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. 2019 Oct 24;10:2148. doi: 10.3389/fimmu.2019.02148

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Copyright © 2019 Agarwal, Loder, Cholok, Li, Bian, Yalavarthi, Li, Carson, Hwang, Marini, Pagani, Edwards, Delano, Standiford, Knight, Kunkel, Mishina, Ward and Levi.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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NET-induced NETosis augments inflammation. (A) In vivo experimental design with Cl-Amidine; (B) Cl-Amidine significantly reduces normalized neutrophil (1.0 vs. 0.31, p < 0.05) and macrophage (1.0 vs. 0.32, p < 0.05) counts in the immobile hindlimb of DNase I-treated mice (n = 4) 48 h after injury vs. untreated immobile plus DNase I-treated controls (n = 3); (C) Cl-Amidine significantly reduces normalized neutrophil (1.0 vs. 0.40, p < 0.05) and macrophage (1.0 vs. 0.31, p < 0.05) counts in the mobile hindlimb (n = 5) 48 h after injury vs. untreated mobile controls (n = 5); (D) Cl-Amidine does not reduce normalized neutrophil (1.0 vs. 0.96, p = 0.79) or macrophage (1.0 vs. 1.35, p = 0.08) counts in the immobile hindlimb without DNase I (n = 5) 48 h after injury vs. untreated immobilized controls (n = 5); (E) H3-Cit immunostaining and DAPI showing NETs in the mobile hindlimb 48 h after injury; (F) H3-Cit immunostaining and DAPI showing NETs in the mobile hindlimb with Cl-Amidine 48 h after injury; (G) In vitro experimental set-up with Cl-Amidine; (H) H3-Cit and DAPI showing 2° NETs with or without Cl-Amidine after mechanical disruption of 1° NETs; (I) Number of 2° NETs normalized to the number of DAPI+ cells per hpf with Cl-Amidine (46.6 vs. 9.1, p < 0.05); (J) Number of compound 2° NETs normalized to the number of all NETs per hpf with Cl-Amidine (92.7 vs. 5.4, p < 0.05); (K) Number of DAPI+ nuclei normalized to the number of 2° NETs per hpf with Cl-Amidine (2.9 vs. 0.9, p < 0.05); (L) In vivo experimental-set up with delayed Cl-Amidine; (M) Cl-Amidine significantly reduces normalized neutrophil (1.0 vs. 0.27, p < 0.05) and macrophage (1.0 vs. 0.20, p < 0.05) counts in the immobile hindlimb of DNase I-treated mice 48 h after injury vs. immobile DNase I-treated controls; (N) Cl-Amidine significantly reduces normalized neutrophil (1.0 vs. 0.06, p < 0.05) and macrophage (1.0 vs. 0.14, p < 0.05) counts in the mobile hindlimb 48 h after injury vs. untreated mobile controls. Scale bars are 200 μm. All in vivo studies had n ≥ 3/group. All in vitro studies had n = 15 hpf/group. *p < 0.05.