title: Anoxia and glucose supplementation preserve neutrophil viability and function.
abstract: Functional studies of human neutrophils and their transfusion for clinical purposes have been hampered by their short life span after isolation. Here, we demonstrate that neutrophil viability is maintained for 20 hours in culture media at 37°C under anoxic conditions with 3 mM glucose and 32 μg/mL dimethyloxalylglycine supplementation, as evidenced by stabilization of Mcl-1, proliferating cell nuclear antigen (PCNA), and pro-caspase-3. Notably, neutrophil morphology (nucleus shape and cell-surface markers) and functions (phagocytosis, degranulation, calcium release, chemotaxis, and reactive oxygen species production) were comparable to blood circulating neutrophils. The observed extension in neutrophil viability was reversed upon exposure to oxygen. Extending neutrophil life span allowed efficient transfection of plasmids (40% transfection efficiency) and short interfering RNA (interleukin-8, PCNA, and Bax), as a validation of effective and functional genetic manipulation of neutrophils both in vitro and in vivo. In vivo, transfusion of conditioned neutrophils in a neutropenic guinea pig model increased bacterial clearance of Shigella flexneri upon colonic infection, strongly suggesting that these conditioned neutrophils might be suitable for transfusion purposes. In summary, such conditioning of neutrophils in vitro should facilitate their study and offer new opportunities for genetic manipulation and therapeutic use.
authors.affiliation: Investigation Clinique et Accès aux Ressources Biologiques, Institut Pasteur, Paris, France;
authors.forename: Valérie
authors.initials: V
authors.lastname: Monceaux
authors.affiliation: Unité de Pathogénie Microbienne Moléculaire, Institut Pasteur, Paris, France, INSERM Unité 786, Paris, France;
authors.forename: Clarisse
authors.initials: C
authors.lastname: Chiche-Lapierre
authors.affiliation: Medizinische Klinik m.S. Infektiologie und Pulmologie Charité-Universitätsmedizin Berlin, Berlin, Germany;
authors.forename: Catherine
authors.initials: C
authors.lastname: Chaput
authors.affiliation: INSERM Unité 1016, Département Immunologie-Hematologie, Institut Cochin, Paris, France;
authors.forename: Véronique
authors.initials: V
authors.lastname: Witko-Sarsat
authors.affiliation: Ultrapole, Institut Pasteur, Paris, France;
authors.forename: Marie-Christine
authors.initials: MC
authors.lastname: Prevost
authors.affiliation: Conway Institute, University College Dublin, Belfield, Dublin, Ireland;
authors.forename: Cormac T
authors.initials: CT
authors.lastname: Taylor
authors.affiliation: Investigation Clinique et Accès aux Ressources Biologiques, Institut Pasteur, Paris, France;
authors.forename: Marie-Noelle
authors.initials: MN
authors.lastname: Ungeheuer
authors.affiliation: Unité de Pathogénie Microbienne Moléculaire, Institut Pasteur, Paris, France, INSERM Unité 786, Paris, France; Collège de France, Paris, France; and.
authors.forename: Philippe J
authors.initials: PJ
authors.lastname: Sansonetti
authors.affiliation: Unité de Pathogénie Microbienne Moléculaire, Institut Pasteur, Paris, France, INSERM Unité 786, Paris, France; Laboratoire de Thérapie Cellulaire, Gustave Roussy Cancer Campus, Villejuif, France.
authors.forename: Benoit S
authors.initials: BS
authors.lastname: Marteyn
mesh_terms: D000818:Animals; D000890:Anti-Infective Agents / Q000378:metabolism; D017209:Apoptosis / Q000187:drug effects; D015415:Biomarkers / Q000378:metabolism; D001803:Blood Transfusion; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D002469:Cell Separation; D048430:Cell Shape / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D005947:Glucose / Q000494:pharmacology*; D006168:Guinea Pigs; D006801:Humans; D000860:Hypoxia / Q000473:pathology*; D009504:Neutrophils / Q000166:cytology* / Q000187:drug effects / Q000648:ultrastructure; D010100:Oxygen / Q000494:pharmacology; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism; D014162:Transfection; D051028:bcl-2-Associated X Protein / Q000378:metabolism
chemical_list: D000890:Anti-Infective Agents; D015415:Biomarkers; D018809:Proliferating Cell Nuclear Antigen; D051028:bcl-2-Associated X Protein; D005947:Glucose; D010100:Oxygen