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Analysis of Sterols in Human T Helper Cells / Eylan Yutuc
Swansea University Author: Eylan Yutuc
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PDF | E-Thesis – open access
Copyright: The author, Eylan Yutuc, 2017. This thesis is released under a Creative Commons Attribution 4.0 International (CC BY 4.0) License. Third party content is excluded for use under the license terms.
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DOI (Published version): 10.23889/SUthesis.60641
Abstract
Oxysterols are oxidised cholesterol species which can be in the form of one or more hydroxyl, carbonyl, epoxide and carboxylic acid of cholesterol and its precursors. They are intermediates in bile acid and steroid hormone biosynthesis pathway and were initially thought not to have any biological ac...
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Swansea
2017
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| Institution: | Swansea University |
| Degree level: | Doctoral |
| Degree name: | Ph.D |
| Supervisor: | Wang, Yuqin ; Griffiths William J. |
| URI: | https://cronfa.swan.ac.uk/Record/cronfa60641 |
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| Abstract: |
Oxysterols are oxidised cholesterol species which can be in the form of one or more hydroxyl, carbonyl, epoxide and carboxylic acid of cholesterol and its precursors. They are intermediates in bile acid and steroid hormone biosynthesis pathway and were initially thought not to have any biological activity but now emerged to have roles in lipid homeostasis, embryonic development and immunity. Specifically, activation of macrophages leads to 25-hydroxycholesterol (25-HC) production which contributes to suppression of viral entry and replication. In addition, 7α,25-dihydroxycholesterol (7α,25-DiHC) has been identified as a ligand for Epstein-Barr virus-induced gene 2 which influences B-cell migration and function. Key to coordinating effective immune responses are CD4 T cells. However, studies involving these cells and oxysterols are currently limited, especially in human CD4+ T cells. Moreover, there are currently no optimised mass spectrometry methods for sterol analysis of T cells. This study was inspired with the identification of autoimmune-associated TH17 cells and sterol ligand-activated RORγt as its master regulator, prompting a need for a method in analysing sterols in T cells. Here, in vitro cultures of cells isolated from healthy human donors were utilised to study and profile CD4+ T cell sterol production using an enzyme assisted derivatisation method and HPLC-ESI-MSn analysis. The function of the produced sterols and the mechanisms of production were also investigated using inhibitors of cholesterol biosynthetic pathway and downstream T cell receptor signalling. In addition, different subsets of CD4 T cells were also screened for sterols to link sterol production and immune function. Several oxysterols were detected in activated CD4+ T cells, including 25-HC, 7α,25-DiHC and 24S,25-epoxycholesterol (24S,25-EC), which increases with time but were absent in resting cells. The production of 25HC was seen to be unaffected by suppression of cholesterol biosynthesis compared to 24S,25-EC. Activation of CD4+ T cells stimulates several intracellular signalling pathways, and the PKC and MAPK pathways were found to have most influence in sterol production. However, the relationship between interferon and 25-HC in human CD4+ T cells was not observed in the current study. Lastly, differentiation of naïve CD4+ T cells towards the Treg-polarising conditions saw increased production of 7α,24S,25-trihydroxycholeterol not seen in either TH0- or TH2-polarising conditions. This study paves the way to progress the field of sterolomics and T cell immunology and to understand further the crosstalk between sterol metabolism and immune function. |
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| Item Description: |
ORCiD identifier: https://orcid.org/0000-0001-9971-1950 |
| Keywords: |
Sterols, Cholesterol, CD4, T cells, Oxysterol, LCMS, Mass Spectrometry |
| College: |
Faculty of Medicine, Health and Life Sciences |