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. 2017 Nov 30;7:16628. doi: 10.1038/s41598-017-16778-4

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© The Author(s) 2017

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 license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license 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 license, visit http://creativecommons.org/licenses/by/4.0/.

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HMGB1 is associated with NETs. (A) Cross-section (top panel) and 3D-reconstruction (bottom panel) of NETs interacting with THP-1 cells. NETs were stained green; chromatin DNA, blue. Plasma membrane of THP-1 cells was stained red to outline the cells. THP-1 macrophages closely interacted with NETs. Data are representative of three independent experiments. (B) NET-associated HMGB1 was released along with NETs after addition of mineral particles, and digestion of NETs removed the presence of HMGB1. Data are representative of three independent experiments. (C) Percentage of neutrophils forming NETs in response to particle stimuli in presence or absence of the neutrophil elastase inhibitor GW311616A for 2 hours. Data are shown as means ± SEM and the results of at least three independent experiments. **p < 0.005, vs. resting (control) neutrophils. ^## p < 0.005, as compared to particle-stimulated neutrophils. (D) Detection of freed NET-bound HMGB1 in culture medium by ELISA after DNase treatment. Data are shown as means ± SEM and the results of at least three independent experiments. **p < 0.005, vs. particle-stimulated, non-DNase-treated neutrophils without GW311616A treatment. ^## p < 0.005, vs. particle-stimulated, DNase-treated neutrophils without GW311616A treatment. (E) Proteins in culture supernatant of particle-treated neutrophils or THP-1 cells were precipitated and subjected to immunoblotting. HMGB1 was detected only in culture medium of particle-treated neutrophils upon DNase treatment to solubilize extracellular NETs. Data are representative of three independent experiments.