Enhanced capture of preeclampsia-derived extracellular vesicles from maternal plasma by monocytes and T lymphocytes

Abstract

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_NP) and those with PE (EV_PE), 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_PE was higher than of EV_NP 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.

Keywords: Circulating EVs; Cytochalasin D; EV uptake; Macrophages; Placenta; Placenta-associated miRNA; Preeclampsia; Wortmannin.