La inducción de la inflamación intestinal murino por transferencia adoptiva de Efector CD4<sup> +</sup> CD45RB<sup> Alta</sup> T células en ratones inmunodeficientes
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
Hay muchos modelos diferentes de animales disponibles para el estudio de la patogénesis de las enfermedades humanas inflamatorias del intestino (IBD), cada uno con sus propias ventajas y desventajas. Se describe aquí un modelo de colitis experimental que se inicia por la transferencia adoptiva de células de alta T CD4 + CD45RB singeneicos bazo en ratones receptores deficientes de células T y B. La población de células de alta T CD4 + CD45RB que consiste en gran parte de las células efectoras no tratados previamente es capaz de inducir la inflamación intestinal crónica, muy parecidas a aspectos clave de la EII humana. Este método puede ser manipulado para estudiar aspectos de la aparición de la enfermedad y la progresión. Además, puede ser utilizado para estudiar la función de innato y adaptativo, y las poblaciones de células inmunes reguladoras, y el papel de la exposición ambiental, es decir, la microbiota, en la inflamación intestinal. En este artículo se expone la metodología para la inducción de la colitis con un protocolo paso a paso. Esta incLudes un video de demostración de los aspectos técnicos clave necesarios para desarrollar con éxito este modelo murino de colitis experimental con fines de investigación.
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
Aquí se describe un protocolo paso a paso la inducción de la inflamación del colon en ratones mediante la transferencia adoptiva de células T CD4 + CD45RB + en ratones inmunodeficientes. Se utilizó bazo C57BL / 6 donantes y Rag1 singénico – / – ratones receptores, aunque otras cepas (por ejemplo, BALB / c, 129S6 / SvEv, diabético no obeso (NOD)) modelos y genéticos de la inmunodeficiencia (por ejemplo, SCID, Rag2 – / -) también se p…
Declarações
The authors have nothing to disclose.
Acknowledgements
Este trabajo fue apoyado por la Asociación Americana de Gastroenterología (AGA) Investigación estudiosos Premio y la enfermedad de Crohn y Colitis Foundation of America (CCFA) Premio Desarrollo Profesional (a ENS), NIH NIDDK F30 DK089692 (ECS), y la Universidad de Carolina del Norte Centro de Biología Gastrointestinal y Grant Enfermedades P30 DK34987 (Histología Core). El Fondo para el flujo de Citometría de UNC es apoyado en parte por una subvención del NCI Core Support Center (P30CA016086) al Centro Integral del Cáncer de la UNC Lineberger. Damos las gracias a Lucas B. Borst de North Carolina State University College de Medicina Veterinaria por su ayuda con el análisis histopatológico e inmunohistoquímica.
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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