Modelling bovine tuberculosis infection in stem cell-derived macrophages

多能干细胞的增殖和分化与CRISPR/Cas9基因编辑相结合，正在彻底改变医学和农业中开发疗法和药物的前景。在这个项目中，我们将应用这种转化技术来研究病原体牛分枝杆菌与其在哺乳动物中的主要靶宿主细胞巨噬细胞1之间的相互作用。M.牛结核病（TB）在牛和人类中引起显著的经济和健康问题。由于牛结核病和人结核病具有高度相似的免疫学和病理学特征，因此了解结核分枝杆菌的早期相互作用。牛及其宿主细胞将为动物和人类的疾病控制提供重要的见解。肺泡巨噬细胞是第一个遇到M.牛，是决定感染结果的关键。研究这些早期相互作用需要可靠的巨噬细胞来源，但是，当从活体动物中获得时，它们由于遗传背景或健康状况而变化，并且由于其有限的增殖能力和培养寿命而产生经常性成本。相比之下，多能干细胞无限增殖，易于进行遗传修饰，并且可以被诱导分化为巨噬细胞2。我们最近建立了从猪多能干细胞获得巨噬细胞的能力。将这种方法转化为从牛多能干细胞产生巨噬细胞3，为研究巨噬细胞与M.牛为了了解宿主-病原体相互作用，我们将使用流式细胞术，基因表达分析和功能测定。此外，我们还将使用无标记光学分析技术（拉曼）来研究M.牛这在以前已经被用来区分休眠和活跃的M。结核病，并将使我们能够确定是否M。牛的代谢在宿主巨噬细胞内改变。这些综合分析将揭示通过基因编辑进行操纵的途径。该项目的目标是：1。从牛多能干细胞中分化出巨噬细胞2。为了比较M.在离体和体外衍生的牛巨噬细胞中的牛感染3.目的：利用影像学技术分析细胞内M。牛4.对牛巨噬细胞M进行功能分析.通过基因编辑进行牛的相互作用。

The propagation and differentiation of pluripotent stem cells in tandem with CRISPR/Cas9 gene editing is revolutionising prospects for developing therapies and drug development in medicine and agriculture. In this project we will apply this transformational technology to study the interaction between the pathogen Mycobacterium bovis and its primary target host cell in mammals, the macrophage 1. M. bovis causes Tuberculosis (TB) in cattle and humans with significant economic and health concerns. Since bovine and human TB present with highly similar immunology and pathology, understanding the early interactions of M. bovis with its host cells will provide important insights for disease control in both animals and humans. The alveolar macrophage is the first host immune cell that encounters M. bovis and is pivotal in defining the outcome of infection. Studying these early interactions requires a reliable source of macrophages, but, when obtained from live animals they vary due to genetic background or health status, and incur recurrent costs due to their limited proliferative capacity and lifespan in culture. Pluripotent stem cells, by contrast, proliferate indefinitely, are readily amenable to genetic modification, and can be induced to differentiate into macrophages 2. We recently established the capability to derive macrophages from porcine pluripotent stem cells. Translation of this methodology to generate macrophages from bovine pluripotent stem cells3 provides new and exciting opportunities to study the interaction of macrophages with M. bovis. In order to understand the host-pathogen interaction we will use flow cytometry, gene expression analyses and functional assays. Alongside this we will use a label-free optical analysis technique (Raman), to investigate the intracellular life of M. bovis. This has been used previously to distinguish between dormant and active M. tuberculosis and will allow us to determine whether M. bovis metabolism is altered within host macrophages. These combined analyses will reveal pathways for manipulation through gene editing. The objectives of the project are:1. To differentiate macrophages from bovine pluripotent stem cells2. To compare M. bovis infection in ex vivo and in vitro derived bovine macrophages3. To use imaging to analyse intracellular M. bovis4. To functionally analyse bovine macrophage- M. bovis interactions through gene editing.