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HbF rescues dyserythropoiesis in SCD
sequestration in the pool of SCD dead cells under hypox- ia could account for the lower HSP70 nuclear content of these cells and the subsequent lower GATA-1 nuclear lev- els. As the molecular event that initiates HSP70 trapping in SCD, namely HbS polymerization, occurs in cells with high cellular concentration of HbS, required for polymer formation, our results show the absence of cell death in the early stages of differentiation where the intracellular concentration of HbS is likely insufficient to induce sick- ling. Likewise, low levels of HbF are less protective against cell death, since a minimal threshold of intracellu- lar HbF is needed for a protective polymer-inhibiting effect.41 At physiological levels of hypoxia, there is how- ever not an exclusive selection of F-cells, as significant amounts of cells with no/low HbF were found to com- plete erythroid differentiation. Further studies are needed to fully address the biological mechanisms underlying this observation and the commitment of erythroid pro- genitors to the F lineage in SCD.
Finally, this study sheds light on the importance of applying partial hypoxia during erythroid differentiation in vitro in order to mimic in vivo conditions in SCD. In our view, this together with cell proliferation and apoptosis are important parameters to consider in assessing the beneficial impact of therapeutic approaches in SCD such as HbF induction,10,20,42 anti-sickling molecules such as voxelotor43,44 or gene therapy aiming at expressing a ther- apeutic b-globin.45 In addition, specific targeting of inef- fective erythropoiesis should presumably have a major beneficial clinical impact.
In summary, our study shows that HbF has a dual ben- eficial effect in SCD by conferring a preferential survival of F-cells in the circulation and by decreasing ineffective erythropoiesis. These findings thus bring new insights into the role of HbF in modulating clinical severity of anemia in SCD by both regulating red cell production and red cell destruction.
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CD
Figure 6. Effect of fetal hemoglobin induction by pomalidomide or CRISPR/Cas9 on terminal erythroid differentiation of sickle cell disease erythroblasts. (A) Percentage of cells expressing fetal hemoglobin (F-cells) at day (D) 7 and D9 of phase II of culture of culture in patient erythroblasts under normoxia (PN), hypoxia (PH), normoxia with POM [PN(POM)] and hypoxia with POM [PH(POM)] (n=4). (B) Percentage of F-cells at D7 and D9 of phase II of culture in patient erythroblasts under normoxia (PN) and hypoxia (PH) treated with guide RNA (gRNA) targeting the LRF binding site (-197) or an unrelated locus as control (AAVS1) (n=4). Genome editing efficiency was 56.1 ± 9.6% and 79.2± 2.8% for -197 and AAVS1 samples, respectively. (C) Percentage of apoptotic cells measured by flow cytometry in PN, PH, PN(POM) and PH(POM) at D7 and D9 of phase II of culture of culture (n=4). (D) Percentage of apoptotic cells at D7 and D9 of phase II of culture in patient ery- throblasts under normoxia (PN) and hypoxia (PH) treated with gRNA targeting the LRF binding site (-197) or an unrelated locus as control (AAVS1) (n=4). Horizontal bars represent the mean of each group; *P<0.05, Mann-Whitney test (A, B and D).
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