Induktion av murina tarminflammation med adoptiv överföring av Effector CD4<sup> +</sup> CD45RB<sup> Hög</sup> T-celler i möss med immunbrist
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
Det finns många olika djurmodeller tillgängliga för att studera patogenesen av mänskliga inflammatoriska tarmsjukdomar (IBD), alla med sina egna fördelar och nackdelar. Vi beskriver här en experimentell kolit modell som initieras genom adoptiv överföring av syngena mjält CD4 ^ CD45RB hög T-celler i T och B-cellsbrist mottagarmöss. CD4 + CD45RB hög T-cellpopulation som till stor del består av naiva effektorceller kan inducera kronisk tarminflammation, nära påminner viktiga aspekter av mänskligt IBD. Denna metod kan manipuleras för att studera aspekter av sjukdomsdebut och progression. Dessutom kan den användas för att studera funktionen av medfödda, adaptiv och regulatoriska immuncellpopulationer, och den roll som miljöexponeringar, dvs mikroorganismer, i tarminflammation. I denna artikel illustrerar vi metodiken för att inducera kolit med en steg-för-steg-protokoll. Denna incLudes en video demonstration av viktiga tekniska aspekter som krävs för att framgångsrikt utveckla denna musmodell av experimentell kolit för forskningsändamål.
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
Här beskriver vi en steg-för-steg-protokoll inducerande colonic inflammation hos möss genom adoptiv överföring av CD4 + CD45RB + T-celler in i möss med immunbrist. Vi använde C57BL / 6 donator mjältar och syngena Rag1 – / – mottagarmöss, även om andra stammar (t.ex. BALB / c, 129S6 / SvEv, icke-feta diabetiska (NOD)) och genetiska modeller av immunbrist (t.ex. SCID, Rag2 – / -) kan också användas 4,14-16. Det är väl …
Divulgaciones
The authors have nothing to disclose.
Acknowledgements
Detta arbete stöddes av amerikanska Gastroenterological Association (AGA) Forskning Scholars Award och Crohns och kolit Foundation of America (CCFA) Career Development Award (till ENS), NIH NIDDK F30 DK089692 (till ECS) och University of North Carolina Center for Gastrointestinal Biologi och sjukdom Grant P30 DK34987 (Histologi Kärna). UNC flödescytometri Core Facility stöds delvis av en NCI Center Kärna Support Grant (P30CA016086) till UNC Lineberger Comprehensive Cancer Center. Vi tackar Luke B. Borst från North Carolina State University College of Veterinary Medicine för hans hjälp med histopatologisk analys och immunohistokemi.
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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