Multicellular Derived S100A8/A9 Maintains Megakaryocytes in an Immature State in Immune Thrombocytopenia

Immune thrombocytopenia (ITP) is a common acquired autoimmune bleeding disorder characterized by reduced platelet count. Impaired maturation of megakaryocyte (MK) plays an important role, although the exact mechanisms are not fully understood. In this study, we performed 10x Genomics single-cell sequencing (SC-seq) on bone marrow samples from ITP patients and validated the data through in vitro models of megakaryocyte generation and an ITP mouse model. The results showed elevated levels of S100A8/A9 in ITP patients and it was further confirmed using RNA-seq data in the GEO database. Cell communication analysis via SC-seq revealed that neutrophils, megakaryocytes, platelets, and hematopoietic stem cells can produce S100A8/A9 and act on the TLR4 receptor of megakaryocytes. An in vitro ITP system constructed using patient plasma and primary CD34+ cells demonstrated that impaired megakaryocyte maturation and platelet reduction could be reversed by inhibiting S100A8/A9. Single-cell sequencing analysis of the in vitro system further classified megakaryocytes into four types: Immature MK, Cycling MK, Immune MK, and Thrombopoiesis-biased MK, and Pseudo-time analysis revealed S100A8/A9 decreases gradually with the maturation of megakaryocytes. High levels of S100A8/A9 were found to maintain megakaryocytes in an Immature MK state. Pathway analysis identified inhibition of the MAPK signaling pathway in the ITP group, regulated by the Raf protein, and blocking S100A8/A9 restored the MAPK pathway. Active and passive ITP mouse models treated with S100A8/A9 inhibitors demonstrated increased platelet recovery rate and the number of mature megakaryocytes in the bone marrow, highlighting the potential therapeutic value of S100A8/A9 inhibitors in ITP patients.