Regulation of Wound Detection in Animal Tissues

描述（由申请人提供）：伤口检测和愈合是一个休眠的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外发现伤口，并在几分钟内迁移到伤口上。在空间上传播组织中损伤发生的地点和时间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（包括白细胞、内皮细胞和上皮细胞）的行为，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（如血管舒张、白细胞募集等）的持续时间和幅度与组织损伤的程度相适应。利用斑马鱼尾鳍损伤实验，我们最近发现上皮NADPH氧化酶DUOX产生过氧化氢（H2O2）梯度，从伤口边缘延伸到组织约200um。这种梯度对于白细胞的快速伤口募集是必需的。然而，目前尚不清楚H2O2等反应性化学物质是如何被利用作为特定的伤口信号的，因为H2O2的分子靶标选择性很小，而且会损伤细胞。我们假设，在组织内，H2O2的作用范围和/或细胞选择性的精确时空控制使其能够作为特定的信号。来

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.