Department of Molecular Genetics and Microbiology, Duke University School of Medicine, Durham, NC, USA.
Public Health Research Institute, New Jersey Medical School, Rutgers, The State University of New Jersey, Newark, NJ, USA.
Nat Methods. 2018 Dec;15(12):1098-1107. doi: 10.1038/s41592-018-0215-8. Epub 2018 Nov 30.
A central and critical structure in tuberculosis, the mycobacterial granuloma consists of highly organized immune cells, including macrophages that drive granuloma formation through a characteristic epithelioid transformation. Difficulties in imaging within intact animals and caveats associated with in vitro assembly models have severely limited the study and experimental manipulation of mature granulomas. Here we describe a new ex vivo culture technique, wherein mature, fully organized zebrafish granulomas are microdissected and maintained in three-dimensional (3D) culture. This approach enables high-resolution microscopy of granuloma macrophage dynamics, including epithelioid macrophage motility and granuloma consolidation, while retaining key bacterial and host characteristics. Using mass spectrometry, we find active production of key phosphotidylinositol species identified previously in human granulomas. We also describe a method to transfect isolated granulomas, enabling genetic manipulation, and provide proof-of-concept for host-directed small-molecule screens, identifying protein kinase C (PKC) signaling as an important regulator of granuloma macrophage organization.
结核分枝杆菌中存在一个重要且关键的结构，即分枝杆菌肉芽肿，它由高度组织化的免疫细胞组成，包括巨噬细胞，巨噬细胞通过特征性的上皮样转化来驱动肉芽肿的形成。由于完整动物体内的成像困难以及体外组装模型的局限性，严重限制了对成熟肉芽肿的研究和实验操作。在这里，我们描述了一种新的离体培养技术，其中成熟的、完全组织化的斑马鱼肉芽肿被微分离并在三维（3D）培养中维持。这种方法能够对肉芽肿巨噬细胞动力学进行高分辨率显微镜观察，包括上皮样巨噬细胞的运动和肉芽肿的巩固，同时保留关键的细菌和宿主特征。通过质谱分析，我们发现了先前在人类肉芽肿中鉴定出的关键磷酯酰肌醇物种的活性产生。我们还描述了一种转染分离的肉芽肿的方法，从而能够进行基因操作，并提供了宿主定向小分子筛选的概念验证，确定蛋白激酶 C（PKC）信号作为肉芽肿巨噬细胞组织的重要调节剂。
