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
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 高 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 高 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
Representative Results
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 |
References
- Xavier, R. J., Podolsky, D. K. Unravelling the pathogenesis of inflammatory bowel disease. Nature. 448 (7152), 427-434 (2007).
- Cho, J. H., Brant, S. R. Recent insights into the genetics of inflammatory bowel disease. Gastroenterology. 140 (6), 1704-1712 (2011).
- Jostins, L., et al. Host-microbe interactions have shaped the genetic architecture of inflammatory bowel disease. Nature. 491 (7422), 119-124 (2012).
- Powrie, F., Leach, M. W., Mauze, S., Caddle, L. B., Coffman, R. L. Phenotypically distinct subsets of CD4+ T cells induce or protect from chronic intestinal inflammation in C. B-17 scid mice. Int Immunol. 5 (11), 1461-1471 (1993).
- Ostanin, D. V., et al. T cell transfer model of chronic colitis: concepts, considerations, and tricks of the trade. Am J Physiol Gastrointest Liver Physiol. 296 (2), 135-146 (2009).
- Ma, B. W., et al. Routine habitat change: a source of unrecognized transient alteration of intestinal microbiota in laboratory mice. PLoS One. 7 (10), e47416 (2012).
- Read, S., Powrie, F. Induction of inflammatory bowel disease in immunodeficient mice by depletion of regulatory T cells. Curr Protoc Immunol. Chapter 15 (Unit 15 13), (1999).
- Maillard, M. H., et al. The Wiskott-Aldrich syndrome protein is required for the function of CD4(+)CD25(+)Foxp3(+) regulatory T cells. J Exp Med. 204 (2), 381-391 (2007).
- Hegazi, R. A., et al. Carbon monoxide ameliorates chronic murine colitis through a heme oxygenase 1-dependent pathway. J Exp Med. 202 (12), 1703-1713 (2005).
- Kole, A., et al. Type I IFNs regulate effector and regulatory T cell accumulation and anti-inflammatory cytokine production during T cell-mediated colitis. J Immunol. 191 (5), 2771-2779 (2013).
- Kobayashi, T., et al. NFIL3-deficient mice develop microbiota-dependent, IL-12/23-driven spontaneous colitis. J Immunol. 192 (4), 1918-1927 (2014).
- Steinbach, E. C., et al. Innate PI3K p110delta Regulates Th1/Th17 Development and Microbiota-Dependent Colitis. J Immunol. 192 (8), 3958-3968 (2014).
- Kobayashi, T., et al. NFIL3 is a regulator of IL-12 p40 in macrophages and mucosal immunity. J Immunol. 186 (8), 4649-4655 (2011).
- Leach, M. W., Bean, A. G., Mauze, S., Coffman, R. L., Powrie, F. Inflammatory bowel disease in C.B-17 scid mice reconstituted with the CD45RBhigh subset of CD4+ T cells. Am J Pathol. 148 (5), 1503-1515 (1996).
- Powrie, F., et al. Inhibition of Th1 responses prevents inflammatory bowel disease in scid mice reconstituted with CD45RBhi CD4. T cells. Immunity. 1 (7), 553-562 (1994).
- Read, S., Malmstrom, V., Powrie, F. Cytotoxic T lymphocyte-associated antigen 4 plays an essential role in the function of CD25(+)CD4(+) regulatory cells that control intestinal inflammation. J Exp Med. 192 (2), 295-302 (2000).
- Rogers, G. B., et al. Functional divergence in gastrointestinal microbiota in physically-separated genetically identical mice. Sci Rep. 4, 5437 (2014).
- Fukata, M., et al. The myeloid differentiation factor 88 (MyD88) is required for CD4+ T cell effector function in a murine model of inflammatory bowel disease. J Immunol. 180 (3), 1886-1894 (2008).
- Kurtz, C. C., et al. Extracellular adenosine regulates colitis through effects on lymphoid and nonlymphoid cells. Am J Physiol Gastrointest Liver Physiol. 307 (3), 338-346 (2014).
- Naganuma, M., et al. Cutting edge: Critical role for A2A adenosine receptors in the T cell-mediated regulation of colitis. J Immunol. 177 (5), 2765-2769 (2006).
- Ranatunga, D. C., et al. A protective role for human IL-10-expressing CD4+ T cells in colitis. J Immunol. 189 (3), 1243-1252 (2012).
- Srikrishna, G., et al. Carboxylated glycans mediate colitis through activation of NF-kappa. B. J Immunol. 175 (8), 5412-5422 (2005).
- Wang, F., et al. IFN-gamma-induced TNFR2 expression is required for TNF-dependent intestinal epithelial barrier dysfunction. Gastroenterology. 131 (4), 1153-1163 (2006).
