用于研究呼吸道病毒感染期间先天免疫细胞活化的非接触式共培养模型
1Department of Otolaryngology, Yong Loo Lin School of Medicine,National University of Singapore, 2Department of Microbiology and Immunology, Yong Loo Lin School of Medicine,National University of Singapore, 3Haartman Institute, Medicum,University of Helsinki, 4Department of Otolaryngology, Head and Neck Surgery, National University Hospital,National University Health System, 5Agency for Science, Technology and Research (A*STAR),Singapore Immunology Network (SIgN), 6NUHS Infectious Diseases Translational Research Program, Yong Loo Lin School of Medicine,National University of Singapore
Summary
该协议详细研究了病毒感染的鼻上皮细胞与先天细胞活化之间的早期相互作用。免疫细胞的单个亚群可以根据其对病毒感染的反应而激活来区分。然后可以进一步研究它们以确定它们对早期抗病毒反应的影响。
Abstract
在病毒感染期间，鼻上皮层和先天免疫细胞之间的早期相互作用仍然是一个探索不足的领域。先天免疫信号传导在病毒感染中的重要性已大大增加，因为表现出高先天性T细胞活化的呼吸道感染患者显示出更好的疾病结果。因此，剖析这些早期先天免疫相互作用可以阐明控制它们的过程，并可能促进潜在的治疗靶点和策略的发展，以抑制甚至预防病毒感染的早期进展。该协议详细介绍了一种多功能模型，可用于研究病毒感染的气道上皮细胞分泌的因子中先天免疫细胞的早期串扰，相互作用和激活。使用H3N2流感病毒（A/ Aichi / 2 / 1968）作为代表性病毒模型，使用流式细胞术分析了共培养外周血单核细胞（PBMC）的先天细胞活化，以研究由上皮释放的可溶性因子激活的细胞亚群以响应病毒感染。结果验证了分化细胞亚群的门控策略，揭示了PBMCs活化群体及其与对照和感染上皮的串扰之间的明显差异。然后可以进一步分析激活的亚群以确定其功能以及细胞特异性的分子变化。这种串扰研究的结果可能会发现对激活重要先天细胞群很重要的因素，这些因素有利于控制和抑制病毒感染的进展。此外，这些因素可以普遍适用于不同的病毒性疾病，特别是新出现的病毒，以减轻这些病毒首次在幼稚人群中传播时的影响。
Introduction
呼吸道病毒可能是最普遍的病原体之一，造成严重的医疗保健和经济负担。从每年定期爆发的新流行毒株（如H1N1、H5N1、H3N2、MERS、COVID-19）到季节性流感毒株，病毒对公共卫生构成持续威胁。虽然疫苗构成了应对这些全球公共卫生挑战的主要部分，但令人清醒的是，这些对策只是响应1，2。此外，在新的传染性菌株的出现与其疫苗的成功开发之间出现延迟是不可避免的3，导致遏制病毒传播的现有措施非常有限。
这些拖延因给社会造成的经济和社会代价而进一步凸显。仅季节性流感每年就造成约80亿美元的间接成本、32亿美元的医疗费用和3.63万人死亡4。这是在考虑资助疫苗开发所需的研究成本之前。流行病的暴发对社会的影响甚至更加严重，而全球化的速度每年都在加快，严重急性呼吸系统综合征冠状病毒2（SARS-CoV-2）的出现和迅速传播造成的全球破坏就证明了这一点， 5，6，7。
最近的研究表明，具有更多活化先天性T细胞群的感染患者往往具有更好的疾病结果8，9，10。此外，先天性T细胞群被分类为多个亚组：粘膜相关不变T（MAIT）细胞，Vδ1 γδ T细胞，Vδ2 γδ T细胞和自然杀伤T（NKT）细胞。这些先天性T细胞亚群在其群体内也表现出异质性，增加了参与先天免疫应答的细胞群11之间相互作用的复杂性。因此，激活这些先天性T细胞的机制和先天性T细胞特定亚群的知识可能提供不同的研究途径，以减少这些病毒对人类宿主的感染作用，特别是在疫苗开发期间。
受流感感染的上皮细胞产生迅速激活先天性T细胞的因子12，13，14。基于这一发现，这种非接触式空气 – 液体界面（ALI）共培养模型旨在模拟感染的鼻上皮层和PBMCs在早期感染期间的早期化学相互作用（由受感染的上皮层释放的可溶性因子介导）。鼻上皮层（在膜插入物上培养）和PBMC（在下面的腔室中）之间的物理分离以及上皮完整性可防止PBMCs被病毒直接感染，从而可以详细研究上皮衍生的可溶性因子对PBMCs的影响。因此，可以进一步研究所确定的因素在诱导适当的先天性T细胞群方面的治疗潜力，从而可以预防流感感染。因此，本文详细介绍了建立共培养的方法，用于研究上皮衍生的可溶性因子的先天性T细胞活化。
Protocol
注：有关此协议中使用的介质配方，请参阅 表 1 。注意：已发现在12孔透孔上生长的hNECS可生长成更优化的厚度，使可溶性因子在感染流感病毒时容易到达基底室。因此，建议使用12孔大小的转孔进行共培养。 1. 建立3T3供料层 从冻结的库存中建立 从冷冻储液中解冻NIH / 3T3成纤维细胞的冷冻管。将冷冻管的内容物加入2 mL完全Dulbecco的最小必需培养?…
Representative Results
虽然传统的T细胞构成了针对病毒感染的适应性免疫反应的主要库，以促进病毒清除，但先天性T细胞群在更广泛的谱系中起作用，以抑制病毒载量，以便在后期有效清除。因此，该方案专门创造了一种强大的条件来研究流感感染后的先天性T细胞，它们的活化及其功能群体，而不需要来自同一供体的上皮和免疫细胞样本。该协议也可以应用于其他病毒，尽管它可能仅限于具?…
Discussion
针对病毒的先天免疫反应是抗病毒管理中一个尚未得到充分研究的领域。气道上皮细胞和先天免疫细胞协同工作以抑制感染期间的病毒复制，此外，如果病毒载量未受到控制，则作为过度反应的决定因素12，13，17。然而，开发用于研究上皮 – 先天免疫串扰的相关人类模型以研究先天免疫细胞的激活以赋予适当的抗病毒反应仍然…
Disclosures
The authors have nothing to disclose.
Acknowledgements
我们要感谢新加坡国立大学耳鼻喉科和微生物学和免疫学系的研究人员，感谢他们在hNEC培养和病毒培养相关工作方面的帮助。我们还要感谢国立大学医院耳鼻喉科的外科医生和外科团队，感谢他们协助提供研究所需的细胞和血液样本。
这项研究由国家医学研究委员会资助，新加坡No。NMRC/CIRG/1458/2016（致德云·王）和MOH-OFYIRG19may-0007（致谭启森）。Kai Sen Tan是2019年欧洲过敏和临床免疫学（EAACI）研究奖学金支持的获得者。
Materials
| 0.5% Trypsin-EDTA | Gibco | 15400-054 | |
| 0.5 M Ethylenediaminetetraacetic acid (EDTA), pH 8.0, RNase-free | Thermofisher | AM9260G | 0.5M EDTA |
| 1.5 mL SafeLock Tubes | Eppendorf | 0030120086 | 1.5mL Centrifuge Tube |
| 10 mL K3EDTA Vacutainer Tubes | BD | 366643 | 10mL Blood Collection Tubes |
| 10x dPBS | Gibco | 14200-075 | |
| 10x PBS | Vivantis | PC0711 | |
| 12-well Plate | Corning | 3513 | |
| 12-well Transwell Insert | Corning | 3460 | membrane insert |
| 1x FACS Lysing Solution | BD | 349202 | |
| 2.0 mL SafeLock Tubes | Eppendorf | 0030120094 | 2 mL centrifuge tube |
| 24-well Plate | Corning | 3524 | |
| 24-well Transwell Insert | Corning | 3470 | |
| 3% Acetic Acid with Methylene Blue | STEMCELL Technologies | 07060 | |
| 3,3',5-triiodo-l-thyronine | Sigma | T-074 | |
| 37% Formaldehyde Solution w 15% Methanol as Stabilizer in H2O | Sigma | 533998 | |
| 5810R Centrifuge | Eppendorf | 5811000320 | |
| 5 mL polypropylene tubes (flow tubes) | BD | 352058 | |
| 70 µm Cell Strainer | Corning | 431751 | |
| A-4-62 Rotor | Eppendorf | 5810709008 | |
| Accutase | Gibco | A1110501 | Cell Dissociation Reagent |
| Antibiotic-Antimycotic | Gibco | 15240-062 | |
| Avicel CL-611 | FMC Biopolymer | NA | Liquid Overlay |
| Bio-Plex Manager 6.2 Standard Software | Bio-Rad Laboratories, Inc | 171STND01 | Multiplex Manager Software |
| Butterfly Needle 21 G | BD | 367287 | |
| Cholera Toxin | Sigma | C8052 | |
| Crystal Violet | Merck | C6158 | |
| Cytofix/Cytoperm Solution | BD | 554722 | Fixation and Permeabilization Solution |
| Dispase II | Sigma | D4693 | Neutral Protease |
| DMEM/High Glucose | GE Healthcare Life Sciences | SH30243.01 | |
| DMEM/Nutrient Mixture F-12 | Gibco-Invitrogen | 11320033 | |
| dNTP Mix | Promega | U1515 | dNTP Mix |
| EMEM (w L-Glutamine) | ATCC | 30-2003 | |
| EVOM voltohmmeter device | WPI, Sarasota, FL, USA | 300523 | |
| FACS Lysing Solution | BD | 349202 | 1x Lysing Solution |
| Falcon tube 15 mL | CellStar | 188271 | 15 mL tube |
| Falcon tube 50 mL | CellStar | 227261 | 50 mL Tube |
| Fast Start Essential DNA Probes Master | Roche | 6402682001 | qPCR Master Mix |
| Ficoll Paque Premium | Research Instruments | 17544203 | Density Gradient Media |
| H3N2 (A/Aichi/2/1968) | ATCC | VR547 | |
| H3N2 M1 Forward Primer Sequence | Sigma | 5'- ATGGTTCTGGCCAGCACTAC-3' | |
| H3N2 M1 Reverse Primer Sequence | Sigma | 5'- ATCTGCACCCCCATTCGTTT-3' | |
| H3N2 NS1 Forward Primer Sequence | Sigma | 5'- ACCCGTGTTGGAAAGCAGAT-3' | |
| H3N2 NS1 Reverse Primer Sequence | Sigma | 5'- CCTCTTCGGTGAAAGCCCTT-3' | |
| Heat Inactivated Fetal Bovine Serum | Gibco | 10500-064 | |
| hNESPCs | Human Donors | NA | |
| Human Epithelial Growth Factor | Gibco-Invitrogen | PHG0314 | |
| Hydrocortisone | STEMCELL Techonologies | 7925 | Collected from nasal biopsies during septal deviation surgeries |
| Insulin | Sigma | I3536 | |
| Lightcycler 96 | Roche | 5815916001 | qPCR Instrument |
| Live/DEAD Blue Cell Stain Kit *for UV Excitation | Thermofisher | L23105 | Viability Stain |
| MILLIPLEX MAP Human Cytokine/Chemokine Magnetic Bead Panel II – Premixed 23 Plex | Merck Pte Ltd | HCP2MAG-62K-PX23 | Immunology Multiplex Assay |
| Mitomycin C | Sigma | M4287 | |
| M-MLV 5x Buffer | Promega | M1705 | RT-PCR 5x Buffer |
| M-MLV Reverse Transcriptase | Promega | M1706 | Reverse Transcriptase |
| N-2 supplement | Gibco-Invitrogen | 17502-048 | |
| NIH/3T3 | ATCC | CRL1658 | |
| Perm/Wash Buffer | BD | 554723 | Permeabilization Wash Buffer |
| PneumaCult-ALI 10x Supplement | STEMCELL Techonologies | 5001 | |
| PneumaCult-ALI Basal Medium | STEMCELL Techonologies | 5001 | |
| PneumaCult-ALI Maintenance Supplement (100x) | STEMCELL Techonologies | 5001 | |
| Random Primers | Promega | C1181 | Random Primers |
| Recombinant Rnasin Rnase Inhibitor | Promega | N2511 | RNase Inhibitor |
| RNA Lysis Buffer | Qiagen | Part of 52904 | |
| RPMI 1640 (w L-Glutamine) | ATCC | 30-2001 | |
| STX2 electrodes | WPI, Sarasota, FL, USA | STX2 | Electrode |
| T25 Flask | Corning | 430639 | |
| T75 Flask | Corning | 430641U | |
| TPCK Trypsin | Sigma | T1426 | |
| Trypan Blue | Hyclone | SV30084.01 | |
| Viral RNA Extraction Kit | Qiagen | 52904 | Viral RNA Extraction Kit |
| V-Shaped 96-well Plate | Corning | 3894 | |
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Cite This Article
Lew, Z. Z. R., Liu, J., Ong, H. H., Tan, V. J., Luukkainen, A., Ong, Y. K., Thong, M., Puan, K. J., Chow, V. T. K., Tan, K. S., Wang, D. Y. Contact-Free Co-Culture Model for the Study of Innate Immune Cell Activation During Respiratory Virus Infection. J. Vis. Exp. (168), e62115, doi:10.3791/62115 (2021).