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. 2018 Aug 22;8:12612. doi: 10.1038/s41598-018-31087-0

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© The Author(s) 2018

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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NEC induces neutrophil activation and possibly NET formation. (A,E) Quantification of extracellular DNA, including neutrophil extracellular traps (NETs), was visualized by SYTOX Orange staining. NEC animals demonstrated a relevant increase in NETs, which was significantly reduced in the absence of PAD (blue). Human subjects with NEC had similar NET scores. (B,C,F) Neutrophil elastase (NE) and myeloperoxidase (MPO), both markers of neutrophil activation, increased with time in NEC animals in comparison to controls, and were reduced in animals with PAD inhibition (blue). (D,G) Quantification of the NET marker citrullinated histone H3 (H3cit) showed an elevation in animals, as well as humans with NEC. The surrogate marker was significantly reduced in the PAD inhibition group (blue), with H3cit levels in animals with PAD inhibition being close to controls (p = 0.075). Total number of subjects: controls n = 10, 1d NEC n = 6, 2d NEC n = 8, 4d NEC n = 10, 6d NEC n = 7, PAD inhibition n = 10, humans n = 9. Statistics: Mann-Whitney test and ANOVA.