Background: Anemia is a frequent diagnosis in premature and critically ill neonates, and anemic hypoxia is known to regulate erythropoiesis. However, the role of phlebotomy-induced anemia (PIA) associated hypoxia on erythropoiesis in neonatal mouse developing liver is unclear. In this study, we are investigating the effects of PIA on hypoxia in the liver and its erythropoiesis response. Objective: Investigate the hypothesis that severe anemia in neonatal murine pups can cause chronic hypoxia and alters the erythropoietic niche in the liver. Design/Methods: C57BL/6 pups were rendered in 2 groups (n=6 each): (1) naïve controls; (2) severely anemic (phlebotomy P2,4,6,8,10; -hct 20-24%). On P11, the liver tissue was extracted and used for single-cell RNA sequence analysis, mRNA analysis, protein analysis, and flow cytometry. For scRNA-seq analysis, liver tissues from both groups were processed to generate stable cDNA libraries (10x Genomics), sequenced on a NextSeq500 (Illumina) then analyzed. CD71+ erythroid cells were separated from liver tissue and measured the mRNA and protein expression levels of HIF-1α, HIF-2 α, HIF-3α, hemoglobin genes (HBB-b1, HBB-b2, HBA-a1, HBA-a2 and HBB-Y. Liver-derived cells were stained with antibodies against Lineage, CD45, TER119, CD71 and cells were acquired by BD LSRII-flow cytometry followed by FlowJo for gating the erythroid cells. Results: The scRNA-UMAP showed that PIA enriched the numbers of GYPA+ erythroid cells cluster with increased expression of hemoglobin chain DEGs (HBB-b1, HBB-b2, HBA-a1, HBA-a2) in severe anemic murine neonatal liver than control. Anemic-liver-derived CD71+ erythroid cells showed significantly increased mRNA and protein expression HIF-3α with decreased HIF-1α and HIF-2 expression in comparison to control indicating that PIA may induce chronic hypoxia in these cells. The mRNA and protein expression of hemoglobin genes (HBB-b1, HBB-b2, HBA-a1, HBA-a2) also significantly increased in anemic-liver-derived erythroid cells indicating that PIA may induce the hemoglobin synthesis. Consistent with scRNA analysis, flow cytometry also showed higher frequencies (%) of CD71+ erythroid cells were significantly increased in anemic liver, and flow gating with TER119 indicates the presence of immature erythrocytes than mature RBC, showing that neonatal anemic hypoxia triggers stressed erythropoiesis with deficiency of maturation.
Conclusion(s): Severe anemia-associated chronic hypoxia state in the liver-derived erythroid cells leads to increased expression of HIF-3α and alters the erythropoietic niche with increased production of erythroid cells with maturation deficits.
