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EASTBIO Modelling bovine tuberculosis infection in stem cell-derived macrophages


Project Description

Royal (Dick) School of Veterinary Studies / The Roslin Institute

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 project objectives are:

1. To differentiate macrophages from bovine pluripotent stem cells
2. To compare M. bovis infection in ex vivo and in vitro derived bovine macrophages
3. To use imaging to analyse intracellular M. bovis
4. To functionally analyse bovine macrophage- M. bovis interactions through gene editing.

Training
Laboratory work in this project will involve state-of-the-art training in PSC propagation and CRISPR/Cas9 gene-editing (Burdon lab); training in techniques for the isolation of ex vivo bovine macrophages, M. bovis infection and immunological assays (Hope lab) and training in optical imaging (Raman) and analysis in the Dholakia lab at St Andrews. The student will attend weekly research seminars at the Roslin Institute. Opportunities to develop presentation and communication skills will include: presentations at regular lab meetings, one-on-one meetings with supervisor(s) on a 1-2 week basis, critical review of research papers at journal clubs, written skills developed in preparation of PhD annual reports, manuscript preparation, and poster and oral presentations at internal seminars and UK/international meetings. Additional Courses covering Research and Career Management, Personal Effectiveness and Networking are provided by the University of Edinburgh, and are offered during the course of research, tailored to student needs.

Eligibility:
All candidates should have or expect to have a minimum of an appropriate upper 2nd class degree. To qualify for full funding students must be UK or EU citizens who have been resident in the UK for 3 years prior to commencement.

Funding Notes

Applications:
Completed application form along with your supporting documents should be sent to our PGR student team at

References:
Please send the reference request form to two referees. Completed forms for University of Edinburgh, Royal (Dick) School of Veterinary Studies and the Roslin Institute project should be returned to by the closing date: 5th January 2020.

It is your responsibility to ensure that references are provided by the specified deadline.
Download application and reference forms via:
View Website

References

1. Queval, C. J. et al. The Macrophage: A Disputed Fortress in the Battle against Mycobacterium tuberculosis. Front. Microbiol. 8, 2284 (2017).
2. Takata, K. et al. Induced-Pluripotent-Stem-Cell-Derived Primitive Macrophages Provide a Platform for Modeling Tissue-Resident Macrophage Differentiation and Function. Immunity 47, 183-198.e6 (2017).
3. Bogliotti, Y. S. et al. Efficient derivation of stable primed pluripotent embryonic stem cells from bovine blastocysts. Proc. Natl. Acad. Sci. U. S. A. 115, 2090–2095 (2018).

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