Local and systemic circadian cues coordinately regulate innate immunity via an epigenetic circuit.

The circadian clock represents one of most ancient evolutionarily conserved physiological processes, permitting anticipation of the external environment. We have recently developed a model using mice to study circadian responses in the lung, using aerosolised LPS (mimicking bacterial cell wall) to target the lung. This insult generates an inflammatory response, with greatly exaggerated responses at dawn vs dusk. Our studies suggest that the epithelial Clara cells of the lung may be important regulators of innate immunity, so we used genetic targeting in mice to "knock-out" the clockwork specifically in these cells. Disruption of the Clara cell clockwork caused dramatic increases in inflammatory responses to LPS, with a candidate mediator, the chemokine CXCL5 emerging. Removal of natural circulating levels of Gc following adrenalectomy (ADX) eliminated circadian responses to LPS in the lung, pointing to Gc hormones as key regulators. Since CXCL5 had previously been identified as strongly regulated by glucocorticoid (Gc) hormones, we checked whether rhythmical repression by the glucocorticoid receptor (GR) may be involved.

In normal mice, at dusk the immuno-suppressive GR complex maximally binds to regulatory regions in the CXCL5 gene at the time of the natural nocturnal rise in Gc hormone levels, which coincides with the nadir of LPS response. This is compatible with the idea that a rhythmic (repressive) hormone signal regulates circadian lung immune responses. Fascinatingly, when we knocked out the clockwork of the Clara cells, GR repression was lost, as was rhythmic binding to the CXCL5 gene, despite the fact that these animals had normal rhythmic adrenal function! This shows that a local clock in the lung controls a rhythmic epigenetic mechanism essential for normal immune responses. The key question now is how does this clock-driven epigenetic circuit regulate Gc repression of innate immune responses?

We will explore this in 3 ways.

First, using ADX mice, we will deliver timed aerosolised Gc signals in or out of phase with the main body clockwork. This will establish whether the Gc rhythm entrains a local rhythm within the lung, timing immune responses, and whether these responses can be set out of phase with the rest of the body's clockwork. We will extend this by using genetic targeting to disrupt the GR in Clara cells, and ask whether GR signaling within these cells is essential for normal timing of circadian immune responses.

Our second goal is to map the full repertoire of GR target genes in this lung model to reveal the full extent by which the clock-work is coupled to immune responses. To assess this, we will use a "genome-wide" method to detect circadian patterns of GR binding, comparing normal and mice in which the Clara-cell clock-work has been targeted. We will test the exciting idea that a core component of the circadian clock (Cryptochome, Cry) is the pathway that couples the core clock to GR activity, and that the rhythmic Cry signal blocks GR action on target genes.

Finally, we return to the intact animal to test the consequences of targeting pathways emerging from Aim 2 for the control of innate responses to environmental challenge.

This study will therefore reveal novel interactions between an ancient energy and stress response system, present in all vertebrate lineages (glucocorticoids), and circadian clocks which serve as key agents for environmental anticipation. Although the close coupling of these 2 systems has only recently been defined, it makes excellent biological sense. Since glucocorticoids are widely used in medicine, it is important to define their basic biological mechanism of action with the circadian clockwork in the control of normal mammalian physiology.

Circadian clocks are tightly conserved through evolution, and are essential in permitting anticipation of environmental change (light/dark). We have pioneered studies of clock control of immunity, using the lung as a model. We discovered striking clock-regulated inflammatory reactions, and show that this is driven by specialized bronchial epithelial cells (Clara cells). Disruption of the clock in these cells ablated the circadian rhythm in inflammatory responses, but also exacerbated the inflammatory reaction. This appeared to be driven, at least in part, by the chemokine CXCL5. Moreover, we discovered a critical role for glucocorticoids (Gc) in maintaining time of day regulation of the lung inflammatory response.

We now seek to define how local oscillators in the lung operate in conjunction with systemic circadian signals (Gc). This will be achieved using local Gc delivery to the lung by nebulisation, and also by deleting the glucocorticoid receptor (GR) in Clara cells. The identification of CXCL5 as a Gc-regulated, circadian controller of pulmonary inflammation prompts us to define how the circadian, and Gc pathways converge. We will address this using chromatin immunoprecipitation (ChIP) analysis in intact lung, subjected to inflammatory, and glucocorticoid challenge, in wild-type, and Clara cell disrupted clock animals. We will then apply ChIP-sequencing, and transcriptome mapping. We will specifically identify the GR cistrome, and how it is regulated by the clock, and by inflammation.
We will exploit these discoveries using in-vivo studies to measure the expression, regulation, and cellular distribution of candidate clock "pioneer" transcription factors in the lung. Again, we will address how these are affected by time of day, and by induced inflammation. Taken together, our studies will define how the circadian machinery operating in an intact animal, comprising both local clocks, and systemic clock signals, regulates organ-specific immunity.

The research questions posed within this proposal are of major interest to ACADEMIC GROUPINGS in Biological and Biomedical Sciences. The academic community will benefit from elucidation of novel mechanisms whereby innate immunity interacts with the circadian clockwork on a molecular and anatomical level. Examination of the adverse effects of disrupted clock function presents clear implication to human health and welfare. 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 2-4 high-quality primary research papers.

All findings will be of high interest to the GENERAL PUBLIC due to the prevalence of 24hr lifestyles in our modern society. 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. brain awareness week, annual science open days at the UoM, Café Scientifique presentations), as well as through mass media. For example, several of our recent papers have been reported widely in national and international newspapers, on local radio, and on the intranet following press releases issued by the University of Manchester and BBSRC.

The proposed research is of interest to PHARMACEUTICAL COMPANIES due to direct implications for human metabolic disease. Pharmaceutical industry investment into circadian biology is rapidly growing due to the fact that circadian dysfunction has been linked to sleep disorders, mental health disorders, cancer, inflammation, and aging. 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 taken a major lead within the extensive community of researchers at the University of Manchester by developing significant interactions and links with GSK and a joint ChronoBiology Programme.

Industrial interest is evidenced by the substantive contributions to this IPA application (£70K cash, £139K support in kind). The Faculty of Life Science at Manchester has taken a strong proactive role in developing links with major pharmaceutical companies, enhancing public communication of science, as well as identification and development of commercialisation opportunities. There are dedicated members of staff employed within the Faculty to assist in these areas.

As an indication of potential wider uptake of our research, UoM researchers have recently engaged in a novel 3-way chronobiological programme with GSK researchers and McLaren Technology, to exploit expertise using devices to track human physiology remotely. The clear end point here is develop approaches using novel short-acting drugs timed to the correct circadian phase, to optimize efficacy and therapeutic index. Our studies will provide key enabling data for such approaches.