Inflammatory therapeutics and the role of the circadian clock

慢性炎症在人类疾病中非常普遍。炎症信号对细胞生物钟产生广泛的影响，因此在炎症组织中显示昼夜节律振荡的基因数量是健康状态下的五倍。这是因为炎症深刻地重新连接了受影响细胞内的昼夜节律耦合。总的来说，生物钟控制着不同器官系统中高达25%的代谢途径，可以预测环境中的可预测变化，例如睡眠/觉醒周期以及从进食到禁食状态的伴随变化。我们提出，对急性炎症挑战的初始适应性反应需要生物能量需求的重大变化，这是由生物钟调节的，由核心时钟成分、能量代谢调节因子和炎症信号（如糖皮质激素受体；GR）之间的串扰介导。在慢性炎症中，这种反应变得不适应，并施加生物能量成本，导致炎症消退和有机体能量代谢的后果。关键的是，它还将改变对治疗干预的反应。我们将在四个相互关联的目标中解决这一核心假设。Aim 1将定义炎症对核心时钟组件Cryptochrome （CRY）和REVERB功能的影响；GR与CRY和REVERB结合，从而调节核心生物钟阶段，同时也是炎症信号传导和能量代谢的主要内源性调节剂。我们使用重要阶段显微镜进行测试，允许在单细胞水平上分析运输和分子相互作用。GR与REVERB和CRY之间的分子相互作用将使用荧光互相关光谱（FCCS）和FRET进行研究。由此，我们将定义细胞中相互作用发生的位置，昼夜节律阶段如何调节GR的分子功能，以及炎症信号如何影响GR：生物钟界面。目标2将定义炎症如何重新连接组织内代谢物和基因表达的节律曲目。我们将使用计算方法来建立预测模型，我们将使用遗传和药理学干预直接测试。作为该方法的一个例子，我们最近发现神经酰胺，胰岛素作用的有效调节剂，在活动性类风湿关节炎患者中获得强烈的昼夜节律振荡。目的3将研究肝脏对肺或肢体关节慢性炎症的反应。我们将揭示触发肝脏昼夜变化的体液信号。通过结合代谢组学和转录组学模型，我们旨在建立功能网络，能够解释炎症状态下新节律过程的出现。这些适应性的生理作用将通过基因方法（AAV6传递CRISPR，和/或shRNA，以及白蛋白靶向重组）靶向紧急肝脏反应来测试。目的4将研究将昼夜节律逻辑嵌入抗炎药物治疗的转化潜力，以优化疗效，同时最大限度地减少脱靶效应。我们将使用在Aim 2（炎症病灶）和Aim 3（肝脏）中获得的基因表达数据，为改变给药时间的治疗试验设计提供信息。作为起点，我们将使用糖皮质激素，利用我们最近发现的GR功能的严格昼夜节律调节，但我们期待后续出现其他有希望的药物靶点。其他环境挑战，如改变喂养协议或照明条件，以改变时钟相位也将进行测试。因此，我们将确定炎症如何重新连接时钟，以及其中对炎症持续性，代谢后果和药物反应的影响。

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.