Inflammatory therapeutics and the role of the circadian clock

Chronic inflammation is highly prevalent in human disease. Inflammatory signals exert extensive effects on the cellular circadian clockwork, so that up to five times as many genes show a circadian oscillation in inflamed tissue than in healthy states. This occurs because inflammation profoundly re-wires circadian coupling within affected cells. Overall, the circadian clock controls up to 25% of metabolic pathways in diverse organ systems, acting to anticipate predictable changes in the environment for example sleep/wake cycles with attendant changes from fed to fasted state. We propose that an initial adaptive response to an acute inflammatory challenge requires major changes in bioenergetic demands, which is regulated by the circadian clock, mediated by cross-talk between core clock components, regulators of energy metabolism, and inflammatory signals (eg the glucocorticoid receptor; GR). In chronic inflammation this response becomes maladaptive and imposes a bioenergetic cost, with consequences for inflammatory resolution and organismal energy metabolism. Critically, it will also modify the response to therapeutic intervention.

We will address this core hypothesis in four interlinked aims.
Aim 1 will define the impact of inflammation on the function of the core clock components Cryptochrome (CRY) and REVERB; and their interactions with GR. The GR binds to both CRY and REVERB, and thereby regulates both the core circadian clock phase, and also serves as the major endogenous regulator of inflammatory signaling, and energy metabolism. We test this using vital stage microscopy, allowing analysis of trafficking and molecular interaction, at the single cell-level. Molecular interactions between GR and both REVERB and CRY will be pursued using fluorescence cross correlation spectroscopy (FCCS), and FRET. From this, we will define where in the cell the interactions take place, how circadian phase regulates the molecular function of the GR, and how inflammatory signaling impacts on the GR:circadian clock interface.

Aim 2 will define how inflammation re-wires the rhythmic repertoire of metabolites and gene expression within tissues. We will use computational approaches to build predictive models, which we will directly test using genetic and pharmacological intervention. As an example of the approach, we have recently discovered that ceramides, potent regulators of insulin action, acquire a strong circadian oscillation in patients with active rheumatoid arthritis.
Aim 3 will investigate hepatic responses to chronic inflammation in lung or limb joint. We will reveal humoral signals responsible for triggering hepatic circadian change. By combining metabolomic and transcriptomic models, we aim to build functional networks, capable of explaining the emergence of newly rhythmic processes under the inflamed state. The physiological role of these adaptations will be tested by targeting emergent hepatic responses with genetic approaches (AAV6 delivered CRISPR, and/or shRNA, and albumincre targeted recombination).

Aim 4 will investigate the translational potential of embedding circadian logic into anti-inflammatory drug treatment in order to optimize efficacy, and simultaneously minimize off-target effects. We will use the gene expression data acquired in Aim 2 (inflammatory focus), and Aim 3 (liver) to inform the design of therapeutic trials of altered timing of drug administration. As a starting point, we will employ glucocorticoids, capitalizing from our recent discovery of tight circadian regulation of GR function, but we anticipate following up other promising drug targets that emerge. Other environmental challenges such as altered feeding protocols or lighting conditions to shift the clock phase will also be tested.

Thus, we will identify how inflammation re-wires the clock, and the implications therein for inflammatory persistence, metabolic consequences and drug response.

Chronic, unresolving inflammation plays a major role in human disease, yet remains a therapeutic challenge. Key problems are lack of drug efficacy, and the significant metabolic disturbance that accompanies both the chronic disease state (notably accelerated cardiovascular risk) and long-term anti-inflammatory treatment. We have recently discovered that inflammation leads to reprogramming of the circadian clock. The mechanism involves destabilisation of the clock mechanism, as well as disturbance of the intricate cross-talk between components of the circadian clock (e.g. CRY, REVERB) and the glucocorticoid receptor. Reprogramming of the clock is likely an important feature of the inflammatory response at sites of inflammation, to loosen circadian control of inflammatory and metabolic pathways; however, progression to a systemic level can drive profound disturbance. To decipher how cellular clock dynamics and the clock:GR interface are altered by inflammation, we will use systems microscopy approaches (e.g. FCS, FCCS), functional imaging and RNA-SEQ on cell and tissue slice models. We will define molecular mechanisms and functional consequences of inflammatory reprogramming of the clock within foci of inflammation (lung and joint). To achieve this we will use novel in vivo imaging, with time series RNA-SEQ and metabolomics. This generates highly dimensional data, to which computational analyses can be used to build predictive causation models. We will extend these studies to investigate how localised sites of inflammation can disrupt the liver metabolic/circadian processes. These studies will identify pathways of destructive or emergent rhythms, which will be tested pharmacologically or genetically (using AAV delivery of CRISPR/Cas9). Finally, we will investigate how basal and disease-induced rhythmicity can be capitalised on for timed delivery of anti-inflammatory drugs, with the goal of optimizing therapeutic index.

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 inflammation, circadian biology, energy homeostasis, glucocorticoid action, and sensitivity, and obesity-related pathology. As such, research findings will impact greatly 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 4-8 high-quality primary research papers.

Our findings will be of interest to the GENERAL PUBLIC due to the prevalence of chronic inflammatory disease, and the very widespread usage of therapeutic glucocorticoids. 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 activities (e.g. cafe scientifique), as well as through mass media. For example, Ray, Loudon, Bechtold and Gibbs all 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, or to mitigate the metabolic consequences of such inflammation.

Pharmaceutical industry investment into the problems resulting from chronic inflammation is rapidly growing due to the fact that the prevalence is increasing, and therapeutic options, or mitigating factors remain elusive. In the context of "building partnerships" to enhance take-up and impact, thereby contributing to the economic competitiveness of the United Kingdom", our laboratories have a successful track record of engagement with GSK on inflammation, and glucocorticoid projects, and regular communication will ensure our research findings are taken-up. Ray is an investigator on the NorthWest England Clinical Pharmacology Scheme, an industry/academic partnership.
Benefits of this research to the UK ECONOMY are immediate in terms of research activity, capacity building, and generation of intellectual property. In addition, chronic inflammation and its sequelae including metabolic disorders (obesity, cardiovascular disease, diabetes etc) are, and will continue to be, a massive burden on the national health care service.

This proposal also offers a unique and significant opportunity for high-level in vivo training of the associated post-doctoral scientist, and any PhD 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. Numerous undergraduate and Master's degree students will be exposed to this research and 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. Patients suffering with the associated co-morbidities of chronic inflammation, including accelerated cardiovascular disease urgently require new thinking to inform treatment development. Within the proposal, there are numerous pathways where therapeutic interest is already clear (e.g.modified GR ligands, for which we already have patent protection).