Functional and molecular diversity of skin fibroblasts in systemic sclerosis.

Systemic sclerosis (SSc), also called scleroderma, is an uncommon autoimmune rheumatic condition leading to thickened skin (sclerosis) with fibrosis, or scarring, in the skin and vital organs such as the lungs and heart, kidneys and bowel. These complications are responsible for major impact of the disease on almost all aspects of everyday life and in the most severe cases can lead to death. The pattern and severity of skin thickening can be used to identify cases most at risk of severe internal organ involvement and reduced long-term survival. However, the relationship between skin disease and overall outcome is imprecise because the basic mechanisms of disease in scleroderma remain poorly understood. The extent of skin involvement helps to classify patients, and distinct trajectories of skin involvement are associated with differing prognosis and survival. As a result, scleroderma remains a challenging disease to treat and is responsible for more deaths than any other rheumatic disease.
Although scleroderma is rare, the processes that have malfunctioned and that determine severity of the disease are likely to be relevant to many commoner conditions such as lung and liver fibrosis, or excessive scarring after surgery, burns or skin wounding. In this way scleroderma offers a possible route to understand and ultimately prevent or treat these other important disorders.
Previous work in scleroderma has suggested that a key type of cell in the skin and internal organs called the fibroblast acts as a driver of fibrosis in skin disease. Fibroblasts can be grown from small pieces of skin (biopsies) taken under local anaesthetic from scleroderma patients. However only some of the fibroblasts grow out of the biopsy and recent research suggests that these may not be the most important cells determining skin fibrosis. This has limited previous studies because there are likely to be many different types of fibroblasts that have different roles in normal healthy skin and in diseases such as scleroderma.
New laboratory techniques provide exciting tools to distinguish between the different populations of fibroblasts in skin and we have developed a new method to isolate different populations of fibroblasts from skin biopsies in scleroderma. This is a major step forward because unlike other cell types, such as immune cells or blood cells, fibroblasts examined using traditional methods and laboratory tools are often indistinguishable from each other. The present application will use modern scientific approaches to examine gene and protein expression and to define the distinct types of fibroblasts in the skin, and further understand their potential importance in the scleroderma disease process. We will ask patients with scleroderma attending our routine clinic to donate small samples of skin and will analyse each cell in the biopsy individually to identify the types of cells present. We expect to find small groups of cells within the biopsy expressing similar proteins. The technique we propose to use has already been applied in rheumatoid arthritis, another autoimmune disease affecting the joints, as well as in healthy skin.
We fully expect that there will be different fibroblast populations, and that these will have distinct properties at different stages of scleroderma. Definition of the detailed properties and function of these cells will have implications on future treatment targets and determine which of the emerging therapies for scleroderma will work best for an individual patient. This is vital considering the very high clinical burden of scleroderma and current variation in response to available drugs.
As well as providing important information about scarring and fibrosis, this work will also have wider implications on other common autoimmune diseases affecting the skin, such as psoriasis, where there may be overlap in the types of cells driving the disease, and similar treatments may be applicable.

Systemic sclerosis (SSc) has high unmet medical need and only limited therapeutic options. Fibroblasts have a pivotal role in skin fibrosis in SSc. Explant cultures from early stage diffuse SSc show a marked profibrotic phenotype, and gene expression profiles are dominated by a TGFb signature. Recent therapeutic trials have reversed this phenotype in explant cells, but the molecular response has not always been reflected by clinical improvement in skin, suggesting that other fibroblast subpopulations may be important. We recently identified a second population of fibroblasts which remain in the skin after outgrowth of explant cells. This late resident population is profibrotic but much less motile or contractile and, we hypothesise, highly relevant to pathogenesis of SSc. Our proposal will define subpopulations of SSc fibroblasts using molecular techniques for single cell analysis of gene and protein expression in skin biopsies of well characterised SSc cases.
Our aims are (1) to define differences in functional properties and gene expression between early explant and late resident fibroblasts including bulk RNA sequencing, providing a functional anchor for the remainder of this project. (2) To analyse fibroblast subpopulations from whole skin using scRNAseq utilising biopsies from different stages of disease (early and late), and different antibody status (ATA and ARA) (n=16) and healthy controls (n=4). Creation of t-SNE plots will help identify clusters of cells, and key gene and protein expressions to differentiate them. (3) To further characterise the skin biopsies by identifying where the different subpopulations of fibroblasts are localised in using confocal image analysis.
This project will use approaches already established in rheumatoid arthritis and healthy skin to identify subpopulations of fibroblasts, and their discriminating markers. This will define relevance to SSc and potentially identify new therapeutic targets for this challenging disease.

The main beneficiaries from this research will be:

1.Patients with scleroderma and related connective tissue diseases.
Scleroderma and related connective tissue diseases are uncommon, affecting approximately 1 in 10,000 individuals in the UK but represent a very important chronic disease population through its high case-specific mortality (more than half of cases diagnosed with scleroderma eventually die as a direct result of the disease) and because of a major burden of non-lethal complications including pain, digital ulceration and psychosocial impact.

2. General public stakeholders associated with the themes of Lifelong health and wellbeing and ageing.
Vascular disease and fibrosis or scarring are core processes in scleroderma that lead to significant morbidity and mortality. These processes and their assessment have much in common with other conditions. Fibrosis is a common pathway to damage and dysfunction in almost all body systems and has particular relevance to cardiovascular disease, lung disease and kidney pathology. The application will explore the fundamentals of skin biology in disease, and the findings are likely to be relevant to both normal skin, and disease in other autoimmune settings.

3. Academic/private sector collaboration and future benefits for the economy.
There is an important unmet medical need in scleroderma and related diseases, which has the potential to benefit from progress in this area both from an economic and commercial benefit. The development of new indications for medications that have been utilised for other indications (repurposing) is important and there may also be economic benefits from developing improved ways of assessing fibrosis, and ultimately treating and reversing fibrosis that emerge from this study.

4. International academic colleagues working in scleroderma, fibrosis and autoimmunity.
The sponsor is an international leader in the field of scleroderma and fibrosis and has a strong track record of translational research which includes preclinical studies, clinical trials and outcomes research. The academic community will benefit from work outlined in this proposal through interaction at regular international meetings and collaborative endeavours through organisations including the EULAR scleroderma trial and research group (EUSTAR).

5.Personal Development
This fellowship would give me the opportunity to develop my skills as a researcher. I will have the opportunity to develop my skills in laboratory techniques, bioinformatics, critical appraisal, as well as patient recruitment and scientific communication. Upon completion of a PhD and my specialty training, I hope to be competitive to apply for further funding to enable me to continue to combine my interest in translational research in the field of rheumatology and connective tissue diseases. I hope to continue to make contributions to the field of research and clinical care in Rheumatology, as well as act as a mentor or supervise to my junior colleagues. This fellowship would therefore be an important step in the direction of achieving my career goals.