Chin J Schisto Control ›› 2021, Vol. 33 ›› Issue (2): 177-.

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PD-1/PD-L1 expression and its interaction with interferon-γ in Toxoplasma gondii-infected mice at middle and late pregnancy

XUE Sa, ZENG Yu-Lu, BI Xiang-Lian, LU Yun-Yu, ZHANG Da-Yi, ZHANG Li-Lin, HAN Xue, YANG Jun, FU Xiao-Yin, LIU Deng-Yu#br#   

  1. Department of Parasitology, School of Preclinical Medicine, Guangxi Medical University, Nanning 530021, Guangxi Zhuang Autonomous Region, China
  • Online:2021-04-30 Published:2021-04-30

PD-1/PD-L1在弓形虫感染小鼠妊娠中晚期的表达及与IFN-γ的相互调节作用

薛飒,曾雨露,闭香连,卢韵宇,张大义,张立林,韩雪,杨军,傅晓茵*,刘登宇*   

  1. 广西医科大学基础医学院寄生虫学教研室(南宁530021)
  • 作者简介:薛飒,女,硕士研究生。研究方向:病原生物学
  • 基金资助:
    国家自然科学基金(81760370);广西自然科学基金(2017GXNSFBA198154)

Abstract: Objective To explore the dynamic expression of programmed cell death?1 (PD?1) and its ligand PD?L1 at the maternal?fetal interface of mice post?infection with Toxoplasma gondii at early pregnancy and examine its interaction with interferon?γ (IFN?γ). Methods A total of 20 mice at day 0 of pregnancy were randomly assigned into 4 groups, including the 12?day pregnancy control group (12 dpn group), 12?day pregnancy and infection group (12 dpi group), 18?day pregnancy control group (18 dpn group) and 18?day pregnancy and infection group (18 dpi group), respectively. On the 6th day of the pregnancy, mice in the 12 dpi and 18 dpi groups were injected intraperitoneally with 150 tachyzoites of the T. gondii PRU strain, while mice in the 12 dpn and 18 dpn groups were injected with the same volume of PBS. All mice in the four groups were sacrificed on 12th and 18th day of the pregnancy, and the number of placenta and fetus was counted and the weight of placenta and fetus was measured. Then, the placental and uterine tissues of the pregnant mice in each group were sampled for pathological examinations. The mRNA expression of PD?1, PD?L1, T. gondii surface antigen SAG?1 and IFN?γ genes was quantified using a quantitative real?time PCR (qPCR) assay, and the correlation between PD?1 and IFN?γ expression was examined. In addition, the 12 dpn group, 12 dpi group, 18 dpn group, 18 dpi group, PBS negative control of the 12 pdi group and PBS negative control of the 18 dpi group were assigned, and the PD?1 expression was determined in the uterine and placenta tissues of the pregnant mice. Results Adverse pregnant outcomes were seen in mice in the 12 dpi and 18 dpi groups, including placental dysplasia and fetal maldevelopment, and the placental weights and fetal body weights were significantly lower in mice in the 12 dpi and 18 dpi groups than those in the 12 dpn and 18 dpn groups (t = 5.52, 11.44, 12.63 and 11.67, all P < 0.01). The histopathological examinations showed that the decidua and junctional regions of the placental tissues were loosely connected in the 12 dpi and 18 dpi groups, and a large number of inflammatory cells infiltration and congestion were seen in the placental and uterine tissues. qPCR assay detected significant differences in PD?1, PD?L1, IFN?γ and SAG?1 expression in the placental and uterine tissues among the 12 dpn, 12 dpi, 18 dpn and 18 dpi groups (F = 22.48, 51.23, 9.61, 47.49, 16.08, 21.52, 28.66 and 238.90, all P < 0.05), and the PD?1, PD?L1, IFN?γ and SAG?1 expression was all significantly higher in the placental and uterine tissues of mice in the 12 dpi group than in the 12 dpn group (all P values < 0.05). The PD?1 and PD?L1 expression was significantly lower in the placental tissues of mice in the 18 dpi group than in the 18 dpn group (all P values < 0.05), and the IFN?γ and SAG?1 expression was significantly higher in the placental and uterine tissues of mice in the 18 dpi group than in the 18 dpn group (all P values < 0.05), while the PD?1 and PD?L1 expression was significantly lower in the placental and uterine tissues of mice in the 18 dpi group than in the 12 dpi group (all P values < 0.05). Immunohistochemical staining showed PD?1 expression in the inflammatory cells of the placental tissues of mice in the 12 dpi group, and no apparent PD?1 expression in the 18 dpi group, while strongly positive PD?1 expression was found in the uterine epithelium of mice in the 12 dpi group, and mildly strong expression was in the 18 dpi group. In addition, the IFN?γ mRNA expression was positively correlated with the PD?1 mRNA expression in placental (rs = 0.99, P < 0.01) and uterine tissues of mice in the 12 dpi group (rs = 0.97, P < 0.01) and in placental (rs = 0.82, P < 0.01) and uterine tissues of mice in the 18 dpi group (rs = 0.81, P < 0.01). Conclusions Following T. gondii infection at early pregnancy, the PD?1 and PD?L1 expression shows a remarkable rise at middle pregnancy and a reduction at late pregnancy in placental and uterine tissues of mice, which appears the same tendency with IFN?γ expression during the same time period, and PD?1 expression positively correlates with IFN?γ expression. The dynamic expression of PD?1 and PD?L1 on the maternal?fetal interface of mice may be mutually mediated by IFN?γ induced by T. gondii infection.

Key words: Toxoplasma gondii, Pregnancy, Mice, PD?1/PD?L1, Interferon?γ

摘要: 目的 探讨程序性死亡蛋白(programmed death protein?1, PD?1)及其配体PD?L1在小鼠妊娠早期弓形虫感染后母胎界面的动态表达,及其与γ干扰素(interferon?γ,IFN?γ)的相互调节作用。方法 将20只孕0 d母鼠随机平均分为4组,即孕12 d对照组(12 dpn组)、孕12 d感染组(12 dpi组)和孕18 d对照组(18 dpn组)、感染组(18 dpi组)。在妊娠第6天时对12 dpi组、18 dpi组孕鼠给予150个弓形虫PRU株速殖子腹腔注射,12 dpn组、18 dpn组孕鼠则注射等量PBS。于小鼠妊娠第12天和第18天分别处死4组小鼠,计数胎盘、胎儿数量并称重。取各组孕鼠胎盘和子宫组织观察其组织病理变化,采用实时荧光定量聚合酶链反应(qPCR)检测胎盘、子宫组织中PD?1与PD?L1、弓形虫表面抗原SAG?1及细胞因子IFN?γ mRNA表达水平,并分析PD?1与IFN?γ表达的相关性。设置12 dpn、12 dpi、18 dpn、18 dpi组以及12 dpi PBS阴性对照组、18 dpi PBS阴性对照组,采用免疫组织化学染色检测孕鼠子宫、胎盘组织中PD?1表达水平。结果  12 dpi组和18 dpi组孕鼠均出现胎盘和胎儿发育不良等不良妊娠结局,且胎盘重量和胎儿体质量均低于12 dpn组、18 dpn组 (t = 5.52、11.44、12.63、11.67,P 均< 0.01)。组织病理观察结果显示,12 dpi组和18 dpi组孕鼠胎盘组织蜕膜及连接区连接疏松,胎盘及子宫组织中均见大量炎性细胞浸润和淤血。经qPCR检测发现,12 dpn、12 dpi、18 dpn组和18 dpi组小鼠胎盘和子宫组织中PD?1、PD?L1、IFN?γ和SAG?1表达均存在差异(F = 22.48、51.23、9.61、47.49、16.08、21.52、28.66、238.90,P 均< 0.05),其中12 dpi组小鼠胎盘(P均 < 0.05)及子宫组织中(P均 < 0.05)中PD?1、PD?L1、IFN?γ和SAG?1表达水平均较12 dpn组升高。与18 dpn组相比,18 dpi组小鼠胎盘组织中PD?1和PD?L1表达水平均下降(P 均< 0.05),胎盘和子宫组织中IFN?γ和SAG?1表达水平均升高(P均< 0.05)。与12 dpi组相比,18 dpi组孕鼠胎盘和子宫中PD?1和PD?L1表达水平均下降(P均< 0.05)。免疫组化染色结果显示,PD?1在12 dpi组孕鼠胎盘组织中浸润的炎性细胞上有表达,在18 dpi组中表达不明显;PD?1在12dpi组孕鼠子宫上皮呈强阳性表达,而在18 dpi组呈弱阳性表达。相关分析结果显示,12 dpi组孕鼠胎盘(rs = 0.99,P <0.01)、子宫(rs = 0.97,P <0.01),18 dpi组孕鼠胎盘(rs = 0.82,P < 0.05)、子宫(rs = 0.81,P <0.05)中 IFN?γ与PD?1 mRNA表达水平均呈正相关。结论 小鼠妊娠早期感染弓形虫后,胎盘和子宫中PD?1/PD?L1表达呈孕中期显著上升、孕晚期下降的动态变化,且与同期 IFN?γ表达趋势一致,其中PD?1与IFN?γ表达呈正相关;故推测在孕鼠妊娠中、晚期,母胎界面PD?1/PD?L1通路与弓形虫感染所诱导的IFN?γ有相互调节关系。

关键词: 刚地弓形虫, 妊娠, 小鼠, PD?1/PD?L1, γ干扰素

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