JoVE Journal > Immunology and Infection
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Abstract
Introduction
Protocol
Results
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Immunology and Infection

抗原驱动结肠炎模型的开发由抗原呈递细胞T细胞抗原研究的介绍

Published: September 18, 2016
doi:
10.3791/54421
, , ,
1APC Microbiome Institute,University College Cork, 2Division of Gastroenterology and Hepatology,University Hospital Basel, 3Institute of Microbiology and Biotechnology,University of Ulm

Summary

In this antigen-driven colitis model, OT-II CD4+ T cells expressing a red fluorescent protein were adoptively transferred into RAG-/- mice that express a green fluorescent protein in mononuclear phagocytes (MPs). The hosts were challenged with Escherichia coli (E.coli) expressing the ovalbumin protein (OVA) fused to a cyan fluorescent protein (CFP).

Abstract

Inflammatory bowel disease (IBD) is a chronic inflammation which affects the gastrointestinal tract (GIT). One of the best ways to study the immunological mechanisms involved during the disease is the T cell transfer model of colitis. In this model, immunodeficient mice (RAG-/- recipients) are reconstituted with naive CD4+ T cells from healthy wild type hosts.

This model allows examination of the earliest immunological events leading to disease and chronic inflammation, when the gut inflammation perpetuates but does not depend on a defined antigen. To study the potential role of antigen presenting cells (APCs) in the disease process, it is helpful to have an antigen-driven disease model, in which a defined commensal-derived antigen leads to colitis. An antigen driven-colitis model has hence been developed. In this model OT-II CD4+ T cells, that can recognize only specific epitopes in the OVA protein, are transferred into RAG-/- hosts challenged with CFP-OVA-expressing E. coli. This model allows the examination of interactions between APCs and T cells in the lamina propria.

Introduction

肠道是暴露于外部环境的人体最大的表面上。居民微生物的广大阵列殖民人体的肠道内形成的肠道菌群（或微生物）。这估计是由最多百万亿微生物细胞和构成生物学1-3知人口最稠密的细菌栖息地之一。在GIT细菌定植肠道的利基在那里生存繁衍4。作为回报，菌群赋予了未编码它的基因组中1个附加的功能特征的主机。例如微生物群刺激上皮细胞的增殖，通过自身产生承载不能生产维生素，调节代谢和防止病原体4-6。鉴于这种有利的关系，一些作者认为人是“超生物体”或“holobionts”是细菌和人类基因7,8的混合。鉴于（人）主机上的微生物的有利影响，肠道免疫系统需要耐受共生微生物，使他们能够管腔存在，而且杀了，从腔侧9-11入侵的病原体。肠道免疫系统已开发机制无害的和潜在有害的管腔微生物来区分;然而这些机制尚未很好地理解12。维护肠道完整性要求严格调节免疫稳态保持宽容和豁免权13之间的平衡。在免疫稳态不平衡有助于肠道疾病的诱导如炎性肠病（IBD）3,14。

有两种主要类型的IBD：克罗恩病（CD）和溃疡性结肠炎（UC）。患者与这些疾病通常患有直肠出血，严重腹泻和腹痛15,16。 IBD的单一原因仍未知的，但遗传因素，环境因素和错调免疫反应的组合可能是疾病发展的15关键事件。

鸡传染性法氏囊病动物模型已经使用了超过50年。在过去几十年中新的IBD模型系统已被开发来测试关于IBD 17,18的发病机制中的各种假说。慢性结肠炎的最佳表征的模型是诱导T细胞稳态19,20的破坏的T细胞转移模型。该模型涉及到从免疫活性小鼠转印幼稚T细胞分化成缺乏T和B细胞（如RAG – / –和SCID小鼠）的主机16,21。疾病在该模型的发展是通过评估腹泻的存在下，体力活动减少和体重的损失为3-10周监测。这就是所谓的消耗综合征16。相比于健康小鼠移植主机的结肠组织是thickeR，短重16。使用T细胞转移模型中，也可以理解的T细胞群如何不同可以向IBD 22的发病机制。 T细胞转移模型不分析抗原特异性方式在疾病过程中APC和T细胞之间的相互作用。它已经表明，骨髓细胞和淋巴样细胞之间的相互作用可能是负责肠道炎症23的发展。尽管IBD的许多方面都得到了澄清，仍然需要清楚地理解，导致疾病发展的初始事件。

它已经表明，在不存在的微生物转移结肠炎不能建立24。最近，一些理论认为IBD可以针对共生细菌25的免疫应答的结果。作者也提出了共生菌是必不可少的诱发炎症在远端肠26，在无菌（GF）的动物的肠道免疫系统通常受损27,28，但这些小鼠中完全胜任肠道免疫系统29的发展无特定病原体动物细菌结果的混合物的定植。因此，该微生物群似乎是在IBD的发病机理的一个关键因素，无论是作为可诱发或防止肠炎症30,31的发展的机制。当前理论认为IBD是微生物不平衡，称为生态失调，遗传倾向的患者32的结果，但是它是目前尚不清楚，如果生态失调是原因或疾病12的结果。考虑到微生物在IBD的发展中的作用， 在体外实验表明，CD4 + T细胞可以通过肠道细菌33,34脉冲式装甲运兵车被激活。

此外，已经表明，从抗原不同共生细菌物种，例如大肠杆菌大肠杆菌，类杆菌，真杆菌和变形杆菌 ，能够激活CD4 + T细胞35。这表明，细菌抗原给T细胞的呈现是重要的，对IBD的发展。为了减少通过在疾病过程中的微生物衍生的多种抗原的复杂性，在大肠杆菌菌株已创建产生该OVA抗原。转移结肠炎诱导OVA特异性T细胞注射到RAG – / –动物的OVA表达E.殖民大肠杆菌 。

这种模型是基于最近的证据表明，CX 3 CR1 +国会议员，在结肠固有层（CLP）36的主要细胞亚群，与CD4 + T细胞转移过程中相互作用肠炎37。国会议员采样为颗粒性抗原肠腔，如细 ​​菌，使用他们的树突36，38,39。以往的研究表明，国会议员也可以占用可溶性抗原，如OVA，引入到肠腔40,41。给出的丰度的CX 3 CR1 +国会议员中电的，可能的是这些细胞可以品尝管腔细菌和与CD4的T细胞相互作用。小鼠共焦成像与大肠杆菌定植OVA特异性CD4 + T细胞移植大肠杆菌 CFP-OVA，表明CX 3 CR1 +国会议员都在抗原驱动结肠炎的发展过程中与OT-II CD4 + T细胞接触。这种模式使肠道装甲运兵车和具体仅在肠腔特定的抗原表达细菌性T细胞的抗原呈递过程的研究。

Protocol

小鼠繁殖并保存在乌尔姆大学（德国乌尔姆）的动物设施无特定病原体（SPF）的条件下。所有的动物实验是根据当地的动物使用和保护委员会和国家动物福利法的指导进行。 1. pCFP-OVA质粒的构建扩增使用引物Ova_SpeI_fw（3'- GACCAACTAGTATGGAATTTTGTTTTGATGTATT-5'）和Ova_ClaI_rev（3'- GACCAGATCGATTAAGGGGAAACACATCTGCC-5'）37使用质粒的pCI-OVA（氨苄青霉素抗性）42</…

Representative Results

要建立抗原驱动结肠炎模型中的E.大肠杆菌菌株 已构建了包含在其中在强组成型启动子P 超 （ 图1A）的控制被表示为在CFP的基因融合到为鸡卵清蛋白和所述融合构建体的编码序列的质粒。荧光显微镜显示重组大肠杆菌大肠杆菌 pCFP-OVA，但不是亲本大肠杆菌大肠杆菌 DH10B，表达CFP（ 图1B）。 CFP-OVA-生产E.?…

Discussion

与所有其他的模式，上述的抗原驱动结肠炎模型可呈现在执行该技术的研究者必须意识到几个问题。当注射OT-II /红+ CD4 + T CD62L +细胞在主机上，研究者必须非常温柔细心针插入腹腔。不这样做可能会导致在小鼠可能导致死亡，或这将不会引起任何疾病的细胞的皮下给药的肠的撕裂。

通常情况下，老鼠会在细胞转移后的第一周体重增加。研究者应当在?…

Disclosures

The authors have nothing to disclose.

Acknowledgements

JHN is supported by the Swiss National Foundation (SNSF 310030_146290).

Materials

LB Broth, Miller (Luria-Bertani) Difco 244620
Rotary Shake Reiss Laborbedarf e. K. Model 3020 GFL
2 mm gap couvettes  Peqlab Biotechnologie GmbH 71-2020
Glycerol Sigma-Aldrich G5516-100ML
Gene Pulser Xcell system  BioRad Laboratories GmbH 1652660
LB Agar, Miller (Luria-Bertani) Difco 244510
Ampicillin Sigma-Aldrich A9393-5G
SOC Medium Sigma-Aldrich S1797-100ML
High Pure Plasmid Isolation Kit Roche 11754777001
Agarose Carl Roth GmbH & Co 3810.1
EDTA Sigma-Aldrich E9884-100G
Tris-HCl Sigma-Aldrich T5941
Glacial acetic acid Sigma-Aldrich 537020 
Gel chamber  PEQLAB Biotechnology GmbH 40-0708
Loading Dye Thermo Fisher R0611
GeneRuler 1 kb DNA Ladder  Thermo Fisher SM0312
Ethidium bromide solution Carl Roth GmbH & Co. KG 2218.3
Photo-documentation system  Decon Science Tech GmbH DeVision G 
DNA sequencing  MWG-Biotech GmbH
Phosphate buffered saline (PBS) Biochrom L182-50
Fluorescent microscope  Zeiss HBO 100
Mini-PROTEAN Tetra System Bio-Rad Laboratories GmbH 1658005
PageRuler Prestained Protein Ladder  Fermentas, St. Leon-Rot, Germany
IstanBlue Solution Expedeon, Cambridgeshire, United Kingdom
Nitrocellulose membrane  Macherey-Nagel GmbH & Co. KG 741280
Electro blotter  Biometra GmbH 846-015-600
Bovine Serum Albumins (BSA) Sigma-Aldrich A6003-25G
Anti-Ovalbumin antibody  Abcam ab181688
Anti-rabbit IgG  HRP Sigma-Aldrich A0545 
Pierce ECL Plus Western Blotting Substrate Pierce Biotechnology, Thermo Fischer Scientific Inc 32132
Forene Abbott 2594.00.00
FBS Invitrogen 10500-064
Falcon Cell Strainers Fischer Scientific  08-771-19
Ammonium chloride Sigma-Aldrich 254134-5G
Tris Base Sigma-Aldrich 10708976001
CD4+ CD62 L+ T isolation kit  Miltenyi Biotec 130-093-227 
MACS LS Columns  Miltenyi Biotec 130-042-401
MACS MS Columns  Miltenyi Biotec 130-042-201
MidiMACS Separator Miltenyi Biotec 130-042-302
MiniMACS Separator Miltenyi Biotec 130-042-102
MACS MultiStand Miltenyi Biotec 130-042-303
Feeding Needle 20G SouthPointe Surgical Supply, Inc FN-7903
Formalin solution, neutral buffered, 10% Sigma-Aldrich HT501128
Paraffin Sigma-Aldrich 1496904
Hematoxylin Sigma-Aldrich H9627
Eosin Y Sigma-Aldrich 230251 
Dithiothreitol Sigma-Aldrich D9779 
Collagenase type VIII Sigma-Aldrich C-2139
Roswell Park Memorial Institute (RPMI) medium AppliChem A2044, 9050
Percoll (density 1.124 g/ml) Biochrome L-6145
Sodium azide Sigma-Aldrich 438456
Mouse BD Fc Block BD Pharmingen 553141
FITC-conjugated mAb binding Vß 5.1, 5.2  BD Pharmingen 553189
APC-conjugated mAb binding CD4 GK1.5  eBioscience 17-0041-83
FACS Calibur  BD Biosciences
FCS Express V3 software DeNovo
Meta scanning confocal microscope  Zeiss LSM 710 
Zeiss Workstation Zeiss LSM 7
Zeiss ZEM software  Zeiss v4.2.0.121
Maxisorp immuno plates  NUNC, Roskilde 442404
Streptavidin conjugated alkaline phosphatase Jackson Immuno Research 016-050-084
Alkaline phosphatase substrate 4-Nitrophenyl phosphate disodium salt hexahydrate Sigma-Aldrich 71768-5G
mAb R4-6A2 BD Biosciences 551216
mAb XMG1.2  BD Biosciences 554410
TECAN microplate-ELISA reader Tecan
EasyWin software Tecan
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Tags

Antigen-driven ColitisAntigen Presenting CellsT CellsInflammatory Bowel DiseaseLamina PropriaT Cell Transfer Colitis ModelMononuclear PhagocytesCD4 T CellsEpithelial Cell Removal
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Rossini, V., Radulovic, K., Riedel, C. U., Niess, J. H. Development of an Antigen-driven Colitis Model to Study Presentation of Antigens by Antigen Presenting Cells to T Cells. J. Vis. Exp. (115), e54421, doi:10.3791/54421 (2016).
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