The circadian clock as a therapeutic target for inflammatory arthritis

Rheumatoid arthritis (RA) is a common, chronic inflammatory disease affecting over 0.6 million people in the UK. At present, there is no cure for RA and an urgent requirement for improved therapeutic options. Inflammation of the joints causes pain and loss of movement, eventually leading to disability. Patients suffering with RA report daily rhythms in symptom severity, with joint pain and stiffness more severe in the morning. Clinical studies have demonstrated that this day-night variation is accompanied by corresponding fluctuations in levels of inflammatory markers detected in the blood. Taken together, this indicates that inflammatory pathways underlying this debilitating condition are more active at specific times of the day. I now show that rhythmic circadian (circa 24h) oscillations in inflammatory markers and joint swelling are robustly replicated in a mouse model of chronic inflammatory arthritis. These observations advocate the involvement of the circadian clock in disease expression, suggesting an exciting new approach to target pathogenic mechanisms. Indeed, I have recently shown that small, drug-like molecules targeting the core clockwork have unexpected anti-inflammatory efficacy in vitro. I now seek to exploit these discoveries with the explicit goal of early clinical translation.

Specifically, I will address the hypothesis that the circadian clock within the immune system controls disease expression and treatment responsiveness of inflammatory arthritis, and is a valid novel therapeutic target. I will use a well-established pre-clinical mouse model of inflammatory arthritis to track temporal changes in the inflammatory environment within affected joints. Parameters assessed will include: cytokine levels; expression of adhesion molecules; and numbers and activation states of infiltrating leukocytes. This will provide for the first time a complete understanding of how, and to what extent, the clock controls localised immune processes underlying inflammatory arthritis.

Subsequent studies will employ previously validated transgenic mice which lack a functional clock in subsets of immune cells critical to the development of RA (CD4+ T lymphocytes or macrophages). This will provide unique insight into the role of individual cellular timers on disease rhythmicity and disease progression. These studies will define the relative significance of each cell type on the generation of rhythmic inflammatory signals. Further, it is predicted that this work will reveal the critical importance of robust localised clocks in the repressive control of processes underlying inflammatory arthritis, thereby supporting the concept that the clock is a valid target for treatment of this disease.

Finally, I will test the potential of novel clock-acting compounds for the management of inflammatory arthritis. These studies will expand on my prior work, which demonstrated effective anti-inflammatory action of clock activators in joint-derived cells. Ex vivo approaches will be taken to explore compound efficacy and mechanism of action on target cells. In vivo experiments will test efficacy in a pre-clinical model of RA, measuring effects on inflammatory markers, limb swelling and joint histology. Subsequently I will translate my findings to human disease through the application of these compounds to inflammatory cells derived from arthritic patients. These studies will provide proof-of-principle data to support drug targeting of the clock as a new therapeutic avenue for the management of inflammatory arthritis.

This project will investigate how the clock impacts on pathogenic pathways underlying inflammatory arthritis, and test novel clock-modulatory ligands for efficacy in treating this disorder.

To track temporal changes in the inflammatory environment within sites of disease, joints from arthritic mice (collagen induced arthritis) will be harvested at different circadian phases and analysed for local cytokine production (ELISA, Bioplex array, QPCR) and cellular infiltrates (flow cytometry). Temporal tracking of gene transcription in FACS purified cultures of cells from inflamed joints will identify effector cells orchestrating rhythmic production of inflammatory signals.

To interrogate the role of individual cellular timers on disease rhythmicity and progression, arthritis will be induced in mice with targeted clock deletions in inflammatory cells (T cells or macrophages). Disease rhythmicity (temporal tracking of inflammatory markers, twice daily assessments of paw swelling) and severity (daily scoring, terminal histological assessment of joints) will be monitored. The contribution of circadian fluctuations in circulating glucocorticoids (GCs) to the entrainment of peripheral inflammatory oscillators will be addressed via application of corticosterone pellets to arthritic animals. The effects of clamping GC levels at the night-time high on rhythmic cytokine production will be tracked pre- and post- clamp.

To validate targeting the clockwork as a treatment for inflammatory arthritis, clock-acting compounds will be tested ex vivo using murine inflammatory cells (macrophages, T-cells and synoviocytes). In subsequent intervention studies, novel compounds will be administered to mice after arthritis induction. Disease severity, inflammatory markers and histological joint damage will be assessed. Finally, in translational studies I will test the efficacy of these compounds in inflammatory cells isolated from arthritic patients and healthy controls.

The questions posed within this proposal are of major interest to ACADEMIC GROUPINGS in biological, biomedical and clinical sciences. This academic community will benefit from elucidation of novel mechanisms by which the circadian clockwork interacts with the immune system on a molecular and anatomical level. Examination of the adverse effects of a disrupted clock function on inflammation, and defining how targeting the clock represents a potential avenue for the treatment of rheumatoid arthritis presents clear translational application to human health and welfare. As such, research findings will greatly impact on the HEALTH CARE COMMUNITY. I will disseminate my findings by publishing primary papers and reviews in high impact journals and presenting work at national and international meetings. I anticipate that the proposed work will produce 2-3 high quality primary research papers.

All findings will be of interest to the GENERAL PUBLIC due to the prevalence of rheumatoid arthritis in our aging population. At its most basic, the work will engage sections of the populous who wish to learn about their health and human physiology. Research findings will be delivered to the general public through public engagement events (e.g. Cafe Scientifique at local schools and colleges), as well as through mass media. The University of Manchester has an excellent Press office, regularly issuing press releases regarding high impact publications from the Faculty. This often leads to reporting in national and international newspapers, on local radio and on the intranet.

The proposed research is of major interest to PHARMACEUTICAL COMPANIES due to direct implications for human inflammatory diseases affecting the joints, but also other organs. Pharmaceutical investment into circadian biology is rapidly growing due to the fact that circadian dysfunction has been linked to sleep disorders, mental health disorders, cancer, inflammation and aging. I already have established collaborations within GSK, and these links have proved fruitful. The bi-directional transfer of information is valuable to both parties, and my own previous research has helped to inform the direction of projects run by GSK. Continued industrial interest is evidenced in this application by the provision of access to tool compound libraries by GSK.

The Faculty of Medical and Human Sciences at Manchester has taken a strong proactive role in developing links with major pharmaceutical companies, enhancing public communication of science, as well as identification and development of commercialisation opportunities. There are dedicated members of staff employed within the faculty to assist in these areas. A recent success is the establishment of the Manchester Centre for Collaborative Inflammation Research (MCCIR). This Centre draws in substantial cash investment from both GSK and AZ in inflammation research. My programme of work will link to the Centre, maximising commercial exploitation potential.

This proposal also offers a significant opportunity for high level in vivo training of the TECHNICIAN and any PhD/Masters STUDENTS joining for related work. This is a significant benefit as a lack of in vivo research training has been highlighted as a weakness in UK bioscience.