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.

Keywords: EVs sorting; extracellular vesicles; flow cytometry; infectious associated diseases; neutrophils; size exclusion chromatography.

FIGURE 1

Production and characterization of neutrophil‐derived extracellular vesicles (NDEVs). (A) Preparation and purification of NDEVs: NDEVs were collected from purified neutrophil supernatants (basal or stimulated cells). Then, they were purified by serial centrifugation (to remove dead cells and apoptotic bodies), ultrafiltration, size exclusion chromatography and a second ultrafiltration. Purified NDEVs were numerated to generate aliquots of 1000 NDEV/μl. (B) Images of NDEVs by transmission electron microscopy. (C) Effect of Triton 0.5X buffer and 0.1‐μm filtration on NDEVs by flow cytometry. (D) Western blot (protein load in μg of NDEV). (E) Size distribution by tunable resistive pulse sensing (qNano) with p200 nanopores. GAPDH, glyceraldehyde‐3 phosphate deshydrogenase

FIGURE 2

Linearity of neutrophil‐derived extracellular vesicles (NDEVs) detection. (A) NDEVs were supplemented in large‐extracellular vesicle (EV)‐free plasma and stained with Annexin V FITC and PE antibodies. Three antibodies were tested on large EV‐free plasma supplemented with NDEV: CD15, CD66b and CD66c. (B) NDEVs concentrations between 50 and 800 Annexin V + FITC NDEV/μL obtained by serial dilutions of NDEVs spiked‐in plasma. A regression curve for each antibody was generated using three independent experiments. AnnV, AnnexinV

FIGURE 3

Impact of washing step (SEC) on neutrophil‐derived extracellular vesicles (NDEVs) detection. (A) Representative graphs of CD15, CD66b and CD66c detection on extracellular vesicles (EVs) in no‐wash (no SEC) and wash (SEC) conditions. Blue dots represent supplemental AnnV+/PE+ NDEVs detected in the wash protocol compared to red dots detected in both conditions. (B, C) PE median fluorescence intensity background noise (Annexin V FITC‐/PE‐ events) and percentage of double‐positive NDEVs detected among Annexin V+ FITC NDEVs between no wash and wash conditions for the nine antibodies tested. Ab, antibody; AnnV, Annexin V; SEC, size exclusion chromatography

FIGURE 4

Impact of antibody combinations on neutrophil‐derived extracellular vesicles (NDEVs) detection. The three best antibodies for the detection of NDEVs, CD15, CD66b and CD66b were associated at their optimal concentrations with the same PE fluorochrome in combination with AnnV FITC and were compared to CD15 staining alone. (A) PE median fluorescence intensity (MFI) of double‐positive NDEV‐detected, (B) PE MFI background noise, (C) separation index of NDEVs. SI was calculated using the formula: (PE MFI NDEV‐PE MFI background noise)/2SD PE fluorescence background noise. (D) Percentage of double‐positive NDEVs detected. This figure is the result of five independent experiments. MFI, median fluorescence intensity; SEC, size exclusion chromatography

FIGURE 5

Neutrophil‐derived extracellular vesicles (NDEVs) enumeration in sepsis patients. Ten platelet‐free plasma samples from sepsis patients were stained according to the current or new combination strategy. For the current strategy, platelet‐free plasma (PFP) was stained with AnnV‐FITC, CD15‐PE and CD41‐APC without a washing step. New combination strategy: PFP stained with AnnV‐FITC, CD15‐PE, CD66b‐PE, CD66c‐PE and CD41‐APC with a washing step. Representative graphs of Annexin V FITC+ NDEVs (A, B) and platelet EVs (E, F). The percentages indicated on the graph correspond to the proportion of NDEVs or platelet EVs among total AnnexinV‐stained EV. (C, G) Comparison of the separation index for NDEVs and platelet EV, respectively. (D, H) NDEVs and platelet‐EVs concentrations, respectively. (I) Ratio between platelet EVs and NDEVs quantification. AnnV, AnnexinV; PEV, platelet EVs

FIGURE 6

Neutrophil‐derived extracellular vesicles (NDEVs) sorting in COVID‐19 plasma. For each plasma sample, three pre‐analytical conditions were tested: Annexin V FITC/CD15 PE staining without washing (n = 5), Annexin V FITC/CD15 PE staining, washing (n = 10), Annexin V FITC/CD15 CD66b CD66c PE staining (n = 10) and washing. For the three conditions, CD41 APCs were also added to the sample to exclude NDEVs CD41+ (n = 5). (A) Illustration of the dot plot showing NDEVs sorting by the three conditions. (B) Number of NDEVs sorted per minute