Sulfated Colominic Acid Enhances CXCR4 Function and Promotes Hematopoietic Stem /Progenitor Egress from Bone Marrow.
Authors: Kubonishi Shiro , Katayama Yoshio , Yamaguchi Shinya , Tamamura Hirokazu , Fujii Nobutaka , Watanabe Takeshi , Arenzana-Seisdedos Fernando , Ikeda Kazuma , Tanimoto Mitsune 
Published: April 2020

Abstract
Hematopoietic progenitors (HPCs) can be mobilized from bone marrow (BM) compartment to the blood by G-CSF. In this process, CXCR4, a cognitive receptor for chemokine SDF-1/CXCL12, and CD26/dipeptidyl peptidase (DPP) IV play critical roles. Colominic acid (CA) is a polymer of N-acetylneuraminic acid, and it has been reported that sulfated colominic acid (SCA) can inhibit HIV entry, the step which requires CXCR4 and CD26 as co-receptors. Thus, we hypothesized that SCA would modulate HPC egress from BM. First, we injected SCA into C57BL/6 mice (100mg/kg, i.v.) and observed rapid HPC mobilization (assessed by CFU-Cs/mL blood, PBS 42±10, SCA 215±50, n=9–12, p&lt;0.01, peaked at 30 min after injection). CA displayed no mobilization, suggesting that the sulfation is critical for its action. G-CSF-induced HPC mobilization was strongly enhanced by the addition of single dose of SCA (CFU-Cs/ml blood, PBS vs SCA: 225±57 vs 1950±485 in 2 day-G-CSF, n=5, p&lt;0.01; 1950±400 vs 8233±1225 in 4 day-G-CSF, respectively, n=6, p&lt;0.001). To evaluate the stem cell activity in mobilized blood by 2 day-G-CSF+SCA, we mixed the same volume of blood from CD45.2 mice treated with 2 day-G-CSF+SCA and CD45.1 mice treated with 4 day-G-CSF, and injected into lethally irradiated CD45.1 mice. The ratio of CFU-C injected (2 day-G-CSF+SCA: 4 day-G-CSF) was 1.12±0.03 and the ratio of peripheral blood leukocytes derived from engrafted stem cells originated from each donor 12 weeks after transplantation was 2.16±0.54, n=3, p=0.11). These results suggest that 2 day administration of G-CSF together with a single dose of SCA may be sufficient for clinical mobilization and that the addition of SCA in full-term G-CSF administration may overcome poor mobilization cases. To elucidate the mechanism of SCA action on HPCs, we first focused on CD26/DPPIV. Pretreatment of mice with a DPPIV inhibitor Diprotin A strongly inhibited the increase of leukocytes but did not alter HPC mobilization. In addition, SCA-induced mobilization in CD26-deficient mice was similar to that in wild-type (WT) controls (WT vs CD26−/−: 356±80 vs 444±86 CFU-Cs/ml blood, n=4). These results suggest that, in contrast to G-CSF-induced mobilization, CD26 is not important for SCA action. We then next evaluated the functional alteration of CXCR4 by SCA. Flow cytometric analyses revealed that SDF-1 bound more efficiently (2.5 times) to HPC cell line FDCP-mix treated with SCA compared to those treated with PBS (n=3, p&lt;0.01). Strikingly, SCA treatment strongly (4.3 times) enhanced FDCP-mix transwell migration toward SDF-1 (n=8, p&lt;0.05) which was completely blocked by a CXCR4 inhibitor. SDF-1 ELISA with K15C as a capture antibody revealed that non-truncated (active) form of SDF-1 was increased 5 times in serum after SCA injection (n=5, p&lt;0.0001) whereas no change was observed in BM extracellular fluid. Together, we propose that SCA induces HPC mobilization by enhancing CXCR4 function and by altering the gradient of its ligand, active SDF-1, toward circulation.