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 vari...
| Main Authors: | , , , , , , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Wiley
2022-04-01
|
| Series: | Journal of Extracellular Vesicles |
| Subjects: | |
| Online Access: | https://doi.org/10.1002/jev2.12204 |
| _version_ | 1828414386081890304 |
|---|---|
| author | Amandine Bonifay Stéphane Robert Belinda Champagne Paul‐Rémi Petit Aude Eugène Corinne Chareyre Anne‐Claire Duchez Mélanie Vélier Shirley Fritz Loris Vallier Romaric Lacroix Françoise Dignat‐George |
| author_facet | Amandine Bonifay Stéphane Robert Belinda Champagne Paul‐Rémi Petit Aude Eugène Corinne Chareyre Anne‐Claire Duchez Mélanie Vélier Shirley Fritz Loris Vallier Romaric Lacroix Françoise Dignat‐George |
| author_sort | Amandine Bonifay |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-12-10T13:25:09Z |
| format | Article |
| id | doaj.art-12117ce56d534d6fac6a5cdf99098b32 |
| institution | Directory Open Access Journal |
| issn | 2001-3078 |
| language | English |
| last_indexed | 2024-12-10T13:25:09Z |
| publishDate | 2022-04-01 |
| publisher | Wiley |
| record_format | Article |
| series | Journal of Extracellular Vesicles |
| spelling | doaj.art-12117ce56d534d6fac6a5cdf99098b322022-12-22T01:47:12ZengWileyJournal of Extracellular Vesicles2001-30782022-04-01114n/an/a10.1002/jev2.12204A new strategy to count and sort neutrophil‐derived extracellular vesicles: Validation in infectious disordersAmandine Bonifay0Stéphane Robert1Belinda Champagne2Paul‐Rémi Petit3Aude Eugène4Corinne Chareyre5Anne‐Claire Duchez6Mélanie Vélier7Shirley Fritz8Loris Vallier9Romaric Lacroix10Françoise Dignat‐George11Aix‐Marseille University, C2VN, INSERM 1263, INRA 1260 Marseille FranceAix‐Marseille University, C2VN, INSERM 1263, INRA 1260 Marseille FranceAix‐Marseille University, C2VN, INSERM 1263, INRA 1260 Marseille FranceDepartment of Hematology and Vascular Biology CHU La Conception, APHM Marseille FranceDepartment of Hematology and Vascular Biology CHU La Conception, APHM Marseille FranceAix‐Marseille University, C2VN, INSERM 1263, INRA 1260 Marseille FranceAix‐Marseille University, C2VN, INSERM 1263, INRA 1260 Marseille FranceAix‐Marseille University, C2VN, INSERM 1263, INRA 1260 Marseille FranceDepartment of Hematology and Vascular Biology CHU La Conception, APHM Marseille FranceAix‐Marseille University, C2VN, INSERM 1263, INRA 1260 Marseille FranceAix‐Marseille University, C2VN, INSERM 1263, INRA 1260 Marseille FranceAix‐Marseille University, C2VN, INSERM 1263, INRA 1260 Marseille FranceAbstract 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.https://doi.org/10.1002/jev2.12204EVs sortingextracellular vesiclesflow cytometryinfectious associated diseasesneutrophilssize exclusion chromatography |
| spellingShingle | Amandine Bonifay Stéphane Robert Belinda Champagne Paul‐Rémi Petit Aude Eugène Corinne Chareyre Anne‐Claire Duchez Mélanie Vélier Shirley Fritz Loris Vallier Romaric Lacroix Françoise Dignat‐George A new strategy to count and sort neutrophil‐derived extracellular vesicles: Validation in infectious disorders Journal of Extracellular Vesicles EVs sorting extracellular vesicles flow cytometry infectious associated diseases neutrophils size exclusion chromatography |
| title | A new strategy to count and sort neutrophil‐derived extracellular vesicles: Validation in infectious disorders |
| title_full | A new strategy to count and sort neutrophil‐derived extracellular vesicles: Validation in infectious disorders |
| title_fullStr | A new strategy to count and sort neutrophil‐derived extracellular vesicles: Validation in infectious disorders |
| title_full_unstemmed | A new strategy to count and sort neutrophil‐derived extracellular vesicles: Validation in infectious disorders |
| title_short | A new strategy to count and sort neutrophil‐derived extracellular vesicles: Validation in infectious disorders |
| title_sort | new strategy to count and sort neutrophil derived extracellular vesicles validation in infectious disorders |
| topic | EVs sorting extracellular vesicles flow cytometry infectious associated diseases neutrophils size exclusion chromatography |
| url | https://doi.org/10.1002/jev2.12204 |
| work_keys_str_mv | AT amandinebonifay anewstrategytocountandsortneutrophilderivedextracellularvesiclesvalidationininfectiousdisorders AT stephanerobert anewstrategytocountandsortneutrophilderivedextracellularvesiclesvalidationininfectiousdisorders AT belindachampagne anewstrategytocountandsortneutrophilderivedextracellularvesiclesvalidationininfectiousdisorders AT paulremipetit anewstrategytocountandsortneutrophilderivedextracellularvesiclesvalidationininfectiousdisorders AT audeeugene anewstrategytocountandsortneutrophilderivedextracellularvesiclesvalidationininfectiousdisorders AT corinnechareyre anewstrategytocountandsortneutrophilderivedextracellularvesiclesvalidationininfectiousdisorders AT anneclaireduchez anewstrategytocountandsortneutrophilderivedextracellularvesiclesvalidationininfectiousdisorders AT melanievelier anewstrategytocountandsortneutrophilderivedextracellularvesiclesvalidationininfectiousdisorders AT shirleyfritz anewstrategytocountandsortneutrophilderivedextracellularvesiclesvalidationininfectiousdisorders AT lorisvallier anewstrategytocountandsortneutrophilderivedextracellularvesiclesvalidationininfectiousdisorders AT romariclacroix anewstrategytocountandsortneutrophilderivedextracellularvesiclesvalidationininfectiousdisorders AT francoisedignatgeorge anewstrategytocountandsortneutrophilderivedextracellularvesiclesvalidationininfectiousdisorders AT amandinebonifay newstrategytocountandsortneutrophilderivedextracellularvesiclesvalidationininfectiousdisorders AT stephanerobert newstrategytocountandsortneutrophilderivedextracellularvesiclesvalidationininfectiousdisorders AT belindachampagne newstrategytocountandsortneutrophilderivedextracellularvesiclesvalidationininfectiousdisorders AT paulremipetit newstrategytocountandsortneutrophilderivedextracellularvesiclesvalidationininfectiousdisorders AT audeeugene newstrategytocountandsortneutrophilderivedextracellularvesiclesvalidationininfectiousdisorders AT corinnechareyre newstrategytocountandsortneutrophilderivedextracellularvesiclesvalidationininfectiousdisorders AT anneclaireduchez newstrategytocountandsortneutrophilderivedextracellularvesiclesvalidationininfectiousdisorders AT melanievelier newstrategytocountandsortneutrophilderivedextracellularvesiclesvalidationininfectiousdisorders AT shirleyfritz newstrategytocountandsortneutrophilderivedextracellularvesiclesvalidationininfectiousdisorders AT lorisvallier newstrategytocountandsortneutrophilderivedextracellularvesiclesvalidationininfectiousdisorders AT romariclacroix newstrategytocountandsortneutrophilderivedextracellularvesiclesvalidationininfectiousdisorders AT francoisedignatgeorge newstrategytocountandsortneutrophilderivedextracellularvesiclesvalidationininfectiousdisorders |