S100A8/A9-mediated Disruption Of The Bone Marrow Microenvironment In Immune Thrombocytopenia

BackgroundImmune thrombocytopenia(ITP)is a prevalent acquired autoimmune bleeding disorder,comprising one-third of all bleeding disorders.Current research suggests that the pathophysiology of ITP is primarily attributed to the impaired maturation of megakaryocytes and an increased destruction of platelets.However,the exact mechanisms responsible for these phenomena are not yet fully understood.MethodsWe investigated the molecules that induce ITP by utilizing 10x single cell sequencing analysis on the bone marrow of ITP patients.We collected a total of 48 samples of ITP patients and 20 samples of normal controls to detect the S100A8/A9 level and verify their correlation with ITP.Additionally,we collected RNA-seq data of ITP from two centers through the Gene Expression Omnibus(GEO)database and reanalyzed the data to further validate the molecules’role in ITP.To conduct in vitro experiments,we constructed an in vitro model of megakaryopoiesis by incubating stem cells with ITP plasma,interfering with target genes,labeling CD41~+,CD42~+,CD61~+using flow cytometry,and measuring ploidy number and plate production to verify the effect of target genes on megakaryopoiesis.We also performed single cell sequencing of the in vitro system to analyze the pathways affecting the inhibition of megakaryopoiesis in megakaryocyte precursor cells and hematopoietic stem cells.These findings were further validated through Western-Blot analyses in megakaryocyte lines and primary systems.Finally,we constructed both active and passive ITP models to validate the role of target genes in vivo.We analyzed possible intercellular interactions by single-cell sequencing to refine our understanding of the underlying mechanisms.ResultsOur single-cell sequencing analysis of bone marrow samples from patients with ITP revealed elevated expression levels of S100A8/A9 in megakaryocyte precursor cells and stem cells.Additionally,we performed a secondary analysis of RNA-seq data from two foreign centers by searching the GEO database(GSE23754、GSE46922、GSE23754)and found that S100A8/A9 levels were increased in patients with ITP.Quantitative PCR(q PCR)analysis revealed that both S100A8(1.867 vs.0.988,P<0.01)and S100A9(1.978 vs.1.024,P<0.01)m RNA levels were significantly elevated in ITP patients compared to healthy controls.Concurrently,we assessed the S100A8/A9 protein concentrations in plasma samples using enzyme-linked immunosorbent assay(ELISA)and discovered that S100A8/A9 protein levels were also markedly higher in ITP patients relative to healthy controls(423.4 vs.220.5,P<0.01).To investigate the effect of S100A8/A9 on megakaryocyte maturation and platelet production,we performed in vitro studies using plasma from ITP patients.We observed a decrease in the number of ploidy and CD41~+,CD42~+,CD61~+cells,platelet production,and megakaryocyte maturation in the experimental group,which was reversed using S100A8/A9 inhibitors.Single cell sequencing analysis of the experimental group of megakaryocyte precursor cells revealed inhibition of the MAPK pathway.To validate this finding,we performed Western blot analysis after blocking S100A8/A9 levels in primary megakaryocyte systems and cell lines,which led to MAPK pathway reactivation.Our results suggest that the MAPK pathway may regulate megakaryocyte maturation in the ITP state via S100A8/A9.To further investigate the therapeutic potential of S100A8/A9 inhibitors,we constructed both active and passive ITP models in mice.Treatment with S100A8/A9 inhibitors led to a significant increase in the rate of platelet recovery compared to controls.Immuno-fluorescence and pathological HE is staining of bone marrow sections from mice also showed an increase in the number of mature megakaryocytes after treatment with S100A8/A9 inhibitors.Single-cell sequencing analysis of mice in the active ITP model before and after drug administration also showed that the MAPK signaling pathway was restored in mice after the application of S100A8/A9 inhibitors.ConclusionPatients diagnosed with ITP exhibit elevated levels of S100A8/A9.This protein complex may impede megakaryocyte maturation and consequently contribute to the development of ITP via the inhibition of the MAPK pathway.Thus,S100A8/A9 inhibitors have the potential to serve as a therapeutic agent for the treatment of ITP.