JoVE Journal > Immunology and Infection
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Abstract
Introduction
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Immunology and Infection

实时测量中性粒细胞的线粒体生物能量特征

Published: June 02, 2023
doi:
10.3791/64971
, ,
1Department of Immunology, School of Medicine,UConn Health, 2Arthritis and Clinical Immunology Program,Oklahoma Medical Research Foundation, 3Department of Microbiology and Immunology,University of Oklahoma Health Science Center

Summary

我们描述了使用代谢细胞外通量分析仪测量小鼠和人中性粒细胞以及HL60细胞线粒体呼吸的逐步方案。

Abstract

中性粒细胞是人类的第一道防线，也是最丰富的白细胞。这些效应细胞执行吞噬作用和氧化爆发等功能，并产生中性粒细胞细胞外陷阱（NET）用于微生物清除。对中性粒细胞代谢活动的新见解挑战了它们主要依赖于糖酵解的早期概念。代谢活动的精确测量可以揭示中性粒细胞的不同代谢需求，包括生理条件和疾病状态下的三羧酸（TCA）循环（也称为克雷布斯循环），氧化磷酸化（OXPHOS），磷酸戊糖途径（PPP）和脂肪酸氧化（FAO）。本文描述了在代谢细胞外通量分析仪上进行代谢通量分析，测量小鼠骨髓来源中性粒细胞、人血源性中性粒细胞和中性粒细胞样HL60细胞系的耗氧率（OCR）作为线粒体呼吸指标的分步方案和先决条件。该方法可用于量化中性粒细胞在正常和疾病条件下的线粒体功能。

Introduction

线粒体在细胞生物能量学中起主要作用，其通过氧化磷酸化（OXPHOS）产生三磷酸腺苷（ATP）。除此之外，线粒体的作用延伸到活性氧的产生和解毒，细胞质和线粒体基质钙调节，细胞合成，分解代谢以及细胞内代谢物的运输1。线粒体呼吸在所有细胞中都是必不可少的，因为它们的功能障碍会导致代谢问题2，包括心血管疾病3 和多种神经退行性疾病，如年龄相关性黄斑变性4，帕金森氏症和阿尔茨海默氏症5，以及Charcot-Marie-Tooth疾病2 A（CMT2A）6。

对中性粒细胞的电子显微镜研究表明，线粒体7相对较少，并且由于线粒体呼吸速率非常低，它们严重依赖糖酵解来产生能量8。然而，线粒体对中性粒细胞功能至关重要，例如趋化性9和细胞凋亡10,11,12。之前的一项研究揭示了具有高膜电位的人中性粒体中性粒体网络。线粒体膜电位丢失是中性粒细胞凋亡的早期指标10。用线粒体解偶联剂羰基氰化物间氯苯基腙（CCCP）处理显示出对趋化性的显着抑制，以及线粒体形态的变化9,10。

虽然中性粒细胞的主要能量来源是糖酵解，但线粒体通过推动嘌呤能信号传导的第一阶段来提供启动中性粒细胞激活的 ATP，从而促进 Ca2+ 信号传导，放大线粒体 ATP 的产生，并启动中性粒细胞功能反应13。线粒体呼吸链功能障碍导致有毒活性氧（ROS）的过量产生并导致致病性损害14,15,16。NETosis是形成中性粒细胞细胞外陷阱（NET）的过程，是中性粒细胞的关键特性，可帮助它们对抗病原体17，并导致许多病理状况，包括癌症，血栓形成和自身免疫性疾病18。线粒体来源的 ROS 有助于 NETosis19，线粒体 DNA 可以是NETs 18 的组成部分，线粒体稳态改变会损害 NETosis 20,21,22,23,24。此外，在正常分化或成熟期间，中性粒细胞代谢重编程通过限制糖酵解活性而被逆转，并且它们参与线粒体呼吸并动员细胞内脂质25,26。

代谢细胞外通量分析仪可以连续监测和量化活细胞线粒体呼吸和糖酵解。该分析仪利用一个 96 孔板格式的传感器盒和两个荧光团来量化氧 （O2） 浓度和 pH 值变化。在测定过程中，传感器盒位于细胞单层上方，并形成~200nm高的微室。分析仪中的光纤束用于激发荧光团并检测荧光强度变化。自动计算 O2 浓度和 pH 值的实时变化，并显示为耗氧速率 （OCR） 和细胞外酸化速率 （ECAR）。传感器盒上有四个端口，允许在测定测量期间将多达四种化合物加载到每个孔中。该协议侧重于使用代谢细胞外通量分析仪量化小鼠和人中性粒细胞的线粒体呼吸，以及中性粒细胞样HL60细胞。

Protocol

肝素化全血样本是在获得知情同意后从健康人类捐赠者那里获得的，经康涅狄格大学卫生机构审查委员会根据赫尔辛基宣言批准。所有动物实验都遵循康涅狄格大学卫生机构动物护理和使用委员会（IACUC）的指导方针，并根据美国国立卫生研究院实验动物护理和使用指南中概述的标准，获得了康涅狄格大学健康IACUC对啮齿动物的使用批准。本研究使用6周龄的雄性C57BL / 6小鼠。 <s…

Representative Results

显示代表性的OCR动力学表明线粒体呼吸变化响应寡霉素，FCCP和鱼藤酮/抗霉素A小鼠中性粒细胞（图3A），人中性粒细胞（图3B）以及未分化和分化的HL60细胞（图3C）的混合物。在所有细胞中，寡霉素处理通过抑制ATP合酶的质子通道来降低OCR值;FCCP处理通过增加电子流量和耗氧量来恢复OCR值，以维持膜电位并实现最大呼吸;和鱼藤酮/?…

Discussion

使用代谢细胞外通量分析仪测量中性粒细胞线粒体呼吸的标准程序受到许多因素的限制，包括细胞数量、细胞生长和活力。在该测定中，每种化合物浓度因细胞的类型和来源而异。寡霉素和鱼藤酮/抗霉素A在大多数细胞类型中大多以相似的浓度使用。然而，由于FCCP诱导的最大呼吸频率因不同细胞而异，因此需要仔细滴定FCCP以优化浓度42。在优化期间执行 FCCP 的顺序添加也更好。?…

Disclosures

The authors have nothing to disclose.

Acknowledgements

我们感谢UConn Health免疫学系的Anthony T. Vella博士和Federica Aglianoin博士在使用代谢细胞外通量分析仪方面的培训，以及UConn Health免疫学系的Lynn Puddington博士对这些仪器的支持。我们感谢康涅狄格大学医学院的日内瓦·哈吉斯博士在科学写作和编辑本手稿方面的帮助。这项研究得到了美国国立卫生研究院，国家心脏，肺和血液研究所（R01HL145454），国家普通医学科学研究所（R35GM147713和P20GM139763），UConn Health的启动基金以及美国免疫学家协会的职业再入奖学金的支持。

Materials

37 °C non-CO2 incubator Precision Economy Model 2EG Instrument
Biorender Software Application
Centrifuge Eppendorf Model 5810R Instrument
Corning Cell-Tak Cell and Tissue Adhesive Corning 102416-100 Reagent
EasySep Magnet STEMCELL 18000 Magnet
EasySepMouse Neutrophil Enrichment kit STEMCELL 19762A Reagents
Graphpad Prism 9 Software Application
Human Serum Albumin Solution (25%) GeminiBio 800-120 Reagents
Ketamine (VetaKet) DAILYMED NDC 59399-114-10 Anesthetic
PBS Cytiva SH30256.01 Reagents
Plate buckets Eppendorf UL155 Accessory
PolymorphPrep PROGEN 1895 (previous 1114683) polysaccharide solution
Purified mouse anti-human CD18 antibody Biolegend 302102 Clone TS1/18
RPMI 1640 Medium Gibco 11-875-093 Reagents
Seahorse metabolic extracellular flux analyzer Agilent XFe96 Instrument
Seahorse XF Cell Mito Stress Test Kit Agilent 103015-100 mitochondrial stress test Kit
Swing-bucket rotor Eppendorf A-4-62 Rotor
Vactrap 2 Vacum Trap Fox Lifesciences 3052101-FLS Instrument
Wave Software Application
XF 1.0 M Glucose Solution Agilent 103577-100 Reagent
XF 100 mM Pyruvate Solution Agilent 103578-100 Reagent
XF 200 mM Glutamine Solution Agilent 103579-100 Reagent
XF DMEM medium Agilent 103575-100 Reagent
XFe96 FluxPak Agilent 102601-100 Material
Xylazine (AnaSed Injection) DAILYMED NDC 59399-110-20 Anesthetic
DOWNLOAD MATERIALS LIST

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Keywords: Mitochondrial RespirationNeutrophilsHL60 CellsMetabolic Extracellular Flux AnalyzerMitochondrial FunctionCell Seeding DensityAssay MediumMouse NeutrophilsHuman NeutrophilsDifferentiated HL60 CellsUndifferentiated HL60 CellsBackground Correction
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Pulikkot, S., Zhao, M., Fan, Z. Real-Time Measurement of the Mitochondrial Bioenergetic Profile of Neutrophils. J. Vis. Exp. (196), e64971, doi:10.3791/64971 (2023).
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