关键词: 刚地弓形虫, 滋养层细胞, 白细胞免疫球蛋白样受体B4, 细胞因子, 不良妊娠

Abstract:

Objective To investigate the changes in the expression of leukocyte immunoglobulin-like receptor subfamily B member 4 (LILRB4) on the surface of trophoblast cells following Toxoplasma gondii infection during pregnancy, and to examine its effects on trophoblast cell functions and pregnancy outcomes. Methods Human primary trophoblast cells were incubated in cell culture dishes at a density of 1 × 10⁷ cells per dish and divided into control and infected groups. Cells in the infected group were infected with T. gondii at a ratio of 1:1, incubated with APC-conjugated anti-human LILRB4 monoclonal antibody, fixed, permeabilized, incubated with APC-Cy7-conjugated anti-human cytokeratin 7 (CK7) monoclonal antibody and Alexa Fluor 488-conjugated anti-human vimentin monoclonal antibody, and the expression of LILRB4 was detected by flow cytometry. HTR-8/SVneo cells in the logarithmic growth phase were seeded into 24-well plates at a density of 2 × 10⁵ cells per well and divided into control and infected groups. Cells in the infected group were infected with T. gondii at a ratio of 1:1, incubated with mouse anti-human LILRB4 monoclonal antibody (1:200 dilution) and Elab Fluor 647-conjugated goat anti-mouse IgG monoclonal antibody (1:200 dilution) and enveloped. Images were photographed with a confocal laser scanning microscope, and the immunofluorescence intensity of LILRB4 protein was analyzed using the Image J software. Forty female and 20 male wild-type mice of the C57BL/6J strain and 40 female and 20 male LILRB4^-/- mice were randomly caged overnight, and presence of copulatory plugs in female mice on morning of the following day was considered gestational day 0. Wild-type pregnant mice were randomly assigned to wild-type control and infected groups, while LILRB4^-/- pregnant mice were randomly divided into LILRB4^-/- control and infected groups, with 6 mice in each group. Pregnant mice in wild-type and LILRB4^-/- infected groups were intraperitoneally injected with 300 T. gondii tachyzoites on day 8 of gestation, while animals in the control groups received the same volume of physiological saline. On day 14 of gestation, all mice were sacrificed and their uteri were dissected to evaluate placental and fetal development. The expression of LILRB4 protein was detected using immunohistochemistry in mouse placental tissues in wild-type control and infected groups, and the percentage of positive LILRB4 protein expression was analyzed with the software Image J. The relative expression of LILRB4 protein was determined using Western blotting in mouse placental tissues in wild-type control and infected groups. Western blotting was used to detect the relative protein expression levels of interleukin-10 (IL-10) and IL-12 in mouse placental tissues of wild-type control group, wild-type infected group, and LILRB4^-/- infected group. HTR-8/SVneo cells were seeded into cell culture dishes at a density of 1 × 10⁷ cells per dish and divided into control, infected, and LILRB4 blockade and infected group. Cells in the LILRB4 blockade and infected group were pretreated with LILRB4 neutralizing antibody for 2 hours, while cells in the infected and LILRB4 blockade and infected groups were infected with T. gondii at a ratio of 1:1. The relative expression of IL-10 and IL-12 proteins was quantified using Western blotting in HTR-8/SVneo cells in control, infected and LILRB4 blockade and infected groups. The invasive ability of HTR-8/SVneo cells was evaluated using Transwell assay following T. gondii infected. All statistical analyses were performed using the software GraphPad Prism 9.0. Difference of means between groups was tested for statistical significance with independent-sample t-test, and multiple-group comparisons were conducted with one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test. Results Flow cytometry detected a higher percentage of LILRB4-positive human primary trophoblast cells in the control group than in the infected group [(26.10 ± 1.99)% vs. (18.60 ± 1.13)%; t = 15.00, P < 0.01], and immunofluorescence staining revealed that the mean fluorescence intensity of LILRB4 protein was lower in the infected group than in the control group [(122.56 ± 5.24) vs. (149.27 ± 3.50); t = 5.36, P < 0.05]. Pregnant mice in the wild-type and LILRB4^-/- control groups presented shiny fur, normal mental state, and well placental and fetal development, while pregnant mice in the wild-type and LILRB4^-/- infected groups exhibited remarkable low spirits, shaggy and rough fur, placental ischemia, and poor fetal growth. The mouse placental [(54.82 ± 7.12) mg vs. (72.51 ± 1.11) mg; t = 4.25, P < 0.05] and fetal weights [(140.59 ± 3.19) mg vs. (201.03 ± 17.37) mg; t = 5.92, P < 0.01] were lower in the wild-type infected group than in the wild-type control group, and the mouse placental [(41.24 ± 2.80) mg vs. (54.82 ± 7.12) mg; t = 3.07, P < 0.05] and fetal weights [(68.25 ± 11.55) mg vs. (140.59 ± 3.19) mg; t = 10.45, P < 0.01] were lower in the LILRB4^-/- infected group than in the wild-type infected group. Immunohistochemical staining showed that the LILRB4 protein was highly positive in mouse placental tissues in the wild-type control group, with positive LILRB4 protein expression primarily found in cell membranes, and low LILRB4 protein expression was found in the wild-type infected group. The proportion of positive LILRB4 protein expression was lower in the wild-type infected group than in the wild-type control group [(16.13 ± 2.55)% vs. (36.64 ± 6.62)%; t = 5.00, P < 0.01]. Western blotting showed that the relative protein expression of LILRB4 in mouse placental tissues was higher in the wild-type control group than in the wild-type infected group [(1.15 ± 0.05) vs. (0.78 ± 0.10); t = 5.40, P < 0.05], and the relative IL-10 protein expression was lower in mouse placental tissues in the wild-type infected group than in the wild-type control group [(0.93 ± 0.09) vs. (1.28 ± 0.16); Tukey’s post hoc test, P < 0.05], and lower in the LILRB4^-/- infected group than in the wild-type infected group [(0.61 ± 0.10) vs. (0.93 ± 0.09); Tukey’s post hoc test, P < 0.05]. The relative IL-12 protein expression was higher in mouse placental tissues in the wild-type infected group than in the wild-type control group [(1.08 ± 0.11) vs. (0.55 ± 0.18); Tukey’s post hoc test, P < 0.05], and higher relative IL-12 protein expression was detected in the LILRB4^-/- infected group than in the wild-type infected group [(1.67 ± 0.29) vs. (1.08 ± 0.11); Tukey’s post hoc test, P < 0.05]. The relative IL-10 protein expression was lower in HTR-8/SVneo cells in the infected group than in the control group [(0.85 ± 0.05) vs. (1.15 ± 0.06); Tukey’s post hoc test, P < 0.05], and lower in the LILRB4 blockade and infected group (0.72 ± 0.04) than in the infected group (Tukey’s post hoc test, P < 0.05), and the relative IL-12 protein expression was higher in HTR-8/SVneo cells in the infected group than in the control group [(0.89 ± 0.10) vs. (0.52 ± 0.14); Tukey’s post hoc test, P < 0.05], and higher in the LILRB4 blockade and infected group than in the infected group [(1.21 ± 0.04) vs. (0.89 ± 0.10); Tukey’s post hoc test, P < 0.05]. In addition, the counts of invasive HTR-8/SVneo cells were lower in the infected group than in the control group [(178 ± 21) vs. (278 ± 18); t = 45.60, P < 0.01], and lower in the LILRB4 blockade and infected group (119 ± 9) than in the infected group (t = 5.50, P < 0.05). Conclusion T. gondii infection may significantly downregulate LILRB4 protein expression in human trophoblast cells and mouse placental tissues, and downregulation of LILRB4 promotes IL-12 expression and inhibits IL-10 production, thereby attenuating the invasive ability of trophoblast cells.

Key words: Toxoplasma gondii, Trophoblast cells, LILRB4, Cytokine, Adverse pregnancy