Glutathione peroxidase 4 and vitamin E control reticulocyte maturation, stress erythropoiesis and iron homeostasis

Abstract

Glutathione peroxidase 4 (GPX4) is unique as it is the only enzyme that can prevent detrimental lipid peroxidation in vivo by reducing lipid peroxides to the respective alcohols thereby stabilizing oxidation products of unsaturated fatty acids. During reticulocyte maturation, lipid peroxidation mediated by 15-lipoxygenase in humans and rabbits and by 12/15-lipoxygenase (ALOX15) in mice was considered the initiating event for the elimination of mitochondria but is now known to occur through mitophagy. Yet, genetic ablation of the Alox15 gene in mice failed to provide evidence for this hypothesis. We designed a different genetic approach to tackle this open conundrum. Since either other lipoxygenases or non-enzymatic autooxidative mechanisms may compensate for the loss of Alox15, we asked whether ablation of Gpx4 in the hematopoietic system would result in the perturbation of reticulocyte maturation. Quantitative assessment of erythropoiesis indices in the blood, bone marrow (BM) and spleen of chimeric mice with Gpx4 ablated in hematopoietic cells revealed anemia with an increase in the fraction of erythroid precursor cells and reticulocytes. Additional dietary vitamin E depletion strongly aggravated the anemic phenotype. Despite strong extramedullary erythropoiesis reticulocytes failed to mature and accumulated large autophagosomes with engulfed mitochondria. Gpx4-deficiency in hematopoietic cells led to systemic hepatic iron overload and simultaneous severe iron demand in the erythroid system. Despite extremely high erythropoietin and erythroferrone levels in the plasma, hepcidin expression remained unchanged. Conclusively, perturbed reticulocyte maturation in response to Gpx4 loss in hematopoietic cells thus causes ineffective erythropoiesis, a phenotype partially masked by dietary vitamin E supplementation.

Figure 1.

(A-H) Gpx4 is required for stress erythropoiesis in the recovery phase of anemia. Deletion of Gpx4 in the bone marrow (BM) was monitored by PCR using two primers pairs. One detects the deleted allele (509 bp), the other discriminates between the floxed and wild-type (wt) allele. Absence of the floxed allele indicates that deletion was complete. (A) Presence of the wt band indicates that BM cells contain a small proportion of cells of non-hematopoietic origin. (B) Quantification of Gpx4 mRNA in the bone marrow by quantitative RT-PCR. (C) Detection of GPX4 protein by Western blot analysis. (D) Temporal scheme of bone marrow transplantation (BMT), tamoxifen treatment (TAM) and determination of red blood cell (RBC) parameters. Lethally irradiated mice were reconstituted with 10⁶ BM cells of Gpx4^wt/wt; Cre ERT2 (designated wt, black columns, n=10) and Gpx4^fl/fl;Cre ERT2 mice (designated k.o., grey columns, n=19). Mice were fed a tamoxifen citrate containing diet for three weeks. Blood was drawn before (left columns, - TAM), at the last day of (0), and 3, 6, and 9 weeks after tamoxifen administration (+TAM), and (E) erythrocyte counts, (F) hemoglobin, (G) hematocrit, and (H) reticulocyte counts were determined. (I-M) Vitamin E depletion in the diet severely aggravates the anemia caused by Gpx4-deficiency in hematopoietic cells. (I) Temporal scheme of BMT, tamoxifen administration, vitamin E depletion and determination of red blood parameters. Lethally irradiated mice were reconstituted with BM cells of Gpx4^wt/wt;Cre-ERT2 (designated wt, black columns) and Gpx4^fl/fl;Cre ERT2 mice (designated k.o., grey and white columns). After tamoxifen administration for three weeks, mice were allowed to recover for 12 weeks before the vitamin E-depleted diet was started (− vitE, n=7 for wt, n=7 for k.o.) or the normal diet continued (+ vitE, n=3 for wt, n=7 for k.o.). Blood was drawn before vitamin E depletion (time point 9 weeks in Figure 1D-H), and 26 (black and grey columns) and 54 days [white columns], n=3) after starting the vitamin E-depleted diet for the determination of (J) erythrocyte counts, (K) hemoglobin, (L) hematocrit levels, and (M) reticulocyte counts. Red blood parameters of blood taken 54 days after starting the vitamin E-depleted diet were unaltered as compared to the earlier time point. N-R) Administration of a vitamin E-enriched diet. (N) Temporal scheme of feeding the mice a vitamin E-enriched diet. (R) A 5-fold increase of α-tocopherol in the diet reduced the degree of reticulocytosis but had not impact on (O) erythrocyte counts, (P) hemoglobin and (Q) hematocrit levels. S-W) White blood counts and red blood parameters and in Gpx4^fl/fl;LysMCre and control mice. There is no difference in (S) white blood cell (WBC) counts, (T) erythrocytes, (U) hemoglobin, (V) hematocrit, and (W) reticulocyte counts between Gpx4^fl/fl;LysMCre and control mice.

Figure 2.

Relative increase in immature erythroid precursor cells in the bone marrow and spleen of mice with Gpx4-deficiency in the hematopoetic system. Representative fluorescence-activated cell sorting (FACS) staining of (A-F) BM and (G-L) spleen cells with CD44-PE-Cy7 and Ter119-PE (A-C) as well as with CD71-PE-Cy7 and Ter119-PE antibodies (D-F, J-L). The gate set by forward sideward scatter (FSC) and lineage marker-negative cells (G-I) illustrates the increase in extramedullary erythropoiesis in the spleen of mice with Gpx4-deficient hematopoietic cells kept on a (H) normal or (I) on a vitamin E-depleted diet as compared to (G) Gpx4 wt mice kept on a normal diet. The shift towards immature erythroid precursor cells is strongly increased under combined Gpx4- and vitamin E-deficiency (C, F, L).

Figure 3.

Ineffective erythropoiesis in mice with Gpx4-deficiency in the hematopoietic system and severe aggravation by dietary vitamin E deficiency. Total numbers of bone marrow (BM) cells collected from (A) two femura and two tibiae and (B) spleen weights of wild-type (wt) mice (n=8) and of mice with Gpx4-deficient hematopoiesis maintained either on a normal (n=8) or a vitamin E-depleted diet (n=9). The total numbers of proerythroblasts and erythroblasts in the BM and spleen, and of reticulocytes and erythrocytes in the blood were assessed as described in the Online Supplementary Materials and Methods. Comparative quantification of the total numbers of (F-H) proerythroblasts (n=8 for each condition), of (I-K) erythroblasts (n=8 for each condition), (L) reticulocytes and (M) erythrocytes (both n=42 wt; n=23 k.o., normal diet; n=16 k.o. minus vitE). Proerythroblasts in the BM and spleen were quantified separately in G and H, and erythroblasts in J and K, respectively. The significance was calculated by the Mann-Whitney test. The erythropenia caused by Gpx4-deficiency in hematopoietic cells is compensated to a large extent by increased extramedullary erythropoiesis and strongly elevated reticulocyte counts. Under combined Gpx4- and vitamin E-deficiency, the number or erythrocytes is strongly decreased, but the number of reticulocytes is not significantly higher than in wt mice. This points to a loss of erythroid progenitor cells at the proerythroblast and/or erythroblast stage in addition to the reticulocyte maturation defect under these conditions.

Figure 4.

Extramedullary hematopoiesis in lethally irradiated wild-type (wt) mice reconstituted with Gpx4-deficient BM cells. (A-F) Histological sections of the spleen of (A,D) Gpx4 wt mice kept on a normal diet and (B,E) of mice with Gpx4-deficient hematopoiesis maintained either on a (C) normal or on a (F) vitamin E-depleted diet, stained with Perls’ blue stain (A-C) or by immunohistochemistry with anti-Ter119 antibody (D-F). The splenic red pulp is increased in mice with Gpx4-deficient hematopoiesis (B,E) and the white pulp is almost completely dissipated when vitamin E is additionally depleted (C,F). (A) Iron deposits derived from erythrocyte turnover are clearly visible in the red pulp of wt mice kept on a normal diet, but (B,C) are only faintly visible in the periphery of follicles in the white pulp of mice with Gpx4-deficient hematopoiesis. Iron deposits are decreased rather than increased in the spleen of severely anemic mice arguing against increased hemolysis as the cause of anemia.

Figure 5.

(A-C) Gpx4-deficiency in hematopoietic cells causes a reticulocyte maturation defect that is to a large extent compensated by vitamin E in vivo. Representative fluorescence-activated cell sorting (FACS) stainings of peripheral blood cells of (A) Gpx4 wt mice kept on a normal diet and of mice with Gpx4-deficient hematopoiesis maintained either on (B) a normal or on (C) a vitamin E-depleted diet (C), stained with Mitotracker Deep Red (MTDR), thiazol orange (TO), and CD71-PE-Cy7 and Ter119-PE antibodies. The experiment is described and quantitatively evaluated in the Online Supplementary Figure 5. Ter119-positive cells were gated and plotted as shown in A-C. Immature reticulocytes (CD71^high) are shown in green, mature reticulocytes (CD71^low) in blue and erythrocytes (CD71^neg/Ter119⁺) in purple. CD71^high cells were subdivided into immature and highly immature reticulocytes based on MTDR and TO staining. (C) Under combined Gpx4- and vitamin E-deficiency the fraction of highly immature reticulocytes was strongly increased. D-F) Lipid peroxidation is increased in Gpx4-deficient reticulocytes and erythrocytes. Peripheral blood cells of the mice shown in A-C mice were stained with anti-CD71-PE-Cy7- and anti-Ter119-APC-Cy7-antibodies and with 2 μM C11-Bodipy(581/591). Ter119-positive cells were gated and the increase in % green fluorescence-positive cells was measured in CD71^high cells (immature reticulocytes, D), in CD71^low cells (mature reticulocytes, E) and in CD71neg cells (erythrocytes, F) upon excitation at 488 nm. Wild-type, normal diet, (n=2); k.o., normal diet (n=2); k.o., minus vitamin E (n=3). The significance was calculated using an unpaired T-Test. Note the high degree of lipid peroxidation in Gpx4-deficient immature reticulocytes upon feeding a vitamin E-depleted diet. G-L) Ultrastructural analysis of red blood cells from the mice shown in A-C. Remnants of mitochondria (Mi) are marked by white arrows. Under Gpx4-deficiency (H and K), and more so under combined Gpx4- and vitamin E-deficiency (I and L) large unphagocytosed vesicles containing mitochondria accumulated in reticulocytes (R). Blood pellets were processed for transmission electron microscopy as described in the Online Supplementary Materials and Methods.

Figure 6.

Iron overload in the liver and simultaneous iron demand for erythropoiesis in mice with a Gpx4-deficient hematopoietic system. In the liver of mice with Gpx4-proficient and Gpx4-deficient hematopoiesis only the wild-type (wt) allele of Gpx4 is detected (A), and the same levels of Gpx4 mRNA (B) and protein (C) are expressed. (D-K, V) Parameters of iron metabolism in the liver: Non-heme hepatic iron (D), ferroportin mRNA (E), heme oxygenase-1 mRNA (F), thiobarbituric acid reactive substances (TBARS)(G), hepcidin mRNA (H), Smad6 mRNA (I), Smad7 mRNA (J), and ID1 mRNA (K). The proteins ferroportin (FPN), heme oxygenase (HO-1), and ferritine light chain (FTL) are expressed at higher level in mice with a Gpx4-deficient hematopoietic system (V). Parameters of splenic and peripheral iron metabolism: non-heme splenic iron (L), splenic Erfe mRNA (M), plasma iron (N), plasma ferritine (O), plasma EPO (P), plasma ERFE (Q), plasma hepcidin (R), plasma bilirubin total (S), plasma bilirubin direct (T), plasma bilirubin indirect (U). Increased hepatic non-heme iron and TBARS as well as elevated ferroportin and heme oxygenase-1 expression point to hepatic iron overload, whereas highly increased EPO and ERFE levels in the plasma and elevated Erfe mRNA expression levels are strong indicators of severe iron demand in the erythropoietic system. Note that hepcidin expression is not down regulated despite the strong erythropoietic iron demand. For HO-1/actin ratio in the liver and duodenal ferroportin expression, see Online Supplementary Figure S7A-B.

Figure 7.

Proposed model for the role of 12/15-lipoxygenase, GPX4 and vitamin E during reticulocyte maturation.