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

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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.