Cellular, Molecular and Biomedical Sciences Graduate Program, University of Vermont, Burlington, VT, United States of America.
The Department of Biomedical and Health Sciences, University of Vermont, Burlington, VT, United States of America.
PLoS One. 2024 Mar 7;19(3):e0299294. doi: 10.1371/journal.pone.0299294. eCollection 2024.
Dendritic cell (DC) activation is marked by key events including: (I) rapid induction and shifting of metabolism favoring glycolysis for generation of biosynthetic metabolic intermediates and (II) large scale changes in gene expression including the upregulation of the antimicrobial enzyme inducible nitric oxide synthase (iNOS) which produces the toxic gas nitric oxide (NO). Historically, acute metabolic reprogramming and NO-mediated effects on cellular metabolism have been studied at specific timepoints during the DC activation process, namely at times before and after NO production. However, no formal method of real time detection of NO-mediated effects on DC metabolism have been fully described. Here, using Real-Time Extracellular Flux Analysis, we experimentally establish the phenomenon of an NO-dependent mitochondrial respiration threshold, which shows how titration of an activating stimulus is inextricably linked to suppression of mitochondrial respiration in an NO-dependent manner. As part of this work, we explore the efficacy of two different iNOS inhibitors in blocking the iNOS reaction kinetically in real time and explore/discuss parameters and considerations for application using Real Time Extracellular Flux Analysis technology. In addition, we show, the temporal relationship between acute metabolic reprogramming and NO-mediated sustained metabolic reprogramming kinetically in single real-time assay. These findings provide a method for detection of NO-mediated metabolic effects in DCs and offer novel insight into the timing of the DC activation process with its associated key metabolic events, revealing a better understanding of the nuances of immune cell biology.
树突状细胞 (DC) 的激活标志着关键事件的发生，包括：(I) 快速诱导和代谢转移，有利于糖酵解生成生物合成代谢中间产物，以及 (II) 基因表达的大规模变化，包括抗菌酶诱导型一氧化氮合酶 (iNOS) 的上调，后者产生有毒气体一氧化氮 (NO)。从历史上看，急性代谢重编程和 NO 对细胞代谢的影响在 DC 激活过程中的特定时间点进行了研究，即在产生 NO 之前和之后的时间点。然而，还没有一种实时检测 NO 对 DC 代谢影响的正式方法得到充分描述。在这里，我们使用实时细胞外通量分析，实验性地建立了 NO 依赖性线粒体呼吸阈值的现象，该现象表明刺激的滴定与 NO 依赖性方式抑制线粒体呼吸是如何不可分割地联系在一起的。作为这项工作的一部分，我们探索了两种不同的 iNOS 抑制剂在实时动力学上阻断 iNOS 反应的效果，并探讨/讨论了使用实时细胞外通量分析技术应用的参数和考虑因素。此外，我们还展示了在单个实时测定中急性代谢重编程和 NO 介导的持续代谢重编程之间的时间关系。这些发现为检测 DC 中的 NO 介导的代谢效应提供了一种方法，并为 DC 激活过程及其相关关键代谢事件的时间安排提供了新的见解，揭示了对免疫细胞生物学细微差别更好的理解。
