The microbiome drives circadian rhythms in inflammation

The circadian clock is an internal biological timer that allows animals to tune their physiology to the 24h environment created by the rotation of Earth on its axis. Inflammation - the response of specialised cells within the body to attack- is regulated by the circadian clock. Human chronic inflammatory diseases, such as rheumatoid arthritis, show day-to-night variation in their symptoms. Patients suffering from rheumatoid arthritis (0.6 million people in the UK) often report pain and joint stiffness is increased during the early morning. We have recently shown in a mouse model of inflammatory arthritis that this is due to the circadian clock directing the inflammatory responses within affected limbs. This localised inflammation shows 24h fluctuations in severity, with significant reductions during the night. We propose that this night-time repression is due to the increased activity of T-regulatory cells (Tregs), immune cells with anti-inflammatory properties. In keeping we have shown that Treg numbers within inflamed joints increase during the night, but fall in the day.

This proposal describes a novel and exciting programme of work which will investigate daily changes in activity of Tregs within inflamed joints.

First we will analyze how timing mechanisms control the activity and movement of Tregs around the body, and into inflamed joints. We will use novel technology that allows us to "paint" cells of interest, and count them as they travel. Then we will investigate how the timing of meals, which is regulated by the clock, orchestrates Treg numbers and trafficking. We have preliminary evidence that the microbial content of the gut, which fluctuates over 24h in response to feeding, directs the clock control of the movement of Tregs around the body. It is known that short chain fatty acids (SCFA), which are produced by gut microbes during the breakdown of fibre, can regulate Treg numbers and function. We will investigate this circuit by using timed-feeding interventions to target the microbes. Then, we will test the idea that the Tregs are directly responding to SCFAs by selectively deleting the SCFA receptor, a sensor, on the surface of the Tregs. Finally, we will test the presence of a timing signal in circulating SCFAs by directly regulating serum levels, and assess the downstream impact on inflammatory disease.

The circadian clock allows organisms to adapt their physiology and behavior to the 24h environment. This internal timer regulates many aspects of physiology, including tissue immune homeostasis. In chronic inflammation, clock oscillations persist within affected tissue, driving rhythmicity in inflammatory pathways. In mouse experimental arthritis (collagen induced arthritis) joint inflammatory pathways are repressed during the night. We propose that this results from the circadian influx of T regulatory cells (Tregs) at night. The immunosuppressive activity of Tregs is influenced by the gut microbiome, which itself is highly circadian rhythmic, with daily changes in bacterial numbers, mucosal proximity, and metabolite production. This project tests the role of microbiome-derived metabolites in conferring circadian behavior to joint Treg cells, and thereby establishing a link between feeding behavior and circadian control of inflammation.

We will assess quantitative and qualitative changes in Tregs within inflamed joints at different phases of the day (flow cytometry, co-culture assays and RNA-Seq). Depletion of Tregs from mice with established inflammatory arthritis will test the hypothesis that Tregs drive diurnal rhythms in inflammation. To examine temporal changes in Treg trafficking we will utilize Kaede mice (in which Tregs can be photo-converted at specific anatomical sites) and adoptive transfer studies. The final workstream will assess the role of the gut microbiome on conferring time-of-day regulation to Treg function and trafficking. We will assess how feeding times and chronic inflammation affect the composition of the microbiome over the course of 24h and its and metabolic outputs (mouse and human). Finally, we will test whether dietary intervention can stabilise Treg activity over the course of 24h thereby improving disease outcome.

The research questions posed within this proposal are of major interest to ACADEMIC GROUPINGS in Biological, Biomedical, and Clinical Sciences. The academic community will benefit from elucidation of mechanisms involved in circadian control of anti-inflammatory processes and the role of the microbiome. As such, research findings will impact on the HEALTH CARE COMMUNITY. We will disseminate findings by publishing primary papers and reviews in high impact journals, and presenting work at national and international meetings. We anticipate that the proposed work will produce 2-3 high-quality primary research papers.

Our findings will be of interest to the GENERAL PUBLIC due to the prevalence of chronic inflammatory disease, such as rheumatoid arthritis. At its most basic, the work will engage sections of the populous who wish to learn about their health, diet, circadian timing, the microbiome and human physiology. Research findings will be delivered to the general public through public engagement activities (e.g. cafe scientifique), as well as through mass media. For example, Ray and Gibbs regularly feature in broadcast and printed media.

The proposed research is of interest to PHARMACEUTICAL COMPANIES due to direct implications for human chronic inflammatory disease, and establishing a strong basis for embedding clock logic into clinical development programmes, as well as identifying novel targets and approaches to tackle chronic inflammation.

Benefits of this research to the UK ECONOMY are immediate in terms of research activity, capacity building, and potential generation of intellectual property. In addition, chronic inflammation is, and will continue to be, a massive burden on the national health care service.

A lack of in vivo research training has been highlighted as a weakness in UK bioscience. This proposal offers a significant opportunity for high-level in vivo training of UNDERGRADUATE AND MASTERS STUDENTS exposed to this research that will gain valuable research skills through lab-based projects.

Although beyond the limits of this grant, our ultimate goal is to deliver novel therapies, which have real benefit for PATIENTS. Within the proposal, there are elements that could potentially be taken forward to develop new therapeutic strategies (e.g. post-biotics or dietary modifications).