| Background:The aberrant expansion of Follicular helper T(Tfh)cells in systemic lupus erythematosus(SLE)is associated with abnormal Germinal center(GCs)reaction and the production of autoantibodies and plays a key role in the occurrence and development of SLE,the specific mechanism remaining unclear.It has been reported that abnormal epigenetic modification is involved in regulating abnormal proliferation and differentiation of Tfh cells in SLE patients.Our team has previously found that SIRT1,as an NAD~+dependent deacetylase,is significantly up-regulated in the CD4~+T cells from both peripheral blood of SLE patients and spleens of MRL/lpr mice.And specific inhibition of SIRT1 results in reduction of serum auto-antibodies and amelioration of lupus phenotypes in MRL/lpr mice,but its mechanism is still unclear.Objective:The purpose of this study was to explore the regulation of SIRT1 on Tfh cell proliferation and differentiation and its potential value in the therapy of SLE.Methods:(1)2m L peripheral blood was collected from each SLE patient and healthy donor.After PBMCs was isolated by density gradient centrifugation,the proportion of Tfh-like(CD4~+CXCR5~+,CD4~+CXCR5~+PD-1~+,CD4~+CXCR5~+PD-1~+BCL-6~+)cells,CD4~+PD-1~+T cells,mean fluorescence intensity(MFI)of SIRT1 and PD-1 in CD4~+T cells and CD4~+CXCR5~+T cells,as well as SIRT1-MFI and BCL-6-MFI in CD4~+CXCR5~+PD-1~+BCL-6~+T cells were detected by flow cytometry.Furtherly,the correlation of SIRT1-MFI and Tfh-like cells with different phenotypes,as well as PD-1 and BCL-6 expression were analyzed.(2)Peripheral blood was collected from healthy volunteers,and na?ve CD4~+T or Total CD4~+T cells were separated from PBMCs using Miltenyi beads.1)on day 0,3 and 5 of Tfh differentiation in vitro,the cells were collected and the expression of SIRT1 was detected by western blot.2)After transfection of na?ve CD4~+T cells with SIRT1-overexpressing lentivirus(LV-SIRT1)or blank lentivirus(LV-Ctrl)for 16 h,cells were induced under Tfh-polarizing condition for another 3days in vitro.The ratio of Tfh cells was detected by flow cytometry,and the activity of cell proliferation and proportion of apoptotic cells was detected by CCK8 kit and Annexin V/7-AAD apoptosis kit,respectively.3)After transfecting na?ve CD4~+T with SIRT1-si RNA and Ctrl-si RNA,cells treated were cultivated under the Tfh-inducing condition in vitro for2 days,the proliferation activity and apoptosis ratio of Tfh-Polarizing cells were detected by CCK8 kit and Annexin V/PI apoptosis kit,respectively.The proportion of Tfh cells was detected by flow cytometry.And the mRNA expressions of SIRT1,IL-21 and BCL-6 in cells were detected by RT-q PCR.4)After transfecting total CD4~+T with SIRT1-si RNA or Ctrl-si RNA,cells were cultured in the presence of anti-CD3/CD28 in vitro for 2 days.The proportion of Tfh cells was detected by flow cytometry.The mRNA expressions of SIRT1,IL-21 and BCL-6 in cells were detected by RT-q PCR.(3)Peripheral blood was collected from healthy volunteers,and na?ve CD4~+T or Total CD4~+T cells were separated from PBMCs using Miltenyi beads.1)In the presence of EX527 or DMSO,na?ve CD4~+T cells were cultured under the Tfh-inducing condition for 72h and Total CD4~+T cells were stimulated and activated with anti-CD3/CD28 for 48h in vitro.Flow cytometry was used to detect the proportion of Tfh or Tfh-like cells.The mRNA expression of IL-21 and BCL-6 were detected by RT-q PCR.2)In the presence of NAM or PBS,na?ve CD4~+T cells were cultured under the Tfh-inducing condition for 72h and Total CD4~+T cells were stimulated and activated with anti-CD3/CD28 for 48h in vitro.Flow cytometry was used to detect the proportion of Tfh or Tfh-like cells.The mRNA expressions of IL-21 and BCL-6 were detected by RT-q PCR.3)After stimulation and activation with anti-CD3/CD28 in the presence of PBS,EX527,NAM,EX572+NAM or SRT1720+NAM for 48 h,the proportion of Tfh-like cells in total CD4~+T cells were detected by Flow cytometry.(4)Peripheral blood was drawn from SLE patients after the informed consent was signed.Total CD4~+T cells were separated from PBMC using magnetic beads.The proportion of Tfh-like cells was detected by flow cytometry after stimulated with anti-CD3/CD28 for 48 h in vitro under the treatment of NAM or PBS.(5)Spleen were isolated from MRL/LPR mice aged 12-16 weeks to obtain splenic cells,and CD4~+T cells were sorted by Miltenyi beads.The proportion of Tfh cells was detected by flow cytometry after stimulated with anti-CD3/CD28 for 48h in vitro under the treatment of NAM or PBS.Results:(1)Compared with healthy controls,there was no significant difference in the proportion of CD4~+CXCR5~+T,CD4~+CXCR5~+PD-1~+T cells(P>0.05,P>0.05),and the proportion of CD4~+PD-1~+T cells and CD4~+CXCR5~+PD-1~+BCL-6~+T cells were significantly increased(P=0.0092,P=0.004).PD-1-MFI was significantly enhanced in CD4~+T and CD4~+CXCR5~+T cells(P=0.0485,P<0.0001,P=0.0275),and BCL-6-MFI was enhanced in CD4~+CXCR5~+PD-1~+T cells without significant difference(P=0.0972).SIRT1-MFI was significantly enhanced in CD4~+T,CD4~+CXCR5~+T,CD4~+CXCR5~+PD-1~+T,CD4~+CXCR5~+PD-1~+BCL-6~+T cells(P=0.0485,P=0.0007,P=0.0022,P=0.0064).There was a positive correlation between SIRT1-MFI in CD4~+T cells and CD4~+PD-1~+T,CD4~+CXCR5~+PD-1~+T cells(r=0.4313,P=0.0122;r=0.4002,P=0.021),but no significant correlation between SIRT1-MFI in CD4~+T cells and CD4~+CXCR5~+T cells(r=0.2942,P>0.05).PD-1-MFI was positively correlated with SIRT1-MFI in CD4~+T cells(r=0.3414,P=0.0518),and there was a strong positive correlation between SIRT1-MFI and BCL-6-MFI,BCL-6~+cells in CD4~+CXCR5~+PD-1~+T cells(r=0.4286,P=0.0149;r=0.6512,P<0.0001).(2)SIRT1 protein level continued to increase during Tfh cell differentiation in vitro.Compared with the control group,the proportion of CD4~+CXCR5~+PD-1~+T cells was significantly increased(P=0.0256),the proliferation activity was significantly increased(P=0.0001),but the proportion of apoptotic cells was not significantly changed(P>0.05)after infected with LV-SIRT1 for 72h.Compared with the control group,the ratios of CD4~+CXCR5~+PD-1~+T cells were significantly decreased(P=0.0089,P=0.0004),and mRNA relative expressions of BCL-6 were significantly down-regulated(P<0.001,P=0.01)in SIRT1-si RNA group,besides,the proliferation activity of Tfh-Polarizing cells was significantly decreased(P=0.0003),but the percentage of apoptotic cells was not significantly changed(P>0.05).(3)Compared with the control group,the ratio of CD4~+CXCR5~+PD-1~+T cells was significantly declined in both EX527and NAM group after culturing na?ve CD4~+T under Tfh-inducing condition for 3 days(P=0.0033,P=0.0433),as well as the mRNA expression of IL-21(P=0.007,P=0.04).Similarly,the proportion of CD4~+CXCR5~+PD-1~+T cells in CD4~+T cells(P=0.0008,P=0.0094)and the mRNA expression of IL-21(P=0.03,P=0.01)were also significantly reduced in NAM and EX527 group.The proportion of CD4~+CXCR5~+PD-1~+T cells in NAM group was significantly lower than that in EX527 group(P<0.0001),and the proportion of CD4~+CXCR5~+PD-1~+T cells in SRT1720+NAM group was significantly higher than that in NAM group(P=0.007),while the proportion of CD4~+CXCR5~+PD-1~+T cells in EX527+NAM group was significantly lower than that in NAM group(P=0.0196).(4)The proportion of CD4~+CXCR5~+PD-1~+T cells in peripheral blood CD4~+T cells of SLE patients was significantly decreased with treatment of NAM in vitro(P=0.007).(5)Compared with the control group,the proportion of Tfh cells in splenic CD4~+T cells of MRL/lpr mice in NAM group was significantly decreased(P=0.0074).Conclusion:SIRT1 is a positive regulator of Tfh cell differentiation and proliferation,and the aberrant overexpression of SIRT1 is closely related to the abnormal proliferation and differentiation of Tfh cells in SLE.Nicotinamide,a natural small-molecule inhibitor of SIRT1,is capable of inhibiting the differentiation and proliferation of Tfh cells.SIRT1 is of the potential to be a therapeutic target of SLE and thus warrants further investigation. |
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