Identification of endogenous ligands for the Retinoid-related Orphan Receptor gamma

Lead Research Organisation: Swansea University
Department Name: Institute of Life Science Medical School

Abstract

In the body's defence against invading pathogens, T cells are essential to protect the body from infection. However, uncontrolled and persistent T cell responses can drive the onset of autoimmunity i.e. the immune system reacts against self tissue. TH17 cells, a specific subset of T cells, have an important role in host defence against certain bacteria and fungi and importantly, are potent inducers of autoimmunity and are implicated in autoimmunity diseases including rheumatoid arthritis and multiple sclerosis. The development of TH17 cells is dependent on RORgammat, a ligand activated transcription factor. For activation, RORgammat requires the binding of a cholesterol derived ligand to initiate the transcription of targeted genes. However, the endogenous ligands of RORgammat which promote TH17 generation and maintains its function are still unknown. Therefore, the goal of this study is to identify endogenous ligands of RORgammat, find out how they regulate TH17 cell development and elucidate the process of their formation and deactivation. These findings will reveal new drug targets and a rationale for therapeutic intervention of autoimmune diseases.

Technical Summary

RORgammat, a nuclear hormone receptor, is important for the development and function of TH17 cells. Although there is some evidence to suggest that oxysterols are natural ligands for RORgammat, the exact endogenous ligands are still unknown. Here, we employ chemical derivatisation combined with liquid chromatography-mass spectrometry (LC-MS) to identify the endogenous ligands of RORgammat. To achieve this we will need to meet three objectives. Objective 1. Quantitative screening of oxysterols during TH cell differentiation under TH1, TH2 and TH17 polarizing conditions to identify the specific oxysterols associated with TH17 cells. This will involve purifying human naïve CD4+ T cells from umbilical cord blood and differentiating the cells in vitro using various combinations of cytokines. The quantitative profile of the oxysterols will be analysed by LC-MS. Objective 2. Investigate the interaction of identified oxysterols with RORgammat and their effect on TH17 cell development. The activities of identified oxysterols towards RORgammat will be first tested using biochemical assays. The effect on TH17 cell development and cytokine production will be examined using flow cytometry, RT-PCR and ELISA. The binding of endogenous ligands to RORgammat will be confirmed by immunoprecipation and detection by LC-MS. Objective 3. Pharmaceutically and genetically manipulating ligand abundance and investigating their effects on TH17 cell development and function. We will use inhibitors to specifically block the enzymes required to produce ligands to find out their effects on suppression on TH17 cell development and TH17-cytokine production. We will also explore the deactivation pathway of the ligands by over-expressing HSD3B7 and analysing the resultant downstream effects.

Planned Impact

The proposed research has academic, commercial and social impact.
The immediate academic beneficiaries include immunologist, nuclear receptor researchers and mass spectroscopists as described in the Academic Beneficiaries section.
Because the proposed research is to identify factors which relate to autoimmune diseases such as rheumatoid arthritis and multiple sclerosis, patients suffering from these diseases will benefit from our findings as will patient groups and charities e.g. Multiple Sclerosis Society, National Rheumatoid Arthritis Society, and ultimately the NHS.
Nuclear receptor RORgammat determines the development of pathogenic cells contributing to autoimmune diseases. Preventing (i) over production of ligands of RORgammat, or (ii) deactivation of ligands, may provide an alternative approach to inhibiting RORgammat for the effective treatment of autoimmune diseases. Therefore pharmaceutical companies will be interested in knowing which molecules are the endogenous ligands regulating the activity of RORgammat, and what are the biosynthetic and catabolic pathway of these ligands. We already collaborate with a commercial partner to quantitatively screen the oxysterols in biological fluids from multiple sclerosis patients using our LC-MS platform. The results from these two projects can be correlated to reveal whether RORgammat ligands play a role in multiple sclerosis. These findings will reveal new drug targets and a rationale for therapeutic intervention of autoimmune diseases. We will patent any intellectual property forthcoming from the present proposal thereby maximising its value to the UK.
The outcome from this project will also have impact in the clinical sector. For example, at the moment there is very little knowledge on what cause multiple sclerosis. Measuring the level of endogenous ligands of RORgammat in multiple sclerosis patients will help clinicians to define the subtype of the diseases, monitor progression and provide personalised medicine.
The outputs of this project will also be of value to the third sector i.e. charities, in directing further areas for their research investment.
The RA employed will work on a multidisciplinary project at the interface of immunology and analytical chemistry. The RA will gain an expert education in biological mass spectrometry, which can in the future be exported to all areas of biological research. The RA will learn to coordinate time between different supervisors and work between two groups. Further, the RA will participate in our meetings with commercial collaborators. The RA will also be involved in the process of applying for patents. The experience and skill gained by working on this project will be useful for all employment sectors.

Publications

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Abdel-Khalik J (2017) Defective cholesterol metabolism in amyotrophic lateral sclerosis. in Journal of lipid research

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Crick PJ (2015) Quantitative charge-tags for sterol and oxysterol analysis. in Clinical chemistry

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Griffiths WJ (2016) New methods for analysis of oxysterols and related compounds by LC-MS. in The Journal of steroid biochemistry and molecular biology

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Griffiths WJ (2017) Cholesterolomics: An update. in Analytical biochemistry

 
Description We quantitatively profiled the oxysterols produced during human Th1, Th2, Th17 and Treg polarised conditions. The cholesterol biosynthesis pathway is activated in all conditions. 24S,25-epoxycholesterol (24S,25-EC), an oxysterol produced from the shunt pathway of cholesterol biosynthesis, is the most abundant of the oxysterols measured. However, we found that 7a,24,25-trihydroxycholesterol (7a,24,25-triHC, chemical name cholest-5-ene-3ß,7a,24,25-tetrol) and its downstream metabolite 7a,24,25-trihydroxycholest-4-en-3-one (7a,24,25-triHC-4-en-3-O) increase significantly in Th17- and Treg-polarising conditions, accompanied by marked increases in oxysterol 7a-hydroxylase (CYP7B1) gene expression. We elucidated the biochemical pathway of metabolism of 24S,25-EC to 7a,24S,25-triHC and 7a,24,25-triHC-4-en-3-O which proceeds via 7a-hydroxy-24S,25-epoxycholesterol (7aH-24S,25-EC) and 7a-hydroxy-24S,25-epoxycholest-4-en-3-one (7aH-24S,25-EC-4-en-3-O), which involves the enzymes CYP7B1 and HSD3B7. 24S,25-EC is a known agonist for liver X receptors (LXRs) and inverse agonist for the retinoid-related orphan receptor gamma (ROR?), but the activities of 7aH-24S,25-EC, 7aH-24S,25-EC-4-en-3-O, 7a,24,25-triHC and 7a,24,25-triHC-4-en-3-O have not been studied before. We found that 7aH-24S,25-EC can induce the expression of LXR target gene ABCG1 in human CD4+ T cells, but 7aH-24S,25-EC-4-en-3-O, 7a,24,25-triHC and 7a,24,25-triHC-4-en-3-O could not. We studied the interactions of oxysterols with LXR and ROR?t using molecular modelling. We found that the conversion of the 3ß-hydroxy group to 3-oxo group disrupts the binding of the oxysterols to both LXR and ROR?t. Our data demonstrates that human CD4+ T cells can generate LXR agonists or ROR?t inverse agonist and also deactivate them locally. LXR and ROR?t have been implicated in regulating the differentiation and function of both Treg and Th17 cells. Our studies, for the first time, identify the metabolic pathway of an endogenous ligand of LXR and ROR? in human T cells and targeting this pathway could be used to modulate the function of Treg or Th17 cells.
Exploitation Route Interleukin-17 (IL-17) is a pro-inflammatory signalling protein which plays a pivotal role in driving autoimmunity and inflammatory diseases. Targeting IL-17 produced by T helper cells has become a clinically validated strategy for successfully treating a variety of autoimmune diseases, evidenced by the high efficacy of Secukinumab (Cosentyx), a human IL-17 neutralising antibody. However, up to now there is no small molecule IL-17 blocker available. Thymus retinoic acid-related orphan receptor gamma (ROR?t), a nuclear hormone receptor which controls both IL-17 production by Th17 cells and conversion of naïve CD4+ T cells into Th17 cells, has emerged as a "hot" drug target for IL-17 inhibitors. Based on the oxysterols we identified in Th17 polarised conditions, we currently use in silico screening to search for specific inverse agonist of ROR?t and inhibitors of CYP7B1 in the ChemBioNet and ZINC compound databases.
Sectors Pharmaceuticals and Medical Biotechnology

 
Description Life Sciences Research Network Wales PhD studentship
Amount £40,000 (GBP)
Organisation Life Sciences Research Network Wales 
Sector Academic/University
Country United Kingdom
Start 04/2017 
End 03/2020
 
Description CYP7B1 knockout mice 
Organisation University Hospital Zurich
Department Department of Ophthalmology
Country Switzerland 
Sector Hospitals 
PI Contribution My team has found that CYP7B1 is induced in certain sub-population of T helper cells. In order to find out the role of CYP7B1 in regulating the balance between pro-inflammation and anti-inflammation T helper cells, we use the CYP7B1 knockout mice and
Collaborator Contribution My collaborator at University Hospital Zurich has CYP7B1 knockout animal and they performed experiment of T helper cell differentiation using wild type and knockout animal.
Impact The research is still on going and no output yet.
Start Year 2016
 
Description Soapbox science 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact Soapbox science Swansea 2016 was held in Swansea's shopping area in the city centre on Saturday September 10th,which buzzing with shoppers, families, tourists and other city dwellers. The event's mission is to raise the profile, and challenge the public's view, of women in science, technology, engineering, maths and medicine (STEMM). I was one of the presenters and talked my research on "cholesterol metabolism and human health".
Year(s) Of Engagement Activity 2016
URL http://soapboxscience.org/soapbox-science-2016-swansea/