肺泡巨噬细胞吞噬作用与小鼠细菌清除
Summary
本文报道了分析小鼠肺泡巨噬细胞吞噬功能和肺细菌清除的常用方法。这些方法研究荧光素异硫氰酸珠的体外吞噬和铜绿假单胞菌绿色荧光蛋白的体内吞噬作用。我们还描述了一种清除小鼠铜绿假单胞菌的方法。
Abstract
肺泡巨噬细胞 (ams) 保护肺的肺泡空间。ams 的吞噬作用在防御入侵病原体、去除死细胞或异物以及解决炎症反应和组织重塑方面发挥着至关重要的作用, 这些过程是由各种表面受体介导的。ams。在这里, 我们报告了分析 ams 的吞噬功能的方法, 使用体外和体内的检测和实验策略, 以区分模式识别受体-, 补体受体, 和 fc 伽玛受体介导吞噬。最后, 我们讨论了建立和表征小鼠铜绿假单胞菌肺炎模型的方法, 以评估细菌在体内的清除率。这些检测是评估 am 功能最常用的方法, 也可用于研究巨噬细胞功能和其他器官的细菌清除。
Introduction
ams 是肺泡中处于休眠阶段的主要脂肪细胞, 也是通过识别和内化吸入病原体和外来颗粒 1, 2 而产生先天免疫反应的主要参与者之一。据报道, ams 对于快速清除铜绿假单胞菌和3,4 肺炎克雷伯菌等多种肺部病原体至关重要, 因此 am 吞噬能力的缺乏往往导致呼吸道疾病。感染, 如急性肺炎, 导致较高的死亡率和发病率。
ams 还通过产生细胞因子和趋化因子 (如 tnf-α和 il-1β) 在肺中引发先天炎症反应, 这些细胞与肺泡环境中的其他细胞相通, 产生趋化因子并诱导炎症中性粒细胞、单核细胞和自适应免疫细胞在肺5。例如, ams 产生的 il-1β有助于促进中性粒细胞趋化因子 cxcl8 从上皮细胞6中释放。此外, ams 还有助于凋亡多核白细胞 (pmn) 的吞噬, 而这种白细胞的失败会导致细胞内酶从 pmn 持续泄漏到周围组织, 从而导致组织损伤和长期炎症。7.,8,9个。
ams 的吞噬作用是通过对病原体表面病原体相关分子模式的直接识别, 通过 ams 的模式识别受体 (prr) 或通过将凋亡的病原体与 ams 的免疫效应受体结合而介导的10. 对于后者, ams 可以通过其 fcγ受体 (fcγr) 识别用免疫球蛋白 (igg) 进行光声的目标, 或通过其补体受体 (cr)11识别含有补体片段 c3b 和 c3bi 的病原体。在补体受体中, 免疫球蛋白超家族 (crig) 的 cr 在组织巨噬细胞12中选择性表达, 最近的一项发现强调了 crig 在铜绿假单胞菌肺炎背景下的作用。13岁
许多原始的研究使用的方法来评估巨噬细胞吞噬, 以描述巨噬细胞功能14,15 的分子机制。然而, 像体内吞噬作用这样的方法需要精确的吞噬量的定量。本文综述了用荧光素异硫氰酸酯 (fitc) 玻璃珠和铜绿绿绿色荧光蛋白 (gfp) 进行体外和体内吞噬的详细方法。此外, 我们还解释了在 prr-、cr-r 和 fcγ r 介导的吞噬作用之间的鉴别方法。最后, 我们报告了一种方法来描述细菌清除在小鼠与铜绿假单胞菌肺炎。
Protocol
该协议遵循东弗吉尼亚医学院动物护理和使用机构委员会 (iacuc) 的指导原则。 1. 荧光珠吞噬作用 根据 iacuc 对动物的伦理安乐死协议, 用 co2 窒息对小鼠进行安乐死 (c57bl/6j, 6周大, 女性)。 将鼠标放在覆盖有纸巾的解剖板上。用爪子把它的爪子固定下来, 四肢展开老鹰, 在门牙下钩住一根绳子, 将它的头向后拉, 这样气管就被垂直和水平地定位。 用7…
Representative Results
我们首先进行了小鼠原发性 ams 吞噬分析的实验。在所有分析中, 我们比较了从 wt 和 trim72 ko 小鼠中分离出的 ams。如图 1a 所示, 荧光显微镜显示, 小鼠原代 ams 在孵育1小时后, 会发生 fitc-玻璃微珠的吞噬作用。图 1b显示流式细胞仪对吞噬性的分析。用显微镜和流式细胞仪测量的吞噬量的定量分别?…
Discussion
在发挥气体交换功能的同时, 肺持续面对外来颗粒、病原体和过敏原。ams 凭借其主要功能, 即吞噬作用, 提供了第一道防线。ams 还与其他免疫细胞协调, 以摧毁病原体和解决炎症。在这里, 我们描述了从小鼠肺分离的 ams 专门评估吞噬性的方法。本手稿中提出的协议解释了在体内和体外对吞噬作用的详细研究, 该研究也可用于研究其他器官的巨噬细胞功能和细菌清除。
所描述的?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作得到 r01hl116826 至 x. zhao 的支持。
Materials
| 18-G Needle | Nipro Medical | CI+1832-2C | Molecular Biology grade |
| 2,7-diaminofluorene (DAF) | Sigma-Aldrich | D17106 | Molecular Biology grade |
| 70% Ethanol | Decon Labs Inc. | 18C27B | Analytical grade |
| 96-well plate | Corning | 3603 | Cell Biology grade |
| ACK lysis buffer | Life Technologies | A10492 | Molecular Biology grade |
| Alexa fluor-488 Zymosan-A-bioparticle | Thermofisher Scientific | Z23373 | Molecular Biology grade |
| C5 deficient serum | Sigma-Aldrich | C1163 | Biochemical reagent |
| Centrifuge | Labnet International | C0160-R | |
| Cytospin 4 Cytocentrifuge | Thermofisher Scientific | A78300101 Issue 11 | |
| DMEM Cell Culture Media | Gibco | 11995-065 | Cell Biology grade |
| FBS | Atlanta Biologicals | S11550 | Cell Biology grade |
| Flow Cytometer | BD Biosciences | FACSCalibur | |
| Flow Jo Software | FlowJo, LLC | ||
| Forceps | Dumont | 0508-SS/45-PS-1 | Suitable for laboratory animal dissection |
| FITC-carboxylated latex beads | Sigma-Aldrich | L4530 | Cell Biology grade |
| GFP-P. aeruginosa | ATCC | 101045GFP | Suitable for cell infection assays |
| Glass bottom dish | MatTek Corp. | P35G-0.170-14-C | Cell Biology grade |
| High-Pressure Syringe | Penn-Century | FMJ-250 | Suitable for laboratory animal use |
| Homogenizer | Omni International | TH-01 | |
| Hydrogen peroxide | Sigma-Aldrich | H1009 | Analytical grade |
| Inverted Fluorescence Microscope | Olympus | IX73 | |
| Ketamine Hydrochloride | Hospira | CA-2904 | Pharmaceutical grade |
| Shandon Kwik-Diff Stains | Thermofisher Scientific | 9990700 | Cell Biology grade |
| LB Agar | Fisher Scientific | BP1425 | Molecular Biology grade |
| LB Broth | Fisher Scientific | BP1427 | Molecular Biology grade |
| MicroSprayer Aerosolizer | Penn-Century | IA-1C | Suitable for laboratory animal use |
| Paraformaldehyde | Sigma-Aldrich | P6148 | Reagent grade |
| PBS | Gibco | 20012-027 | Cell Biology grade |
| rabbit anti-SRBC-IgG | MP Biomedicals | 55806 | Suitable for immuno-assays |
| rabbit anti-SRBC-IgM | Cedarline Laboratories | CL9000-M | Suitable for immuno-assays |
| Scissors | Miltex | 5-2 | Suitable for laboratory animal dissection |
| Small Animal Laryngoscope | Penn-Century | LS-2 | Suitable for laboratory animal use |
| Sodium Dodecyl Sulfate (SDS) | BioRad | 1610301 | Analytical grade |
| Spring Scissors (Med) | Fine Science Tools | 15012-12 | Suitable for laboratory animal dissection |
| Spring Scissors (Small) | Fine Science Tools | 91500-09 | Suitable for laboratory animal dissection |
| sheep red blood cells (SRBCs) | MP Biomedicals | 55876 | Washed, preserved SRBCs |
| Urea | Sigma-Aldrich | U5378 | Molecular Biology grade |
| Xylazine | Akorn Animal Health | 59399-110-20 | Pharmaceutical grade |
References
- Hussell, T., Bell, T. J. Alveolar macrophages: plasticity in a tissue-specific context. Nature Reviews Immunology. 14, 81-93 (2014).
- Belchamber, K. B. R., Donnelly, L. E. Macrophage Dysfunction in Respiratory Disease. Results and Problems in Cell Differentiation. 62, 299-313 (2017).
- Broug-Holub, E., et al. Alveolar macrophages are required for protective pulmonary defenses in murine Klebsiella pneumonia: elimination of alveolar macrophages increases neutrophil recruitment but decreases bacterial clearance and survival. Infection and Immunity. 65, 1139-1146 (1997).
- Knapp, S., et al. Alveolar macrophages have a protective antiinflammatory role during murine pneumococcal pneumonia. American Journal of Respiratory and Critical Care Medicine. 167, 171-179 (2003).
- Bhatia, M., Zemans, R. L., Jeyaseelan, S. Role of chemokines in the pathogenesis of acute lung injury. American Journal of Respiratory Cell and Molecular Biology. 46, 566-572 (2012).
- Marriott, H. M., et al. Interleukin-1beta regulates CXCL8 release and influences disease outcome in response to Streptococcus pneumoniae, defining intercellular cooperation between pulmonary epithelial cells and macrophages. Infection and Immunity. 80, 1140-1149 (2012).
- Greenlee-Wacker, M. C. Clearance of apoptotic neutrophils and resolution of inflammation. Immunological Reviews. 273, 357-370 (2016).
- Haslett, C. Granulocyte apoptosis and its role in the resolution and control of lung inflammation. American Journal of Respiratory and Critical Care Medicine. 160, 5-11 (1999).
- Cox, G., Crossley, J., Xing, Z. Macrophage engulfment of apoptotic neutrophils contributes to the resolution of acute pulmonary inflammation in vivo. American Journal of Respiratory Cell and Molecular Biology. 12, 232-237 (1995).
- Groves, E., Dart, A. E., Covarelli, V., Caron, E. Molecular mechanisms of phagocytic uptake in mammalian cells. Cellular and Molecular Life Sciences. 65, 1957-1976 (2008).
- Mosser, D. M., Zhang, X. Measuring Opsonic Phagocytosis via Fcγ Receptors and complement receptors on macrophages. Current Protocols in Immunology. , (2011).
- He, J. Q., Wiesmann, C., van Lookeren Campagne, M. A role of macrophage complement receptor CRIg in immune clearance and inflammation. Molecular Immunology. 45, 4041-4047 (2008).
- Nagre, N., et al. Inhibition of Macrophage Complement Receptor CRIg by TRIM72 Polarizes Innate Immunity of the Lung. American Journal of Respiratory Cell and Molecular Biology. 58 (6), 756-766 (2018).
- Miksa, M., Komura, H., Wu, R., Shah, K. G., Wang, P. A Novel Method to Determine the Engulfment of Apoptotic Cells by Macrophages using pHrodo Succinimidyl Ester. Journal of Immunological Methods. 342 (1-2), 71-77 (2009).
- Su, H., Chen, H., Jen, C. J. Severe exercise enhances phagocytosis by murine bronchoalveolar macrophages. Journal of Leukocyte Biology. 69, 75-80 (2001).
- Amiel, E., Lovewell, R. R., O’Toole, G. A., Hogan, D. A., Berwin, B. Pseudomonas aeruginosa. evasion of phagocytosis is mediated by loss of swimming motility and is independent of flagellum expression. Infection and Immunity. 78, 2937-2945 (2010).
- Giannoni, E., Sawa, T., Allen, L., Wiener-Kronish, J., Hawgood, S. Surfactant Proteins A and D Enhance Pulmonary Clearance of Pseudomonas aeruginosa. American Journal of Respiratory Cell and Molecular Biology. 34, 704-710 (2006).
Cite This Article
Nagre, N., Cong, X., Pearson, A. C., Zhao, X. Alveolar Macrophage Phagocytosis and Bacteria Clearance in Mice. J. Vis. Exp. (145), e59088, doi:10.3791/59088 (2019).