Foxo1 Involved In Regulation Of Proliferation And Apoptosis Of Natural Regulatory T Cells

Background:Regulatory T cells(Tregs)are a subpopulation of T cells that are due to the same expression of CD4 and CD25.In addition,Tregs express the biomarker forkhead box P3(Foxp3)transcription factor.Treg differs from CD4+ in conventional T cells(Tcon)because of the low or no expression of CD127(IL-7R α).Tregs play an important role in autoimmune diseases,cancer,and organ transplantation.Tregs are the front-runners in the race for therapeutic immune-regulation due to their ability to suppress effector T cells,which are known to play an important role in preventing autoimmunity.In recent years,it has been reported that nTregs can also effectively prevent the rejection of transplanted allografts in experimental models.Naturally,n Tregs were used in hematopoieticstem cell transplantation to inhibit graft-versus-host disease(GVHD).However,the widespread clinical use of Tregs has been limited by the low number of these cells in the peripheryand immune homeostas.Therefore,there is a great need for understanding the mechanism of proliferation and immune homeostasis in nTregs to prevent the rejection of transplanted allografts.Interleukin-7(IL-7)is a hematopoietic growth factor that plays a critical role in regulating the homeostasis of T cells.The control of IL-7 signaling is mainly dependent on the IL-7 receptor(IL-7R),which is a heterodimer that consists of the IL-7 receptor alpha(IL-7Rα,CD127)and common gamma chain receptor.Therefore,CD127 plays a key role in modulating the homeostasis of T-cells.However,the molecular mechanism by which CD127 controls Treg cell proliferation and homeostasis remains unknown.Forkhead box protein o1(Foxo1)is a transcription factor and is characterized by a distinct fork headdomain.It was shown that Foxo1-deficient mice developed a fatal inflammatory disorder.Indeed,a growing body of research suggests that Foxo1 plays an important role in the immune system,including peripheral T-cell homeostasis.Foxo1 can modulate the expression of IL-7Rα,which is expressed in T-cells.Furthermore,Foxo1 can regulate the expression of Aven,which is an adaptor protein that has been implicated in anti-apoptotic signaling.Therefore,we speculated that Foxo1 modulates Treg cell homeostasis and functions via CD127 and Aven.Objective:To investigate the expression of Foxo1 and Aven by plasmids construction and siRNA transient transfections,we further study how Foxo1 can regulate the proliferation and apoptosis of nTreg cells by affecting the expression of CD127 and Aven.Methods:1.Sorting and Flow cytometrySpleen lymphocytes were isolated from Foxp3-GFP mice.The mice were killed by neck dislocation after following 4.5 mg/kg pentobarbital sodium via intraperitoneal injection in sterile environments.The spleen was removed for broking,and the cell lymphocyte suspension was obtained.Next,the cells were separated by lymphocyte separation liquid,and the cells were resuspended in FACS buffer(PBS)containing 1% BSA and 2% FBS for sorting by FACSCanto.The sorted cells were cultured in 1640 medium containing 20% FBS for further experiments.Cell staining was performed in FACS buffer(PBS)containing 1% BSA and 2% FBS.The cells were stained with phycoerythrin-conjugated anti-mouse ICOS antibody,Allophycocyanin conjugated anti-mouse CD127 antibody,phycoerythrin-conjugated anti-mouse CD103 antibody antibody or Allophycocyanin conjugated anti-mouse CD25 antibody at 4°C for 30 min and were then washed with PBS containing 2% BSA or FBS(wt/vol).Cells were collected using a FACSCanto and analyzed by FlowJo.2.Plasmid Construction and Transient TransfectionsInvitrogen provided Foxo1 and Aven small hairpin RNA(shRNA).With reference to the target gene,mouse Foxo1 Sense 5’-GGG GTA TGG CCG AAG CGC CCC AGG-3’,antisense 5’-TTA GCC TGA CAC CCA GCT GAG AGC-3’,mouse Aven Sense5’ –GGG GTA TGT TCG AAG CAC GT-3’,antisense 5’-TCA GGA AAT CAT GCT GTA GAG CA-3’.A series of chemically synthesized oligonucleotides was spliced by polymerase chain reaction(PCR)to obtain gene sequences,and these sequences were then inserted into the pCMV5 vector.The integrity of all constructs was confirmed by DNA sequencing.Using the Nucleofector Program U-25,2 mg of plasmid was transfected into 1×106 cells using the Amaxa Basic Nucleofector kit,according to the manufacturer’s protocol.3.Quantitative PCRTotal RNA was extracted in Trizol LS reagent and reverse-transcribed using a PrimeScript RT reagent kit.Next,cDNA was amplified(38 cycles of 95°C for 20 seconds,62°C for 15 seconds,and 72°C for 20 seconds)with SYBR qPCR SuperMix according to the manufacturer’s instructions,using gene-specific sets of primers: for mouse Foxo1 gene,5’ – TGT TTG ATT CAT TTC CTT TGG T-3’ and 5’ –TGA TTT TCT CCG CTT ACT GTT G-3’;mouse CD127 gene,5’-AAA AGT AAA GCA TGA TGT GGC C-3’ and 5’ –TTG AAG TAA TCG TTA TGG GGA A-3’;mouse Icos gene,5’ – CAT TCC CAA CAC GAA CAC CTA A-3’ and 5’ –TCT TCA CCC CCA GAA AAC ACA G-3’;mouse Aven gene,5’-GGG ACC AGG AAC CAG AAA AAG A-3’ and 5’ – TAC ACA GAA GGC AAC CAG CAT T-3’;mouse IL-2 gene,5’-GAT GAA CTT GGA CCT CTG CG-3’ and 5’-AGG GCT TGT TGA GAT GAT GC-3’;mouse IL-4 gene,5’-CAT CCT GCT CTT CTT TCT CG-3’ and 5’-CCT TCT CCT GTG ACC TCG TT-3’;mouse IL-7 gene,5’-GTT ATG GCA AAG CCA GAG CG-3’ and 5’-TGC GGG AGG TGG GTG TAG TC-3’;mouse IL-15 gene,5’-CCC CTT CTG TCC AGC CAC TC-3’ and 5’-TCC CGT CTT CG TCC AA TCT-3’.Mouse Bcl2 gene,5’-GC TAC CGT CGT GAC TTC GC-3’ and 5’-ATC CCA GCC TCC GTT ATC C-3’.For each gene,the mRNA level was normalized against GAPDH expression in the respective cDNA preparation.Controls were set to 1.0,and each gene induction was calculated as the fold difference compared to controls.Each reported expression value represents the average of 3 independent experiments.4.Western Blot AnalysisCells were collected and dissolved in RIPA buffer(1% sodium deoxycholate,1% Triton X-100,150 mM NaCl,0.1% SDS,10 mM Tris-HCl pH 7.2),Total proteins were separated on 10% SDS-PAGE and transferred onto polyvinylidene fluoride(PVDF)membranes by electroblotting for 1 hour(110 V).The PVDF membranes were blocked in Tris-buffered saline containing 5% nonfat milk and incubated with primary antibody in Tris-buffered saline and Tween(TBST)with 5% nonfat milk over-night at 4°C.Primary antibodies against the following proteins were used: Erk1/2,p-Erk1/2,Akt,p-Akt,Stat5,p-Stat5,Aven and GAPDH;Foxo1,p-Foxo1,CD127 and Bcl2.The membranes were washed three times for 5 min with TBST and were then incubated with the appropriate secondary antibody in TBST with 5% nonfat milk for 1 hour at room temperature.Protein binding was visualized using an enhanced chemiluminescence kit and X-ray films.5.Chromatin Immunoprecipitation Assays and Real-Time PCRChromatin immunoprecipitation assays were performed using a ChIP assay kit according to the manufacturer’s protocol.Cells(1×106)were fixed in formaldehyde(1%)in medium and were then incubated for 10 min at room temperature.The pellets were washed with cold PBS,and the fixed cells were sonicated(15 cycles of 30-second sonication with 60 seconds of cooling in an ice-water bath)using a MISONIX XL-2000 at a power setting of 15,to shear the DNA in SDS lysis buffer.After preclearing by shaking with protein A or G,these lysates were incubated with 4 mg of antibodies or normal IgG at 4°C for 16 hours.The immuno-precipitated DNA was purified using a QIAquick PCR purification kit according to the manufacturer’s protocol.Next,the purified DNA was used as a template for semi-quantitative and real-time PCRs with specific pairs of primers: for the mouse Aven promoter,5’-TTT GAG CCA AGG TTC TAA CAA A-3’ and 5’ – CCA ATA CTA ACA TCA CGG AGG G-3’.6.Detection of Cell Apoptosis by Flow CytometryCell apoptosis was detected using the Annexin V-FITC/PI apoptosis detection kit.Treg cells were seeded into 6-well culture plates(4.0 × 103 cells/cm2)and cultured for 24 h.The medium was removed after these groups were treated,and the cells were rinsed once with 0.1 M PBS.Cells were passaged using 0.25% trypsinization for 2 min and collected in centrifuge tubes.Cells were centrifuged at 1500 r/min for 5 min,and the supernatants were discarded.Binding Buffer(200 μL)was added into each tube and vortexed.Annexin V-FITC(5 μL)was added for 10 min at room temperature in the dark,and the tubes were centrifuged at 1000 r/min for 5 min.Supernatants were discarded,and 200 μL Binding Buffer was added to resuspend the cells.PI(5 μL)was added to the mixture for testing.7.MTT AssayCells were cultured in a 96-well culture plate at a density of 1 × 104 cells/cm2 for 1-6 days.Each group was pretreated with different reagents for 4 h.Reagents and 40 μM TBHP were added,and 24 h later,the media were removed.DMEM(100 μL)with 10% MTT was added to each well and kept at 37°C;4h later,the media was discarded.DMSO was added and placed on a shaker in the dark for 10 min.The absorption was measured at 492 nm using a Bio-Rad 400 microplate reader.Results:1.To investigate the role of Foxo1 on IL-7Rα expression in Treg cells,Treg cells were transfected with Foxo1 siRNA or control siRNA.We found that Foxo1 mRNA expression decreased by more than 50% when using Foxo1 siRNA compared to control siRNA.Consistent with these findings,Foxo1 protein expression was downregulated in Treg cells that were treated with Foxo1 siRNA compared with control siRNA.Unexpectedly,CD127 protein expression was downregulated in Treg cells-treated with Foxo1 siRNA compared with control siRNA.Conversely,Foxo1 over-expression increased the expression of CD127 in Treg cells,which suggested that Foxo1 plays an important role in CD127 expression.2.To test the role of Foxo1 in activating Treg cells,we detected CD103 and inducible co-stimulatory molecule(ICOS)by FCM,they have been described to identify activated Treg cells.CD127 was also detected by FCM in Treg cells.We found that CD127,CD103 and ICOS showed little change in Treg cells treated with Foxo1 siRNA and control siRNA,and the Median Fluorescence Intensity(MFI)of CD127 showed no significant difference between Foxo1 siRNA-treated cells and control si RNA cells.However,CD127,CD103 and ICOS expression was significantly increased in Foxo1 over-expression Treg cells,and the MFI of CD127 in Foxo1 over-expressed cells was 2.6 times higher than control.CD25 and Foxp3 showed little change in Treg cells in both Foxo1 knockdown and over-expressed cells.These findings suggested that Treg cells can be activated by over-expression Foxo1,And maintain the stability of its cell phenotype.3.Treg cells were stimulated with CD3,CD28 and IL-7 to activate cell proliferation,and the cell proliferation rate was detected using MTT.We found that the cell proliferation rate was higher in the Foxo1 over-expression cells than in the Foxo1 siRNA cells.But the cell proliferation rate decreased following treatment with the anti-CD127 antibody in the Foxo1 over-expression cells.Similarly,Treg cells were incubated without IL-7,the cell proliferation rate decreased,which suggested that Foxo1 controls Treg cell proliferation by regulating IL-7/CD127 signaling.4.Treg cells were transfected with Foxo1-plasmid prior to incubation with CD3,CD28 and IL-7,and apoptosis was measured according to the instructions of the PI-Annexin V apoptosis detection kit.We found that Foxo1 over-expression inhibited apoptosis.Conversely,cell apoptosis was enhanced in Treg cells after transfection with Foxo1 siRNA.However,IL-7 or CD127 demonstrated no apoptotic role in the presence of Foxo1,suggesting that Foxo1 inhibits Treg cell apoptosis via other signaling pathways.In view of the opinion that Foxo1 may regulate the expression of the anti-apoptotic protein Aven,we tested the hypothesis that Foxo1 inhibits Treg cell apoptosis by regulating Aven expression.We first observed that Foxo1 can bind to the promoter of Aven as assessed by the Chip assay.Furthermore,we found that Aven mRNA expression was inhibited in Treg cells treated with Foxo1 siRNA compared with control.Conversely,Foxo1 over-expression increased the mRNA levels of Aven in Treg cells.Consistent with these findings,the protein expression of Aven showed the same trend at the mRNA level.This finding indicated that Foxo1 regulates Aven expression.We detected the mRNA and protein levels of Bcl2 when Foxo1 was over-expressed and knocked down,whereas at the mRNA and protein levels,there were no differences between the conditions.Bcl2 might be not regulated by Foxo1.5.To detect the role of Aven in Treg cell apoptosis,Treg cells were transfected with Aven siRNA or control siRNA.Compared to Treg cells that were treated with control siRNA,Aven expression was downregulated in Treg cells treated with Aven siRNA at the mRNA and protein levels.The flow cytometry results showed that apoptosis was enhanced in Treg cells transfected with Aven siRNA.When Aven was over-expressed in Treg cells,we found that Aven expression was upregulated at the mRNA and protein levels and that cell apoptosis was attenuated,suggesting that Aven has anti-apoptotic properties in Treg cells.Furthermore,Treg cells are mainly divided into two subgroups: n Treg and i Treg.We also investigated the apoptosis of i Treg with Aven over-expression and knockdown.These results indicated that iTreg cells were similar with nTreg cells,such that apoptosis in both cell types could be regulated by Aven.Conclusions:1.Foxo1 can activate nTreg cells,Foxo1 overexpression can upregulate the expression of CD127 in nTreg cells.2.Foxo1 can promote the proliferation of nTreg cells by activating IL-7 / IL-7Rα signaling pathway.3.Foxo1 can activate the expression of anti-apoptotic gene Aven,which plays an important role in the apoptosis of nTreg cells,indicating that Foxo1 through Aven expression inhibit nTreg cell apoptosis.