Principal Supervisor: Dr Ewan RossLink opens in a new window

Co-supervisor: Dr Douglas Browning

PhD project title: Investigating the host pathogen interaction between Salmonella and the bone marrow stem cell niche

University of Registration: University of Aston

Project outline:

The bone marrow stem cell niche is a critically important structure for regulating the activation and differentiation of haematopoietic stem cells (HSCs) for both normal homeostasis, as well as in times of stress such as injury or infection¹. Control of this niche is critical to ensure that overproduction of immune cells does not occur leading to blood borne conditions such as leukaemias. It is now clear that this important niche is more responsive to environmental signals than first thought, as it can be modulated by soluble factors released during inflammation as well as in direct contact with pathogens². Indeed it is hypothesised that recurrent exposure to pathogens can ‘educate’ this bone marrow niche, making it more responsive to infections and allowing it to generate the necessary precursor cells to fight the invading organism more efficiently. This host-pathogen interaction has important implications for our understanding of how our environment affects our immune response, and informs on how our stem cells have the capacity to adapt and are altered after exposure to distinct pathogens,

We have previously demonstrated that systemic exposure to the bacteria Salmonella directly modulates the bone marrow niche environment, causing activation and expansion of naïve HSCs³. Importantly, after resolution of the infection, subsets of these progenitors have altered differentiation potential depending on the length of time of exposure to the pathogen. More recently, preliminary work has demonstrated that mesenchymal stromal cells (MSCs), key regulatory cells of the BM niche can be directly infected with Salmonella, and we believe these cells act as a reservoir of infection, even after the systemic infection has been resolved. How the function of MSCs are altered after infection is an intriguing question and has important implications in our understanding of how the BM niche is regulated and how it can be influenced by changes to these supporting cells.

In this project, you will investigate the direct physiological effects of Salmonella infection on MSC biology using a multidisciplinary approach. This novel project will use our in house expertise and provide training in molecular biology, microbiology, stem cell biology and the immune response. You will monitor the effects of Salmonella infection on primary BM MSCs using fluorescently labelled bacteria and assess changes to the important functions of MSCs; using diagnostic assays to measure changes in their ability to modulate other immune cells (T cell suppression, generation of regulatory T cells, macrophage polarisation), secrete soluble factors (cytokines, chemokines and extracellular vesicles) and support the in vitro expansion of HSCs in our bone marrow niche models. As part of this study, you will also engineer new variants of laboratory Salmonella strains for use in these assays. Engineering new fluorescently tagged Salmonella will allow a dynamic tracking of how this bacteria interacts and infects MSCs, where it resides in the cell and how long it can remain within a cell.

This cross-discipline project is ideal for someone who has a strong interest in how pathogens interact with cells of the immune system and is keen to learn new technical skills in molecular biology, immunology and the application of cell based assays.

 

References:

1. Haematopoietic stem cell activity and interactions within the niche. Pinho S. and Frenette P.S. Nat Rev Molec Cell Biol. 2019. https://www.nature.com/articles/s41580-019-0103-9
2. The Role of the Bone Marrow Microenvironment in the Response to Infection. Johnson CB, Zhang J, Lucas D. Front Immunol. 2020 Nov 25;11:585402 PMID: 33324404 https://www.frontiersin.org/articles/10.3389/fimmu.2020.585402/full
3. Resolving Salmonella infection reveals dynamic and persisting changes in murine bone marrow progenitor cell phenotype and function. Ross EA et al. Eur J Immunol. 2014 Aug;44(8):2318-30. PMID: 24825601 https://onlinelibrary.wiley.com/doi/10.1002/eji.201344350
   
   

BBSRC Strategic Research Priority: Understanding the rules of life - Immunology, Microbiology, and Stem Cells

Techniques that will be undertaken during the project:

Cell Culture: The project will predominantly use primary human bone marrow mesenchymal stem cells (MSCs). Full training in culturing these cells and diagnostic assays to monitor their growth, functional activity and differentiation will all be provided.

Imaging and analysis: Assessing how Salmonella interacts with MSCs will be primarily monitored using microscopy. Fluorescently labelled bacteria allow real-time analysis of the infection and colonisation of the MSCs. You will use a range of techniques to assess changes to MSCs functionality and physiology. Flow cytometry will be used routinely to monitor alterations to MSCs physiology including cell surface phenotyping, mitochondrial activity and how they influence other immune cells in co-culture. Changes in gene expression linked to the induction of differentiation of naïve MSCs to stromal precursors will be assessed using qPCR. Changes in soluble factors release will be quantified using ELISAs.

Molecular Biology: You will use PCR, DNA manipulation, purification and cloning to generate new strains of Salmonella. Importantly, new tagged versions of this bacteria will be produced to allow precise evaluation of its interaction with the host cells

 

Contact: Dr Ewan RossLink opens in a new window