Molecular mechanisms retaining macrophages at sites of inflammation

细胞如何在我们的体内移动并到达合适的目的地是生物学的一个基本主题。我们已经确定了许多线索，细胞跟随这些线索到达它们在体内的必要位置。然而，人们对将它们留在这些地方的信号知之甚少。细胞在其正确作用部位的保留与整个生物学相关，在发育过程中至关重要，对我们身体的正常维持以及衰老和疾病都是至关重要的。在免疫反应中，保留尤其关键：在细胞重新招募后，例如在组织损伤部位，未能保留细胞可能会破坏对感染的免疫反应或对修复造成负面影响。与此同时，由于一些免疫细胞在我们体内产生的负面影响，过度滞留可能会导致组织损伤。了解这些过程可以为适当地刺激或抑制免疫反应提供干预点，并提供对我们如何将细胞保持在正确位置的更一般的理解。免疫细胞功能障碍可导致多种人类疾病，包括慢性炎症性疾病(如COPD)、癌症和神经变性。幸运的是，免疫细胞可以接受药物干预，因此识别新的药物靶点在这一医学领域特别有用。果蝇(果蝇)含有一组血细胞，与我们体内称为巨噬细胞的白细胞非常相似。由于它们的遗传冗余、最高的遗传学和成像能力，与其他多细胞生物相比，它们是理解免疫系统调节的一个极好的(在伦理上不那么具有挑战性的)模型。我们最近的工作发现了一种新的分子，能够在伤口处保留巨噬细胞，称为SIMU。SIMU与损伤部位受损细胞表面的分子相互作用。我们建议SIMU帮助免疫细胞附着在伤口上，将它们保留在这些关键位置，直到它们分解的时候。在这个建议中，我们希望了解SIMU如何促进巨噬细胞在损伤部位的保留。我们将使用苍蝇遗传学与实时成像技术相结合，研究发育中的胚胎中巨噬细胞对损伤的反应。使用高功率激光，我们可以重复地缠绕苍蝇胚胎的表面，巨噬细胞对其做出快速反应。使用荧光标记，我们可以可视化苍蝇巨噬细胞迁移到伤口上，并使用我们从伤口招募和扩散的分析来了解哪些基因在这个过程中很重要，以及它们是如何相互作用的。同时，我们的目标是以一种不偏不倚的方式寻找新的分子参与者：我们将确定哪些蛋白质在健康细胞受到挑战后增加或减少，这些细胞的类型与在伤口发现的死亡细胞相同。这也将使我们能够看到哪些信号蛋白通过一种称为磷酸化的修饰而改变。然后，我们将回到我们的创伤分析，以确定哪些新的监管机构在管理伤口保留方面是重要的。我们现在知道，免疫细胞未能保留在炎症部位可能会产生负面后果，因此我们还将利用这些新分子的识别来检查它们对后续巨噬细胞行为的影响。这项工作结合在一起，将使我们能够组装一张在受伤时保留免疫细胞的关键调控图。我们不偏不倚的方法也将使我们能够识别这一过程的新的和令人兴奋的调节因素，而我们对濒死细胞和免疫细胞之间的接触如何改变后者的行为的特征将提供对这些机制的重要性的洞察。最终，这项研究将提高我们对生物学基本领域的理解，并有可能发现操纵人类患者免疫细胞功能的新药物靶点。

How cells move within our bodies and reach their appropriate destination is a fundamental topic in biology. We have identified many cues that cells follow to reach their necessary locations in the body. However, far less is known about the signals that keep them in those places. The retention of cells at their correct site of action has relevance across biology, being critical during development, for normal maintenance of our bodies, and in ageing and disease. Retention is particularly key during immune responses: following recruitment of cells, e.g. to sites of tissue damage, failing to retain cells could undermine immune responses to infection or negatively affect repair. At the same time, excessive retention could cause tissue damage due to the negative effects some immune cells exert within our bodies. Understanding these processes can provide points of intervention to stimulate or dampen immune responses as appropriate, as well as provide a more general understanding of how we keep cells in their correct locations. Immune cell dysfunction contributes to a wide range of human diseases including chronic inflammatory conditions (e.g. COPD), cancer and neurodegeneration. Fortunately, immune cells are amenable to pharmacological interventions so identification of new drug targets is of particular use in this area of medicine.Fruit flies (Drosophila) contain a population of blood cells very similar to the white blood cells known as macrophages within our own bodies. Due to their genetic redundancy, superlative genetics and imaging capabilities, fruit flies they make an excellent (and less ethically challenging) model compared to other multicellular organisms to understand regulation of the immune system. Our recent work has uncovered a novel molecule able to retain macrophages at wounds called Simu. Simu interacts with molecules present on the surfaces of damaged cells at sites of damage. We propose Simu helps immune cells to adhere to wounds, retaining them at these critical locations until it is time for them to depart.In this proposal we wish to understand how Simu facilitates retention of macrophages at sites of damage. We will use fly genetics in combination with real-time imaging of macrophage responses to injury in developing embryos. Using high-powered lasers we can reproducibly wound the surface of fly embryos, towards which macrophages mount a rapid response. Using fluorescent markers, we can visualise fly macrophages as they migrate to wounds and use our assays of recruitment and dispersal from wounds to understand which genes are important in this process and how they interact with each another. In parallel, we aim to find new molecular players in an unbiased fashion: we will identify which proteins are increased or decreased following challenge of healthy cells with dying cells of the type found at wounds. This will also enable us to see which signalling proteins are altered via a modification called phosphorylation. We will then return to our wounding assays to determine which new regulators are important in governing retention at wounds. We now know that there can be negative consequences to failed retention of immune cells at sites of inflammation, therefore we will also capitalise on the identification of these new molecules to examine their impact on subsequent macrophage behaviour.Taken together this work will enable us to assemble a map of key regulators that retain immune cells at injuries. Our unbiased approaches will also enable identification of new and exciting regulators of this process, while our characterisation of how contact between dying cells and immune cells can change the behaviour of the latter will provide an insight into the importance of these mechanisms. Ultimately this research will improve our understanding of a fundamental area of biology and has the potential to uncover new drug targets for the manipulation of immune cell function within human patients.