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Targeting the Role of Myeloid Steroid Metabolism in Chronic Inflammation


Project Description

Background
Myeloid derived cell populations such as macrophages play a critical role in the early innate immune response, whilst dendritic cells are essential in bridging the progression to an adaptive immune response. Steroid hormones such as glucocorticoids (GCs) and Vitamin D possess both immunomodulatory and anti-inflammatory properties and play a role in regulating macrophage and dendritic cell differentiation and function. This project aims to better characterise the mechanism whereby endogenous and therapeutic steroid hormone metabolism mediates the anti-inflammatory actions of GCs and steroid hormones in myeloid cells using in vitro cell populations and murine model of chronic inflammation. Preliminary studies using targeted deletion of pre-receptor GC metabolism in myeloid cell populations suggest that steroid metabolism in these cells mediates increasing in severity of chronic polyarthritis reported in the mice with a global disruption of pre-receptor glucocorticoid metabolism. Consequently, this project aims to better characterise the precise role of GC, and steroid metabolism more widely, to the progression of inflammation in the TNF-tg murine model of chronic polyarthritis. These data will inform whether future studies aiming to target pre-receptor activated GCs to macrophages or dendritic cells may be effective in the treatment of chronic inflammatory disease.

Aims
1. Isolate, differentiate and characterise primary cultures of dendritic cells and M1/M2 polarised macrophages from the TNF-tg mouse
2. Using well established primary murine cultures of dendritic cells and M1/M2 polarised macrophages, characterise pre-receptor steroid metabolism and regulation and assess their functional effects in relation to key pro-inflammatory and inflammatory resolution action
3. Examine whether these processes can be targeted in vivo to develop novel strategies to treat chronic inflammatory disease

Research Methods
Bench work will be performed on the 2nd floor of the Institute of Metabolism and Systems Research in the Hardy lab. Leukocyte synovial infiltration will be assessed in the TNF-tg and KBXN serum transfer models of polyarthritis. Macrophage subsets at sites of inflammation will be determined by FACS following tissue digests as previously reported. Morphology and numbers will be assessed in paraffin embedded sections of inflamed joints using immunohistochemistry. Primary cultures of murine macrophages will be generated in vitro using Ficoll separation and macs positive selection of cd14+ monocytes. Adherent macrophages are then incubated with IL-4 and IL-13 in order to obtain M2 polarized macrophages or with IFN-gamma and LPS for classical macrophage activation (M1). Primary human myeloid cultures will be generated to validate findings in mice. Steroid metabolism wuill be assessed by scanning thin layer chromatography and Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS). The actions of pre-receptor metabolised steroids (corticosterone, oestrogen, vitamin D) on inflammatory cytokine production (TNFα, IL-6, IL-10) by RT-PCR and ELISA. Functional effects on differentiation will be assessed by FACS analysis of differentiation markers (CD64, CXCL10, CD86, CD206, CD23, CD200R). Phagocytosis and efferocytosis will be assessed using standardised kits. Animal experiments to generate cell cultures and for assessment of pathophysiology will be performed under Dr Hardy’s project licence P51102987. Both the animal model and methods of scoring inflammation are currently well established at the Birmingham BMSU.

Significance of Research
In this project, we aim to determine whether modulation of pre-receptor steroid metabolism can influence macrophage polarisation and function during acute and chronic inflammation. Using chemicals that modify these processes we will examine whether we can favour anti-inflammatory pro-resolution pathways in macrophages.

Person Specification
Applicants should have a strong background in cell biology, and preferably with myeloid cell culture and analysis of gene expression. Experience with animal models of inflammation would be advantageous but not essential. They should have a commitment to endocrinology, immunology and metabolism research and hold or realistically expect to obtain at least an Upper Second Class Honours Degree in Biosciences, Medical Sciences or Cancer Biology.

Informal enquiries should be directed to Dr Rowan Hardy, email

To be considered for this studentship, please send the following documents to Viktorija Ziabliceva, email :
• A detailed CV, including your nationality and country of birth;
• Names and addresses of two referees;
• A covering letter highlighting your research experience/capabilities;
• Copies of your degree certificates with transcripts;
• Evidence of your proficiency in the English language, if applicable.


Funding Notes

Self-funded applications only.

References

Sagmeister M*, Taylor A, Fenton A, Wall N, Nightingale P, Cockwell P, Hardy RS#*, Harper L. Amplification of cortisol signalling in chronic kidney disease is associated with inflammation, cardiovascular pathology and impaired glycaemic control. 2018. Manuscript accepted for publication. Clin Endocrinol (Oxf). 2018 Oct 25. doi: 10.1111/cen.13889. [Epub ahead of print].
Hardy RS, Fenton C, Croft AP, Naylor AJ, Begum R, Desanti G, Buckley CD, Lavery G, Cooper MS, Raza K. 11Beta-Hydroxysteroid Dehydrogenase Type 1 Regulates Synovitis, Joint destruction, and Systemic Bone Loss in Chronic Polyarthritis. J Autoimmun. 2018 Aug;92:104-113. doi: 10.1016/j.jaut.2018.05.010. Epub 2018 Jun 8
Hardy RS*, Hussain Z, Doig C, Morgan S, Pearson MJ, Naylor A, Jones SW, Filer A, ,Stewart PM, Buckley CD, Lavery GG, Cooper MS, Raza K, 11beta-Hydroxysteroid Dehydrogenase within Muscle Protects Against the Adverse Effects of Local Inflammation, J Path, 2016 Dec;240(4):472-483. doi: 10.1002/path.4806.
Hardy RS, Cooper MS. Unravelling how glucocorticoids work in rheumatoid arthritis. Nat Rev Rheumatol. 2018 Oct;14(10):566-567. PMID: 30181582 DOI: 10.1038/s41584-018-0079-4
Hardy RS, Zhou H, Seibel MJ, Cooper MS. Glucocorticoids and bone: consequences of endogenous and exogenous excess and replacement therapy. Endocr Rev. 2018 Jun 13. doi: 10.1210/er.2018-00097. [Epub ahead of print] PMID: 29905835

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