在臭氧暴露后阿尔韦拉巨噬细胞的Vivo评估中
Summary
本手稿描述了一个协议，用于确定暴露于臭氧（一种标准空气污染物）是否会损害体内的藻体细胞体细胞化。该协议利用常用试剂和技术，可适用于多种肺损伤模型，以确定对肺泡巨噬细胞细胞病的影响。
Abstract
臭氧 （O3） 是一种标准空气污染物，它加剧和增加慢性肺病的发病率.O3接触已知会引起肺部炎症，但很少知道接触如何改变对炎症解决重要的过程。细胞凋亡是一个解析过程，其中巨噬细胞噬菌体细胞。此协议的目的是测量O3诱发肺损伤和炎症后的肺泡巨噬细胞细胞化。介绍了几种测量细胞化的方法;然而，大多数需要外生操作。这里详细介绍了在O3接触后24小时测量体内海藻巨噬细胞的一种协议，它避免了对巨噬细胞的体细胞的体外操作，并用作一种简单的技术，可用于准确表示此解析过程。该协议是一种技术上非密集且相对便宜的方法，涉及全身O3吸入，随后在全身麻醉下对凋亡细胞（即Jurkat T细胞）的造血性吸入。然后，通过从支气管（BAL）洗浴中收集的巨噬细胞的光学显微镜评估来测量阿尔韦拉尔巨噬细胞。最后通过计算细胞指数来测量细胞病。总体而言，概述的方法量化肺在体内的排卵细胞活性，同时也有助于分析O3或其他吸入性侮辱对健康的负面影响。
Introduction
肺部经常受到环境侮辱，包括空气颗粒、病毒、细菌和氧化气体，这些气体会引发肺部炎症1，2，3。这些侮辱会损害气体交换，并诱发不可逆转的组织损伤4，5。阿尔韦拉巨噬细胞，约占在母体和人体肺中发现的免疫细胞的95%，是环境侮辱1、2、2、后肺部炎症的重要调节者。3，4，5.在宿主防御过程中，通过噬菌体和消除病原体，阿尔韦拉尔巨噬细胞是必不可少的。最近，阿尔韦拉尔巨噬细胞已被证明促进组织平衡和通过排卵细胞病6，7的炎症的解决。肝细胞化是一种吞噬过程，其中巨噬细胞吞噬和消除凋亡细胞8，9，10。埃塞洛西病还导致生产介质（即IL-10、TGF-+、PGE2和一氧化氮），进一步增强过程，导致炎症9、10、11的解决 ，12，16，18.这个过程是必要的，以防止继发性坏死和促进组织平衡12，13，14。几项研究已经将受损的肺细胞病与各种慢性肺病联系起来，包括哮喘、慢性阻塞性肺病和特发性肺纤维化8、9、15、 16，17.
O3是一种标准空气污染物，它加剧和增加慢性肺病的发病率19、20、21。O3诱发肺部炎症和损伤，已知会损害肺泡巨噬细胞噬菌体细胞化细菌病原体22，23。然而，目前还不清楚O3是否损害藻体细胞细胞化。调查O3诱导的肺泡巨噬细胞病变，将提供潜在的洞察力，了解接触如何导致慢性肺病的发病率和恶化。下面描述是一个简单的方法，以评估急性O3暴露后雌性小鼠肺部的肺泡细胞化。
与该领域常用的其他排卵虫化方案不同，该方法具有多种优势，无需使用昂贵的荧光染料、广泛的流式细胞测量和对藻体巨噬细胞的外体操作24 ， 25.此外，该方案还测量肺小食道在肺微环境中的肺细胞细胞化，从而影响巨噬细胞功能。
Protocol
所有方法均已获得东卡罗来纳大学机构动物护理和使用委员会（IACUC）的批准。 1. 臭氧 （O3） 和过滤空气暴露（第 1 天） 将最多 12 只 8-12 周大的雌性 C57BL/6J 小鼠放入钢笼（带 12 个独立的隔间），带钢丝网盖放入 O3暴露室。 将温度计与保持架放在曝光室中，以准确记录温度和湿度。 打开连接到设备的氧气和紫外线 （UV） 光。注：?…
Representative Results
O3接触已知可诱发肺部炎症和损伤，并且需要排卵细胞化来维持组织平衡。C57BL/6J雌性小鼠暴露于过滤空气（FA）或1ppm O3 3 3小时，并在接触后24小时进行尸检，以检查肺部炎症和损伤。与FA对照组相比，O3暴露小鼠在空气空间中的巨噬细胞和嗜中性粒细胞明显增加（图1A，B）。此外，O3-暴露小鼠的BAL蛋白显?…
Discussion
Efferocytosis是一个抗炎过程，其中巨噬细胞清除细胞和碎片，以及产生多个抗炎介质9，10，11，12，16 ， 18.多模型的细胞化提供了洞察宏噬细胞是如何在炎症6，7的分辨率的关键细胞。最近，慢性肺…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项研究由健康影响研究所沃尔特·罗森布利斯奖和NIEHS R01ES028829（到K.M.G）资助。我们要感谢DianneWalters博士（ECU生理学系）协助她获得有代表性的阿尔韦拉尔巨噬细胞图像。
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. |
References
- 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).
Cite This Article
Hodge, M. X., Reece, S. W., Madenspacher, J. H., Gowdy, K. M. In Vivo Assessment of Alveolar Macrophage Efferocytosis Following Ozone Exposure. J. Vis. Exp. (152), e60109, doi:10.3791/60109 (2019).