流式细胞术分析鉴定鼠肺先天免疫细胞和适应性免疫细胞
Summary
在这项研究中，我们提出了一种有效且可重复的方案来分离小鼠呼吸系统的免疫群体。我们还提供了一种方法，使用基于9色的流式细胞术面板来鉴定驻留在健康小鼠肺部的所有先天性和适应性免疫细胞。
Abstract
呼吸道与外部环境直接接触，需要精确调节的免疫系统来提供保护，同时抑制对环境抗原的不良反应。肺宿主着几个先天性和适应性免疫细胞群，这些免疫细胞提供免疫监视，但也介导保护性免疫反应。这些细胞保持健康的肺免疫系统平衡，也参与几种病理状况，如哮喘，感染，自身免疫性疾病和癌症。表面和细胞内蛋白质的选择性表达为肺的免疫细胞提供独特的免疫表型特性。因此，流式细胞术在稳态和病理条件下鉴定此类细胞群方面起着重要作用。本文提出了一种方案，描述了一种一致且可重复的方法，用于在稳态条件下鉴定驻留在健康小鼠肺部的免疫细胞。然而，该协议也可用于识别各种疾病模型中这些细胞群的变化，以帮助识别肺免疫景观中的疾病特异性变化。
Introduction
鼠呼吸道含有独特的免疫系统，负责对抗病原体和维持免疫稳态。肺免疫系统由在表型、功能、起源和位置方面具有显著异质性的细胞群体组成。常驻肺泡巨噬细胞 （AMs） 主要起源于胎儿单核细胞，位于肺泡腔1，而骨髓来源的间质巨噬细胞 （IM） 位于肺实质2。IM可以通过CD206的表达式进一步细分。CD206+ IM位于支气管周围和血管周围区域，而CD206-IM位于肺泡间质3。最近提出了IM的几个子分类3，4，5，6。虽然IM的研究少于AM，但最近的证据表明它们在调节肺免疫系统中起着至关重要的作用7。此外，CD206还以替代激活的AM8表示。
肺树突状细胞（DC）是肺免疫细胞的另一组异质性，就其功能特性，位置和起源而言。在肺中已经描述了四种DC亚类：常规CD103 + DC（也称为cDC1），常规CD11b + DC（也称为cDC2），单核细胞衍生的DC（MoDC）和浆细胞样DCs9，10，11，12，13。前三个子类可以定义为主要的组织相容性复合物（MHC）II + CD11c + 9，10，14，15。浆细胞样 DC 表达 MHC II，CD11c 中度阳性，但表达高水平的 B220 和 PDCA-19，13，16。在幼稚的鼠肺中，CD103 DC和CD11b DC位于气道间质中，而浆细胞样DC位于肺泡间质17中。
单核细胞的两个主要群体在稳态下驻留在肺中：经典单核细胞和非经典单核细胞。经典的单核细胞是Ly6C+ ，对最初的炎症反应至关重要。相比之下，非经典单核细胞是Ly6C– ，并被广泛视为抗炎细胞3，16，18。最近，描述了另一个CD64 + CD16.2 + 单核细胞群体，其起源于Ly6C– 单核细胞并产生CD206 + IMs3。
嗜酸性粒细胞主要出现在蠕虫感染或过敏性疾病期间的肺部。然而，在稳定状态期间，肺实质中存在少量嗜酸性粒细胞，称为常驻嗜酸性粒细胞。与常驻嗜酸性粒细胞相反，炎症性嗜酸性粒细胞存在于肺间质和支气管肺泡灌洗 （BAL） 中。在家尘螨（HDM）小鼠模型中，炎症性嗜酸性粒细胞在抗原介导的刺激后被招募到肺部。有人提出，居民嗜酸性粒细胞可能通过抑制对HDM19的T辅助剂2（Th2）致敏而在过敏中具有调节作用。
与其他肺髓细胞相反，中性粒细胞表达Ly6G但不表达CD68，其特征在于CD68-Ly6G +免疫表亲型的特征16，20，21。可视化研究表明，在稳态期间，肺在血管内隔室中保留了一池嗜中性粒细胞，并容纳了相当数量的血管外嗜中性粒细胞22。与嗜酸性粒细胞类似，在稳定状态下的BAL中找不到嗜中性粒细胞;然而，几种形式的免疫刺激，如LPS激发，哮喘或肺炎，驱动嗜中性粒细胞进入肺泡腔，导致它们存在于BAL21，22，23中。
大量肺 CD45+ 细胞代表自然杀伤 （NK）、T 细胞和 B 细胞，大多数骨髓标志物均为阴性24。在幼稚小鼠的肺部，可以根据CD11b和MHC II18的表达来鉴定这三种细胞类型。大约25%的肺CD45 +细胞是B细胞，而NK细胞在肺部的百分比高于其他淋巴和非淋巴组织24，25，26。在肺T细胞中，相当一部分是CD4-CD8–，在呼吸道感染中起重要作用26。
由于肺宿主非常复杂和独特的免疫系统，因此已经开发出几种用于识别肺免疫细胞的门控策略并报告了16，18，20，27。本文描述的门控策略提供了一种全面且可重复的方法，使用9种标志物鉴定多达12种不同的肺髓系和非髓系免疫群体。已使用其他标记来验证结果。此外，还提供了一种详细的方法来制备单细胞悬浮液，该单细胞悬浮液可最大限度地减少细胞死亡，并允许鉴定肺免疫细胞区室的最完整特征。应该注意的是，肺非免疫细胞的鉴定，如上皮细胞（CD45-CD326 + CD31–），内皮细胞（CD45-CD326-CD31 +）和成纤维细胞需要不同的方法28，29。此处描述的协议和方法不包括此类人群的识别。
Protocol
该协议中描述的所有研究和实验都是根据Beth Israel Deaconess医疗中心的机构动物护理和使用委员会（IACUC）的指南进行的。使用六到十周龄的C57BL / 6小鼠，无论男女，都用于开发该协议。 1. 手术切除和组织准备 通过腹腔内注射1mL三溴乙醇（根据标准方案制备; 材料表）。注意：在肺部研究中应避免CO2 窒息，因为它可能导致肺损伤并改变肺免疫细?…
Representative Results
门控策略我们的门控策略的第一步是排除碎屑和双层（图1A）。仔细排除双联对于避免假阳性人群至关重要（补充图S2）。然后，使用CD45 +鉴定免疫细胞，CD45 +是造血细胞的标志物（图1B）。可以添加活死染色剂以排除死细胞。然而，该协议导致<5%的CD45 + 细胞死亡（图1C），而更多的CD45…
Discussion
肺免疫细胞的鉴定可能具有挑战性，因为与驻留在其他组织中的对应物相比，肺中存在多种免疫细胞类型及其独特的免疫表型特征。在几种病理条件下，具有不同表型特征的细胞出现在肺部。例如，博来霉素诱导的肺损伤导致肺泡腔中循环单核细胞来源的巨噬细胞募集，在那里它们可以停留长达一年，甚至在博来霉素诱导的纤维化后持续存在。与组织驻留的AM相反，循环的单核细胞衍生巨噬细胞?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作得到了NIH拨款R01CA238263和R01CA229784（VAB）的支持。
Materials
| 10 mL syringe plunger | EXELINT | 26265 | |
| 18 G needles | BD Precision Glide Needle | 305165 | |
| 21 G needles | BD Precision Glide Needle | 305195 | |
| 50 mL conical tubes | Falcon | 3520 | |
| 70 μm cell strainer | ThermoFisher | 22363548 | |
| 96-well plates | Falcon/corning | 3799 | |
| ACK Lysing Buffer | ThermoFisher | A10492-01 | |
| anti-mouse CD11b | Biolegend | 101215 | For details see Table 2 |
| anti-mouse CD11c | Biolegend | 117339 / 117337 | For details see Table 2 |
| anti-mouse CD45 | Biolegend | 103115 | For details see Table 2 |
| anti-mouse CD64 | Biolegend | 139319 | For details see Table 2 |
| anti-mouse CD68 | Biolegend | 137009 | For details see Table 2 |
| anti-mouse GR-1 | Biolegend | 108433 | For details see Table 2 |
| anti-mouse Siglec F | Biolegend | 155503 | For details see Table 2 |
| AVERTIN | Sigma-Aldrich | 240486 | |
| B220 | Biolegend | 103228 | For details see Table 2 |
| Bovine Serum Albumin (BSA) | Sigma-Aldrich | 9048-46-8 | |
| CD103 | Biolegend | 121405 / 121419 | For details see Table 2 |
| CD24 | Biolegend | 138503 | For details see Table 2 |
| CD3 | Biolegend | 100205 | For details see Table 2 |
| Centrifuge | |||
| Collagenase Type 1 | Worthington Biochemical Corp | LS004196 | |
| CX3CR1 | Biolegend | 149005 | For details see Table 2 |
| DNase I | Millipore Sigma | 10104159001 | |
| Ethanol | |||
| F4/80 | Biolegend | 123133 | For details see Table 2 |
| FcBlock (CD16/32) | Biolegend | 101301 | For details see Table 2 |
| Fetal Bovine Serum | R&D Systems | ||
| Fine Serrated Forceps | Roboz Surgical Instrument Co | ||
| Foxp3 / Transcription Factor Staining Buffer Set | ThermoFisher | 00-5523-00 | |
| Futura Safety Scalpel | Merit Medical Systems | SMS210 | |
| Live/Dead Fixable Far Read Dead Cell Stain Kit | ThermoFisher | L34973 | For details see Table 2 |
| MERTK | Biolegend | 151505 | For details see Table 2 |
| MHC-II | Biolegend | 107621 | For details see Table 2 |
| NK1.1 | Biolegend | 108705 | For details see Table 2 |
| Orbital Shaker | VWR | Model 200 | |
| Petri dish | Falcon | 351029 | |
| Refrigerated benchtop centrifuge | SORVAL ST 16R | ||
| Small curved scissor | Roboz Surgical Instrument Co |
References
- Guilliams, M., et al. Alveolar macrophages develop from fetal monocytes that differentiate into long-lived cells in the first week of life via GM-CSF. Journal of Experimental Medicine. 210 (10), 1977-1992 (2013).
- Tan, S. Y., Krasnow, M. A. Developmental origin of lung macrophage diversity. Development. 143 (8), 1318-1327 (2016).
- Schyns, J., et al. Non-classical tissue monocytes and two functionally distinct populations of interstitial macrophages populate the mouse lung. Nature Communications. 10 (1), 3964 (2019).
- Gibbings, S. L., et al. Three unique interstitial macrophages in the murine lung at steady state. American Journal of Respiratory Cell and Molecular Biology. 57 (1), 66-76 (2017).
- Ural, B. B., et al. Identification of a nerve-associated, lung-resident interstitial macrophage subset with distinct localization and immunoregulatory properties. Science Immunology. 5 (45), 8756 (2020).
- Chakarov, S., et al. Two distinct interstitial macrophage populations coexist across tissues in specific subtissular niches. Science. 363 (6432), (2019).
- Liegeois, M., Legrand, C., Desmet, C. J., Marichal, T., Bureau, F. The interstitial macrophage: A long-neglected piece in the puzzle of lung immunity. Cellular Immunology. 330, 91-96 (2018).
- Stouch, A. N., et al. IkappaB kinase activity drives fetal lung macrophage maturation along a non-M1/M2 paradigm. Journal of Immunology. 193 (3), 1184-1193 (2014).
- Kopf, M., Schneider, C., Nobs, S. P. The development and function of lung-resident macrophages and dendritic cells. Nature Immunology. 16 (1), 36-44 (2015).
- Plantinga, M., et al. Conventional and monocyte-derived CD11b(+) dendritic cells initiate and maintain T helper 2 cell-mediated immunity to house dust mite allergen. Immunity. 38 (2), 322-335 (2013).
- Liu, H., et al. Dendritic cell trafficking and function in rare lung diseases. American Journal of Respiratory Cell and Molecular Biology. 57 (4), 393-402 (2017).
- Cook, P. C., MacDonald, A. S. Dendritic cells in lung immunopathology. Seminars in Immunopathology. 38, 449-460 (2016).
- Guilliams, M., Lambrecht, B. N., Hammad, H. Division of labor between lung dendritic cells and macrophages in the defense against pulmonary infections. Mucosal Immunology. 6 (3), 464-473 (2013).
- Nobs, S. P., et al. PPARγ in dendritic cells and T cells drives pathogenic type-2 effector responses in lung inflammation. Journal of Experimental Medicine. 214 (10), 3015-3035 (2017).
- Aegerter, H., et al. Influenza-induced monocyte-derived alveolar macrophages confer prolonged antibacterial protection. Nature Immunology. 21 (2), 145-157 (2020).
- Misharin, A. V., Morales-Nebreda, L., Mutlu, G. M., Budinger, G. R., Perlman, H. Flow cytometric analysis of macrophages and dendritic cell subsets in the mouse lung. American Journal of Respiratory Cell and Molecular Biology. 49 (4), 503-510 (2013).
- Hoffmann, F. M., et al. Distribution and interaction of murine pulmonary phagocytes in the naive and allergic lung. Frontiers in Immunology. 9, 1046 (2018).
- Yu, Y. R., et al. A protocol for the comprehensive flow cytometric analysis of immune cells in normal and inflamed murine non-lymphoid tissues. PLoS One. 11 (3), 0150606 (2016).
- Mesnil, C., et al. Lung-resident eosinophils represent a distinct regulatory eosinophil subset. Journal of Clinical Investigation. 126 (9), 3279-3295 (2016).
- Zaynagetdinov, R., et al. Identification of myeloid cell subsets in murine lungs using flow cytometry. American Journal of Respiratory Cell and Molecular Biology. 49 (2), 180-189 (2013).
- Tavares, A. H., Colby, J. K., Levy, B. D., Abdulnour, R. E. A model of self-limited acute lung injury by unilateral intra-bronchial acid instillation. Journal of Visualized Experiments: JoVE. (150), e60024 (2019).
- Kreisel, D., et al. In vivo two-photon imaging reveals monocyte-dependent neutrophil extravasation during pulmonary inflammation. Proceedings of the National Academy of Sciences of the United States of America. 107 (42), 18073-18078 (2010).
- Krishnamoorthy, N., et al. Neutrophil cytoplasts induce TH17 differentiation and skew inflammation toward neutrophilia in severe asthma. Science Immunology. 3 (26), (2018).
- Ascon, D. B., et al. Normal mouse kidneys contain activated and CD3+CD4- CD8- double-negative T lymphocytes with a distinct TCR repertoire. Journal of Leukocyte Biology. 84 (6), 1400-1409 (2008).
- Wang, J., et al. Lung natural killer cells in mice: phenotype and response to respiratory infection. Immunology. 137 (1), 37-47 (2012).
- Cowley, S. C., Meierovics, A. I., Frelinger, J. A., Iwakura, Y., Elkins, K. L. Lung CD4-CD8- double-negative T cells are prominent producers of IL-17A and IFN-gamma during primary respiratory murine infection with Francisella tularensis live vaccine strain. Journal of Immunology. 184 (10), 5791-5801 (2010).
- Gibbings, S. L., Jakubzick, C. V., Alper, S., Janssen, W. Isolation and characterization of mononuclear phagocytes in the mouse lung and lymph nodes. In Lung innate immunity and inflammation. Methods in Molecular Biology. 1809, (2018).
- Singer, B. D., et al. Flow-cytometric method for simultaneous analysis of mouse lung epithelial, endothelial, and hematopoietic lineage cells. American Journal of Physiology. Lung Cellular and Molecular Physiology. 310 (9), 796-801 (2016).
- Matsushima, S., et al. CD248 and integrin alpha-8 are candidate markers for differentiating lung fibroblast subtypes. BMC Pulmonary Medicine. 20 (1), 21 (2020).
- Cong, J., Wei, H. Natural killer cells in the lungs. Frontiers in Immunology. 10, 1416 (2019).
- Daubeuf, F., et al. A fast, easy, and customizable eight-color flow cytometric method for analysis of the cellular content of bronchoalveolar lavage fluid in the mouse. Current Protocols in Mouse Biology. 7 (2), 88-99 (2017).
- Yi, S., et al. Eosinophil recruitment is dynamically regulated by interplay among lung dendritic cell subsets after allergen challenge. Nature Communications. 9 (1), 3879 (2018).
- Langlet, C., et al. CD64 expression distinguishes monocyte-derived and conventional dendritic cells and reveals their distinct role during intramuscular immunization. Journal of Immunology. 188 (4), 1751-1760 (2012).
- Moran, T. P., Nakano, H., Kondilis-Mangum, H. D., Wade, P. A., Cook, D. N. Epigenetic control of Ccr7 expression in distinct lineages of lung dendritic cells. Journal of Immunology. 193 (10), 4904-4913 (2014).
- Schyns, J., Bureau, F., Marichal, T. Lung interstitial macrophages: past, present, and future. Journal of Immunology Research. 2018, 5160794 (2018).
- Krljanac, B., et al. RELMalpha-expressing macrophages protect against fatal lung damage and reduce parasite burden during helminth infection. Science Immunology. 4 (35), (2019).
- Ginhoux, F., Guilliams, M. Tissue-resident macrophage ontogeny and homeostasis. Immunity. 44 (3), 439-449 (2016).
- Svedberg, F. R., et al. The lung environment controls alveolar macrophage metabolism and responsiveness in type 2 inflammation. Nature Immunology. 20 (5), 571-580 (2019).
- Yona, S., et al. Fate mapping reveals origins and dynamics of monocytes and tissue macrophages under homeostasis. Immunity. 38 (1), 79-91 (2013).
- Misharin, A. V., et al. Monocyte-derived alveolar macrophages drive lung fibrosis and persist in the lung over the life span. Journal of Experimental Medicine. 214 (8), 2387-2404 (2017).
- Koch, C. M., Chiu, S. F., Misharin, A. V., Ridge, K. M. Lung Interstitial Macrophages: Establishing Identity and Uncovering Heterogeneity. American Journal of Respiratory Cell and Molecular Biology. 57 (1), 7-9 (2017).
Cite This Article
Christofides, A., Cao, C., Pal, R., Aksoylar, H. I., Boussiotis, V. A. Flow Cytometric Analysis for Identification of the Innate and Adaptive Immune Cells of Murine Lung. J. Vis. Exp. (177), e62985, doi:10.3791/62985 (2021).