POS0199 ANTI-β2GLYCOPROTEIN I-INDUCED NEUTROPHIL EXTRACELLULAR TRAPS CAUSE ENDOTHELIAL ACTIVATION

Le résumé fourni par la source

Background: Neutrophil extracellular trap (NET) formation, a process known as NETosis, is recognized as contributor in the pathogenesis of antiphospholipid syndrome (APS). However, the specific role of anti-β2glycoprotein I antibodies (aβ2GPI)-induced NETs in promoting a procoagulant and proinflammatory phenotype in endothelial cells (ECs) has never been addressed. Objectives: This study investigated whether aβ2GPI-induced NETs can activate ECs and whether aβ2GPI-induced NET and phorbol myristate acetate (PMA)-induced NET have different proteomic profiles. Methods: Healthy donors (HD) neutrophils were stimulated with aβ2GPI isolated from a pool of primary APS patient sera by affinity chromatography, normal human IgG or PMA. NETs were stained with anti-neutrophil elastase and DAPI, and the ability of aβ2GPI to bind NETs and inhibit DNA degradation was investigated. Following stimulation with aβ2GPI, aβ2GPI-induced NETs, and PMA-induced NETs, we assessed endothelial cell (EC) activation by analyzing the expression of ICAM-1 (Intercellular Adhesion Molecule 1), VCAM-1 (Vascular Cell Adhesion Molecule 1), and tissue factor (TF) using flow cytometry. EC dysfunction was evaluated by examining the release of extracellular microvesicles (EMVs) CD31+/Annexin V+ through flow cytometry and NanoSight analysis. Additionally, mass spectrometry-based proteomics was conducted on aβ2GPI-induced and PMA-induced NETs. Results: Unlike normal IgG, aβ2GPI induced NETosis and bound to NETs by colocalizing with the neutrophil elastase signal at 93.6 % without preventing NET degradation. Compared with unstimulated EC, aβ2GPI-induced NET triggered a robust expression of TF, VCAM and ICAM in EC with a change-fold MFI of 6 (SE 0.1), 4.2 (SE 0.09), 2.3 (SE 0.09). VCAM-1 and ICAM-1 were higher expressed in EA.hy926 treated with aβ2GPI-induced NET than those treated with aβ2GPI (p < 0.0001 in all instances) (Figure 1). aβ2GPI induced a significant increase in EMVs CD31+/Annexin V+ compared to untreated samples and those treated with NETs. Fifty-six proteins were identified, 7 resulted upregulated in aβ2GPI-induced NET and downregulated in PMA-induced ones. GO enrichment analysis revealed that proteins upregulated in aβ2GPI-induced NET were enriched for ubiquitin protein ligase binding and SLC2A4 translocation to the plasma membrane. Notably, triosphosphate isomerase 1 (TPI1), 14-3-3 epsilon protein (YWHAE) and tubulin alpha-1B (TUBA1B) proteins, upregulated in aβ2GPI-induced NET, showed functional relationships among themselves at network analysis that were distinct from other proteins, indicating unique interconnections within some aβ2GPI-induced NET proteins that differentiate them from PMA-induced NET proteins (Figure 2). TPI1, a glycolytic enzyme, is the target of autoantibodies (anti-TPI1) that have been found in the cerebrospinal fluid of patients with systemic lupus erythematosus (SLE), particularly those with focal neuropsychiatric SLE (NPSLE). The 14-3-3 protein, mainly the epsilon isoform (YWHAE), plays a role in cell survival and protects the brain from ischemia-induced damage caused by ischemic stroke. Also, TUBA1B, a cytoskeleton protein, resulted to be upregulated in patients with non-aPL-related myocardial infarction and ischemic stroke. Conclusion: In conclusion, our findings reveal that aβ2GPI-induced NETs induce endothelial cell activation and tissue factor (TF) expression, unveiling a novel mechanism linking NETosis to thrombotic pathogenesis in APS. Moreover, our results underscore the compositional and functional differences between aβ2GPI-induced NETs and PMA-induced NETs. Proteins upregulated in aβ2GPI-induced NETs exhibit distinct functional characteristics, particularly in promoting glycolytic activity. This metabolic shift toward glycolysis is pivotal in the processes of autoimmunity and thrombosis. REFERENCES: NIL . Acknowledgements: NIL . Disclosure of Interests: None declared . © The Authors 2025. This abstract is an open access article published in Annals of Rheumatic Diseases under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Neither EULAR nor the publisher make any representation as to the accuracy of the content. The authors are solely responsible for the content in their abstract including accuracy of the facts, statements, results, conclusion, citing resources etc.

Ce résumé expose les affirmations des auteurs. BNTIC ne l’interprète pas comme une validation indépendante des résultats.