Reticulocyte mitochondrial retention increases reactive oxygen species and oxygen consumption in mouse models of sickle cell disease and phlebotomy-induced anemia

Abstract

•Reticulocytes and erythrocytes containing mitochondria in sickle cell disease mice exhibit elevated levels of reactive oxygen species (ROS) and increased oxygen consumption.•Stress erythropoiesis in phlebotomized, anemic AA mice increases the fraction of reticulocytes and erythrocytes that retain mitochondria and high levels of ROS, regardless of an underlying hemolytic disorder.•The oxygen consumption rate increases in mitochondria-retaining erythrocytes and reticulocytes. Sickle cell disease (SCD) is caused by a mutation of the β-globin gene that results in the production of hemoglobin S (HbS). People with SCD experience anemia, severe acute pain episodes, persistent chronic pain, multiorgan damage, and a reduced life span. The pathophysiology of SCD caused by the polymerization of HbS on deoxygenation results in red cell deformability and the generation of reactive oxygen species (ROS). These 2 factors lead to red cell fragility and hemolysis. Reticulocytosis is an independent predictor of disease morbidity and mortality in SCD. We previously established that humans and mice with SCD exhibit abnormal mitochondrial retention in erythrocytes increasing ROS-associated hemolysis. Here, we investigated the hypothesis that mitochondrial retention and increased ROS are a consequence of stress erythropoiesis. Our results show clearly that stress erythropoiesis in phlebotomized, anemic AA mice results in mitochondrial retention and increased ROS in reticulocytes. We observed that elevated mitochondrial retention in reticulocytes also alters oxygen consumption and potentially contributes to increased HbS polymerization and red blood cell hemolysis. Therefore, these events occurring due to stress erythropoiesis contribute significantly to the pathology of SCD and suggest new therapeutic targets. Sickle cell disease (SCD) is caused by a mutation of the β-globin gene that results in the production of hemoglobin S (HbS). People with SCD experience anemia, severe acute pain episodes, persistent chronic pain, multiorgan damage, and a reduced life span. The pathophysiology of SCD caused by the polymerization of HbS on deoxygenation results in red cell deformability and the generation of reactive oxygen species (ROS). These 2 factors lead to red cell fragility and hemolysis. Reticulocytosis is an independent predictor of disease morbidity and mortality in SCD. We previously established that humans and mice with SCD exhibit abnormal mitochondrial retention in erythrocytes increasing ROS-associated hemolysis. Here, we investigated the hypothesis that mitochondrial retention and increased ROS are a consequence of stress erythropoiesis. Our results show clearly that stress erythropoiesis in phlebotomized, anemic AA mice results in mitochondrial retention and increased ROS in reticulocytes. We observed that elevated mitochondrial retention in reticulocytes also alters oxygen consumption and potentially contributes to increased HbS polymerization and red blood cell hemolysis. Therefore, these events occurring due to stress erythropoiesis contribute significantly to the pathology of SCD and suggest new therapeutic targets. Sickle cell disease (SCD) is an inherited blood disorder that affects millions of people worldwide [1Ballas SK. The cost of health care for patients with sickle cell disease.Am J Hematol. 2009; 84: 320-322Crossref PubMed Scopus (57) Google Scholar,2Kauf TL Coates TD Huazhi L Mody-Patel N Hartzema AG. The cost of health care for children and adults with sickle cell disease.Am J Hematol. 2009; 84: 323-327Crossref PubMed Scopus (278) Google Scholar] and is caused by a point mutation within the β-globin gene, resulting in a substitution of valine for glutamic acid at position 6 in the β-globin chain. The abnormal β-globin chain combines with α-globin resulting in the production of hemoglobin S (HbS). HbS polymerizes on deoxygenation resulting in red cell fragility, hemolysis, and anemia. In addition, those with SCD suffer from acute and chronic pain episodes, multisystem organ damage, and a shorter lifespan [3Jagadeeswaran R Rivers A. Evolving treatment paradigms in sickle cell disease.Hematology. 2017; 2017: 440-446Crossref PubMed Scopus (14) Google Scholar]. Traditionally, these complications of SCD were believed to be solely due to HbS polymerization, leading to an abnormal cell shape and blockage of capillaries. However, it is now understood that the overproduction of reactive oxygen species (ROS) in SCD red blood cells (RBCs) causes damage to lipids, proteins, and DNA [4Kohen R Nyska A. Invited review: oxidation of biological systems: oxidative stress phenomena, antioxidants, redox reactions, and methods for their quantification.Toxicol Pathol. 2002; 30: 620-650Crossref PubMed Scopus (1811) Google Scholar], thereby contributing to SCD pathology. In addition, excessive ROS in the bloodstream leads to several pathologies, including endothelial damage, accelerated hemolysis, hypercoagulability, and SCD-related vasoocclusion [5Nur E Brandjes DP Schnog JJB et al.Plasma levels of advanced glycation end products are associated with haemolysis-related organ complications in sickle cell patients.Br J Haematol. 2010; 151: 62-69Crossref PubMed Scopus (34) Google Scholar, 6Singer S Ataga K. Hypercoagulability in sickle cell disease and beta-thalassemia.Curr Mol Med. 2008; 8: 639-645Crossref PubMed Scopus (17) Google Scholar, 7Hebbel RP Osarogiagbon R Kaul D. The endothelial biology of sickle cell disease: inflammation and a chronic vasculopathy.Microcirculation. 2004; 11: 129-151Crossref PubMed Google Scholar, 8Kato GJ Hebbel RP Steinberg MH Gladwin MT. Vasculopathy in sickle cell disease: Biology, pathophysiology, genetics, translational medicine, and new research directions.Am J Hematol. 2009; 84: 618-625Crossref PubMed Scopus (246) Google Scholar]. Unlike most cells in the body, RBCs lack organelles because they mainly transport oxygen and carbon dioxide through hemoglobin [9Nandakumar SK Ulirsch JC Sankaran VG. Advances in understanding erythropoiesis: evolving perspectives.Br J Haematol. 2016; 173: 206-218Crossref PubMed Scopus (94) Google Scholar,10Martino S Arlet J Odièvre M et al.Deficient mitophagy pathways in sickle cell disease.Br J Haematol. 2021; 193: 988-993Crossref PubMed Scopus (10) Google Scholar]. Formed in the bone marrow, RBC precursors progress through distinct stages, from proerythroblasts to orthochromatic erythroblasts, before the removal of nuclei and organelles that characterizes the maturation of reticulocytes to RBCs. As a part of terminal erythrocyte differentiation and quality control, selective autophagy (mitophagy) rids the reticulocytes of mitochondria [11Ashrafi G Schwarz TL. The pathways of mitophagy for quality control and clearance of mitochondria.Cell Death Differ. 2013; 20: 31-42Crossref PubMed Scopus (1092) Google Scholar]. Initial findings indicate that abnormally retained mitochondria contribute to SCD pathogenesis by increasing the intracellular oxygen consumption and ROS in the RBCs of patients with SCD [12Jagadeeswaran R Lenny H Vazquez B et al.The abnormal presence of mitochondria in circulating red blood cells cause an increased oxygen consumption rate, ros generation and hemolysis in patients with sickle cell disease.Blood. 2017; 130: 2237PubMed Google Scholar]. This study aims to determine the role of stress erythropoiesis in erythrocyte mitochondrial retention. We hypothesized that mitochondrial retention, increased ROS levels, and elevated oxygen consumption in reticulocytes and erythrocytes in SCD are the consequences of stress erythropoiesis. To investigate our hypothesis, we compared these parameters in SCD mice; phlebotomized, anemic AA mice; and healthy control AA mice. Our results establish that stress erythropoiesis is a significant contributing factor to the pathology of SCD. SCD (B6; 129-Hba tm1 (HBA) Tow Hbb tm2 (HBG1, HBB*) Tow/Hbb tm3 (HBG1, HBB) Tow/J) and Hb AA (B6;129-Hbatm1(HBA)Tow Hbb tm3 (HBG1, HBB) Tow) 8- to 10-week-old mice, both male and female, were used in this study. Biological sex differences have not been reported as a variable in SCD pathophysiology [13Rivers A Vaitkus K Ruiz MA et al.RN-1, a potent and selective lysine-specific demethylase 1 inhibitor, increases γ-globin expression, F reticulocytes, and F cells in a sickle cell disease mouse model.Exp Hematol. 2015; 43 (546–553.e3)Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar,14Cui S Lim KC Shi L et al.The LSD1 inhibitor RN-1 induces fetal hemoglobin synthesis and reduces disease pathology in sickle cell mice.Blood. 2015; 126: 386-396Crossref PubMed Scopus (65) Google Scholar]. These mice contain the human α, human γ, and βS or βA globin genes knocked into the appropriate mouse globin locus. The mice containing βS genes (HbSS) exhibit pathology similar to that of the patients with SCD, whereas mice containing βA genes (HbAA) are healthy. A model of stress erythropoiesis and reticulocytosis was created in HbAA mice by performing repeated phlebotomies. The mice were bled daily for 4 days at 10% of their body weight, and the blood samples were collected on the fifth day. All procedures involving mice were carried out in accordance with the protocol (AN189029) approved by the Institutional Animal Care and Use Committee at the University of California, San Francisco, an AAALAC-accredited institution. Peripheral blood was assayed by standard flow cytometry using the BD LSR Fortessa (BD Biosciences). Samples were stained with APC-conjugated CD71 (R17217) antibody to differentiate RBCs and reticulocytes, and tetramethylrhodamine methyl ester (TMRM) was used to measure the mitochondria levels and CM-H2DCFDA [15George A Pushkaran S Konstantinidis DG et al.Erythrocyte NADPH oxidase activity modulated by Rac GTPases, PKC, and plasma cytokines contributes to oxidative stress in sickle cell disease.Blood. 2013; 121: 2099-2107Crossref PubMed Scopus (136) Google Scholar] to quantify the ROS levels. All labels were acquired from Invitrogen. FACS data were analyzed using FlowJo software [16FlowJoTM Software MAC Version 10.8.1. Becton, Dickinson and Company, Ashland, OR2022Google Scholar]. The oxygen consumption rate (OCR) was measured as a metric of mitochondrial function and oxidative phosphorylation. Peripheral blood was obtained, and 4 subpopulation fractions were isolated by FACS using a BD Aria II. Fractions were sorted following staining with FITC-conjugated CD71 (R172147) and MitoTracker Deep Red. The Aria was supported by DIVA 9. The total RBCs and fractions from each group were then analyzed using the Agilent Seahorse XFe24-extracellular flux analyzer, with cells suspended in a Seahorse XFe Base Medium with supplements [17AgilentSeahorse XFe96 Analyzer.2022https://www.agilent.com/en/product/cell-analysis/real-time-cell-metabolic-analysis/xf-analyzers/seahorse-xfe96-analyzer-740879Google Scholar]. OCR was monitored in real time. Basal OCR, ATP production (after oligomycin), maximal respiration (after FCCP), and reserve capacity (after rotenone) were measured following Agilent's mitochondrial stress test procedures. Peripheral blood was obtained by retro-orbital bleeding from anesthetized AA control, phlebotomized anemic AA, and SCD mice. Following euthanasia, the bone marrow cells were obtained by flushing excised femurs with phosphate-buffered saline. The cells were stained immediately for flow cytometry. Erythroid progenitors were measured from the bone marrow as previously described [18Liu J Zhang J Ginzburg Y et al.Quantitative analysis of murine terminal erythroid differentiation in vivo: novel method to study normal and disordered erythropoiesis.Blood. 2013; 121: e43-e49Crossref PubMed Scopus (165) Google Scholar]. ROS and mitochondrial stains were added for the FACS analysis. Statistical analyses were performed using GraphPad Prism (version 9.0; GraphPad Software). Qualitative data are expressed as percentages, medians, or exact values as stated. An unpaired t-test or one-way analysis of variance was used as appropriate. The results are presented as percentage or median ± SD. p < 0.05 was considered statistically significant. The mitochondria levels were measured by flow cytometric analysis using a cell-permeable, cationic, red-orange, fluorescent mitochondrial probe TMRM (Figure 1A, B). We observed elevated levels of mitochondria-positive RBCs in the phlebotomized anemic AA model compared with control AA (control AA: 1.198% ± 1.118; anemic AA: 3.953% ± 2.148; p < 0.05), but lower than SCD mice (SCD: 12.52% ± 4.868; p = 0.01). The levels of mitochondria-positive reticulocytes determined for the anemic AA mice were not significantly different than that of the SCD mice (Anemic AA: 21.18% ± 3.271; SCD: 31.87% ± 12.12; p = 0.1291) but were significantly higher than that of the control group (control AA: 2.956% ± 1.890, n = 5; p < 0.0001). This indicates that stress erythrocytosis from anemia increases the percentage of reticulocytes with mitochondria. The ROS levels of mitochondria-positive RBCs and reticulocytes were similar in SCD, phlebotomized anemic AA, and control AA mice (Figure 1C). Mitochondria-negative RBCs of SCD mice had significantly elevated ROS compared with control AA (p < 0.05) and anemic AA (p < 0.01) mice (control AA: 784 ± 436.6; SCD: 1677 ± 643.4; anemic AA: 211 ± 69.39). SCD mice also displayed significantly higher ROS levels than both control AA and anemic AA groups across the total peripheral RBCs and reticulocytes: (control AA: 814.8 ± 448.0; SCD: 2068 ± 482.0; anemic AA: 285.5 ± 95.17; control AA/SCD: p < 0.01; SCD/anemic AA: p < 0.0001). These data indicate that the presence of mitochondria in control AA, SCD, or anemic AA mice led to increased ROS levels. The percentage of mitochondria-positive RBC precursors (TMRM+) in the bone marrow was determined to evaluate the trend in precursor mitochondrial retention (Figure 2B). Across all groups, the levels of mitochondria-positive precursors decreased as differentiation proceeded from populations IV-VI. We observed similar levels of mitochondria-positive precursors in populations I-V and a significant elevation in the population VI of the anemic AA group compared with AA controls (p < 0.05). Elevated levels of mitochondria-positive mature RBCs were observed in anemic AA mice compared with controls (p < 0.05). SCD mice had an increased level of mitochondria-positive RBCs; however, there was no statistically significant difference compared with controls. These data suggest that more mitochondria-positive RBCs are being released into the peripheral circulation in the SCD model when compared with control.Figure 2ROS and mitochondrial content in RBC progenitors of control AA, phlebotomized anemic AA, and SCD mice. (A) Representative erythropoiesis profiles of the bone marrow of control AA, SCD, and anemic AA mice. Populations I-VI correspond to pro-erythroblasts, basophilic erythroblasts, polychromatic erythroblasts, orthochromatic erythroblasts, reticulocytes, and mature RBCs, respectively. (B) Quantitative bar graphs show the percentage of ROS-generating (ROS+) precursors of populations I-VI in the bone marrow of each group. (C) Quantitative bar graphs show the percentage of mitochondria-containing (TMRM+) precursors of populations I-VI in the bone marrow of each mouse group. Control AA: n = 3; SCD: n = 3; Anemic AA: n = 4; *p < 0.05, unpaired two-tailed t-test comparisons between the groups.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To evaluate the differences in ROS generation between the bone marrow populations, the percentage of RBC precursors containing ROS (ROS+) was determined (Figure 2C). There were no significant differences between the levels of ROS+ precursors of the 3 groups in populations I-V. There was drastic decline in ROS+ in all groups between populations V and VI. The level of ROS+ precursors was significantly elevated in the mature RBCs (population VI) of the SCD mice compared with control AA and anemic AA groups (p < 0.05). We investigated mitochondrial RBC energy metabolism by analyzing OCR. Basal and maximal OCR (pmol/min) of RBCs were significantly higher in SCD and anemic AA mice compared with AA controls (Figure 3A; basal: (control AA: 0.7333 ± SCD: ± anemic AA: ± p = control AA/SCD: p = anemic AA: p = maximal (control AA: ± SCD: ± anemic AA: ± p = control AA/SCD: p = control AA: p = There were no significant differences between OCR of SCD and anemic AA and maximal oxygen consumption of peripheral blood RBCs and reticulocytes from control AA, SCD, and phlebotomized anemic AA mice. (A) The bar graphs OCR = and maximal OCR = levels of peripheral RBCs from SCD, control AA, and anemic AA mice. Representative Seahorse flux graphs show OCR levels at different and between each group. Control AA: n = 3; SCD: n = Anemic AA: n = *p < 0.05, < < (B) Representative graphs show OCR of mitochondria-positive and RBCs and reticulocytes from an SCD N = 3 mice. (C) Quantitative bar graphs show the maximal respiration of and RBCs and reticulocytes of SCD and anemic AA mice. N = 3 mice Large Image Figure ViewerDownload Hi-res image Download (PPT) The maximal respiration of erythroid fractions from SCD and phlebotomized anemic AA mice was analyzed in Figure and In both groups, mitochondria-positive reticulocytes the maximal OCR, by mitochondria-positive RBCs, RBCs, and reticulocytes. the mitochondria-positive SCD mice a higher maximal OCR than anemic AA mice with anemic AA: ± SCD: ± p < RBCs with AA: ± SCD: ± p < 0.05). There were no significant differences between OCR of the fractions AA: ± SCD: ± reticulocytes AA: ± SCD: ± Our data clearly show that reticulocytes with mitochondria contribute a significant of ROS and oxygen regardless of the presence of been for that reticulocytosis is an independent predictor of morbidity in SCD et of in children with sickle cell J Med. PubMed Scopus Google Reticulocytosis and anemia are associated with an increased of and in the of the of Sickle J Hematol. PubMed Scopus Google Scholar], and the results from the presented establish ROS and oxygen consumption as a for the of reticulocytes and associated We that abnormally retained mitochondria in RBCs of patients and mice with SCD are associated with elevated levels of ROS [12Jagadeeswaran R Lenny H Vazquez B et al.The abnormal presence of mitochondria in circulating red blood cells cause an increased oxygen consumption rate, ros generation and hemolysis in patients with sickle cell disease.Blood. 2017; 130: 2237PubMed Google R Vazquez M et of LSD1 and reduces mitochondrial retention and associated ROS levels in the red blood cells of sickle cell Hematol. 2017; Full Text Full Text PDF PubMed Scopus Google Scholar]. we and groups reported that reticulocytes from patients with SCD also had elevated levels of ROS S Arlet J Odièvre M et al.Deficient mitophagy pathways in sickle cell disease.Br J Haematol. 2021; 193: 988-993Crossref PubMed Scopus (10) Google R Lenny H Vazquez B et al.The abnormal presence of mitochondria in circulating red blood cells cause an increased oxygen consumption rate, ros generation and hemolysis in patients with sickle cell disease.Blood. 2017; 130: 2237PubMed Google R Vazquez M et of LSD1 and reduces mitochondrial retention and associated ROS levels in the red blood cells of sickle cell Hematol. 2017; Full Text Full Text PDF PubMed Scopus Google Scholar]. In this we data that mitochondrial retention stress independent of the presence of hemoglobin mouse were used to evaluate peripheral blood and RBC in mitochondrial retention precursors are between the and the mature RBC This leads to elevated ROS, reticulocytes in the blood or bone marrow therapeutic to SCD. In the bone marrow of anemia, the generation of ROS was to a role in and was associated with the of the R and in bone marrow associated with ROS in J PubMed Scopus Google Scholar]. In with SCD, the RBC levels are lower when compared with SCD. and used to SCD, in RBC mitochondria. an for SCD, is an acid that and in erythrocytes F G et for sickle cell disease: more than Hematol. PubMed Scopus Google Scholar, V JC and PubMed Scopus Google Scholar, et al.Erythrocyte redox and in sickle cell disease.Blood. 2008; PubMed Scopus Google Scholar]. an is for role in oxidative and the transport chain. in the alters the redox in leading to an in ROS and oxidative The presence of mitochondria in RBCs and our findings of increased ROS this but with been to mitochondrial in and ROS Gladwin MT. for mitochondrial oxidative PubMed Scopus Google Scholar]. of mitochondrial RBC not been Therefore, of to mitochondrial in RBCs is of oxygen consumption in RBCs was used to mitochondrial function in SCD, phlebotomized, and control mouse with flow cytometry and we that reticulocytes with mitochondria had an elevated OCR in both phlebotomized and SCD mouse We also observed higher and maximal oxygen consumption in SCD and phlebotomized anemic AA mice compared with controls. There are of increased oxygen consumption by mitochondria leading to cell increased mitochondrial oxygen consumption to a mitochondrial leads to and contributes to disease M E M M to increased oxygen induces of and oxidative 2013; PubMed Scopus Google Scholar]. In increased and maximal respiration of mitochondria in peripheral blood cells were associated with M E M M to increased oxygen induces of and oxidative 2013; PubMed Scopus Google Scholar]. We that increased oxygen consumption in SCD reticulocytes led to intracellular and increased in HbS our described method of OCR analysis from patients with SCD for Unlike to ROS, that be used to increased oxygen consumption have not been to OCR, also all of mitochondria in RBCs, be the of that RBC RN-1 or increased RBC reduced ROS, and mitochondrial retention in a sickle mouse model independent of Hb F A Vaitkus K V et al.The LSD1 inhibitor RN-1 the fetal of hemoglobin synthesis in 2016; PubMed Scopus Google Scholar]. However, that ROS or OCR have than that is the disease mitochondria are retained in the Our data indicate that disease that a of RBCs in the bone marrow cause and erythrocyte mitochondrial retention and ROS and OCR. from is associated with erythropoiesis erythropoiesis. In a in and capacity on erythrocyte an of the oxidative and disease activity and the presence of mitochondria-positive RBCs have been RBCs with mitochondria a that in cells and A mitochondria to the 2021; Full Text Full Text PDF PubMed Scopus Google S J AA et mitochondrial retention in human 2021; Full Text Full Text PDF PubMed Scopus Google Scholar]. This the by et that SCD erythrocyte mitochondrial retention as M M et of mitochondria in mature red blood cells from patients with sickle cell disease.Br J Haematol. PubMed Scopus (10) Google Scholar], that mitochondrial be in or erythropoiesis. These similar findings in indicate the for research in erythrocyte mitochondrial retention in disease In into mitochondrial retention, ROS and OCR of mitochondria-positive and fractions of peripheral blood and RBC precursor populations in a of SCD pathogenesis and RBC In addition, these data the of and mitophagy as therapeutic for hemolytic anemia, blood and hemolysis to to the understanding of the of mitochondrial in the peripheral blood that be to and pain events in SCD. This study was supported by from the of

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