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 Benzocaine biological activity 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 Pentagastrin web 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.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.
