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.
Det er mange forskjellige dyremodeller er tilgjengelige for å studere patogenesen av human inflammatoriske tarmsykdommer (IBD), hver med sine egne fordeler og ulemper. Vi beskriver her en eksperimentell kolitt modell som er initiert av adoptiv overføring av syngene milt CD4 + CD45RB høy T-celler til T og B-celle mangel resipientmus. CD4 + CD45RB høy T-celle-populasjon som i stor grad består av naive effektor-celler er i stand til å indusere kronisk intestinal inflammasjon, nærmere ligner viktige aspekter av human IBD. Denne metoden kan bli manipulert for å studere aspekter ved sykdomsdebut og progresjon. I tillegg kan den brukes til å studere funksjonen av medfødt, adaptiv, og regulatoriske immuncellepopulasjoner, og rollen til miljømessige eksponeringer, dvs. bakterieflora i tarmbetennelse. I denne artikkelen vil vi illustrere metoden for å indusere kolitt med en trinn-for-trinn-protokollen. Dette includes en video demonstrasjon av viktige tekniske aspekter som kreves for å lykkes med å utvikle denne murine modell av eksperimentell kolitt for forskningsformål.
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.
Her beskriver vi en trinn-for-trinn-protokollen induserende tykktarmsbetennelse hos mus ved adoptiv overføring av CD4 + CD45RB + T-celler i immundefekte mus. Vi brukte C57BL / 6 donor spleens og syngen Rag1 – / – resipientmus, selv om andre stammer (f.eks BALB / c, 129S6 / SvEv, ikke-overvektige diabetikere (NOD)) og genetiske modeller av immunsvikt (f.eks SCID, Rag2 – / -) kan også anvendes 4,14-16. Det er godt etablert at bakg…
The authors have nothing to disclose.
Dette arbeidet ble støttet av amerikanske Gastroenterologisk Association (AGA) Forskning Scholars Award og Crohns og kolitt Foundation of America (CCFA) Career Development Award (til SZS), NIH NIDDK F30 DK089692 (til ECS), og University of North Carolina Center for Gastrointestinal Biology og sykdom Grant P30 DK34987 (Histologi Core). UNC flowcytometrisystemer Kjerne Facility støttes delvis av en NCI senter Kjerne Support Grant (P30CA016086) til UNC Lineberger Comprehensive Cancer Center. Vi takker Luke B. Borst fra North Carolina State University College of Veterinary Medicine for hans hjelp med histopatologisk analyse og immunhistokjemi.
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 |