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‐β2GPI/β2GPI 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‐β2GPI/β2GPI complex stimulated ROS generation without relying on NADPH oxidase, which may participate in NET generation triggered via the anti‐β2GPI/β2GPI complex. In summary, our results indicate that the anti‐β2GPI/β2GPI 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‐β2GPI/β2GPI to stimulate NETosis, demonstrating that anti‐β2GPI/β2GPI is a promising method for triggering NET. Anti‐β2GPI/β2GPI 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‐β2GPI/β2GPI complex in isolation stimulated NETs without relying on p38, AKT, ERK1/2, or zinc signals. The anti‐β2GPI/β2GPI complex stimulated ROS generation without relying on NADPH oxidase, which may participate in NET generation triggered via the anti‐β2GPI/β2GPI complex.

Neutrophils—Important Communicators in Systemic Lupus Erythematosus and Antiphospholipid Syndrome

“…In SLE, LDGs are associated with vascular damage (43, 58) and with disease activity in juvenile lupus (59). In APS, LDGs are enriched especially in patients with high titers of anti-β2-glycoprotein-I (60), antibodies capable of inducing NETosis (61, 62).…”

“…Similarly, patients with primary APS also display enhanced spontaneous NET release (62). Plasma of patients with APS (both primary and secondary) as well as aPL alone can induce NETs from healthy neutrophils (61, 138), and NET release correlates with circulating levels of aPL (62). Deposition of NETs can be found in intervillous tissue of pre-eclampsia pregnancies (139), possibly contributing to the pregnancy morbidities present in APS.…”

Neutrophil Extracellular Traps (NETs) Take the Central Stage in Driving Autoimmune Responses

“…Furthermore, neutrophils from APS patients were shown to display enhanced spontaneous NET release [134,142]. NET response might be mediated through activation of TLR4 [100,142], together with reactive oxygen species (ROS) [100,142,143]. Remarkably, IgG purified from APS patients induced NET release from healthy neutrophils [142].…”

“…An increase in NET release in response to autoantibodies could be explained by the presence of β2-GP1 on the surface of neutrophils [142]. Several studies observed anti-β2-GP1 to induce NET formation in healthy neutrophils [100,142,143]. Evidences suggest that NETs may directly mediate APS pathology by featuring as a scaffold for platelets to aggregate [144], possibly by presenting a tissue factor [53,100].…”

Neutrophil Extracellular Traps

“…Furthermore, neutrophils from APS patients were shown to display enhanced spontaneous NET release [134,142]. NET response might be mediated through activation of TLR4 [100,142], together with reactive oxygen species (ROS) [100,142,143]. Remarkably, IgG purified from APS patients induced NET release from healthy neutrophils [142].…”