Reducing neutrophil exposure to oxygen allows their basal state maintenance

Abstract

Neutrophils are the most abundant circulating white blood cells and are the central players of the innate immune response. During their lifecycle, neutrophils mainly evolve under low oxygen conditions (0.1–4% O2), to which they are well adapted. Neutrophils are atypical cells since they are highly glycolytic and susceptible to oxygen exposure, which induces their activation and death through mechanisms that remain currently elusive. Nevertheless, nearly all studies conducted on neutrophils are carried out under atmospheric oxygen (21%), corresponding to hyperoxia. Here, we investigated the impact of hyperoxia during neutrophil purification and culture on neutrophil viability, activation and cytosolic protein content. We demonstrate that neutrophil hyper‐activation (CD62L shedding) is induced during culture under hyperoxic conditions (24 h), compared with neutrophils cultured under anoxic conditions. Spontaneous neutrophil extracellular trap (NET) formation is observed when neutrophils face hyperoxia during purification or culture. In addition, we show that maintaining neutrophils in autologous plasma is the preferred strategy to maintain their basal state. Our results show that manipulating neutrophils under hyperoxic conditions leads to the loss of 57 cytosolic proteins during purification, while it does not lead to an immediate impact on neutrophil activation (CD11bhigh, CD54high, CD62Lneg) or viability (DAPI+). We identified two clusters of proteins belonging to cholesterol metabolism and to the complement and coagulation cascade pathways, which are highly susceptible to neutrophil oxygen exposure during neutrophil purification. In conclusion, protecting neutrophil from oxygen during their purification and culture is recommended to avoid activation and to prevent the alteration of cytosolic protein composition.

Isolation methods determine human neutrophil responses after stimulation

“…Thus, we hypothesize that, among various other factors influencing the outcomes, differences in the activation status of neutrophils at the beginning of an experiment likely contribute to the described inconsistencies regarding certain NETosis stimuli. The exact mechanism by which different density gradient methods result in more activated neutrophils remains unknown, but may involve centrifugation shear stress, duration of the isolation process and thus oxygen exposure (43), or osmotic changes. Indeed, a key difference between protocols that resulted in more quiescent neutrophils was the absence of RBC lysis.…”

Neutrophils degranulate GAG-containing proteoglycofili, which block Shigella growth and degrade virulence factors

“…Neutrophils are mainly glycolytic cells 4 and are well-adapted to low-oxygen microenvironments 5 We previously demonstrated that neutrophil viability was maintained when cells were purified and cultured under anoxia 6,7 , allowing the study of their physiology in vitro during extended periods of time. Applying this new method (Fig.…”

Dual proteomic signature of immune cells andYersinia pestisupon blood infection

“…However, this approach would presumably also enrich cell-associated bacteria, and a second orthogonal separation method such as differential centrifugation would be required to separate cell-free and cellassociated bacteria. Moreover, the short life span of neutrophils once blood is drawn from donors at atmospheric level of oxygen, greatly limits truly relevant models of bacteremia (Monceaux et al 2016;Injarabian, Skerniskyte, et al 2021).…”