Page 106 - Haematologica April 2020

S. Altamura et al.
of reticulocyte production exceeded the rate of reticulo- cyte maturation, a disequilibrium that resulted in ineffec- tive erythropoiesis.
Under combined Gpx4- and vitamin E-deficiency the number of proerythroblasts in the spleen was further strongly increased. Within the reticulocyte fraction, there was a pronounced shift towards highly immature reticu- locytes. Yet, production of reticulocytes did not keep up with the production of proerythroblasts as total reticulo- cyte counts were not significantly higher than in wt mice. This suggests that, in addition to the severe reticulocyte maturation defect, erythroid progenitor cells were lost during differentiation from the proerythroblast to the reticulocyte stage. Highest levels of lipid peroxidation, as assessed by C11-Bodipy(581/591)-staining, were observed in immature reticulocytes under combined Gpx4- and vitamin E-deficiency which correlates with the severity of the phenotype. Ultrastructural analysis revealed remnants of mitochondria and ribosomes in reticulocytes of wt mice and mice with Gpx4-deficient hematopoiesis, and a pro- nounced accumulation of large vacuoles containing unphagocytosed mitochondria, when dietary vitamin E was lowered. The data support the model that mitophagy is triggered by lipid oxidation that is kept in check by GPX4. Hence, loss of Gpx4, especially under vitamin E-restricted conditions, leads to uncontrolled lipid peroxi- dation and as a consequence, to severely perturbed mitophagy (Figure 7).
The anemia caused by hematopoietic Gpx4-deficiency shares a number of features with β-thalassemia, for which the term ineffective erythropoiesis has been coined: decreased RBC, elevated reticulocyte counts, overactive extramedullary erythropoiesis, elevated ery- throid progenitors, absence of hemolysis, and systemic iron overload linked to severe iron demand for the ery- throid system.51 This prompted us to study iron metabo- lism in mice with Gpx4-deficient hematopoiesis. Hematopoietic Gpx4-deficiency caused liver iron over- load and oxidative stress, elevated plasma ferritin and iron levels, parameters aggravated by vitamin E depletion. Despite signs of iron overload in the liver and plasma, there was continuous demand for iron in the erythroid system. As a consequence of the anemia, plasma EPO and ERFE levels were elevated. Likewise, Erfe splenic mRNA expression was increased, particularly when Gpx4 dele- tion and vitamin E-deficiency were combined. Since ERFE mirrors the erythropoietic activity and there is virtually no background of extramedullary erythropoiesis in the spleen of wt mice under steady state conditions, spleen Erfe mRNA emerged as the most sensitive parameter of iron demand.
Despite the dramatic increase in ERFE production in mice with combined Gpx4- and vitamin E-deficiency, hepcidin expression in the liver plasma was unchanged. This is reminiscent to what has been observed in β-tha- lassemic Th3/+ mice older than six weeks.57 Suppression of hepcidin expression is only seen when the mice are young and still loading their livers with iron. Once enough iron has been loaded, hepcidin begins to rise, driven by hepatic iron stores, despite high erythroferrone, so that older mice have high liver iron but normal hep- cidin which slows down further iron loading. Thus, hypoxia and elevated iron demand for erythropoiesis decrease hepcidin expression, whereas high plasma and hepatic iron levels counteract the response to ERFE in the
liver. An alternative explanation is that a yet-unrecog- nized factor induced in response to oxidative stress or a direct oxidative modification of ERFE counteracts the action of ERFE on hepcidin expression.
The pronounced similarity between anemia caused by hematopoietic Gpx4-deficiency and β-thalassemia raises the question whether underlying pathogenic principles are shared between both conditions. A common denom- inator is by no doubt the involvement of oxygen radicals. In case of Gpx4-deficiency, they arise from increased lipid peroxidation, in case of β-thalassemia from inappropriate folding of globin chains and hemoglobin assembly. This liberates oxygen, heme and iron thus favoring the pro- duction of oxygen radicals through non-enzymatic autox- idation. There is ample evidence in the literature that increased lipid peroxidation in RBC and decreased lipid- soluble antioxidant levels in the plasma as well as in ery- throcytes are consistent features of β-thalassemia.59-63 Administration of vitamin E normalized the plasma oxi- dant/antioxidant balance and vitamin E content of ery- throcytes, yet, the clinical benefit of vitamin E adminis- tration remained limited due to the persistence of iron- overload in affected patients.62-65 Taken together, per- turbed reticulocyte maturation through uncontrolled lipid peroxidation may be the underlying cause of ineffective erythropoiesis in both conditions.
It still remains unclear to which extent lipoxygenase- mediated enzymatic and/or non-enzymatic mechanisms contribute to lipid peroxidation in the anemia described here. Lipoxygenases lower the threshold for non-enzy- matic autoxidation29 and may be required for initiating lipid oxidation in mitochondrial membranes. In the absence of 12/15-lipoxygenase other lipoxygenases may functionally compensate for the loss of the missing enzyme. A scenario of non-enzymatic lipid peroxidation is, however, also conceivable. Once lipid peroxidation is triggered, the process may be self-sustaining due to the high concentration of iron, heme compounds and oxygen rendering enzymatic lipid peroxidation dispensable.
Another critical determinant of physiological reticulo- cyte maturation is the cholesterol concentration in the reticulocyte membrane as well as in the plasma. Holm et al. have shown in an elegant study that mice lacking apolipoprotein E and high-density lipid protein receptor I (SR- BI) are unable to expel autophagocytosed organelles and accumulate autophagolysosomes in their reticulocytes.66 These mice lack mature erythrocytes and their gas trans- port relies exclusively on reticulocytes. Remarkably, the block in terminal reticulocyte maturation is cell-non- autonomous in these mice and reversible: transfusion of these reticulocytes into wt mice or hematopoietic recon- stitution of lethally irradiated wt mice with apoliporotein E- and SR-BI-deficient BM cells completely normalized reticulocyte maturation. The phenotype could also be reversed through sequestration of free cholesterol: administration of cyclodextrin led to immediate expul- sion of stored autophagolysosomes in vivo and in vitro and normalized the phenotype. Contrary to the apolipoprotein E- and SR-BI-model of Holm et al., we are dealing in our model with a cell-autonomous effect of Gpx4-deficiency to which cell-nonautonomous factors like the plasma concentration of vitamin E also contribute to a significant extent. In both conditions, the precise molecular underpinnings underlying the defects in reticu- locyte maturation await to be elucidated.
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