Summary

Die Induktion von Murine Darmentzündungen durch adoptiven Transfer von Effektor CD4<sup> +</sup> CD45RB<sup> Hoch</sup> T-Zellen in immundefizienten Mäusen

Published: April 21, 2015
doi:

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

HINWEIS: Stellen Sie sicher, dass alle Tier Protokolle werden von und in Übereinstimmung mit Institutional Animal Care und Verwenden Committee (IACUC) Vorschriften und der National Research Council Handbuch für die Pflege und Verwendung von Labortieren zugelassen. Spendermäusen kann entweder männlich oder weiblich ist, aber Empfängermäuse sollte männlich. Wenn weibliche Empfänger zu verwenden sind, müssen die Spendermäuse female 5 sein. Pflegen Kolonien regelmäßig, unsteril Betten und nic…

Representative Results

Ca. 10 x 10 6 CD4 + CD45RB hoch -T-Zellen aus 10 Milz von erwachsenen C57BL / 6 Spendermäusen sind zuverlässig isoliert. Diese Anzahl wird in Abhängigkeit von dem Alter und dem Stamm des Spendermaus und dem Können des Forschers variiert. Beim 4 x 10 5 C57BL / 6 CD4 + CD45RB hoch -T-Zellen in C57BL / 6 Rag1 übertragen – / – Empfängermäuse, klinischen Anzeichen einer Krankheit entstehen rund 5 Wochen nach der Sättigung oder früh…

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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Cite This Article
Steinbach, E. C., Gipson, G. R., Sheikh, S. Z. Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice. J. Vis. Exp. (98), e52533, doi:10.3791/52533 (2015).

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