PI3K-AKT Pathway Mediates the Imbalance of Macrophage Polarization in Immune Thrombocytopenia Patients

Background: Immune thrombocytopenia (ITP) is one of the hemorrhagic diseases that pose serious health hazards to humans. Contributing to impaired platelet production, defective megakaryocytopoiesis is considered to be impacted by components in the bone marrow (BM) microenvironment. Our serial studies have identified that M1 MФs and M2 MФs exerted opposing effects on hematopoiesis and PI3K-AKT pathway was essential for M2 MФs to support megakaryopoiesis (2018BJH, 2021STTT). Thrombopoietin (TPO), a critical cytokine for HSCs maintenance and megakaryopoiesis, is thought to be related to multiple signaling pathways throughout the process of thrombopoiesis, including PI3K-AKT pathway. However, the role of PI3K-AKT pathway-mediated BM MΦ polarization in regulating megakaryopoiesis and approach for regulating MΦ polarization in ITP patients remains to be further elucidated.

Aims: To investigate the effect of PI3K in BM MФs on megakaryopoiesis and platelet production in vivo. Moreover, to determine the status and function of M1/M2 MΦ polarization in regulating hematopoiesis in patients with ITP. Furthermore, to uncover the therapeutic potential of modulating the imbalanced polarization of MΦs in ITP patients by TPO.

Methods: CRISPR/Cas9 technology was adopted to construct BM MФ specific PI3K-knockout mice (genotype: flox/flox; lyz2-Cre) and then RNA-seq was performed on sorted MKs from PI3K-knockout mice and control mice (genotype: flox/flox), respectively. The functions of BM MKs from ITP patients and their age-matched healthy controls (HCs) were evaluated by apoptosis, platelet production and MK ploidy percentage. In order to determine the status and the roles of MΦs in ITP patients, the M1/M2 MՓ polarization state, PI3K-AKT pathway as well as the functions of BM MΦs from ITP patients and HCs were evaluated. The coculture experiments of BM HSCs and MKs with BM MΦs from HCs or ITP patients treated with or without TPO were performed, respectively.

Results: Compared with control mice, BM MФ specific PI3K-knockout mice manifested an abnormal M1/M2 ratio and a sharp decline in megakaryopoiesis and platelet production. RNA-seq indicated that the platelet activation pathway and its initiator (vWF-GP1b/GPV) was down-regulated in MKs of PI3K-knockout mice. Subsequently, the result above was validated in the clincal cohorts. Compared with HCs, impaired megakaryopoiesis and aberrant M1 (CD68+CCR2+) / M2 (CD163+CX3CR1+) polarization were observed in ITP patients. Notably, higher levels of p-PI3K and p-AKT were detected in BM MΦs of HCs. Meanwhile, dysfunctional BM-MФs with decreased migration and phagocytosis from ITP patients showed damaged hematopoiesis-supporting ability as well (decreased proliferation, increased apoptosis, and low CFU plating efficiency of CD34+ cells; elevated apoptosis, declined platelet production and polyploidy percentage of MKs). To further explore the repairing effect of TPO, the primary BM MФs from ITP patients were treated by TPO in vitro and then aforementioned functions of BM MФs were improved. One point of note, the M1/M2 ratio of TPO-treated MФs was also decreased with higher levels of p-PI3K and p-AKT.

Conclusions: The current study was an extension to our previous research (2021STTT), which demonstrated for the first time that PI3K-AKT pathway played a critical role in M2 MФs supporting megakaryopoiesis. This study further revealed that knocking out PI3K specifically on BM MФ inhibited platelet production and activation in vivo. Additionally, abnormally polarized BM MФs with impaired hematopoiesis-supporting abilities might contribute to the occurrence of ITP, which could be ameliorated by TPO. In all, our study indicated a novel mechanism that TPO might modulate MФ polarization through PI3K-AKT pathway, thereby improving thrombopoiesis in ITP patients.

Keywords: Immune thrombocytopenia, PI3K-AKT pathway, MΦ polarization, Megakaryocytopoiesis, Thrombopoietin