Aging-Related Changes in Erythropoietic Activity and Iron Metabolism in a Mouse Model of Congenital Erythrocytosis with Human Gain-of-Function Erythropoietin Receptor

We previously created and characterized a mouse model of congenital erythrocytosis with low erythropoietin (EPO) levels from a gain-of-function mutation of the human erythropoietin receptor gene (mtHEPOR) (Divoky et al. PNAS. 2001; 98:986; Divoky et al. JMM Berl. 2016; 94:597). These mice develop fetal erythrocytosis, followed by transient amelioration of erythrocytosis in perinatal life, and reappearance at 3-6 weeks of age. Similarly, erythrocytosis is observed in heterozygous mtHEPOR patients postnatally but not at birth. We previously reported dynamic changes of the erythron with iron homeostasis during ontogenesis in these mice (Kralova et al. Blood 2017; 130: 170). We observed that while perinatal mtHEPOR mice exhibit relative iron deficiency, aged mice had iron overload. Here, we evaluated developmentally-determined factors associated with hyperactivation of EPOR signaling which could cause a transition from iron deficiency (neonates) to hyperferremia and increased iron deposition (aged mice). To assess the consequences of different levels of EPOR-JAK2-STAT5 signaling, we studied hetero- and homozygous mtHEPOR mice that differ in their severity of erythrocytosis. We found that prenatally and perinatally, mtHEPOR hetero- and homozygous mice have increased erythroferrone (Erfe) transcripts and reduced hepcidin, consistent with the known inverse correlation between Erfe and hepcidin and in accordance with increased numbers of immature erythroid progenitors in the fetal hepatic circulation. At birth, previously normal Epo expression decreased and remained low in adulthood. Iron deficiency, observed in mtHEPOR hetero- and homozygotes at postnatal day 7, was likely related to increased iron consumption by augmented erythropoiesis at this stage. Postnatally, hepcidin levels increased in mutant mice, accompanied by low Erfe induction and iron accumulation in the liver and spleen as reflected by the upregulation of hepatic Bmp6 expression in mature adult (aged ~6.5 months) and old (~16 months) mtHEPOR homozygotes. We hypothesized that this could be a consequence of diminished iron consumption due to a progressive decline of erythropoiesis in mtHEPOR mice, possibly mediated by premature aging of erythroid progenitors with cell-autonomously increased proliferative history and/or increased inflammation. Indeed, young mutant erythrocytes had decreased erythrocyte survival and expression of a senescent marker CD47, an inhibitor of erythrocytes' phagocytosis. Additionally, a progressive decline in the percentage of Ter119-positive bone marrow cells and immature erythroblasts was observed in mtHEPOR hetero- and homozygotes with aging. Clonogenic assays of old mice revealed suppression of early (BFU-E) and late (CFU-E) erythroid progenitors and myeloid bias of hematopoiesis, paralleled by the up-regulation of PU.1 expression, elevation of platelet counts, and an increase in megakaryocytes chiefly in the bone marrow of mtHEPOR homozygotes. Serum levels of inflammatory cytokines did not indicate systemic inflammation; however, induced transcripts of IL-6, Inf-γ, Tgf-β, and Tnf-α, mainly in mtHEPOR homozygotes showed local bone marrow inflammatory stress. These data indicate progressive attenuation of erythroid drive in mtHEPOR homozygotes, and less so in mtHEPOR heterozygotes, paralleled by a decline in hematocrit levels with aging. In response to attenuated erythropoietic activity, iron consumption was reduced in mtHEPOR mice, leading to iron accumulation in the liver and spleen accompanied by markedly increased hepcidin synthesis. Our data suggest that even in the absence of systemic inflammation, albeit with possible paracrine inflammatory signals, known to affect bone marrow remodeling and hematopoietic aging, life-lasting prolonged activation of EPOR-JAK2-STAT5 signaling promoted exhaustion of erythroid progenitors and resulted in an age-related decline of accelerated erythropoiesis in this mouse model of congenital erythrocytosis with human gain-of-function EPOR. Grant support: Czech grant agencies projects GA17-05988S, NV19-07-00412 and LTAUSA17142, Palacky University project IGA_LF_2021_004. Disclosures No relevant conflicts of interest to declare.