Inflammatory therapeutics and the role of the circadian clock

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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.