Impact of hematopoietic lineage-specific deletion of CYB5R3 on steady-state and stress erythropoiesis In Vivo

Abstract Background: Cytochrome b5 reductase 3 (CYB5R3) is a reductase that modulates oxidative signaling by reducing substrates such as heme in hemoproteins (i.e. hemoglobin, soluble guanylyl cyclase) and lipid-soluble antioxidants (i.e. vitamin E, and coenzyme Q). The CYB5R3 T117S polymorphism, which occurs almost exclusively and with high frequency (23% minor allele) in persons of African ancestry, decreases CYB5R3 activity by roughly 50%. We recently showed that hydroxyurea, which is widely used for managing anemia in sickle cell disease, is less effective at inducing fetal hemoglobin when CYB5R3 is deficient. Our in vitro data using erythroid cells demonstrates that loss of CYB5R3 tempers hemoglobin formation and erythrocyte maturation, which can be partially rescued by hemin treatment. However, a causal role for CYB5R3 in the case of in vivo erythropoiesis remains unexplored. In this study, we employed a novel mouse model with hematopoietic compartment-specific deletion of CYB5R3 to assess its effects on steady-state and stress erythropoiesis in vivo. Methodology:We crossed CYB5R3 flox/+ mice with a well-established Vav1cre strain to delete CYB5R3 throughout the hematopoietic compartment, generating CYB5R3 flox/flox;Vav1Cre knockout (KO) and CYB5R3 flox/flox control (WT) mice. Age-matched male and female KO and WT mice were analyzed for hematological parameters using peripheral blood at baseline. We then used different challenges to stimulate stress-erythropoiesis and probe the effect of CYB5R3 on recovery of blood cell levels: 1) sub-lethal irradiation (5Gy, 1 wk); 2) hypoxia (10% oxygen and 90% nitrogen, 3 wk); 3) splenectomy (2 mo); and 4) hypoxia at 2 months post-splenectomy (3 wk). Result: Baseline hematological parameters comparing CYB5R3 KO and CYB5R3 WT mice were similar. We did observe methemoglobinemia in KO vs. WT mice, a known phenotype with diminished CYB5R3 activity. Using irradiation to ablate bone marrow and peripheral mature blood cells, we found that red blood cells (RBC), hemoglobin (Hb) and hematocrit (Hct) were significantly decreased in CYB5R3 KO vs. CYB5R3 WT mice (RBC: -26.47% vs. -11.82%, P = 0.0405; Hb: -25.56% vs -12.98%, P = 0.0484; Hct: -27.77% vs. -13.74%, P = 0.0521) after 7 days. Next, we used chronic hypoxia (3 weeks) to stimulate stress erythropoiesis in bone marrow and spleen. RBCs, Hb, and Hct were significantly induced by hypoxia, with induction lower primarily in the male KO vs. WT mice (RBC: 26.59% vs. 38.69%, P = 0.0405; Hb: 32.77% vs 43.30%, P = 0.0299; Hct: 44.77% vs. 54.20%, P = 0.0288). We used splenectomy to eliminate splenic compensation and found a significant drop in the percentage of RBC, Hb, and Hct at normoxia, after 2 months of the procedure, in the KO vs. WT mice (RBC: -3.974% vs. 5.553%, P = 0.0162; Hb: -4.459% vs 3.693%, P = 0.0154; Hct: -4.025% vs. 4.194%, P = 0.0269). Splenectomy, as reported by others, also triggered leukocytosis and thrombocytosis in both groups. Lastly, splenectomized mice were subjected to chronic hypoxia (3 wk), after which hematological parameters were again measured. When comparing KO to WT mice, there was lower induction of RBC, Hb, and Hct and a greater induction of platelets (RBC: 14.37% vs. 24.21%, P = 0.0379; Hb: 20.33% vs 32.84%, P = 0.0179; Hct: 27.60% vs. 41.98%, P = 0.0218; platelets: 26.25% vs. 3.405%, P = 0.0092). When we performed sex-specific analysis, the effect seen was more severe in male KO vs. WT mice (RBC: 6.207% vs. 25.40%, P = 0.0267; Hb: 14.93% vs 38.61%, P = 0.0226; Hct: 23.30% vs. 49.17%, P = 0.0208; platelets: 21.58% vs. -4.186%, P = 0.0429), in comparison to the females. Conclusion: Our combined findings suggest that CYB5R3 in the hematopoietic compartment is required for robust stress-induced hemoglobin formation and erythroid differentiation, especially in hypoxic males. Additionally, we found that splenectomy exacerbated CYB5R3 KO effects in hypoxia-induced erythropoiesis. Overall, we conclude that CYB5R3 plays a critical role in erythropoiesis under stress conditions when compared to steady-state conditions. CYB5R3 deficiency may aggravate susceptibility to hypoxic stress (i.e. physical exertion or hemorrhage) with potentially life-threatening consequences in individuals harboring the T117S polymorphism.