An Unbalanced Monocyte Macrophage Polarization in the Bone Marrow Microenvironment of Patients with Poor Graft Function after Allogeneic Hematopoietic Stem Cell Transplantation

Background

Poor graft function (PGF) remains a life-threatening complication following allogeneic hematopoietic stem cell transplantation (allo-HSCT), and the underlying mechanisms have not yet been elucidated. Considerable evidence from murine studies has demonstrated that effective hematopoiesis depends on the specific bone marrow (BM) microenvironment, where hematopoietic stem cells (HSCs) reside. In this regard, we previously reported that PGF patients had impaired BM endosteal cells and endothelial progenitor cells, as well as dysregulated T cell responses in the BM microenvironment, which may be involved in the occurrence of PGF (BBMT 2013; BMT2016; Oncotarget 2016; BBMT 2016; Blood 2016; J Transl Med2017). Macrophage is one of the important components of BM immune microenvironment. Meanwhile, Murine studies suggest that macrophages regulate the retention of HSCs by regulating osteoblastic cell activity and nestin-expressing cells. Increasing evidence shows that BM resident macrophages are indispensable for HSCs function and BM erythroid output. However, little is known about the quantity and function of BM macrophages and whether the unbalanced BM macrophages directly interact with HSCs in PGF patients post allo-HSCT.

Aims

To compare the number and function of BM macrophages between patients with PGF and GGF after allo-HSCT. Moreover, to investigate whether the unbalanced BM macrophages directly interact with HSCs in PGF patients.

Methods

This prospective nested case-control study enrolled 30 patients with PGF, 60 matched patients with good graft function (GGF), defined as persistent successful engraftment after allotransplant, and 30 healthy donors (HD). Standard monocyte subsets, defined by cluster of differentiation CD14 and CD16, were quantified by flow cytometry. In addition, based on the phenotype of polarized macrophages in vitro, classically activated inflammatory macrophage (M1:CD68+CCR2+) and alternatively activated anti-inflammatory macrophage (M2:CX3CR1+CD163+/CD206+) from BM samples were analyzed by flow cytometry. BM CD14+ monocytes were isolated from BM mononuclear cell (BMMNCs) and were cultured with 100 ng/ml recombinant human M-CSF for 10 days to obtain macrophages. The functions of BM derived macrophages were evaluated by migration assay and phagocytosis assay. CD34+ cells from healthy donors were co-cultured with BM derived macrophages from PGF patients, and the levels of reactive oxygen species (ROS) and apoptosis in CD34+ cells were analyzed by flow cytometry after 5 days co-culture.

Results

Alterations in standard monocyte subsets (classical, intermediate and non-classical) were found when comparing subjects among PGF, GGF and normal controls. In particular, elevated intermediate and non-classical monocyte subsets were observed in PGF patients when compared with those in GGF patients. Moreover, PGF patients displayed an imbalanced M1/M2 ratio compared with the GGF and normal controls, attributable to a reduction in M2 and an increase in M1. Dysfunctional BM derived macrophages, which were characterized by impaired proliferation, migration, phagocytosis and higher level of apoptosis, were revealed in patients with PGF. BM CD34+ cells from healthy donors were co-cultured respectively with BM derived macrophages from patients with PGF and normal controls. Apoptosis and intracellular levels of ROS were markedly increased in CD34+ bone marrow cells which were co-cultured with PGF patients' BM derived macrophages compared with normal controls.

Summary/Conclusion

In summary, the current study demonstrated the abnormal number and impaired function of BM macrophages and unbalanced BM macrophages directly interact with HSCs in PGF patients. We speculate that reduction of anti-inflammatory M2 monocytes while increase of pro-inflammatory M1 monocytes in PGF patient BM have a negative impact on HSCs in BM microenvironment. Thus, our results indicate that unbalanced BM M1/M2 and dysfunctional BM macrophages may play an important role in the pathogenesis of PGF following allo-HSCT. Therefore, it would be of value to investigate the appropriate approach to balance the M1/M2 and enhance function of BM macrophages, which may be a promising therapeutic approach for PGF patients.