P < 0.01, ***P < 0.001. Scale bars, 5 µm.