Be due to the suppression of pro-inflammatory cytokine secretion by trophoblasts. While HLA-G expression in IL-10 treated uninfected cells has no difference from uninfected cells without IL-10 treatment. This result showed that IL-10, as a negative regulated factor, may mainly modulate the expression of HLA-G. Interestingly, this down-regulation of HLA-G in the treated infected cells was observed at 36 and 60 hr post infectionIL-10 Protects T. gondii-Infected Trophoblastsand then HLA-G expression was up-regulated compared with the untreated infected group; this is consistent with the data that indicates that IL-10 induces HLA-G expression [19] and with results of our study in vivo [4]. The up-regulation of HLA-G in trophoblast cells by IL-10 at later stages of infection may enhance effects of inhibitory receptors expressed on decidual NK cells. It is likely that IL-10 plays roles both as an anti-inflammatory agent and in inducing HLA-G expression, which may be associated with the improvement of pregnancy outcomes in T. gondii-infected mice [4]. In addition, previous study showed that up-regulation of HLA-G could increase apoptosis of HTR8/svneo trophoblast cells [20]. In our study, similar result was showed that the apoptosis of BeWo cells and primary trophoblast cells were positively with the up-regulation of HLA-G expression; meanwhile, IL-10 treatment could cause a decrease of HLA-G in early stage of infection with T. gondii. These results demonstrated that the apoptosis decreased with IL-10 adding may be partly due to IL-10 induced upregulation of HLA-G. The cellular FLICE inhibitory protein, c-FLIP, an inhibitor of apoptosis, prevents the proteolytic Title Loaded From File activation of pro-caspase-8 by blocking caspase-8 recruitment by Fas associated death domain (FADD) [21,22]. Therefore, in a pro-apoptotic state, one would expect to see a decrease in c-FLIP and, subsequently, an increase in caspase-8 and caspase-3 activities [23,24]. We Title Loaded From File detected the long isoform c-FLIPL and the short isoform c-FLIPs in this study, and we found that T. gondii infection down-regulated levels of both cFLIP isomers, accompanied by an up-regulation of caspase-8, caspase-3. It has been reported that high local concentrations of the pro-caspase zymogen within the DISC death-inducing signaling complex (DISC) leads to auto-processing and activation of caspase-8 [25]. In this study, result showed that active caspase-3 was increased in infected cells compared to uninfected cells. This result suggested T. gondii infection might also directly facilitate the caspase-3 recruitment and activation. However, active caspase-8 was not detected in every group. This result indicated that there was no obvious change of active-caspase-8 among four groups. Therefore, these results indicated that T. gondii induced BeWo and trophoblast cells apoptosis mainly via active caspase-3 pathway induced apoptosis. We also have examined the effects of the recombinant human IL-10 on apoptosis of T. gondii-infected trophoblasts. Treatmentwith IL-10 significantly decreased T. gondii-induced apoptosis, upregulated c-FLIP expression, and down-regulated caspase-8, caspase-3 and active caspase-3 expression. IL-10 may reduce apoptosis through an up-regulation of c-FLIP, which in turn prevents the proteolytic activation of pro-caspase-8 by blocking caspase-8 recruitment by FADD, leading to a down-regulation of caspase-3 activity. These results are in accordance with previous analyses of IL-10 effects on c-FLIP a.Be due to the suppression of pro-inflammatory cytokine secretion by trophoblasts. While HLA-G expression in IL-10 treated uninfected cells has no difference from uninfected cells without IL-10 treatment. This result showed that IL-10, as a negative regulated factor, may mainly modulate the expression of HLA-G. Interestingly, this down-regulation of HLA-G in the treated infected cells was observed at 36 and 60 hr post infectionIL-10 Protects T. gondii-Infected Trophoblastsand then HLA-G expression was up-regulated compared with the untreated infected group; this is consistent with the data that indicates that IL-10 induces HLA-G expression [19] and with results of our study in vivo [4]. The up-regulation of HLA-G in trophoblast cells by IL-10 at later stages of infection may enhance effects of inhibitory receptors expressed on decidual NK cells. It is likely that IL-10 plays roles both as an anti-inflammatory agent and in inducing HLA-G expression, which may be associated with the improvement of pregnancy outcomes in T. gondii-infected mice [4]. In addition, previous study showed that up-regulation of HLA-G could increase apoptosis of HTR8/svneo trophoblast cells [20]. In our study, similar result was showed that the apoptosis of BeWo cells and primary trophoblast cells were positively with the up-regulation of HLA-G expression; meanwhile, IL-10 treatment could cause a decrease of HLA-G in early stage of infection with T. gondii. These results demonstrated that the apoptosis decreased with IL-10 adding may be partly due to IL-10 induced upregulation of HLA-G. The cellular FLICE inhibitory protein, c-FLIP, an inhibitor of apoptosis, prevents the proteolytic activation of pro-caspase-8 by blocking caspase-8 recruitment by Fas associated death domain (FADD) [21,22]. Therefore, in a pro-apoptotic state, one would expect to see a decrease in c-FLIP and, subsequently, an increase in caspase-8 and caspase-3 activities [23,24]. We detected the long isoform c-FLIPL and the short isoform c-FLIPs in this study, and we found that T. gondii infection down-regulated levels of both cFLIP isomers, accompanied by an up-regulation of caspase-8, caspase-3. It has been reported that high local concentrations of the pro-caspase zymogen within the DISC death-inducing signaling complex (DISC) leads to auto-processing and activation of caspase-8 [25]. In this study, result showed that active caspase-3 was increased in infected cells compared to uninfected cells. This result suggested T. gondii infection might also directly facilitate the caspase-3 recruitment and activation. However, active caspase-8 was not detected in every group. This result indicated that there was no obvious change of active-caspase-8 among four groups. Therefore, these results indicated that T. gondii induced BeWo and trophoblast cells apoptosis mainly via active caspase-3 pathway induced apoptosis. We also have examined the effects of the recombinant human IL-10 on apoptosis of T. gondii-infected trophoblasts. Treatmentwith IL-10 significantly decreased T. gondii-induced apoptosis, upregulated c-FLIP expression, and down-regulated caspase-8, caspase-3 and active caspase-3 expression. IL-10 may reduce apoptosis through an up-regulation of c-FLIP, which in turn prevents the proteolytic activation of pro-caspase-8 by blocking caspase-8 recruitment by FADD, leading to a down-regulation of caspase-3 activity. These results are in accordance with previous analyses of IL-10 effects on c-FLIP a.
