| Part I:Deciphering transcriptome alterations in bone marrow hematopoiesis at single-cell resolution in immune thrombocytopeniaBackground:Primary immune thrombocytopenia(ITP)is an acquired autoimmune disorder characterized by reduction in platelet count and increase in the risk of bleeding.It is the most common hemorrhagic disease in clinic practice.Its pathogenesis has been extensively studied,with immune-mediated increase in destruction and decrease in the production of platelets as the accepted mechanisms.Platelet production is a complex biological process that involves hematopoietic stem cell commitment to the megakaryocytic lineage,megakaryocyte maturation,and platelet release.Previously,our group and others have demonstrated that antiplatelet autoantibodies,bone marrow(BM)CD8+ T cells,and tumor necrosis factor-related apoptosisinducing ligand(TRAIL)in BM plasma and megakaryocytes impair megakaryopoiesis in ITP.Recently,Herd et al.reported elevated long-term hematopoietic stem cell(LT-HSC)activation and proliferation in vitro in a murine ITP model.However,whether and how hematopoietic differentiation contributes to the pathogenesis of ITP in humans remain unclear.On the one hand,decrease in platelet count and depletion of immune cells activated hematopoiesis,while on the other hand,hematopoietic stem and progenitor cells(HSPCs)themselves could be targets of autoimmune attack because of the target antigens present on both platelets and the more immature hematopoietic progenitors.In addition,the cellular crosstalk among different subclusters of HSPCs might play critical roles in inducing defective megakaryopoiesis.However,comprehensive documentation of the underlying molecular interactions in HSPCs in ITP remains an open area of investigation.Therefore,we proposed the hypothesis that abnormalities of HSPCs in ITP BM lead to megakaryocytogenesis defects and reduced platelet production,and the examination and intervention of BM HSPCs may become a new target for the diagnosis and treatment of ITP.HSPCs are highly heterogeneous.Single cell technology,especially transcriptome sequencing and its bioinformation analysis strategies,which has incomparable advantages over traditional experimental methods,has made it convenient to directly study heterogeneous cell populations.In recent years,single-cell RNA sequencing(scRNA-seq)has played an important role in the research of stem cell differentiation,embryonic development,and tumor immunity.However,there are still no reports on scRNA-seq of BM HSPCs in ITP.It is necessary to analyze the transcriptome changes of BM HSPCs at the single-cell level and identify the potential pathogenic subpopulations and molecular mechanisms,so as to study the pathogenesis of ITP.Objective:(1)To construct the single-cell transcriptome profile of BM HSPCs in ITP.(2)To investigate the single-cell transcriptome alterations of BM HSPCs in ITP.(3)To determine the cellular and molecular basis for megakaryopoiesis defect in ITP.Methods:(1)Clinical sample collection:Iliac BM samples were obtained from newly diagnosed treatment-naive ITP patients and healthy donors.(2)Single cell RNA library preparation and sequencing:BM CD34+HSPCs were purified using fluorescence-activated cell sorting(FACS).ScRNA-seq libraries were performed using the Chromium Single Cell 3 Solution V3(10x Genomics)and sequenced using Illumina NovaSeq 6000.(3)Processing of single-cell RNA-seq data:The raw files were split using bcl2fastq,read alignment and gene expression quantification processed using Cell Ranger(10x Genomics).In order to find the changes of ITP BM CD34+HSPCs in cell subtypes,gene expression,pseudotime trajectories,transcriptional regulatory networks and cell-cell interactions,Seurat,Harmony,ClusterProfiler,GSEA,pySCENIC,Monocle2,Scanpy,CellPhoneDB and other tools were used for downstream analysis.(4)Antibodies and flow cytometry:Cell surface and intranuclear marker staining was performed according to the manufacturer’s protocol.Flow cytometric analysis(FCA)was performed using the Kaluza Analysis software.(5)In vitro culture and morphological observation:CD9+Lin-CD34+CD45RA-HSPCs,CD9-Lin-CD34+CD45RA-HSPCs,Lin-CD34+CD45RA+CD38+CD9+CD10+HSPCs,and Lin-CD34+CD45RA+CD38+CD161+HSPCs were cultured with StemSpan SFEM II medium added multi-lineage supplemented factors and serum substitute.All cultures were incubated at 37℃ in a humidified chamber in the presence of 5%carbon dioxide.Cell morphology was observed using light microscopy and immunofluorescence microscopy.(6)Statistical analysis:The chi-square(χ2)test,two-tailed Student’s t-test,variable transformation,and analysis of variance(ANOVA),followed by a Scheffe’s post-hoc test were used in R for comparing the experimental groups.Results:(1)Single-cell transcriptomes of BM CD34+HSPCs from ITP patients and healthy controlsAfter quality control and batch effect correction,56,312(ITP,n=28,507;HC,n=27,805)single-cell profiles were included in the downstream analyses.We manually annotated the cell clusters into 15 different cell types with distinct gene expression patterns.These populations included hematopoietic stem cells(HSC),multipotent progenitors(MPP),granulocytemacrophage progenitors(GMP),neutrophil progenitors(NeuP),monocytedendritic-cell progenitors(MDP),eosinophil-basophil-mast-cell progenitors(EBMP),common lymphoid progenitors(CLP),three pre-B cell populations(preB1,preB2,and preB3),natural killer/T cell progenitors(NK/Tp),megakaryocyte-erythroid progenitors(MEP),two MkP populations(MkPl and MkP2),and erythroid progenitor cell populations(EryP).We further analyzed the trajectories in ITP and HC samples separately and obtained similar transition trajectories.However,the immune and transcriptional states differed considerably between ITP and HC,suggesting that immunological interactions within HSPCs should be considered for understanding the pathogenesis of ITP.In addition,the detailed distributions of megakaryocyte and erythrocyte(Mk/Ery)lineage cells along the pseudotime were not entirely consistent,implying that the differentiation potential of HSPCs in ITP might be affected.(2)Transcriptional changes in BM CD34+HSPCs in ITPWe assessed transcriptome alterations in each HSPC subset of ITP,yielding 1,166 differentially expressed genes(DEGs)in total.Among these DEGs,the expression of 33.28%was altered in preB3 and NK/Tp,suggesting that immune cells play an important role in the pathogenesis of ITP.And further analysis revealed that the unique cell-cell interactions and dysregulation of transcriptional regulatory networks may play important roles in this process.(3)HES1 and CD9 were downregulated in ITP patientsDEG analysis indicated that the top upregulated genes in MkPl of ITP included HBB,HBG2,and AHSP,which were associated with erythropoiesis,while the top downregulated genes in MkP2 of ITP included CD9 and TUBB1,which are well-established genes related to thrombopoiesis,suggesting a bias toward erythrocytes in megakaryopoiesis in ITP.We therefore focused on DEGs that affected the differentiation potential,and HES1 and CD9 were selected for further verification.Multi-parameter flow cytometric analysis showed reduction in the number of HES1+ cells and CD9+cells in ITP,confirming the results of our single-cell transcriptome analysis.(4)CD9+Lin-CD34+CD45RA-HSPCs were biased toward megakaryopoiesis in vitro and were defective in ITPLiquid culture assays demonstrated that CD9+Lin-CD34+CD45RA-HSPCs tended to differentiate into megakaryocytes;however,this tendency was not observed in ITP patients and more erythrocytes were produced.The percentage of megakaryocytes differentiated from CD9+Lin-CD34+CD45RA-HSPCs was 3-fold higher than that of the CD9-counterparts from healthy controls,whereas in ITP patients,the percentage decreased to only 1/4th of that in the healthy controls and was comparable to that from the CD9-HSPCs,implying that differentiation of CD9+HSPCs toward the megakaryopoietic lineage was impaired in ITP.CD9 can be used to enrich Mk-biased HSPCs,and CD9+Lin-CD34+CD45RA-HSPCs have the potential to be novel diagnostic and therapeutic targets in ITP.(5)Co-culture with pre-B cells from ITP markedly decreased the generation of megakaryocytes from CD9+Lin-CD34+CD45RA-HSPCsPre-B cells from healthy donors and ITP patients were co-cultured with CD9+Lin-CD34+CD45RA-HSPCs from healthy donors.And the proportion of megakaryocytes in the CD9+Lin-CD34+CD45RA-HSPC progeny decreased significantly in the group co-cultured with pre-B cells from ITP.The data demonstrated that the defective megakaryopoiesis in ITP might be related to aberrant pre-B cells.(6)Cellular heterogeneity in MkP and transcriptional changes in MkP subclusters of ITPWe pooled MkP from all samples of ITP and HC,and these cells were further divided into seven subclusters with different gene expression patterns and functions.The ITP-associated DEGs were MkP subtype-specific,and 98.5%of the upregulated genes and 82.4%of the downregulated genes in these subtype-specific genes occurred in the subcluster possessing dual functions of immunomodulation and platelet generation,suggesting that this subcluster was most associated with ITP.We found that thrombopoiesis-related genes,such as GP9,PF4,CD9,and PPBP,were downregulated significantly in this subcluster,meanwhile,erythropoiesisrelated genes,such as HBB,HBD,CA1,and AHSP,were upregulated significantly,consistent with the previous finding in MkP1 and MkP2.Enrichment analysis revealed that platelet activation and cellular immunity pathways were significantly downregulated in this subcluster of ITP,while RNA splicing and regulation of HSC differentiation were significantly upregulated.Conclusion:Here,we provided a comprehensive single-cell transcriptomic atlas of HSPCs to characterize transcriptome alterations in newly diagnosed treatment-naive ITP.Our analysis revealed a distinct immune ecosystem in ITP BM,characterized by enhancement of immunerelated genes and pathways during hematopoiesis,abundant transcriptome changes in pre-B and NK/Tp subpopulations,activation of immune crosstalk within HSPC clusters,and weakened immunomodulatory functions of an MkP subcluster,along with downregulation of plateletgeneration potential.Functional assays demonstrated that the number of CD9+cells and HES1+ cells in Lin-CD34+CD45RA-HSPCs decreased in ITP,and the differentiation of CD9+Lin-CD34+CD45RA-HSPCs toward the megakaryopoietic lineage was impaired,which might be related to aberrant pre-B cells.In summary,this is the first study to show the significant heterogeneity and disease characteristics of BM CD34+ HSPCs from ITP patients at single-cell resolution using transcriptomic profiling,revealing new insights regarding the pathophysiology of ITP.Part Ⅱ:HDAC3 single-nucleotide polymorphism rs2530223 is associated with increased susceptibility and severity of primary immune thrombocytopeniaBackground:Primary immune thrombocytopenia(ITP)is an acquired autoimmune bleeding disorder,defined as a platelet count<100×109/L without other causes of thrombocytopenia.ITP is the most common clinical hemorrhagic disease,accounting for～30%of all hemorrhagic events and is often a chronic disorder in adults.For years,it is well noted that humoral and cellular immune responses are participating in its pathogenesis,including abnormal T cell differentiation and responses,and autoantibody secretion of plasma cells targeting megakaryocytes and platelets.Although glucocorticoids and other drugs are used to treat ITP,approximately 50%of patients fail to respond to therapy or relapse in a short term.Therefore,it is necessary to further explore the risk factors for ITP to identify novel potential therapeutic targets.The histone deacetylase(HDAC)superfamily comprises 11 HD AC isoforms encoded by the mammalian genome.We previously reported that low-dose chidamide,a selective HDAC inhibitor,restores immune tolerance in patients with ITP.In adult mammals,HDAC3 uniquely regulates environmental challenges,circadian,nutrient,metabolic pathways,and limits autoimmunity,among other homeostatic functions.HDAC3 modulates the functions of immune cells and influences cell homeostasis through systemic immune responses,thereby regulating autoimmunity and inflammation.For example,HDAC3 is an attractive protein target to mediate tolerance induction and reactivity in inflammatory macrophages,and depletion of HDAC3 in macrophages displays an anti-inflammatory histone acetylation gene expression pattern,with a protective effect against inflammation.HDAC3 deletion of T cells impaired their transition to the double-positive stage and displayed downregulation of genes for T cell function and cell cycle.Moreover,HDAC3 deficiency in Tregs perturbed the HDAC3-dependent function of immunosuppressive Tregs,which led to aggravated inflammatory bowel disease(IBD)and other lethal autoimmune outcomes in animals.Therefore,HDAC3 acts as a dynamic chromatin regulator to regulate autoimmunity,but its function in ITP remains to be elucidated.Genetic studies have revealed some genetic variations,especially single nucleotide polymorphisms(SNPs),are related to autoimmune diseases.HDAC3 rs2530223 has been implicated in diabetes mellitus and tumor immunity.Considering the vital immunomodulatory function of HDAC3,we hypothesized that HDAC3 polymorphism may play an important role in ITP pathogenesis.Objective:To investigate the association between HDA C3 rs2530223 polymorphism and the susceptibility or severity or therapeutic response of ITP in the Chinese Han population.Methods:(1)Clinical sample collection:Clinical data and peripheral venous blood samples from ITP patients were collected and stratified according to platelet counts,corticosteroid sensitivity,and refractoriness.Peripheral venous blood samples were collected from age-and sex-matched healthy donors as controls.All participants belonged to the Han population.(2)Determination of HDAC3 gene polymorphism:Peripheral blood mononuclear cells(PBMCs)were isolated from peripheral venous blood samples of ITP patients and healthy donors and genomic DNA was extracted from the PBMCs.Genotyping of the HDAC3 rs2530223 polymorphism was performed using MassARRAY platform.(3)Statistical analysis:The Hardy-Weinberg equilibrium(HWE)of HDAC3 rs2530223 genotypes was calculated in the control group.The chi-squared(χ2)test,Fisher’s exact test,and univariate binary logistic regression analyses were used in SPSS software(version 26.0)for statistical analyses.Results:(1)Study cohortNo significant difference was found in age or sex between ITP patients(n=209)and healthy donors(n=210)(p=0.25 and 0.25,respectively).75.60%of ITP patients had platelet counts<30×109/L(n=158),6.22%were refractory(n=13),and 80.86%received corticosteroid treatment(n=169),of which 86 were corticosteroid-resistant.The HDAC3 SNP rs2530223 conformed to HWE in the healthy control group(p=0.41).(2)Relationship between HDAC3 gene polymorphism and ITP susceptibilityUnder the recessive,dominant,codominant,and allelic models,the genotypic and allelic frequencies of HDAC3 rs2530223 were significantly associated with ITP susceptibility(p=0.036,0.033,0.035,and 0.009,respectively).Individuals with T allele of HDAC3 rs2530223 exhibited a 1.472-fold increased risk of ITP susceptibility(OR 1.472;95%CI 1.100-1.969;p=0.009),while ones with the TT genotype under the codominant and recessive models,and the TC/TT genotypes under the dominant model all revealed increased risk of ITP susceptibility(Dominant:OR 1.965;95%CI 1.046-3.656;p=0.036;Codominant:OR 2.264;95%CI 1.1754.360;p=0.015;Recessive:OR 1.512;95%CI 1.028-2.224;p=0.036).(3)Relationship between HDAC3 gene polymorphism and platelet countWe divided ITP patients into platelet counts<30×109/L(n=158)and platelet counts≥30×109/L(n=51)groups to evaluate the relationship between HDA C3 rs2530223 and platelet count in patients with ITP.Under dominant and codominant models,and statistical analysis revealed different genotypic frequency distributions of HDAC3 rs2530223 between the two groups,suggesting a significant relation with platelet count in patients with ITP(p=0.008 and 0.030,respectively).However,there was no significant difference in the recessive and allele models of HDAC3 rs2530223(p=0.448 and 0.068,respectively).Under the codominant and dominant models,the TC/TT genotypes of HDAC3 rs2530223 are relevant to the in increased risk of P platelet counts<30×109/L(OR 3.932;95%CI 1.426-10.842;p=0.008).(4)Relationship between HDA C3 gene polymorphism and corticosteroid sensitivityWe stratified the patients with corticosteroid therapy into a corticosteroid-sensitive group and a corticosteroid-resistant group(n=83 and 86,respectively),to explore the association between HDAC3 rs2530223 and the susceptibility to corticosteroid treatment.However,there was no significant difference in the allelic or genotypic frequencies of HDAC3 rs2530223 between the corticosteroid-sensitive group and the corticosteroid-resistant group(recessive,p=0.494;dominant,p=0.169;codominant,p=0.110;allele,p=0.968).(5)Relationship between HDAC3 gene polymorphism and ITP refractorinessWe further assigned those patients who responded to medical treatment and splenectomy to the non-refractory group,and the others were assigned to the refractory group(n=196 and 13,respectively).Neither allelic nor genotypic frequencies of HDAC3 rs2530223 were significantly different between the two groups(recessive,p=0.871;dominant,p=0.606;codominant,p=0.753;allele,p=0.547).Conclusion:The results revealed that HDAC3 single-nucleotide polymorphism rs2530223 was associated with increased susceptibility and severity of ITP,but not with refractoriness or corticosteroid sensitivity.HDAC3 may be involved in the molecular pathogenesis of ITP. |
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