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Immunologie et infection

Realtidsmåling af mitokondriel bioenergetisk profil af neutrofiler

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

Vi beskriver trinvise protokoller, der måler mitokondriel respiration af muse- og humane neutrofiler og HL60-celler ved hjælp af den metaboliske ekstracellulære fluxanalysator.

Abstract

Neutrofiler er den første forsvarslinje og de mest rigelige leukocytter hos mennesker. Disse effektorceller udfører funktioner såsom fagocytose og oxidativ burst og skaber neutrofile ekstracellulære fælder (NET’er) til mikrobiel clearance. Ny indsigt i neutrofilers metaboliske aktiviteter udfordrer det tidlige koncept, at de primært er afhængige af glykolyse. Præcis måling af metaboliske aktiviteter kan udfolde forskellige metaboliske krav til neutrofiler, herunder tricarboxylsyre (TCA) cyklus (også kendt som Krebs cyklus), oxidativ phosphorylering (OXPHOS), pentosephosphatvej (PPP) og fedtsyreoxidation (FAO) under fysiologiske forhold og i sygdomstilstande. Dette papir beskriver en trinvis protokol og forudsætninger for at måle iltforbrugshastigheden (OCR) som en indikator for mitokondriel respiration på museknoglemarvsafledte neutrofiler, humane blodafledte neutrofiler og den neutrofillignende HL60-cellelinje ved hjælp af metabolisk fluxanalyse på en metabolisk ekstracellulær fluxanalysator. Denne metode kan anvendes til kvantificering af mitokondriefunktionerne hos neutrofiler under normale og sygdomsbetingelser.

Introduction

Mitokondrier spiller en vigtig rolle i cellebioenergetik, som genererer adenosintrifosfat (ATP) ved oxidativ phosphorylering (OXPHOS). Ud over dette strækker mitokondriernes rolle sig ind i generering og afgiftning af reaktive iltarter, cytoplasmatisk og mitokondriel matrixcalciumregulering, cellulær syntese, katabolisme og transport af metabolitter i cellen1. Mitokondriel respiration er afgørende i alle celler, da deres dysfunktion kan resultere i metaboliske problemer 2, herunder hjerte-kar-sygdomme3 og en lang række neurodegenerative sygdomme, såsom aldersrelateret makuladegeneration4, Parkinsons og Alzheimers sygdomme5 og Charcot-Marie-Tooth sygdom2 A (CMT2A)6.

Elektronmikroskopiske undersøgelser af neutrofiler afslørede, at der er relativt få mitokondrier7, og de er stærkt afhængige af glykolyse for deres energiproduktion, da mitokondrie respirationshastigheder er meget lave8. Imidlertid er mitokondrier afgørende for neutrofile funktioner, såsom kemotakse9 og apoptose10,11,12. En tidligere undersøgelse afslørede et komplekst mitokondrienetværk i humane neutrofiler med højt membranpotentiale. Mitokondriemembranens potentielle tab er en tidlig indikator for neutrofil apoptose10. Behandling med mitokondriel afkobler carbonylcyanid m-chlorphenylhydrazon (CCCP) viste signifikant hæmning i kemotaksi sammen med en ændring i mitokondriemorfologi 9,10.

Selvom den primære energikilde for neutrofiler er glykolyse, tilvejebringer mitokondrier ATP, der initierer neutrofilaktivering ved at brænde den første fase af purinerg signalering, hvilket øger Ca2+ signalering, forstærker mitokondriel ATP-produktion og initierer neutrofile funktionelle reaktioner13. Dysfunktion af mitokondrie-respirationskæden resulterer i overdreven produktion af toksiske reaktive iltarter (ROS) og fører til patogene skader14,15,16. NETosis, som er processen med at danne neutrofile ekstracellulære fælder (NET’er), er en kritisk egenskab ved neutrofiler, der hjælper dem med at bekæmpe patogener17 og bidrager til mange patologiske tilstande, herunder kræft, trombose og autoimmune lidelser18. Mitokondrieafledt ROS bidrager til NETosis19, mitokondrie-DNA kan være en komponent i NETs18, og ændret mitokondriehomeostase svækker NETosis 20,21,22,23,24. Desuden bliver neutrofil metabolisk omprogrammering under normal differentiering eller modning vendt ved at begrænse glykolytisk aktivitet, og de engagerer sig i mitokondriel respiration og mobiliserer intracellulære lipider25,26.

Den metaboliske ekstracellulære fluxanalysator kan kontinuerligt overvåge og kvantificere levende celle mitokondriel respiration og glykolyse. Analysatoren anvender en 96-brønds pladeformatsensorpatron og to fluoroforer til at kvantificere iltkoncentration (O2) og pH-ændringer. Sensorpatronen er over cellemonolaget under analysen og danner et ~ 200 nm højt mikrokammer. De optiske fiberbundter i analysatoren bruges til at excitere fluoroforerne og detektere de fluorescerende intensitetsændringer. Ændringer i realtid iO2-koncentration og pH beregnes automatisk og vises som iltforbrugshastighed (OCR) og ekstracellulær forsuringshastighed (ECAR). Der er fire porte på sensorpatronen, der gør det muligt at indlæse op til fire forbindelser i hver brønd under analysemålingerne. Denne protokol fokuserer på kvantificering af mitokondriel respiration af mus og humane neutrofiler samt de neutrofillignende HL60-celler ved hjælp af den metaboliske ekstracellulære fluxanalysator.

Protocol

Hepariniserede fuldblodsprøver blev opnået fra raske humane donorer efter at have opnået informeret samtykke, som godkendt af Institutional Review Board of UConn Health i overensstemmelse med Helsingfors-erklæringen. Alle dyreforsøg fulgte UConn Health Institutional Animal Care and Use Committee (IACUC) retningslinjer, og godkendelse til brug af gnavere blev opnået fra UConn Health IACUC i henhold til kriterier skitseret i vejledningen til pleje og brug af forsøgsdyr fra National Institutes of Health. C57BL/6-hanm…

Representative Results

Repræsentativ OCR-dynamik vises, hvilket indikerer mitokondrierespirationsændringer som reaktion på oligomycin, FCCP og rotenon/antimycin En blanding af museneutrofiler (figur 3A), humane neutrofiler (figur 3B) og udifferentierede og differentierede HL60-celler (figur 3C). I alle celler nedsætter oligomycinbehandling OCR-værdien ved at hæmme protonkanalen i ATP-syntase; FCCP-behandling gendanner OCR-værdien ved at øge strøm…

Discussion

Standardproceduren, der måler mitokondriel respiration af neutrofiler ved hjælp af den metaboliske ekstracellulære fluxanalysator, er begrænset af mange faktorer, herunder celleantal, cellevækst og levedygtighed. Hver sammensat koncentration varierer mellem typen og kilden til celler i dette assay. Oligomycin og rotenon/antimycin A anvendes mest i en lignende koncentration blandt de fleste celletyper. Da den FCCP-inducerede maksimale respirationsfrekvens varierer mellem forskellige celler, kræves der imidlertid om…

Divulgations

The authors have nothing to disclose.

Acknowledgements

Vi anerkender Dr. Anthony T. Vella og Dr. Federica Aglianoin fra Institut for Immunologi ved UConn Health for deres træning i at bruge den metaboliske ekstracellulære fluxanalysator og Dr. Lynn Puddington i Institut for Immunologi ved UConn Health for hendes støtte til instrumenterne. Vi anerkender Dr. Geneva Hargis fra UConn School of Medicine for hendes hjælp med videnskabelig skrivning og redigering af dette manuskript. Denne forskning blev støttet af tilskud fra National Institutes of Health, National Heart, Lung and Blood Institute (R01HL145454), National Institute of General Medical Sciences (R35GM147713 og P20GM139763), en opstartsfond fra UConn Health og et karriere-genindrejsestipendium fra American Association of Immunologer.

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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Tags

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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