A new strategy to count and sort neutrophil‐derived extracellular vesicles: Validation in infectious disorders

Abstract

Newly recognized polymorphonuclear neutrophil (PMNs) functions include the ability to release subcellular mediators such as neutrophil‐derived extracellular vesicles (NDEVs) involved in immune and thrombo‐inflammatory responses. Elevation of their plasmatic level has been reported in a variety of infectious and cardiovascular disorders, but the clinical use of this potential biomarker is hampered by methodological issues. Although flow cytometry (FCM) is currently used to detect NDEVs in the plasma of patients, an extensive characterization of NDEVs has never been done. Moreover, their detection remains challenging because of their small size and low antigen density. Therefore, the objective of the present study was first to establish a surface antigenic signature of NDEVs detectable by FCM and therefore to improve their detection in biological fluids by developing a strategy allowing to overcome their low fluorescent signal and reduce the background noise. By testing a large panel of 54 antibody specificities already reported to be positive on PMNs, we identified a profile of 15 membrane protein markers, including 4 (CD157, CD24, CD65 and CD66c) never described on NDEVs. Among them, CD15, CD66b and CD66c were identified as the most sensitive and specific markers to detect NDEVs by FCM. Using this antigenic signature, we developed a new strategy combining the three best antibodies in a cocktail and reducing the background noise by size exclusion chromatography (SEC). This strategy allowed a significant improvement in NDEVs enumeration in plasma from sepsis patients and made it feasible to efficiently sort NDEVs from COVID‐19 patients. Altogether, this work opens the door to a more valuable measurement of NDEVs as a potential biomarker in clinical practice. A similar strategy could also be applied to improve detection by FCM of other rare subpopulations of EVs generated by tissues with limited access, such as vascular endothelium, cancer cells or placenta.

Serum‐derived extracellular vesicles: Novel biomarkers reflecting the disease severity of COVID‐19 patients

“…The tetraspanin CD82 is a constituent of tetraspanin‐enriched membrane domains (TEMs, which represent the places of entry/egress of some viruses) but also responsible for immune cells recruitment, migration (Yeung et al., 2018) and acts as a costimulatory molecule for T cells (Earnest et al., 2017; Florin & Lang, 2018; ProteinAtlas: CD82; Sims et al., 2018; Yáñez‐Mó et al., 2009). Finally, we analyzed CD24, which was described as a negative regulator of inflammation, a part of novel CD24/Sig‐10 innate immune checkpoint that regulates inflammation caused by DAMPs (Barkal et al., 2019) and is a protein found on several EV types, including EVs within urine, blood, tears, serum, plasma, saliva, lymphatic exudate, B cells and neutrophils (Altevogt et al., 2021; Ayre et al., 2015; Bonifay et al., 2022; Conzelmann et al., 2020, Ekström et al., 2022; Grigor'eva et al., 2016; Keller et al., 2007; Liu et al., 2022; Park et al., 2021; Sornov et al., 2019).…”

“…(A) gating strategy for EVs is shown from a representative experiment; (B) Concentration of serum extracellular vesicles (EVs) positive for specific EV-associated proteins is shown as Boxplots with median and Tukey Wiskers for each group without removing outliers (see also Supplement Figure 3). *p < 0.05, **p < 0.01, ***p < 0.005 as indicated (Kruskal-Wallis test, Dunn-Bonferroni posttest); (C) The significant (*p < 0.05) correlations between subtypes of EVs are shown as determined by Spearmen correlation test, and the colour and size of the circles represent the level of correlation coefficient et al, 2021;Ayre et al, 2015;Bonifay et al, 2022;Conzelmann et al, 2020,…”

Occult cancer in patients with unprovoked venous thromboembolism: A nested case-control study

“…Numerous techniques are used to determine EVs, including atomic force microscopy, 36 nanotracking analysis, 37 dynamic light scattering, 38 and flow cytometry. 39 , 40 A commonly preferred method for EV quantification is flow cytometry 21 , 41-43 because of its ability to detect, quantify, and characterize EVs simultaneously. Furthermore, flow cytometry is widely available and relatively cost-effective.…”

Neutrophil Extracellular Traps and Neutrophil-Derived Extracellular Vesicles: Common Players in Neutrophil Effector Functions

“…In addition, technical problems such as the limited availability of antibodies and staining techniques with sufficient sensitivity and specificity for newly discovered EV markers compatible with multiplex technologies hamper the development of clinically applicable diagnostics. The first steps towards establishing such methods for detection and characterization have already been taken, e.g., by identifying suitable marker combinations using flow cytometry [193]. Markers of NETosis, on the other hand, may be detected in various, already betterestablished ways.…”