Fan Lin, Shen Hongbo, Huang Huichang, Yang Rui, Yao Lan
Clinic and Research Center of Tuberculosis, Shanghai Key Lab of Tuberculosis, Shanghai Pulmonary Hospital, Tongji University School of Medicine, Shanghai, China.
Unit of Anti-tuberculosis Immunity, Key Laboratory of Molecular Virology and Immunology, Institute Pasteur of Shanghai, Chinese Academy of Sciences, Shanghai, China.
PLoS One. 2017 Mar 23;12(3):e0172549. doi: 10.1371/journal.pone.0172549. eCollection 2017.
Tuberculosis (TB) remains as a leading infectious disease worldwide. Our previous study showed interferon (IFN)-γ and CD3 T cell impairment in patients with severe cavitary pulmonary TB (PTB). However, the cause of the change in immune responses during the progression of TB is still poorly understood. In this study, eight newly diagnosed patients with severe cavitary and mild lesion non-cavity PTB were recruited, and three healthy volunteers were recruited as the control. RNA extracted from blood was tested by whole genome oligo microarrays. A PCR array was used to further test the same samples. Two additional groups of patients were recruited according to the same criteria with healthy control(HC) recruited as well and subjected to peripheral blood mononuclear cell isolation (PBMC)and analysis of TCF-7, β-catenin, cyclin D2, IFN-γ, and tumor necrosis factor (TNF)-α expression in CD14- cells (lymphocytes) and CD14+ cells by quantitative PCR. The changes of expression of β-catenin, CD69+ and IFN-γ by CD3+, CD14- and CD14+ cells in vitro with stimulation of LiCl were tested by flow cytometry. Whole genome oligo microarrays showed a significant decrease in expression of the Wnt signaling pathway in severe PTB patients. Further analysis of the Wnt pathway by PCR array indicated that TCF-7, β-catenin, and cyclin D2 expression was significantly reduced in severe PTB patients compared with mild PTB patients. In the additionally recruited patients, TCF-7, β-catenin, and cyclin D2 were expressed in both CD14+ and CD14- cells, while β-catenin was decreased significantly in CD14- cells compared with CD14+ cells in severe PTB patients, and IFN-γ and TNF-α expression in CD14- cells was also reduced significantly in severe PTB patients. β-catenin can directly trigger T cell activation and IFN-γsecretion in PBMCs stimulated for 24 hours. These findings indicate that Wnt pathway and its key genes, such as β-catenin, were impaired in blood cells of patients with severe PTB. Therefore, Wnt/β-catenin pathway is closely associated with T cell proliferation and TB lesion deterioration.
结核病（TB）仍是全球主要的传染病。我们之前的研究表明，重症空洞型肺结核（PTB）患者存在干扰素（IFN）-γ和CD3 T细胞功能受损。然而，结核病进展过程中免疫反应变化的原因仍知之甚少。在本研究中，招募了8名新诊断的重症空洞型和轻度病变非空洞型PTB患者，并招募了3名健康志愿者作为对照。从血液中提取的RNA通过全基因组寡核苷酸微阵列进行检测。使用PCR阵列进一步检测相同样本。按照相同标准另外招募了两组患者，并招募了健康对照（HC），对外周血单个核细胞进行分离（PBMC），并通过定量PCR分析CD14-细胞（淋巴细胞）和CD14+细胞中TCF-7、β-连环蛋白、细胞周期蛋白D2、IFN-γ和肿瘤坏死因子（TNF）-α的表达。通过流式细胞术检测用LiCl刺激后，体外CD3+、CD14-和CD14+细胞中β-连环蛋白、CD69+和IFN-γ表达的变化。全基因组寡核苷酸微阵列显示重症PTB患者中Wnt信号通路的表达显著降低。通过PCR阵列对Wnt通路进行的进一步分析表明，与轻度PTB患者相比，重症PTB患者中TCF-7、β-连环蛋白和细胞周期蛋白D2的表达显著降低。在另外招募的患者中，TCF-7、β-连环蛋白和细胞周期蛋白D2在CD14+和CD14-细胞中均有表达，而在重症PTB患者中，与CD14+细胞相比，CD14-细胞中的β-连环蛋白显著降低，重症PTB患者CD14-细胞中的IFN-γ和TNF-α表达也显著降低。β-连环蛋白可直接触发PBMC中刺激24小时后的T细胞活化和IFN-γ分泌。这些发现表明，重症PTB患者血细胞中的Wnt通路及其关键基因（如β-连环蛋白）受损。因此，Wnt/β-连环蛋白通路与T细胞增殖和TB病变恶化密切相关。
