I vivo vurdering av Alveolære macrophage Efferocytosis etter ozon eksponering
Summary
Dette manuskriptet beskriver en protokoll for å avgjøre om eksponering for ozon, et kriterium luftforurensning, svekker alveolære macrophage efferocytosis in vivo. Denne protokollen benytter ofte brukte reagenser og teknikker og kan tilpasses flere modeller av lungeskade for å bestemme effekter på alveolære macrophage efferocytosis.
Abstract
Ozon (O3) er et kriterium luft forurensende som forverrer og øker forekomsten av kroniske lungesykdommer. O3 eksponering er kjent for å indusere lungebetennelse, men lite er kjent om hvordan eksponeringen endrer prosesser viktig for oppløsningen av betennelse. Efferocytosis er en oppløsning prosess, der makrofager phagocytize apoptotisk celler. Formålet med denne protokollen er å måle alveolære macrophage efferocytosis etter O3-indusert lungeskade og betennelse. Flere metoder har blitt beskrevet for å måle efferocytosis; men de fleste krever ex vivo manipulasjoner. Beskrevet i detalj her er en protokoll for å måle in vivo alveolære macrophage efferocytosis 24 timer etter O3 eksponering, noe som unngår ex vivo manipulering av makrofager og fungerer som en enkel teknikk som kan brukes til å nøyaktig representere forstyrrelser i Denne løsningsprosessen. Protokollen er en teknisk ikke-intensiv og relativt rimelig metode som involverer hele kroppen O3 inhalasjon etterfulgt av orofaryngeal aspirasjon av apoptotisk celler (dvs. Jurkat T celler) mens under narkose. Alveolære macrophage efferocytosis måles deretter ved lys mikroskopi evaluering av makrofager som er samlet inn fra lunges (BAL-tømming). Efferocytosis måles til slutt ved å beregne en efferocytic indeks. Kollektivt, de skisserte metodene kvantifisere efferocytic aktivitet i lungene in vivo samtidig tjene til å analysere negative helseeffekter av O3 eller andre inhalert fornærmelser.
Introduction
Lunge er stadig utsatt for miljømessige fornærmelser, inkludert luft partikler, virus, bakterier, og oksiderende gasser som utløser lungebetennelse1,2,3. Disse fornærmelser kan kompromittere gassutveksling og indusere irreversible vevsskade4,5. Alveolære makrofager, som utgjør ca 95% av immunceller funnet i murine og menneskelige lunger ved homeostase, er kritiske regulatorer av lungebetennelse etter miljømessige fornærmelser1,2, 3,4,5. Alveolære makrofager er avgjørende i verts forsvaret ved å phagocytizing og eliminere patogener. Nylig, alveolære makrofager har vist å fremme vev homeostase og oppløsningen av betennelse gjennom efferocytosis6,7. Efferocytosis er en phagocytic prosess der makrofager sluker og eliminere apoptotisk celler8,9,10. Efferocytosis også resulterer i produksjon av meglere (dvs. Il-10, TGF-β, PGE2, og nitrogenoksid) som ytterligere utfylle prosessen, noe som resulterer i oppløsningen av betennelse9,10,11 ,12,16,18. Denne prosessen er nødvendig for å forebygge sekundær nekrose og fremme vev homeostase12,13,14. Flere studier har knyttet svekket efferocytosis med ulike kroniske lungesykdommer, inkludert astma, kronisk obstruktiv lungesykdom, og idiopatisk pulmonal fibrose8,9,15, 16,17.
O3 er et kriterium luft forurensende som forverrer og øker forekomsten av kroniske lungesykdommer19,20,21. O3 induserer lungebetennelse og skade og er kjent for å svekke alveolære macrophage fagocytose av bakterielle patogener22,23. Det er imidlertid ukjent om O3 svekker alveolære macrophage efferocytosis. Gransker O3-indusert endringer i alveolære macrophage efferocytosis vil gi potensiell innsikt i hvordan eksponering kan føre til kroniske lungesykdom forekomst og forverring. Beskrevet nedenfor er en enkel metode for å evaluere alveolære macrophage efferocytosis i lungene av kvinnelige mus etter akutt O3 eksponering.
Den skisserte metoden disponerer flere fordeler fremfor andre efferocytosis protokoller som vanligvis brukes i feltet ved å eliminere bruken av kostbare fluorescerende fargestoffer, omfattende strømnings flowcytometri målinger og ex vivo-manipulering av alveolære makrofager24 ,25. I tillegg denne protokollen tiltak alveolære macrophage efferocytosis i sammenheng med lunge mikromiljøet, som kan påvirke macrophage funksjon.
Protocol
Representative Results
Discussion
Efferocytosis er en anti-inflammatorisk prosess der makrofager Clear apoptotisk celler og rusk samt produsere flere anti-inflammatorisk meglere9,10,11,12,16 ,18. Flere modeller av efferocytosis har gitt innsikt i hvordan macrophage er en kritisk celle i oppløsningen av betennelse6,<sup cla…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
Denne studien er finansiert av Health Effects Institute Walter A. Rosenblith Award og NIEHS R01ES028829 (til K. M. G). Vi vil gjerne takke Dr. Dianne Walters (Department of fysiologi, ECU) for hennes hjelp med å skaffe representative bilder av 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. |
Riferimenti
- Puttur, F., Gregory, L. G., Lloyd, C. M. Airway macrophages as the guardians of tissue repair in the lung. Immunology and Cell Biology. , (2019).
- Gregoire, M., et al. Impaired efferocytosis and neutrophil extracellular trap clearance by macrophages in ARDS. European Respiratory Journal. 52 (2), (2018).
- Fan, E. K. Y., Fan, J. Regulation of alveolar macrophage death in acute lung inflammation. Respiratory Research. 19 (1), 50 (2018).
- Michlewska, S., McColl, A., Rossi, A. G., Megson, I. L., Dransfield, I. Clearance of dying cells and autoimmunity. Autoimmunity. 40 (4), 267-273 (2007).
- Bhattacharya, J., Westphalen, K. Macrophage-epithelial interactions in pulmonary alveoli. Seminars in Immunopathology. 38 (4), 461-469 (2016).
- Donnelly, L. E., Barnes, P. J. Defective phagocytosis in airways disease. Chest. 141 (4), 1055-1062 (2012).
- Morimoto, K., Janssen, W. J., Terada, M. Defective efferocytosis by alveolar macrophages in IPF patients. Respiratory Medicine. 106 (12), 1800-1803 (2012).
- Vandivier, R. W., et al. Dysfunctional cystic fibrosis transmembrane conductance regulator inhibits phagocytosis of apoptotic cells with proinflammatory consequences. American Journal of Physiology Lung Cellular and Molecular Physiology. 297 (4), L677-L686 (2009).
- Grabiec, A. M., et al. Diminished airway macrophage expression of the Axl receptor tyrosine kinase is associated with defective efferocytosis in asthma. The Journal of Allergy and Clinical Immunology. 140 (4), 1144-1146 (2017).
- Chen, W., Frank, M. E., Jin, W., Wahl, S. M. TGF-beta released by apoptotic T cells contributes to an immunosuppressive milieu. Immunity. 14 (6), 715-725 (2001).
- Gao, Y., Herndon, J. M., Zhang, H., Griffith, T. S., Ferguson, T. A. Antiinflammatory effects of CD95 ligand (FasL)-induced apoptosis. Journal of Experimental Medicine. 188 (5), 887-896 (1998).
- O’Brien, B. A., Fieldus, W. E., Field, C. J., Finegood, D. T. Clearance of apoptotic beta-cells is reduced in neonatal autoimmune diabetes-prone rats. Cell Death and Differentiation. 9 (4), 457-464 (2002).
- Shen, Z. X., et al. Mineralocorticoid Receptor Deficiency in Macrophages Inhibits Atherosclerosis by Affecting Foam Cell Formation and Efferocytosis. Journal of Biological Chemistry. 292 (3), 925-935 (2017).
- Allard, B., Panariti, A., Martin, J. G. Alveolar Macrophages in the Resolution of Inflammation, Tissue Repair, and Tolerance to Infection. Frontiers in Immunology. 9, 1777 (2018).
- Hamon, R., et al. Bushfire smoke is pro-inflammatory and suppresses macrophage phagocytic function. Science Reports. 8 (1), 13424 (2018).
- Angsana, J., Chen, J., Liu, L., Haller, C. A., Chaikof, E. L. Efferocytosis as a regulator of macrophage chemokine receptor expression and polarization. European Journal of Immunology. 46 (7), 1592-1599 (2016).
- Karaji, N., Sattentau, Q. J. Efferocytosis of Pathogen-Infected Cells. Frontiers in Immunology. 8, 1863 (2017).
- Brouckaert, G., et al. Phagocytosis of necrotic cells by macrophages is phosphatidylserine dependent and does not induce inflammatory cytokine production. Molecular Biology of the Cell. 15 (3), 1089-1100 (2004).
- Gonzalez-Guevara, E., et al. Exposure to ozone induces a systemic inflammatory response: possible source of the neurological alterations induced by this gas. Inhalation Toxicology. 26 (8), 485-491 (2014).
- Robertson, S., et al. CD36 mediates endothelial dysfunction downstream of circulating factors induced by O3 exposure. Toxicological Sciences. 134 (2), 304-311 (2013).
- Kilburg-Basnyat, B., et al. Specialized Pro-Resolving Lipid Mediators Regulate Ozone-Induced Pulmonary and Systemic Inflammation. Toxicological Sciences. 163 (2), 466-477 (2018).
- Jakab, G. J., Spannhake, E. W., Canning, B. J., Kleeberger, S. R., Gilmour, M. I. The effects of ozone on immune function. Environ Health Perspect. 103 Suppl 2, 77-89 (1995).
- Gilmour, M. I., Hmieleski, R. R., Stafford, E. A., Jakab, G. J. Suppression and recovery of the alveolar macrophage phagocytic system during continuous exposure to 0.5 ppm ozone. Experimental Lung Research. 17 (3), 547-558 (1991).
- Nayak, D. K., Mendez, O., Bowen, S., Mohanakumar, T. Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages. Journal of Visualized Experiments. 10 (134), (2018).
- Tao, H., et al. Macrophage SR-BI mediates efferocytosis via Src/PI3K/Rac1 signaling and reduces atherosclerotic lesion necrosis. Journal of Lipid Research. 56 (8), 1449-1460 (2015).
- Coe, L. M., et al. FGF-23 is a negative regulator of prenatal and postnatal erythropoiesis. Journal of Biological Chemistry. 289 (14), 9795-9810 (2014).
- Yong, W. K., Abd Malek, S. N. Xanthohumol induces growth inhibition and apoptosis in ca ski human cervical cancer cells. Evidence-Based Complementary and Alternative Medicine. , 921306 (2015).
- Van de Laar, L., et al. Yolk Sac Macrophages, Fetal Liver, and Adult Monocytes Can Colonize an Empty Niche and Develop into Functional Tissue-Resident Macrophages. Immunity. 44 (4), 755-768 (2016).
- Lavin, Y., et al. Tissue-resident macrophage enhancer landscapes are shaped by the local microenvironment. Cell. 159 (6), 1312-1326 (2014).
- Beattie, L., et al. Bone marrow-derived and resident liver macrophages display unique transcriptomic signatures but similar biological functions. Journal of Hepatology. 65 (4), 758-768 (2016).
- Svedberg, F. R., et al. The lung environment controls alveolar macrophage metabolism and responsiveness in type 2 inflammation. Nature Immunology. , (2019).
- Crowther, J. E., et al. Pulmonary surfactant protein a inhibits macrophage reactive oxygen intermediate production in response to stimuli by reducing NADPH oxidase activity. Journal of Immunology. 172 (11), 6866-6874 (2004).
- Silveyra, P., Floros, J. Genetic variant associations of human SP-A and SP-D with acute and chronic lung injury. Frontiers in Bioscience. 17, 407-429 (2012).
- Schagat, T. L., Wofford, J. A., Wright, J. R. Surfactant protein A enhances alveolar macrophage phagocytosis of apoptotic neutrophils. Journal of Immunology. 166 (4), 2727-2733 (2001).
- Gomez Perdiguero, E., et al. Tissue-resident macrophages originate from yolk-sac-derived erythro-myeloid progenitors. Nature. 518 (7540), 547-551 (2015).
- Nebbioso, A., et al. Time-resolved analysis of DNA-protein interactions in living cells by UV laser pulses. Scientific Reports. 7 (1), 11725 (2017).
- Novak, Z., et al. Efficacy of different UV-emitting light sources in the induction of T-cell apoptosis. Photochemistry and Photobiology. 79 (5), 434-439 (2004).
- Park, Y. J., et al. PAI-1 inhibits neutrophil efferocytosis. Proceedings of the National Academy of Sciences of the United States of America. 105 (33), 11784-11789 (2008).
- Kleeberger, S. R., Reddy, S., Zhang, L. Y., Jedlicka, A. E. Genetic susceptibility to ozone-induced lung hyperpermeability: role of toll-like receptor 4. American Journal of Respiratory Cell and Molecular Biology. 22 (5), 620-627 (2000).
- Wesselkamper, S. C., Chen, L. C., Kleeberger, S. R., Gordon, T. Genetic variability in the development of pulmonary tolerance to inhaled pollutants in inbred mice. American Journal of Physiology-Lung Cellular and Molecular Physiology. 281 (5), L1200-L1209 (2001).
