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.
Er zijn veel verschillende dierlijke modellen voor het bestuderen van de pathogenese van menselijke inflammatoire darmziekten (IBD), elk met zijn eigen voor- en nadelen. We beschrijven hier een experimentele colitis model dat wordt geïnitieerd door adoptieve overdracht van syngenetische milt CD4 + CD45RB- hoge T-cellen in de T- en B-cel deficiënte muizen ontvanger. De CD4 + CD45RB high T-celpopulatie die grotendeels uit naïeve effectorcellen kan induceren chronische darmontsteking, sterk lijken belangrijke aspecten van menselijke IBD. Deze methode kan worden gemanipuleerd om aspecten van de ziekte ontstaan en de progressie bestuderen. Daarnaast kan het worden gebruikt om de functie van aangeboren, adaptief en regelgevende immuuncel populaties en de rol van milieurisico's, dwz de microbiota in intestinale inflammatie bestuderen. In dit artikel tonen we de methode voor het induceren van colitis met een stap-voor-stap protocol. Dit includes een video demonstratie van de belangrijkste technische aspecten die nodig zijn om succesvol te ontwikkelen dit muizenmodel van experimentele colitis voor onderzoeksdoeleinden.
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.
Hier beschrijven we een stap-voor-stap protocol inducerende darm ontsteking bij muizen door adoptieve overdracht van CD4 + CD45RB + T-cellen in immuun deficiënte muizen. We gebruikten C57BL / 6 donor milt en syngene RAG1 – / – ontvangende muizen, hoewel andere stammen (zoals BALB / c, 129S6 / SvEv, non-obese diabetische (NOD)) en genetische modellen van immunodeficiëntie (bijvoorbeeld SCID, RAG2 – / -) kan ook worden gebruikt 4,14-16….
The authors have nothing to disclose.
Dit werk werd ondersteund door de Amerikaanse Gastroenterological Association (AGA) Onderzoek Scholars Award en de Crohn en Colitis Foundation of America (CCFA) Career Development Award (tot SZS), NIH NIDDK F30 DK089692 (ECS), en de Universiteit van North Carolina Centrum voor Gastro-intestinale Biologie en de ziekte van Grant P30 DK34987 (histologie Core). De UNC flowcytometrie Core Facility wordt gedeeltelijk ondersteund door een NCI Center Core ondersteuning Grant (P30CA016086) aan de UNC Lineberger Comprehensive Cancer Center. Wij danken Luke B. Borst uit North Carolina State University College of Veterinary Medicine voor zijn hulp met histopathologische analyse en immunohistochemie.
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 |