Mancuso Silvia, Caliste Mattia, Petretto Andrea, Corsiero Elisa, Grinovero Nicole, Capozzi Antonella, Riitano Gloria, Barbati Cristiana, Truglia Simona, Alessandri Cristiano, Sorice Maurizio, Bombardieri Michele, Conti Fabrizio
Rheumatology Unit, Department of Clinical Internal, Anesthesiologic and Cardiovascular Sciences, Sapienza University of Rome, Rome, Italy.
Centre for Experimental Medicine and Rheumatology, William Harvey Research Institute, Barts and The London School of Medicine and Dentistry, Queen Mary University of London (QMUL), London, UK.
Rheumatology (Oxford). 2025 Aug 1;64(8):4796-4805. doi: 10.1093/rheumatology/keaf204.
Neutrophil extracellular traps (NETs) involvement in antiphospholipid syndrome (APS) pathogenesis is known, but the role of anti-β2glycoprotein I (aβ2GPI) antibodies-induced NETs in triggering a procoagulant and proinflammatory phenotype in endothelial cells (ECs) remains to be evaluated. This study investigated whether NET-aβ2GPI can activate ECs and whether NET-aβ2GPI and NET-phorbol myristate acetate (PMA) have different proteomic profiles.
Healthy donor (HD) neutrophils were stimulated with APS-aβ2GPI, normal human IgG or PMA. NETs were stained with anti-neutrophil elastase and 4'6-diamidino-2-phenylindole (DAPI), and the ability of aβ2GPI to bind NETs and inhibit DNA degradation was investigated. Following aβ2GPI, NET-aβ2GPI and NET-PMA stimuli, we evaluated EC activation investigating intra-cellular adhesion molecule (ICAM), vascular cell adhesion molecule (VCAM) and tissue factor (TF) expression using flow cytometry and reverse transcription-quantitative polymerase chain reaction (RT-qPCR); and EC dysfunction analysing extracellular microvesicles (EMVs) release via flow cytometry and NanoSight analysis. Mass spectrometry-based proteomics was performed on NET-aβ2GPI and NET-PMA.
Unlike normal IgG, aβ2GPI induced NET formation and bound to NETs by colocalizing with the neutrophil elastase signal at 93.6% without preventing NET degradation. Compared with unstimulated EC, NET-aβ2GPI triggered higher mRNA and a robust expression of TF, VCAM and ICAM in EC with a change-fold median fluorescence intensity (MFI) of 6, 4.2 and 2.3. aβ2GPI induced a significant increase in EMVs compared with untreated samples and those treated with NETs. Fifty-six proteins were identified, seven resulted upregulated in NET-aβ2GPI and downregulated in PMA-induced ones. GO enrichment analysis revealed that proteins upregulated in NET-aβ2GPI were enriched for ubiquitin protein ligase binding and SLC2A4 translocation to the plasma membrane.
aβ2GPI-induced NETs can cause EC activation and TF expression.
已知中性粒细胞胞外陷阱（NETs）参与抗磷脂综合征（APS）的发病机制，但抗β2糖蛋白I（aβ2GPI）抗体诱导的NETs在内皮细胞（ECs）中引发促凝血和促炎表型的作用仍有待评估。本研究调查了NET-aβ2GPI是否能激活ECs，以及NET-aβ2GPI和NET-佛波醇肉豆蔻酸酯乙酸酯（PMA）是否具有不同的蛋白质组学特征。
用APS-aβ2GPI、正常人IgG或PMA刺激健康供体（HD）中性粒细胞。用抗中性粒细胞弹性蛋白酶和4',6-二脒基-2-苯基吲哚（DAPI）对NETs进行染色，并研究aβ2GPI结合NETs和抑制DNA降解的能力。在aβ2GPI、NET-aβ2GPI和NET-PMA刺激后，我们通过流式细胞术和逆转录定量聚合酶链反应（RT-qPCR）评估细胞内黏附分子（ICAM）、血管细胞黏附分子（VCAM）和组织因子（TF）的表达来研究EC激活情况；并通过流式细胞术和纳米可视分析来分析细胞外微泡（EMV）释放情况以研究EC功能障碍。对NET-aβ2GPI和NET-PMA进行基于质谱的蛋白质组学分析。
与正常IgG不同，aβ2GPI诱导NET形成，并通过与中性粒细胞弹性蛋白酶信号共定位以93.6%的比例与NETs结合，且不阻止NET降解。与未刺激的EC相比，NET-aβ2GPI在EC中触发更高的mRNA水平以及TF、VCAM和ICAM的强烈表达，中位荧光强度（MFI）变化倍数分别为6、4.2和2.3。与未处理的样本以及用NETs处理过的样本相比，aβ2GPI诱导EMV显著增加。共鉴定出56种蛋白质，其中7种在NET-aβ2GPI中上调而在PMA诱导的NETs中下调。基因本体（GO）富集分析显示，NET-aβ2GPI中上调的蛋白质富含泛素蛋白连接酶结合和溶质载体家族2成员4（SLC2A4）向质膜的转运。
aβ2GPI诱导的NETs可导致EC激活和TF表达。
