Decrease Of CD4～+CD25～+ Regulatory T Cells In The Spleens And Maternal-fetal Interfaces Of Pregnant Mice Infected With Toxoplasma Gondii

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 acute toxoplasmosis during pregnancy is associated with increased adverse pregnancy outcomes such as abortion, fetal death, stillbirth and congenital defects. But to date, whether vertical infection is the only factor contributing to abortion or fetal death during infection is still unclear. Studies in experimental models and human populations showed that T. gondii may result in abortion and fetal resorption without direct transmission of the parasite to the fetus. Thus, in addition to vertical infection, some unknown factors may contribute to the abortion development during T. gondii infection. The maternal immune system has to tolerate the persistence of paternal alloantigen during pregnancy. Several specialized mechanisms have evolved to help the fetus evade maternal immune attack. Accumulating evidence suggest that CD4~+ CD25~+ regulatory T cells play a major role in the maintenance of pregnancy. Investigations in mice revealed that the absence of CD4~+ CD25~+ regulatory T cells during pregnancy would result in fetus rejection, which would be effectively prevented by adoptive transfer of CD4~+ CD25~+ regulatory T cells from normal pregnant mice. Additionally, human studies also documented the importance of CD4~+ CD25~+ regulatory T cells in pregnancy. However, whether CD4~+ CD25~+ Tregs are involved in the pathogenesis of abortion caused by T. gondii has not been reported. Our study focused on the changes of CD4~+ CD25~+ regulatory T cells in pregnant mice infected with T. gondii.In this study, we established C57BL/6 pregnant murine model infected with T. gondii. Control groups were set up at the same time. At each indicated time point, the expression levels of Foxp3 mRNA in splenic cells and placentas were determined by real-time quantitative PCR. The percentages of splenic CD4~+ CD25~+ Foxp3~+ T cells in all splenic cells were measured by Flow cytometry, and the absolute numbers of splenic CD4~+ CD25~+ Foxp3~+ T cells were also assessed. The distribution of Foxp3~+ cells at the maternal-fetal interface was evaluated by immunohistochemistry. In addition, we performed flow cytometric analysis by the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) technique to investigate the apoptosis of splenic CD4~+ CD25~+ T cells, which was further confirmed by Hoechst 33342 staining.The main results we got are as follows:1. Pregnant mice infected with T. gondii presented abnormal pregnancies At 6 days post-infection, all fetuses from the infected mice became necrotic, hemorrhagic, and resorbed. The abortion rates (the ratio of resorption sites to the total number of implantation sites) was significantly higher in infected mice compared with gestational age-matched control mice. The histological analysis revealed hemorrhagic sites and lymphocyte infiltration in the placentas from aborted mice compared to control mice.2. Decreased expression levels of Foxp3 mRNA in splenocytes following infection with T. gondii After an initial decrease at 4 days post-infection, the expression levels of Foxp3 mRNA in pregnant mice exposed to T. gondii reduced further, to significant lower levels than that of control mice at 6 days post-infection. Consistent with the observation in pregnant mice infected with T. gondii, the mRNA levels of Foxp3 in splenocytes from nonpregnant mice decreased significantly following infection.3. Infection with T. gondii reduced the size of the splenic CD4~+ CD25~+ Foxp3~+ T cell population At 2 days post-infection, The percentages of splenic CD4~+ CD25~+ Foxp3~+ T cells in all splenic cells decreased in infected group compared with gestational age-matched control mice, however, there was no statistically significant difference between the two groups. After a dramatic decrease at 4 days post-infection, the percentages of splenic CD4~+CD25~+Foxp3~+ T cells in all splenic cells in T. gondii-infected mice reduced further, to significant lower levels than that of control mice at 6 days post-infection. At 4 days and 6 days post-infection, the absolute numbers of splenic CD4~+ CD25~+ Foxp3~+ T cells were also greatly reduced in infected group compared with control group. A similar kinetics of the percentages of splenic CD4~+ CD25~+ Foxp3~+ T cells in all splenic cells and absolute numbers of splenic CD4~+ CD25~+ Foxp3~+ T cells was also observed in nonpregnant mice during the process of T. gondii infection.4. Foxp3 mRNA levels and Foxp3~+ cell numbers were both down regulated at the feto-maternal interfaces of T. gondii-infected mice By day 6 post-infection, the expression levels of placental Foxp3 mRNA in infected mice decreased significantly when compared with gestational age-matched control mice. Infected mice also presented decreased number of Foxp3~+ cells in placentas when compared to control mice. All these data provided the evidence that infection with T. gondii can lead to a reduction of regulatory T cells at the maternal-fetal interface.5. Splenic CD4~+CD25~+ T cells underwent apoptosis during T. gondii infection A small but significantly higher proportion of splenic CD4~+CD25~+ T cells were TUNEL~+ in pregnant mice infected with T. gondii than in gestational age-matched controls. The higher apoptotic rates in CD4~+ CD25~+ T cells from infected mice were further confirmed by Hoechst 33342 staining. We found that only some necrotic cells with swollen nuclei could be seen in the CD4~+ CD25~+ T cells from uninfected mice, while more apoptotic cells with condensed or fragmented bright nuclei were observed amidst the CD4~+ CD25~+ T cells from T. gondii-infected mice. In conclusion, our study demonstrates that infection with T. gondii can lead to a reduction of CD4~+ CD25~+ regulatory T cells in the spleens and maternal-fetal interfaces of pregnant mice. The reduction of splenic CD4~+ CD25~+ regulatory T cells is associated with apoptosis induced by T. gondii infection. These alterations in CD4~+ CD25~+ regulatory T cells might be involved in the pathogenesis of abortion development caused by T. gondii.