Neutrophils respond to hypoxia through NADPH oxidase and PI3K/AKT/HIF-1α

Objective: Inflamed local lesions often become severely hypoxic.  Inflammation-induced hypoxia induces polymorphonuclear neutrophils (PMN) mediated tissue destruction, as seen in various forms of periodontal diseases.  Previously, we have shown that superoxide release by PMN from patients with localized aggressive periodontitis was significantly elevated in response to hypoxia compared to healthy control individuals. The aim of this study was to elucidate the mechanism of hypoxia-mediated superoxide generation in human PMN (hPMN).

Method: hPMN were isolated from the peripheral blood.  Differentiated PMN-like HL-60 cells were used in transfection experiments.  All experiments were conducted under normoxic or hypoxic conditions over 2-12 hours.  Superoxide generation was measured after stimulation with fMLP (100nM).  The expression of NADPH oxidase components and hypoxia-inducible factor-1α (HIF-1α) were analyzed by qPCR and Western blots.  Activation of PI₃K was evaluated through the phosphorylation of AKT on Western blots.  Cells were treated with specific inhibitors of PI₃K, AKT, and HIF-1α.  Small interfering RNA targeted against HIF-1α (siHIF-1α) was used to transfect and silenced HIF-1α at the gene level.

Result: Superoxide generation by hPMN was significantly increased in 2 hours and steadily elevated over 8 hours of exposure to hypoxia (p<0.01).  mRNA expression of gp91phox, p22phox, p67phox, and p47phox were significantly increased in hPMN (1.7-2.6 -fold; p<0.01) under 8 hours of hypoxia compared to normoxia.  Protein expression of NADPH oxidase components was significantly increased by 1.5-2.6 -fold (p<0.01).  There was a parallel and statistically significant increase in superoxide generation (p<0.01) and expression of NADPH oxidase components (p<0.01) in differentiated PMN-like HL-60 cells under hypoxia over 2-12 hours.  Hypoxia enhanced the phosphorylation of AKT.  Chemical inhibitors and siHIF-1α significantly blocked expression of NADPH oxidase and superoxide generation under hypoxia (p<0.01).

Conclusion: These findings suggest that hypoxia-mediated PMN activation depends on NADPH oxidase assembly and PI₃K/AKT/HIF-1α in PMN.