Uncoupling of CD71 shedding with mitochondrial clearance in reticulocytes in a subset of myelodysplastic syndromes

Abstract

Reticulocytes shed CD71 from the cell membrane and eliminate mitochondria during terminal maturation, but it is unknown whether these two events are coordinated. We demonstrate that timely removal of CD71 is coupled with mitochondrial clearance, which can be disrupted by null mutation of immediate early response gene X-1 (IEX-1), leading to generation of aberrant CD71-positive and mitochondria-negative (CD71+Mito-) reticulocytes. CD71+Mito- reticulocytes were also present in a subset of patients with myelodysplastic syndromes (MDS) in direct proportion to reduced mitochondrial membrane potential (∆ψm). Mitochondrial abnormality caused by either IEX-1 deficiency or agents that dissipate ∆ψm could trigger premature clearance of mitochondria in reticulocytes. Premature clearance of mitochondria or addition of anti-oxidants lowered intracellular reactive oxygen species (ROS) that in turn hindered CD71 shedding and reticulocyte maturation. In contrast, introduction of ROS accelerated CD71 shedding via release of exosomes that contained a high proportion of Fe3+ over Fe2+, suggesting dual functions of CD71 shedding both in removal of toxic Fe3+ from reticulocytes and in limiting importation of Fe3+ into the cells. These observations emphasize the coordination of mitochondrial and CD71 clearance in erythroid terminal maturation and offer new insights into a role for mitochondrial degeneration in the pathogenesis of some MDS-associated anemia.

Conflict of interest statement

Conflict of interest The authors declare that they have no conflict of interest.

Figures

Fig. 1

Impaired erythropoiesis in IEX-1 KO mice. a, b Reticulocytosis in IEX-1 KO mice. Shown in a are representative images of increased reticulocytes marked by Methylene blue (black arrows) and polychromasia stained by Wright-Giemsa (blue arrows) in blood smears from at least 9 mice per group. Scale bar, 20 μm. Peripheral blood cells were stained with Ter119 vs. CD71 and subjected to cell counts by flow cytometry (upper) and statistical analysis of the data (lower) (b). c, d Compensatory expansion of erythroid precursors in KO mice. Bone marrow cells were stained with Ter119 vs. CD71 and the gates I to IV depict erythroid cell populations with an increasing degree of maturation (c). Distributions of cells within individual gates, as defined in the upper, were presented as percentages of cells over total bone marrow cells (lower). Erythropoietin (EPO) was quantified by qRT-PCR in mouse kidneys (d). Data are shown as mean ± SEM for all relevant panels. n = 9, *P < 0.05, **P < 0.01, and ***P < 0.001 compared between WT and KO groups

Fig. 2

Loss of mitochondria in IEX-1 KO reticulocytes while retaining CD71 on the cells. a–c IEX-1 KO CD71+ reticulocytes were absent of mitochondria. Peripheral blood cells of WT and KO mice were stained with Ter119, CD71, and MitoTracker, counted by flow cytometry (upper) and statistically analyzed (lower) (a). IEX-1 KO mice had an abnormal population of CD71+Mito− cells highlighted in red, within Ter119+ RBCs. Data were presented as mean ± SEM (n = 12), **P < 0.01 and ***P < 0.001 compared between WT and KO cells. Ter119+CD71+ reticulocytes were sorted from WT and KO peripheral blood cells for analyzing mitochondrial content by confocal microscopy (b; scale bar, 5 μm) or transmission electron microscopy (c; mitochondria, blue arrows; scale bar, 500 nm). d Accelerated mitochondrial clearance and delayed CD71 shedding in KO reticulocytes during maturation. Ter119+ CD71high reticulocytes were sorted from WT and KO mice 3 days after PHZ treatment and cultured for 2 days. MitoTracker, anti-CD71 antibody, and Methylene blue were then used to stain mitochondria, CD71, and residual RNA, respectively. Scale bar, 20 μm.

Fig. 3

Premature clearance of mitochondria and impaired CD71 shedding only in KO reticulocytes, not erythroblasts. a Reduced mitochondrial membrane potential in the bone marrow erythroblasts and peripheral reticulocytes of IEX-1 KO mice. Ter119+CD71+ cells in the bone marrow (erythroblasts, left panels) or peripheral blood (reticulocytes, right panels) were stained with MitoTracker and JC1 to detect mitochondrial content and membrane potential, respectively. The mean fluorescence intensity (MFI) of MitoTracker and JC1 (red J-aggregate) was analyzed by flow cytometry and presented as mean ± SEM. ns no significance. *P < 0.05 and ***P < 0.001 compared with WT group. b Enhanced clearance of mitochondria by autophagy in KO reticulocytes. Young reticulocytes were matured ex vivo as in Fig. 2d and analyzed by transmission electron microscopy at indicated days: intact mitochondria, blue arrows; mitochondria phagocytosed by autophagosomes, red arrows; scale bar, 1 μm. c Effects of FCCP and wortmannin on mitochondrial clearance and CD71 shedding in WT and KO reticulocytes. Ter119+CD71high reticulocytes were cultured in the presence of 10 μM FCCP or 100 nM wortmannin, followed by flow cytometric analysis of mitochondria and CD71 at indicated days. Data were representative for at least 12 samples per group in all relevant panels

Fig. 4

CD71 shedding is coupled with mitochondrial clearance in a ROS-dependent manner. a–c Exosome release is impaired in IEX-1 KO reticulocytes. Ter119+CD71high reticulocytes were sorted from PHZ-treated mice as above and cultured for 2 days. The size of reticulocytes was analyzed daily by flow cytometry on the basis of forward scatter (a). Exosomes released from 1 × 107 reticulocytes were assessed by the activity of AChE (b). After culturing for 12 h, cells were stained with FITC-anti-CD71 and N-Rh-PE that labeled MVBs to demonstrate their co-localization (c). Scale bar, 5 μm. Images were representative for at least 9 samples per group. d–g Changes of cellular iron concentrations during reticulocyte maturation. Concentrations of total iron, Fe2+, and Fe3+ were measured in reticulocytes before culturing (d) and daily during ex vivo maturation (e). f–i Modulation of ROS regulates CD71 shedding via exosome release. Ter119+CD71high reticulocytes as in a were cultured in the presence of 5 μM antioxidant NAC or 5 mU/mL glucose oxidase (GO), followed by daily measurement of cellular iron concentrations (f, g). Also shown are anti-CD71 staining for visualizing MVBs at 12 h after initial culture (h; scale bar, 5 μm), Ter119 and CD71 expression profiles by flow cytometry (i) with statistical analysis (j) on day 2, and concentrations of indicated irons and ratios of Fe2+ to Fe3+ measured in exosomes released from WT and KO reticulocytes 2 days after ex vivo maturation (k). Data represent as mean ± SEM (n = 12), *P < 0.05, **P < 0.01 and ***P < 0.001 compared between WT and KO cells or between indicated groups

Fig. 5

CD71+Mito− reticulocytes are presented in a majority of MDS patients. a Relative levels of IEX-1 mRNA in human peripheral blood cells in patients and healthy donors. MDS patients with at least a twofold decrease of IEX-1 mRNA compared with the median of healthy controls were defined as MDS patients with low IEX-1. b Reticulocytosis in MDS patients. Peripheral blood cells of healthy donors and MDS patients were stained with CD235a and CD71, followed by flow cytometric analysis. MDS patients exhibited a significantly increased proportion of CD235a+CD71+ reticulocytes compared to healthy controls independent of IEX-1. c Increased CD71+Mito− reticulocytes in MDS patients with a highest level in MDS patients with low IEX-1. d, e Reduced mitochondrial membrane potential Δψm in reticulocytes of MDS patients. Mitochondrial membrane potential Δψm of CD235a+CD71+ reticulocytes was analyzed by flow cytometry using JC1 as Fig. 3a (d). Inverse correlations between mitochondrial membrane potential Δψm and the percentages of CD71+Mito− reticulocytes are analyzed in all healthy donors and MDS patients by coefficient of determination (e). Data represent mean ± SEM, *P < 0.05, **P < 0.01, and ***P < 0.001 compared between indicated groups. (f) A model for CD71 shedding in couple with mitochondrial clearance. Fe3+ released from the CD71/Tf complex by a low pH in the endosomes after endocytosis of CD71/Tf-bounded Fe3+ is converted to Fe2+ by a metalloreductase Steap3 before transported to the cytoplasm by DMT1. An increasing ROS modulates Steap3 activity resulting in a shift of the reductive reaction toward Fe3+. A rise of ROS/Fe3+ would trigger fusion of the endosomes with MVBs followed by exosome excretion. An increase of ROS would also trigger mitophagy to limit ROS production. Altogether, a couple of CD71 shedding with mitochondrial clearance can effectively minimize the toxicity of Fe3+ and ROS during reticulocyte maturation