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

生物钟代表了最古老的进化保守的生理过程之一，允许预期外部环境。我们最近开发了一个模型，使用小鼠研究肺中的昼夜节律反应，使用雾化LPS（模拟细菌细胞壁）靶向肺。这种侮辱会产生炎症反应，在黎明和黄昏时的反应会被大大夸大。我们的研究表明，肺上皮Clara细胞可能是先天免疫的重要调节因子，因此我们在小鼠中使用基因靶向来“敲除”这些细胞中的生物钟。Clara细胞时钟系统的破坏导致对LPS的炎症反应急剧增加，其中出现了一种候选介质，趋化因子CXCL5。肾上腺切除术（ADX）后自然循环Gc水平的去除消除了肺中对LPS的昼夜节律反应，表明Gc激素是关键的调节因子。由于CXCL5先前被认为受糖皮质激素（Gc）激素的强烈调节，我们检查了糖皮质激素受体（GR）的节律性抑制是否可能参与其中。在正常小鼠中，黄昏时，当Gc激素水平在夜间自然升高时，免疫抑制GR复合物与CXCL5基因的调节区域结合最大，这与LPS反应的最低点相吻合。这与节律性（抑制性）激素信号调节肺昼夜免疫反应的观点是一致的。有趣的是，当我们敲除Clara细胞的时钟结构时，GR抑制消失了，与CXCL5基因的节律性结合也消失了，尽管这些动物具有正常的节律性肾上腺功能！这表明，肺中的局部时钟控制着正常免疫反应所必需的节律性表观遗传机制。现在的关键问题是这个时钟驱动的表观遗传回路是如何调节先天免疫反应的Gc抑制的？我们将从三个方面来探讨这个问题。首先，使用ADX小鼠，我们将提供定时雾化Gc信号与主体时钟同步或不同步。这将确定Gc节律是否在肺内携带局部节律，定时免疫反应，以及这些反应是否可以与身体其余部分的生物钟相异。我们将通过使用遗传靶向来破坏Clara细胞中的GR来扩展这一研究，并询问这些细胞中的GR信号是否对昼夜免疫反应的正常时间至关重要。我们的第二个目标是绘制该肺模型中GR靶基因的完整图谱，以揭示时钟工作与免疫反应耦合的全部程度。为了评估这一点，我们将使用“全基因组”方法来检测GR结合的昼夜节律模式，比较正常小鼠和靶向clara -细胞时钟工作的小鼠。我们将测试一个令人兴奋的想法，即生物钟的核心组成部分（Cryptochome, Cry）是将核心时钟与GR活动耦合的途径，而有节奏的哭泣信号会阻止GR对目标基因的作用。最后，我们回到完整的动物来测试Aim 2中出现的靶向途径对环境挑战先天反应控制的后果。因此，这项研究将揭示存在于所有脊椎动物谱系中的古老能量和应激反应系统（糖皮质激素）与作为环境预期关键因素的生物钟之间的新相互作用。尽管这两个系统的紧密耦合直到最近才被定义，但它具有极好的生物学意义。由于糖皮质激素在医学上的广泛应用，确定其与生物钟控制正常哺乳动物生理的基本生物学机制是很重要的。

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.