Name: development of an antigen-driven colitis model to study presentation of antigens by antigen presenting cells to t cells
Uploaded: 2016-09-18T15:00:00.000+00:00
Duration: 6 min 57 s
Description: Watch this Scientific Journal Video about Development of an Antigen-driven Colitis Model to Study Presentation of Antigens by Antigen Presenting Cells to T Cells at JoVE.com

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

The authors have nothing to disclose.

他のすべてのモデルと同様に、上記の抗原駆動型大腸炎モデルは、技術を実行研究者が知っておく必要がありますいくつかの問題を提示することができます。 OT-IIホストで/レッド+ CD4 + T CD62L +細胞を注入すると、研究者は腹腔内に注射針を挿入することは非常に穏やかで注意しなければなりません。これを怠ると、死亡につながる可能性がマウスの腸、または任意の?...

腸は、外部環境にさらされる体の最大の表面です。常駐微生物の広大な配列は、腸内細菌叢（または微生物叢）を形成するために、ヒトの腸にコロニーを形成します。これは、最大100000000000000微生物細胞からなると推定され、生物学1-3で知られている最も人口密度の高い細菌の生息地の1を構成しています。 GITでは細菌は、彼らが生き残ると4を掛け腸のニッチを植民地化。リターンでは、微生物は、そのゲノム1でエンコードされていない追加の機能的特徴を持つホストを付与します。例えば、微生物は、上皮細胞の増殖を刺激し、ホストは自分で作り出すことができないビタミンを生成し、代謝を調節し、病原体4-6から保護します。この有益な関係を考えると、いくつかの著者は、人間が「超生物」または細菌とヒト遺伝子7,8のミックスである「holobionts」であることを示唆しています。 （人間の）ホスト上の微生物叢の有益な影響を考えると、腸管免疫系はルーメンでその存在を可能にするだけでなく、腔側9-11から侵入する病原体を殺すために共生微生物を容認する必要があります。腸管免疫系は無害と潜在的に有害な管腔の微生物を区別するためのメカニズムを開発しました。しかし、これらのメカニズムはまだよく12を理解されていません。腸の整合性を維持することは寛容と免疫の13の間のバランスを保つために厳密に調節免疫恒常性を必要とします。免疫恒常性のアンバランスは、炎症性腸疾患（IBD）3,14などの腸疾患の誘導に寄与する。 クローン病（CD）および潰瘍性大腸炎（UC）：IBDの2つの主要なタイプがあります。これらの疾患を有する患者は、通常、直腸出血、重度の下痢や腹痛15,16苦しみます。 IBDの一つの原因はまだです不明ですが、遺伝的要因の組み合わせ、環境の影響および調節不全免疫応答が疾患の発症15のための重要な事象であるかもしれません。 IBDの動物モデルは、50年以上にわたって使用されてきました。過去数十年間、新しいIBDモデル系は、IBD 17,18の病因に関するさまざまな仮説をテストするために開発されてきました。慢性大腸炎の最もよく特徴付けられたモデルは、T細胞ホメオスタシス19,20の破壊を誘導し、T細胞移入モデルです。 16,21このモデルは、（ - / -およびSCIDマウスのようなRAGなど）TおよびB細胞を欠いている宿主に免疫したマウスからのナイーブT細胞を転送することを含みます。このモデルにおける疾患の発症は、下痢の存在を評価することによって、3-10週間モニター身体活動の減少、及び体重減少です。これには、消耗症候群16と呼ばれています。健康なマウスと比較して移植したホストの大腸組織は、シックですR、16短いと重いです。 T細胞移入モデルを使用して、T細胞集団は、IBD 22の病因に寄与することができる方法の異なる理解することが可能です。 T細胞移入モデルでは、抗原特異的様式で疾患プロセスにおけるAPCおよびT細胞の間の相互作用を分析しません。骨髄細胞およびリンパ系細胞間の相互作用は、腸の炎症23の発症の原因であることが示されています。 IBDの多くの側面が明らかにされてきたが、病気の開発につながる初期のイベントは、まだ明確に理解する必要があります。 微生物転写大腸炎の非存在下で24を確立できないことが示されています。最近、いくつかの説は、IBDは、共生細菌25に対する免疫応答の結果であることを示唆しています。著者らは、共生細菌は、遠位腸内の炎症を誘導するのに不可欠であることも提案しています26。生殖無料（GF）動物では腸管免疫系は、一般的に27,28を低下しているが、完全に有能な腸管免疫系29の開発における特定の病原体を含まない細菌の結果の混合物とこれらのマウスの植民地化。したがって、微生物叢は、いずれかの素因または腸の炎症30,31の開発から保護するメカニズムとして、IBDの病因における重要な要素であると思われます。現在の理論は、遺伝的素因の患者32に、IBDは、腸内毒素症と呼ばれる、微生物の不均衡の結果であることを示唆しているが、それは明らかではないが、まだ腸内毒素症は、原因や病気12の結果である場合。 IBDの発症における微生物の役割を考慮すると、 インビトロ実験において CD4 + T細胞は、腸内細菌33,34でパルスしたAPCにより活性化することができることを示しました。 また、より抗原ことが示されています例えば、Eなど、さまざまな共生細菌種、 大腸菌、バクテロイデス、ユーバクテリウムおよびプロテウスは 、CD4 + T細胞35を活性化することができます。これは、T細胞への細菌抗原の提示は、IBDの開発のために重要であることを示しています。疾患過程における微生物叢によって誘導される複数の抗原の複雑さを軽減するために、 大腸菌株は、OVA抗原を産生するように作成されています。転写性大腸炎は、RAGにOVA特異的T細胞を注射することによって誘導した- / - OVAを発現する大腸菌を保菌動物大腸菌 。 このモデルは、CX 3 CR1 +議員、結腸粘膜固有層（CLP）36の主要な細胞サブセットは、転送37を大腸炎の間にCD4 + T細胞と相互作用していることを示唆している最近の証拠に基づいています。国会議員は、その樹状突起36、38,39を使用して、細菌のような、粒子状抗原のための腸管腔をサンプリング。以前の研究国会議員はまた、腸管腔40,41に導入され、このようなOVAなどの可溶性抗原を、取ることができることを実証しました。 CLPにおけるCX 3 CR1 + MPの存在量を考えると、これらの細胞が管腔細菌をサンプリングし、CD4 T細胞と相互作用することが可能です。 E.が定着OVA特異的CD4 + T細胞を移植したマウスの共焦点イメージングコリ CFP-OVAは、CX 3 CR1 + MPSは抗原駆動型大腸炎の開発中OT-II CD4 + T細胞と接触していることを示しています。このモデルは、腸のAPCおよび唯一の腸管内腔における特定の抗原を発現する細菌に特異的なT細胞間の抗原提示プロセスの研究を可能にします。

<table><tbody><tr><td>LB Broth, Miller (Luria-Bertani)</td><td>Difco</td><td>244620</td><td></td></tr><tr><td>Rotary Shake</td><td>Reiss Laborbedarf e. K.</td><td>Model 3020 GFL</td><td></td></tr><tr><td>2 mm gap couvettes&amp;nbsp;</td><td>Peqlab Biotechnologie GmbH</td><td>71-2020</td><td></td></tr><tr><td>Glycerol</td><td>Sigma-Aldrich</td><td>G5516-100ML</td><td></td></tr><tr><td>Gene Pulser Xcell system&amp;nbsp;</td><td>BioRad Laboratories GmbH</td><td>1652660</td><td></td></tr><tr><td>LB Agar, Miller (Luria-Bertani)</td><td>Difco</td><td>244510</td><td></td></tr><tr><td>Ampicillin</td><td>Sigma-Aldrich</td><td>A9393-5G</td><td></td></tr><tr><td>SOC Medium</td><td>Sigma-Aldrich</td><td>S1797-100ML</td><td></td></tr><tr><td>High Pure Plasmid Isolation Kit</td><td>Roche</td><td>11754777001</td><td></td></tr><tr><td>Agarose</td><td>Carl Roth GmbH &amp;amp; Co</td><td>3810.1</td><td></td></tr><tr><td>EDTA</td><td>Sigma-Aldrich</td><td>E9884-100G</td><td></td></tr><tr><td>Tris-HCl</td><td>Sigma-Aldrich</td><td>T5941</td><td></td></tr><tr><td>Glacial acetic acid</td><td>Sigma-Aldrich</td><td>537020&amp;nbsp;</td><td></td></tr><tr><td>Gel chamber&amp;nbsp;</td><td>PEQLAB Biotechnology GmbH</td><td>40-0708</td><td></td></tr><tr><td>Loading Dye</td><td>Thermo Fisher</td><td>R0611</td><td></td></tr><tr><td>GeneRuler 1 kb DNA Ladder&amp;nbsp;</td><td>Thermo Fisher</td><td>SM0312</td><td></td></tr><tr><td>Ethidium bromide solution</td><td>Carl Roth GmbH &amp;amp; Co. KG</td><td>2218.3</td><td></td></tr><tr><td>Photo-documentation system&amp;nbsp;</td><td>Decon Science Tech GmbH</td><td>DeVision G&amp;nbsp;</td><td></td></tr><tr><td>DNA sequencing&amp;nbsp;</td><td>MWG-Biotech GmbH</td><td></td><td></td></tr><tr><td>Phosphate buffered saline (PBS)</td><td>Biochrom</td><td>L182-50</td><td></td></tr><tr><td>Fluorescent microscope&amp;nbsp;</td><td>Zeiss</td><td>HBO 100</td><td></td></tr><tr><td>Mini-PROTEAN Tetra System</td><td>Bio-Rad Laboratories GmbH</td><td>1658005</td><td></td></tr><tr><td>PageRuler Prestained Protein Ladder&amp;nbsp;</td><td>Fermentas, St. Leon-Rot, Germany</td><td></td><td></td></tr><tr><td>IstanBlue Solution</td><td>Expedeon, Cambridgeshire, United Kingdom</td><td></td><td></td></tr><tr><td>Nitrocellulose membrane&amp;nbsp;</td><td>Macherey-Nagel GmbH &amp;amp; Co. KG</td><td>741280</td><td></td></tr><tr><td>Electro blotter&amp;nbsp;</td><td>Biometra GmbH</td><td>846-015-600</td><td></td></tr><tr><td>Bovine Serum Albumins (BSA)</td><td>Sigma-Aldrich</td><td>A6003-25G</td><td></td></tr><tr><td>Anti-Ovalbumin antibody&amp;nbsp;</td><td>Abcam</td><td>ab181688</td><td></td></tr><tr><td>Anti-rabbit IgG&amp;nbsp; HRP</td><td>Sigma-Aldrich</td><td>A0545&amp;nbsp;</td><td></td></tr><tr><td>Pierce ECL Plus Western Blotting Substrate</td><td>Pierce Biotechnology, Thermo Fischer Scientific Inc</td><td>32132</td><td></td></tr><tr><td>Forene</td><td>Abbott</td><td>2594.00.00</td><td></td></tr><tr><td>FBS</td><td>Invitrogen</td><td>10500-064</td><td></td></tr><tr><td>Falcon Cell Strainers</td><td>Fischer Scientific&amp;nbsp;</td><td>08-771-2</td><td></td></tr><tr><td>Ammonium chloride</td><td>Sigma-Aldrich</td><td>254134-5G</td><td></td></tr><tr><td>Tris Base</td><td>Sigma-Aldrich</td><td>10708976001</td><td></td></tr><tr><td>CD4&lt;sup&gt;+&lt;/sup&gt; CD62 L&lt;sup&gt;+&lt;/sup&gt; T isolation kit&amp;nbsp;</td><td>Miltenyi Biotec</td><td>130-093-227&amp;nbsp;</td><td></td></tr><tr><td>MACS LS Columns&amp;nbsp;</td><td>Miltenyi Biotec</td><td>130-042-401</td><td></td></tr><tr><td>MACS MS Columns&amp;nbsp;</td><td>Miltenyi Biotec</td><td>130-042-201</td><td></td></tr><tr><td>MidiMACS Separator</td><td>Miltenyi Biotec</td><td>130-042-302</td><td></td></tr><tr><td>MiniMACS Separator</td><td>Miltenyi Biotec</td><td>130-042-102</td><td></td></tr><tr><td>MACS MultiStand</td><td>Miltenyi Biotec</td><td>130-042-303</td><td></td></tr><tr><td>Feeding Needle 20G</td><td>SouthPointe Surgical Supply, Inc</td><td>FN-7903</td><td></td></tr><tr><td>Formalin solution, neutral buffered, 10%</td><td>Sigma-Aldrich</td><td>HT501128</td><td></td></tr><tr><td>Paraffin</td><td>Sigma-Aldrich</td><td>1496904</td><td></td></tr><tr><td>Hematoxylin</td><td>Sigma-Aldrich</td><td>H9627</td><td></td></tr><tr><td>Eosin Y</td><td>Sigma-Aldrich</td><td>230251&amp;nbsp;</td><td></td></tr><tr><td>Dithiothreitol</td><td>Sigma-Aldrich</td><td>D9779&amp;nbsp;</td><td></td></tr><tr><td>Collagenase type VIII</td><td>Sigma-Aldrich</td><td>C-2139</td><td></td></tr><tr><td>Roswell Park Memorial Institute (RPMI) medium</td><td>AppliChem</td><td>A2044, 9050</td><td></td></tr><tr><td>Sodium azide</td><td>Sigma-Aldrich</td><td>438456</td><td></td></tr><tr><td>Mouse BD Fc Block</td><td>BD Pharmingen</td><td>553141</td><td></td></tr><tr><td>FITC-conjugated mAb binding V&amp;szlig; 5.1, 5.2&amp;nbsp;</td><td>BD Pharmingen</td><td>553189</td><td></td></tr><tr><td>APC-conjugated mAb binding CD4 GK1.5&amp;nbsp;</td><td>eBioscience</td><td>17-0041-83</td><td></td></tr><tr><td>FACS Calibur&amp;nbsp;</td><td>BD Biosciences</td><td></td><td></td></tr><tr><td>FCS Express V3 software</td><td>DeNovo</td><td></td><td></td></tr><tr><td>Meta scanning confocal microscope&amp;nbsp;</td><td>Zeiss</td><td>LSM 710&amp;nbsp;</td><td></td></tr><tr><td>Zeiss Workstation</td><td>Zeiss</td><td>LSM 7</td><td></td></tr><tr><td>Zeiss ZEM software&amp;nbsp;</td><td>Zeiss</td><td>v4.2.0.121</td><td></td></tr><tr><td>Maxisorp immuno plates&amp;nbsp;</td><td>NUNC, Roskilde</td><td>442404</td><td></td></tr><tr><td>Streptavidin conjugated alkaline phosphatase</td><td>Jackson Immuno Research</td><td>016-050-084</td><td></td></tr><tr><td>Alkaline phosphatase substrate 4-Nitrophenyl phosphate disodium salt hexahydrate</td><td>Sigma-Aldrich</td><td>71768-5G</td><td></td></tr><tr><td>mAb R4-6A2</td><td>BD Biosciences</td><td>551216</td><td></td></tr><tr><td>mAb XMG1.2&amp;nbsp;</td><td>BD Biosciences</td><td>554410</td><td></td></tr><tr><td>TECAN microplate-ELISA reader</td><td>Tecan</td><td></td><td></td></tr><tr><td>EasyWin software</td><td>Tecan</td><td></td><td></td></tr><tr><td>DNase I recombinant</td><td></td><td>4536282001</td><td></td></tr></tbody></table>

development of an antigen-driven colitis model to study presentation of antigens by antigen presenting cells to t cells

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.

抗原駆動型大腸炎モデルEを確立するために、 大腸菌株 CFPの遺伝子は、ニワトリオボアルブミンタンパク質のコード配列に融合され、融合構築物は、強力な構成的プロモーターP ハイパー （ 図1A）の制御下で発現されるプラスミドを含むように構成されています。蛍光顕微鏡は、組換えE.ことを示しています?...

Watch this Scientific Journal Video about Development of an Antigen-driven Colitis Model to Study Presentation of Antigens by Antigen Presenting Cells to T Cells at JoVE.com

Development of an Antigen-driven Colitis Model to Study Presentation of Antigens by Antigen Presenting Cells to T Cells

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).

T細胞への抗原提示細胞による抗原の提示を研究するための抗原駆動性大腸炎モデルの開発

Inflammatory bowel disease (IBD) is a chronic inflammation which affects the gastrointestinal tract (GIT). One of the best ways to study the immunological ...

development-an-antigen-driven-colitis-model-to-study-presentation

Research

JoVE Journal

Immunology and Infection

8.9K ビュー.  University College Cork. 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).

ビデオ: Development of an Antigen-driven Colitis Model to Study Presentation of Antigens by Antigen Presenting Cells to T Cells

Visualization of Estrogen Receptors in Colons of Mice with TNBS-Induced Crohn's Disease using Immunofluorescence

Crohn&#39;s disease is the most diagnosed type of inflammatory bowel disease. Chronic inflammation developing in the intestine leads to peristalsis disorder and damage of intestinal mucosa and seems to be associated with an increased risk of colon neoplastic transformation. Accumulating evidence indicates that estrogens and estrogen receptors affect not only hormone-sensitive tissues, but also other tissues not directly related to estrogens, such as the lungs or colon. Here, we describe the protocol for the successful immunofluorescence staining of estrogen receptors in colon obtained from a murine model of TNBS-induced Crohn&#39;s disease. A detailed protocol for the induction of Crohn&#39;s disease in mice and intestine preparation is provided as well as a step-by-step immunohistochemical procedure using formalin-fixed paraffin-embedded intestine sections. The described methods are not only useful for estrogen receptor detection and estrogen signaling investigation in vivo but can also be applied to for other proteins which may be involved in the development of colitis.

The protocol presents a complete validated TNBS-induced murine model of Crohn&#39;s disease and methods for visualization of estrogen receptors by immunohistochemistry using immunofluorescence of formalin-fixed colon sections embedded in paraffin.

免疫蛍光を用いたTNBS誘発クローン病を用いたマウスの結腸におけるエストロゲン受容体の可視化

Crohn&#39;s disease is the most diagnosed type of inflammatory bowel disease. Chronic inflammation developing in the intestine leads to peristalsis ...

生化学

DNBS/TNBS Colitis Models: Providing Insights Into Inflammatory Bowel Disease and Effects of Dietary Fat

Inflammatory Bowel Diseases (IBD), including Crohn&#39;s Disease and Ulcerative Colitis, have long been associated with a genetic basis, and more recently host immune responses to microbial and environmental agents. Dinitrobenzene sulfonic acid (DNBS)-induced colitis allows one to study the pathogenesis of IBD associated environmental triggers such as stress and diet, the effects of potential therapies, and the mechanisms underlying intestinal inflammation and mucosal injury. In this paper, we investigated the effects of dietary n-3 and n-6 fatty acids on the colonic mucosal inflammatory response to DNBS-induced colitis in rats. All rats were fed identical diets with the exception of different types of fatty acids [safflower oil (SO), canola oil (CO), or fish oil (FO)] for three&#160;weeks prior to exposure to intrarectal DNBS. Control rats given intrarectal ethanol continued gaining weight over the 5 day study, whereas, DNBS-treated rats fed lipid diets all lost weight with FO and CO fed rats demonstrating significant weight loss by 48&#160;hr and rats fed SO by 72&#160;hr. Weight gain resumed after 72&#160;hr post DNBS, and by 5 days post DNBS, the FO group had a higher body weight than SO or CO groups. Colonic sections collected 5&#160;days post DNBS-treatment showed focal ulceration, crypt destruction, goblet cell depletion, and mucosal infiltration of both acute and chronic inflammatory cells that differed in severity among diet groups. The SO fed group showed the most severe damage followed by the CO, and FO fed groups that showed the mildest degree of tissue injury. Similarly, colonic myeloperoxidase (MPO) activity, a marker of neutrophil activity was significantly higher in SO followed by CO fed rats, with FO fed rats having significantly lower MPO activity. These results demonstrate the use of DNBS-induced colitis, as outlined in this protocol, to determine the impact of diet in the pathogenesis of IBD.

DNBS/TNBS induced colitis offers an alternative and inexpensive method to study the pathobiology of inflammatory bowel disease. The protocol discussed in this paper outlines the successful application of DNBS to induce colitis in mice and rats and allows one to thoroughly study host-mediated intestinal responses.

DNBS / TNBS大腸炎モデル：食事脂肪の炎症性腸疾患への洞察とエフェクトを提供

Inflammatory Bowel Diseases (IBD), including Crohn&#39;s Disease and Ulcerative Colitis, have long been associated with a genetic basis, and more recently ...

医学

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages and disadvantages. We describe here an experimental colitis model that is initiated by adoptive transfer of syngeneic splenic CD4+CD45RBhigh T cells into T and B cell deficient recipient mice. The CD4+CD45RBhigh T cell population that largely consists of na&#239;ve effector cells is capable of inducing chronic intestinal inflammation, closely resembling key aspects of human IBD. This method can be manipulated to study aspects of disease onset and progression. Additionally it can be used to study the function of innate, adaptive, and regulatory immune cell populations, and the role of environmental exposures, i.e., the microbiota, in intestinal inflammation. In this article we illustrate the methodology for inducing colitis with a step-by-step protocol. This includes a video demonstration of key technical aspects required to successfully develop this murine model of experimental colitis for research purposes.

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

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.

エフェクターCD4の養子移入によるマウス腸の炎症の誘導<sup&gt; +</sup&gt; CD45RB<sup&gt;高い</sup&gt;免疫不全マウスへのT細胞

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages ...

免疫・感染学

Imaging CD4 T Cell Interstitial Migration in the Inflamed Dermis

The ability of CD4 T cells to carry out effector functions is dependent upon the rapid and efficient migration of these cells in inflamed peripheral tissues through an as-yet undefined mechanism. The application of multiphoton microscopy to the study of the immune system provides a tool to measure the dynamics of immune responses within intact tissues. Here we present a protocol for non-invasive intravital multiphoton imaging of CD4 T cells in the inflamed mouse ear dermis. Use of a custom imaging platform and a venous catheter allows for the visualization of CD4 T cell dynamics in the dermal interstitium, with the ability to interrogate these cells in real-time via the addition of blocking antibodies to key molecular components involved in motility. This system provides advantages over both in vitro models and surgically invasive imaging procedures. Understanding the pathways used by CD4 T cells for motility may ultimately provide insight into the basic function of CD4 T cells as well as the pathogenesis of both autoimmune diseases and pathology from chronic infections.

The mechanisms that govern the interstitial motility of CD4 effector T cells at sites of inflammation are relatively unknown. We present a non-invasive approach to visualize and manipulate in vitro-primed CD4 T cells in the inflamed ear dermis, allowing for study of the dynamic behavior of these cells in situ.

炎症真皮における撮像CD4 T細胞間質への移行

The ability of CD4 T cells to carry out effector functions is dependent upon the rapid and efficient migration of these cells in inflamed peripheral ...

An Efficient and High Yield Method for Isolation of Mouse Dendritic Cell Subsets

Dendritic cells (DCs) are professional antigen-presenting cells primarily responsible for acquiring, processing and presenting antigens on antigen presenting molecules to initiate T-cell-mediated immunity. Dendritic cells can be separated into several phenotypically and functionally heterogeneous subsets. Three important subsets of splenic dendritic cells are plasmacytoid, CD8&#945;Pos and CD8&#945;Neg cells. The plasmacytoid DCs are natural producers of type I interferon and are important for anti-viral T cell immunity. The CD8&#945;Neg DC subset is specialized for MHC class II antigen presentation and is centrally involved in priming CD4 T cells. The CD8&#945;Pos DCs are primarily responsible for cross-presentation of exogenous antigens and CD8 T cell priming. The CD8&#945;Pos DCs have been demonstrated to be most efficient at the presentation of glycolipid antigens by CD1d molecules to a specialized T cell population known as invariant natural killer T (iNKT) cells. Administration of Flt-3 ligand increases the frequency of migration of dendritic cell progenitors from bone marrow, ultimately resulting in expansion of dendritic cells in peripheral lymphoid organs in murine models. We have adapted this model to purify large numbers of functional dendritic cells for use in cell transfer experiments to compare in vivo proficiency of different DC subsets.

Distinct dendritic cell subsets exist as rare populations in lymphoid organs, and therefore are challenging to isolate in sufficient numbers and purity for immunological experiments. Here we describe a high efficiency, high yield method for isolation of all of the currently known major subsets of mouse splenic dendritic cells.

マウスの樹状細胞サブセットの単離のための効率的かつ高収率方法

Dendritic cells (DCs) are professional antigen-presenting cells primarily responsible for acquiring, processing and presenting antigens on antigen ...

Purification of the Membrane Compartment for Endoplasmic Reticulum-associated Degradation of Exogenous Antigens in Cross-presentation

Dendritic cells (DCs) are highly capable of processing and presenting internalized exogenous antigens upon major histocompatibility class (MHC) I molecules also known as cross-presentation (CP). CP plays an important role not only in the stimulation of na&#239;ve CD8+ T cells and memory CD8+ T cells for infectious and tumor immunity but also in the inactivation of self-acting na&#239;ve T cells by T cell anergy or T cell deletion. Although the critical molecular mechanism of CP remains to be elucidated, accumulating evidence indicates that exogenous antigens are processed through endoplasmic reticulum-associated degradation (ERAD) after export from non-classical endocytic compartments. Until recently, characterizations of these endocytic compartments were limited because there were no specific molecular markers other than exogenous antigens. The method described here is a new vesicle isolation protocol, which allows for the purification of these endocytic compartments. Using this purified microsome, we reconstituted the ERAD-like transport, ubiquitination, and processing of the exogenous antigen in vitro, suggesting that the ubiquitin-proteasome system processed the exogenous antigen after export from this cellular compartment. This protocol can be further applied to other cell types to clarify the molecular mechanism of CP.

The method described here is a new vesicle isolation protocol, which allows for the purification of the cellular compartments where exogenous antigens are processed by endoplasmic reticulum-associated degradation in cross-presentation.

外因性の抗原クロス プレゼンテーション内の小胞体関連分解膜コンパートメントの精製

Dendritic cells (DCs) are highly capable of processing and presenting internalized exogenous antigens upon major histocompatibility class (MHC) I ...

Murine Distal Colostomy, A Novel Model of Diversion Colitis in C57BL/6 Mice

Diversion colitis (DC) is a frequent clinical condition occurring in patients with bowel segments excluded from the fecal stream as a result of a diverting enterostomy. The etiology of this disease remains ill-defined but appears to differ from that of classical inflammatory bowel diseases such as Crohn's disease and ulcerative colitis. Research aimed to decipher the pathophysiological mechanisms leading to the development of this disease has been severely hampered by the lack of an appropriate murine model. This protocol generates a murine model of DC that facilitates the study of the immune system's role and its interaction with the microbiome in the development of DC. In this model using C57BL/6 animals, distal parts of the colon are excluded from the fecal stream by creating a distal colostomy, triggering the development of mild to moderate inflammation in the excluded bowel segments and reproducing the hallmark lesions of human DC with a moderate systemic inflammatory response. In contrast to the rat model, a large number of genetically-modified murine models on the C57BL/6 background are available. The combination of these animals with our model allows the potential roles of individual cytokines, chemokines, or receptors of bioactive molecules (e.g., interleukin (IL)-17; IL-10, chemokine CXCL13, chemokine receptors CXCR5 and CCR7, and the sphingosine-1-phosphate receptor 4) to be assessed in the pathogenesis of DC. The availability of congenic mouse strains on the C57BL/6 background largely facilitates transfer experiments to establish the roles of distinct cell types involved in the etiology of DC. Finally, the model offers the opportunity to assess the influences of local interventions (e.g., modification of the local microbiome or local anti-inflammatory therapy) on mucosal immunity in affected and non-affected bowel segments and the on systemic immune homeostasis.

Murine distal colostomy provides a murine model for human diversion colitis, a predominantly lymphocytic colitis in colon segment excluded from the fecal stream.

マウスの遠位人工肛門、転用 c57bl/6 マウス大腸炎の新たなモデル

Diversion colitis (DC) is a frequent clinical condition occurring in patients with bowel segments excluded from the fecal stream as a result of a ...

Investigating Target Gene Function in a CD40 Agonistic Antibody-induced Colitis Model using CRISPR/Cas9-based Technologies

The immune system functions to defend humans against foreign invaders such as bacteria and viruses. However, disorders of the immune system may lead to autoimmunity, inflammatory disease, and cancer. The inflammatory bowel diseases (IBD)-Crohn's disease (CD) and ulcerative colitis (UC)-are chronic diseases marked by relapsing intestinal inflammation. Although IBD is most prevalent in Western countries (1 in 1,000), incident rates are increasing around the world. Through association studies, researchers have linked hundreds of genes to the pathology of IBD. However, the elaborate pathology behind IBD and the high number of potential genes pose significant challenges in finding the best therapeutic targets. Additionally, the tools needed to functionally characterize each genetic association introduce many rate-limiting factors such as the generation of genetically modified mice for each gene. To investigate the therapeutic potential of target genes, a model system has been developed using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated endonuclease (Cas9)-based technologies and a cluster of differentiation 40 (CD40) agonistic antibody. The present study shows that CRISPR/Cas9-mediated editing in the immune system can be used to investigate the impact of genes in vivo. Limited to the hematopoietic compartment, this approach reliably edits the resulting reconstituted immune system. CRISPR/Cas9-edited mice are generated faster and are far less expensive than traditional genetically modified mice. Furthermore, CRISPR/Cas9 editing of mice has significant scientific advantages compared to generating and breeding genetically modified mice such as the ability to evaluate targets that are embryonic lethal. Using CD40 as a model target in the CD40 agonistic antibody-induced colitis model, this study demonstrates the feasibility of this approach.

Here, we describe the methodology to knock out a gene of interest in the immune system using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated endonuclease (Cas9)-based technologies and the evaluation of these mice in a cluster of differentiation 40 (CD40) agonistic antibody-induced colitis model.

The immune system functions to defend humans against foreign invaders such as bacteria and viruses. However, disorders of the immune system may lead to ...

Analysis of Granulocyte-Macrophage Colony-Stimulating Factor-Producing T Helper Cells in a Mouse Model of Contact Hypersensitivity

Parallel to traditional Th1/Th2/Th17/Treg lineages, granulocyte-macrophage colony-stimulating factor-producing T helper (Th-GM) cells have been identified as a distinct subset of T helper cells (GM-CSF+ IFN-&#947;- IL-17A- IL-22- effector CD4+ T cells) in human and mice. Contact hypersensitivity (CHS) is considered an excellent animal model for allergic contact dermatitis (ACD) in human, manifesting an intact T cell-mediated immune response. To provide a standardized and comprehensive assay to analyze the Th-GM cell subset in the T cell-dependent immune response in vivo, a murine CHS model was induced by sensitization/challenge with a reactive, low-molecular-weight, organic hapten, 2,4-dinitrofluorobenzene (DNFB). The Th-GM subset in effector CD4+ T cells generated upon immunization with the hapten was analyzed by flow cytometry. We found that Th-GM was mainly expanded in lesions and draining lymph nodes in the DNFB-induced CHS mouse model. This method can be applied to further study the biology of Th-GM cells and pharmacological research of therapeutic strategies centered on GM-CSF in various conditions, such as ACD.

Here, we present a simple and standardized method of analyzing the granulocyte-macrophage colony-stimulating factor-producing T helper subset in vivo.

Parallel to traditional Th1/Th2/Th17/Treg lineages, granulocyte-macrophage colony-stimulating factor-producing T helper (Th-GM) cells have been identified ...

Induction of Intestinal Inflammation by Adoptive Transfer of CBir1 TCR Transgenic CD4+ T Cells to Immunodeficient Mice

With the increase of incidence, inflammatory bowel diseases (IBD), which are chronic diseases affecting the gastrointestinal tract, impose a considerable health and financial burden on individuals and society. Therefore, it is critical to investigate the mechanisms underlying the pathogenesis and development of IBD. Here, a gut microbiota antigen-specific T cell transfer colitis model is described. CBir1 flagellin has been recognized as the immunodominant gut bacterial antigen in experimental colitis and patients with Crohn&#39;s disease. CBir1 TCR transgenic na&#970;ve CD4+ T cells, specific to CBir1 flagellin, can induce chronic colitis after adoptive transfer into immune-deficient Rag1-/- mice. The disease severity is assessed by histopathology. The CD4+ T cell phenotypes in colonic lamina propria are also determined. This model closely resembles the development of IBD, which provides an ideal murine model for investigating the mechanisms driving the pathogenesis of IBD and testing the potential drugs for treating IBD.

Induction of Intestinal Inflammation by Adoptive Transfer of CBir1 TCR Transgenic CD4+ T Cells to Immunodeficient Mice

In this protocol, a gut microbiota antigen-specific T cell adoptive transfer colitis model is described. CD4+ T cells are isolated from CBir1 TCR transgenic mice. These are specific for an immunodominant gut microbiota antigen CBir1 flagellin, which is transferred into recipient Rag1-/- mice, leading to intestinal inflammation.

免疫不全マウスへのCBir1 TCRトランスジェニックCD4+ T細胞の養子移入による腸管炎症の誘導

With the increase of incidence, inflammatory bowel diseases (IBD), which are chronic diseases affecting the gastrointestinal tract, impose a considerable ...

T細胞への抗原提示細胞による抗原の提示を研究するための抗原駆動性大腸炎モデルの開発

要約

さらに動画を探す

この動画の章

免疫蛍光を用いたTNBS誘発クローン病を用いたマウスの結腸におけるエストロゲン受容体の可視化

DNBS / TNBS大腸炎モデル：食事脂肪の炎症性腸疾患への洞察とエフェクトを提供

エフェクターCD4の養子移入によるマウス腸の炎症の誘導

炎症真皮における撮像CD4 T細胞間質への移行

マウスの樹状細胞サブセットの単離のための効率的かつ高収率方法

外因性の抗原クロスプレゼンテーション内の小胞体関連分解膜コンパートメントの精製

マウスの遠位人工肛門、転用 c57bl/6 マウス大腸炎の新たなモデル

Investigating Target Gene Function in a CD40 Agonistic Antibody-induced Colitis Model using CRISPR/Cas9-based Technologies

Analysis of Granulocyte-Macrophage Colony-Stimulating Factor-Producing T Helper Cells in a Mouse Model of Contact Hypersensitivity

免疫不全マウスへのCBir1 TCRトランスジェニックCD4⁺ T細胞の養子移入による腸管炎症の誘導

T細胞への抗原提示細胞による抗原の提示を研究するための抗原駆動性大腸炎モデルの開発

要約

さらに動画を探す

この動画の章

免疫蛍光を用いたTNBS誘発クローン病を用いたマウスの結腸におけるエストロゲン受容体の可視化

DNBS / TNBS大腸炎モデル：食事脂肪の炎症性腸疾患への洞察とエフェクトを提供

エフェクターCD4の養子移入によるマウス腸の炎症の誘導

炎症真皮における撮像CD4 T細胞間質への移行

マウスの樹状細胞サブセットの単離のための効率的かつ高収率方法

外因性の抗原クロス プレゼンテーション内の小胞体関連分解膜コンパートメントの精製

マウスの遠位人工肛門、転用 c57bl/6 マウス大腸炎の新たなモデル

Investigating Target Gene Function in a CD40 Agonistic Antibody-induced Colitis Model using CRISPR/Cas9-based Technologies

Analysis of Granulocyte-Macrophage Colony-Stimulating Factor-Producing T Helper Cells in a Mouse Model of Contact Hypersensitivity

免疫不全マウスへのCBir1 TCRトランスジェニックCD4+ T細胞の養子移入による腸管炎症の誘導

外因性の抗原クロスプレゼンテーション内の小胞体関連分解膜コンパートメントの精製

免疫不全マウスへのCBir1 TCRトランスジェニックCD4⁺ T細胞の養子移入による腸管炎症の誘導