Title: Neutrophil elastase‐deficient mice form neutrophil extracellular traps in an experimental model of deep vein thrombosis
Abstract: Unlabelled Box<li>•Neutrophil elastase (NE) plays a role in extracellular trap formation (NETosis) triggered by microbes.</li><li>•The contribution of NE was evaluated in mouse NETosis models of sterile inflammation and thrombosis.</li><li>•NE is not required for mouse neutrophil NET production in vitro with non‐infectious stimuli.</li><li>•NE deficiency had no significant effect on thrombosis in the inferior vena cava stenosis model.</li>We thank J. Roes (University College London) and H. Luo (Boston Children's Hospital) for providing neutrophil elastase‐deficient mice to E.R.O. We also thank M. Demers and S. Ling Wong for helpful discussions. This work was supported by the National Heart, Lung, and Blood Institute of the National Institutes of Health grants R01HL102101 and R01HL125501 (to D.D.W.) and 5T32HL066987‐13 (K.M.), and the National Institute of Allergy and Infectious Diseases grant R21AI103407 (to E.R.O.). T.W. was a recipient of a fellowship of the German Cardiac Society (Deutsche Gesellschaft fuer Kardiologie).<li>National Heart, Lung, and Blood InstituteR01HL102101R01HL1255015T32HL066987‐13</li><li>National Institute of Allergy and Infectious DiseasesR21AI103407</li><li>Deutsche Gesellschaft fuer Kardiologie</li> <h3>Summary: Background</h3> Neutrophil serine proteases have been implicated in coagulation and neutrophil extracellular trap (NET) formation. In human neutrophils, neutrophil elastase (NE) translocates to the nucleus during NETosis and cleaves histones, thus aiding in chromatin decondensation. NE<sup>−/−</sup> mice were shown not to release NETs in response to microbes. However, mouse studies evaluating the role of NE in NET formation in sterile inflammation and thrombosis are lacking. <h3>Objective</h3> We wished to establish if neutrophils from NE<sup>−/−</sup> mice have a defect in NETosis, similar to peptidylarginine deiminase 4 (PAD4<sup>−/−</sup>) mice, and how this might have an impact on venous thrombosis, a model where NETs are produced and are crucial to thrombus development. <h3>Methods</h3> We performed <i>in vitro</i>NET assays using neutrophils from wild‐type (WT), NE<sup>−/−</sup>, SerpinB1 (SB1)<sup>−/−</sup> and NE<sup>−/−</sup>SB1<sup>−/−</sup> mice. We compared WT and NE<sup>−/−</sup> animals using the inferior vena cava stenosis model of deep vein thrombosis (DVT). <h3>Results</h3> Neutrophil elastase deficiency resulted in a small reduction in ionomycin‐induced NET formation <i>in vitro</i> without affecting histone citrullination. However, NET production in response to phorbol 12‐myristate 13‐acetate or platelet activating factor was normal in neutrophils from two independent NE‐deficient mouse lines, and in NE<sup>−/−</sup>SB1<sup>−/−</sup> as compared with SB1<sup>−/−</sup> neutrophils. NE deficiency or inhibition did not prevent NETosis <i>in vivo</i> or DVT outcome. <h3>Conclusions</h3> Neutrophil elastase is not required for NET formation in mice. NE<sup>−/−</sup> mice, which form pathological venous thrombi containing NETs, do not phenocopy PAD4<sup>−/−</sup> mice in <i>in vitro</i>NETosis assays or experimental venous thrombosis. Our study suggests that NET‐targeted therapies need to be highly effective to have an impact on DVT.