南方医科大学学报

南方医科大学学报 ›› 2020, Vol. 40 ›› Issue (07): 930-935.doi: 10.12122/j.issn.1673-4254.2020.07.03

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抑制CD96可促进NK细胞分泌IFN-γ并减轻小鼠肺部的衣原体感染

李 静，郑 晶，王敏达，张 艳，江益凡，张小凤，郭 普   

  • 出版日期:2020-07-20 发布日期:2020-07-20
  • 基金资助:

Inhibition of CD96 enhances interferon-γ secretion by natural killer cells to alleviate lung injury in mice with pulmonary Chlamydia muridarum infection

  

  • Online:2020-07-20 Published:2020-07-20

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摘要： 目的 通过调控CD96在C. muridarum肺炎小鼠体内的水平，探讨CD96调控NK细胞功能在衣原体肺部感染中的作用及可能机制。方法 选取雄性BALB/c小鼠45只，采用随机数字表法将小鼠分为Cm组、anti-CD96组及sham组，5只/组，进行3次独立重复实验。采用滴鼻吸入C. muridarum建立肺部感染模型，anti-CD96组：小鼠在感染基础上给予腹腔注射anti-CD96抗体（250 μg/只，每3 d给药1次）；Cm组：腹腔注射生理盐水；sham组：小鼠鼻吸入缓冲液。小鼠每日称重并绘制体质量曲线，造模5 d后，经颈椎脱臼法处死小鼠。采用荧光定量PCR与Western blot法检测CD96的表达；通过HE染色及病理评分评估小鼠肺炎症状；肺组织匀浆检测包涵体形成单位，评估肺部衣原体负荷；采用流式细胞术及ELISA等方法检测肺NK细胞产生IFN-γ的能力，以及NK细胞对巨噬细胞和Th1型细胞的免疫调节能力。结果 C. muridarum感染后第1、3、5天，采用磁珠分离各时间点以及Sham组小鼠肺NK细胞，结果显示NK细胞中CD96 mRNA表达显著降低（P<0.05），NK92细胞系经C. muridarum感染后6、12、24 h，CD96表达均明显下降（P<0.05）；Western blot验证anti-CD96抗体可有效中和小鼠肺组织中CD96表达（P<0.05），自感染后第3天开始anti-CD96组小鼠体质量下降程度较Cm组减轻（P<0.05），肺组织病理切片结果证实，anti-CD96组小鼠肺炎减轻。与Cm组相比，anti-CD96组小鼠肺部衣原体负荷降低（P<0.05）；Anti-CD96组小鼠肺组织中分泌IFN-γ的NK细胞（DX5+IFN-γ+）比例高于Cm组（P<0.05），与Cm组相比，anti-CD96组小鼠肺单个核细胞中IFN-γ的蛋白表达水平也显著升高（P< 0.05）。anti-CD96组与Cm组相比，NK细胞免疫调节作用也显著增强，小鼠肺脏中巨噬细胞比例增加（F4/80+，P<0.05），Th1应答增强（CD3+CD4+IFN-γ+，P<0.05）。结论 抑制CD96可减轻C. muridarum感染小鼠肺炎，可能与其增强NK细胞分泌IFN-γ能力及对固有和适应性免疫的调节作用有关。

关键词: 衣原体, 肺部感染, CD96, NK细胞, 干扰素-γ, 免疫调节

Abstract: Objective To assess the effect of neutralizing CD96 on natural killer (NK) cell functions in mice with pulmonary Chlamydia muridarum infection and explore the possible mechanism. Methods Male BALB/c mice were randomly divided into infection group (Cm group), anti-CD96 treatment group (anti-CD96 group) and control group (n=5). In the former two groups, C. muridarum was inoculated via intranasal administration to establish mouse models of pulmonary C. muridarum infection, and the mice in the control group received intranasal administration of the inhalation buffer. In anti-CD96 group, the mice were injected with anti-CD96 antibody intraperitoneally at the dose of 250 μg every 3 days after the infection; the mice in Cm group received intraperitoneal injections of saline. The body weight of the mice was recorded daily. The mice were sacrificed 5 days after C. muridarum infection, and CD96 expression was detected by quantitative real-time PCR and Western blotting. HE staining and pathological scores were used to evaluate pneumonia of the mice. The inclusion body forming units (IFUs) were detected in the lung tissue homogenates to assess lung tissue chlamydia load. Flow cytometry and ELISA were used to assess the capacity of the lung NK cells to produce interferon-γ (IFN-γ) and regulate macrophages and Th1 cells. Results C. muridarum infection inhibited CD96 expression in NK cells of the mice. Compared with those in Cm group, the mice in antiCD96 mice showed significantly milder lung inflammation (P<0.05) and reduced chlamydia load in the lung tissue (P<0.05). Neutralizing CD96 with anti-CD96 significantly enhanced IFN-γ secretion by the NK cells (P<0.05) and augmented the immunoregulatory effect of the NK cells shown by enhanced responses of the lung macrophages (P<0.05) and Th1 cells (P< 0.05). Conclusion Inhibition of CD96 alleviates pneumonia in C. muridarum-infected mice possibly by enhancing IFN-γ secretion by NK cells and augmenting the immunoregulatory effect of the NK cells on innate and adaptive immunity

Key words: C. muridarum, lung infection, CD96, natural killer cells, interferon-γ, immunoregulation