In vivo-vurdering af alveolær makrofag-Efferocytose efter ozoneksponering
Summary
Dette manuskript beskriver en protokol til bestemmelse af, hvorvidt udsættelse for ozon, et kriterium luftforurenende stof, hæmmer alveolær makrofag efferocytose in vivo. Denne protokol udnytter almindeligt anvendte reagenser og teknikker og kan tilpasses til flere modeller af lungeskade til at bestemme virkninger på alveolær makrofag efferocytose.
Abstract
Ozon (O3) er et kriterium luftforurenende stof, der forværrer og øger forekomsten af kroniske lungesygdomme. O3 eksponering er kendt for at inducere lungebetændelse, men der vides ikke meget om, hvordan eksponeringen ændrer processer, der er vigtige for opløsningen af inflammation. Efferocytose er en afviklingsproces, hvor makrofager phagocytize apoptotiske celler. Formålet med denne protokol er at måle alveolære makrofag-efferocytose efter O3-induceret lungeskade og inflammation. Der er beskrevet flere metoder til måling af efferocytose; de fleste kræver dog ex vivo manipulationer. Beskrevet i detaljer her er en protokol til at måle in vivo alveolær makrofag efferocytose 24 h efter O3 eksponering, som undgår ex vivo manipulation af makrofager og fungerer som en simpel teknik, der kan bruges til præcist at repræsentere perturbationer i denne afviklingsproces. Protokollen er en teknisk ikke-intensiv og relativt billig metode, der involverer hele kroppen O3 indånding efterfulgt af orofaryngeal aspiration af apoptotiske celler (dvs., Jurkat T celler) under generel anæstesi. Alveolær makrofag efferocytose måles derefter ved let mikroskopi evaluering af makrofager indsamlet fra bronalveolær (BAL) skylning. Efferocytose måles endelig ved at beregne et efferocytisk indeks. Tilsammen kvantificerer de skitserede metoder efferocytisk aktivitet i lungerne in vivo og tjener også til at analysere de negative sundhedsmæssige virkninger af O3 eller andre inhalerede fornærmelser.
Introduction
Lungerne er konstant udsat for miljømæssige fornærmelser, herunder luft partikler, vira, bakterier, og oxidant gasser, der udløser lungebetændelse1,2,3. Disse fornærmelser kan kompromittere gasudveksling og fremkalde irreversibel vævsskade4,5. Alveolære makrofager, som udgør ca. 95% af de immunceller, der findes i murine og humane lunger ved homøostase, er kritiske regulatorer af lungebetændelse efter miljømæssige fornærmelser1,2, 3,4,5. Alveolære makrofager er afgørende i værts forsvaret ved fagocyterende og eliminere patogener. For nylig har alveolære makrofager vist sig at fremme vævs homøostase og opløsningen af inflammation gennem efferocytose6,7. Efferocytose er en Fagocytisk proces, hvor makrofager opsluge og eliminere apoptotiske celler8,9,10. Efferocytose resulterer også i produktionen af mediatorer (dvs. Il-10, TGF-β, PGE2, og nitrogenoxid), der yderligere forstærker processen, hvilket resulterer i opløsningen af inflammation9,10,11 ,12,16,18. Denne proces er nødvendig for at forebygge sekundær nekrose og fremme vævs homøostase12,13,14. Flere undersøgelser har forbundet nedsat efferocytose med forskellige kroniske lungesygdomme, herunder astma, kronisk obstruktiv lungesygdom, og idiopatisk pulmonal fibrose8,9,15, 16,17.
O3 er et kriterium luftforurenende stof, der forværrer og øger forekomsten af kroniske lungesygdomme19,20,21. O3 inducerer lungebetændelse og-skade og vides at forringe alveolær makrofag fagocytose af bakterielle patogener22,23. Det er imidlertid ukendt, om O3 hæmmer alveolær makrofag efferocytose. Undersøge O3-induceret ændringer i alveolær makrofag efferocytose vil give potentielle indblik i, hvordan eksponering kan føre til kronisk lungesygdom incidens og eksacerbation. Beskrevet nedenfor er en simpel metode til at evaluere alveolær makrofag efferocytose i lungerne hos hunmus efter akut O3 eksponering.
Den skitserede metode besidder flere fordele i forhold til andre efferocytose protokoller almindeligvis anvendes i marken ved at eliminere brugen af dyre fluorescerende farvestoffer, omfattende flow cytometri målinger, og ex vivo manipulation af alveolære makrofager24 ,25. Desuden, denne protokol foranstaltninger alveolær makrofag efferocytose i forbindelse med lunge mikromiljø, som kan påvirke makrofag funktion.
Protocol
Representative Results
Discussion
Efferocytose er en antiinflammatorisk proces, hvor makrofager klar apoptotiske celler og snavs samt producere flere anti-inflammatoriske mediatorer9,10,11,12,16 ,18. Flere modeller af efferocytosis har givet indsigt i, hvordan makrofag er en kritisk celle i opløsningen af inflammation6,<su…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Denne undersøgelse er finansieret af Health Effects Institut Walter A. Rosenblith Award og NIEHS R01ES028829 (til K. M. G). Vi vil gerne takke Dr. Dianne Walters (Institut for fysiologi, ECU) for hendes hjælp til at opnå repræsentative billeder af alveolære makrofager.
Materials
| Annexin V-FITC Kit | Trevigen | 4830-250-K | The TACS Annexin V-FITC Kit allows rapid, specific, and quantitative identification of apoptosis in individual cells when using flow cytometry. |
| BCL2 Jurkat T Cells | ATCC | ATCC CRL-2899 | The BCL2 Jurkat cell line was derived by transfecting human Jurkat T cells with the pSFFV-neo mammalian expression vector containing the human BCL-2 ORF insert and a neomycin-resistant gene. Has been for models of measuring efferocytosis. |
| Countess II Automated Cell Counter | Thermofisher | AMQAX1000 | It is a benchtop assay platform equipped with state-of-the-art optics, full autofocus, and image analysis software for rapid assessment of cells in suspension. Very easy to use. |
| Cytospin 4 Cytocentrifuge | Thermofisher | A78300003 | Provides economical thin-layer preparations from any liquid matrix, especially hypocellular fluids such as bronchoalveolar lavage fluid. |
| Fetal Bovine Serum, qualified, heat inactivated | Thermofisher | 16140071 | Provides Nutrients to cultured cells for them to grow. It is standard for cell culture. |
| Kwik-Diff Reagent 2, Eosin | Thermofisher | 9990706 | Eosin staining that stains cytoplasm. |
| Kwik-Diff Reagent 1, Fixative | Thermofisher | 9990705 | Fixes cells to be stained by H&E. |
| Kwik-Diff Reagent 3, Methylene Blue | Thermofisher | 9990707 | Methylene Blue staining that stains the nucleus. |
| Penicillin-Streptomycin | Sigma/Aldrich | P0781-100ML | Penicillin-Streptomycin is the most commonly used antibiotic solution for culture of mammalian cells. Additionally it is used to maintain sterile conditions during cell culture. |
| RPMI 1640 Medium, GlutaMAX Supplement | Thermofisher | 61870036 | RPMI 1640 Medium (Roswell Park Memorial Institute 1640 Medium) was originally developed to culture human leukemic cells in suspension and as a monolayer. RPMI 1640 medium has since been found suitable for a variety of mammalian cells, including HeLa, Jurkat, MCF-7, PC12, PBMC, astrocytes, and carcinomas. Helps grow Jurkat T cells fast and efficiently. |
| Stratagene UV Stratalinker 1800 UV Crosslinker | Cambridge Scientific | 16659 | The Stratalinker UV crosslinker is designed to induce apoptosis, crosslink DNA or RNA to nylon, nitrocellulose, or nylon-reinforced nitrocellulose membranes. |
| Teledyne T400 ultraviolet light photometer | Teledyne API | T400 | The Model T400 UV Absorption analyzer uses a system based on the Beer-Lambert law for measuring low ranges of ozone in ambient air. |
| Teledyne T703 Ozone calibrator | Teledyne API | T703 | Provides feedback control of the UV lamp intensity, assuring stable ozone output. |
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