Autoantigen keratin-17 as a key driver of anterior uveitis

Project No: #OxKEN-2023/14
Intake: OxKEN 2023

project overview

The uvea is the vascular and pigmented layer of the eye, lying between the sclera and the retina. It consists of the iris, ciliary body and choroid. The components of the uveal tract have several supportive functions for vision. Inflammation in the uvea (uveitis) is a leading cause of blindness in people of working age, responsible for between 10% to 20% of blindness in the United States and Europe. Anterior uveitis is the most common form of uveitis, with a prevalence of 2 per 1000 population. It has a strong genetic association with the class I MHC allele HLA-B27 and is characterised by a build-up of leukocytes within the anterior chamber of the eye with symptoms including pain, photophobia and reduction in visual acuity.

In our laboratory, we have generated a single cell atlas of the human uveal tract, and demonstrated that the stromal cells of the uvea, in particular the fibroblasts, display marked heterogeneity between the three uveal sites (Figure 1). Iris fibroblasts express high levels of keratin-17 (KRT17), an intermediate filament protein which is also found in skin adnexa such as hair follicles and in the nail bed. Unlike in the iris, keratin-17 is not expressed in either the ciliary body or choroid fibroblasts (Figure 2A). This finding has been validated by RNA in situ hybridisation in human eye tissue (Figure 2B&C). Work by other groups has demonstrated that keratin-17 is an autoantigen in psoriasis. This is of relevance to anterior uveitis as patients with psoriasis are more likely to develop anterior uveitis and nail bed disease than the general population.

Our group has received ethical approval to sample aqueous humour and blood from patients with uveitis. This allows us to investigate the cellular basis of anterior uveitis in detail. The three aims for this project are:

1. Determine whether patients with anterior uveitis have circulating T-lymphocytes which are reactive to keratin-17.

2. Determine whether the aqueous humour of patients with anterior uveitis contains T-lymphocytes reactive to keratin-17.

3. Characterise the T-lymphocyte subsets of the aqueous inflammatory infiltrate from patients with anterior uveitis.

The techniques that will be used to investigate these three aims including spectral cytometry using the Cytek Aurora and single cell transcriptomic analysis on the 10X Chromium platform. Both techniques will allow for extensive phenotyping of the leukocyte populations within the aqueous inflammatory infiltrate. In addition, in vitro cellular assays will be used to test T-cell reactivity and proliferation in response to antigens, including keratin-17 peptides.
This project is an excellent opportunity for a DPhil student to develop skills in experimental and computational techniques, and to drive a project that will advance our knowledge of the pathogenesis of anterior uveitis, and its connection with psoriasis in an eye-skin-joint axis.

Figure 1. Single Cell RNA sequencing data demonstrates that fibroblasts of the iris (green) group separately to those of the ciliary body (orange) and choroid (blue) on single cell RNA sequencing. A: UMAP of fibroblasts, pericytes and endothelial cells from the adult human uvea coloured by tissue of origin. B: UMAPs of stromal cells coloured by canonical markers of fibroblasts (PDGFRA), pericytes (MCAM) and endothelial cells (VWF).

Figure 2. Kertain-17 expression is localised to the human iris.A: Heatmap of top 10 significantly differentially upregulated genes in the fibroblasts of the iris, ciliary body and choroid compared to whole dataset. Keratin -17 marked by red box. B and C: RNA Scope in situ hybridisation for KRT17 (B) and KRT17 and IGFBP5 (C) on human iris and ciliary body FFPE specimens, showing KRT17 expression specifically in the iris, and IGFBP5 expression specifically in the ciliary body.

keywords

Anterior Uveitis, T-lymphocytes, Keratin-17, Psoriasis, Spondyloarthropathy

training opportunities

The student will gain experience of leading a research project where patient samples are taken from bedside-to-bench. It will enable the student to learn a range of state-of-the-art techniques including spectral flow cytometry, bioinformatic single cell RNA sequencing analysis, in vitro culture, and functional cellular assays. The student will be a part of an established team of discovery scientists and clinicians within the Coles-Buckley group based at the Kennedy Institute, who have interest and experience in cross-organ comparison of inflammatory diseases.

The student will present regularly at laboratory and collaborator meetings as well as internal symposia, where they will develop skills in communicating their work to other researchers. They will also be encouraged to submit work to national and international conferences and be supported to write manuscripts for publication. Training is available in systematic literature search methods and the student will produce a literature review in the first part of their DPhil studies, with a view to publication.

key publications

ET, E.C. and M. Zierhut, Vision Loss in Uveitis. Ocul Immunol Inflamm, 2021. 29(6): p. 1037-1039.
Reekie, I.R., et al., The Cellular Composition of the Uveal Immune Environment. Front Med (Lausanne), 2021. 8: p. 721953.
Jin, L. and G. Wang, Keratin 17: a critical player in the pathogenesis of psoriasis. Med Res Rev, 2014. 34(2): p. 438-54.
Yunusbaeva, M., et al., Psoriasis patients demonstrate HLA-Cw*06:02 allele dosage-dependent T cell proliferation when treated with hair follicle-derived keratin 17 protein. Sci Rep, 2018. 8(1): p. 6098.
Denniston, A.K., et al., Aqueous humor suppression of dendritic cell function helps maintain immune regulation in the eye during human uveitis. Invest Ophthalmol Vis Sci, 2012. 53(2): p. 888-96.