HDM过敏原中免疫原RNA物种的识别和特征，可调节嗜酸性肺炎
1Department of Microbiology, Immunology and Molecular Genetics, Long School of Medicine,University of Texas Health San Antonio, 2Department of Clinical Laboratory Sciences, College of Applied Medical Sciences,Jouf University, 3Department of Otolaryngology-Head and Neck Surgery,Xiangya Hospital, Central South University
Summary
环境过敏原，如家庭尘虫（HDM），通常含有微生物物质，激活先天免疫反应，以调节过敏性炎症。此处提出的协议演示了 HDM 过敏原中 dsRNA 物种的识别，并表征了其在调节嗜酸性肺炎症时免疫原性活动。
Abstract
环境过敏原，如家庭尘虫（HDM）通常以复杂形式，既含有过敏性蛋白质，驱动异常2型反应，又含有诱导先天免疫反应的微生物物质。这些过敏原相关的微生物成分在调节过敏性哮喘等2型炎症疾病的发展方面起着重要作用。然而，基本机制基本上仍未确定。此处提出的协议确定了过敏原相关免疫刺激RNA的结构特征和体内活性。具体来说，在HDM引起的过敏性哮喘小鼠模型中，对常见过敏原检查是否存在双链RNA（dsRNA）物种，这些物种可以刺激肺的 IFN 反应，并抑制严重肺嗜酸性病的发展。在这里，我们包括以下三种检测方法：点印显示从包括HDM物种在内的过敏原中分离出的DNA中的dsRNA结构;RT-qPCR测量HDM RNA在小鼠肺部干扰素刺激基因（ISG）表达（ISG）中的活动;FACS分析，分别确定HDM RNA对BAL和肺嗜酸性生物数量的影响。
Introduction
根据Strachan1最初提出的卫生假说，幼儿接触环境微生物因素，如内毒素可以防止过敏性疾病2，3,的发展。在微生物感染期间，例如病毒感染，异核酸（RNA/DNA）的先天免疫检测触发宿主防御反应4,4，5，6。,6然而，免疫原核酸的存在和流行，如长双链RNA（dsRNA）物种在家庭尘虫（HDM）或其他昆虫过敏原仍然未知。该协议旨在确定HDM或昆虫和非昆虫过敏原是否含有长dsRNA物种，可以激活保护性免疫反应，以抵消过敏性哮喘小鼠模型中严重嗜酸性肺炎症的发展。在这里，我们提供三种简单而快速的方法来评估HDM总RNA的结构决定因素，这些决定因素是调节过敏原诱发的嗜酸性肺炎症所需的。
粘膜免疫系统是人体最大的免疫器官，是抵御微生物感染和过敏侮辱的第,一道防线。长dsRNA，许多病毒的复制中间体，被称为病原体相关的分子模式（PAMP），通过像3（TLR3）这样的收费受体（TLR3）有效刺激先天反应，以诱导干扰素刺激基因（ISG）9、10、11、12、13、14,10,11,12,13的表达。我们最近表明，HDM总RNA含有dsRNA结构，当通过心内注射在HDM提取物引起的过敏性哮喘的murine模型中施用时，可调节 ISG 的表达，减少严重的嗜酸性肺炎。肺炎症的严重程度是通过分析支气管（BAL）和肺组织的免疫细胞类型，通过流细胞学16，17，18，19，2016,17,18,19确定肺炎症的严重程度。
该协议包括三种检测：1）使用小鼠单克隆抗体J2快速检测具有RNA点印迹的dsRNA结构，该抗体以与序列无关的方式与dsRNA（±40bp）结合;2） 使用RT-qPCR测量 ISG 的诱导，快速评估小鼠肺部免疫刺激RNA的体内效应;3） 使用流量细胞学分析，在HDM引起的肺部炎症背景下准确定量BAL和肺的嗜酸性。
上述检测不仅可用于研究过敏性肺病，还可用于研究呼吸道细菌和病毒感染。例如，dsRNA特异性J2抗体也可用于其他应用，如免疫仿彩色谱、免疫组织化学、酶链接免疫吸附剂测定（ELISA）和免疫染色21、22、23。21,22,23此外，BAL流体收集下游的几种应用可用于使用ELISA量化可溶性物质，如细胞因子和化疗素，以及气道细胞的转录分析（如道道巨噬细胞）。虽然文献中提供了多种评估肺病的协议，但大多数协议通常侧重于目标验证。此处描述的程序可用于识别环境过敏原中对调节过敏性疾病发展非常重要的成分。
Protocol
这里描述的实验程序得到了德克萨斯大学圣安东尼奥分校机构动物护理和使用委员会的批准。 1. 点印，以显示 HDM 总 RNA 中存在 dsRNA 结构 与过敏原、昆虫和非昆虫过敏原的总RNA分离 将活捉或商业获得的 HDM、昆虫或非昆虫动物放入 50 mL 管中，然后快速用液体-N2 冻结。然后在-70°C储存，以进行后续总RNA分离。注：在这个实验中，HDM，昆虫和非昆虫动物?…
Representative Results
使用 dsRNA 特异性小鼠单克隆抗体 J2（±40bp）对HDM、昆虫和非昆虫小动物中长 dsRNA 结构的存在进行了点印检查。RNase III 用于将 dsRNA 消化成 12~15 bp dsRNA 片段，J2 检测不到这些片段（图 1）。 RT-qPCR分析了HDM总RNA以剂量依赖的方式刺激小鼠肺部先天免疫反应的能力（图2，上部）。RNase III 治疗废除了 HDM 总 RNA 的免疫刺激活性，表明 HDM ?…
Discussion
目前的协议描述了如何评估过敏原相关微生物RNA的免疫刺激特性及其对过敏性哮喘小鼠模型中嗜酸性肺炎症发展的影响。虽然长dsRNA被称为许多病毒的复制中间体，可以有力地激活哺乳动物细胞中的干扰素反应，它们在HDM过敏原中的存在一直未知，直到我们最近的工作15。本手稿中介绍的RNA点印迹、RT-qPCR和FACS分析的组合，可能为解剖先天成分（如环境过敏原中的 dsRNA 物种）提供…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
我们感谢卡拉·戈雷纳女士在流动细胞学方面提供技术援助。L.S. 得到中国奖学金理事会和湖南省研究生创新基金会 （CX201713068） 的支持。H.H.A. 由沙特阿拉伯萨卡卡朱夫大学应用医学学院临床实验室科学系提供支持。X.D.L. 由 UT 健康圣安东尼奥医学院创业基金和马克斯和明奈沃尔克基金支持。
Materials
| 0.40 µm Falcon Cell Strainer | Thermo Fisher Scientific | 08-771-1 | |
| 1 mL syringes | Henke Sass Wolf | 5010.200V0 | |
| 15 mL Tube | TH.Geyer | 7696702 | |
| 50 mL Tube | TH.Geyer | 7696705 | |
| 70% ethanol | Decon Labs | 2701 | |
| Absolute Counting Beads | Life Technologies Europe B.V. | C36950 | |
| ACK-RBC lysing buffer | Lonza | 10-548E | |
| Amersham Hybond-N+ Membrane | GE Healthcare | RPN203B | |
| Ant | San Antonio | Note: Locally collected | |
| Antibody dilution buffer | (see Table 5 for recipe) | ||
| Anti-Mouse CD11b V450 Rat (clone M1/70) | BD Bioscience | 560456 | 1 to 200 dilution |
| Anti-Mouse CD11c PE-Cy7 (clone N418) | BioLegend | 117317 | 1 to 200 dilution |
| Anti-Mouse CD19 Alexa Flour 647 (clone 1D3) | eBioscience | 15-0193-81 | 1 to 200 dilution |
| Anti-Mouse CD3e APC (clone 145-2C11) | Invitrogen | 15-0031-81 | 1 to 200 dilution |
| Anti-Mouse CD45 APC-Cy7 (clone: 30-F11) | BioLegend | 103130 | 1 to 200 dilution |
| Anti-Mouse Fixable Viabillity Dye eFluor 506 | Invitrogen | 65-0866-14 | 1 to 200 dilution |
| Anti-Mouse IgG (H+L), AP Conjugate | Promega | S3721 | |
| Anti-Mouse Ly-6G FITC (clone RB6-8C5) | Invitrogen | 11-5931-82 | 1 to 200 dilution |
| Anti-Mouse MHC II APC-eFluor 780 (clone M5/114.15.2) | eBioscience | 47-5321-80 | 1 to 200 dilution |
| Anti-Mouse Siglec-F PE (clone E50-2440) | BD Pharmingen | 552126 | 1 to 200 dilution |
| BCIP/NBT substrate | Thermo Fisher Scientific | PI34042 | |
| Blocking Buffer | (see Table 5 for recipe) | ||
| Cannual, 20G X 1.5” | CADENCE SCIENCE | 9920 | |
| Centrifuge | Thermo Fisher Scientific | 75004030 | |
| CFX384 Touch Real-Time PCR Detection System | Bio-Rad Laboratories | 1855485 | |
| Chloroform | Thermo Fisher Scientific | C298-500 | |
| Cockroach | Greer Laboratories | B26 | |
| Counting beads | Thermo Fisher Scientific | 01-1234-42 | |
| D. farinae | Greer Laboratories | B81 | |
| D. pteronyssinus | Greer Laboratories | B82 | |
| Denville Cell Culture Plates with lid, 96 well cell culture plate | Thomas Scientific | 1156F03 | |
| Digital Dry Bath – Four Blocks | Universal Medical, Inc. | BSH1004 | |
| Earthworm | San Antonio | Note: Locally collected | |
| Ethylenediaminetetraacetic acid (EDTA) | Sigma-Aldrich | E6511 | |
| FACS buffer | (see recipe in Table 5) | ||
| Falcon Round-Bottom Polypropylene Tubes, 5 mL | STEMCELLTM TECHNOLOGIES | 38056 | |
| Flow cytometer (BD FACS Celesta) | BD Biosciences | ||
| Fly | Greer Laboratories | B8 | |
| Forceps | Roboz Surgical Instrument | RS-5135 | |
| Hemocytometer | Hausser Scientific | 3110 | |
| HT-DNA | Sigma | D6898 | |
| In Vivo MAb anti-mouse CD16/CD32 (clone: 2.4G2) | Bio X Cell | BE0307 | |
| iScript cDNA Synthesis Kit | Bio-Rad Laboratories | 1708891 | |
| Isoflurane | Abbott Labs | sc-363629Rx | |
| Isopropanol | Thermo Fisher Scientific | BP2618500 | |
| J2 anti-dsRNA monoclonal antibody | SCICONS | 10010200 | |
| Lung digestion solution | (see recipe in Table 5) | ||
| Lysing Matrix D | MP Biomedicals | 116913050-CF | |
| Lysing Matrix D, 2 mL tube | MP Biomedicals | SKU:116913100 | |
| Mice (female, 8-12 weeks old, C57BL/6J) | Jackson Laboratory | #000664 | |
| Microcentrifuge tube 1.5 mL | Sigma-Aldrich | 30120.094 | |
| Microscope | Olympus | CK30 | |
| Mini-BeadBeater | Homogenizers | SKU:BS:607 | |
| Mini-Beadbeater-16 | Biospec | 607 | |
| Mosquito | Greer Laboratories | B55 | |
| NanoDrop 2000C | Thermo Scientific Spectophotometer Medex Supply | TSCND2000C | |
| Needle, 21 G x 1 1/2 in | BD Biosciences | 305167 | |
| Non-fat milk | Bio-Rad Laboratories | 1706404 | |
| Nylon string | Dynarex | 3243 | |
| Phosphate-buffered Saline (PBS) | Lonza | BE17-516F | |
| RNase III | Thermo Fisher Scientific | AM2290 | |
| RNase T1 | Thermo Fisher Scientific | AM2283 | |
| Scissors | Roboz Surgical Instrument | RS-6802 | |
| Shaker or Small laboratory mixer | Boekel Scientific | 201100 | |
| SPHERO AccuCount Fluorescent | Spherotech | ACFP-70-5 | 1 to 10 dilution |
| Spider | San Antonio | Note: Locally collected | |
| TBS | (see recipe in Table 5) | ||
| TBS-T | (see recipe in Table 5) | ||
| Total cell medium | (see recipe in Table 5) | ||
| TRIzol Reagent | Thermo Fisher Scientific | 15596018 | |
| Tween 20 | Sigma-Aldrich | P9416 | |
| UV Stratalinker 2400 UV | LabX | 20447 | |
| Wasp | San Antonio | Note: Locally collected |
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Citazione di questo articolo
Alanazi, H. H., She, L., Li, X. Identification and Characterization of Immunogenic RNA Species in HDM Allergens that Modulate Eosinophilic Lung Inflammation. J. Vis. Exp. (159), e61183, doi:10.3791/61183 (2020).