Modelling bovine tuberculosis infection in stem cell-derived macrophages

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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.