Identifikation og karakterisering af immunogeneRNA-arter i HDM-allergener, der modulerer eosinofil lungebetændelse
Summary
Miljømæssige allergener såsom husstøvmider (HDM) indeholder ofte mikrobielle stoffer, der aktiverer medfødte immunreaktioner for at regulere allergisk inflammation. Den protokol, der præsenteres her, viser identifikationen af dsRNA-arter i HDM-allergener og karakterisering af deres immunogene aktiviteter i modulerende eosinofil lungebetændelse.
Abstract
Miljømæssige allergener såsom husstøvmider (HDM) er ofte i komplekse former, der indeholder både allergiske proteiner, der driver afvigende type 2-reaktioner og mikrobielle stoffer, der fremkalder medfødte immunresponser. Disse allergen-associerede mikrobielle komponenter spiller en vigtig rolle i reguleringen af udviklingen af type 2 inflammatoriske tilstande såsom allergisk astma. De underliggende mekanismer er dog stort set udefinerede. Den protokol, der præsenteres her, bestemmer de strukturelle egenskaber og in vivo-aktiviteten af allergenrelateret immunostimulerende RNA. Specifikt undersøges almindelige allergener for tilstedeværelsen af dobbeltstrengede RNA-arter (dsRNA), der kan stimulere IFN-reaktioner i lungerne og begrænse udviklingen af svær lungeeosinofili i en musemodel af HDM-induceret allergisk astma. Her har vi medtaget følgende tre analyser: Dot blot for at vise dsRNA-strukturerne i total RNA isoleret fra allergener, herunder HDM-arter, RT-qPCR til måling af aktiviteterne i HDM RNA i interferonstimulerende gener (ISGs) udtryk i muselunger og FACS-analyse for at bestemme virkningerne af HDM RNA på antallet af eosinofile i bal og lunge.
Introduction
På grundlag af den hygiejnehypotese , der oprindeligt blev foreslået af Strachan1, kan udsættelse for mikrobiel miljøfaktorer såsom endotoxin beskytte mod udviklingen af allergiske lidelser2,3. Under mikrobielle infektioner, fx virusinfektioner, udløser den medfødte immundetektion af udenlandske nukleinsyrer (RNA/DNA) værtsforsvarsrespons4,,5,6. Det er dog fortsat uvist, om der findes og prævalenser af immungene nukleinsyrer såsom lange dobbeltstrengede RNA-arter (dsRNA) i husstøvmider (HDM) eller andre insektalrogener. Denne protokol er designet til at afgøre, om HDM eller insekt- og ikke-insektallergener indeholder lange dsRNA-arter, der kan aktivere en beskyttende immunrespons for at modvirke udviklingen af alvorlig eosinofil lungebetændelse i en musemodel af allergisk astma. Her tilbyder vi tre enkle og hurtige metoder til at evaluere de strukturelle determinanter i HDM total RNA, som er nødvendige for at regulere allergeninduceret eosinofil lungebetændelse.
Slimhinden immunsystemet er den største immunorgan i kroppen og fungerer som den første linje i vært forsvar mod både mikrobielle infektioner og allergiske fornærmelser7,8. Den lange dsRNA, replikation mellemliggende af mange vira, er kendt for at fungere som en patogen-associeret molekylære mønster (PAMP) til potent stimulere medfødte reaktioner via Toll som receptor 3 (TLR3) at fremkalde udtryk for interferon stimulerede gener (ISGs)9,10,,11,,12,13,14. Vi har for nylig vist, at HDM total RNA indeholdt dsRNA-strukturer, som upregulated udtrykket af ISGs og reduceret svær eosinofil lungebetændelse, når det administreres via intratracheal instillation i en murine model af allergisk astma induceret af HDMekstrakter 15. Sværhedsgraden af lungebetændelse bestemmes ved at analysere immuncelletyperne i bronskoalveolær lavage (BAL) og lungevæv via flowcytometri16,17,18,19,20.
Denne protokol omfatter tre analyser: 1) hurtig påvisning af dsRNA-strukturer med RNA-punkt blot ved hjælp af et monoklonalt museantistof J2, som specifikt binder sig til dsRNA (≥40bp) på en sekvensuafhængig måde; 2) hurtig evaluering af in vivo-virkninger af immunstimulerende RNA i muselunger ved at måle induktion af ISG’er ved hjælp af RT-qPCR; 3) nøjagtig kvantificering af eosinofile i BAL og lunge i forbindelse med HDM-induceret lungebetændelse ved hjælp af flow cytometri analyse.
Ovenstående analyser kan bruges til at studere ikke kun allergiske lungesygdomme, men også respiratoriske bakterielle og virale infektioner. For eksempel kan det DSRNA-specifikke J2-antistof også anvendes til andre formål såsom immunoaffinitetkromatografi, immunhistokemi, enzymbundet immunosorbensanalyse (ELISA) og immunstaining21,22,23. Desuden kan flere anvendelser neden for BAL væske indsamling anvendes til kvantificering opløselige indhold såsom cytokiner og chemokines ved hjælp af ELISA, og transskriptionel profilering af celler i luftvejene (f.eks alveolær makrofager). Selv om der er en række protokoller til rådighed i litteraturen til at evaluere lungesygdomme, de fleste af disse protokoller fokuserer ofte på målet validering. De procedurer, der er beskrevet her, kan anvendes til at identificere komponenter i miljømæssige allergener, der er vigtige for at regulere udviklingen af allergiske sygdomme.
Protocol
Representative Results
Discussion
Den nuværende protokol beskriver, hvordan man vurderer de immunstimulerende egenskaber af allergen-associerede mikrobielle RNA og deres indvirkning på udviklingen af eosinofil lungebetændelse i en musemodel af allergisk astma. Selv om lange dsRNA’er er kendt som replikationsmellemprodukter af mange vira, der potent kan aktivere interferonresponser i pattedyrceller, har deres tilstedeværelse i HDM-allergener været ukendt indtil vores seneste arbejde15. Kombinationen af RNA-dot blot, RT-qPCR og…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Vi takker Ms Karla Gorena for teknisk bistand i flow cytometri. L.S. støttes af China Scholarship Council og Hunan Provincial Innovation Foundation for Postgraduate (CX201713068). H.H.A. er støttet af Institut for Kliniske Laboratorievidenskaber, College of Applied Medical Sciences, Jouf University, Sakaka, Saudi-Arabien. X.D.L. støttes af UT Health San Antonio School of Medicine Startup Fund og Max og Minnei Voelcker Fund.
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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