Steady State Differences In Metabolic Properties Of Bone Marrow Versus Spleen Erythroid Cells

Erythropoiesis in adult mice occurs principally in the bone marrow, while stress erythropoiesis is mostly localized in the spleen. Although both bone marrow and spleen produce fully functional mature red blood cells, we noticed that the rate of maturation of erythroid precursors (based on the ratio of mature/immature erythroid cells) is over 10 times higher in the spleen as compared to the bone marrow in C57BL6 mice (3.7 ± 0.6 in the spleen as compared to 0.4 ± 0.1 in the bone marrow, n ≥ 10 mice). Cell cycle analysis of erythroid precursors revealed that bone marrow erythroid cells cycle up to 3 times more than their spleen counterparts (88 ± 1% vs 25 ± 7% of cells in the S phase, n=3). As reactive oxygen species (ROS) influence cell cycle, we measured ROS levels by flow cytometry using the CM-H₂DCFDA probe. To our surprise, we found ROS levels to decrease (rather than increase) progressively in the bone marrow erythroid cells as they mature and accumulate hemoglobin. Interestingly, the levels of ROS were twice as high in the spleen erythroid cells as compared to erythroid cells in the bone marrow. As mitochondria are a major site of ROS production we measured mitochondrial mass by flow cytometry using Mitotracker Green. Mitochondrial mass was found to be two fold lower in the spleen erythroid cells as compared to the bone marrow. In agreement with these findings, qRT-PCR expression analysis of different antioxidant enzymes such as gluthathione peroxidases Gpx1 and Gpx4 showed higher levels in the bone marrow as compared to the spleen erythroid precursors. In particular, Gpx1 expression increased ten fold during erythroid maturation in the bone marrow while in the spleen the expression of Gpx1 did not change significantly. Together these results suggest erythroid metabolic profile is distinct in the spleen as compared to the bone marrow at the steady state. In order to compare homeostatic versus stress erythropoiesis we analyzed bone marrow and spleen from Foxo3^-/- and Th3/+ thalassemic mice, two models of ineffective erythropoiesis with different degrees of severity and splenomegaly. As anticipated Foxo3^-/- and Th3/+ erythroid precursors displayed decreased rate of maturation as compared to wild type cells in both bone marrow (0.3 ± 0.02 and 0.12 ± 0.01 for Foxo3^-/- and Th3/+ respectively, n ≥ 10) and spleen (2.1 ± 0.3 and 0.42 ± 0.1 for Foxo3^-/- and Th3/+ respectively, n ≥ 10 mice per group) and cell cycle analysis showed an increased number of cells in the S phase in both Foxo3^-/- and Th3/+ spleen erythroid cells as compared to wild type (63 ± 2% and 79 ± 2% respectively, n=3). Unexpectedly however, ROS levels in both Foxo3^-/- and Th3/+ spleen erythroid cells were decreased as compared to their wild type counterparts. The observed inverse correlation between cell cycling and ROS levels was further supported by expression analysis of Gpx1 and Gpx4, the levels of which were increased in Foxo3^-/- as compared to wild type spleen erythroid cells. Collectively our results highlight the different erythroid metabolic conditions in the bone marrow versus spleen under both homeostatic and disease states. Further investigations elucidating differences in metabolic conditions and properties of erythroid cells in bone marrow versus spleen should improve our understanding of generation of erythroid cells under stress and the production of erythroid cells in vitro.