Abstract:

Objective To investigate the effect of Toxoplasma gondii infection during pregnancy on CD73 expression in trophoblasts and decidual immune cells and to examine the association of CD73 expression with adverse pregnancy. Methods Four healthy decidua and villi tissue specimens in normal abortions at early pregnancy were collected. Decidual mononuclear cells were isolated with Ficoll-Paque lymphocyte separation solutions, and trophoblasts were isolated with the Percoll method. Purified decidual mononuclear cells and trophoblasts were divided into the control and infection groups, with 1 × 10⁷ cells in each group, and cells in the infection group were infected with T. gondii at a ratio of 3∶1 for 24 hours. Then, the CD73 expression was detected on the surface of trophoblasts, decidual natural killer (dNK) cells, decidual macrophage (dMφ) and decidual dendritic cell (dDC) using flow cytometry. Trophoblasts were divided into the control and infection groups, with 1 × 10⁷ cells in each group, and cells in the infection group were infected with T. gondii at a ratio of 1∶1 for 24 hours. Then, total protein was extracted from cells, and the relative CD73 protein expression was determined in trophoblasts using Western blotting post-infection with T. gondii. Sixty wild-type female mice of the C57BL/6J strain and 30 male mice of the C57BL/6J strain, and 60 female CD73^-/- mice and 30 male CD73^-/- mice were randomly caged overnight at a female to male ratio of 2∶1, respectively. Wild-type pregnant mice were randomly divided into the Wild-type control and Wild-type infection groups, and CD73^-/- pregnant mice were randomly divided into the CD73^-/- control and infection groups. Pregnant mice in the two infection groups were intraperitoneally injected with 400 T. gondii tachyzoites on day 8 of pregnancy, and pregnant mice in the two control groups were injected with the same volume of physiological saline. Mouse uterus was dissected on day 14 of pregnancy, and the placentas and fetal mice were observed, with the percentage of fetal abnormality estimated. The CD73 expression was detected in placental tissues using immunohistochemical staining. All statistical analyses were performed using the software GraphPad Prism 9.0, and differences of means were tested for statistical significance with Student’s t test for independent samples. Results Flow cytometry detected that the proportion of CD73-positive trophoblasts in total trophoblasts was (39.25 ± 1.43)% in the control group and was higher than that in the infection group (31.27 ± 2.79)% in the infection group (t = 5.31, P < 0.05). The proportions of CD73-positive cells were (38.17 ± 7.71)%, (25.80 ± 6.85)% and (9.39 ± 0.75)% on the surface of dDCs, dMφ and dNK cells in the control group higher, all were higher than that in the infection group (36.33 ± 7.64)%, (20.58 ± 5.18)% and (7.74 ± 1.08)% in the infection group (t = 4.25, 5.24 and 3.57; P < 0.05). Western blotting determined that the relative CD73 protein expression was 0.79 ± 0.07 in trophoblasts in the control group and 0.38 ± 0.05 in the infection group (t = 24.80, P < 0.01), and immunohistochemical staining showed that wild-type control placental tissue-positive cells were brown in color and expressed centrally on the lateral side of the placenta, especially near blood vessels, wild-type infected group had fewer positive cells were fewer in the wild-type infected group than in the control group, the grayscale value of CD73 expression was 9.79 ± 0.30 in mouse placental tissues in the wild-type infection group and 20.20 ± 4.60 in the wild-type control group (t = 3.89, P < 0.05). The placentas and fetuses of wild-type control and CD73^-/- control groups were well-developed, shiny, and the uterus was round and full, whereas pregnant rats of wild-type infected and CD73^-/- infected groups were obviously ischemic with severe bruising, and the fetuses and placentas were not shaped, and the fetuses were absorbed, and the placentas were underdeveloped. The mouse placenta and fetal body weights were (0.05 ± 0.01) g and (0.07 ± 0.03) g in the wild-type infection group, and (0.07 ± 0.01) g and (0.13 ± 0.08) g in the wild-type control group (t = 3.22 and 5.36, both P < 0.01). The mouse placenta and fetal body weights were (0.07 ± 0.04) g and (0.12 ± 0.01) g in the CD73^-/- control group, which were comparable to those in the wild-type control group (t = 0.62 and 0.13, both P > 0.05), and were (0.03 ± 0.00) g and (0.01 ± 0.00) g in the CD73^-/- infection group, which significantly reduced as compared with those in the wild-type infection group (t = 3.10 and 5.08, both P < 0.01). The proportions of fetal abnormality were 0 in the wild-type control group and CD73^-/- group, (55.73 ± 0.18)% in the wild-type infection group, which was significantly higher than in the wild-type control group (t = 7.79, P < 0.01), and was (76.00 ± 0.20)% in the CD73^-/- infection group, which significantly increased as compared with that in the wild-type infection group (t = 2.44, P < 0.05). Conclusion T. gondii infection at early pregnancy may remarkably down-regulate CD73 expression on the surface of trophoblasts and decidual immune cells (dNK cells, dMφ and dDC) in the maternal-fetal microenvironment, and CD73 down-regulation is strongly associated with adverse pregnancy caused by T. gondii infection.

Key words: Toxoplasma gondii, Adverse pregnancy, CD73, Trophoblast, Decidual immune cell, Immune microenvironment