Intermittent Hypoxia Affects the Spontaneous Differentiation In Vitro of Human Neutrophils into Long-Lived Giant Phagocytes

Previously we identified, for the first time, a new small-size subset of neutrophil-derived giant phagocytes (G phi) which spontaneously develop in vitro without additional growth factors or cytokines. G phi are CD66b(+)/CD63(+)/MPO+/LC3B(+) and are characterized by extended lifespan, large phagolysosomes, active phagocytosis, and reactive oxygen species (ROS) production, and autophagy largely controls their formation. Hypoxia, and particularly hypoxia/reoxygenation, is a prominent feature of many pathological processes. Herein we investigated G phi formation by applying various hypoxic conditions. Chronic intermittent hypoxia (IH) (29 cycles/day for 5 days) completely abolished G phi formation, while acute IH had dose-dependent effects. Exposure to 24 h (56 IH cycles) decreased their size, yield, phagocytic ability, autophagy, mitophagy, and gp91-phox/p22-phox expression, whereas under 24 h sustained hypoxia (SH) the size and expression of LC3B and gp91-phox/p22-phox resembled G phi formed in normoxia. Diphenyl iodide (DPI), a NADPH oxidase inhibitor, as well as the PI3K/Akt and autophagy inhibitor LY294002 abolished G phi formation at all oxygen conditions. However, the potent antioxidant, N-acetylcysteine (NAC) abrogated the effects of IH by inducing large CD66b(+)/LC3B(+)G phi and increased both NADPH oxidase expression and phagocytosis. These findings suggest that NADPH oxidase, autophagy, and the PI3K/Akt pathway are involved in G phi development.