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

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.

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.

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.