Summary

诱导小鼠肠道炎症由效应CD4的过继转移<sup> +</sup> CD45RB<sup>高</sup> T细胞免疫缺陷小鼠

Published: April 21, 2015
doi:

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

有可用于研究人炎性肠疾病(IBD),每个都有其自己的优点和缺点的发病许多不同的动物模型。我们在这里描述通过过继转移的同源脾的CD4 + CD45RB T细胞的成T和B细胞缺陷型受体小鼠启动的一个实验性结肠炎模型。在CD4 + CD45RB T细胞群在很大程度上是由幼稚效应细胞能够诱导的慢性肠道炎症,非常类似于人类IBD的关键环节。这种方法可以被操纵来研究疾病发作和进展的方面。此外,它可以被用来研究先天,自适应的功能,以及调节免疫细胞群,和环境暴露的作用,也就是说 ,该微生物群,在肠道炎症。在这篇文章中,我们阐述了诱导结肠炎一步一步的协议的方法。这INC吕代的成功开发实验性结肠炎的研究目的这个小鼠模型所需要的关键技术环节的视频演示。

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

注:确保所有的动物协议通过并符合机构动物护理和使用委员会(IACUC)的规定和国家研究委员会的指南实验动物的护理和使用的批准。供体小鼠可以是男性或女性,但受体小鼠应该是雄性。如果女性收件人使用,供体小鼠必须是女性5。使用常规的,非无菌床上用品和非酸化水中维持菌落,因为这些可能会影响肠道菌群,并且因此,小鼠5,6的结肠炎的表型。 <p class="jove_t…

Representative Results

约10×10 6 CD4 + CD45RB 高Tg从成年C57BL / 6小鼠捐助10脾脏细胞是可靠的隔离。这个数目将根据患者的年龄的供体小鼠的应变和研究者的熟练程度而有所不同。当4×10 5只C57BL / 6的CD4 + CD45RB 高 T细胞转移到C57BL / 6 RAG1 – / -受体小鼠,疾病的临床症状出现围绕周5后饱食或更快,如果小鼠在遗传上易感更严重的疾病( 图2,3)11,12。</sup…

Discussion

在这里,我们描述了一个一步一步的协议,通过过继转移CD4 + CD45RB + T细胞的免疫缺陷小鼠诱导小鼠结肠的炎症。我们使用的C57BL / 6供体脾和同系RAG1 – / –受体小鼠,尽管其它菌株( 例如 ,BALB / c小鼠,129S6 / SvEv,非肥胖糖尿病(NOD))免疫缺陷的和遗传模型( 例如 ,SCID, 的Rag2 – / – ),也可以使用4,14-16。它是公认的背景品系?…

Disclosures

The authors have nothing to disclose.

Acknowledgements

这项工作是由美国胃肠病协会(AGA)研究学者奖和克罗恩和美国(CCFA)职业发展奖(以ENS),美国国立卫生研究院NIDDK F30 DK089692(以ECS)结肠炎基金会,以及北卡罗莱纳州中心大学的胃肠生物学支持与疾病格兰特P30 DK34987(组织学核心)。在UNC流式细胞仪核心设施是由NCI中心的核心支持格兰特(P30CA016086)的UNC莱恩伯格综合癌症中心支持的一部分。我们感谢卢克B.博斯特从兽医北卡罗莱纳州立大学对他的帮助与组织病理学分析和免疫组化。

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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Cite This Article
Steinbach, E. C., Gipson, G. R., Sheikh, S. Z. Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice. J. Vis. Exp. (98), e52533, doi:10.3791/52533 (2015).

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