The Role Of Toxoplasma Gondii Excreted-secreted Antigens In Some Subpopulations Of Murine Immune Cells

Toxoplasma gondii is an opportunistic parasite with an extensive range of warm-blooded animal hosts, including humans. In an immunocompetent host infection often goes asymptomatic and unnoticed. However, maternal infection of T.gondii during pregnancy is associated with increased adverse pregnancy outcomes such as abortion, fetal death, stillbirth and congenital defects. Our previous study has demonstrated that infection with T. gondii can lead to a reduction of CD⁴⁺CD²⁵⁺ regulatory T cells in the spleens and the maternal-fetal interfaces of pregnant mice. Since accumulating evidence suggests that CD⁴⁺CD²⁵⁺ regulatory T cells play a major role in the maintenance of pregnancy, these alterations in CD⁴⁺CD²⁵⁺ regulatory T cells might be involved in the pathogenesis of abortion development caused by T. gondii. The excreted-secreted antigens (ESA) are expressed at both the tachyzoite and encysted bradyzoite stages of T. gondii. They represent the majority of the T. gondii circulating antigens in sera from hosts with acute T. gondii infection. ESA have been shown as important components in the process of invasion and replication of T. gondii within host cells. Recent evidence in pregnant mice indicates that T. gondii ESA may result in abortion and reduction of CD⁴⁺CD²⁵⁺ regulatory T cells, which is similar to that of the T. gondii infection. Up to now, few studies involve in the impact of this pooled ESA in fetus development and immune cells. Our study focus on the role of the function of CD⁴⁺CD²⁵⁺ regulatory T cells in abortion caused by T. gondii ESA treatment and the changes of CD⁴⁺T cells, CD8+T cells and NK cells in mice treated with T. gondii ESA.In this study, C57BL/6 murine pregnant model were established and treated with T. gondii ESA to verify whether fetal rejection could be prevented by adoptive transfer of Treg cells from normal pregnant mice. Then, the C57BL/6 normal murine model were established and treated with T. gondii ESA. The numbers of splenic CD⁴⁺T cells, CD⁴⁺CD²⁵⁺Foxp3+T cells, CD8+T cells and NK cells in all splenic cells were measured by flow cytometry, and the functions of CD⁴⁺CD²⁵⁺ regulatory T cells and NK cells were also assessed. In addition, the flow cytometric analysis was performed to investigate the apoptosis of splenic CD⁴⁺T cells and NK cells. The expression levels of Fas and caspase3 mRNA in splenic CD⁴⁺T cells were determined by RT-PCR. Finally, the influences of T. gondii ESA on CD⁴⁺T cells in different cell culture system were assessed by flow cytometry in vitro.The main results we got are as follows:1. Adoptive transfer of Tregs significantly diminished the abortion rates in ESA-treated pregnant mice As previous described by other groups and us, high abortion incidents were observed in both ESA-treated mice and T. gondii infected mice. The transfer of CD⁴⁺CD²⁵⁺Treg cells from normal pregnant mice significantly reduced the abortion rates in the ESA-treated mice. However, transfer of CD⁴⁺CD²⁵⁺Treg cells failed to prevent abortion in T. gondii infected pregnant mice. 2. Treatment with T. gondii ESA reduced the sizes of the splenic CD⁴⁺T cells and CD⁴⁺CD²⁵⁺ Foxp3+ T cells, but increase the splenic NK cells At 2 days post-treatment, the percentages of splenic CD⁴⁺T cells and CD⁴⁺CD²⁵⁺Foxp3+ T cells in all splenic cells tended to decreased in ESA treated mice compared with that in control mice. However, there was no statistically significant difference between the two groups. After a dramatic decrease, the percentages of splenic CD⁴⁺T cells and CD⁴⁺CD²⁵⁺Foxp3+ T cells in all splenic cells in ESA-treated mice reduced further at 4 days post-treatment, and reached significant lower levels than that of control mice at 6 days post-treatment. The percentages of splenic CD8+T cells in all splenic cells decreased a little in ESA treated group, but there was no statistically significant difference, when compared with that of control mice. However, the percentages of splenic NK cells in all splenic cells increased obviously in ESA-treated mice.3. The suppression function of CD⁴⁺CD²⁵⁺ T regulatory cells was decreased in T. gondii ESA treated mice CD⁴⁺CD²⁵⁺T cells from ESA treated mice could suppress the proliferation of immunocytes caused by anti-CD3 stimulation. At a serial treated days, 65.86%, 51.34% and 52.22% inhibition were observed when the CD⁴⁺CD25-T cells were co-cultured with CD⁴⁺CD²⁵⁺T cells from mice treated with ESA for 2, 4, and 6 days, respectively, which were significant lower than that of 76.28% in control. In addition, the cytokines IFN-γand IL-4 released by CD⁴⁺CD25-T cells in the co-culture supernatant were tested. The levels of IFN-γreleased by CD⁴⁺CD25-T cells when co-cultured with CD⁴⁺CD²⁵⁺T cells from ESA-treated mice were higher than that in controls, and they were in a time-dependent manner. These demonstrated that T. gondii ESA can decrease the suppressive functions of CD⁴⁺CD²⁵⁺ regulatory T cells.4. T. gondii ESA enhanced NK cell cytotoxicity against target cells At 2 days post-treatment, the activity of NK cells in splenic cells increased, when compared with that of control mice. Then, at 4 days post-treatment, the activity of NK cells in splenic cells increased further. However, at 6 days post treatment, the cytotoxicity of NK cells decreased, it was still higher than that in control mice. Consistent with the observation in vivo, when compared with that of control, the cytotoxicity of NK cells which were co-cultured with T. gondii ESA increased in vitro.5. Treatment with ESA induced the apoptosis of the splenic CD⁴⁺T cells The apoptotic rates of the splenic CD⁴⁺CD²⁵⁺T cells and CD⁴⁺CD25-T cells increased obviously in mice treated with ESA for 2, 4, and 6 days, when compared with that of control mice. However, there was no statistically significant difference of apoptotic rates in splenic NK cells between the ESA treated mice and the control mice. In addition, the expression levels of Fas and caspase3 mRNA in splenic CD⁴⁺T cells were determined by RT-PCR. After an initial increase at 2 days and 4 days post-treatment, the expression levels of Fas mRNA in splenic CD⁴⁺T cells from ESA treated mice increased further, to significant high levels than that of control mice at 6 days post-treatment. The mRNA levels of caspase3 in CD⁴⁺T cells from ESA treated mice increased significantly following the ESA treatment.6. ESA indirectly induced the apoptosis of the CD⁴⁺T cells in vitro when splenic mononuclear cells (SMCs) were co-cultured with T. gondii ESA, the apoptotic rates of CD⁴⁺T cells increased compared with that of control. However, when isolated CD⁴⁺T cells were co-cultured with T. gondii ESA, there were no significant apoptosis of CD⁴⁺T cells compared with that in control.In conclusion, our study gives the evidence that the decrease of CD⁴⁺CD²⁵⁺ Tregs contributes to the abortion caused by T. gondii ESA treatment. Our study also demonstrates that treatment with T. gondii ESA can lead to a reduction of murine splenic CD⁴⁺T cells both quantitatively and functionally. The reduction of CD⁴⁺T cells is not due to a direct destruction of T.gondii ESA but rather to an indirect effect. It may be associated with Fas/caspase3 mediated apoptosis which induced by ESA treatment. However, ESA treatment can increase the number of splenic NK cells and enhance the cytotoxicity of these cells.