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Characteristics of CD25 and Foxp3 Expression in Uterine Natural Killer Cells in Both Murine Model of Abortion and Human

Author: ChenLinLin
Tutor: LinZuo
School: Jinan University
Course: Developmental Biology
Keywords: abortion immune tolerance feto-maternal interface CD25 Foxp3 human mice
CLC: R714.2
Type: Master's thesis
Year: 2011
Downloads: 27
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Abstract

Spontaneous abortion is common during early pregnancy and the reasons for its occurrence are more complicated. In addition to genetic factors, endocrine disorders, infections, systemic diseases and genital deformities, immune factors are considered to be the main reason for recurrent spontaneous abortion during early-stage pregnancy in recent years. Although the strategy has been improved significantly under the efforts of scientific researchers in prevention and treatment of pregnancy-related diseases, it is difficult to further improve the efficacy limited by the fact that the molecular mechanism of diseases has not been fully elucidated. Under this condition, it is helpful for us to further understand the mechanism by which pregnancy tolerance is established and it is necessary for us to investigate the possible functions of T cells and NK cells which may play critical roles in affecting the outcome of pregnancy physiologically or pathologically.CBA/JxDBA/2J mice is a classic animal model of spontaneous abortion. The spontaneous resorption rate of embryos in CBA/J×DBA/2J mice was significantly higher than that of CBA/JxBALB/c mice with identical genotype (both H-2k×H-2d), but the pathogenesis has not been fully elucidated.Foxp3 is expressed in human CD4+CD25+ T cells and also expressed in the CD8+CD25-T cells. In mice, Foxp3 is specifically expressed in CD4+CD25+ T cells. The level of Foxp3 expression in CD4+ T cells was significantly higher than that of CD8+T cells. Foxp3 is believed to play an important role in the maintenance of normal pregnancy. It may be involved in establishment of local immune tolerance at the feto-maternal interface. CD25 and Foxp3 are also expressed in NK cells from both human and mice. It is necessary to further investigate the relationship between pregnancy maintenance and functional molecule or cytokine production such as CD25, Foxp3, TNF-α, IL-4, TGF-βand IL-10.Objectives1. To investigate the relationship between the status of CD25 and Foxp3 expression and murine spontaneous abortion.2. To analyze the effect of CD25 and Foxp3 on the establishment of human pregnancy.Materials and Methods1. CBA/JxDBA/2J mating combination was established and CBA/JxBALB/c was used as the normal control. Each female CBA/J mouse was co-caged with a male DBA/2J or BALB/c mouse. Twelve mice were used in both the experimental group and the control group. The females were killed on gestational day 12.5 to remove the uterus and calculate the percentage of embryo resorption. Magnetic affinity cell sorting was used to isolate uterine natural killer (NK) cells, and flow cytometry was performed to determine the status of CD25 and Foxp3 expression. The resorption rate of embryos and the proportion of NK cells producing the above-mentioned molecules were compared between CBA/JxDBA/2J and CBA/JxBALB/c mice at day 12.5 of gestation. Each experiment was repeated for 4 times.2. CBA/J×DBA/2J mating combination was established and CBA/JxBALB/c was used as the normal control. Eighteen mice were used in each group. The placentas were collected from the CBA/J×BALB/c mice to isolate mononuclear cells under sterile conditions. CD49b+ CD25+ Foxp3+ NK cells were purified using magnetic affinity cell sorting (MACS). The purified cells were cultured in RPMI1640 medium for about 24h. Eighteen pairs of mice from CBA/JxDBA/2J mating combination were divided into two groups, nine pairs in each group. Adoptive transfer of CD49b+CD25+Foxp3+ NK cells was performed at day 4.5 of gestation in CBA/JxDBA/2J mice by intravenous injection. The control group was injected with the same volume of sterile PBS. The females were killed at day 12.5 of gestation. The embryo resorption rate in both groups were calculated and compared. 3. Placental cells were colleted from 5 cases of spontaneous abortion patients and used as the experimental group. Placental cells from 5 cases of induced abortion patients were used as control group. Flow cytometry was performed to examine the status of CD25 and Foxp3 expression in human uterine NK cells.4. Data were statistically analyzed by SPSS13.0 software.χ2 test (Chi-square test) was performed to compare the resorption rate of embryo between murine groups. Independent sample t test was used to compare the percentage of CD25- and Foxp3-producing cells between both murine groups and human groups. P<0.05 is defined as significant difference. Data were presented as mean±standard deviation (x±s).ResultsThe resorption rate was significantly higher in CBA/J×DBA/2J mice than that of CBA/JxBALB/c mice (23.4%±15.2% versus 5.5%±5.1%;P<0.01), whereas the CD25+ and Foxp3+ NK cell percentages were significantly lower in CBA/JxDBA/2J mice than that of CBA/JxBALB/c mice (4.8%±2.8% and 10.5%±6.3% for CD25,0.3%±0.2% and 7.4%±4.2% for Foxp3, respectively, P<0.01 for both). It was shown in further experiments that no CD3 or CD4 molecules were produced by these cells. The percentage of embryo loss on gestational day 12.5 was significantly decreased in CBA/JxDBA/2J mice after the transfer of exogenous CD49b+CD25+Foxp3+ NK cells (8.0%±4.5% in the cell-transfer group and 24.7%±13.8% in control PBS group, respectively) (P<0.01). The CD25+ and Foxp3+ NK cell percentage in the patients who suffer from recurrent spontaneous abortion was significantly lower than that of normal control (29.68%±6.45% and 55.62%±6.22% for CD25, 29.26%±10.58% and 46.24%±9.95% for Foxp3, respectively, P<0.05 for both). ConclusionsThese results suggest that sufficient expression of CD25 and Foxp3 molecules by uterine NK cells may be correlated with the increased incidence of embryo resorption in CBA/J×DBA/2J model and increased abortion rate in some patients who suffer from recurrent spontaneous abortion. In addition, subfertility in CBA/J×DBA/2J mice may be partially attributed to insufficiency of CD25+ and Foxp3+ NK cells recruited into the pregnant uterus. Adoptive transfer of CD49b+CD25+Foxp3+NK cells could decrease the percentage of embryo loss in CBA/J×DBA/2J mice. Since CD25 and Foxp3 are expressed by CD3-CD4-CD49b+CD25+Foxp3+ cells, similarly as that in regulatory T cells, this result suggests that uterine NK cells might take place some roles played by regulatory T cells to a certain extent.

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