The PI3K–AKT pathway mediates the imbalance of bone marrow macrophage polarization in patients with immune thrombocytopenia

BACKGROUND: Immune thrombocytopenia (ITP), characterized by accelerated destruction and insufficient production of platelets, is commonly thought to be an immune-mediated disorder. The unsatisfactory treatment outcomes associated with the disorder indicate that the pathogenic mechanism underlying ITP requires further investigation. M1 macrophages (MФs) have inhibitory effects on haematopoietic stem cells (HSCs) and megakaryocytes (MKs), whereas M2 MФs have opposite effects. However, whether aberrant MΦ polarization in bone marrow (BM) is involved in the occurrence of ITP remains elusive. METHODS: CRISPR/Cas9 technology was used to construct BM MФ-specific PI3K-knockout mice, and RNA sequencing (RNA-seq) was performed on the sorted MKs. The functions of BM MKs were evaluated by apoptosis, platelet production, and the percentage of MK ploidy. M1/M2 MΦ polarization; the PI3K–AKT pathway in MΦs; the phagocytosis, migration, HSC-supporting and MK-supporting abilities of BM MΦs were evaluated. The cytokines in the BM plasma or culture supernatant were detected by ELISA. RESULTS: Compared with control mice, BM MФ-specific PI3K-knockout mice manifested an abnormal M1/M2 ratio and a sharp decrease in megakaryopoiesis and platelet production. RNA-seq indicated that the activity levels of the platelet activation pathway and its initiator (vWF-GPIb) were decreased in the MKs of the PI3K-knockout mice. The results were subsequently validated in clinical cohorts. Compared with those in healthy controls (HCs), impaired megakaryopoiesis and increased BM M1/M2 ratio with decreased levels of p-PI3K and p-AKT in MФs were observed in patients with ITP. Moreover, dysfunctional BM-MФs from patients with ITP could be restored by TPO in vitro. Mechanistically, vascular endothelial growth factor (VEGF) seemed to be the pivotal mediator in the crosstalk whereby MФs influenced MK maturation by increasing vWF secretion. CONCLUSIONS: Our findings revealed that increased BM M1/M2 MФ ratio with downregulation of the PI3K–AKT pathway is associated with impaired haematopoiesis, which may be related to the occurrence of ITP. Moreover, we found that TPO improved the functions of BM MФs from patients with ITP by restoring MФ polarization in vitro, which may partially explain why patients with ITP respond better to corticosteroid combined with TPO therapy.