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Medicina

基于光片显微镜的变应性炎症体内模型中肺呼名人淋巴细胞的高级成像

Published: April 16, 2019
doi:
10.3791/59043
, , , , , ,
1Department of Medicine 1,University Hospital of Erlangen, 2Department of Medicine 3,University Hospital of Erlangen

Summary

这里介绍的协议允许在体内炎症条件下对原代人类淋巴细胞的肺归巢能力进行定性。通过化学清除肺组织的光片荧光显微镜, 可以对过敏性炎症小鼠模型中通过人工免疫细胞的选择性转移的肺浸润进行成像和定量。

Abstract

高度激活的免疫细胞的大量组织积累是各种慢性炎症性疾病的标志, 在受影响患者的临床管理中成为一个有吸引力的治疗靶点。为了进一步优化旨在调节促炎症免疫细胞病理不平衡组织浸润的治疗策略, 对疾病和器官特异性有更好的认识将是特别重要的。外周血淋巴细胞的归巢特性。这里描述的实验方案允许监测肺积累荧光标记和采用转移的人类淋巴细胞的背景下, 纸张引起的肺部炎症。与经常用于分析免疫细胞迁移和趋化性的标准体外检测不同, 现在引入的体内设置考虑到了肺特定的组织方面以及复杂炎症的影响发生在活的小鼠有机体的场景。此外, 三维横截面光片荧光显微镜成像不仅提供了浸润免疫细胞的定量数据, 而且还描述了免疫细胞在炎症肺内的定位模式。总的来说, 我们能够为慢性炎症性肺病领域的免疫学研究引入一种具有高价值的创新技术, 通过遵循所提供的分步协议, 可以很容易地应用。

Introduction

典型的肺部炎症性疾病, 如过敏性哮喘和慢性阻塞性肺病 (copd), 是众所周知的驱动, 增加招募活性淋巴细胞到肺组织1,2。淋巴细胞释放的细胞因子 (例如, IL-4、IL-5、IL-9、IL-13、ifn-Γ和 TNF-α) 进一步促进先天免疫细胞的趋化和适应性免疫细胞, 诱导成纤维气道重塑或直接损害肺实质 2.到目前为止, 负责肺组织内淋巴细胞病理积累的潜在机制还没有完全了解。与描述为肠道和皮肤归巢的组织选择性 T 细胞印迹相比, 肺树突状细胞 (Dc) 显然能够获得优先肺浸润的原始外周 T 细胞, 至少部分是通过诱导 CCR4 表达在淋巴细胞表面3。除了 ccr4 外, 气道浸润 t 细胞的特征还包括趋化因子受体 ccr5 和 cxcr3 的表达特别增加, 在外周血中的 t 细胞为 1,4,5。总体而言, 现有数据与 t 淋巴细胞在生理或炎症条件下肺归巢的概念是一致的, 即在生理或炎症条件下, t 淋巴细胞的肺归巢涉及许多不同的趋化因子受体及其各自的配体, 因此关键取决于控制先天免疫细胞和适应性免疫细胞之间的协作1。特别是在病原体或过敏原接触的初始阶段, 先天免疫系统的细胞通过立即释放不同的趋化因子4、ccl1、ccl17 等不同的趋化因子, 对 TLR刺激或 ige 介导的交联做出反应,Ccl22、ccl20、cxcl10 和 pgd21、6、7。作为一个主要的例子, PGD2与趋化剂受体 CRTh2 之间的相互作用被认为是特别重要的 th2 细胞的趋化性, 因此在哮喘的临床管理中表现为有希望的治疗靶点。事实上, 与安慰剂第8组 相比, 中度哮喘患者在使用选择性 crth2 拮抗剂治疗后一秒钟 (fev 1) 症状改善, 强制呼气量显著增加 (fev 1),9。在炎症反应的一个更进步的状态下, 已经被招募的 T 细胞能够通过释放 IL-4 和 IL-13 作为肺 Dc 的有效刺激物, 进一步扩增肺淋巴细胞的积累。随后, 这些骨髓源性先天细胞以依赖 stat6 的方式调节 ccl17 和 ccl22 的表达, 以 1,10,11的方式。 虽然描述的情况的复杂性仍然阻碍了对 T 细胞肺归巢的全面了解, 但它为潜在的炎症性或过敏性肺病的优化治疗控制提供了大量的分子靶点。因此, 迫切需要创新的实验技术, 这些技术能够进一步深化和补充我们在 T 细胞趋化和肺定位领域的知识。

由于人体内淋巴细胞的肺归巢受到多种细胞、体液和物理参数1的影响, 现有的大多数实验方法都无法模拟这一免疫学过程的整体复杂性。相反, 许多标准的方案, 以分析肺归巢选择性地集中在一个特定的方面涉及淋巴细胞吸引, 粘附, 迁移和保留的级联。除了对外周或肺浸润淋巴细胞整合素和趋化因子受体的 mRNA 或蛋白表达模式进行纯描述性测定, 以及对血液中各自趋化因子水平的补充测量,支气管肺泡灌洗 (bal) 或肺组织12,13, 14,15, 在体外细胞培养检测允许淋巴细胞粘附或趋化的功能表征在确定的实验条件下 16,17,18。原则上, 静态体外粘附检测监测培养的淋巴细胞与内皮单层或玻璃滑块的结合能力, 并与重组内皮粘附分子 (如 MAdCAM-1, VCAM-1) 结合, 而标准的体外化疗通常是为了量化淋巴细胞在经井系统19中沿着趋化因子梯度迁移的能力而进行的。这两种体外设置都能控制实验条件的调节和调节, 但另一方面缺乏已知的重要变量, 这些变量对体内趋化和淋巴细胞的粘附产生了重要影响。静态细胞培养检测主要无视永久血流19引起的剪切力的影响, 并可能忽略周围免疫环境和相互作用的非淋巴细胞免疫细胞的参与,两者都存在于一个活的有机体中。为了克服这些限制, 对静态体外趋化或粘附检测结果的解释需要在流动条件下的动态粘附实验中进一步验证, 在流动条件下, 在 20、21和炎症器官病理的体内模型19。事实上, 从动物研究中分析不同肺部疾病的定义模型中的转基因小鼠, 可以得出有关炎症或过敏条件下调节 t 细胞肺归巢的重要结论.22,23. 野生小鼠和有特定感兴趣基因缺乏缺陷的小鼠肺浸润淋巴细胞的定量比较是定义特定细胞影响的一个成熟且广泛使用的工具T 细胞分布的疾病驱动模式上的途径或受体。然而, 与之前讨论过的体外细胞培养检测不同的是, 基于经典动物模型的研究设计缺乏分析和监测直接来自炎症性肺患者血液或 BAL 的原代人类 T 细胞的能力疾病。因此, 它仍然具有挑战性的功能验证功能指定的肺部疾病是否能够印迹人类淋巴细胞的优先肺取向, 以及临床参数可能对这种情况的影响多大。最近, 在炎症性肠病 (IBD) 的背景下引入了一种非常优雅的体内方法, 它能够克服其中的大部分限制, 并为肠道淋巴细胞归巢的高级翻译研究开辟了新的途径 24.利用溶剂型组织清除方案, 然后横截面光片荧光显微镜作为一种强大的成像工具, 可以直观地显示采用转移的人 T 细胞的浸润和分布在结肠免疫缺陷小鼠的肠道 24。特别是, 该实验设置实现了两项主要创新: (1) 在实验定义的体内条件下, 可以对原代人体免疫细胞进行分析;(2) 患病器官的相当大的区域 (约1.5 厘米 x1.5 厘米) 可以以高分辨率成像, 然后进行三维重建。此外, 最近的几项研究成功地确定了使用溶剂型组织清除和光片荧光显微镜作为先进的肺成像25,26的重要工具。为了从肺免疫学领域的这一技术进步中受益, 我们现在采用了肺归巢分析系统。

这里提出的协议提供了一个逐步介绍如何净化和荧光标记原代人类 T 细胞转移到小鼠诱发肺部炎症, 而且, 详细介绍了随后的过程的光片荧光显微镜成像, 包括器官制备和图像处理。总体而言, 我们希望通过引入一个复杂但可行的实验模型来监测人类淋巴细胞肺在体内的归巢, 以支持未来炎症或过敏性肺病领域的翻译研究。

Protocol

涉及动物的实验是根据 Erlangen 有关地方当局批准的议定书进行的 (德国 Würzburg, Regierung von Unterfranken)。老鼠被安置在特定的无病原体条件下。人类血液的收集得到了地方道德委员会和埃尔朗根-纽伦堡大学机构审查委员会的批准。每个病人都给予了书面知情同意。 1. 诱导小鼠过敏性肺炎症 请注意:如前面的研究27所述, 以下实验…

Representative Results

该协议描述了一个实验小鼠模型, 它允许监测和量化通过光片荧光显微镜在肺中通过传递的人 t 淋巴细胞的积累。图 1A提供了实验计划的体内步骤的示意图概述。为了保证可靠的结果, 它是非常重要的, 以确保一个良好的质量的隔离和荧光标记的人类 CD4+ t 细胞, 这将随后转移到小鼠。如图 1b、c所示, 上述微珠基细胞富集…

Discussion

这里描述的实验设置提供了一个机会来监测在体内炎症条件下的原代人类免疫细胞的肺归巢能力, 从而相关地补充了经典的体外粘附和趋化。检测。考虑到肺的具体解剖器官特征、免疫细胞归巢的重要方面 (包括目标器官内的趋化和细胞分布) 以及获得的数据的临床相关性和可转移性, 我们采取了三个技术关键特征的优势: (1) 对活小鼠机体内原代人细胞的分析;(2) 纸张介导的肺?…

Declarações

The authors have nothing to disclose.

Acknowledgements

作者感谢 DFG 合作研究中心 SFB 1181 和 TRR 241 的资助。光学成像中心 erlangen (OICE), 特别是 Ralf Palmisano、Philip Tripal 和 Tina Fraaß (DFG CRC 1181 Z2 项目) 被公认为光片荧光显微成像的专家技术支持。

Materials

Agarose NEEO Ultra Carl Roth GmbH + Co. KG, Karlsruhe, Germany 2267.4
AlexaFlour594 anti-human CD45 antibody BioLegend, San Diego, USA 304060
Ammonium chloride Carl Roth GmbH + Co. KG, Karlsruhe, Germany K2981
Cannula 21 G Becton, Dickinson and Company, Franklin Lakes, USA 301300
Cell proliferation dye eflour670 eBioscience Inc., San Diego, USA 65-0840-85
CD4 MicroBeads, human Miltenyi Biotech GmbH, Bergisch-Gladbach, Germany 130-045-101
EDTA (ethylenediaminetetraacetic acid) Carl Roth GmbH + Co. KG, Karlsruhe, Germany 8043.1
Potassium-EDTA blood collection tube, 9 ml Sarstedt AG & Co., Nümbrecht, Germany 21066001
Ethly cinnamate (ECi) Sigma-Aldrich, Steinheim, Germany 112372-100G
Ethanol ≥ 99.5 % (EtOH) Carl Roth GmbH + Co. KG, Karlsruhe, Germany 5054.3
FBS (fetal bovine serum) Good Forte PAN-Biotech GmbH, Aidenbach, Germany P40-47500
Filter 100 µm  VWR International Germany GmbH, Darmstadt, Germany 732-2758
Imaris Image Analysis Software 9.0.2 Bitplane AG, Zurich, Switzerland n.a.
ImspectorPro software Abberior Instruments GmbH, Göttingen, Germany n.a.
Ketamin  Inresa Arzneimittel GmbH, Freiburg, Germany 3617KET-V
LaVision UltraMicroscope II LaVision BioTec GmbH, Bielefeld, Germany n.a.
MACS MultiStand Miltenyi Biotech GmbH, Bergisch-Gladbach, Germany 130-042-303
Multifly cannula 20 G Sarstedt AG & Co., Nümbrecht, Germany 851638035
30 G needle B. Braun Melsungen AG, Melsungen, Hessen, Germany 9161502
Neubauer counting chamber neoLab Migge GmbH, Heidelberg, Germany C-1003
Pattex Glue Henkel AG & Co, Düsseldorf, Germany PSK1C
LS column Miltenyi Biotech GmbH, Bergisch-Gladbach, Germany 130-042-401
Lymphocyte Separation Media (Density 1,077 g/ml) anprotec AC-AF-0018
RPMI medium  (Gibco) Life Technologies GmbH,
Darmstadt, Germany 61870-010
Papain Merck 1,071,440,025
PBS Dulbecco (phosphate buffered saline) Biochrom GmbH, Berlin, Germany L182-10
PerCP/Cy5.5 anti-human CD4 BioLegend, San Diego, USA 317428
PerCP/Cy5.5 mouse IgG2b, κ isotype Ctrl BioLegend, San Diego, USA 400337
PFA (paraformaldehyde) Carl Roth GmbH + Co. KG, Karlsruhe, Germany 0335.1
Potassium hydrogen carbonate Carl Roth GmbH + Co. KG, Karlsruhe, Germany P7481
Serological pipette 10 ml Sarstedt AG & Co., Nümbrecht, Germany 86.1254.001 
Syringe 1 ml B. Braun Melsungen AG, Melsungen, Hessen, Germany 9166017V
Syringe 5 ml Becton, Dickinson and Company, Franklin Lakes, USA 260067
Syringe 20 ml Becton, Dickinson and Company, Franklin Lakes, USA 260069
Tube 1.5 ml Sarstedt AG & Co., Nümbrecht, Germany 72,706,400
Tube 2 ml Sarstedt AG & Co., Nümbrecht, Germany 72.695.400 
Tube 2 ml, brown Sarstedt AG & Co., Nümbrecht, Germany 72,695,001
Tube 15 ml Sarstedt AG & Co., Nümbrecht, Germany 62.554.502 
Tube 50 ml Sarstedt AG & Co., Nümbrecht, Germany 62.547.254 
QuadroMACS Separator Miltenyi Biotech GmbH, Bergisch-Gladbach, Germany 130-090-976
Xylazin (Rompun 2%) Bayer Vital GmbH, Leverkusen, Germany KPOBD32
DOWNLOAD MATERIALS LIST

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Keywords: Lung HomingHuman LymphocytesAllergic InflammationLight-sheet MicroscopyT Cell Lung HomingInflammatory Lung DiseasesPapainFicollPBMCCD4+ T CellsCell Proliferation DyeAdoptive Transfer
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Schulz-Kuhnt, A., Zundler, S., Grüneboom, A., Neufert, C., Wirtz, S., Neurath, M. F., Atreya, I. Advanced Imaging of Lung Homing Human Lymphocytes in an Experimental In Vivo Model of Allergic Inflammation Based on Light-sheet Microscopy. J. Vis. Exp. (146), e59043, doi:10.3791/59043 (2019).
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