Page 103 - Haematologica April 2020

GPX4 controls reticulocyte maturation
macrophages (BMEIM) and spleen red pulp macrophages (RPM) by flow cytometry. The number of BMEIM was slightly reduced, while the number of RPM remained unaffected in the absence of Gpx4 (Online Supplementary Figure S4A-C). Importantly, erythrocyte and reticulocyte counts were unaltered as well as hemoglobin and hemat- ocrit values (Figure 1T-W) indicating that GPX4 in macrophages plays a minor if any role in the development of the erythropenia. This strongly argues for a cell- autonomous action of GPX4 in the erythroid system.
Vitamin E-deficiency severely aggravates erythropenia caused by Gpx4-deficiency
In cell culture and in certain tissues (endothelium, T cells, hepatocytes) the phenotype of Gpx4-deficiency can be partially or completely masked by vitamin E in vivo. Vitamin E thus acts as a full or partial backup system for GPX4 in some cell types in vitro and in vivo. To exclude unintended side effects of Cre and Cre inducers or activa- tors with vitamin E deficiency, Gpx4 was deleted prior to vitamin E deprivation. Feeding the vitamin E-depleted diet for three to four weeks to mice with Gpx4-deficiency severely aggravated erythropenia. Despite the strong decrease in RBC count as well as hemoglobin and hemat- ocrit levels, reticulocyte counts were increased only to a small and non-significant extent (Figure 1I-M). This indi- cates that the anemia caused by Gpx4-deficiency could no longer be compensated by increased production of reticu- locytes when the dietary vitamin E level was reduced. In wt control mice, dietary vitamin E deprivation had no impact on RBC parameters (Online Supplementary Figure S5). An increase of the vitamin E (i.e. α-tocopherol) con- centration to the 5-fold level in the diet had no impact on erythrocyte counts, hemoglobin and hematocrit values but led to a significant decrease in reticulocyte counts (Figure 1N-R) corroborating the fact that reticulocyte counts respond more sensitively to external factors with impact on erythropoiesis than do other RBC parameters.
Ineffective erythropoiesis in mice with Gpx4-deficient hematopoietic cells
To get deeper insight into the dynamics of Gpx4-defi- cient erythropoiesis, we quantified the percentage of ery- throid precursor cells in the BM and spleen by fluores- cence-activated cell sorting (FACS) analysis (Figure 2) and calculated the total numbers of erythroid precursors in these organs (Figure 3A-B). Cell suspensions of BM and spleen were stained with antibodies for CD44 or CD71 and Ter119. Erythroid precursor cells were classified into proerythroblasts based on the expression level of CD44 or CD71 and Ter119. These analyses revealed a marked increase in extramedullary erythropoiesis in the spleen of mice with Gpx4-deficient hematopoiesis (Figure 2H,K), and a pronounced shift towards immature precursors cells in the BM and spleen when vitamin E was additionally depleted (Figure 2 F,L). The dynamic changes became more apparent when the total numbers of erythroid pre- cursors were calculated under the different conditions. In mice with Gpx4-deficient hematopoietic cells the total number of proerythroblasts increased by a factor of 1.7- fold under a normal diet, and to about 4-fold when vita- min E was additionally depleted (Figure 3F). This increase was due to an increase of proerythroblasts in the spleen (Figure 3H). The number of erythroblasts in the spleen of these mice also increased significantly, and again to a
much higher extent when vitamin E was depleted (Figure 3K). Under both conditions of Gpx4-deficiency (normal versus vitamin E-depleted diet) the number of erythrob- lasts in the spleen increased at the expense of erythrob- lasts in the BM leaving the total number of erythroblasts virtually unchanged (Figure 3 I-K).
The ratio of erythrocytes to proerythroblasts illustrates the efficacy of erythropoiesis. It decreased from about 3400 in wt mice to 1340 in mice with Gpx4-deficient hematopoiesis (Figure 3C-D). While the total number of reticulocytes increased by almost 3-fold and the ratio of reticulocytes to total proerythroblasts increased from 140 in wt mice to 240 in mice with Gpx4-deficient hematopoiesis, RBC counts decreased to 65% (Figure 3L- M) indicating that maturation of reticulocytes to erythro- cytes is defective under Gpx4-deficiency.
Under combined Gpx4- and vitamin E-deficiency, the ratio of erythrocytes to proerythroblasts decreased to about 250 (Figure 3E). Erythrocyte counts dropped to less than 30% (compared to wt) (Figure 3M), while the num- ber of reticulocyte counts increased only by about 1.3 fold (Figure 3L). The only moderate and non-significant increase in reticulocyte counts is in stark contrast to the severe anemia under these conditions. The ratio of reticu- locytes to total proerythroblasts decreased from 240 in mice with Gpx4-deficient hematopoiesis to 45 in mice with combined Gpx4- and vitamin E-deficiency suggest- ing that erythroid progenitor cells were lost during differ- entiation from proerythroblasts to reticulocytes, in addi- tion to the reticulocyte maturation defect observed under these conditions. Mice with Gpx4-deficiency in hematopoietic cells (on a normal diet) showed an increase in the size of the red pulp and the number of Ter119-pos- itive cells (Figure 4B,E) corroborating data showing inef- fective erythropoiesis. Yet, iron deposits in the red pulp stemming from physiological RBC turnover diminished (Figure 4B), arguing against hemolysis as the cause of ane- mia. Upon Gpx4-deficiency and vitamin E depletion, the white pulp dispersed to a large extent and was intermin- gled with Ter119-positive erythroid cells (Figure 4C,F).
Impaired reticulocyte maturation and increased lipid peroxidation in Gpx4-deficient erythroid cells and aggravation of the phenotype by vitamin E-depletion
Next, we attempted to define the maturation state of the reticulocytes in the anemic mice. To this end, periph- eral blood cells were stained with CD71, Ter119, Mitotracker Deep Red, and thiazol orange. Representative examples of mice are depicted in Figure 5A-C (quantifica- tion of total numbers of mature, immature and highly immature reticulocytes in the Online Supplementary Figure S6A-F). Deletion of Gpx4 increased not only the total number of reticulocytes, but also that of each fraction: 2.1- fold of mature, 3.5-fold of immature, and 4.2-fold of high- ly immature reticulocytes (Online Supplementary Figure S6D-F). The differentially higher increase in more imma- ture reticulocytes led to a general shift towards more immature reticulocytes. Under combined Gpx4 and vita- min E deficiency, the number of highly immature reticulo- cytes increased 7-fold, while the total number of mature reticulocytes decreased by 20%. The failure of reticulo- cytes to undergo maturation was associated with an increase in lipid peroxidation of reticulocytes and erythro- cytes as revealed by Bodipy 581/591-C11-staining. Lipid peroxidation was particularly pronounced in immature
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