Imaging Immunometabolism in live animals during host defense

项目摘要 巨噬细胞生物学的进展揭示了代谢途径在控制其活化方面起着关键作用 在宿主防御和组织稳态过程中的表型和效应子功能。体外研究导致了 对免疫代谢的深入了解，然而，我们对代谢变化的功能相关性的理解 在天然间质组织内的巨噬细胞中仍然有限。在这里，我们提出荧光寿命 代谢辅酶NAD（P）H和FAD的成像显微镜（FLIM）是一种强大的基于成像的工具， 在活的有机体中原位探测细胞内代谢的时间和空间变化。基于成像 这些方法允许在它们的天然微环境中维持高度可塑的巨噬细胞，并检查它们的 在生理和临床相关背景下的细胞内代谢。斑马鱼是一种已建立的体内模型 免疫和炎症，与人类免疫系统和基因组高度相似。由于光学 在幼虫阶段清晰，它很容易与大多数成像方式相结合。临床治疗与管理 由热损伤引起的皮肤伤口是困难的，并且患者处于进一步遇到的高风险中。 常见的院内病原体如铜绿假单胞菌引起的并发症。在这里，我们将采用 FLIM研究巨噬细胞对假单胞菌应答的活化和功能的代谢调节 aerodosa-infected烧伤创面，使用斑马鱼幼虫作为我们的体内系统。在目标I中，我们将开发工具， 评估活生物体内原位巨噬细胞代谢的空间和时间变化。在Aim II中，我们将 研究铜绿假单胞菌使用的机制，如最近发现的微生物氧脂素， 在受损组织的宿主防御过程中调节巨噬细胞炎症，并影响整个伤口 治愈免疫代谢的体外研究支持调节代谢途径的前景， 治疗益处。然而，在不了解代谢如何的情况下， 调节体内免疫细胞功能。这项研究将证明，成像的内源性荧光， 代谢辅酶是一种有价值的非侵入性和无标记的方法，可以填补我们理解中的这些空白 更好地为新疗法的发展提供信息。

PROJECT SUMMARY Advances in macrophage biology have revealed that metabolic pathways play key roles to control their activation phenotype and effector function during host defense and tissue homeostasis. In vitro studies led to fundamental insights into immunometabolism, however our understanding of the functional relevance of metabolic changes in macrophages within native interstitial tissues remains limited. Here, we propose that fluorescence lifetime imaging microscopy (FLIM) of metabolic coenzymes NAD(P)H and FAD is a powerful imaging-based tool to probe the temporal and spatial changes in intracellular metabolism in situ in a live organism. Imaging-based approaches allow for maintaining highly plastic macrophages in their native microenvironment and examine their intracellular metabolism in physiological and clinically relevant contexts. Zebrafish is an established in vivo model of immunity and inflammation, with high similarity to the human immune system and genome. Due to the optical clarity at larval stage it readily combines with most imaging modalities. Clinical treatment and management of cutaneous wounds caused by thermal injury is difficult, and patients are at high risk to encounter further complications due to common nosocomial pathogens, such as Pseudomonas aeruginosa. Here, we will employ FLIM to study the metabolic regulation of activation and function of macrophages responding to Pseudomonas aeruginosa-infected burn wounds, using larval zebrafish as our in vivo system. In Aim I, we will develop tools to assess the spatial and temporal changes in macrophage metabolism in situ in a live organism. In Aim II, we will investigate the mechanisms Pseudomonas aeruginosa employs, such as recently identified microbial oxylipins, to modulate macrophage inflammation during host defense at damaged tissues, and impact overall wound healing. In vitro studies in immunometabolism support the prospects of modulating metabolic pathways for therapeutic benefits. However, the therapeutic potential remains unclear without understanding how metabolism regulates immune cell function in vivo. This study will demonstrate that imaging the endogenous fluorescence of metabolic coenzymes is a valuable non-invasive and label-free approach to fill these gaps in our understanding and better inform the development of new therapies.