Abstract: Objective　To investigate the regulatory role of the programmed cell death protein 1 (PD⁃1) and its ligand programmed cell death protein ligand 1 (PD⁃L1) signaling on the subtypes and functions of natural killer (NK) cells at the maternal⁃fetal interface during the second trimester in mice following Toxoplasma gondii infection during the first trimester. Methods　Twelve 6⁃ to 8⁃week⁃old female mice of the C57BL/6J strain were divided into a control group and an infection group, of 6 mice in each group. On the 6.5th day of pregnancy (Gd6.5), each pregnant mouse in the infection group was intraperitoneally injected with 150 tachyzoites of the Toxoplasma gondii PRU strain, while mice in the control group were injected with an equal volume of physiological saline. On the 12.5th day of pregnancy (Gd12.5), uterus and placenta tissues were sampled from pregnant mice for pathological observations, and the mRNA expression levels of PD⁃1, PD⁃L1, and tumor necrosis factor⁃α (TNF⁃α) were quantified in uterus and placenta tissues. The PD⁃1 and DX5 expression was measured on NK cells at the maternal⁃fetal interface using flow cytometry. In addition, the in vitro JEG⁃3 trophoblast cells and NK⁃92MI cells co⁃culture system was established as the control group, and the addition of T. gondii tachyzoites in the co⁃culture system served as the infection group. The PD⁃1, PD⁃L1, and DX5 mRNA expression was quantified in cells using real⁃time fluorescence quantitative reverse transcription PCR (RT⁃qPCR) assay, and the TNF⁃α concentration was measured in the cell culture supernatant using enzyme⁃linked immunosorbent assay (ELISA). Results　On Gd12.5, clear and intact cellular structures of placental decidual tissues were seen in pregnant mice in the control group, with no remarkable abnormal changes found in the uterine columnar epithelial cells, and inflammatory cell infiltration and blood stasis at varying degrees were found in uterine and placental tissues from pregnant mice in the infection group. The relative PD⁃1, PD⁃L1, and TNF⁃α mRNA expression was (1.004 ± 0.004), (1.001 ± 0.001), and (1.001 ± 0.001) in uterine tissues from pregnant mice in the control group and (2.480 ± 0.720), (3.355 ± 0.920), and (2.391 ± 0.073) in the infection group, respectively. The relative PD⁃1, PD⁃L1, and TNF⁃α mRNA expression was (1.007 ± 0.010), (1.006 ± 0.006), and (1.001 ± 0.001) in the uterine tissues in the control group and (6.948 ± 1.918), (3.225 ± 1.034), and (1.536 ± 0.150) in the infection group, respectively. The relative PD⁃1, PD⁃L1, and TNF⁃α mRNA expression was higher in both the uterine (t = 3.55, 4.43 and 33.02, all P values < 0.05) and placental tissues (t = 5.36, 3.72 and 6.18, all P values < 0.05) in the infection group than in the control group. Flow cytometry showed that the proportions of PD⁃1+ NK cells, PD⁃1+ DX5+ NK cells, and DX5+ NK cells were (12.200 ± 1.082)%, (9.373 ± 7.728)%, and (44.000 ± 4.095)% in uterine tissues from pregnant mice in the control group, and (21.733 ± 1.630)%, (18.767 ± 1.242)%, and (73.367 ± 0.611)% in the infection group, respectively. The proportions of PD⁃1+ NK cells, PD⁃1+ DX5+  NK cells, and DX5+ NK cells were (1.100 ± 0.510)%, (2.277 ± 1.337)%, and (96.167 ± 2.831)% in placental tissues from mice in the control group, and (26.867 ± 9.722)%, (23.433 ± 6.983)%, and (82.467 ± 2.248)% in the infection group, respectively. The proportions of PD⁃1+ NK cells (t = 8.45, P < 0.05) and DX5+ NK cells (t = 12.29, P < 0.05) were higher in uterine tissues from pregnant mice in the infection group than in the control group, and no significant difference was seen in the proportion of PD⁃1+DX5+ NK cells (Z = -1.09, P > 0.05). The proportions of PD⁃1+ NK cells (t = 4.58, P < 0.05) and PD⁃1+ DX5+ NK cells (t = 5.15, P < 0.05) were higher in placental tissues from pregnant mice in the infection group than in the control group, while the proportion of DX5+ NK cells was lower in the infection group than in the control group (t = -6.56, P < 0.05). RT⁃qPCR assay revealed that the relative PD⁃1, PD⁃L1, and DX5 mRNA expression was (1.010 ± 0.005), (1.002 ± 0.003), and (1.001 ± 0.001) in the JEG⁃3 cells and NK92MI cells co⁃culture system and (3.638 ± 1.258), (0.397 ± 0.158), and (4.267 ± 1.750) in the control group, and ELISA measured that the TNF⁃α concentration was higher in the cell culture supernatant in the infection group [(22.056 ± 3.205) pg/mL] than in the control group [(12.441 ± 0.001) pg/mL] (t = 5.20, P < 0.05). The PD⁃1(t = 3.62, P < 0.05) and DX5 mRNA expression (t = 3.23, P < 0.05) was higher in the infection group than in the control group, and the PD⁃L1 mRNA expression was lower in the infection group than in the control group (t = -6.63, P < 0.05). Conclusions　Following T. gondii infection, both PD⁃L1 expression and PD⁃1 expression on DX5+ NK cells at the maternal⁃fetal interface are upregulated in mice during the second trimester; however, the proportion of DX5+ NK cells decreases. These findings suggest that PD⁃1/PD⁃L1 signaling may suppress NK cell functions by modulating DX5+ NK cell subsets.

Key words: Toxoplasma gondii, Pregnancy, Programmed cell death protein 1, Programmed cell death protein ligand 1, Natural killer cell, Mouse

摘要： 目的　探讨小鼠孕早期感染弓形虫后，孕中期母胎界面程序性死亡蛋白1（programmed cell death protein⁃1, PD⁃1）及其程序性死亡蛋白配体1（programmed cell death protein ligand 1, PD⁃L1）通路对自然杀伤（natural killer，NK）细胞亚型和功能的调节作用。方法　将12只雌性C57BL/6J小鼠（6～8周龄）随机分为对照组和感染组，每组6只。妊娠第6.5天（Gd6.5）时，每只感染组孕鼠均采用腹腔注射方式感染150只弓形虫PRU株速殖子，对照组小鼠腹腔注射等量生理盐水。妊娠第12.5天（Gd12.5）时取孕鼠子宫和胎盘组织观察其病理变化，检测组织中PD⁃1、PD⁃L1及肿瘤坏死因子（tumor necrosis factor，TNF）⁃α mRNA表达水平，并采用流式细胞术检测母胎界面NK细胞上PD⁃1和DX5表达水平。此外，设置JEG⁃3滋养层细胞与NK92MI细胞体外共培养体系为对照组，在此基础上加入弓形虫速殖子作为感染组，通过实时荧光定量PCR（real⁃time quantitative reverse transcription polymerase chain reaction，RT⁃qPCR）法检测细胞PD⁃1、PD⁃L1及DX5 mRNA表达水平，采用酶联免疫吸附试验（enzyme⁃linked immunosorbent assay，ELISA）检测细胞培养上清液中TNF⁃α浓度。结果　Gd12.5时，对照组孕鼠胎盘蜕膜组织细胞结构清晰完整，子宫柱状上皮细胞无明显异常改变；感染组孕鼠子宫和胎盘组织均出现不同程度炎症细胞浸润和淤血。对照组孕鼠子宫组织中PD⁃1、PD⁃L1、TNF⁃α mRNA相对表达水平分别为1.004 ± 0.004、1.001 ± 0.001、1.001 ± 0.001，感染组分别为2.480 ± 0.720、3.355 ± 0.920、2.391 ± 0.073；对照组胎盘组织中PD⁃1、PD⁃L1、TNF⁃α mRNA相对表达水平分别为1.007 ± 0.010、1.006 ± 0.006、1.001 ± 0.001，感染组分别为6.948 ± 1.918、3.225 ± 1.034、1.536 ± 0.150；与对照组相比，感染组子宫组织（t = 3.55、4.43、33.02，P均< 0.05）和胎盘组织中PD⁃1、PD⁃L1、TNF⁃α mRNA表达水平（ t = 5.36、3.72、6.18，P均< 0.05）均升高。流式细胞术检测结果显示，对照组孕鼠子宫组织中PD⁃1+ NK细胞、PD⁃1+ DX5+ NK细胞和DX5+ NK细胞比例分别为（12.200 ± 1.082）%、（9.373 ± 7.728）%、（44.000 ± 4.095）%，感染组分别为（21.733 ± 1.630）%、（18.767 ± 1.242）%、（73.367 ± 0.611）%；对照组胎盘组织中PD⁃1+ NK细胞、PD⁃1+ DX5+ NK细胞和DX5+ NK细胞比例分别为（1.100 ± 0.510）%、（2.277 ± 1.337）%、（96.167 ± 2.831）%，感染组分别为（26.867 ± 9.722）%、（23.433 ± 6.983）%、（82.467 ± 2.248）%。与对照组相比，感染组小鼠子宫组织中PD⁃1+ NK细胞（t = 8.45，P < 0.05）、DX5+ NK细胞比例（t = 12.29，P < 0.05）升高，PD⁃1+ DX5+ NK细胞比例无显著变化（Z = -1.09，P > 0.05）；胎盘组织中PD⁃1+ NK细胞（t = 4.58，P < 0.05）、PD⁃1+ DX5+ NK细胞比例升高（t = 5.15，P < 0.05），而DX5+ NK细胞比例下降（t = -6.56，P < 0.05）。JEG⁃3细胞与NK92MI细胞体外共培养体系检测结果显示，对照组细胞上PD⁃1、PD⁃L1、DX5 mRNA相对表达水平分别为1.010 ± 0.005、1.002 ± 0.003、1.001 ± 0.001，感染组分别为3.638 ±1.258、0.397 ± 0.158、4.267 ± 1.750；对照组和感染组细胞培养上清液中TNF⁃α浓度分别为（12.441 ± 0.001）、（22.056 ± 3.205） pg/mL；与对照组相比，感染组PD⁃1（t = 3.62，P < 0.05）、DX5 mRNA（t = 3.23，P < 0.05）表达均上调，PD⁃L1 mRNA表达下调（t = -6.63，P < 0.05），且细胞培养上清液中TNF⁃α浓度升高（t = 5.20，P < 0.05）。结论　刚地弓形虫感染后，小鼠孕中期母胎界面中PD⁃L1表达水平及DX5+ NK细胞PD⁃1表达水平均上调、DX5+ NK细胞比例下降，提示PD⁃1/PD⁃L1通路可能通过调控DX5+ NK细胞亚群抑制NK细胞功能。

关键词: 刚地弓形虫, 妊娠, 程序性死亡蛋白1, 程序性死亡蛋白受体1, 自然杀伤细胞, 小鼠