Neutrophil HIF-1α stabilization is augmented by mitochondrial ROS produced via the glycerol 3-phosphate shuttle

Abstract

Neutrophils are predominantly glycolytic cells that derive little ATP from oxidative phosphorylation; however, they possess an extensive mitochondrial network and maintain a mitochondrial membrane potential. Although studies have shown neutrophils need their mitochondria to undergo apoptosis and regulate NETosis, the metabolic role of the respiratory chain in these highly glycolytic cells is still unclear. Recent studies have expanded on the role of reactive oxygen species (ROS) released from the mitochondria as intracellular signaling molecules. Our study shows that neutrophils can use their mitochondria to generate ROS and that mitochondrial ROS release is increased in hypoxic conditions. This is needed for the stabilization of a high level of the critical hypoxic response factor and pro-survival protein HIF-1α in hypoxia. Further, we demonstrate that neutrophils use the glycerol 3-phosphate pathway as a way of directly regulating mitochondrial function through glycolysis, specifically to maintain polarized mitochondria and produce ROS. This illustrates an additional pathway by which neutrophils can regulate HIF-1α stability and will therefore be an important consideration when looking for treatments of inflammatory conditions in which HIF-1α activation and neutrophil persistence at the site of inflammation are linked to disease severity.

Graphical abstract

Figure 1.

Hypoxia induces the production of mROS in neutrophils, which augments HIF-1α stabilization. (A-B) Neutrophils cultured in normoxia (21% O₂, red bars), 10% O₂ (light green bars), or hypoxia (1% O₂, blue bars) were stained with redox-sensitive dyes and fluorescence intensity analyzed using flow cytometry. (A) Neutrophil mROS levels were determined using MitoSOX Red after 1 hour in culture, n = 7. (B) Overall cellular ROS levels were quantified with DCF staining following treatment with fMLF, n = 8. (C,D) Untreated neutrophils (filled bars) and neutrophils treated with MitoTEMPO (open bars) were aged for 20 hours in normoxia or hypoxia and apoptosis determined through morphology, n = 7. Representative images at ×40 original magnification show hypoxia (top), hypoxia with MitoTEMPO (bottom). Red arrows indicate apoptotic cells. (E) Neutrophils were cultured for 4 hours in normoxia, 10% O₂, or hypoxia, sonication lysed, and proteins separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Membranes probed for HIF-1α relative to β-actin loading control, n = 4. (F) Neutrophils were cultured with or without rotenone (Rot, 2 μM), oxaloacetate (Oxa, 2 μM), antimycin A (AA, 10 ng/mL), DPI (10 μM), myeloperoxidase inhibitor-I (MPOi, 100 μM), or MitoTEMPO (MT, 100 μM) before lysis, SDS-PAGE, and membranes probed for HIF-1α relative to β-actin loading control, n = 6. Data represented as mean ± SEM. P values determined by (A-C) 2-way ANOVA or (E-F) 1-way ANOVA, *P < .05, **P < .01, *P < .001, ****P < .0001.

Figure 2.

Neutrophil hypoxic mROS release is driven by flux through the glycerol 3-phosphate shuttle. (A-K) Neutrophils were cultured in normoxia (21% O₂, red bars) and hypoxia (1% O₂, blue bars). (A) Mitochondrial membrane potential was determined by TMRM staining in neutrophils aged for 1 or 4 hours, n = 6. (B) Neutrophils cultured for 4 hours were lysed, proteins separated by SDS-PAGE, and membranes probed for GPD2, n = 3. (C) Neutrophils were treated with the GPD2 inhibitor iGP-1 or the protonophore CCCP membrane potential measured using TMRM dye, n = 6 (normoxia/hypoxia 1000 μm iGP-1 n = 4). (D) Neutrophils were treated with the glycolytic inhibitor 2-DG and membrane potential measured using TMRM dye, n = 3. (E-F) Neutrophils were lysed after 4 hours in culture, proteins separated by SDS-PAGE and membranes probed for HIF-1α expression, n = 4. (G) Neutrophils aged for 1 hour were treated with iGP-1 or the ROS scavenger MitoTEMPO (MT) stained with MitoSOX dye to assess mROS production, n = 6. (H-I) Apoptosis rates were determined in neutrophils aged for 20 hours by morphology (H), n = 3, and Annexin-TO-PRO3 positivity (I), n = 3. Representative images at ×40 original magnification show hypoxia (top) and hypoxia with 1 mM iGP-1 treatment. Red arrows indicate apoptotic cells. (J) Neutrophils were treated for 1 hour with iGP-1 in normoxia or hypoxia before infection with heat-killed CTFR-labeled S. aureus (SH1000 - MOI 1:1) and phagocytic uptake quantified after 5 minutes by flow cytometry, n = 5. (K) Neutrophil release of MPO was quantified following 4 hours of incubation in normoxia or hypoxia with iGP-1 and priming stimulation with granulocyte macrophage colony-stimulating factor (10 ng/mL) and fMLF (100 nM), n = 6; untreated, n = 3. (L) Schematic representation of the role of the glycerol-3-phosphate shuttle in the stabilization of HIF-1α. Data represent mean ± SEM. P values determined by (A,J,K) 2-way ANOVA, (C-E,H-I) paired t tests, or (F-G) 1-way ANOVA with Tukey’s multiple comparisons, **P < .01. ETC, electron transport chain.