Die Induktion von Murine Darmentzündungen durch adoptiven Transfer von Effektor CD4<sup> +</sup> CD45RB<sup> Hoch</sup> T-Zellen in immundefizienten Mäusen
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
Es gibt viele verschiedene Tiermodelle zum Untersuchen der Pathogenese von menschlichen entzündlichen Darmerkrankungen (IBD), die jeweils ihre eigenen Vor- und Nachteile vorhanden. Wir beschreiben hier eine experimentelle Colitis-Modell, das durch adoptiven Transfer von syngenen Milz CD4 + CD45RB hoch -T-Zellen in T-und B-Zell-defizienten Mäusen Empfänger eingeleitet wird. Die CD4 + CD45RB hoch T-Zellpopulation, die größtenteils aus naiven Effektorzellen induzieren kann chronische Darmentzündung, sehr ähnlich wichtige Aspekte der menschlichen IBD. Dieses Verfahren kann manipuliert werden, um Aspekte der Krankheit Beginn und Fortschreiten zu untersuchen. Zusätzlich kann es für die Funktion der angeborenen, adaptive und regulatorische Immunzellpopulationen, und die Rolle von Umweltfaktoren, das heißt, die Mikroflora im Darmentzündung zu untersuchen. In diesem Artikel erläutern wir die Methode zur Induzierung ulcerosa mit einer Schritt-für-Schritt-Protokoll. Diese includes eine Video-Demonstration der wichtigsten technischen Aspekte erforderlich, um diese Mausmodell der experimentellen Kolitis zu Forschungszwecken erfolgreich zu entwickeln.
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
Hier beschreiben wir eine Schritt-für-Schritt-Protokoll Induzieren Kolonentzündung in Mäusen durch adoptiven Transfer von CD4 + CD45RB + T-Zellen in immundefizienten Mäusen. Wir verwendeten C57BL / 6 Spender Milz und syngenen Rag1 – / – Empfängermäuse, obwohl auch andere Stämme (zB BALB / c, 129S6 / SvEv, nicht-übergewichtigen diabetischen (NOD)) und genetische Modelle der Immunschwäche (zB SCID, Rag2 – / -), können ebenfalls verwe…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Diese Arbeit wurde vom amerikanischen Gesellschaft für Gastroenterologie (AGA) Forschung Wissenschaftler-Preis und Crohn und Colitis Foundation of America (CCFA) Career Development Award (den SZS), NIH NIDDK F30 DK089692 (ECS) und Universität von North Carolina Center for Gastrointestinal Biology unterstützt und Disease Grants P30 DK34987 (Histologie Core). Der UNC-Durchflusszytometrie Core Facility ist teilweise durch eine NCI-Center Core-Unterstützung Grant (P30CA016086) an den UNC Lineberger Comprehensive Cancer Center unterstützt. Wir danken Lukas B. Borst von North Carolina State University College of Veterinary Medicine für seine Hilfe bei histopathologischen Analyse und Immunhistochemie.
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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