Regulation of Wound Detection in Animal Tissues

描述（由申请人提供）：伤口检测和愈合呈现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稳态。白细胞在几秒钟内就能从几百微米的距离检测到伤口，并在几分钟内迁移到伤口。在空间上传播关于何时何地在组织中发生损伤的信息的机制仍然很少被研究和理解。通过协调受伤组织中不同细胞类型的行为（包括白细胞、内皮细胞和上皮细胞），这些机制控制炎症反应长度和时间尺度，并保证炎症事件的持续时间和幅度（例如血管舒张、白细胞募集等）。与组织损伤的程度相应地缩放。使用斑马鱼尾鳍创伤测定，我们最近发现上皮NADPH氧化酶DUOX产生过氧化氢（H2 O2）的梯度，其从创伤边缘延伸到组织中高达~200 μ m。这种梯度是白细胞快速伤口募集所必需的。然而，仍然不清楚如何利用诸如H2 O2的反应性化学物质作为特定的伤口信号，H2 O2表现出很小的分子靶向选择性并且可以损伤细胞。我们假设在组织内，H2 O2的作用范围和/或细胞选择性的精确空间和时间控制允许它作为一个特定的信号。到 了解如何H2 O2介导的伤口检测，因此，我们建议调查H2 O2产生的位置和时间，多远和速度，它通过组织传播，以及在哪里，何时，以及通过哪些信号通路不同的细胞类型响应it. The斑马鱼尾鳍创伤试验是一个很好的脊椎动物模型系统的成像伤口反应和分子扰动的药理学和反向遗传学。为了系统地解决创伤反应的时间和空间动力学，我们将使用转基因斑马鱼与无处不在的，内皮，上皮和白细胞特异性表达的荧光报告H2 O2，其可能的上游激活剂钙（Ca 2+），和下游效应NF B。使用生物传感器成像和分子扰动在活斑马鱼，我们将解决的基本问题有多远和快H2 O2，一种新的旁分泌信号，在组织中旅行，以及这种氧化化学物质是如何能够介导特定的细胞反应。此外，我们将询问H2 O2模式的长度和时间尺度如何由DUOX激活剂Ca 2+调节。最后，我们将推导出合作或行动的H2 O2从他们的转录签名使用微阵列/生物信息学的下游途径。从NF开始B是一种中枢炎症调节因子，我们将对这些通路的时空激活进行成像，并通过H2 O2梯度探测它们的调节。 公共卫生相关性：该提案研究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，定时和缩放的炎症反应，例如从附近血管快速招募白色血细胞。了解白色血细胞募集的信号通路， 损伤部位将有助于更好地理解在高度炎症性疾病和癌症期间炎症事件的病理性失调。这可能会导致这些疾病的新治疗方法。

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.