Regulation of Wound Detection in Animal Tissues

描述(由申请人提供)：伤口检测和愈合是休眠形态发生程序的连续步骤，以恢复损伤后的屏障功能和组织动态平衡。白细胞在几秒钟内检测到数百微米外的伤口，并在几分钟内迁移到伤口上。关于在组织中何时何地发生损伤的信息在空间上传播的机制仍然鲜有研究和了解。通过协调受伤组织中不同类型细胞的行为(包括这些机制控制炎症反应的长度和时间尺度，并保证炎症事件(如血管扩张、白细胞募集等)的持续时间和幅度。根据组织损伤的程度进行适当的调整。利用斑马鱼尾鳍损伤试验，我们最近发现，上皮NADPH氧化酶DUOX产生一个从伤口边缘延伸到组织中的过氧化氢(H_2O_2)梯度，最高可达~200微米。这一梯度是白细胞在伤口快速募集所必需的。然而，目前仍不清楚像过氧化氢这样的活性化学物质是如何被用作特定的创伤信号的，因为它表现出很小的分子靶标选择性，并且可以损害细胞。我们假设，在组织内，对过氧化氢S作用范围和/或细胞选择性的精确时空控制使其能够充当特定的信号。至 为了了解过氧化氢是如何介导伤口检测的，我们建议调查过氧化氢在哪里和何时产生，它在组织中传播的距离和速度有多远，以及不同类型的细胞在哪里、何时和通过哪些信号通路对其做出反应。斑马鱼尾鳍损伤试验是一种优秀的脊椎动物模型系统，可用于创伤反应的成像以及通过药理学和反向遗传学进行分子扰动。为了系统地解决创伤反应的时间和空间动态，我们将使用转基因斑马鱼，该转基因斑马鱼普遍表达针对过氧化氢、其可能的上游激活剂钙(Ca2)和下游效应物NfB的荧光报告，内皮、上皮和白细胞特异性表达。利用生物传感器成像和活斑马鱼的分子扰动，我们将解决基本问题，即过氧化氢(一种新的旁分泌信号)在组织中传播的距离和速度有多快，以及这种氧化性化学物质如何能够中介特定的细胞反应。进一步，我们将询问双氧水模式的长度和时间尺度是如何受到DUOX激活剂钙的调节的。最后，我们将利用微阵列/生物信息学从它们的转录特征中推断出与过氧化氢合作或作用于过氧化氢下游的途径。从中枢炎症调节因子NFB开始，我们将成像这些通路的时空激活，并探索它们受过氧化氢梯度的调节。 与公共健康相关：该提案调查了动物组织如何检测它们何时何地受伤，以及它们如何相应地触发适当的定向、定时和规模的炎症反应，例如从附近血管快速招募白细胞。了解支持白细胞募集的信号电路 损伤部位将有助于更好地理解高炎性疾病和癌症期间炎性事件的病理去调节。这可以为这些疾病带来新的治疗方法。

DESCRIPTION (provided by applicant): Wound detection and healing present consecutive steps of a dormant morphogenetic program to restore barrier function and tissue homeostasis after injury. Leukocytes detect a wound within seconds from hundreds of micrometers away, and migrate to the wound within minutes. The mechanisms that spatially propagate the information on where and when an injury has occurred in a tissue remain little studied and understood. By coordinating the behavior of different cell types in the wounded tissue (incl. leukocytes, endothelial and epithelial cells), these mechanisms control length- and time- scales of inflammatory responses, and warrant that duration and amplitude of inflammatory events (e.g. vasodilation, leukocyte recruitment, etc.) scale appropriately with the extent of tissue damage. Using the zebrafish tail fin wounding assay, we recently found that the epithelial NADPH oxidase DUOX generates a gradient of hydrogen peroxide (H2O2) that extends up to ~200 um from the wound margin into the tissue. This gradient is required for rapid wound recruitment of leukocytes. However, it remains still unclear how a reactive chemical such as H2O2, which exhibits little molecular target selectivity and that can damage cells, is harnessed as a specific wound signal. We hypothesize that within tissues, the precise spatial and temporal control of H2O2's range of action and/or cell selectivity allows it to act as a specific signal. To understand how H2O2 mediates wound detection, we thus propose to investigate where and when H2O2 is generated, how far and fast it propagates through the tissue, and where, when, and via which signaling pathways different cell types respond to it. The zebrafish tail fin wounding assay represents an excellent vertebrate model system for imaging wound responses and for molecular perturbation by pharmacology and reverse genetics. To systematically address temporal and spatial dynamics of wound responses, we will use transgenic zebrafish with ubiquitous, endothelial, epithelial, and leukocyte specific expression of fluorescence reporters for H2O2, its likely upstream activator calcium (Ca2+), and downstream effectors NF¿B. Using biosensor imaging and molecular perturbation in live zebrafish, we will address fundamental questions of how far and fast H2O2, a novel paracrine signal, travels in tissues, and how this oxidizing chemical is able to mediate specific cellular responses. Further, we will interrogate how length and timescales of H2O2 patterns are regulated by the DUOX activator Ca2+. Finally, we will deduce pathways that cooperate or act downstream of H2O2 from their transcriptional signature using microarray/bioinformatics. Starting with NF¿B, a central inflammatory regulator, we will image the spatiotemporal activation of these pathways, and probe their regulation by the H2O2 gradient. PUBLIC HEALTH RELEVANCE: The proposal investigates how animal tissues detect when and where they are wounded, and how they trigger appropriately directed, timed and scaled inflammatory responses accordingly, such as rapid recruitment of white blood cells from nearby blood vessels. Understanding the signaling circuitry that underlies white blood cell recruitment to injury sites will help to better understand the pathologic deregulation of inflammatory events during hyper-inflammatory disease and cancer. This can lead to novel treatments for these conditions.