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Influence of chidamide on histone acetylation andactivation of CD4+and CD8+T cells in vitroObjective:Histone deacetylase inhibitor(HDACi)can increase the histone acetylation levels in human cells.The genes of HIV-1 reservoirs in resting CD4+T cells increase with high levels of histone acetylation,which activate the HIV-1 reservoirs and eliminate them to achieve functional cure of AIDS.Chidamide is the first selective HDACi subtype independently developed by China that can be orally administered.Whether chidamide can increase the expression of genes related to cell activation,and activate CD4+and CD8+T cells due to high levels of histone acetylation remain unknown.The global activation of CD4+and CD8+T cells may promote HIV-1 infection and subsequent CD4+T cells depletion to produce toxic reactions.Hence,it is imperative to evaluate the influence of chidamide on histone acetylation and activation of CD4+and CD8+T cells in HIV-1 infected individuals in order to ensure the prerequisite in functional cure of AIDS.Methods:(1)Peripheral blood mononuclear cells(PBMCs)were isolated and cultured from six HIV-1 infected individuals with plasma HIV-1 RNA less than 20 copies/mL after antiretroviral therapy in vitro.(2)The same sample was classified into two groups according to the principle of self-control:Experimental and Control.The experimental group included different concentrations of chidamide(500 nmol/L,1000 nmol/L,1500 nmol/L and 2000 nmol/L),while the control group included 0.02%DMSO.(3)The histone(H3 and H4)acetylation levels in CD4+and CD8+T cells in PBMCs were detected by flow cytometry at three time intervals:at 0 hour,after 24 hours and after 72 hours.(4)The activation markers(CD69,CD38 and HLA-DR)on CD4+and CD8+T cells in PBMCs were detected by flow cytometry at three time intervals:at 0 hour,after 24 hours and after 72 hours.(5)The cell viability was detected by MTT assay at three time intervals:after 24 hours,48 hours and 72 hours.Results:(1)The histone(H3 and H4)acetylation levels in CD4+and CD8+T cells after PBMCs were exposed to different concentrations of chidamide(500 nmol/L,1000 nmol/L,1500 nmol/L and 2000 nmol/L)in the experimental group were significantly higher than those in the control group at 24 and 72 hours respectively(P<0.05).However,in the experimental group,there were no significant differences in histone(H3 and H4)acetylation levels in CD4+and CD8+T cells between different concentrations of chidamide(P>0.05).(2)There were no statistically significant changes in activation markers(CD69,CD38 and HLA-DR)on CD4+T cells between the experimental group and the control group after PBMCs were exposed to different concentrations of chidamide(500 nmol/L,1000 nmol/L,1500 nmol/L and 2000 nmol/L)at 24 and 72 hours(P>0.05).But the activation markers(CD38 and HLA-DR)on CD8+T cells after PBMCs were exposed to 1500 nmol/L and 2000 nmol/L chidamide in the experimental group were significantly higher than those in the control group at 72 hours(P<0.05).(3)On exposure to 500 nmol/L chidamide for 24 hours,48 hours and 72 hours,the cell viability was more than 90%,and there were no significant differences in cell viability at three time intervals(P>0.05).Conclusion:(1)Chidamide may have the ability to activate the HIV-1 reservoirs in vitro.(2)500 nmol/L chidamide does not cause the toxicity of abnormal immune activation in vitro.Part ? Study on the activation of HIV-1 reservoirs inPBMCs by chidamide in vitroObjective:Different concentrations of chidamide(500 nmol/L,1000 nmol/L,1500 nmol/L and 2000 nmol/L)can increase the histone(H3 and H4)acetylation levels in CD4+and CD8+ T cells in HIV-1 infected individuals in vitro,but 500 nmol/L chidamide does not cause the abnormal immune activation on CD4+and CD8+T cell and the cytotoxicity is relatively small.Thus,500 nmol/L chidamide not only has the potential ability to activate the HIV-1 reservoirs in vitro,but also has relatively good safety.But the histone(H3 and H4)acetylation levels in PBMCs can be effectively increased by chidamide in vitro,and then activate the HIV-1 reservoirs in PBMCs remain unknown.In this part,we will increase the samples and choose a lower concentration(250 noml/L)of chidamide to further explore whether the histone(H3 and H4)acetylation levels in PBMCs in HIV-1 infected individuals can be increased in vitro,and the HIV-1 reservoirs can be activated with the high levels of histone acetylation.Therefore,it provides an important basis for further research to evaluate the activation of HIV-1 reservoirs by chidamide in vivo.Methods:(1)Peripheral blood mononuclear cells(PBMCs)were isolated and cultured from 20 HIV-1 infected individuals with plasma HIV-1 RNA less than 20 copies/mL after antiretroviral therapy in vitro.(2)The same sample was classified into two groups according to the principle of self-control:Experimental and Control.The experimental group included 250 nmol/L and 500 nmol/L chidamide,while the control group included 0.02%DMSO.(3)The histone(H3 and H4)acetylation levels in PBMCs were detected by flow cytometry at two time intervals:at 0 hour and after 72 hours.(4)The HIV-1 DNA in PBMCs were detected by PCR-fluorescence probe method at 72 hours.Results:(1)The histone(H3 and H4)acetylation levels after PBMCs were exposed to 250 nmol/L and 500 nmol/L chidamide were significantly higher than those in the control group at 72 hours respectively(P<0.05).However,there were no significant differences in histone(H3 and H4)acetylation levels in PBMCs between 250 nmol/L and 500 nmol/L chidamide(P>0.05).(2)At 72 hours,compared with the control group,50%(10/20)of HIV-1 DNA in PBMCs decreased in 250 nmol/L chidamide group;55%(11/20)of HIV-1 DNA in PBMCs decreased in 500 nmol/L chidamide group.The percentage of decreasing numbers of HIV-1 DNA in PBMCs in 500 nmol/L chidamide group was higher than that in 250 nmol/L chidamide group.But there were no significant differences between the two groups(P=1.000).Conclusion:Chidamide may activate and reduce the HIV-1 reservoirs in PBMCs in vitro.Part ? Study on the activation of HIV-1 reservoirs inPBMCs and resting CD4+T cells by chidamide in vivoObjective:Chidamide may activate and reduce the HIV-1 reservoirs in PBMCs while increasing histone(H3 and H4)acetylation levels in HIV-1 infected individuals in vitro.It is not clear whether chidamide can increase the histone(H3 and H4)acetylation levels in HIV-1 infected individuals in vivo,and then activate the HIV-1 reservoirs.Evaluating the above roles of chidamide and its influence on virological and immunological indicators of HIV-1 infected individuals in vivo is the key to achieving functional cure of AIDS.In this part,it will provide theoretical basis and technical support for the final use of chidamide for functional cure of AIDS in vivo.Methods:(1)Fourteen HIV-1 infected individuals with plasma HIV-1 RNA less than 20 copies/mL after antiretroviral therapy were selected.(2)Participants in the study were randomly(according to 1:1)divided into chidamide group or placebo group,7 cases in each group.(3)Chidmide group:12 weeks of chidamide treatment based on current antiretroviral therapy;Placebo group:12 weeks of placebo treatment based on current antiretroviral therapy.(4)According to the follow-up plan(0,4,8,12,16 and 24 weeks),the clinical and laboratory indices were detected regularly.Results:(1)In chidamide group,the increase of histone(H3 and H4)acetylation levels in PBMCs and CD4+T cells in HIV-1 infected individuals were more obvious than those in placebo group.(2)In chidamide group,HIV-1 DNA in resting CD4+ T cells at 8 weeks and 16 weeks was lower than that at 0 weeks(baseline)(P<0.05).(3)The central memory CD4+T cells in chidamide group had a lower trend than those in placebo group.(4)The coreceptor CXCR4 on CD4+T cells in chidamide group had a lower trend than that in placebo group.(5)Chidamide had little effect on plasma HIV-1 RNA,coreceptor CCR5,suppressors(CTLA-4,PD-1 and TIM-3)on CD4+T cells,activation markers(CD69,CD38 and HLA-DR)on CD4+and CD8+T cells and Treg cells in HIV-1 infected individuals in vivo.Conclusion:(1)Chidamide may activate and reduce the HIV-1 reservoirs in resting CD4+ T cells in vivo.(2)The detection of HIV-1 reservoirs in resting CD4+T cells is more sensitive and specific than that in PBMCs.(3)Chidamide doesn't cause the toxicity of abnormal immune activation or immunosuppression of CD4+and CD8+T cells while activating the HIV-1 reservoirs in vivo. |
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