Winter Hephzibah E, Murrieta-Coxca José M, Álvarez Daniel, Henao-Restrepo Julián, Fuentes-Zacarías Paulina, Arcila-Barrera Sebastian, Steiniger Frank, Groten Tanja, Markert Udo R, Morales-Prieto Diana M
Placenta Lab, Department of Obstetrics, Jena University Hospital, Jena, Germany.
Placenta Lab, Department of Obstetrics, Jena University Hospital, Jena, Germany; Grupo Reproducción, Departamento Microbiología y Parasitología, Facultad de Medicina, Universidad de Antioquia UdeA, Medellín, Colombia.
J Reprod Immunol. 2025 Feb;167:104417. doi: 10.1016/j.jri.2024.104417. Epub 2024 Dec 15.
Released from trophoblast and other fetal cells, placental extracellular vesicles (EVs) reach the maternal peripheral blood and modulate immune responses. Increased EVs in plasma of preeclampsia (PE) patients indicate their involvement in the etiology of this condition. This study addresses the uptake of plasma EVs by peripheral blood mononuclear cells (PBMCs) and explores the underlying internalization mechanisms. Plasma EVs were isolated from women with normotensive pregnancy (EV) and those with PE (EV), and characterized by cryo-transmission electron microscopy, nanoparticle tracking analysis, Western blotting, flow cytometry, and micro bicinchoninic acid assay (micro-BCA). To investigate whether the origin of PBMCs affects uptake, samples from males, pregnant women, and non-pregnant women were included. Primary PBMCs and macrophages derived from the human leukemia monocytic cell line THP-1 were incubated with PKH-stained EVs, and uptake was assessed by flow cytometry and confocal microscopy. Key molecules involved in monocyte differentiation and macrophage function were evaluated in EV-treated cells using LEGENDplex™ assay and real-time polymerase chain reaction (RT-PCR). Independent of the PBMC source, EVs were mostly captured by monocytes and in a lower proportion by T lymphocytes. Capture of EV was higher than of EV in primary T lymphocytes, monocytes, and THP-1-derived macrophages. After inhibition by Wortmannin and Cytochalasin D, EV internalization by THP-1-derived macrophages was significantly inhibited but not completely abolished. No defined polarization profile of treated THP-1-derived macrophages could be identified. These findings provide evidence of EV modifications in PE, which enhance their uptake by monocytes and other immune cells, mainly through phagocytosis and endocytosis.
胎盘细胞外囊泡（EVs）由滋养层细胞和其他胎儿细胞释放，进入母体外周血并调节免疫反应。子痫前期（PE）患者血浆中EVs增加表明其参与了该疾病的病因。本研究探讨外周血单个核细胞（PBMCs）对血浆EVs的摄取，并探索其潜在的内化机制。从血压正常的孕妇（EV）和PE患者（EV）中分离血浆EVs，并通过冷冻透射电子显微镜、纳米颗粒跟踪分析、蛋白质免疫印迹、流式细胞术和微量双辛可宁酸测定法（micro-BCA）进行表征。为了研究PBMCs的来源是否影响摄取，纳入了男性、孕妇和非孕妇的样本。将源自人白血病单核细胞系THP-1的原代PBMCs和巨噬细胞与PKH染色的EVs孵育，并通过流式细胞术和共聚焦显微镜评估摄取情况。使用LEGENDplex™检测和实时聚合酶链反应（RT-PCR）评估EV处理细胞中参与单核细胞分化和巨噬细胞功能的关键分子。无论PBMCs来源如何，EVs大多被单核细胞捕获，T淋巴细胞捕获的比例较低。在原代T淋巴细胞、单核细胞和THP-1衍生的巨噬细胞中，EV的捕获率高于EV。用渥曼青霉素和细胞松弛素D抑制后，THP-1衍生的巨噬细胞对EV的内化显著受到抑制，但并未完全消除。未发现处理后的THP-1衍生巨噬细胞有明确的极化特征。这些发现提供了PE中EV修饰的证据，这些修饰主要通过吞噬作用和内吞作用增强了单核细胞和其他免疫细胞对它们的摄取。
