Macrophage Metabolism After Target Cell Ingestion Regulates Anti-Inflammatory Reprogramming

摘要 炎症是肺部疾病中普遍存在的一个组成部分，涉及白细胞在肺内的聚集。 空域。为了消解炎症，必须去除死亡和濒临死亡的白细胞，并产生 必须关闭炎性细胞因子。巨噬细胞(Mϕ)是这些过程的关键协调者， 然而，重新编程炎性Mϕ以发挥这些作用的触发因素仍然不完全清楚。 清除死单元已被证明提供了一个重要的重新编程信号，但我们限制了 了解死亡细胞吞噬促进M-ϕ功能重定向的精确机制。 最近的研究表明，细胞代谢在决定M-ϕ功能中起着关键作用。摄取的目标 细胞提供了各种大分子的明确来源，这些大分子必须被Mϕ消化。然而，很少有研究 已经做了评估这个过程产生的细胞内代谢物或考虑随后的 免疫后果。 我们建议首次全面研究Mϕ在靶细胞摄取后的代谢和 降解，并假设细胞内代谢物水平的变化控制摄取驱动的Mϕ 重新编程。我们的初步研究已经确定了一个有希望的分子靶标--多胺，它是 吞噬凋亡靶细胞后，M-ϕ显著增加。多胺有一种已知的抗- 炎症功能，包括抑制许多促炎细胞因子。我们建议测试一下 假设M-ϕ在靶细胞摄取后上调多胺合成是抑制 一种关键的Mϕ细胞因子IL-1β，对体内肺部炎症的消退很重要。 在K99指导阶段，我将以我研究Mϕ生物学和靶细胞的经验为基础 通过学习与细胞代谢相关的技术，包括无偏代谢组学和使用 用于测量代谢流量的同位素代谢物。在这一阶段，我将：1)完成全面、 对摄取目标细胞后产生的代谢物进行综合评估，重点是多胺， 并评估这些代谢物是否来自消化的靶细胞材料，以及2)具体评估 精氨酸酶-1在炎性M-ϕ调节多胺合成中的作用同时，我将丰富我的 通过参加期刊俱乐部、研讨会、国家会议、课程和 由实习生咨询委员会每半年进行一次评估。R00独立阶段将允许我 在我继续研究Mϕ多胺如何影响细胞因子产生的同时建立我的实验室 靶细胞摄取，以及4)Mϕ多胺对炎症消退和肺修复的影响。 总的来说，这项建议将加强我目前的专业知识，解决目标领域的一个关键未知问题 细胞清除，并提供必要的基础，以确立我作为一名独立的研究科学家。

ABSTRACT Inflammation is a ubiquitous component of lung disease involving accumulation of leukocytes in the airspaces. In order for inflammation to resolve, dead and dying leukocytes must be removed and production of inflammatory cytokines must be turned off. Macrophages (Mϕ) are key orchestrators of these processes, however the triggers that reprogram inflammatory Mϕ to perform these roles remain incompletely understood. Clearance of dead cells has been shown to provide an important reprogramming signal, but we have limited knowledge of the precise mechanisms by which dead cell ingestion facilitates redirection of Mϕ function. Recent work has demonstrated a key role for cellular metabolism in determining Mϕ function. Ingested target cells provide a clear source of varied macromolecules that must be digested by Mϕ. However, little research has been done to assess intracellular metabolites produced by this process or to consider the subsequent immune consequence. We propose the first comprehensive study of Mϕ metabolism following target cell ingestion and degradation, and hypothesize that changes in the levels of intracellular metabolites control ingestion-driven Mϕ reprogramming. Our preliminary studies have identified a promising molecular target, polyamines, which are dramatically increased in Mϕ following the ingestion of apoptotic target cells. Polyamines have a known anti- inflammatory function including suppression of numerous pro-inflammatory cytokines. We propose to test the hypothesis that upregulation of polyamine synthesis by Mϕ following target cell ingestion is critical to suppress a key Mϕ cytokine IL-1β, and important for the resolution of lung inflammation in vivo. During the K99 mentored phase, I will build upon my experience studying Mϕ biology and target cell clearance by learning techniques related to cellular metabolism including unbiased metabolomics and the use of isotope metabolites to measure metabolic flux. During this phase, I will: 1) complete a comprehensive, integrated assessment of the metabolites produced following ingestion of target cells, focusing on polyamines, and assess whether these metabolites derive from digested target cell material, and 2) specifically assess the role of arginase-1 in regulating polyamine synthesis by inflammatory Mϕ. Simultaneously, I will enrich my professional development by participating in journal clubs, seminars, national conferences, coursework, and having semi-annual evaluations by a trainee advisory committee. The R00 independent phase will allow me to establish my laboratory as I continue investigation into: 3) how Mϕ polyamines affect cytokine production after target cell ingestion, and 4) the effects of Mϕ polyamines on resolution of inflammation and lung repair. Collectively, this proposal will enhance my current expertise, address a critical unknown in the field of target cell clearance, and provide the necessary foundation to establish myself as an independent research scientist.