M2 Macrophages, but Not M1 Macrophages, Support Megakaryopoiesis Via up-Regulating PI3K-AKT Pathway

Background

Megakaryopoiesis and platelets production intensely depend on bone marrow(BM) microenvironment. Our previous studies found that impaired BM microenvironment and dysfunctional megakaryopoiesis are responsible for the occurrence of prolonged isolated thrombocytopenia (PT), which is defined as the engraftment of all peripheral blood cell lines other than a platelet count less than 20×10⁹/L or a dependence on platelet transfusions for more than 60 days following allo-HSCT(BBMT 2014; BBMT 2017; Brit J Haematol 2018; Am J Hematol 2018). As an important component of the BM microenvironment, macrophages (MՓs) are heterogeneous and polarized into classically activated (M1) MՓs and alternatively activated (M2) MՓs with distinct phenotypes and function. Although inconsistent effect of BM MՓs was reported on megakaryopoiesis, the functional role of M1 and M2 MՓs and related pathway in regulating megakaryopoiesis and its effect on PT patients post-allotransplant remain to be elucidated.

Aims

To address the roles of M1 MФs and M2 MФs in regulating megakaryopoiesis as well as PI3K-AKT pathway in the process. Moreover, polarization status and the function of BM MФs in regulating megakaryopoiesis were evaluated in PT patients.

Methods

This prospective nested case-control study enrolled 12 patients with PT, 24 matched patients with good graft function (GGF), defined as persistent successful engraftment after allotransplant, and 12 healthy donors (HD). BM standard monocyte subsets and M1/M2 MՓs polarization state were analyzed by flow cytometry. To generate M1 and M2 MՓs, both primary BM MՓs and THP1 cell lines were treated with LPS and IFN-γ or with IL-4 and IL-13. The functions of BM MՓs were evaluated by migration, phagocytosis and cytokine secretion assay. The sorted CD34+ cells from HD were co-cultured with BM MՓs from PT and GGF patients or M1 and M2 MՓs respectively for megakaryopoiesis. The quantification of the megakaryocytes(MKs), MKs apoptosis, MKs polyploidy distribution, colony-forming unit MK(CFU-MK) efficiency, and platelet production were analyzed in the coculture system. To understand the underlying mechanism of MՓs polarization in regulating MKs, RNA-seq analyses were performed in BM MՓs from PT and GGF patients. In addition, M1 and M2 MՓs were treated with the chemical inhibitors and lentivirus for PI3K-AKT pathway.

Results

Elevated intermediate and non-classical monocyte subsets were found in PT patients when compared with those in GGF patients. Moreover, PT patients displayed increased M1 and reduced M2 MՓs, resulting an unbalanced M1/M2 polarization, compared with GGF and HD. BM MՓs from PT patients, with high TNF-α levels and low TGF-β levels, showed decreased megakaryopoiesis-supporting ability. No significant differences in migration and phagocytosis function of MՓs among the three groups. RNA sequencing of BM MՓs showed down-regulated PI3K-AKT pathway in MՓs of PT patients compared with GGF. Consistently, the phosphorylation levels of AKT decreased significantly in MՓs of PT patients, suggesting that PI3K-AKT pathway may functionally regulate megakaryopoiesis-supporting ability of MՓs. Subsequently, BM-M2 and THP1-M2 showed superior effect on megakaryopoiesis-supporting ability compared with BM-M1 and THP1-M1. Specifically, the BM CD34+ cells cocultured with M2 MՓs demonstrated significant increased percentages of MKs and MK polyploidy, CFU-MK efficiency, and platelet count compared with those cocultured with M1 MՓs. Preventing PI3K-AKT pathway by PI3K inhibitor or Akt inhibitor significantly reduced the megakaryopoiesis-supporting ability of M2 MՓs. Moreover, knockdown of AKT1 induced the impairment of megakaryopoiesis-supporting ability via suppressing M2 MՓs polarization, which could be attenuated by AKT1 overexpression complementarily.

Summary/Conclusion

The current study demonstrated the polarization status of MՓs modulates their ability to support megakaryopoiesis. M2 MՓs, but not M1 MՓs, support megakaryopoiesis via up-regulating PI3K-AKT pathway. Defective M2 MՓs polarization via down-regulating PI3K-AKT pathway may be responsible for the pathogenesis of PT post-allotransplant, which provides a promising therapeutic target for PT patients.