Screening for, and characterisation of, novel immune cell extravasation genes in Drosophila, mice and man

炎症反应在许多正常的生理反应中都是关键的，例如在伤口或感染部位。在这种情况下，如果炎症延迟或不充分，那么对健康的后果可能是灾难性的。相反，如果炎症反应过度或消退缓慢，那么“慢性”炎症也可能导致多种病理，从组织瘢痕到动脉粥样硬化甚至癌症。更好地了解炎症细胞是如何被招募到目标组织的，将有助于指导我们如何调节炎症反应来治疗这些疾病。炎症反应中关键的“限速”步骤之一是白细胞从循环中穿过血管壁（称为“外渗”）向损伤或感染部位移动。然而，尽管在这一领域进行了数十年的研究，白细胞招募这一步骤的许多方面仍然知之甚少，还有很多东西需要学习；这在一定程度上是因为在分子水平上解剖血管壁是一个高度复杂的过程，并且在传统哺乳动物模型（例如小鼠）的不透明组织中成像非常困难。在布里斯托尔，我们最近开发了一种新的模型，用果蝇来研究免疫细胞的“外渗”。果蝇为这项研究提供了许多优势，它们在基因上“易于控制”（易于突变和研究单个基因），而且是半透明的（使我们能够使用显微镜观察这些运动）。由于果蝇在很大程度上具有“开放式循环”，没有离散的血管，因此它们以前没有被认为是该领域的有用模型。然而，我们已经确定了蛹生命中的一段时期，免疫细胞在翅膀的“静脉”中循环，激光诱导的伤口会触发免疫细胞离开静脉，向损伤处移动。我们已经使用该模型确定了一种新的受体蛋白（Tre1），它对免疫细胞外渗至关重要，令人兴奋的是，哺乳动物中的等效基因（GPR84）已被证明是小鼠中性粒细胞外渗所必需的（我们在QMUL的初步研究），并且与人类患者的几种炎症有关。我们的研究结果表明，果蝇可能提供了一种强大的快速通道方法，可以识别免疫细胞（或血管细胞）中需要的更多基因，以进行炎症的关键步骤。在这里，我们建议使用我们的苍蝇模型来启动这样的“筛选”，并在新孵化的成虫中开发一个更快的筛选过程，最终搜索整个基因组。对苍蝇的研究也将提供像Tre1这样的蛋白质如何在分子水平上起作用的新细节。在QMUL，我们将通过探索等效基因在哺乳动物外渗过程中的功能来补充这项工作，并研究这些基因如何影响重要的生理和病理过程，如皮肤伤口的愈合和腹膜炎。最终，我们想要确定在果蝇和小鼠研究中突出的哪些基因最有希望作为临床炎症调节的靶点。为了将我们的发现扩展到翻译，我们将在人类细胞中进行研究，我们在人类血管或免疫细胞中敲除候选基因/信号通路，以确定这如何影响白细胞外溢。除此之外，我们将使用“人口健康”方法来搜索人类遗传学数据，以确定哪些候选基因与人类疾病相关。综上所述，我们建议采用多学科、跨机构和多生物的方法来发现更多在免疫细胞外渗中具有重要功能的基因，这将为临床炎症性疾病提供潜在的治疗靶点。我们设想这种多模式的方法将比单一模型的研究更有见地。

An inflammatory response is pivotal in many normal physiological responses, for example wherever there is a wound or site of infection. In such scenarios, if inflammation is delayed or insufficient then the consequences to health can be catastrophic. Conversely, if the inflammatory response is excessive or slow to resolve, then 'chronic' inflammation can also lead to several pathologies, ranging from tissue scarring through to atherosclerosis or even cancer. A better understanding of how inflammatory cells are recruited to their target tissues will help guide us how the inflammatory response might be modulated therapeutically to treat these conditions.One of the key 'rate-limiting' steps in an inflammatory response is the movement of leukocytes (white blood cells) from the circulation across blood vessel walls (known as 'extravasation') to sites of damage or infection. However, despite decades of research in this field, many aspects of this step in leukocyte recruitment remain poorly understood and there is still much to learn; this is in part because breaching of vessel walls is a highly complex process to dissect at the molecular level and it is incredibly difficult to image in opaque tissues of traditional mammalian models (e.g. mouse).In Bristol we have recently developed a new model in which to study immune cell 'extravasation' using the fruitfly, Drosophila. Fruitflies offer many advantages for this research, being genetically 'tractable' (easy to mutate and study individual genes) and also translucent (enabling us to watch these movements live using microscopy). Since fruitflies largely have an 'open circulation' without discrete blood vessels, they were not previously considered a useful model for this field. However, we have identified a period in pupal life when immune cells circulate through the wing "veins" and a laser-induced wound triggers immune cells to leave the veins and move towards the damage. We have already used this model to identify one novel receptor protein (Tre1) that is essential for immune cell extravasation, and excitingly, the equivalent gene in mammals (GPR84) has proven to be required for neutrophil extravasation in mice (our pilot studies in QMUL), and is associated with several inflammatory conditions in human patients.Our findings suggest that Drosophila may provide a powerful fast-track approach to identify more genes required in immune cells (or vessel cells) for this pivotal step in inflammation. Here we propose to use our fly model to initiate such a 'screen' and develop an even faster screening process in newly hatched adults to ultimately search through the whole genome. Studies in the fly will also provide new detail on how proteins like Tre1 function at the molecular level. At QMUL, we will complement this work by exploring how the equivalent genes function in mammals during extravasation and investigate how these genes might impact on important physiological and pathological processes, such as healing of a skin wound and during peritonitis.Ultimately, we want to identify which of the genes highlighted by our studies in fly and mouse are most promising as targets for modulation of inflammation in the clinic. To extend our findings towards translation, we will undertake studies using human cells where we knockdown candidate genes/signalling pathways in human vessel or immune cells to determine how this impacts leukocyte extravasation. Alongside this, we will use "population health" approaches to search human genetics data to identify which of our candidate genes are associated with human disease.In summary, we propose to employ a multi-disciplinary, cross-institutional and multi-organism approach to uncover more genes with important functions in immune cell extravasation, that will provide potential therapeutic targets for inflammatory disorders in the clinic. We envision this multi-modal approach will be far more insightful than studies in a single model alone.