SDF-1/CXCL12 Modulates Mitochondrial Respiration of Hematopoietic Stem and Progenitor Cells in a Bi-Phasic Manner

SDF-1, also known as CXCL12, is a potent and well-characterized chemokine required for the homing and engraftment of hematopoietic stem and progenitor cells (HSPCs) during hematopoietic stem cell transplantation (Sharma et al., 2011, Stem Cells Dev., 6:933) as well as HSC maintenance in the bone marrow (Greenbaum et al., 2013, Nature, 495:227). Data from our group has shown that in an SDF-1/CXCL12 transgenic mouse model, Lineage^- Sca-1⁺ c-Kit⁺ (LSK) bone marrow cells have reduced mitochondrial membrane potential versus wild-type (Mantel et al., 2010, Cell Cycle, 9:2008). These results suggest that SDF-1/CXCL12 potentially functions to keep mitochondrial respiration low in HSPCs in the bone marrow. Low mitochondrial metabolism helps to maintain low levels of reactive oxygen species (ROS), which can promote differentiation and be detrimental to HSCs in the bone marrow (Mantel, et. al., 2015, Cell 161:1553). To test whether SDF-1/CXCL12 regulates mitochondrial metabolism, we first employed the human leukemia cell line HL-60 as a model of hematopoietic progenitor cells in vitro. HL-60 cells are known to express high levels of the SDF-1/CXCL12 receptor, CXCR4. First, HL-60 cells were treated with 50ng/ml SDF-1/CXCL12 for 2 and 24 hours, respectively. After 2 and 24 hours, the oxygen consumption rates (OCR), a measure of mitochondrial respiration, were analyzed by the Seahorse Bioscience XF96 Extracellular Flux Analyzer. The OCR of HL-60 cells treated for 2 hours was significantly reduced as compared to untreated control. Conversely, cells treated with SDF-1/CXCL12 for 24 hours had significantly increased OCR versus untreated control. Also, HL-60 cells treated for 2 hours with SDF-1/CXCL12 had significantly reduced mitochondrial membrane potential, while cells treated for 24 hours had significantly increased mitochondrial membrane potential. These results suggest a novel function of SDF-1/CXCL12 in regulating mitochondrial respiration. Furthermore, after two hour of SDF-1/CXCL12 treatment, OCR associated mitochondrial ATP production is decreased versus untreated control. On the other hand 24 hours of treatment produces an increase in OCR associated mitochondrial ATP production. These results begin to establish a role for SDF-1/CXCL12 in regulating mitochondrial oxidative phosphorylation (OXPHOS) and in an SDF-1/CXCL12-CXCR4 mediated process. To begin understanding the mechanism by which SDF-1/CXCL12 regulates mitochondrial respiration, HL-60 cells were again treated for 2 and 24 hours, respectively, with 50ng/ml of SDF-1/CXCL12 and mitochondrial mass was assayed using the mitochondrial specific fluorescent dye MitoTracker Green as measured by flow cytometry. Mitochondrial mass was significantly reduced in cells treated for 2 hours while cells treated for 24 hours had significantly increased levels of mitochondrial mass, suggesting that changes in mitochondrial respiration could be accounted for by changes in mitochondrial content. Furthermore, lineage^- primary mouse bone marrow cells were treated with 50ng/ml of SDF-1/CXCL12 for 2 and 24 hours, respectively, ex vivo. The OCR of treated lineage^- cells was assayed for using the Seahorse Bioscience XF96 Extracellular Flux Analyzer. Much like the HL-60 cells, lineage^- bone marrow cells treated for 2 hours had significantly reduced OCR, while lineage^- cells treated for 24 hours had significantly increased OCR. Taken together our results suggest that SDF-1/CXCL12 functions to regulate mitochondrial respiration by regulating mitochondrial oxidative phosphorylation, ATP production and mitochondrial content in a bi-phasic manner. The in vitro data obtained from experiments with HL-60 cells and ex vivo studies with lineage^- primary bone marrow cells are first examples of a role for SDF-1/CXCL12 as a regulator of mitochondrial respiration in immature hematopoietic cells.