Induktion af tarmbetændelse ved adoptivoverførsel af CBir1 TCR transgene CD4+ T-celler til immundefekte mus
Summary
I denne protokol beskrives en tarmmikrobiota-antigenspecifik T-celleadoptel overførsel colitis-model. CD4+ T-celler isoleres fra CBir1 TCR-transgene mus. Disse er specifikke for et immundominerende tarmmikrobiota-antigen CBir1 flagellin, som overføres til modtager Rag1-/- mus, hvilket fører til tarmbetændelse.
Abstract
Med stigningen i forekomsten pålægger inflammatoriske tarmsygdomme (IBD), som er kroniske sygdomme, der påvirker mave-tarmkanalen, en betydelig sundhedsmæssig og økonomisk byrde for enkeltpersoner og samfund. Derfor er det afgørende at undersøge de mekanismer, der ligger til grund for patogenesen og udviklingen af IBD. Her beskrives en tarmmikrobiota-antigenspecifik T-celleoverførselskolitismodel. CBir1 flagellin er blevet anerkendt som det immundominerende tarmbakterieantigen i eksperimentel colitis og patienter med Crohns sygdom. CBir1 TCR transgene naϊve CD4 + T-celler, der er specifikke for CBir1 flagellin, kan fremkalde kronisk colitis efter adoptiv overførsel til immunmangelfulde Rag1-/- mus. Sygdommens sværhedsgrad vurderes af histopatologi. CD4 + T-cellefænotyperne i colonic lamina propria bestemmes også. Denne model ligner meget udviklingen af IBD, som giver en ideel murinmodel til undersøgelse af de mekanismer, der driver patogenesen af IBD og tester de potentielle lægemidler til behandling af IBD.
Introduction
Inflammatoriske tarmsygdomme (IBD), hovedsageligt herunder Crohns sygdom (CD) og ulcerøs colitis (UC), er karakteriseret ved kronisk, recidiverende-remitterende betændelse i mave-tarmkanalen, der påvirker millioner over hele verden1. Flere faktorer har været impliceret i udviklingen og patogenesen af IBD, herunder genetisk modtagelighed, tarmmikrobiota, immunresponser, kost og livsstil2. Den nøjagtige mekanisme for IBD er dog stadig ikke helt forstået.
En af de særlige interesser er samspillet mellem tarmmikrobiota og værtsimmunresponser i reguleringen af tarmbetændelse3. Tarmmikrobiota tilvejebringer en række immunstimulerende molekyler og antigener, som kan aktivere immunresponser4. Mens balancen mellem effektor T-celler og regulatoriske T-celler (Tregs) er kritisk for at opretholde intestinal homeostase, bidrager det overdrevne tarmslimhinde-CD4 + T-cellerespons på tarmmikrobiotaantigener til tarmbetændelse5,6,7. Som et immundominerende tarmmikrobiotaantigen har CBir1 flagellin været relateret til patogenesen af human CD8,9. Desuden inducerer overførsel af CBir1 TCR transgene (Tg) T-celler tarmbetændelse hos immunmangelfulde mus6, der ligner den humane IBD, hvilket indikerer, at denne T-celleoverførselsmodel hjælper med at undersøge mekanismerne for human IBD.
Dette arbejde beskriver den detaljerede protokol for inducering af colitis hos Rag1-/- mus ved adoptivoverførsel af CBir1 TCR Tg naϊve CD4+ T-celler og vurdering af sygdommens sværhedsgrad. Desuden vises de forventede resultater, og de kritiske trin i proceduren og fejlfinding diskuteres, hvilket vil hjælpe forskere med at undersøge mekanismerne for patogenese af tarmbetændelse og teste de potentielle lægemidler til behandling af IBD.
Protocol
Representative Results
Discussion
Selvom hvert trin er afgørende for reproducerbarheden af denne colitis-model, er der flere kritiske trin. Modtageren Rag-/- mus skal modtage tilstrækkelig levedygtige naϊve CD4 + T-celler til at fremkalde tarmbetændelse. Vi brugte milt til isolering af naive CD4 + T-celler i stedet for MLN’er. Fordi udbyttet af naive CD4 + T-celler i MLN’er er meget lavere end i milt. CD62L udtrykkes stærkt i naive T-celler, og CD44 og CD25 er aktiveringsmarkørerne for T-cel…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
Dette arbejde blev delvist støttet af National Institutes of Health-tilskud DK125011, AI150210 og DK124132, University of Texas System STARs-prisen (Y.C.) og James W. McLaughlin Fellowship Fund fra University of Texas Medical Branch i Galveston (W.Y.). Figur 1 blev oprettet med BioRender.com.
Materials
| 0.22 µm vacuum-driven disposable bottle top filter | MilliporeSigma | SCGPS05RE | |
| 100x Penicillin-Streptomycin | Corning | 30-002-CI | |
| 100-µm strainer | BD Biosciences | 352360 | |
| 3-mL Transfer Pipette | Fisherbrand | 13-711-9CM | |
| Anti-Mouse CD16/32 | Biolegend | 101302 | |
| Anti-Mouse CD25-Percp/Cy5.5 | Biolegend | 102030 | |
| Anti-mouse CD3-Percp/Cy5.5 | Biolegend | 100327 | |
| Anti-Mouse CD4 APC | Biolegend | 100516 | |
| Anti-Mouse CD4 Magnetic Particles | BD Biosciences | 551539 | |
| Anti-Mouse CD4-BV421 | Biolegend | 100544 | |
| Anti-Mouse CD62L-PE | Biolegend | 104408 | |
| Anti-Mouse Foxp3-PE | ThermoFisher | 12-5773-82 | |
| Anti-Mouse IFNγ-FITC | Biolegend | 505806 | |
| Anti-Mouse IL-17A-PE/Cy7 | Biolegend | 506922 | |
| Automated Cell Counter | Bio-rad | TC20 | |
| Brefeldin A | BD Biosciences | 555029 | |
| BSA | Fisher Bioreagents | BP1600-1 | |
| C tube | Miltenyi | 130-093-237 | |
| Cell Separation Magnet | BD Biosciences | 552311 | |
| Collagenase IV | Sigma-Aldrich | C5138 | |
| DAPI | Sigma-Aldrich | D9542 | |
| Dissociator Machine | Miltenyi | 130-096-427 | |
| DNase I | Sigma-Aldrich | ||
| EDTA | Corning | 46-034-CI | |
| EDTA (0.5 M, PH 8.0) | Corning | 46-034-CI | |
| FBS | R&D Systems | S11550 | |
| Flow cytometer | BD Biosciences | LSD Fortessa | |
| Heat Lamp | CoverShield | BR40 | |
| Hematoxylin and Eosin (H&E) Stain Kit | Abcam | ab245880 | |
| Insulin Syringes | BD Biosciences | 329412 | |
| Ionomycin | ThermoFisher | I24222 | |
| Live/dead Fixable Near-IR Dead Cell Stain kit | ThermoFisher | L10119 | |
| MaxQ 6000 Incubated/Refrigerated Stackable Shakers | ThermoFisher | SHKE6000 | |
| NH4Cl | Thermo Scientific | A687-500 | |
| Percoll | GE Healthcare | 17-0891-01 | |
| Phorbol-12-myristate 13-acetate | Sigma-Aldrich | P8139 | |
| RPMI 1640 Medium | Cytiva HyClone | SH3002702 | |
| Sorter | BD Biosciences | Arial Fusion | |
| Tissue Automatic Processor | ThermoFisher | STP120 | |
| Tissue Embedding/Processing Cassette | Fisher Healthcare | 22048142 | |
| Tris Base | Thermo Scientific | BP154-1 | |
| True-Nuclear Transcription Factor Buffer Set (including Perm Buffer) | Biolegend | 424401 |
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