American Society of Hematology

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.

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.

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