Role Of SAHA In Modulating T Lymphocytes Function And The Underlying Molecular Mechanism

Objective: Histone deacetylase inhibitors(HDACI) are a class of chemicals which modulat gene expression at chromatin level.The genes regulated by HDACi are associated with cell cycle arrest,cell differentiation and apoptosis.HDACI have been demonstrated to have anti-tumor activity in multiple organic and hematological tumors.Thus,research of HDACI emphasize on treatment of tumors.Most recently,HDACI have been shown to have a serious of immunoregulatory activity,such as amelioration of symptom in autoimmune disease models, regulating function of inherent immunity and inhibition of proinflammatory cytokine.In view of the key role in immune response, T lymphocytes have been shown to have strong modulatory effects in Inflammatory and immune diseases and transplantation immunity.However,reports which aim at the effects of HDACI on T lymphocytes function and transplantation immunity are rarely seen.The exact molecular mechanism is remain unclear.In this report,the effects of SAHA on T cells proliferation,activation,differentiation as well as genes expression are investigated in vitro.SAHA is applied in a mouse heterotopic cardiac transplantation modle to observe their activity against allograft rejection in vivo.The effects and exact molecular mechanism of SAHA is investigated and discussed later in this paper..Methods: (1)CD4⁺ and CD8⁺ T lymohocytes were isolated and purified from C57BL/6 mouse spleens by magnetic activated cell sorting.T cells were activated by concanavalin A or plate-bounded anti-CD3 plus CD28 in the presence of varying concentrations of SAHA. Tritiated thymide assya was employed to evaluate the inhibitory effect of SAHA on T cells proliferation after 3d cultivation. Flow cytometr was used to analyze T cells apoptisis by staining T cells with Annexin V and PI. Transcription factors expression following T cells activation such as NF-κB,NFAT and et cetera was detected by Western Blot.(2)CD4⁺T cells isolated by MACS were activated and incubated with varying concentration of SAHA.The effects of SAHA on Treg differentiaon were investigated by FCM analysis of Foxp3 protein level.Treg and Teff were isolated by MACS and activated in the presence of SAHA.FOXP3 expression was analyzed by quantitative PCR to understand whether SAHA promotes Treg expansion and conversion of Teff to Treg.FCM was used to estimated the effect of SAHA on suppression function of Treg by CFSE staining.The gene expression involved in T helper cells differention was explored by quantitative PCR. (3) CD4⁺ T cells were isolated and purified by magnetic activated cell sorting.SAHA was added in culture solution during the induction of Na?ve CD4⁺ T helper cells to Th17 cells. FCM analysis of IL-17A was used to evaluate the effect of SAHA on Th17 differentiation.Gene expression involved in Th17 differentiation was examined by quantitative PCR. (4)The ability of i.p. administered SAHA and RPM to prevent allograft rejection was assessed in a mouse heterotopic cardiac transplant model(BALB/C→C57)by allograft survival and histopathology. Intragraft proinflammatory gene expression was examined by quantitative PCR.FCM was used to analyse the percentage of Foxp3⁺T cells in CD4⁺T cells in thymus, draining lymph node and spleen with or without SAHA treatment. The effect of SAHA on conversion of CD4⁺CD25^-T cells to regulatory cells was investigated in a adoptive transfer model in vivo.Suppression function of SAHA-treated Treg was assessed by CFSE staining in vitro.(5)HDAC1-10 expression in CD4⁺CD25⁺ T cells from SAHA and DMSO treated mouse spleen was examined by quantitative PCR before or after activation.HDAC involved in T regulatory differentiation and function was picked out for further investigation.siRNA was designed to konck down HDAC expression in Jurkat T cells and gene expression associated with Treg function was evaluated by quantitative PCR.Results:(1)The ability of SAHA on T cells proliferation,activation and differentiation:SAHA inhibits CD4⁺ and CD8⁺ T cells proliferation in a dose dependent manner.CD69 expression at 2h,6h and 12h after T cell activation was not affected by SHAH treatment.However,CD25 expression as well as NF-κB was significantly suppressed in the presence of high concentration of SAHA,indicates that T cell activation was interupt in advanced stage. Proinflammatory cytokines such as IL-2,IFN-γ,IL-12,TNF-αandIL-10 were significantly blocked in SAHA-treated groups.FCM analysis revealted that SAHA induced CD4⁺T cells apoptosis in a time and dose dependent manner.(2)The effect of SAHA on Treg cells differentiation: With an increase concentration of SAHA,we found that the proportion of foxp3⁺T cells in CD4⁺ T cells significantly decreased.Similar results were ovserved in FOXP3 gene expression of CD4⁺T cells.Interestingly,0.1μM SAHA moderately increase the Foxp3⁺ populations of CD4⁺ T cells yet failed to enhance Foxp3 gene expression.Moreover, quantitative PCR analysis revealed that SAHA treatment neither favor CD4⁺CD25⁺ T cells expansion nor promote the conversion of of CD4⁺CD25-T cells to Foxp3⁺Treg. Futher detects showed that 0.1μM SAHA selectively induced apoptosis of Teff but not Treg,which may explained the promotion of Treg proportion. Although SAHA failed to induce Treg differentiation,low dose of SAHA was related with enhanced suppression function of Treg.And further detects showed that there was a direct correlation between the enhanced suppression function and CTLA-4 expression.(3) The effect of SAHA on Th17 cells differentiation: SAHA(0.1-1μM) was shown to have significant inhibitory effect on Th17 differentiation.IL-17A,IL-17F as well as STAT3 mRNA expression was suppressed by 0.1μM SAHA treatment,while RORγt expression was not affected,indicated that SAHA may inhibit Th17 differentiation through STAT3 pathway.Interestingly,compared with control group,FOXP3 expression was up-regulated in SAHA-treated Th17 cells,indicated that SAHA may modulate the balance of Treg and Th17.(4)In mouse heterotopic cardiac transplantation model, vehicle treated allografts were rejected within 7 d after transplantation.Compare with vehicle treated group, a daily dose of 50mg/kg SAHA produced MST of 16d. 0.1mg/kg RPM was considered to be subtherapeutic and produced MST of 10d. Nevertheless,when a daily dose of 50mg/kg SAHA was combined with low dose RPM, it produced a significant prolongation of graft survival compared to the vehicle.The transplanted grafts were examined histopathologically 7d after transplantation.In the vehicle control,interstitial cell infiltration was severe,and the structure of the myocardium was damaged.Administration of 50mg/kg of SAHA kept the structure of the myocardium intact but failed to suppress interstitial cell infiltration completely.However,combination of 50mg/kg SAHA and low dose RPM not only protected the structure of the myocardium but also prevented interstitial cell infiltration. Intragraft gene expression revealed that SAHA significantly blocked CD4,CD11b,IL-17,INF-γexpression.And FOXP3,CTLA-4 and IL-10 expression was up regulated by SAHA treatment.These data indicates that SAHA not only promotes the treg differentiation but also inhibits Th1 and Th17 response in vivo. (5)SAHA treatment dramatic promoted the population of CD4⁺Foxp3⁺ T cells in thymus, draining lymph nodes and spleen,indicated that SAHA favored Treg differentiation in vivo.Next,SAHA was found not to promote the conversion of CD4⁺CD25^-T cells to Foxp3⁺Treg in a adoptive transfer model,indicated that SAHA enhanced the thymic production of nature Treg.Furthermore,CD4⁺CD25⁺Treg cells freshly isolated from SAHA treated mice showed significantly enhanced suppression function. Amazingly,a daily dose of 50 mg/kg SAHA had no effect on the prolongation of MST in IL-^2-/- mice.Since IL-2 is essential for T regulatory development,these data together revealed that SAHA probably prevent allograft rejection by promoting the numer of thymic nature Treg enhancing their suppression function.(5) Quantitative PCR analysis revealed that after T cells activation,HDAC1,HDAC2,HDAC3 and HDAC7 were up regulated significantly.However,there was no difference between SAHA and DMSO treated mice,indicated that they were related with Treg cells activation.HDAC9 expression was down regulated after activation,and there was a significant deviation of HDAC9 expression between SAHA and DMSO treated Treg cells isolated from spleen,indicated that HDAC9 might be involved in Treg development.siRNA was designed to knock down of HDAC9 expression in Jurkat T cells.And down regulation of HDAC9 leaded to significantly increase of CTLA-4 and FOXP3 expression,suggested that HDAC9 plays a key role in Treg function. Conclusion: The effect of SAHA on T lymphocytes function and allograft rejection was investigated in this paper.SAHA was demonstrated to have varying modulating effect on T cells function.High concentration of SAHA was related with remarkably T cells apoptosis as well as significantly inhibitory effect on T cells prolieferation,T cells activation and multiple proinflammatory gene expression in vitro.While low concentration of SAHA was shown to enhance suppression function of Treg and modulat the balance of Treg and Th17.In vivo studies revealed that SAHA prevented allograft rejection by promoting the thymic production of nature Treg and enhancing their suppression function. And HDAC9 was probably to paly a key role in Treg function.The effect of SAHA against allograft rejection was demonstrated in this paper,and its molecular mechnism was discussed,which provide evidence for HDACI application in organ transplantation.