Project Outline

Control of the immune system activity is crucial to the maintenance of health, regenerative responses in healthy ageing but it also plays a role in the induction of disease when controlled poorly. Macrophages are a key immune cell that controls pro inflammatory and anti-inflammatory responses and by doing so are crucial cells for the maintenance of health and homeostasis. The phenotype of macrophages can change in response to their microenvironment and extracellular vesicles (EV) appear able to control this phenotype but the mechanisms remain poorly defined. Intercellular communication mediated through extracellular vesicle (EV) release is only recently acknowledged as a novel mechanism of cellular crosstalk1. Because of their diverse biomolecular cargo, EV are capable of transmitting complex instructions to the recipient cells beyond the ‘classical’ soluble factor signalling.

Our previous research suggests that EV are metabolically-active extracellular compartments, rich in bioactive lipid mediators of inflammation and active enzymes responsible for their synthesis2. Such EV may act as extracellular messengers capable of modulating immune responses in recipient cells and tissues. Our work shows that the balance between the carried enzymes and their bioactive products within EV may determine the outcome (‘activation’ or ‘silencing’) in the recipient cells of the immune system, such as macrophages. Why is this important? Understanding how this balance of enzymes and bioactive lipids maintains health and is altered in disease and communicated in the surrounding tissues will highlight the importance of EV-mediated signalling in both health and disease.

Our recent work identified an intact sphingolipid enzymatic pathway in EV isolated from brain tissue, a pathway known to play an important role in inflammation. Sphingolipids are integral components of cell membranes with bioactive properties involved in regulation of many aspects of cell fate and function3. These lipids, as well as the enzymes responsible for their biosynthesis, are highly enriched in the central nervous system where they regulate a variety of cellular processes in physiological and pathophysiological conditions. We have also identified that brain-derived EV are enriched in different sphingolipid classes, with both pro-inflammatory and anti-inflammatory properties. However, to date, the role of bioactive sphingolipids and enzymes responsible for their synthesis in EV-mediated immunomodulation within the central nervous system remains ill-defined.

This innovative research proposal aims to understand the balance of these enzymes and bioactive lipids in EV in healthy and pathological brain and how this may be altered to promote either neuro-inflammation or tissue homeostasis. The fundamental nature of lipid signalling will further pave the way to a better understanding of neurodegenerative disorders and will define potential novel targets for future therapeutic approaches.

To answer these questions, this project will use a broad range of bioanalytical techniques including cell culture, nanoparticle isolation and analysis, mass spectrometry and data analysis, protein biochemistry, enzyme activity assays, flow cytometry and assays of immune cell function and inflammation.

References

¹ Van Niel, G., D’Angelo, G., & Raposo (2018). Shedding light on the cell biology of extracellular vesicles. Nature Reviews: Molecular Cell Biology: DOI: 10.1038/nrm.2017.125

² Grant, L., Milic, I. & Devitt, A. (2019). Apoptotic cell derived extracellular vesicles: structure-function relationships. Biochemical Society Transactions: 47(2): 509-516.

³ Verderio, C., Gabrielli, M., Giussani, P., (2018) Role of sphingolipids in the biogenesis and biological activity of extracellular vesicles, J Lipid Res, 59(8),1325-1340.

Techniques

• Mass spectrometry of lipids and proteins: to profile and relatively quantify lipid and protein profiles of cells and derived EV with a focus on immunomodulating enzymes
• Cell culture: Cell culture of a range of cell lines and primary cells for the isolation of EV, isolation of primary cells from human peripheral blood for the immune-system crosstalk functional studies.
• Vesicle analyses: isolation and analysis of EV structure and function using Exoid tenable resistive pulse sensing, nano flow cytometry, analysis of immune-modulating capability in vitro using established functional assays.
• Imaging and analysis: Flow cytometry, Confocal microscopy, automated cell imaging for functional studies
• Protein biochemistry: Western blotting, Cytokine assays, Enzyme activity assays