Laboratory of Medical Biotechnology, Research Institute of Biochemistry, Federal Research Center of Fundamental and Translational Medicine, Novosibirsk, Russia.
Scientific Department, Ural Research Institute for Phthisiopulmonology, National Medical Research Center of Tuberculosis and Infectious Diseases of Ministry of Health of the Russian Federation, Yekaterinburg, Russia.
Int J Mycobacteriol. 2020 Apr-Jun;9(2):176-184. doi: 10.4103/ijmy.ijmy_39_20.
Previously, the ex vivo cultures of alveolar macrophages were developed from the surgical samples of the lungs in patients with pulmonary tuberculosis (TB) to establish the unique features of Mycobacterium tuberculosis (Mtb) lifestyle in host cells, but the question has remained whether Mtb-infected cells are isolated from the human lungs or they may be the result of Mtb phagocytosis in ex vivo culture. The study was aimed to investigate Mtb uptake by TB patients' cells after ex vivo expansion.
Alveolar macrophages were infected with the Mtb clinical isolates in ex vivo culture, and the acid-fast Mtb loads in the cells were analyzed. Immunofluorescent staining and the examination of cytological and histological preparations by confocal microscopy were applied to detect Mtb ligands and macrophage surface markers.
The studies shown the lack of Mtb uptake by patients' alveolar macrophages during experimental infection with highly virulent Mtb clinical isolates containing pathogen-associated molecular patterns lipoarabinomannan and Ag38 at all used multiplicity of infection including a very high dose of infection. This fact was probably determined by the absence of pattern recognition receptors CD14, TLR2, and CD11b on the plasma membrane of human cells, likely, as a result of cellular processing from the resected lung tissues of patients.
The findings indicate that alveolar macrophages with single Mtb or Mtb in colonies, including those with cord-morphology, found in the ex vivo cell cultures of all TB patients examined, were isolated from the lungs, and they characterize the Mtb infection in patients at the time of surgery.
此前，通过对肺结核（TB）患者肺部的手术样本进行肺泡巨噬细胞的体外培养，以建立分枝杆菌（Mtb）在宿主细胞中生活方式的独特特征，但仍存在疑问，即感染 Mtb 的细胞是从人肺中分离出来的，还是可能是体外培养中 Mtb 吞噬作用的结果。本研究旨在调查 TB 患者细胞在体外扩增后对 Mtb 的摄取情况。
将 Mtb 临床分离株感染肺泡巨噬细胞进行体外培养，并分析细胞内抗酸 Mtb 负荷。应用免疫荧光染色和共聚焦显微镜检查细胞学和组织学标本，检测 Mtb 配体和巨噬细胞表面标志物。
研究表明，在使用包括高感染剂量在内的所有感染复数实验性感染高毒力 Mtb 临床分离株时，这些分离株含有病原体相关分子模式脂阿拉伯甘露聚糖和 Ag38，患者肺泡巨噬细胞并未摄取 Mtb。这一事实可能是由于人细胞的质膜上缺乏模式识别受体 CD14、TLR2 和 CD11b，可能是由于从患者切除的肺组织中进行了细胞处理。
这些发现表明，从所有检查的 TB 患者的体外细胞培养中分离出的单个 Mtb 或含有菌落的 Mtb（包括具有索状形态的菌落）的肺泡巨噬细胞是从肺部分离出来的，它们代表了手术时患者的 Mtb 感染情况。
