Fetal resorption rates and the levels of TSH, TT4 and TgAb in serum
Fetal resorption rates were increased in mTg group compared to the control group (45.63% vs. 3.1%; P<0.05 Fig1). TSH and TT4 levels in both control group and mTg group did not show significant differences (P<0.05 Table 1). Serum TgAb ELISA test showed that TgAb of the mice in mTg group was enhanced and significantly higher than in the control group, P<0.05 Table 2).
Flow cytometry to determine cell expression
Treg cells in the Placenta and Spleen
The proportion of Treg cells in the placenta and spleen was evaluated in mTg group and control group by flow cytometry. The results in Fig.2 showed that the percentage of Treg cells in the placenta in mTg group was lower than that of control group (3.39 ± 1.82% vs. 6.70 ± 1.79%; P<0.05). No significant differences were found in the frequency of CD4+CD25-Foxp3+ and CD4+CD25-Foxp3- as percentage of CD4+ T cells between mTg group and control group (91.23 ± 2.45% vs. 87.60 ± 2.26%; P>0.05) and 5.47 ± 2.21% vs. 5.71% ± 1.79%; P >0.05). In accordance with the following results, we also found that the proportion of Treg cells in the spleen in mTg group was lower than that of control group (2.91 ± 0.76% vs. 4.72 ± 0.57%; P<0.05). Meanwhile, no significant differences were found in the frequency of CD4+CD25-Foxp3+ as a percentage of CD4+T cells between mTg group and con group (87.12 ± 3.34% vs. 88.33 ± 3.22%; P>0.05). There was also no apparent difference in the frequency of CD4+CD25-Foxp3-as a percentage of CD4+ T cells between mTg group and control group (8.96 ± 1.27% vs. 6.94 ± 1.19%; P>0.05).
Th17 cells in placenta and spleen
The proportion of placental Th17 cells in CD4+ subsets in the mTg group was higher than that of Control group (2.27 ± 0.07% vs. 1.37 ± 0.09%; P = 0.031) (Fig.3.A,C), whereas the comparison of splenic Th17 cells between mTg group and Control group did not show statistically significant increases (2.09 ± 0.07% vs. 1.76 ± 0.13%; P>0.05) (Fig.3.B,D)
The ratio of Treg/Th17 in the placenta and spleen
As shown in Figures 3E and F, the ratio of placental Treg/IL-17 was reduced in mTg group compared to Control group (1.49 ± 0.31% vs. 4.89 ± 0.24%; P<0.05). Moreover, the ratio of Treg/IL-17 in the spleen in mTg group was significantly lower than that of Control group (1.39 ± 0.29% vs. 4.45 ± 0.36%; P<0.05).
PD-1 expression in CD4+T cell subsets in the placenta and spleen
The expression of PD-1 in placenta and spleen was investigated by flow cytometry. Our results revealed that PD-1 expression in mTg group was reduced in CD4+ T cell subset in the placenta compared with the control (6.51 ± 1.56% vs. 18.22 ± 2.12%; P<0.05) (Fig.4.A,C). PD-1 expression in mTg group was reduced in CD4+ T cell subset in the spleen compared with the control (8.52 ± 1.36%; P<0.05 vs. 20.22 ± 2.98%) (Fig.4.B,D).
PD-1 expression in Treg cells in placenta and spleen
We determined the expression of PD-1 in CD4+CD25+Foxp3+, CD4+CD25-Foxp3+ and CD4+CD25-Foxp3- cell subsets in the placenta and spleen by flow cytometry. The results showed that PD-1 expression on the placental Treg cells’ surface in mTg group was significantly lower than that of Control group (3.45 ± 0.96% vs. 37.60 ± 5.26%; P<0.01) (Fig.4.E,G), it was similar result in the spleen (2.45 ± 1.16% vs. 33.60 ± 3.26%; P<0.01) (Fig.4.F,H). In the placenta, the percentage of PD-1 in CD4+CD25-Foxp3+andCD4+CD25-Foxp3- in mTg group and Con group was (6.71% ± 0.99% vs. 4.57 ± 0.81%; P>0.05) and (7.60 ± 1.16% vs. 5.23 ± 1.05%; P>0.05) respectively; it was similar results in the spleen (7.72 ± 1.44% vs. 5.17 ± 1.28%; P>0.05) and (6.60 ± 1.27% vs. 4.23 ± 1.15%; P>0.05)
PD-L1 expression in placenta and spleen in Treg cells
Our results revealed that placental PD-L1 expression in CD4+ T cell subsets in mTg group was significantly reduced compared to that of Control group (15.11 ± 3.22% vs. 31.15 ± 2.46%; P<0.05) (Fig.5.A,C),it was similar results in spleen 18.45 ± 2.92% vs. 36.15 ± 3.76%; P<0.05) (Fig.5.B,D).
PD-L1 expression in CD4+T cell subset in placenta and spleen
The results clearly showed that PD-L1 expression in the placental Treg cell surface in mTg group was significantly lower than that of Control group (15.79 ± 4.56% vs. 41.15 ± 4.06%; P<0.01) (Fig.5.E,G). But there were no significant differences of PD-L1 expression in CD4+CD25-Foxp3+ cells and CD4+CD25-Foxp3- cells in the placenta of mTg group and Con group (5.37 ± 2.17% vs. 7.91 ± 1.99% and 8.33 ± 1.65% vs.10.10 ± 2.47%; P>0.05). PD-L1 expression on splenic Treg cell surface in mTg group was significantly decreased in comparison with control group (19.19 ± 4.10% vs. 43.67 ± 3.76%; P<0.01) (Fig.5.F,H). There were no significant differences of PD-L1 expression in CD4+CD25-Foxp3+ cells and CD4+CD25-Foxp3- cells in spleen of mTg group and Control group (8.99 ± 1.01% vs. 9.13 ± 1.87% and 7.07 ± 3.92% vs. 7.70 ± 2.91%; P>0.05).
Real-time PCR analysis of mRNA expression
Real-time amplification curve and corresponding amplification curve of each primer
The amplification power curve is S-shaped, with obvious exponential expansion period and plateau period. The whole curve runs smoothly (Fig.6.A), showing that the amplification result is ideal. The melting curve was analyzed by real-time PCR, the melting temperature was uniform, and the shape of the peak was sharp (Fig.6.B). It was confirmed that each primer was the only amplicon in the corresponding amplification product.
The mRNA level of Treg specific transcription factors and representative cytokines
The expression level of Treg cell-specific transcription factor Foxp3 and representative cytokine TGF-β mRNA in placental cells of mTg group was significantly lower than that of Control group (0.59 ± 0.17 vs. 1.01 ± 0.21; P<0.05) (Fig.7.A) and (0.73 ± 0.12 vs. 1.91 ± 0.15; P<0.05) (Fig.7.B). Similarly, the results clearly show that the mRNA level of specific transcription factor Foxp3 and the mRNA level of TGF- β in the spleen were consistent with that of the placenta (0.57 ± 0.31 vs. 1.18 ± 0.11; P<0.05) (Fig.7.C) and (0.53 ± 0.21 vs. 1.42 ± 0.35; P<0.05) (Fig.7.D).
The mRNA level of specific transcription factor RORγt and IL-17A in placenta and spleen
The expression level of Th17 cell-specific transcription factor RORγt and representative cytokine IL-17A mRNA in placental cells of mTg group were significantly higher than that of Control group (0.99 ± 0.05 vs. 0.37 ± 0.06; P<0.05) (Fig.7.E) and (1.07 ± 0.11 vs. 0.56 ± 0.07; P<0.05) (Fig.7.F).The expression levels of Th17 cell-specific transcription factor RORγt and representative cytokine IL-17A mRNA in spleen cells of mTg group were higher than those in Control group, but there were no statistical difference (1.13 ± 0.06 vs. 0.81 ± 0.13; P>0.05) (Fig.7.G) and (0.91 ± 0.07 vs. 0.72 ± 0.11; P>0.05) (Fig.7.H)
The mRNA level of PD-1 and PD-L1 in placenta and spleen
The mRNA levels of PD-1 and PD-L1 in the placenta and spleen in the mTg group and the Control group were determined by real-time RT-PCR. The mRNA level of PD-1 and PD-L1 in placenta in mTg group were significantly lower than that in the Control group (0.63 ± 0.13 vs. 1.51 ± 0.29; P<0.05) (Fig.7.I) and (1.55 ± 0.31 vs. 2.23 ± 0.19; P<0.05) (Fig.7.J), it was similar results in spleen (0.59±0.13 vs. 1.33 ± 0.31; P<0.05) (Fig.7.K) and (1.09 ± 0.35 vs. 2.11 ± 0.12; P<0.05) (Fig.7.L)