Imaging Immunometabolism in live animals during host defense

项目总结 巨噬细胞生物学研究进展表明，代谢途径在控制巨噬细胞活化中起着关键作用。 寄主防御和组织动态平衡中的表型和效应器功能。体外研究导致了基本的 对免疫代谢的洞察，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的表达仍然有限。在这里，我们提出了荧光寿命 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 在活体中原位探索细胞内代谢的时间和空间变化。基于图像的 方法允许在其自然微环境中保持高度可塑性的巨噬细胞，并检查其 生理和临床相关环境中的细胞内代谢。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。由于光学系统 在幼虫期的清晰度，它很容易与大多数成像方式相结合。慢性阻塞性肺疾病的临床治疗与管理 热损伤引起的皮肤创伤很难，患者有很高的风险进一步遭遇 常见医院病原体引起的并发症，如铜绿假单胞菌。在这里，我们将雇用 研究假单胞菌对巨噬细胞激活和功能的代谢调节 铜绿假单胞菌感染的烧伤创面，使用幼虫斑马鱼作为我们的体内系统。在AIM I中，我们将开发工具来 评估活生物体中巨噬细胞原位代谢的空间和时间变化。在AIM II中，我们将 研究铜绿假单胞菌的作用机制，如最近发现的微生物氧化脂， 在宿主防御受损组织的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控代谢途径的前景 治疗方面的好处。然而，在不了解新陈代谢是如何进行的情况下，治疗潜力仍然不清楚。 在体内调节免疫细胞的功能。这项研究将证明，成像的内源荧光 代谢辅酶是一种有价值的非侵入性和无标记的方法来填补我们理解中的这些空白 并更好地为新疗法的开发提供信息。

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.