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Our aim is to dissect epigenetic and chromatin modification systems that underlie inflammatory musculoskeletal diseases such as Rheumatoid arthritis and Ankylosing spondylitis.

Towards this goal we have started to analyse systematically the effect of epigenetic modifications using chemical and genetic tools on various immune cell populations of importance in disease, such as macrophages, Tcell subsets or Natural Killer (NK) cells.

EPIGENETICS, GENETICS AND MUSCULOSKELETAL AUTOIMMUNE DISEASE

For most autoimmune diseases, genetic evidence from monozygotic and dizygotic twin studies show concordance rates below 50%, suggesting that additional mechanisms exist which potentially link individual susceptibility and environmental factors such as lifestyle (for example, smoking or stress), infection or xenobiotic exposure. Genome-wide association studies (GWASs), have provided a wealth of possible genetic factors contributing to the phenotypic diversity of syndromes such as Rheumatoid arthritis (RA) and ankylosing spondylitis (AS).

Rheumatoid arthritis (RA) is a chronic inflammatory disease characterised by synovial cartilage and bone destruction. The pathogenesis of disease in RA is attributed to the production of proinflammatory cytokines from activated cells that infiltrate the synovial tissues from the blood (T cells, macrophages, plasma cells, NK cells) together with resident cell types (fibroblasts and endothelium). Multiple studies addressing chromatin and DNA modifications in several autoimmune diseases have clearly shown that tissue-specific epigenetic modifications play a role in autoimmune disease. For example, DNA methylation in RA is impaired in peripheral blood mononuclear cells, and particularly in CD4+ T cells, rendering them more autoreactive.

EPIGENETICS OF NK CELLS IN INFLAMMATORY AUTOIMMUNE DISEASES

Natural killer (NK) cells participate in the clearance of virally-infected, aberrant or transformed cells following sensing of target cells through activating receptors or following pro-inflammatory stimulation. In rheumatoid arthritis (RA), NK cells are readily detectable in the synovial tissue of early stage patients, and constitute around 20% of all lymphocytes in the synovial fluid of established RA patients. NK cells also trigger osteoclastogenesis and bone destruction in RA.

Therefore, strategies aimed at reducing NK cell function may prove beneficial in treating RA. Our aim is to identify epigenetic pathways that can be manipulated to reduce NK cell activation in autoimmunity. Currently, we have identified a number of epigenetic pathways that can reduce NK cell mediated osteoclastogenesis. Specifically, we have shown that NK cells treated with JQ1, a BET bromodomain inhibitor, have a reduced capacity to induce osteoclastogenesis when cultured with monocytes.

BET inhibition

BET bromodomain inhibition reduces NK cell mediated osteoclastogenesis. Following pre-treatment of NK cells with +JQ1 there is a reduction in the ability to induce osteoclasts from monocytes co-cultures (Observed as a reduction in the formation of F-actin rings (green stain)). The non-active stereoisomer –JQ1 is used as a control.

EPIGENETICS OF T CELLS IN INFLAMMATORY AUTOIMMUNE DISEASES

We have previously shown that epigenetic modification in suppressive T cells, called regulatory T cells (Tregs), underpins their defective function in RA. Specifically, methylation of a single CpG within an NFAT transcription factor binding site in the Ctla4 promoter results in reduced Ctla4 transcription and protein expression. [Cribbs et al., 2014] Ultimately this led to a failure to induce the immunosuppressive indoleamine-2,3 dioxygenase (IDO) pathway in antigen presenting cells (APCs) and consequently an inability to suppress effector T cells.

This suggests that epigenetic modifications in Tregs from RA patients underpin reduced suppression. This provides a solid rationale for identifying epigenetic pathways that can be manipulated to restore Treg suppressive function. The goal of this project is to generate a list of druggable epigenetic targets that can be manipulated to increase Treg function and/or decrease effector T cell function in patients with autoimmune diseases, such as RA.

Nature reviews

"Who knows why regulatory T cells are defective in RA...IDO" Nature reviews rheumatology, 2014 [Bernard, 2014]

EPIGENETICS AND MACROPHAGES IN RHEUMATOID ARTHRITIS

Within the synovial lining of an RA joint macrophages produce copious amounts of cytokines, such as TNFα and IL-6. These cytokines not only amplify inflammation but also influence other cell types, such as fibroblasts and osteoclasts, to produce cartilage and bone degrading enzymes. Although anti-inflammatory cytokines are also present in the synovium, RA is typically known to have an imbalance of pro- to anti-inflammatory cytokines, driving disease pathology.

Although current treatments, such as disease modifying anti-rheumatic drugs (DMARDs) and biologics, are successful at reducing inflammation, none achieve disease remission and are often accompanied by significant side effects [2]. Even with successful treatments, approximately 30% of patients do not respond to any current treatments, suggesting a need to identify new therapeutic targets.

Research has identified that wide spread changes in chromatin modification patterns in RA synovial fibroblasts could impact gene expression associated with RA. Studies have found that the histone acetyltransferase (HAT) to histone deacetylase (HDAC) ratio in synovial fibroblasts was skewed favourably towards HAT activity, resulting in an increase of specific gene expression. However, there is still much that is unknown about the synovial macrophage epigenetic landscape, despite being one of the main effector cells in the RA and their contribution to driving disease pathology.

Small molecule inhibitor

We use in our studies small molecule inhibitors targeting epigenetic modulators as a tool to understand the essential regulators of inflammation. By initially screening in vitro models of RA with a focused library of tool compounds we identified which epigenetic modulators are important for the production of pro-inflammatory cytokines from macrophages. Isolated macrophages from the synovial fluid of RA patients were then exposed to these hits and a reduction in pro-inflammatory gene expression signatures was observed.

Using this approach we identified several classes of epigenetic modifiers that are able to reduce proinflammatory cytokine production from macrophages isolated from synovial fluid from RA patients. We currently study in detail the underlying chromatin mechanisms leading to altered pro-inflammatory behaviour.