1.Multifaceted Characterization Of The Signatures Of Mesenchymal Stem/Stromal Cells In Acquired Aplastic Anemia 2.Leptin-mediated Pro-inflammatory Environment In Acquired Aplastic Anemia

Background:Although the detailed mechanism of acquired aplastic anemia(AA)remains unknown,there is a consensus that hyperactive immune response targeting hematopoietic stem/progenitor cells(HSPCs)plays a key role in the onset and development of AA.Hematological recovery from intensive immunosuppressive therapy(IST)provides powerful evidence for the immune-mediated pathogenesis.While,the "culprits" that trigger the activation of immune response are still unclear.Mesenchymal stem cells,as the main bone marrow stromal cells,have pivotal role in the immunoregulatory process.However,the precise characteristics and alternations of MSCs during acquired aplastic anemia(AA)remain obscure.Objective:To compare the biological functions and differentially expressed genes between AA-MSC and HD-MSC,and explore how AA-MSC contributed to the hyperimmune status of AA.Methods:In this study,we originally collected samples from both healthy donors(HD)and AA patients to dissect the hematological changes.To systematically evaluate the biological defects of AA-MSCs,we analyzed alterations in cellular morphology,immunophenotype,multi-lineage differentiation,cell migration,cellular apoptosis and chromosome karyocyte,together with the immunosuppressive effect on the activation,proliferation and differentiation of T lymphocytes.With the aid of RNA sequencing and bioinformatic analysis,we try to compare the differences between AA-MSCs and PHD-MSCs upon the molecular genetics,especially the immune-associated gene expression pattern.Results:Compared with HD,AA patients showed higher percentage of CD8+T cells(29.02%vs 19.68%,P<0.001),and the ratio of CD4+T cells to CD8+T cells was significantly decreased(1.43 vs 2.54,P<0.001).In addition,the contents of Th1(CD4+IFN?+IL-4-T)cells and Tc1(CD8+IFN?+IL-4-T)cells were both increased in AA patients(13.71%vs 10.76%and 25.82%vs 18.43%;P<0.001 for both comparisons).The ELISA assays indicated that the concentrates of IFN-?,TNF-?,IL-6 and IL-8 in plasma were higher in AA patients than those in HD,while the content of IL-10 was remarkedly lower in AA patients.Although with similar immunophenotype and chromosome karyotype to HD-MSCs,AA-MSCs showed distinguishable morphology and multiple distinct characteristics including impaired migration capacity,slower proliferation,increased apoptosis and extensive cell senescence.In addition,the immunosuppressive effects on lymphocytes were significantly impaired in AA-MSCs.What's more,the profile of gene expression was distinct between AA-MSC and HD-MSC by using RNA sequencing(RNA-seq).A total number of 163 genes(Fold change AA/HD>2)and 129 genes(Fold change AA/HD<-2)were highly enriched in AA and HD groups,respectively.The gene ontology(GO)analysis showed that the differentially expressed genes(DEGs)were principally associated with immunoregulation(e.g.,immune system process)and cellular process(e.g.,cell cycle,cell migration).Accordingly,biological process(metabolic pathways,cell cycle,apoptosis,etc.),MAPK signaling pathway,Wnt signaling pathway,mTOR signaling pathway and PI3K/Akt signaling pathway were enriched as well by utilizing KEGG pathway analysis.Conclusions:Herein,we systematically investigated the signatures and efficacy of MSCs to dissect the alterations occurred in AA both at the cellular and molecular levels.Differ from HD-MSCs,AA-MSCs exhibited multifaceted defects in biological characteristics and alterative molecular genetics.Our findings have provided systematic and overwhelming new evidence for the defects of AA-MSCs.Background:Acquired aplastic anemia(AA)is mainly caused by abnormal immune activation which induces the apoptosis of hematopoietic stem cells.The impaired immunoregulatory capacity of AA-derived mesenchymal stem cell(AA-MSC)may contribute to the hyperimmune status of AA.Additionally,enhanced adipogenesis of AA-MSCs which causes the "fatty marrow" indicates their functional defects in hematopoietic support.However,whether the adipokines,especially leptin,in the bone marrow environment are secondary to the abnormal immune status or directly induce the development of AA,is not known.Objective:To compare the differences of leptin concentrates among AA patients,myelodysplasia syndrome(MDS)patients and healthy donors(HDs),and explore the leptin-mediated dysfunctions of T cells in AA patients.Methods:After collecting bone marrow(BM)samples from AA patients,MDS patients and HDs,we detected the concentrates of leptin and evaluated their correlation with regulatory T cells(CD4+CD25briCD127dim).Then,we detected the relative expression of leptin receptor(Lepr)mRNA of bone marrow mononuclear cells(BMMNCs)from AA patients and HDs.We also analyzed the mean fluorescence intensity(MFI)of Lepr upon different T-cell subsets from AA patients and HD.The proliferation,activation and intracellular cytokines of T cells from AA patients and HD was measured with or without the presence of leptin.In addition,the effect of leptin on the induction of CD4+Foxp3+T cells was explored.Results:The concentrates of leptin in BM from AA was significantly higher than that in HD and MDS(13.07±2.09 ng/ml vs 3.90±0.71 ng/ml vs 4.47±0.98 ng/ml;P=0.001 and P=0.03).And,the leptin concentrates in AA patients was negatively related to the proportions of regulatory T cells(R=-0.70,P=0.005).The adipogenesis of MSC was remarkedly enhanced in AA-MSC.What's more,the relative expression of leptin mRNA together with the leptin concentrates in the culture medium were significantly higher in AA-MSC than that in HD-MSC.We also found that the relative expression of Lepr mRNA was higher in BMMNCs form AA patients than that from HD(12.21±2.30 v s 1.42±0.52,P=0.003).Furthermore,the MFI of CD8+T cells,CD4+CD25-T cells and CD4+CD25+T cells was significantly higher in AA patients(54.22±4.65 vs 40.45±3.23,P=0.02;117.40±9.83 vs 65.79±6.62,P<0.001;209.20±23.36 vs 142.10±14.61,P=0.02).In vitro,leptin could promote the proliferation and activation of CD4+and CD8+T cells from AA patients.While,leptin could only promote the expression of CD25 in CD4+and CD8+T cells and exerted no effect on the proliferation of T cells from HD.Additionally,leptin could promote the secretion of IFN-? of CD4+and CD8+T cells from both AA patients and HD,while had no effect on the expression of IL-4.Finally,leptin inhibited the induction of CD4+Foxp3+T cells from CD4+CD25-T cells.Conclusion:The enhanced adipogenesis of AA-MSC induced the fatty marrow of AA.And,excessive adipocytes in AA-BM secreted superabundant leptin which contributed to the hyperimmune status of AA.