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.

分枝杆菌肉芽肿是结核病的核心和关键结构，由高度组织化的免疫细胞组成，包括通过特征性上皮样转化驱动肉芽肿形成的巨噬细胞。与体外组装模型相关的完整动物和注意事项中成像的困难严重限制了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，完全组织的斑马鱼肉芽肿被显微解剖并保持在三维（3D）培养中。这种方法可以高分辨率肉芽肿巨噬细胞动力学的显微镜，包括上皮样巨噬细胞的运动性和肉芽肿合并，同时保留关键的细菌和宿主特征。使用质谱法 我们发现先前在人类肉芽肿中发现的关键磷酸tidylinositol种类的活跃生产。我们还描述了一种方法，可以转染分离的肉芽肿，实现基因操作，并为宿主导向的小分子筛查提供概念验证，确定蛋白激酶C（PKC）信号为肉芽肿巨噬细胞组织的重要调节剂。