弓形虫感染对小鼠和人子宫自然杀伤细胞的影响

目的　观察弓形虫感染对小鼠和人子宫自然杀伤细胞（uterine natural killer cells， uNK cells）比例、数量、分化和功能的影响，探讨uNK细胞在弓形虫感染致早孕流产中的作用。方法　妊娠第6.5天给予孕鼠腹腔注射弓形虫速殖子，建立孕早期弓形虫感染致流产小鼠模型，妊娠第9.5天分离小鼠子宫淋巴细胞。取人正常妊娠孕早期流产新鲜蜕膜组织，分离人子宫淋巴细胞，与弓形虫RH株速殖子体外共培养48 h（弓形虫与子宫淋巴细胞比例分别为0.5∶1、1∶1、2∶1）。采用流式细胞术检测小鼠uNK细胞表型（CD122、NK1.1、DX5）、人uNK细胞表型（CD3、CD56、CD11b、CD27）及胞内细胞因子γ⁃干扰素（interferon⁃γ， IFN⁃γ）和肿瘤坏死因子⁃α（tumor necrosis factor⁃α， TNF⁃α）表达水平。磁珠分选小鼠和人uNK细胞，以小鼠YAC⁃1或人K562细胞作为靶细胞、以小鼠或人uNK细胞作为效应细胞，采用乳酸脱氢酶（lactate dehydrogenase， LDH）释放法检测效应细胞/靶细胞比例分别为1∶1、5∶1、10∶1、20∶1时uNK细胞的杀伤功能。结果　妊娠第9.5天，弓形虫感染组小鼠流产率较对照组显著上升（83.02% vs. 3.51%；[χ2] = 71.359，P < 0.001）。与对照组比较，弓形虫感染组小鼠uNK细胞绝对数量显著降低[（4 547 ± 1 610）个/只 vs.（8 978 ± 3 339）个/只；U = 2.000，P < 0.05]、细胞表面NK1.1表达水平显著下降[（74.53 ± 8.37）% vs.（93.00 ± 1.11）%；U = 0.000，P < 0.05]、NK1.1⁃DX5⁃细胞比例显著上升[（20.10 ± 8.03）% vs.（5.04 ± 0.68）%；U = 0.000，P < 0.05]、NK1.1+DX5+细胞比例显著下降[（21.70 ± 12.48）% vs.（45.75 ± 2.26）%；U = 0.000，P < 0.05]、IFN⁃γ表达水平显著升高[（16.74 ± 1.36）% vs.（8.13 ± 1.90）%；U = 0.000，P < 0.05]，但TNF⁃α表达水平无显著改变[（67.98 ± 9.20）% vs.（52.93 ± 10.42）%；U = 2.000，P > 0.05]。弓形虫感染组小鼠uNK细胞表现出较强杀伤能力，且杀伤作用随着效应细胞/靶细胞比例升高逐渐增强；效应细胞/靶细胞比例为1∶1、5∶1、10∶1、20∶1时，感染组uNK细胞对YAC⁃1细胞的杀伤活性分别为2.30%、4.32%、8.12%和12.65%，对照组分别为1.21%、1.63%、2.51%和3.22%。将人子宫淋巴细胞与弓形虫RH株速殖子共培养48 h后，弓形虫感染组人uNK细胞在子宫淋巴细胞中所占比例较对照组显著降低[弓形虫2∶1组 vs. 对照组：（6.61 ± 1.75）% vs.（17.48 ± 4.81）%；F = 7.307，P < 0.01]、绝对数量显著减少[弓形虫2∶1组vs. 对照组：（12 104 ± 5 726）个/孔 vs.（65 285 ± 21 810）个/孔；H =11.540，P < 0.01]。弓形虫感染组uNK细胞中，CD56brightCD16⁃调节型NK细胞亚群比例较对照组减少（弓形虫2∶1组vs. 对照组：（25.25 ± 5.90）% vs.（36.03 ± 4.51）%；F = 3.213，P > 0.05]、CD56dimCD16+杀伤型NK细胞亚群比例增加[弓形虫2∶1组vs. 对照组：（11.15 ± 2.15）% vs.（7.09 ± 2.24）%，F = 2.992，P > 0.05]，两者比值显著降低[弓形虫2∶1组vs. 对照组：（2.37 ± 0.92）vs.（5.58 ± 2.39）；H = 8.228，P < 0.05]；CD11b+CD27⁃细胞比例显著上升[弓形虫2∶1组vs. 对照组：（30.28 ± 6.91）% vs.（17.48 ± 4.67）%；H = 6.556，P < 0.05]，IFN⁃γ [弓形虫2∶1组vs. 对照组：（14.13 ± 1.28）% vs.（15.19 ± 1.64）%；F = 1.639，P > 0.05]、TNF⁃α表达水平无显著改变[弓形虫2∶1组vs. 对照组：（54.76 ± 10.02）% vs.（50.33 ±3.67）%；F = 0.415，P > 0.05]。弓形虫感染组人uNK细胞具有较强杀伤能力，且杀伤作用随着效应细胞/靶细胞比例升高逐渐增强；效应细胞/靶细胞比例为1∶1、5∶1、10∶1、20∶1时，感染组uNK细胞对K562细胞的杀伤活性分别为11.90%、28.11%、49.91%和73.35%，对照组分别为11.21%、21.63%、33.51%和48.22%。结论　弓形虫感染对小鼠和人uNK细胞分化和分泌功能产生了不同影响，但均可引起小鼠和人uNK细胞绝对数量减少、杀伤功能增强。

Effects of Toxoplasma gondii infection on mouse and human uterine natural killer cells

Objective　To examine the effects of Toxoplasma gondii infection on the proportion, quantity, differentiation and function of mouse and human uterine natural killer cells (uNK cells), so as to explore the role of uNK cells in abortion of early pregnancy caused by T. gondii infection. Methods　Pregnant mice were injected intraperitoneally with T. gondii tachyzoites on day 6.5 of pregnancy, and the abortion mouse model caused by T. gondii infections was constructed. Mouse uterine lymphocytes were isolated on day 9.5 of pregnancy. Human uterine lymphocytes were isolated from fresh human decidual specimens after abortion in normal early pregnancy and co⁃cultured with tachyzoites of the T. gondii RH strain for 48 h at T. gondii/uterine lymphocytes ratios of 0.5∶1, 1∶1 and 2∶1. The phenotypes of mouse uNK cells (CD122, NK1.1, DX5) and human uNK cells (CD3, CD56, CD11b, CD27) and the expression of intracellular cytokines interferon⁃γ (IFN⁃γ) and tumor necrosis factor⁃α (TNF⁃ α) were detected by flow cytometry. Mouse and human uNK cells were sorted by magnetic beads, and the cytotoxicity of uNK cells was tested using the lactate dehydrogenase (LDH) release assay at effector/target cell ratios of 1∶1, 5∶1, 10∶1 and 20∶1 with mouse or human uNK cells as effector cells and mouse YAC⁃1 cells or human K562 cells as target cells. Results　On day 9.5 of pregnancy， the mouse abortion rate was significantly higher in the infected group than that in the control group (83.02% vs. 3.51%; [χ2] = 71.359, P < 0.001). Significantly lower absolute number of uNK cells [(4 547 ± 1 610) cells/mouse vs. (8 978 ± 3 339) cells/mouse; U = 2.000， P < 0.05], lower NK1.1 expression on uNK cell surface [(74.53 ± 8.37)% vs. (93.00 ± 1.11)%; U = 0.000, P < 0.05], higher proportion of NK1.1⁃DX5⁃ cells [(20.10 ± 8.03)% vs. (5.04 ± 0.68)%; U = 0.000, P < 0.05], lower proportion of NK1.1+DX5+ cells [(21.70 ± 12.48)% vs. (45.75 ± 2.26)%; U = 0.000, P < 0.05] and higher IFN⁃γ expression [(16.74 ± 1.36)% vs. (8.13 ± 1.90)%; U = 0.000, P < 0.05] were detected in the infected group than in the control group, while no significant difference was seen in TNF⁃α expression between the two groups [(67.98 ± 9.20)% vs. (52.93 ± 10.42)%; U = 2.000, P > 0.05]. The mouse uNK cells showed a strong cytotoxicity in the infected group, and the cytotoxicity gradually increased with the effector/target cell ratio. The cytotoxicity of uNK cells against YAC⁃1 cells was 2.30%, 4.32%, 8.12% and 12.65% in the infected group and 1.21%, 1.63%, 2.51% and 3.22% in the control group at effector/target cell ratios of 1∶1, 5∶1, 10∶1 and 20∶1, respectively. Following co⁃culture of human uterine lymphocytes and tachyzoites of the T. gondii RH strain for 48 h, the proportion [TOX 2∶1 group vs. control group: (6.61 ± 1.75)% vs. (17.48 ± 4.81)%; F = 7.307, P < 0.01], and absolute number of human uNK cells in uterine lymphocytes of human uNK cells in uterine lymphocytes [TOX 2∶1 group vs. control group: (12 104 ± 5 726) cells/well vs. (65 285 ± 21 810) cells/well; H = 11.540, P < 0.01] were significantly lower in the infected group than in the control group. A lower proportion of CD56brightCD16⁃ NK cells [TOX 2∶1 group vs. control group: (25.25 ± 5.90)% vs. (36.03 ± 4.51)%; F = 3.213, P > 0.05] and higher proportion of CD56dimCD16+ NK cells [TOX 2∶1 group vs. control group: (11.15 ± 2.15)% vs. (7.09 ± 2.24)%; F = 2.992, P > 0.05] were detected in uNK cells in the infected group than in the control group, and the ratio of CD56brightCD16⁃ cells/CD56dimCD16+ cells was significantly lower in the infected group than in the control group [TOX 2∶1 group vs. control group: (2.37 ± 0.92) vs. (5.58 ± 2.39); H = 8.228, P < 0.05]. In addition， the proportion of CD11b+CD27⁃ cells in human uNK cells was significantly higher in the infected group than in the control group [TOX 2∶1 group vs. control group: (30.28 ± 6.91)% vs. (17.48 ± 4.67)%; H = 6.556, P < 0.05]， while no significant differences were found between the two groups in terms of IFN⁃γ [TOX 2∶1 group vs. control group: (14.13 ± 1.28)% vs. (15.19 ± 1.64)%; F = 1.639, P > 0.05] or TNF⁃α expression [TOX 2∶1 group vs. control group: (54.76 ± 10.02)% vs. (50.33 ± 3.67)%; F = 0.415, P > 0.05]. Human uNK cells presented a strong cytotoxicity in the infected group, and the cytotoxicity gradually increased with the effector/target cell ratio. The cytotoxicity of human uNK cells against K562 cells was 11.90%, 28.11%, 49.91% and 73.35% in the infected group and 12.21%, 21.63%, 33.51% and 48.22% in the control group at effector/target cell ratios of 1∶1, 5∶1,10∶1 and 20∶1, respectively. Conclusions　T. gondii infection presents diverse effects on the differentiation and secretion ability of mouse and human uNK cells. However, T. gondii infection causes a reduction in the absolute number and enhances the cytotoxicity of both mouse and human uNK cells.