Low-dose Chidamide Restores Immune Tolerance In ITP By Modulating NTreg Cells And CTLA4 Expression

Primary immune thrombocytopenia(ITP)is the most common clinical autoimmune hemorrhagic disease.Immune intolerance to auto-antigens,excessive destruction of platelets mediated by humoral and cellular immunity,and insufficient production of platelets due to abnormal quantity and quality of megakaryocytes are the main pathogenesis of ITP.Natural CD4+CD25+Foxp3+regulatory T cells(nTregs)can inhibit the proliferation and expansion of T cells,which is necessary to maintain immune tolerance.The decline in the number and function of natural CD4+CD25+Foxp3’regulatory T cells plays an important role in the pathogenesis of ITP.In recent years,a large number of studies have found that histone deacetylase inhibitor(HDACi)has immunoregulatory activity.Low-dose HDACi can inhibit inflammatory reaction in juvenile idiopathic arthritis patients,inflammatory bowel disease and colitis animal models,and regulate the activity of immune cytokines.Low-dose HDACi can also up-regulate Foxp3+expression and the number and function of natural regulatory T cells so as to alleviate the response of graft-versus-host disease(GVHD).Until now,no studies have confirmed whether histone deacetylase inhibitors have the above effects in ITP.In this study,ITP active model mice were treated with low-dose HDACi,chidamide.Flow cytometry was used to detect the proportion of natural CD4+CD25+Foxp3 Treg cells in spleen cells of ITP mice model.It was found that low-dose HDACi treatment could significantly increase the platelet level in peripheral blood of ITP mice models.Further studies showed that the number and percentage of CD4+CD25+Foxp3 Treg cells in ITP mice model were significantly increased after low-dose HDACi treatment.Cytotoxic T lymphocyte associated antigen 4(CTLA4)is highly expressed on the surface of activated T-regulatory cells.It is highly homologous to CD28 and can also bind to B7.CTLA4 binds to B7 and reduces T-cell response.Therefore,CTLA4 can competently bind to B7 molecule with CD28,thereby blocking the co-stimulating pathway of T cell activation and exerting a negative regulatory effect on T cell activation.Based on previous studies,flow cytometry was used to detect the expression of CTLA4 on the surface of CD4+T cell membrane in spleen cells of ITP mice model after treatment with low-dose histone deacetylase inhibitor chidamide.The expression of CTLA4 gene in spleen cells of ITP mice model was detected by QT-PCR.It was found that the expression of CTLA4 on the surface of CD4+T cell membrane in spleen cells of ITP mice model was significantly increased by the treatment of low-dose HDACi chidamide.The results of QT-PCR showed that CTLA4 gene expression in splenocytes of active ITP mice was significantly increased after treatment with low-dose chidamide.Subsequently,peripheral blood monocytes,PBMCs were isolated from ITP patients and healthy control group,and cultured in vitro.Low-dose chidamide was added into the culture system.The effect of low-dose chidamide on CD4+CD25+Foxp3+Treg cells in PBMC culture system was detected.CD4+CD25+Treg cells and CD4+CD25-effector T cells were sorted by immunomagnetic beads isolation method.The inhibition effect of low-dose chidamide on CD4+CD25+Treg cells to the proliferation of effector T cells was detected by flow cytometry.We also studied the expression of CTLA4 in CD4+CD25+Foxp3+Treg cells of ITP patients before and after treatment with chidamide.It was found that low-dose chidamide could significantly increase the percentage and number of CD4+CD25+Foxp3 Treg cells in PBMC culture system of ITP patients.Low-dose chidamide can significantly promote the expression of CTLA4 in peripheral blood CD4+CD25+Foxp3+Treg cells of ITP patients.Low-dose chidamide can significantly increase the inhibitory effect of CD4+CD25+Treg cells on the proliferation of effector T cells,that is,to enhance the immunosuppressive effect of CD4+CD25+Treg cells.Finally,Chromatin immunoprecipitation(ChIP)technique was used to detect the acetylation level of CTLA4 genomic histone H3K27 in peripheral blood mononuclear cells of ITP patients and healthy normal controls.It was found that the acetylation level of PBMC histone H3K27 in ITP patients was significantly lower than that in normal controls.We isolated peripheral blood mononuclear cells from ITP patients,cultured in vitro,and treated with low-dose chidamide.The results showed that low-dose chidamide could significantly enhance the level of acetylated histone H3K27 in PBMC of ITP patients.To sum up,low-dose chidamide can promote the production of CD4+CD25+Foxp3+Treg cells in ITP animal model,increase the platelet level in peripheral blood of ITP animal model,promote the transformation of peripheral blood T cells to CD4 CD25+Foxp3+Treg cells,and enhance the immunosuppressive function of Treg cells in ITP patients.Further study found that ITP patients had lower acetylated histone H3K27 in CTLA4 gene PBMC than that in healthy controls.This abnormality may lead to the decrease of CTLA4 gene expression,the impairment of immune tolerance,and participated in the pathogenesis of ITP.Chidamide can up-regulate the level of acetylated histone H3K27 in CTLA4 gene in PBMC of ITP patients and induce immune tolerance.Therefore,chidamide can be used in the clinical treatment of ITP,which is expected to improve the prognosis of ITP patients.Part Ⅰ:Therapeutic Effect of Low-dose Chidamide on Active ITP Mice Model and Its Effect on CD4 CD25 Foxp3 Treg Cell Growth and CTLA4 Gene ExpressionObjectiveTo determine whether low-dose HDACi’s have therapeutic effects on ITP mice model.The number and proportion of Treg cells and the expression of CTLA4 on the surface membrane of CD4+T cell were detected to further discover the specific mechanism of histone deacetylase inhibitors on ITP treatment.Methods1.To establish active ITP mice model,the mice were then randomly divided into control group and chidamide treatment group.The control group was given intragastric administration of phosphate buffer solution(PBS)and the treatment group was given intragastric administration of chidamide(0.1mg/kg).The peripheral blood routine of mice was tested once a week.2.Four weeks later,mice were executed to prepare splenocyte suspension.Flow cytometry was used to detect the proportion of natural regulatory T cells(CD4+CD25+ Foxp3+ nTreg cells)and the expression of CTLA4 in CD4+T cells.The expression of CTLA4 mRNA in spleen cells of mice was detected by QT-PCR.Results1.Low-dose chidamide significantly increased the platelet level in peripheral blood of ITP mice model,reduced the hemorrhage-related mortality and improved the overall survival of ITP mice model.2.Low-dose chidamide significantly increased the number and proportion of natural CD4+CD25+Foxp3+regulatory T cells in spleen cells of ITP animal model.3.Low-dose chidamide significantly increased CTLA4 expression in CD4+T cells of splen ocytes of ITP mice model.4.The expression of CTLA4 gene in splenocytes of ITP mice model was significantly up-regulated by low-dose chidamide treatment.ConclusionLow-dose chidamide has a significant therapeutic effect on ITP mice.It can up-regulate the number and proportion of Treg and the expression of CTLA4,thus promote immune tolerance in ITP.Part Ⅱ Effects of Low-dose Chidamide on Treg Cell Formation,CTLA4 Expression and Immunosuppressive Function of Treg cells in ITP PatientsObjectiveTo study the effects of low-dose HDACi’s on Treg cell production and immunosuppressive function of Treg cells in PBMC culture system of peripheral blood of ITP patients.To study the expression of CTLA4 in Treg cells by low-dose chidamide treatment.Methods1.Peripheral blood of ITP patients and healthy control group was collected and PBMC cells were separated.2.Phytohemagglutinin and IL-2 were used to stimulate lymphocyte growth in vitro,and PBMC cell culture system in vitro was established3.PBMC cells from ITP patients and healthy controls were co-cultured with low-dose chidamide and collected for flow cytometry.4.Treg cells were labeled with PEcy5-CD4,FITC-CD25 and APC-Foxp3 monoclonal antibodies.Flow cytometry was used to detect the proportion of CD4+CD25+Foxp3+Treg cells in PBMC cells5.Treg cells were labeled with PEcy5-CD4,FITC-CD25,APC-Foxp3 monoclonal antibodies,CTLA4 was labeled with PE-CTLA4 monoclonal antibodies,and CTLA4 expression in CD4+CD25+Foxp3+Treg cells was detected by flow cytometry.6.CD4+CD25+Treg cells and CD4+CD25-effector T cells were sorted by immunomagnetic beads,and treated with low-dose chidamide to detect the inhibitory effect of CD4+CD25+Treg cells on the proliferation of effector T cells.Results1.Low-dose chidamide can significantly increase the proportion and number of CD4+CD25+Foxp3+Treg cells in PBMC culture system of ITP patients2.Low-dose chidamide can significantly promote the expression of CTLA4 in peripheral blood CD4+CD25+Foxp3+Treg cells of ITP patients3.Low-dose chidamide can significantly enhance the inhibitory effect of CD4+CD25+ Treg cells on the proliferation of effector T cells,that is,to enhance the immunosuppressive effect of CD4+CD25+Treg cells.ConclusionLow-dose chidamide can promote the proportion and number of CD4+CD25+Foxp3+Treg cells in ITP PBMC culture system,promote the expression of CTLA4 in ITP patients’peripheral blood CD4+CD25+Foxp3+Treg cells,and enhance the immunosuppressive effect of CD4+CD25+Treg cells to promote immune tolerance.Part III Low-Dose Chidamide Induces Immune Tolerance in ITP Patients by Modulation of Acetylation level of Histone H3K27 in CTLA4 GeneObjectiveTo study the acetylation level of CTLA4 genomic protein H3K27 in peripheral blood PBMCs of ITP patients and healthy controls.Peripheral blood PBMCs from ITP patients were isolated and cultured in vitro,and treated with low-dose chidamide.The effect of histone deacetylase inhibitor on the acetylation level of CTLA4 genomic protein H3K27 in PBMC culture system of ITP patients was detected.Methods1.Peripheral blood of ITP patients and healthy control group was collected and PBMC cells were separated.2.Chromatin immunoprecipitation(ChIP)was used to detect the acetylation level of histone H3K27 in CTLA4 gene in ITP patients and healthy control PBMC cells.3.PBMCs from ITP patients were cultured in vitro and treated with w-dose chidamide.The acetylation level of histone H3K27 in CTLA4 gene in PBMC cells before and after treatment with chidamide was detected by ChIP.Results1.ITP patients had less acetylated histone H3K27 in CTLA4 gene in PBMC cells than that of healthy controls.2.Low-dose chidamide treatment can significantly increase the level of acetylated histone H3K27 in CTLA4 gene in peripheral blood PBMC cells of ITP patients,increase the expression of CTLA4 in peripheral blood PBMC cells of ITP patients,and promote immune tolerance.Conclusion1.The acetylated histone H3K27 in CTLA4 gene in PBMC cells of ITP patients was significantly lower than that of healthy controls.This abnormality may lead to the decrease of CTLA4 gene expression level,the decrease of patients’ immune tolerance,and participate in the pathogenesis of ITP.2.Low-dose chidamide can up-regulate the acetylated histone H3K27 in CTLA4 gene in PBMC of ITP patients,correct the abnormal acetylation of CTLA4 gene histone H3K27 in ITP patients,and induce immune tolerance..3.Low-dose chidamide can be used in the clinical treatment of ITP,and it is expected to improve the prognosis of ITP patients.