BACKGROUND
regulatory t cells  are a subpopulation of t cells that are thought to be derived from the same lineage as naïve cd <dig> cells due to the same expression of cd <dig> and cd <dig>  <cit> .
in addition, tregs express the biomarker forkhead box p <dig>  transcription factor  <cit> .
it has been reported that tgf-β is essential for tregs to differentiate from naïve cd <dig> cells and is important in maintaining treg homeostasis  <cit> .
tregs can be divided into two subsets: naturally occurring or thymus-derived treg  cells and t regulatory type  <dig> cells.
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  <cit> .
in recent years, it has been reported that ntregs can also effectively prevent the rejection of transplanted allografts in experimental models  <cit> .
naturally, ntregs were used in hematopoietic stem cell transplantation to inhibit graft-versus-host disease   <cit> .
however, the widespread clinical use of tregs has been limited by the low number of these cells in the periphery and immune homeostas  <cit> .
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- <dig>  is a hematopoietic growth factor that plays a critical role in regulating the homeostasis of t cells  <cit> .
the control of il- <dig> signaling is mainly dependent on the il- <dig> receptor , which is a heterodimer that consists of the il- <dig> receptor alpha  and common gamma chain receptor  <cit> .
therefore, cd <dig> plays a key role in modulating the homeostasis of t-cells.
however, the molecular mechanism by which cd <dig> controls treg cell proliferation and homeostasis remains unknown.
forkhead box protein o <dig>  is a transcription factor and is characterized by a distinct fork head domain.
it was shown that foxo1-deficient mice developed a fatal inflammatory disorder  <cit> .
indeed, a growing body of research suggests that foxo <dig> plays an important role in the immune system, including peripheral t-cell homeostasis  <cit> .
foxo <dig> can modulate the expression of il-7rα, which is expressed in t-cells  <cit> .
furthermore, foxo <dig> can regulate the expression of aven  <cit> , which is an adaptor protein that has been implicated in anti-apoptotic signaling  <cit> .
therefore, we speculated that foxo <dig> modulates treg cell homeostasis and functions via cd <dig> and aven.
in the present study, we found that foxo <dig> over-expression resulted in treg cell proliferation by activating il-7/cd <dig> signaling.
in addition, foxo <dig> over-expression suppressed treg cell apoptosis by regulating aven expression.
taken together, these data suggest that foxo <dig> is required for treg cell proliferation and apoptosis by controlling cd <dig> and aven expression.
methods
mice
foxp3-gfp mice were obtained from jackson laboratories and were bred according to the experimental animal centre of the third military medical university and housed in a specific pathogen-free facility.
mice ages 6– <dig> weeks were used for the experiment.
the experimental protocol was approved by the ethics committee of the third military medical university.
sorting and flow cytometry
spleen lymphocytes were isolated from foxp3-gfp mice.
the mice were killed by neck dislocation after following  <dig>  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  containing 1% bsa and 2% fbs for sorting by facscanto .
the sorted cells were cultured in  <dig> medium containing 20% fbs for further experiments.
cell staining was performed in facs buffer  containing 1% bsa and 2% fbs.
the cells were stained with phycoerythrin-conjugated anti-mouse icos antibody , allophycocyanin conjugated anti-mouse cd <dig> antibody , phycoerythrin-conjugated anti-mouse cd <dig> antibody  antibody or allophycocyanin conjugated anti-mouse cd <dig>  antibody at 4 °c for 30 min and were then washed with pbs containing 2% bsa or fbs .
cells were collected using a facscanto  and analyzed by flowjo.
plasmid construction and transient transfections
invitrogen provided foxo <dig> and aven small hairpin rna .
with reference to the target gene, mouse foxo <dig> 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  to obtain gene sequences, and these sequences were then inserted into the pcmv <dig> vector  <cit> .
the integrity of all constructs was confirmed by dna sequencing.
using the nucleofector program u- <dig>  2 mg of plasmid was transfected into 1 ×  <dig> cells using the amaxa basic nucleofector kit , according to the manufacturer’s protocol.
quantitative pcr
total rna was extracted in trizol ls reagent  and reverse-transcribed using a primescript rt reagent kit .
next, cdna was amplified  with sybr qpcr supermix  according to the manufacturer’s instructions, using gene-specific sets of primers: f for mouse foxo <dig> gene, 5′ – tgt ttg att cat ttc ctt tgg t -3′ and 5′ –tga ttt tct ccg ctt act gtt g -3′; mouse cd <dig> 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- <dig> gene, 5′- gat gaa ctt gga cct ctg cg -3′ and 5′- agg gct tgt tga gat gat gc -3′; mouse il- <dig> gene, 5′- cat cct gct ctt ctt tct cg -3′ and 5′- cct tct cct gtg acc tcg tt -3′; mouse il- <dig> gene, 5′- gtt atg gca aag cca gag cg -3′ and 5′- tgc ggg agg tgg gtg tag tc-3′; mouse il- <dig> gene, 5′- ccc ctt ctg tcc agc cac tc -3′ and 5′- tcc cgt ctt cg tcc aa tct-3′.
mouse bcl <dig> 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  <dig> , and each gene induction was calculated as the fold difference compared to controls.
each reported expression value represents the average of  <dig> independent experiments.
western blot analysis
cells were collected and dissolved in ripa buffer , total proteins were separated on 10% sds-page and transferred onto polyvinylidene fluoride  membranes by electroblotting for 1 h .
the pvdf membranes  were blocked in tris- buffered saline containing 5% nonfat milk and incubated with primary antibody in tris-buffered saline and tween  with 5% nonfat milk over-night at 4 °c.
primary antibodies against the following proteins were used: erk1/ <dig>  p-erk1/ <dig>  akt, p-akt, stat <dig>  p-stat <dig>  aven and gapdh ; foxo <dig>  p-foxo <dig> , cd <dig>  and bcl <dig> .
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 h at room temperature.
protein binding was visualized using an enhanced chemiluminescence kit  and x-ray films.
chromatin immunoprecipitation assays and real-time pcr
chromatin immunoprecipitation assays were performed using a chip assay kit  according to the manufacturer’s protocol.
cells  were fixed in formaldehyde  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  using a misonix xl- <dig>  at a power setting of  <dig>  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 h. 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′
detection of cell apoptosis by flow cytometry
cell apoptosis was detected using the annexin v-fitc/pi apoptosis detection kit.
sh-sy5y cells were seeded into 6-well culture plates  and cultured for 24 h. the medium was removed after these groups were treated, and the cells were rinsed once with  <dig>  m pbs.
cells were passaged using  <dig> % 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  was added into each tube and vortexed.
annexin v-fitc  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  was added to the mixture for testing.
mtt assay
cells were cultured in a 96-well culture plate at a density of 1 ×  <dig> cells/cm <dig> for 1– <dig> 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  with 10% mtt was added to each well and kept at 37 °c; 4 h later, the media was discarded.
dmso was added and placed on a shaker in the dark for 10 min.
the absorption was measured at 570 nm using a bio-rad  <dig> microplate reader .
experiments were repeated  <dig> times.
statistical analyses
all data were analyzed using the spss  <dig>  statistical software .
the data are presented as the mean ± s.d.
of three independent experiments; student’s t-test was used for comparisons.
p <  <dig>  was considered significant.
RESULTS
foxo <dig> regulates cd <dig> expression in treg cells
to investigate the role of foxo <dig> on il-7rα expression in treg cells, treg cells were transfected with foxo <dig> sirna or control sirna.
we found that foxo <dig> mrna expression decreased by more than 50% when using foxo <dig> sirna compared to control sirna .
consistent with these findings, foxo <dig> protein expression was downregulated in treg cells that were treated with foxo <dig> sirna compared with control sirna .
unexpectedly, cd <dig> protein expression was downregulated in treg cells-treated with foxo <dig> sirna compared with control sirna .
conversely, foxo <dig> over-expression increased the expression of cd <dig> in treg cells , which suggested that foxo <dig> plays an important role in cd <dig> expression.fig.
<dig> detection of foxo <dig> and cd <dig> after knockdown and over-expression of foxo <dig> in treg cells.
a expression of mrna and protein of foxo <dig> and cd <dig> in treg cells, 48 h after transfection with foxo <dig> sirna.
b expression of mrna and protein of foxo <dig> and cd <dig> in treg cells 48 h after transfection with over-expression plasmid of foxo <dig>  treg cells stimulated with anti-cd <dig>  and anti-cd <dig>  in medium during culture.
data are presented as the mean + standard deviation .
*p <  <dig> ; **p <  <dig>
fig.
<dig> detection of cell surface molecules and signaling pathway molecules after knockdown and over-expression of foxo <dig> in treg cells.
a, representative expression of the foxo <dig>  cd <dig>  cd <dig>  icos, foxp <dig> or cd <dig> in treg cells 48 h after transfection with foxo <dig> sirna by flow cytometry .
b representative expression of the foxo <dig>  cd <dig>  cd <dig>  icos, foxp <dig> or cd <dig> in treg cells 48 h after transfection with over-expression plasmid of foxo <dig> by flow cytometry .
c detected of median fluorescence intensity  for cd <dig> in treg cells 48 h after transfection with foxo <dig> sirna and foxo <dig> over-expression plasmid by flow cytometry.
d representative western blot of p-erk1/ <dig>  total erk1/ <dig>  p-akt, total akt, p-stat <dig>  total stat <dig>  p-foxo <dig> and total foxo <dig> in treg cells 48 h after transfection with foxo <dig> sirna and over-expression plasmid of foxo <dig>  gadph was used as a control.
treg cells stimulated with anti-cd <dig>  and anti-cd <dig>  in medium during culture.
e expression of mrna for il- <dig>  il- <dig>  il- <dig> and il- <dig> in treg cells, 48 h after transfection with foxo <dig> sirna and foxo <dig> over-expression plasmid.
all experiments were repeated at least three times.
**p <  <dig> , n.s: no significance
foxo <dig> controls treg cell proliferation by regulating cd <dig> expression
to test the role of foxo <dig> in activating treg cells, we detected cd <dig> and inducible co-stimulatory molecule  by fcm, they have been described to identify activated treg cells  <cit> .
cd <dig> was also detected by fcm in treg cells.
we found that cd <dig>  cd <dig> and icos showed little change in treg cells treated with foxo <dig> sirna and control sirna, and the median fluorescence intensity  of cd <dig> showed no significant difference between foxo <dig> sirna-treated cells and control sirna cells .
however, cd <dig>  cd <dig> and icos expression was significantly increased in foxo <dig> over-expression treg cells, and the mfi of cd <dig> in foxo <dig> over-expressed cells was  <dig>  times higher than control .
cd <dig> and foxp <dig> showed little change in treg cells in both foxo <dig> knockdown and over-expressed cells .
in addition, intracellular signaling molecules associated with the activity of treg cells, including p-erk1/ <dig>  p-akt, p-foxo <dig> and p-stat <dig>  demonstrated no change .
these findings suggested that treg cells can be activated by over-expression foxo <dig>  cytokines that affect treg cell activities, such as il- <dig>  il- <dig>  il- <dig> and il- <dig>  <cit> , were detected using quantitative pcr.
however, at the mrna level of the genes, there were no differences when foxo <dig> was over-expressed and knocked down .
next, treg cells were stimulated with cd <dig>  cd <dig> and il- <dig> to activate cell proliferation, and the cell proliferation rate was detected using elisa.
we found that the cell proliferation rate was higher in the foxo <dig> over-expression cells than in the foxo <dig> sirna cells .
but the cell proliferation rate decreased following treatment with the anti-cd <dig> antibody in the foxo <dig> over-expression cells .
similarly, treg cells were incubated without il- <dig>  the cell proliferation rate decreased , which suggested that foxo <dig> controls treg cell proliferation by regulating il-7/cd <dig> signaling.fig.
<dig> proliferation and apoptosis after knockdown or over-expression of foxo <dig> in treg cells.
a treg cells growth were measured for 6 days using a mtt assay.
treg cells of every group  except foxo1-plasmid  was stimulated with anti-cd <dig> , anti-cd <dig>  and il- <dig>  during culture, and anti-cd <dig> and anti-cd <dig> was only added in the foxo1-plasmid  group.
in addition, anti-cd <dig>  was added to the foxo1-plasmid + cd <dig> group.
these results are presented as the mean ± s.d.
of the values obtained in three independent experiments.
b representative result of apoptosis in treg cells based on the above groups 48 h after stimulation
foxo <dig> controls treg cell apoptosis by regulating aven expression
treg cells were transfected with foxo1-plasmid prior to incubation with cd <dig>  cd <dig> and il- <dig>  and apoptosis was measured according to the instructions of the pi-annexin v apoptosis detection kit.
we found that foxo <dig> over-expression inhibited apoptosis .
conversely, cell apoptosis was enhanced in treg cells after transfection with foxo <dig> sirna .
however, il- <dig> or cd <dig> demonstrated no apoptotic role in the presence of foxo <dig> , suggesting that foxo <dig> inhibits treg cell apoptosis via other signaling pathways.
in view of the opinion that foxo <dig> may regulate the expression of the anti-apoptotic protein aven  <cit> , we tested the hypothesis that foxo <dig> inhibits treg cell apoptosis by regulating aven expression.
we first observed that foxo <dig> 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 foxo <dig> sirna compared with control .
conversely, foxo <dig> 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 foxo <dig> regulates aven expression.
the il-7/cd <dig> axis is important for t-cell homeostasis, which can be also mediated by bcl <dig>  we detected the mrna and protein levels of bcl <dig> when aven was over-expressed and knocked down, whereas at the mrna and protein levels, there were no differences between the conditions .
bcl <dig> might be not regulated by foxo <dig> fig.
<dig> detection of aven after knockdown and over-expression of foxo <dig> in treg cells.
a level of foxo <dig> enrichment at the aven gene promoters in treg cells in which foxo <dig> was knocked down and over-expressed.
expression of the  mrna and  protein of foxo <dig> and aven in treg cells 48 h after transfection with foxo <dig> sirna and over-expression.
expression of the  protein and  mrna of bcl <dig> in treg cells 48 h after transfection with foxo <dig> sirna and over-expression.
cells was stimulated with anti-cd <dig> , anti-cd <dig>  and il- <dig>  during culture.
data are presented as the mean + standard deviation .
*p <  <dig> ; **p <  <dig> , ***p <  <dig>
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: ntreg and itreg.
we also investigated the apoptosis of itregs 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 .fig.
<dig> detection of apoptosis after knockdown and over-expression of aven in treg cells.
expression of the  mrna and protein of aven in treg cells 48 h after transfection with aven sirna or over-expression plasmid of foxo <dig>  c representative result of apoptosis in ntreg and itreg cells 48 h after transfection with aven sirna or over-expression plasmid of foxo <dig>  treg cells were stimulated with anti-cd <dig>  and anti-cd <dig>  in medium during culture.
data are presented as the mean + standard deviation .
***p < <dig>
discussion
the forkhead box  family genes have important functions in many biological processes in human and mice.
the mutations of fox genes caused many human diseases, such as glaucoma, cancer, language disorders and immune diseases  <cit> .
foxn <dig>  a member of the fox family, involved in the differentiation and development of human central nervous system  <cit> , and palys important roles for the differentiation and growth of t cell.
alterations of foxn <dig> result in the failure of generating mature and functional thymocytes  <cit> .
foxo <dig> was also an important regulatory factor for the differentiation and function of t cells  <cit> .
in the present study, we showed that foxo <dig> activated the il-7/cd <dig> signaling pathway, resulting in increased treg proliferation.
in addition, foxo <dig> activated aven expression, which plays an important role in cell apoptosis in treg cells, suggesting that foxo <dig> inhibits treg cell apoptosis via aven expression.
these results indicated that foxo <dig> plays a key role in controlling treg cell proliferation and apoptosis by regulating cd <dig> and aven expression.
many studies have shown that foxo <dig> plays an important role in t-cell homeostasis  <cit> .
foxo <dig> regulates the expression of cd <dig> by binding to the promoter of the cd <dig> gene, and deletion of the foxo <dig> gene can block the expression of cd <dig> in t-cells  <cit> .
in this study, we found that foxo <dig> regulates cd <dig> expression in treg cells.
this result is consistent with the result obtained in previous reports.
activation of the il-7/cd <dig> signaling pathway can promote treg cell proliferation.
therefore, foxo <dig> is involved in the regulation of treg cell proliferation by regulating cd <dig>
in addition, we found that foxo <dig> regulates cell apoptosis in treg cells.
however, cd <dig> was confirmed to not be involved in the cell apoptosis in treg cells.
therefore, foxo <dig> regulates other molecules to inhibit cell apoptosis.
aven is a novel molecule and can bind to the proapoptotic apaf- <dig> protein to prevent the oligomerization of apaf- <dig> in the intrinsic apoptosis pathway  <cit> .
recently, it was shown that aven contributes to the anti-apoptotic properties of this protein  <cit> .
we found that foxo <dig> can regulate aven expression in treg cells, which inhibits cell apoptosis.
these data indicated that foxo <dig> is involved in the regulation of treg cell apoptosis via aven.
in conclusion, we demonstrated that foxo <dig> over-expression in the treg cells results in a significant enhancement of cell proliferation due to the activation of the il-7/cd <dig> signaling pathway.
in addition, foxo <dig> over-expression in treg cells inhibits cell apoptosis by regulating aven expression.
collectively, foxo <dig> is required for treg cell proliferation and apoptosis via cd <dig> and aven expression.
CONCLUSIONS
our results suggest that foxo <dig> is a positive regulatory factor for ntreg cell activation.
it promotes the proliferation of treg cells and inhibits apoptosis via the aven signaling pathway.
these data suggest that foxo <dig> might be a promising target for activating ntreg cells in vivo and in vitro.
basic and clinical research of treatment drugs provides a useful reference.
abbreviations
foxo1forkhead box protein o 1
gvhdgraft-versus-host disease
il-7interleukin-7
il-7rinterleukin- <dig> receptor
tregregulatory t

 regulatory t cells are a subpopulation of t cells that are thought to be derived from the same lineage as naïve cd cells due to the same expression of cd and cd. in addition, tregs express the biomarker forkhead box p transcription factor. it has been reported that tgf-β is essential for tregs to differentiate from naïve cd cells and is important in maintaining treg homeostasis. tregs can be divided into two subsets: naturally occurring or thymus-derived treg cells and t regulatory type cells. 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, ntregs were used in hematopoietic stem cell transplantation to inhibit graft-versus-host disease. however, the widespread clinical use of tregs has been limited by the low number of these cells in the periphery and 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- is a hematopoietic growth factor that plays a critical role in regulating the homeostasis of t cells. the control of il- signaling is mainly dependent on the il- receptor, which is a heterodimer that consists of the il- receptor alpha and common gamma chain receptor. therefore, cd plays a key role in modulating the homeostasis of t-cells. however, the molecular mechanism by which cd controls treg cell proliferation and homeostasis remains unknown. forkhead box protein o is a transcription factor and is characterized by a distinct fork head domain. it was shown that foxo1-deficient mice developed a fatal inflammatory disorder. indeed, a growing body of research suggests that foxo plays an important role in the immune system, including peripheral t-cell homeostasis. foxo can modulate the expression of il-7rα, which is expressed in t-cells. furthermore, foxo can regulate the expression of aven, which is an adaptor protein that has been implicated in anti-apoptotic signaling. therefore, we speculated that foxo modulates treg cell homeostasis and functions via cd and aven. in the present study, we found that foxo over-expression resulted in treg cell proliferation by activating il-7/cd signaling. in addition, foxo over-expression suppressed treg cell apoptosis by regulating aven expression. taken together, these data suggest that foxo is required for treg cell proliferation and apoptosis by controlling cd and aven expression. methods mice foxp3-gfp mice were obtained from jackson laboratories and were bred according to the experimental animal centre of the third military medical university and housed in a specific pathogen-free facility. mice ages 6– weeks were used for the experiment. the experimental protocol was approved by the ethics committee of the third military medical university. sorting and flow cytometry spleen lymphocytes were isolated from foxp3-gfp mice. the mice were killed by neck dislocation after following  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 containing 1% bsa and 2% fbs for sorting by facscanto. the sorted cells were cultured in medium containing 20% fbs for further experiments. cell staining was performed in facs buffer containing 1% bsa and 2% fbs. the cells were stained with phycoerythrin-conjugated anti-mouse icos antibody, allophycocyanin conjugated anti-mouse cd antibody, phycoerythrin-conjugated anti-mouse cd antibody antibody or allophycocyanin conjugated anti-mouse cd antibody at 4 °c for 30 min and were then washed with pbs containing 2% bsa or fbs. cells were collected using a facscanto and analyzed by flowjo. plasmid construction and transient transfections invitrogen provided foxo and aven small hairpin rna. with reference to the target gene, mouse foxo 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 to obtain gene sequences, and these sequences were then inserted into the pcmv vector. the integrity of all constructs was confirmed by dna sequencing. using the nucleofector program u- 2 mg of plasmid was transfected into 1 ×  cells using the amaxa basic nucleofector kit, according to the manufacturer’s protocol. quantitative pcr total rna was extracted in trizol ls reagent and reverse-transcribed using a primescript rt reagent kit. next, cdna was amplified with sybr qpcr supermix according to the manufacturer’s instructions, using gene-specific sets of primers: f for mouse foxo gene, 5′ – tgt ttg att cat ttc ctt tgg t -3′ and 5′ –tga ttt tct ccg ctt act gtt g -3′; mouse cd 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- gene, 5′- gat gaa ctt gga cct ctg cg -3′ and 5′- agg gct tgt tga gat gat gc -3′; mouse il- gene, 5′- cat cct gct ctt ctt tct cg -3′ and 5′- cct tct cct gtg acc tcg tt -3′; mouse il- gene, 5′- gtt atg gca aag cca gag cg -3′ and 5′- tgc ggg agg tgg gtg tag tc-3′; mouse il- gene, 5′- ccc ctt ctg tcc agc cac tc -3′ and 5′- tcc cgt ctt cg tcc aa tct-3′. mouse bcl 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, and each gene induction was calculated as the fold difference compared to controls. each reported expression value represents the average of independent experiments. western blot analysis cells were collected and dissolved in ripa buffer, total proteins were separated on 10% sds-page and transferred onto polyvinylidene fluoride membranes by electroblotting for 1 h. the pvdf membranes were blocked in tris- buffered saline containing 5% nonfat milk and incubated with primary antibody in tris-buffered saline and tween with 5% nonfat milk over-night at 4 °c. primary antibodies against the following proteins were used: erk1/ p-erk1/ akt, p-akt, stat p-stat aven and gapdh; foxo p-foxo, cd and bcl. 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 h at room temperature. protein binding was visualized using an enhanced chemiluminescence kit and x-ray films. chromatin immunoprecipitation assays and real-time pcr chromatin immunoprecipitation assays were performed using a chip assay kit according to the manufacturer’s protocol. cells were fixed in formaldehyde 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 using a misonix xl- at a power setting of 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 h. 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′ detection of cell apoptosis by flow cytometry cell apoptosis was detected using the annexin v-fitc/pi apoptosis detection kit. sh-sy5y cells were seeded into 6-well culture plates and cultured for 24 h. the medium was removed after these groups were treated, and the cells were rinsed once with  m pbs. cells were passaged using % 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 was added into each tube and vortexed. annexin v-fitc 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 was added to the mixture for testing. mtt assay cells were cultured in a 96-well culture plate at a density of 1 ×  cells/cm for 1– 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 with 10% mtt was added to each well and kept at 37 °c; 4 h later, the media was discarded. dmso was added and placed on a shaker in the dark for 10 min. the absorption was measured at 570 nm using a bio-rad microplate reader. experiments were repeated times. statistical analyses all data were analyzed using the spss statistical software. the data are presented as the mean ± s.d. of three independent experiments; student’s t-test was used for comparisons. p <  was considered significant.  foxo regulates cd expression in treg cells to investigate the role of foxo on il-7rα expression in treg cells, treg cells were transfected with foxo sirna or control sirna. we found that foxo mrna expression decreased by more than 50% when using foxo sirna compared to control sirna. consistent with these findings, foxo protein expression was downregulated in treg cells that were treated with foxo sirna compared with control sirna. unexpectedly, cd protein expression was downregulated in treg cells-treated with foxo sirna compared with control sirna. conversely, foxo over-expression increased the expression of cd in treg cells, which suggested that foxo plays an important role in cd expression.fig. detection of foxo and cd after knockdown and over-expression of foxo in treg cells. a expression of mrna and protein of foxo and cd in treg cells, 48 h after transfection with foxo sirna. b expression of mrna and protein of foxo and cd in treg cells 48 h after transfection with over-expression plasmid of foxo treg cells stimulated with anti-cd and anti-cd in medium during culture. data are presented as the mean + standard deviation. *p < ; **p <  fig. detection of cell surface molecules and signaling pathway molecules after knockdown and over-expression of foxo in treg cells. a, representative expression of the foxo cd cd icos, foxp or cd in treg cells 48 h after transfection with foxo sirna by flow cytometry. b representative expression of the foxo cd cd icos, foxp or cd in treg cells 48 h after transfection with over-expression plasmid of foxo by flow cytometry. c detected of median fluorescence intensity for cd in treg cells 48 h after transfection with foxo sirna and foxo over-expression plasmid by flow cytometry. d representative western blot of p-erk1/ total erk1/ p-akt, total akt, p-stat total stat p-foxo and total foxo in treg cells 48 h after transfection with foxo sirna and over-expression plasmid of foxo gadph was used as a control. treg cells stimulated with anti-cd and anti-cd in medium during culture. e expression of mrna for il- il- il- and il- in treg cells, 48 h after transfection with foxo sirna and foxo over-expression plasmid. all experiments were repeated at least three times. **p < , n.s: no significance foxo controls treg cell proliferation by regulating cd expression to test the role of foxo in activating treg cells, we detected cd and inducible co-stimulatory molecule by fcm, they have been described to identify activated treg cells. cd was also detected by fcm in treg cells. we found that cd cd and icos showed little change in treg cells treated with foxo sirna and control sirna, and the median fluorescence intensity of cd showed no significant difference between foxo sirna-treated cells and control sirna cells. however, cd cd and icos expression was significantly increased in foxo over-expression treg cells, and the mfi of cd in foxo over-expressed cells was times higher than control. cd and foxp showed little change in treg cells in both foxo knockdown and over-expressed cells. in addition, intracellular signaling molecules associated with the activity of treg cells, including p-erk1/ p-akt, p-foxo and p-stat demonstrated no change. these findings suggested that treg cells can be activated by over-expression foxo cytokines that affect treg cell activities, such as il- il- il- and il-, were detected using quantitative pcr. however, at the mrna level of the genes, there were no differences when foxo was over-expressed and knocked down. next, treg cells were stimulated with cd cd and il- to activate cell proliferation, and the cell proliferation rate was detected using elisa. we found that the cell proliferation rate was higher in the foxo over-expression cells than in the foxo sirna cells. but the cell proliferation rate decreased following treatment with the anti-cd antibody in the foxo over-expression cells. similarly, treg cells were incubated without il- the cell proliferation rate decreased, which suggested that foxo controls treg cell proliferation by regulating il-7/cd signaling.fig. proliferation and apoptosis after knockdown or over-expression of foxo in treg cells. a treg cells growth were measured for 6 days using a mtt assay. treg cells of every group except foxo1-plasmid was stimulated with anti-cd, anti-cd and il- during culture, and anti-cd and anti-cd was only added in the foxo1-plasmid group. in addition, anti-cd was added to the foxo1-plasmid + cd group. these results are presented as the mean ± s.d. of the values obtained in three independent experiments. b representative result of apoptosis in treg cells based on the above groups 48 h after stimulation foxo controls treg cell apoptosis by regulating aven expression treg cells were transfected with foxo1-plasmid prior to incubation with cd cd and il- and apoptosis was measured according to the instructions of the pi-annexin v apoptosis detection kit. we found that foxo over-expression inhibited apoptosis. conversely, cell apoptosis was enhanced in treg cells after transfection with foxo sirna. however, il- or cd demonstrated no apoptotic role in the presence of foxo, suggesting that foxo inhibits treg cell apoptosis via other signaling pathways. in view of the opinion that foxo may regulate the expression of the anti-apoptotic protein aven, we tested the hypothesis that foxo inhibits treg cell apoptosis by regulating aven expression. we first observed that foxo 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 foxo sirna compared with control. conversely, foxo 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 foxo regulates aven expression. the il-7/cd axis is important for t-cell homeostasis, which can be also mediated by bcl we detected the mrna and protein levels of bcl when aven was over-expressed and knocked down, whereas at the mrna and protein levels, there were no differences between the conditions. bcl might be not regulated by foxo fig. detection of aven after knockdown and over-expression of foxo in treg cells. a level of foxo enrichment at the aven gene promoters in treg cells in which foxo was knocked down and over-expressed. expression of the mrna and protein of foxo and aven in treg cells 48 h after transfection with foxo sirna and over-expression. expression of the protein and mrna of bcl in treg cells 48 h after transfection with foxo sirna and over-expression. cells was stimulated with anti-cd, anti-cd and il- during culture. data are presented as the mean + standard deviation. *p < ; **p < , ***p <  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: ntreg and itreg. we also investigated the apoptosis of itregs 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.fig. detection of apoptosis after knockdown and over-expression of aven in treg cells. expression of the mrna and protein of aven in treg cells 48 h after transfection with aven sirna or over-expression plasmid of foxo c representative result of apoptosis in ntreg and itreg cells 48 h after transfection with aven sirna or over-expression plasmid of foxo treg cells were stimulated with anti-cd and anti-cd in medium during culture. data are presented as the mean + standard deviation. ***p < discussion the forkhead box family genes have important functions in many biological processes in human and mice. the mutations of fox genes caused many human diseases, such as glaucoma, cancer, language disorders and immune diseases. foxn a member of the fox family, involved in the differentiation and development of human central nervous system, and palys important roles for the differentiation and growth of t cell. alterations of foxn result in the failure of generating mature and functional thymocytes. foxo was also an important regulatory factor for the differentiation and function of t cells. in the present study, we showed that foxo activated the il-7/cd signaling pathway, resulting in increased treg proliferation. in addition, foxo activated aven expression, which plays an important role in cell apoptosis in treg cells, suggesting that foxo inhibits treg cell apoptosis via aven expression. these results indicated that foxo plays a key role in controlling treg cell proliferation and apoptosis by regulating cd and aven expression. many studies have shown that foxo plays an important role in t-cell homeostasis. foxo regulates the expression of cd by binding to the promoter of the cd gene, and deletion of the foxo gene can block the expression of cd in t-cells. in this study, we found that foxo regulates cd expression in treg cells. this result is consistent with the result obtained in previous reports. activation of the il-7/cd signaling pathway can promote treg cell proliferation. therefore, foxo is involved in the regulation of treg cell proliferation by regulating cd in addition, we found that foxo regulates cell apoptosis in treg cells. however, cd was confirmed to not be involved in the cell apoptosis in treg cells. therefore, foxo regulates other molecules to inhibit cell apoptosis. aven is a novel molecule and can bind to the proapoptotic apaf- protein to prevent the oligomerization of apaf- in the intrinsic apoptosis pathway. recently, it was shown that aven contributes to the anti-apoptotic properties of this protein. we found that foxo can regulate aven expression in treg cells, which inhibits cell apoptosis. these data indicated that foxo is involved in the regulation of treg cell apoptosis via aven. in conclusion, we demonstrated that foxo over-expression in the treg cells results in a significant enhancement of cell proliferation due to the activation of the il-7/cd signaling pathway. in addition, foxo over-expression in treg cells inhibits cell apoptosis by regulating aven expression. collectively, foxo is required for treg cell proliferation and apoptosis via cd and aven expression.  our results suggest that foxo is a positive regulatory factor for ntreg cell activation. it promotes the proliferation of treg cells and inhibits apoptosis via the aven signaling pathway. these data suggest that foxo might be a promising target for activating ntreg cells in vivo and in vitro. basic and clinical research of treatment drugs provides a useful reference. abbreviations foxo1forkhead box protein o 1 gvhdgraft-versus-host disease il-7interleukin-7 il-7rinterleukin- receptor tregregulatory t 
ntreg cells were transfected separately with foxo and aven small-interfering rna or over-expression plasmid. forkhead box protein o and il-7rα are closely related to the homeostasis of treg cells. foxo strengthened the proliferative ability of treg cells by activating il-7/cd signaling. the results in this study indicated that foxo is a positive regulatory factor for the proliferation and activity of treg cells. foxo might be a potential target for the activation of ntreg cells in vivo and in vitro. treg cellsfoxo1avenapoptosishttp://dx.doi.org/ /501100001809national natural science foundation of china81273258liu hong issue-copyright-statement© the author 2017 regulatory t cells play important roles in autoimmune diseases, cancer, and organ transplantation. in addition, foxo suppressed treg cell apoptosis by regulating aven expression. the expression of cell surface molecules and apoptosis was confirmed by flow cytometry 3- - diphenyltetrazolium bromide for cell proliferation assays. however, the mechanism underlying treg proliferation and activation remains unclear.
 regulatory t cells play important roles in autoimmune diseases, cancer, and organ transplantation. forkhead box protein o and il-7rα are closely related to the homeostasis of treg cells. however, the mechanism underlying treg proliferation and activation remains unclear. here, we evaluated how the over-expression of foxo affects treg cell proliferation via intracellular signaling. ntreg cells were transfected separately with foxo and aven small-interfering rna or over-expression plasmid. the expression of signaling pathway genes and cd was confirmed using rt-qpcr and western blot analysis. the expression of cell surface molecules and apoptosis was confirmed by flow cytometry 3- - diphenyltetrazolium bromide for cell proliferation assays.  foxo strengthened the proliferative ability of treg cells by activating il-7/cd signaling. in addition, foxo suppressed treg cell apoptosis by regulating aven expression.  the results in this study indicated that foxo is a positive regulatory factor for the proliferation and activity of treg cells. foxo might be a potential target for the activation of ntreg cells in vivo and in vitro. keywords treg cellsfoxo1avenapoptosishttp://dx.doi.org/ /501100001809national natural science foundation of china81273258liu hong issue-copyright-statement© the author 2017 