Proteolytic neutralization of extracellular histones by neutrophil elastase is enhanced by heparin

Background: Release of extracellular histones during cell death or neutrophil extracellular trap formation is linked to initiation and progression of several acute inflammatory diseases. Presence and proteolysis status of extracellular histones are associated with disease severity and the risk of thromboembolic events. Targeting extracellular histones by proteolysis or complexation constitutes a potential therapeutic option. Neutrophil elastase (NE), released during neutrophil activation or neutrophil extracellular trap formation, is associated with extracellular histone plasma levels in severe intensive care unit COVID-19 patients and other acute inflammatory diseases. Objectives: We aimed to investigate the ability of NE to proteolyze extracellular histones, characterize the effect of proteolyzed histones on inflammation and cytotoxicity, and explore the influence of heparin on proteolysis. Methods: Human-activated NE in the presence or absence of heparin was used to proteolyze extracellular histones. Reaction mixtures were analyzed by Western blot analysis, mass spectrometry, and in vitro cell-based systems to assess TLR4 activation with human embryonic kidney (HEK)-blue cells and cell cytotoxicity with human vascular endothelial (EA.hy926) cells. Results: All classes of extracellular histones could be cleaved by NE, which resulted in multiple histone fragments. Histone H3 and H4 proteolysis significantly reduced their cytotoxic potential but did not reduce TLR4 activation. Addition of heparin significantly increased the rate of proteolysis of histones by NE and significantly reduced histone-mediated cytotoxicity. Conclusion: NE can proteolyze extracellular histones, resulting in reduced cytotoxicity and inflammation in vitro. Histone proteolysis can be enhanced by the addition of heparins, indicating a potential combinatory treatment of histone-mediated events by heparins and NE in acute inflammatory diseases.