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，允许对外部环境的预期。我们最近开发了一种模型，用小鼠研究肺部的昼夜节律反应，使用雾化内毒素(模仿细菌细胞壁)来靶向肺。这种侮辱产生了一种炎症反应，在黎明和黄昏之间的反应非常夸张。我们的研究表明，肺的上皮Clara细胞可能是先天免疫的重要调节器，因此我们在小鼠身上使用了基因靶向来“敲除”这些细胞中的特定发条。Clara细胞发条的破坏导致对内毒素的炎症反应急剧增加，出现了一种候选的介质，趋化因子CXCL5。肾上腺切除术(ADX)后清除自然循环中的GC水平消除了肺内对LPS的昼夜节律反应，表明GC激素是关键的调节因素。由于CXCL5先前已被鉴定为受糖皮质激素(GC)强烈调节，我们检查了是否可能涉及糖皮质激素受体(GR)的节律性抑制。在正常小鼠中，在黄昏时，免疫抑制GR复合体最大限度地与CXCL5基因的调节区结合，此时GC激素水平自然上升，这与内毒素反应的最低点相吻合。这与节律性(抑制性)激素信号调节昼夜节律的肺部免疫反应的观点是一致的。有趣的是，当我们敲除Clara细胞的发条时，GR抑制就消失了，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，肺部的局部时钟控制着一种对正常免疫反应至关重要的有节奏的表观遗传机制。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GC抑制先天免疫反应的？我们将从三个方面来探讨这个问题。首先，使用ADX小鼠，我们将提供与主体发条同步或异相的定时气雾化GC信号。这将确定GC节律是否在肺内引起局部节律，对免疫反应进行计时，以及这些反应是否可以与身体的其他发条系统同步。我们将通过使用基因靶向来干扰Clara细胞中的GR来扩展这一过程，并询问这些细胞中的GR信号是否对正常的昼夜免疫反应时间至关重要。我们的第二个目标是绘制这个肺模型中GR靶基因的完整图谱，以揭示时钟功与免疫反应耦合的完整程度。为了评估这一点，我们将使用一种“全基因组”方法来检测GR结合的昼夜节律模式，比较正常小鼠和以Clara细胞时钟工作为目标的小鼠。我们将测试令人兴奋的想法，即生物钟(Cryptochome，Cry)的核心组件是将核心时钟耦合到GR活动的途径，以及有节奏的Cry信号阻止GR对靶基因的作用。最后，我们回到完好的动物身上，测试目标2中出现的靶向通路对控制对环境挑战的先天反应的后果。因此，这项研究将揭示存在于所有脊椎动物谱系(糖皮质激素)中的古老能量和应激反应系统与作为环境预期关键因素的生物钟之间的新的相互作用。虽然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，在控制正常哺乳动物生理的过程中，确定它们的基本生物学作用机制是非常重要的。

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.