Induzione di Murine infiammazione intestinale per trasferimento adottivo di Effector CD4<sup> +</sup> CD45RB<sup> Alto</sup> T Cells in topi immunodeficienti
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
Ci sono molti modelli animali differenti disponibili per lo studio della patogenesi delle malattie infiammatorie croniche intestinali umane (IBD), ognuno con i suoi vantaggi e svantaggi. Descriviamo qui un modello di colite sperimentale che è iniziata da trasferimento adottivo di cellule della milza alta T CD4 + CD45RB singenici in T e cellule B topi riceventi deficienti. La popolazione di cellule T CD4 + alta CD45RB che consiste in gran parte di cellule effettrici naïve è in grado di indurre infiammazione intestinale cronica, molto simili aspetti chiave di IBD umana. Questo metodo può essere manipolato per studiare gli aspetti di insorgenza della malattia e la progressione. Inoltre può essere utilizzata per studiare la funzione di innata, adattabile e popolazioni regolamentazione cellule immunitarie, e il ruolo delle esposizioni ambientali, cioè, il microbiota, infiammazione intestinale. In questo articolo si illustra il metodo per indurre la colite con un protocollo passo-passo. Questo incLudes un video dimostrativo di aspetti tecnici chiave necessari per sviluppare con successo questo modello murino di colite sperimentale per scopi di ricerca.
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
Qui si descrive un protocollo step-by-step indurre infiammazione del colon nei topi mediante trasferimento adottivo di cellule CD4 + T + CD45RB in topi immunodeficienti. Abbiamo usato milza C57BL / 6 donatori e Rag1 singenico – / – topi riceventi, anche se altri ceppi (ad esempio, BALB / c, 129S6 / SvEv, non-obesi diabetici (NOD)) Modelli e genetici di immunodeficienza (ad esempio, SCID, Rag2 – / -) può essere utilizzato anche 4,14-16….
Disclosures
The authors have nothing to disclose.
Acknowledgements
Questo lavoro è stato sostenuto da Gastroenterologiche American Association (AGA) Research Scholars Award e morbo di Crohn e Colite Foundation of America (CCFA) Career Development Award (a SOI), NIH NIDDK F30 DK089692 (ECS), e University of North Carolina Centro di Biologia gastrointestinale e la malattia di Grant P30 DK34987 (Istologia Core). Il Fondo UNC Citometria a flusso Core è sostenuto in parte da un nucleo di supporto NSC Centro Grant (P30CA016086) alla UNC Lineberger Comprehensive Cancer Center. Ringraziamo Luca B. Borst da North Carolina State University College di Medicina Veterinaria per il suo aiuto con analisi istopatologica e immunoistochimica.
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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