| Toxoplasma gondii (T. gondii) is an obligate intracellular protozoan pathogen able to probably invade any cell with nuclear. T. gondii infection is generally asymptomatic in immunocompetent persons but can cause severe disease, such as pneumonia and encephalitis, in immunocompromised hosts. Most researchers prefer to define the fetal-maternal symbiosis as a tolerant state. As a unique subpopulation of T cells, CD4+CD25+ regulatory T cells were claimed to be important players in the tolerance towards the fetus bearing alloantigens. Our previous study demonstrated that CD4+CD25+ regulatory T cells were involved in the pathogenesis of abortion caused by T. gondii. Foxp3 gene, a master regulator of Tregs, its expression levels decreased in splenocytes and placentas of the infected mice. Moreover, T. gondii or T. gondii excreted-secreted antigens (ESA) could also result in the decrease of CD4+CD25+ regulatory T cells and the foetal loss.T. gondii infection may result in maternal immune deregulation and cause a variety of syndromes during pregnancy, such as miscarriage, spontaneous abortion, or fetal teratogenesis. Moreover, the severity of congenital toxoplasmosis depends on the stage of pregnancy at which infection takes place. Although previous reports have indicated that the abortion is closely dependent on the timing of maternal infection during pregnancy, the molecular mechanism remains unclear. This study was focused on the effects of T. gondii excreted-secreted antigens(ESA) on the pregnant outcomes and the CD4+CD25+Foxp3+ regulatory T cells at the different stages of pregnancy. In these study, T. gondii excreted-secreted antigens were injected intraperitoneally into pregnant mice at gestational day 5 (G5), gestational day 10 (G10), gestational day 15 (G15), respectively. The animals were sacrificed at gestational day 18 (G18). The results showed that all the fetuses and placentas became necrotic and haemorrhagic, and were resorbed after the administration of T. gondii ESA intraperitoneally at G5 (G5 ip), with the abortion rate of nearly 100%. Some embryos and placentas exhibited a necrotic and haemorrhagic appearance after the administration of T. gondii ESA intraperitoneally at G10 (G10 ip), with the abortion rate up to 56.20%.However, after the treatment of T. gondii ESA at G15, there was no visible fetal abnormality in pregnant mice, which was consistent with the mice in control group. These results indicated that injection of T. gondii ESA at different time results in the different consequence of pregnancy. Moreover, the abortion is depedent on the injection with ESA, independent of vertical transmission. We found that treatment of T. gondii ESA at early (G5) and intermediate (G10) stages of pregnancy could also lead to the decrease of CD4+CD25+Foxp3+ T cells. However, after the injection of T. gondii ESA at late pregnancy (G15), the percentage of CD4+CD25+Foxp3+ T cells were not decreased compared with that of control. The phenomenon could also be observed in the inguinal lymph nodes and peripheral blood lymphocytes (PBL), suggesting that T. gondii ESA could induce global changes of CD4+CD25+Foxp3+ T cells.Next, we tested whether the regulatory function of these cells from the injected group of mice had been damaged by evaluating the suppressing proliferation of CD4+CD25+T cells in vitro and Th2/Thl-like responses in vivo. We obtained purified CD4+CD25+ T cells from the normal pregnant mice and the mice with T. gondii ESA-injection at G5, G10 and G15, respectively. The decreased suppressive ability of CD4+CD25+ T cells was observed in mice with ESA-injection at G5 and G10. However, the inhibitory capacity of the CD4+CD25+ T cells was not decreased after the injection of T. gondii ESA at G15. Due to the capacity of CD4+CD25+ Treg cells controlling potentially detrimental IFN-y reactions during pregnancy, we detected the serum level of IFN-y after the treatment of T. gondii ESA. We found that the serum levels of IFN-y were up to 448.3 pg/ml at G5 ip, suggesting that the activity of CD4+CD25+ Tregs on the suppression of IFN-γ production was impaired. As expected, in all groups of mice, the serum IL-4 levels were not affected obviously. Taken together, the results demonstrated that the frequency and function of CD4+CD25+Foxp3+ T cells were diminished after treatment of T. gondii ESA at early and intermediate stages of pregnancy.To verify whether the diminished capacity of Tregs at G5 was causally associated with the fetal loss, we adoptively transferred CD4+CD25+T cells isolated from the spleens of normal pregnant mice (NP), pregnant mice injected with T. gondii ESA at G5 or those at G15 into T. gondii ESA-injected pregnant mice at G5, respectively. We found that the adoptive transfering of CD4+CD25+ T cells from pregnant mice injected with T. gondii ESA at G5 failed to prevent the abortion. Interestingly, more than 50% abortion could be prevented by adoptive transfering of CD4+CD25+ T cells from pregnant mice injected with T. gondii ESA at G15 and normal pregnant mice (NP). Taking together, these data supports that the diminished capacity of CD4+CD25+Tregs caused by T. gondii ESA at early pregnancy directly leads to abortion.To investigate whether apoptosis contributes to the reduction of CD4+CD25+ Tregs during T. gondii ESA injection at different stages of pregnancy, we performed flow cytometric analysis on splenocytes. T. gondii ESA treatment both at G5 ip and G10 ip, but not at G15 ip, could significantly induce apoptosis of CD4+CD25+ Tregs compared with the control. In line with the apoptosis, both mRNA and protein levels of activated Caspase-3 in CD4+CD25+ Tregs from mice with T. gondii ESA treatment at G5 ip and G10 ip, but not at G15 ip, were increased significantly. Also, the expression of Bcl-2 was decreased at G5 ip and G10 ip, but not at G15 ip. However, the levels of Bax presented no obvious changes among the groups. Therefore, T. gondii ESA treatment at G5 ip and G10 ip leads to the apoptosis of splenic CD4+CD25+ Tregs by down-regulating the expression of Bcl-2, but not up-regulating the expression of Bax. |
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