Reticulocytosis from Stress Erythropoiesis Is a Major Source of Erythrocyte Mitochondrial Retention, Oxygen Consumption and Reactive Oxygen Species in a SCD Mouse Model

Introduction: People with Sickle Cell Disease (SCD) experience severe pain episodes and multi-organ damage. SCD is caused by a mutation of the β-globin gene which results in Hemoglobin S (Hb S). The polymerization of Hb S when deoxygenated is the primary catalyst for activating the pathophysiology of the disease and its hemolytic anemia. The associated reticulocytosis is an independent predictor of disease morbidity and mortality. Our group reported that humans and mice with SCD exhibit erythrocyte mitochondrial retention that increases reactive oxygen species (ROS) associated hemolysis and increases oxygen consumption rate (OCR) that could lead to increases in Hb S polymerization. Objective: Determine if pathologic mitochondrial retention, ROS, and OCR are universal consequences of stress erythropoiesis. Methods: In this study, we performed phlebotomy on healthy Townes (Hb AA) mice to create a model of erythrocyte mitochondria retention with Hb A. Peripheral blood of phlebotomized Townes (Hb AA), SCD, and non-phlebotomized Townes Hb AA mice were evaluated with CD-71, tetramethylrhodamine methyl ester (TMRM) or MitoSOX™Red and CM-H2DCFDA. The separation of mitochondria positive (mito+, TMRM+ or MitoSOX™+) versus mitochondria negative (mito- , TMRM– or MitoSOX™Red-) fractions of RBCs and reticulocytes was carried out by fluorescent activated cell sorting (FACS). ROS levels were measured by utilizing the commercially available cellular ROS probe CM-H2DCFDA. OCR was measured using a Seahorse XFe96-extracellular flux analyzer. Erythrocyte precursors were evaluated by FAC using CD44 vs FSC. Results: We observed that levels of mito+ RBCs in the phlebotomy model were higher than controls, but lower than SCD mice. Levels of mito+ reticulocytes of the phlebotomy group were similar to the SCD group and significantly higher than the control group (Table 1). We observe that across all groups of mice, ROS was higher in mito+ RBCs and reticulocytes compared to mito- RBCs and reticulocytes. There were no significant differences in median ROS intensity of mito+ RBCs and reticulocytes between the three mice groups. Mito- RBCs and reticulocytes of SCD mice had significantly elevated ROS (Table 1). OCR (pmol/min) was significantly higher in SCD and phlebotomized mice RBCs compared to controls: (Basal: (Control: 0.7333 ± 1.250, n=3; SCD: 17.05 ± 3.465, n=2; Phlebotomy: 10.55 ± 2.758, n=2; p=0.004; Control/SS: p=0.0041; Phlebotomy/control: p=0.0108); Maximal Respiration: (Control: 1.833 ± 0.3786, n=3; SCD: 19.10 ± 3.960, n=2; Phlebotomy: 12.85 ± 1.344, n=2; p=0.002; Control/SS p=0.0038; Control/Phlebotomy: p=0.0007)). We separated peripheral blood from SCD mice into four groups. Reticulocytes with mitochondria had the highest basal and maximal oxygen consumption rate: (Basal respirations: 1) reticulocytes-mito+ (2.2 ± 1.266), 2) RBCs-mito+ (1.4 ± 0.7810), 3) reticulocytes-mito- (0 ± .3215), and 4) RBCs-mito- (0 ± 0.5), n=3; p<.01); Maximal respirations: 1) reticulocytes-mito+ (12.73 ± 4.7), 2) RBCs-mito+ (4.5 ± 1.3), 3) reticulocyte-mito- (0 ± 0.35 and 4) RBCs-mito- (0 ± 0.43, n=3; p<.01)). Our evaluation of reticulocytes and erythrocytes with retained mitochondria in spleen and bone marrow showed an increased fraction proportion compared to controls. In spleen and bone marrow SCD (Hb SS) and phlebotomize mice in population VI (RBC) had significantly higher levels of mitochondrial intensity: ((Spleen (Control (Hb AA): 6.415 ± 1.817, n=2; SCD (Hb SS): 30.20 ± 4.525, n=2; Hb AA with phlebotomy: 10.92 ± 2.209, n=4; p=0.0007); Bone marrow (Control (Hb AA): 8.360 ± 2.744, n=2; SCD (Hb SS): 183.0 ± 57.98, n=2; Hb AA with phlebotomy: 18.30 ± 1.590, n=4; p=0.0002)). Conclusion: These findings indicate that reticulocytes with mitochondria contribute a significant amount of ROS and oxygen consumption regardless of the presence of Hb S. Acute anemia induces rapid expansion of erythroid precursors and accelerated differentiation to replenish erythrocytes regardless of the existence of an underlying hemolytic disorder. This finding supports the clinical data that reticulocytosis is an independent predictor of disease morbidity. Research into erythrocyte mitochondrial retention is essential in identifying new therapeutic targets for SCD.