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. 2022 Mar 4;9:839475. doi: 10.3389/fmed.2022.839475

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Copyright © 2022 Murru, Allard, Paré, Vaillancourt, Boyer, Cayer, Vitry, Landry, Labrecque, Robitaille, Branch, Girard and Fernandes.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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Viability and function of prednisone and G-CSF GC neutrophils. (A) Proportion of apoptosis and necrosis determined by Annexin-V and 7-AAD staining of neutrophils isolated from non-stimulated, healthy donors (-/-), and prednisone or G-CSF GCs after leukapheresis on the day of collection. (B) Intracellular calcium levels measured with a spectrofluorometer after fMLF stimulation as in “Materials and Methods.” The kinetics of the change in intracellular calcium concentration is shown. For (A,B), neutrophils of 10 non-stimulated, healthy donors, 9 prednisone and 9 G-CSF GCs were analyzed. (C) Chemotaxis toward fMLF expressed as the maximal proportion of migrated neutrophils [non-stimulated, healthy donors (n = 3), prednisone (n = 9) and G-CSF GC donors (n = 9)]. (D) Phagocytosis of non-opsonized pHRodo Red zymosan conjugated bioparticles is expressed as the proportion of phagocytic neutrophils [non-stimulated, healthy donors (n = 10), prednisone (n = 7) and G-CSF GC donors (n = 9)]. (E) LPS-induced IL-8 release by neutrophils determined by ELISA (n=8 for all experimental conditions). (F) Intracellular ROS production induced by fMLF [non-stimulated, healthy donors (n = 10), prednisone and G-CSF GC neutrophils (n = 9)] or PMA [non-stimulated. healthy donors (n = 10), prednisone (n = 7) and G-CSF GC neutrophils (n = 8)]. Data are expressed as a MFI ratio of stimulated cells/cells incubated in DMSO (diluent). A ratio of 1 corresponds to the absence of an increase in ROS production compared to the DMSO-treated neutrophils (dotted line). (G) Extracellular ROS production measured with cytochrome c by neutrophils incubated with DMSO, fMLF [non-stimulated, healthy donors (n = 10), prednisone GCs (n = 7), G-CSF GCs (n = 9)] or PMA [non-stimulated, healthy donors (n = 10), prednisone (n = 7), G-CSF (n = 8)] expressed as the concentration of superoxide produced. Statistical analysis: Dunn's and Dunnett's multiple comparison test performed to compare neutrophils from non-stimulated, healthy donors with prednisone and G-CSF GC neutrophils, *p-value < 0.05; **p-value < 0.01.