Short-term fasting releases the hematopoietic brake to promote bone marrow erythropoiesis Free

Erythropoietin (EPO) and erythropoiesis-stimulating agents are primary treatments for anemia in chronic kidney disease. However, their clinical utility is significantly limited by high cost, patient compliance challenges, adverse cardiovascular effects associated with frequent injections, and inherent hyporesponsiveness. These constraints underscore an urgent need for cost-effective, safer, and more potent therapeutic alternatives. Here, we demonstrate that a short-term fasting regimen (3 days) in humans significantly increased red blood cell (RBC) counts with enhanced functionality to youthful levels, particularly in individuals with baseline cytopenia. To identify a more feasible fasting duration capable of augmenting erythropoiesis, we performed a fasting time course in mice. Remarkably, a single 6-hour fasting was sufficient to increase the production of RBCs in both young and aged mice. This effect was sustained throughout 15 days of refeeding, equivalent to approximately 2% of the mouse lifespan. Flow cytometry analysis revealed that short-term fasting selectively increased terminally differentiated erythrocytes within the bone marrow (BM), without altering splenic erythropoiesis. Flow cytometry and single-cell RNA sequencing demonstrated that short-term fasting promoted the self-renewal of BM megakaryocyte-erythroid progenitors (MEPs), accelerated erythroblast maturation (with transcriptional changes preceding surface marker alterations, confirmed by RNA velocity), and increased proportions of MEPs and downstream erythroid cells during refeeding. Hematopoietic stem cell pools and megakaryocyte lineages were not compromised. Surprisingly, short-term fasting-induced erythropoiesis occurred independently of canonical Epo-EpoR signaling or hypoxia. Transcriptomic profiling of mouse BM MEPs post-fasting revealed significant alterations in cell cycle pathways. BrdU/Ki67 assays confirmed accelerated MEP cycling without disrupting quiescence, aligning with downregulation of cell cycle inhibitory regulators. Bulk transcriptomic analysis revealed that short-term fasting profoundly downregulated the G1-S checkpoint gene Ms4a3 in MEPs. Functional studies confirmed that nutrient starvation in an in vitro mouse erythroid cell model (mouse erythroleukemia cells, MEL), or MS4A3 silencing in MEL cells, human cord blood-derived HSC erythroid progenitors (HUDEP-2), and primary mouse MEPs significantly enhanced erythroid differentiation and maturation, establishing MS4A3 downregulation as a key facilitator. Further in vivo studies showed that short-term fasting activates autophagy in mouse MEPs. The erythropoietic response to short-term fasting phenocopied rapamycin-induced effects and was abolished by pharmacological (3-methyladenine, 3-MA) or genetic (Atg5 or Atg7 knockout) inhibition of autophagy in mice. In autophagy-deficient MEPs in mice, fasting failed to modulate Ms4a3 expression or accelerate the G1-S transition. Thus, short-term fasting downregulates MS4A3 via autophagy to enhance erythropoiesis, independent of EPO elevation. Proteomic analysis in starved MEL cells ± 3-MA identified the transcription factor JunD as a potential autophagy-sensitive regulator of Ms4a3. Short-term fasting reduced JunD protein levels in wild-type mouse BM MEPs, but not in autophagy-deficient MEPs, and autophagy mediated JunD degradation. Combined Cleavage Under Targets & Tagmentation (CUT&Tag) and qPCR analyses confirmed JunD as a direct transcriptional activator of Ms4a3 in mouse MEPs. JunD silencing reduced Ms4a3 expression and promoted erythroid lineage progression in both MEL cells and primary MEPs.

In summary, we identify a novel mechanism whereby short-term fasting potently augments erythropoiesis. Short-term fasting triggers autophagy-mediated degradation of the transcription factor JunD, leading to suppression of its target, MS4A3, a critical negative regulator of erythroid maturation. Rather than activating the energy-intensive canonical EPO signaling “gas pedal”, short-term fasting disables the JunD-MS4A3 “brake” operating under steady-state conditions, enabling energy-efficient erythropoiesis. These findings reveal a non-invasive, physiological intervention to significantly improve RBC output and propose novel therapeutic strategies targeting the autophagy-JunD-MS4A3 axis for erythroid disorders, particularly EPO-insensitive anemias.