Anti-β2GPI/β2GPI induces human neutrophils to generate NETs by relying on ROS

Abstract

Neutrophils participate in the regulation of pathogens by phagocytosis as well as by generating neutrophil extracellular traps (NETs). Antiphospholipid antibodies, particularly those targeting beta-2-glycoprotein I (β2GPI), stimulate monocytes, platelets, and endothelial cells with prothrombotic participation. This study aimed to explore NET generation in response to anti-β2GPI/β2GPI. A series of experiments involving the separation of primary human leukocytes, NETosis quantification using propidium iodide, exploration of NETosis by fluorescence microscopy, western blotting, examination of free Zn²⁺ using FluoZin-3, and reactive oxygen species (ROS) examination with dihydrorhodamine 123 were performed in this study. We found that anti-β2GPI/β2GPI triggered NETosis, resembling phorbol 12-myristate 13-acetate (PMA)-induced NETosis in magnitude and morphology. The anti-β₂ GPI/β₂ GPI complex in isolation stimulated NETs without relying on p38, protein kinase B (AKT), extracellular signal-related kinase (ERK) 1/2, and zinc signals. NET generation was unaffected by the NADPH oxidase suppressor DP1. The anti-β₂ GPI/β₂ GPI complex stimulated ROS generation without relying on NADPH oxidase, which may participate in NET generation triggered via the anti-β₂ GPI/β₂ GPI complex. In summary, our results indicate that the anti-β₂ GPI/β₂ GPI complex reinforced NET generation by relying on ROS. THE SIGNIFICANCE OF THE PAPER IN THE CONTEXT OF CURRENT KNOWLEDGE: Neutrophils as one of the first lines of defence and essential in the response to pathogen invasion. They eradicate bacteria via phagocytosis or by releasing antimicrobial proteins in degranulation. In this study, we explored the capability of anti-β₂ GPI/β₂ GPI to stimulate NETosis, demonstrating that anti-β₂ GPI/β₂ GPI is a promising method for triggering NET. Anti-β₂ GPI/β₂ GPI induced ROS generation without relying on NADPH oxidase, which contributes to NETosis independently of ERK1/2, Zn²⁺ , or AKT. Our results showed that anti-β2GPI/β2GPI triggered NETosis, resembling PMA-induced NETosis in magnitude as well as morphology. The anti-β₂ GPI/β₂ GPI complex in isolation stimulated NETs without relying on p38, AKT, ERK1/2, or zinc signals. The anti-β₂ GPI/β₂ GPI complex stimulated ROS generation without relying on NADPH oxidase, which may participate in NET generation triggered via the anti-β₂ GPI/β₂ GPI complex.

Keywords: anti-β2GPI/β2GPI; neutrophil extracellular traps; primary human leukocytes; propidium iodide; reactive oxygen species.