Erythropoietin Exposure Impairs B Cell Development and Promotes Myeloid-Imprinted Biphenotypic Progenitors

Erythropoietin (EPO) is the major regulator of erythropoiesis, with levels increasing in response to anemia or hypoxia. Recombinant human EPO (rhEPO) is commonly used to treat anemia in patients with chronic kidney disease or cancer. Beyond its role in erythropoiesis, EPO exerts immunomodulatory effects. Emerging evidence suggests that EPO modulates other hematopoietic lineages, such as megakaryocytes while negatively impacting B-cell development. However, its influence on lympho-hematopoiesis remains insufficiently explored. We investigated the effects of EPO on B lymphopoiesis in the context of hyper-EPOemia-induced stress hematopoiesis. Using an EPO supplementation model in C57BL/6 mice, we demonstrate that EPO impairs B lymphopoiesis during Pre-Pro-B to Pro-B transition, by downregulating essential receptor like IL-7 receptor. Conversely, EPO promotes early B-cell development at the common lymphoid progenitor (CLP) and Pre-Pro-B stages. Remarkably, EPO induces the emergence of atypical B-cell precursors with a myeloid imprint, including CSF1R/CD115-expressing CLPs and CD11b and CD16/32-expressing Pre-Pro-B cells. Gene expression profiling revealed that EPO reprograms early B-cell precursors, upregulating a myeloid transcriptional signature while downregulating B-lymphoid identity genes as for instance genes targeted by key transcription factors IRF4, FOXO1, ERG and IRF8. Additionally, EPO exposure impairs hematopoietic stem and progenitor cell proliferation, induces oxidative stress and disrupts iron homeostasis. We additionally observed lymphoid over myeloid progenitor imbalance in the bone marrow and increased extramedullary hematopoiesis in the spleen. In conclusion, hyper-EPOemia reprograms B lymphopoiesis and promotes the emergence of mixed-lineage myeloid-like B cells. Future studies will explore EPO's effects on lymphoid progenitor fate under hypoxic and infectious conditions. Overall design: For each cell population, batches of 100 cells (4–5 replicates) were sorted by FACS in 96-well V-bottom plate wells containing 2 mL of lysis buffer (Ultra-pure Water, 10% Triton X-100, RNasinPlus 40Uµ/l; Promega). After evaporation of the lysis buffer at 95ºC for 3 min, first strand cDNA synthesis was performed using the Maxima H Minus Reverse Transcriptase (Thermo Fisher), E3V6NEXT primers (specifiµc primer for each well) and Template Switching Primer: E5V6NEXT. Resulting cDNAs were pooled and purified by the DNA Clean and Concentrator TM-5 kit (Zymo research) and treated with Exonuclease I (New England Biolabs) before amplification using the Advantage 2 PCR kit (Clontech) and the SINGV6 primer (95°C for 1 min, 15 cycles at 95°C for 15 s, 65°C for 30 s, 68°C for 6 min and 72°C for 10 min). PCR products were purified by Agencourt AMPure XP (Beckman Coulter). Libraries were prepared using the Illumina DNA Prep kit (Illumina) according to the manufacturer's guidelines and sequenced on a NextSeq 2000 (Illumina) at the Genome East Platform of the IGBMC (Strasbourg, France). Image analysis and base calling were performed using RTA 2.7.7 and Bcl-2fastq 2.17.1.14 software and adapter dimer reads were removed using Dimer Remover (https://sourceforge.net/projects/dimerremover/).