| Toxoplasma gondii (T. gondii) is a worldwide distributed obligate intracellular protozoan. It can infect all warm-blooded animals, including humans. Most T. gondii strains has been grouped into three genotypes by PCR-restriction fragment length polymorphism (RFLP), and RH strain which has been internationally recognized as virulent strain belongs to type I genotype. The distribution of T. gondii genotypes varies in different regions. Recent studies have shown that prevalence of T. gondii in China had limited genotypes, Chinal genotype was the dominant genotype, accounting for 75% of all isolated strains. T. gondii is an important opportunistic pathogen, whose infection is generally asymptomatic in immunocompetent persons but can cause serious disease in immunocompromised hosts, i.e. in AIDS or organ transplant patients. In these conditions, reactivation of a latent infection can result in toxoplasmic encephalitis (TE). T. gondii infection in pregnant woman may cause miscarriage, premature birth, stillbirth, birth defects, and the severity of congenital toxoplasmosis is related to the infection time of the pregnancy stage. As a distinctive subpopulation of T cells, CD4+CD25+regulatory T cells (Tregs) were considered as important regulatory players during pregnancy.The excreted-secreted antigens (ESA) are soluble antigens that are secreted or excreted by parasites. Our previous study found that ESA of RH strain could decrease the number of CD4+CD25+ regulatory T cells of pregnant mice in vivo, and the expression of their functional molecule-Foxp3 declined because of the apoptosis. These findings indicated that apoptosis of regulatory T cells induced by T. gondii ESA was an important cause of abortion. Many studies suggested that estrogen could increase the expression of Foxp3, and inhibit the apoptosis of some cells. However, whether estrogen played a role in apoptosis induced by ESA or not remained unclear. So this study focused on figuring out this issue.In this study, firstly we identified the genotype of RH stain and Chinal stain by RFLP-PCR. Then we prepared ESAs of RH strain and Chinal strain by serum-free culturing method. After removing endotoxin we determinated the concentration of protein. To test whether ESA could induce apoptosis of regulatory T cells, we cultured splenocytes and added ESA of different strains at different doses sustaining different time. The results showed that ESA could induce apoptosis of CD4+CD25+ regulatory T cells, and the degree of apoptosis enhanced when the dose of ESA was increased or stimulation time was prolonged. More importantly, RH strain ESA could cause higher apoptosis than Chinal strain at the same dose with the same stimulating time.To identify the factors in the process of apoptosis induced by ESA, we analyzed the IFN-y levels of cell culture supernatant by ELISA. The IFN-y level increased in ESA treatment group compared with control, and the IFN-y level of RH group was higher than that of China1 group. In order to prove the role of IFN-y in Treg apoptosis, we added anti-IFN-y antibody to culture, the apoptosis of regulatory T cells decreased. Then we used the IFN-y knockout mice, compared to the wildtype mice, apoptosis of regulatory T cells induced by ESA reduced.As one of the female hormone, estrogen not only can regulate female characteristics, but also can inhibit cell apoptosis. To investigate the ability of estrogen in suppressing apoptosis, we added estradiol to cell culture, after a certain time, added ESA. The result showed that estradiol could reduce apoptosis induced by ESA. To further verify this result, we sorted CD4+CD25+ regulatory T cells from the culture and a higher expression of Bcl-2 and lower expression of Caspase3 was observed in estradiol group. Then we used estrogen receptor blocker to interrupt ER action and found that the apoptosis rised in the blocking group. According to these results, we basically proved that estrogen could reduce apoptosis induced by ESA. |
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