The Role of Macrophages in Bone Marrow Injury and Hematopoietic Reconstitution

Introduction Successful homing, engrafment and effective hematopoietic recovery after hematopoietic stem cell transplantation (HSCT) are strictly regulated by various hematopoietic microenvironment cells. Increasing evidence shows that macrophages (MФs), one of the most important component niche cells are crucial for the haematopoietic regulation. MФs depletion can enhance hematopoietic stem cell mobilization. Our previous study showed that MФs ameliorate bone marrow inflammatory injury and promote hematopoiesis in mice after allo-HSCT, but its role in syngeneic HSCT and acute bone marrow injury is still unknown. Our aim is to explore the role of macrophages in acute bone marrow injury and hematopoietic reconstitution after isogenic hematopoietic stem cell transplantation and sublethal dose irradiation in vivo.

Methods BALB/c male mice at 8-10 weeks were irradiated with 60 Co 7.5 Gy and 3.0 Gy, respectively, and then isogenic hematopoietic stem cell transplantation model and sublethal-dose bone marrow injury model were constructed. The transplantation model mice were randomly divided into total body irradiation group (TBI group), bone marrow cell transplantation group (BMT group), bone marrow cell transplantation + Clodronate Liposomes injection group (BMT+Clod-Lip group), bone marrow cell transplantation + PBS Liposomes injection group ( BMT + PBS-Lip group), and normal control group (Normal group). The sublethal-dose experimental mice were randomly divided into the total body irradiation group (TBI group), the whole body irradiation + Clodronate Liposomes injection group (TBI+Clod-Lip group), the whole body irradiation + PBS Liposomes injection group (TBI+PBS-Lip group), and normal control group (Normal group). Mice in Clod-Lip group were injected with Clodronate Liposomes for several specific times to deplete macrophages until the specimens were obtained. Mice in PBS-Lip group were injected PBS Liposomes as controls.Then, the living conditions and body weight changes of the mice were observed and the survival rates of mice in different experimental groups were recorded. Peripheral blood and bone marrow in each group were collected at the corresponding detection time, blood routine analyzer was used to detect blood routine changes, HE staining was used to observe bone marrow damage, and flow cytometry was used to analyze changes in macrophages, hematopoietic stem/progenitor cells and their subgroups such as myeloid cells, megakaryocytes, and nucleated red blood cells in bone marrow.

Results Depletion of bone marrow macrophages could reduce the survival rate of hematopoietic stem cell transplantation mice. The pathological results of bone marrow showed that bone marrow injury were heaviest on the 7th day in all three transplantation groups, and then gradually alleviated. The recovery of the BMT+Clod-Lip group was inferior to that of the BMT+PBS-Lip group and the BMT group at the corresponding time point. Depletion of macrophages increased the percentage of myeloid cells in the bone marrow and the number of white blood cells in the peripheral blood, reduced the total number of bone marrow cells, the proportion of hematopoietic stem cells and megakaryocytes in the bone marrow, and delayed recovery of red blood cells and platelets in peripheral blood; Depletion of bone marrow macrophages could also reduce survival rate of sublethal dose irradiation mice, delayed the repair of pathological damage of bone marrow, and increase the proportion of progenitor cells, CMP, GMP, myeloid cells and the number of peripheral white blood cells ,increase the proportion of hematopoietic stem cell apoptosis, reduce the total number of bone marrow cells, the proportion of hematopoietic stem cells, MEP, megakaryocytes and nucleated red blood cells in the bone marrow, delayed peripheral blood recovery of red blood cells and platelets.

Conclusion In the isogenic hematopoietic stem cell transplantation model and the sublethal dose irradiation mouse model, the removal of mouse bone marrow macrophages could affect the survival rate of transplanted mice, aggravate the pathological damage of bone marrow, increase the number of GMP and white blood cells, and reduce the total number of bone marrow cells, the number of hematopoietic stem cells and MEP cells. Macrophage depletion was not conducive to the recovery of peripheral blood red blood cells and platelets.