P103: THE ABNORMAL PRESENCE OF MITOCHONDRIA IN CIRCULATING SCD RED BLOOD CELLS ASSOCIATED WITH STRESS ERYTHROPOIESIS
Authors: J Ramasamy, A Gallivan, L Hong, H Horneman, R Molokie, A Diamond, D Lavelle, A Rivers
Journal: HemaSphere (2022)

Sickle Cell Disease (SCD) is an inherited blood disorder that affects millions of people worldwide, and it is caused by a mutation of the β-globin gene which results in the polymerization of HbS when deoxygenated. Sickle cell patients experience severe pain, multi organ damage, and shortened life span. Changes arising from sickle hemoglobin (HbS) inside the red blood cell (RBC) leads to an imbalance of the oxidative reactions which increases reactive oxygen species (ROS) formation. Consent was obtained from people with SCD and people with SCD. Our lab discovered that SCD patients have a significant fraction of RBCs retaining-mitochondria which were associated with excessive levels of ROS1. We also noticed and increase in immature mitochondria containing reticulocytes in peripheral blood of SCD patient compared to control subjects. The percentage of mitochondria retaining RBCs directly correlated with absolute number of reticulocytes (n=18,r=0.69, P< 0.001). As would be expected we were able to link mitochondria-retention with hemolysis. In addition when we investigated energy metabolism we made the novel finding that SCD RBC have increase oxygen consumption rate (OCR). Mitochondrial-RBC retention and markers of hemolysis (serum bilirubin, and LDH) were compared by using Pearson's correlation coefficient analysis. The total serum bilirubin was directly associated with the percentage of mitochondria-containing RBCs in SCD patients' blood samples (r=0.31, n=41, p<0.04). A subgroup analysis showed a strong association between bilirubin and mitochondria-retaining RBCs in SCD patients who were not taking Hydroxyurea (r=0.55, n=17, P<0.02). There was a less significant association of percentage of mitochondria-containing RBCs in SCD with serum LDH (r=0.02, n=40, P<0.2) when all patients were analyzed, however, a strong association was seen with serum LDH and mitochondria retaining RBCs in SCD patients who were not taking Hydroxyurea (r=0.67, n=17, P<0.002). Lactate dehydrogenase (LDH) is a known biomarker for hemolysis-and associated disease complications and early mortality in patients with SCD. Percentage of mitochondria retaining RBCs also directly correlated with absolute number of reticulocytes (n=18,r=0.69, P< 0.001).We next investigated the mitochondrial RBCs energy metabolism in SCD as compared to controls in both human and mice. RBCs obtained from Townes SCD or control mice were seeded in an Agilent Seahorse XF 24-well plate using the Seahorse XF Base Medium with supplements and we monitored the OCR and extracellular acidification rate (ECAR), in real time by Seahorse XFe-extracellular flux analyzer. OCR is significantly higher in sickle cell RBCs of both human and mice origin compared to control. OCR was reduced to normal levels with oligomycin which is a known blocker of mitochondrial respiration. The mechanism responsible for mitochondrial retention in SCD is unknown. We further investigated that stress erythropoiesis is responsible for abnormal mitochondrial retention in erythrocytes. Terminal erythrocyte precursors and reticulocyte from bone marrow and spleen and peripheral blood had ROS and mitochondrial content by FACS2. Phlebotomized showed similar increased in erythrocyte mitochondrial retention and similar pattern of precursor erythrocyte in bone marrow to SCD mice precursors. Conclusion: Our data suggest a novel pathophysiology of stress erythropoiesis in SCD. References 1. Jagadeeswaran, R. et al. Pharmacological inhibition of LSD1 and mTOR reduces mitochondrial retention and associated ROS levels in the red blood cells of sickle cell disease. Exp. Hematol.50, 46–52 (2017). 2. Liu, J. et al. Quantitative analysis of murine terminal erythroid differentiation in vivo: novel method to study normal and disordered erythropoiesis. Blood121, e43–e49 (2013).