| Objective:Toxoplasma gondii infection in pregnant women leads to unfavorable pregnancy outcomes, and inflammatory responses contribute to the pathologic changes induced by this parasite infection. Tregs as well as other T subsets at fetal-maternal interface are crucial factors in maintaining normal pregnancy. It has reported that the reduction of splenic CD4+CD25+ regulatory T cells (Tregs) were associated with the abortion caused by this parasite. However, whether Th17 cells play important roles in this process is unknown. In the present study, we detected the percentages of Tregs and Thl7 cells in spleen and placenta, and the ratio of Treg/Th17 using a model of pregnant mice infected with Toxoplasma gondii.Methods:Toxoplasma gondii RH trophozoites, which were maintained by passage once every 54 hours in mice, were obtained from the peritoneal fluid of intraperitoneally-infected mice. The experimental pregnant mice were inoculated intraperitoneally (i.p.) with 200 tachyzoites in 200μl sterile PBS at gestational day 8 (gd 8), and control group with 200μl sterile PBS. The mice were killed at 6 day post-infection (6 dpi), uteri and spleen were removed, and the total numbers of implantations and resorption sites (abortions) were recorded. The frequencies of Tregs and Th17 cells from spleen and placenta were detected by flow cytometric analysis. In addition, the expression of related cytokine secretion (TGF-β, IL-17A), key transcription factors (Foxp3, RORyt) and IL-6 were examined by real-time PCR and ELISA.Results:①The number of living embryos of normal group were 45, and the number of death embryos and absorption embryos were 2, then the stillborn rate was 0.4%. But the infection group was 3,50,94.3% respectively and the stillborn rate was significantly higher than that of normal group (P<0.001)②The number of Tregs in spleen and placenta. The percentage of Tregs to CD4 positive splenocytes in experiment group was (20.30±6.37)%, the control group was (37.16±21.34)%. The differences between the two groups was significant (P<0.01). The percentage of Tregs to total splenocytes in experiment group was (0.19±0.13)%, the control group was (0.55±0.46)%. The differences between the two groups was significant (P<0.01). The percentage of Treg cells to CD4 positive placental cells in experiment group was (34.49±12.71)%, the control group was (56.89±17.10)%. The differences between the two groups was significant (P< 0.05). The percentage of Tregs to total placental cells in experiment group (0.55±0.52)% was significantly higher than the control group (0.31±0.39)%, (P< 0.05). Moreover, the percentage of Tregs in placenta was much higher than that in spleen (P< 0.05), indicating that Tregs may play important roles in abortion caused by Toxoplasma gondii infection.③The number of Th17 cells in spleen and placenta. The percentage of Th17 cells to CD4 positive splenocytes in experiment group was (29.39±9.29)%, the control group was (12.39±8.40)%. But the differences between the two groups have no significance. The percentage of Th17 cells to total splenocytes in experiment group was (0.36±0.12)%, the control group was (0.18±0.12)%. But the differences between the two groups have no significance. The percentage of Th17 cells to CD4 positive placental cells in experiment group (46.62±16.51)% was significantly lower than the control group (14.44±7.43)%, (P< 0.05). The percentage of Th17 cells to total placental cells in experiment group was (0.54±0.34)%, the control group was (0.08±0.07)%. The differences between the two groups was significant (P< 0.05). Moreover, the percentage of Th17 cells in placenta was much higher than that in spleen(P<0.05), indicating that Th17 cells may play important roles in abortion caused by Toxoplasma gondii infection.④The relative expression levels of IL-6,RORct and Foxp3 in the normal group were 0.45±0.09,0.37±0.16,0.61±0.12 respectively, the infection group were 0.6±0.09, 0.79±0.48,0.50±0.09 and there was significant difference between the two groups (P< 0.05, P< 0.05, P< 0.05). During pregnancy Toxoplasma gondii infection could increase the expression of the mice placental Th17 transcription factor and reduce the expression of placental Treg transcription factor. It will result in breakage of the Th17/Treg balance which the normal pregnant was need.⑤The average mRNA expression of IL-17 and TGF-βin infected group were 1.00±0.27,0.057±0.023 respectively, but the control group were 0.63±0.28,0.10±0.04. The difference between the two groups was significant (P<0.05, P<0.05). The average protein expression of IL-17 and TGF-P in infected group were 45.06±10.68 pg/ml, 22.92±6.01 pg/ml respectively, but the control group were 16.03±5.82 pg/ml, 62.14±11.02pg/ml. The difference between the two groups was significant (P< 0.05, P< 0.05). During pregnancy Toxoplasma gondii infection could increase the expression of the mice placental Th17 cytokines and reduce the expression of placental Treg cytokines. It will also result in breakage of the Th17/Threg balance which the normal pregnant was need.⑥The ratio of Treg/Thl7 was significantly decreased in placenta and spleen of infected group(0.368±0.086,0.323±0.065) compared with control group(3.276±0.872, 3.507±1.133) (P< 0.05, P< 0.05). Moreover, the ratio of TGF-β/IL-17 at protein level and mRNA level and Foxp3/RORyt at mRNA level was also significantly decreased in placenta of infected group compared with control group (P< 0.01,P< 0.01,P< 0.01).Conclusion:All above results showed that Toxoplasma gondii infection during early pregnancy could change some important immune moleculares expression and the normal Treg/Th17 balance was broken. This change will result in abnormal pregnancy. These molecular mechanisms may play an important role in human abnormal pregnancy caused by Toxoplasma gondii infection during early pregnancy. We hope our present research would bring new means to furtherly discuss the molecular immune mechanism underlying abnormal pregnancy caused by Toxoplasma gondii infection and finally find the effect means to block the vertical transmission and decrease the intrauterine infection of Toxoplasma gondii. |
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