Ture of naive CD4+CD252CD45RO2 T cells and allogeneic CD40-activated B cells and filled histogram indicates staining obtained from the isotype-matched mAb for staining antibodies. Data were shown in Mean+SEM, n = 6. (B) 3H-thymidine incorporation of CD4hiCD25+ regulatory T cells in suppressive MLR at different regulatory T cells: responsders ratio. Data show Mean+SEM, n = 6. All data shown are from 3 independent experiments. NS, not significant, one way ANOVA with Tukey’s pairwise comparisons. doi:10.1371/journal.pone.0067969.gknowledge, is the first report concerning TLR5-related signals in iTregs. Here we found an increase of TLR5 expression in CD4hiCD25+ regulatory T cells. This was probably the consequence of CD4+ T cell activation during the co-culture. NF-kB and AP-1 binding sites are situated around the promoter region of TLR5 locus [37]. NF-kB and AP-1 are synthesized during T cell activation [38,39] and may bind to the promoter of TLR5, resulting in the transcription of TLR5. Interestingly, TLR5 can also activate the synthesis of NF-kB and AP-1 [14], thus it is possible that TLR5 was activated during the co-culture and positively feedback to the TLR5 expression. Since TLR5 expression was also up regulated inTLR5 Enhances Induced Treg Proliferationresting nTregs [22], it is possible that Foxp3 also up regulate the TLR5 expression but the precise mechanism remains to be investigated. In this study, we further found that blockade of TLR5 using anti-TLR5 blocking antibody reduced the proliferation of CD4hiCD25+ regulatory T cells through S phase arrest but did not increase the apoptosis of CD4hiCD25+ regulatory T cells or CD4+CD252 T cells. Since TLR5 was reported to be antiapoptotic [40], it was surprising that blockade of TLR5 did not increase the apoptosis of the cells. This may be explained by the observation from our previous investigation that large amount of IL-2 was produced by the CD40-activated B cells [28], thus it is possible that these IL-2 molecules rescued the CD4+ T cells from apoptosis. The S phase arrest of the CD4hiCD25+ regulatory T cells may be explained by the associated reduction of the ERK1/2 Title Loaded From File phosphorylation after TLR5 blockade. It is known that S phase exit or G2/M phase entry is controlled by cdk2 and cyclin A [41], the cdk2 is in turn activated by cdc25a [42], which could be activated and phosphorylated by p-ERK1/2 [43]. Therefore, it is speculated that the reduced ERK1/2 phosphorylation in the CD4hiCD25+ regulatory T cells decreased the expression and activation of cdc25a, thus in turn, the cdk2 activation, causing S phase arrest. However, the precise molecular mechanism between the reduced ERK1/2 phosphorylation and the S phase arrest remains to be elucidated. In addition, the reduced proliferation of the CD4hiCD25+ regulatory T cells may also be the result of reduced Ificant effect on this proliferation index. Control includes both sham and production of different cytokines. It was reported that stimulation of TLR5 using flagellin resulted in IL-8 production in epithelial cells and gastric cancer cells, increasing the proliferation of these cells [44,45], and the production of IFN-c [46]. Therefore, it is possible that TLR5-related signals may enhance 23977191 the production of IFN-c, which in turn increases the proliferation of CD4hiCD25+ regulatory T cells. However, the relative importance between cell cycle control and cytokine production in regulating the proliferation of the CD4hiCD25+ regulatory T cells remains to be elucidated. Our results demonstrated that TLR5 is not involved.Ture of naive CD4+CD252CD45RO2 T cells and allogeneic CD40-activated B cells and filled histogram indicates staining obtained from the isotype-matched mAb for staining antibodies. Data were shown in Mean+SEM, n = 6. (B) 3H-thymidine incorporation of CD4hiCD25+ regulatory T cells in suppressive MLR at different regulatory T cells: responsders ratio. Data show Mean+SEM, n = 6. All data shown are from 3 independent experiments. NS, not significant, one way ANOVA with Tukey’s pairwise comparisons. doi:10.1371/journal.pone.0067969.gknowledge, is the first report concerning TLR5-related signals in iTregs. Here we found an increase of TLR5 expression in CD4hiCD25+ regulatory T cells. This was probably the consequence of CD4+ T cell activation during the co-culture. NF-kB and AP-1 binding sites are situated around the promoter region of TLR5 locus [37]. NF-kB and AP-1 are synthesized during T cell activation [38,39] and may bind to the promoter of TLR5, resulting in the transcription of TLR5. Interestingly, TLR5 can also activate the synthesis of NF-kB and AP-1 [14], thus it is possible that TLR5 was activated during the co-culture and positively feedback to the TLR5 expression. Since TLR5 expression was also up regulated inTLR5 Enhances Induced Treg Proliferationresting nTregs [22], it is possible that Foxp3 also up regulate the TLR5 expression but the precise mechanism remains to be investigated. In this study, we further found that blockade of TLR5 using anti-TLR5 blocking antibody reduced the proliferation of CD4hiCD25+ regulatory T cells through S phase arrest but did not increase the apoptosis of CD4hiCD25+ regulatory T cells or CD4+CD252 T cells. Since TLR5 was reported to be antiapoptotic [40], it was surprising that blockade of TLR5 did not increase the apoptosis of the cells. This may be explained by the observation from our previous investigation that large amount of IL-2 was produced by the CD40-activated B cells [28], thus it is possible that these IL-2 molecules rescued the CD4+ T cells from apoptosis. The S phase arrest of the CD4hiCD25+ regulatory T cells may be explained by the associated reduction of the ERK1/2 phosphorylation after TLR5 blockade. It is known that S phase exit or G2/M phase entry is controlled by cdk2 and cyclin A [41], the cdk2 is in turn activated by cdc25a [42], which could be activated and phosphorylated by p-ERK1/2 [43]. Therefore, it is speculated that the reduced ERK1/2 phosphorylation in the CD4hiCD25+ regulatory T cells decreased the expression and activation of cdc25a, thus in turn, the cdk2 activation, causing S phase arrest. However, the precise molecular mechanism between the reduced ERK1/2 phosphorylation and the S phase arrest remains to be elucidated. In addition, the reduced proliferation of the CD4hiCD25+ regulatory T cells may also be the result of reduced production of different cytokines. It was reported that stimulation of TLR5 using flagellin resulted in IL-8 production in epithelial cells and gastric cancer cells, increasing the proliferation of these cells [44,45], and the production of IFN-c [46]. Therefore, it is possible that TLR5-related signals may enhance 23977191 the production of IFN-c, which in turn increases the proliferation of CD4hiCD25+ regulatory T cells. However, the relative importance between cell cycle control and cytokine production in regulating the proliferation of the CD4hiCD25+ regulatory T cells remains to be elucidated. Our results demonstrated that TLR5 is not involved.