Regulation of circadian timers in a peripheral tissue the lung and identification of cellular and in vivo physiological pathways

Lead Research Organisation: University of Manchester
Department Name: Life Sciences


This project aims to discover how circadian timers regulate an important physiological pathway in the lung. A number of diseases are known to have a circadian basis, and in the lung, inflammatory diseases such as asthma are known have a strong circadian component. In addition, many of the agents responsible for inflammatory responses (cytokines) are also driven by circadian clocks. We will start by defining which cell types in the lung contain circadian oscillators. This builds on our preliminary data which suggest that there may be specific timer cells in the lung which are also involved in tissue responses to external insult and inflammatory responses. We will then collect cells from a transgenic mouse in which a clock gene promoter has been adapted to drive luciferase, and monitor light emissions from these cells in culture to track the underlying circadian clock. Once we have established these culture conditions, we will examine how a hormone (glucocorticoid) resets the phase of the circadian oscillator. We will also examine the same cells from another mouse strain in which a normally expressed receptor for a peptide (VPAC2) has been disrupted. This mouse is known to be arrhythmic, but it is believed that circadian oscillators in peripheral tissues are still active, but unsynchronized. By culturing these cells, we aim to see whether they can be re-synchronised by glucocorticoids. Once we have developed these methods, we will proceed to study genes driven by the circadian clock (so-called clock controlled genes). Our first candidates are a family of genes called CCAAT enhancer binding proteins or C/EBPs, three members of which operate in the lung. C/EBP's are important as they may drive rhythms of cytokine activity in lung cells and hence circadian inflammatory responses. We will use a variety of methods including suppression of C/EBP genes with a technique called siRNA, and then seeing whether we can block the rhythmical activity of a cytokine gene called interleukin-6, which our preliminary data already shows is circadian regulated. Our studies on living mice will focus on two questions. First, we aim to re-set the lung clock in mice by treating them once a day with a specially formulated glucocorticoid in an aerosol spray (nebulised), so that only the lung cells are targeted. We will then see whether we can re-set lung oscillators in normal and VPAC2 mutant mice. We will next test whether the severity of the lung inflammatory responses to external insult is dependent on the circadian clock, and for this study we will use normal mice and animals bearing a mutation of the Clock gene (clk/clk mice) which renders individual circadian oscillators arrhythmic. We need to distinguish whether circadian timers in the lung contribute to the response or whether there other contributions from elsewhere in the body. In other to test this, we aim to re-set the phase of the lung clock from the rest of the body using nebulised glucorticoids given to two groups of animals at opposite phases of the circadian cycle, 12 hours apart. We will then challenge the animals with an external insult which we know will elicit an inflammatory response, so that we can test whether the nature of the tissue response is controlled by a timing system.

Technical Summary

Endogenous circadian oscillators govern many aspects of physiology. It is now clear that in addition to the central, light-entrained master oscillator of the suprachiasmatic nucleus many peripheral tissues express core clock component proteins, and exhibit endogenous clock activity. Coordination of the clocks throughout the organism is achieved by neuroendocrine (glucocorticoid) and autonomic mechanisms. Glucocorticoids are widely used therapeutically, and exert pleiotrophic effects in many tissues. Of relevance here is their use to treat lung disease, their functional interaction with the C/EBP family of transcription factors, and their entraining activity on the peripheral tissue core clock. Little is known of the consequences arising from peripheral clock rhythms. However, many phenomena have a diurnal rhythm, including lung function, and lung disease (asthma). We intend to identify lung timer cells, determine how they are entrained, and discover their physiological output pathways. Initial work will use genetically modified mice to establish the identity of primary, lung oscillating cells. Preliminary data suggests that these will be type II pneumocytes, and Clara cells. Both cell types express clock proteins, and co-express the GR. Importantly Clara cells have an important role in bronchial epithelial repair, and anti-inflammation. The Clara cell specific protein CC10 protects the airway from inflammatory insult, and is regulated by C/EBPs. C/EBPs are known to show a marked circadian rhythm in expression in other tissues, but have not been analysed in the lung. We will use primary cell culture approaches with clock reporter gene mice to allow longitudinal measurement of clock oscillations, and the response of these to glucocorticoid manipulation. Our second aim will be to address the role of C/EBPs as clock controlled genes mediating the effects of the core clock on peripheral output pathways. We will use cell line models and primary bronchial epithelial cells from genetically modified mice to identify oscillations in C/EBP expression over time. The same models systems will be used to test the requirement of C/EBP oscillations for the observed rhythmical expression of IL-6. Our third aim is to identify circadian lung function in living animals, and explore the role of glucocorticoids in entraining the lung rhythm. These studies rely on state-of-the-art in-vivo physiology measurement available by collaboration with GSK, Stevenage. The collaboration also allows us to administer, to the lung, topical glucocorticoid with known pharmacokinetic profiles to locally, and in a time dependent manner, activate the glucocorticoid receptor. Our initial studies will address the role of glucocorticoids on the phase of the lung clock (by examination of lung tissue), and will also track fine changes in lung function (by Buxco measurement). Later studies will use a well-characterised lung inflammatory challenge (ovalbumin sensitisation, and tracheal instillation) to determine the effect of circadian phase on the response. This would, for the first time, link clock activity with an important, integrated organ response to external insult.
Description We have identified a critical role for the epithelial cells that line the airways of the lung in regulating immune responses by time of day. These cells act as critical local "pacemakers" driving overall lung immunity. We have been able to disrupt the clockwork in these cells and this has severely impaired inflammatory responses. We then went on to discover the genetic mechanisms and from this identify a key clock gene RevErba. When we targeted this gene, responses to inflammatory stimuli were greatly augmented - indicating that this gene acts as a natural brake on the system preventing over-elaboration of an inflammatory response. We have then gone on to define how this operates within the cell, and shown that REVERBa protein impairs action of inflammatory pathways in a highly selective manner - targeting only one cytokine (chemical message from inflamed cells).
Exploitation Route This work has highlighted novel Therapeutic options from the use of small molecule activators of circadian clock proteins. Our group was the first to publish in this area, and we continue to retain an active interest
Sectors Healthcare,Pharmaceuticals and Medical Biotechnology

Description This research has lead to new studies involving industry (GSK), and a new programme searching for novel drugs that act on the circadian clock. Briefly, we have now embarked on a programme to identify novel ligands for a clock gene - reverb-alpha - as this exhibits potent anti-inflammatory action.
First Year Of Impact 2011
Sector Healthcare,Pharmaceuticals and Medical Biotechnology