Molecular and metabolic influences on the activation of monocytes and macrophages at single-cell resolution

项目概要/摘要 单核细胞和巨噬细胞在不同的过程中发挥作用，从稳态 维持免疫反应和组织再生。这些功能是 通过以下机制与细胞代谢协调并受到细胞代谢的强烈影响 在巨噬细胞群体中进行了越来越多的研究和表征。然而，这样的 研究掩盖了细胞间的变异，这是巨噬细胞的固有特性 多样性。事实上，单细胞转录组学数据已经证明巨噬细胞 连续梯度比离散状态更好地描述偏振 适合隔离和群体分析。然而，转录测量是 不足以表征塑造单核细胞的代谢和蛋白质网络 巨噬细胞多样性。了解这些网络如何控制巨噬细胞 极化和功能，我们建议直接量化蛋白质和调节信号 （例如关键调节因子的定位，例如 NF-κB）在原代人单核细胞中和 巨噬细胞对生理相关的代谢环境做出反应。 此外，我们将把这种单细胞分析扩展到这些细胞对 病原体相关分子模式和损伤相关分子模式。 这些数据将使我们能够识别驱动单核细胞和 巨噬细胞对代谢状态和分子模式的反应。随后，我们 将通过药理学和遗传扰动来测试这些网络。我们是独一无二的 由于我们最近开创了量化方法，因此我们有能力开展这项研究 许多单细胞中存在数千种蛋白质。此外，我们还有所需的 分析代谢系统的专业知识（包括有氧糖酵解，这通常是 与巨噬细胞激活相关）并开发新的数据算法 分析。该项目将增进我们对巨噬细胞免疫代谢的理解 和极化，将引入更灵敏、更准确的单细胞方法 分析，并将提供原理证明的可能性证明 单细胞分辨率下蛋白质介导的分子机制。我们坚信 实现这些目标将对生物医学研究产生变革性影响 并将提供新的、更好的治疗策略。

Project Summary/Abstract Monocytes and macrophages function in diverse processes, from homeostatic maintenance to immune responses and tissue regeneration. These functions are coordinated with and strongly influenced by cellular metabolism via mechanisms that are increasingly studied and characterized in populations of macrophages. However, such studies mask the cell-to-cell variation which is an inherent property of macrophage diversity. Indeed, single-cell transcriptomics data have demonstrated that macrophage polarization is better described by continuous gradients rather than by discrete states amenable to isolation and population analysis. Yet, transcriptional measurements are insufficient to characterize the metabolic and protein networks that shape monocyte and macrophage diversity. To understand how these networks control macrophage polarization and functions, we propose to directly quantify proteins and regulatory signals (such as localization of key regulators, e.g., NF-κB) in primary human monocytes and macrophages responding to physiologically relevant metabolic environments. Furthermore, we will extend this single-cell analysis to the responses of these cells to pathogen-associated molecular patterns and damage-associated molecular patterns. These data will enable us to identify likely regulatory networks driving monocyte and macrophage responses to metabolic states and molecular patterns. Subsequently, we will test these networks via pharmacological and genetic perturbations. We are uniquely positioned to perform this research since we recently pioneered methods for quantifying thousands of proteins across many single cells. Furthermore, we have the required expertise in analyzing metabolic systems (including aerobic glycolysis, which is frequently associated with macrophage activation) and developing new algorithms for data analysis. This project will advance our understanding of macrophage immunometabolism and polarization, will introduce methods for more sensitive and accurate single-cell analysis, and will provide a proof-of-principle demonstration of the possibility to identify protein-mediated molecular mechanisms at single-cell resolution. We strongly believe that attaining these goals will have a transformative impact on biomedical research and will inform new and better therapeutic strategies.