Evaluation of NETosis Inhibitors in a Laboratory-Developed ex-vivo Human Neutrophil Model

Neutrophils are central players in the innate immune system, primarily defending the host through phagocytosis, degranulation, and the release of inflammatory mediators. In 2004, a fourth mechanism was identified: the formation of neutrophil extracellular traps (NETs).1 These extracellular web-like structures are composed of decondensed chromatin fibers (cell free DNA (cfDNA)) decorated with histones and granular enzymes such as neutrophil elastase (NE), myeloperoxidase (MPO), and cathepsin G.1 The release process, termed NETosis, involves reactive oxygen species (ROS) generation, chromatin decondensation facilitated by peptidyl-arginine deiminase 4 (PAD4), and the rupture of nuclear and plasma membranes, allowing the extrusion of NETs into the extracellular space.2 Although initially described as a defense mechanism against pathogens, NETosis is now recognized as a double-edged sword. Excessive or dysregulated NET formation contributes to sterile inflammation, promotes thrombosis, and may lead to tissue damage in various pathologies, including acute respiratory distress syndrome (ARDS) and autoimmune diseases.3 These detrimental effects have sparked growing interest in pharmacological inhibition of NETosis as a therapeutic strategy. Among
the agents under investigation, Cl-amidine, an irreversible PAD4 inhibitor, and sivelestat, a selective NE inhibitor, have shown potential in limiting NET formation in preclinical studies.4 In this work, we aim to evaluate the inhibitory effects of Cl-amidine and sivelestat on NETosis using an ex vivo laboratory-developed model, offering insight into their potential therapeutic utility.