PB0853 - An in-house ex-vivo NETs induction model for the evaluation of potential NETosis inhibitors

Background: NETosis is a form of neutrophil death involved in the pathogenesis of thrombosis and disseminated intravascular coagulation by promoting a pro-inflammatory and a procoagulant state leading to multiorgan failure.

Aims: This study aims to evaluate the capacity of a novel NETosis monitoring assay to detect the potency of compounds known to be NETosis inhibitors.

Methods: We developed an ex-vivo model to evaluate the NETosis and the impact of sivelestat and Cl-Amidine on the NETosis process. Neutrophils were extracted from whole blood of an healthy donor. Tumor necrosis factor-alpha was used to trigger NETosis. NETs monitoring was performed using specific fluorescent markers targeting DNA and neutrophile elastase (NE). Readings were performed every 2 minutes for 8 hours on a plate reader. Results were expressed as the percentage of DNA/NE released as a function of time. Time to cross 50% release (T50) and area under the curve (AUC), representing total NETosis potential, were computed.

Results: The AUC of all inhibitor concentrations showed significant differences on ex-vivo NETosis induction compared to positive-controls although never reaching the negative-control. There were two different kinetic profiles for the two markers analyzed. The DNA release kinetic is described by a sigmoid correlation, while the release of NE is more describe by first order association kinetics model. (Figure 1) There is also a dose-dependent effect of sivelestat, which is not observed with Cl-Amidine, suggesting some mechanism of action rapidly reach their maximal effect on NETosis inhibition (Table 1).

Conclusion(s): We successfully developed an ex-vivo model to analyze the NETosis. We demonstrated a strong impact of the inhibitors on NETosis with two different fluorescent markers. Both the initiation and the amplitude of NETosis is reduced with the sivelestat in a dose-dependent manner. This in-vitro model could be used as a monitoring tool to assess NETosis and help in the development of NETosis inhibitors.