Induktion af murine tarmbetændelse ved adoptiv overførsel af effektor CD4<sup> +</sup> CD45RB<sup> Høj</sup> T celler i immunsvækkede mus
Summary
Here, we present a protocol to induce colonic inflammation in mice by adoptive transfer of syngeneic CD4+CD45RBhigh T cells into T and B cell deficient recipients. Clinical and histopathological features mimic human inflammatory bowel diseases. This method allows the study of the initiation of colonic inflammation and progression of disease.
Abstract
Der er mange forskellige dyremodeller til rådighed til at studere patogenesen af humane inflammatoriske tarmsygdomme (IBD), hver med sine egne fordele og ulemper. Vi beskriver her en eksperimentel colitis model, der er initieret af adoptiv overførsel af syngene milt CD4 + CD45RB høj T-celler i T og B-celle-mangel recipientmus. CD4 + CD45RB high T-cellepopulation, der i vid udstrækning består af naive effektorceller er stand til at inducere kronisk intestinal inflammation, der ligner vigtige aspekter af human IBD. Denne metode kan manipuleres til at studere aspekter af sygdommen indtræden og progression. Derudover kan den bruges til at studere funktionen af medfødte, adaptive, og regulatoriske immuncellepopulationer, og den rolle, miljømæssige eksponeringer, dvs. mikrobiota i tarmbetændelse. I denne artikel illustrerer vi metoden til at inducere colitis med en trin-for-trin-protokol. Denne incLudes en video demonstration af vigtig tekniske aspekter, der er nødvendige til at udvikle denne musemodel af eksperimentel colitis til forskningsformål.
Introduction
The inflammatory bowel diseases (IBD) Crohn’s disease and ulcerative colitis result from an incompletely defined and complex interaction between host immune responses, genetic susceptibility, environmental factors, and the enteric luminal contents1. Recent genome-wide association studies report associations between immune cell regulatory genes and IBD susceptibility2,3. Both innate and adaptive immune cell intrinsic genes are represented in these studies, indicating a central role for these cell populations in IBD pathogenesis.
There currently exist more than 50 animal models of human IBD. While no one model perfectly phenocopies human IBD, many are useful for studying various aspects of human disease, including disease onset and progression and the wound-healing response. In the method described here, intestinal inflammation is initiated with syngeneic splenic CD4+CD45RBhigh T cell adoptive transfer into T and B cell deficient recipient mice4. The CD4+CD45RBhigh T cell population contains mainly naïve T cells primed for activation that are capable of inducing chronic small bowel and colonic inflammation. This method allows the researcher to modify key experimental variables, including both innate and adaptive immune cell populations, to answer biologically relevant questions relating to disease pathogenesis. Additionally, this method provides precise initiation of disease onset and a well-characterized experimental time course. This permits the kinetic study of clinical features of disease progression in mice. Intestinal inflammation induced by this method shares many features with human IBD, including chronic large and small bowel transmural inflammation, pathogenesis driven by cytokines such as TNF and IL-12, and systemic symptoms such as wasting5. Thus, it is an ideal model system for studying the pathogenesis of human IBD.
The method here describes in detail the protocol for inducing experimental colitis by adoptive transfer of CD4+CD45RBhigh T cells into Rag1-/- mice. We discuss key technical steps, expected results, optimization, and trouble-shooting. We address the required elements for the successful development of this murine model of intestinal inflammation for research purposes.
Protocol
Representative Results
Discussion
Her beskriver vi en trin-for-trin-protokollen inducere colon inflammation i mus ved adoptiv overførsel af CD4 + CD45RB + -T-celler i immunsvækkede mus. Vi brugte C57BL / 6 donor milt og syngene RAG1 – / – recipientmus, selv om andre stammer (fx BALB / c, 129S6 / SvEv, ikke-fede diabetiske (NOD)) og genetiske modeller af immundefekt (fx SCID, rag2 – / -) kan også anvendes 4,14-16. Det er velkendt, at baggrunden stammen påvirker…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Dette arbejde blev støttet af American Gastroenterologisk Association (AGA) Forskning Scholars Award og Crohns og Colitis Foundation of America (CCFA) Karriereudvikling Award (til SZS), NIH NIDDK F30 DK089692 (ECS), og University of North Carolina Center for Gastrointestinal Biologi og Sygdom Grant P30 DK34987 (Histologi Core). UNC Flowcytometri Core Facility støttes delvist af et NCI center Core Support Grant (P30CA016086) til UNC Lineberger Comprehensive Cancer Center. Vi takker Luke B. Borst fra North Carolina State University College of Veterinary Medicine for hans hjælp med histopatologisk analyse og immunhistokemi.
Materials
| Name of Reagent/ Equipment | Company | Catalog Number | Comments/Description |
| 10x PBS | Gibco | 14200075 | |
| 12x75mm round-bottom tube | Falcon | 352052 | |
| 15 ml conical | Corning | 430790 | |
| 26g x 3/8 Needle | BD Biosciences | 305110 | |
| 50 ml conical | Corning | 430828 | |
| 70 um Cell Strainer | Fisherbrand | 22363548 | |
| BD IMagnet | BD Biosciences | 552311 | |
| β-mercaptoethanol | Thermo Scientific | 35602 | |
| CD4-FITC IgG2b | eBioscience | 11-0041 | |
| CD45RB-PE IgG2a | BD Pharminogen | 553101 | |
| Complete Media | RPMI-1640, 1% Pen/Strep, 10% FBS, 0.0004% β-ME | ||
| FACS tube + strainer | BD Falcon | 352235 | |
| Glass Microscope Slides | Fisherbrand | 12550A3 | |
| Heat-inactivated FBS | Gemini | 100-106 | |
| Labeling Buffer | 1x PBS, 0.5% BSA, 2 mM EDTA | ||
| Lysis Buffer | 0.08% NH4Cl, 0.1% KHCO3, 1 mM EDTA | ||
| MoFlo XDP | Beckman Coulter | ||
| Mouse CD4 T lymphocyte Enrichment Set – DM | BD Biosciences | 558131 | |
| Mouse IgG2a-PE | BD Pharminogen | 553457 | |
| Mouse IgG2b-FITC | eBioscience | 11-4732 | |
| Pasteur pipet | Fisherbrand | 13-678-20D | |
| Penicillin-Streptomycin Solution, 100X | Corning Cellgro | 30-002-CI | |
| Petri Dish | Fisherbrand | 875713 | |
| Pure Ethanol 200 Proof | Decon Labs | 2705-HC | |
| RPMI-1640 | Gibco | 11-875-093 | |
| Syringe | BD Biosciences | 309597 | |
| Trypan blue | Corning Cellgro | 25-900-CI | |
| Wash Media | RPMI-1640, 1% Pen/Strep, 0.0004% β-ME |
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