Thanks to Brad Lackford for assistance optimizing some of the techniques described in this manuscript.

マクロファージ細胞株の1メンテナンス<ol><li> RAW264.7を成長させ、DMEM中J774A.1細胞株は、5％CO 2存在下、37ºCで10％ウシ胎児血清（FBS）を補充した。 （これは厳密には必要ではないが）無菌性を維持するために、メディアへの1％ペニシリン/ストレプトマイシン（ペニシリン/ストレプトマイシン）を追加します。 </li><li> 重要なステップ：マクロファージは効率的なトランスフェクションおよびsiRNAによる遺伝子ノックダウンのために健全な状態で成長していることを確認します。これらのマクロファージラインは最高のトランスフェクション効率を示し、この段階で強固なトランスフェクション後の生存を維持するように、新鮮解凍後通路3と10との間の細胞を使用する。両方が大幅に減少するノックダウントランスフェクション効率および遺伝子として、高い継代数（&gt; 20）を使用しないでください。 </li><li>細胞には80％以上のコンフルエンスに到達しなくなるまで最適化されたsiRNAトランスフェクションのために、プレートを20％コンフルエントで細胞を1日か2日成長する。生い茂った細胞はefficトランスフェクトしませんiently。 </li></ol> 2.トランスフェクションのための96ウェルシャトルシステムのプログラミング<ol><li>シャトルに接続されたコンピュータ上の96ウェルシャトルソフトウェアを起動します。左上のファイルメニューから新しいパラメータファイルを選択します。 </li><li>表示された96ウェルプレート回路図にトランスフェクションされる井戸を強調表示します。緑色のボックスが選択されたウェルが示されます。 </li><li> （J774A.1ためRAW264.7またはDS-130用のDS-136）が使用されてマクロファージ細胞株に対応したプログラムを選択し、[ 適用]を選択し、図解さ96ウェルプレートの下に位置しています。プログラムを選択した後、回路図、プレート上のウェルが濃い青色に着色されることに注意してください。白丸は、プログラムを割り当てられていないとショックを受けることはありません井戸を示している。 </li></ol> 3.トランスフェクションのための試薬を調製する<ol><li> SF細胞株を、96ウェルのNucleofector™溶液を混合することにより、作業のNucleofector™溶液を調製囲まれた補助チューブの全内容を持つ。混合試薬は使用前に室温で平衡化させる。将来の使用のために、最大3ヶ月間4ºCでの過剰完全なヌクレオ™ソリューションに保管してください。 </li><li>前加温トランスフェクションのためにマクロファージを準備する前に、37ºCでDMEM、RPMI-1640、0.25％トリプシン/ EDTA、リン酸緩衝生理食塩水（PBS）。 </li><li>氷上で解凍し、トランスフェクションのためのsiRNAを準備します。 siRNAの凍結融雪の数を最小限にするために、一定分量でそれらが使用されて初めて、新たなsiRNAを凍結する。 </li><li>最初の実験では、（20μMストックから1:10に希釈）2μmの比較的高い用量でのsiRNAを使用しています。一般的に、より高い用量が他の細胞型に比べてマクロファージにおける効率的な遺伝子ノックダウンのために必要とされているがその後、より低用量までノックダウン強固な遺伝子を誘導するsiRNAを滴定。 注：siRNAは、多くの供給源から入手可能である。かなり強固な遺伝子ノックダウンを得ることができる各遺伝子を標的と4 siRNAのプールですsiGenome SMARTPOOLsを使用。陰性対照として、様々な非標的とするsiRNAプールまたはsiRNAのいずれかを使用します。 </li><li>トランスフェクション効率を監視するには、実験的なsiRNAと並列にpmaxGFP制御（Amaxa社キットで提供される）プラスミドまたは蛍光標識されたsiRNAのいずれかをトランスフェクト。 GFPプラスミド（顕微鏡によってアッセイまたはトランスフェクションの翌日フローサイトメトリー）および蛍光標識されたsiRNA（すぐにトランスフェクションおよび復旧後、フローサイトメトリーによってアッセイ）の&gt; 99％のトランスフェクションの30〜50％のトランスフェクションを期待しています。 </li></ol> 4.トランスフェクションのためのマクロファージの準備<ol><li>トリプシン処理により細胞培養皿から付着したマクロファージを外します。培地を吸引除去し、10mlのPBSで細胞を2回洗浄します。細胞がデタッチし始めるまで、PBSを除去した後、1~2分間予め温めておいた0.25％トリプシン/ EDTA（1ミリリットル/ 10cmプレート）で細胞をインキュベートする。優しくプレートをタップして、ソリューを移動トリプシン処理を最大化するインキュベーション中る。 </li><li>インキュベーション後、滅菌チューブにピペッティングして細胞を、9ミリリットルのDMEM + FBS培地を追加し、上下穏やかに混合し、収集します。 </li><li>血球計数器を用いて、単離されたマクロファージの数を数える。 10cmプレートあたりおよそ千万マクロファージを期待しています。必要なマクロファージの総数を計算する。 96ウェルシャトルの各は200,000細胞を必要とします。ピペッティングの損失を可能にするために、細胞のいくつかの余分な井戸」の価値を追加することを忘れないでください。 </li><li>遠心管に細胞の計算された数をピペット。室温で150×gで10分間細胞を遠心します。これはマクロファージの健康とトランスフェクション効率を低下させるよう早すぎる遠心しないでください。 </li></ol> siRNAによるマクロファージの5ヌクレオ<ol><li> （試薬の損失を最小限に抑えながら、混合促進する）丸底プレートの無菌の96ウェルを使用して、転送ように各ウェルに各siRNAの2μlを添加する精製カートリッジ。 注：siRNAの数に応じて、これはマクロファージ遠心分離工程の間に達成することができる。 </li><li>遠心分離した細胞から培地を吸引除去する。静かにペレット化した200,000個の細胞あたり20μlのを使用して、ヌクレオ™ソリューションSF（サプリメント溶液を含む）で細胞を懸濁します。再懸濁した後、最適なトランスフェクションのためにすぐに細胞を使用しています。 </li><li>無菌トラフに再懸濁した細胞を移す。 siRNAを含む96ウェルプレートの各ウェルにマルチチャンネルピペット、転送再懸濁細胞混合物の20μlのを使用して。優しく上下にピペッティングして混ぜる。 </li><li>転送96ウェルNucleocuvette™プレートへの細胞/ siRNAの混合物20μlを重要なステップ：これはヌクレオ中にエラーを誘発することができるように転写工程中に気泡を発生しないようにしてください。 </li><li>トランスフェクションプレートの実験試料のすべてを配置した後に、設けられた蓋と版で96ウェルプレートをカバーyが優しくサンプルから任意の気泡を除去するために硬い表面の上をタップします。 </li><li> 96ウェルヌクレオシャトルに蓋をし、96ウェルプレートを置き、「アップロードおよび開始」プログラム画面の右下に選択します。ヌクレオに先立ち、結果を保存するためにプログラムのプロンプトに従ってください。 注：クレオプロセスの間に、首尾よくトランスフェクトウェルは、プラス記号を持つ緑の円で画面上の96ウェル回路図に描かれます。マイナス記号を持つ赤い円は、気泡や誤ペッティング試薬の正しいボリュームをに、最も可能性の高いエラーを示します。 </li></ol> 6.回復とマクロファージのめっき<ol><li>すぐにヌクレオの後、マルチチャンネルピペットを用いて、各ウェルにRMP1-1640メディアを予め温めた（37℃）の80μlを添加する。メディアはゆっくりと、この段階では非常にデリケートであるトランスフェクションされた細胞、と混合することができ、各ウェルの側にメディアを追加します。このうように、速すぎて、メディアを追加しないでください大幅に生存能力を減少させる。 </li><li> 2分間、5％CO 2、37ºCインキュベーター中にトランスフェクト試料およびRPMI-1640を有する96ウェルプレートを置く。これは、細胞が前にさらなる操作まで回復することを可能にすることが重要です。 </li><li>インキュベーターから96ウェルプレートを取り外し、慎重にマルチチャンネルピペットを用いて、96ウェル組織培養プレートに各ウェルからの混合物を移す。 </li><li>マルチチャンネルピペットを用いて、各ウェルに予め温めた（37℃）DMEM + FBS培地100μlを加える。 </li><li> （この時間範囲は一般に良好に機能するが、個々のsiRNAのための正確なタイミングを最適化する）24〜36時間5％CO 2で37ºCインキュベーター中でサンプルを96ウェル組織培養プレートをインキュベートする。 </li></ol> LPSによるマクロファージの刺激7. <ol><li> 24-36時間後クレオインキュベーション後、新鮮な培地中で、LPS（または他のPAMP）を用意。すべてのウェルのための十分な新鮮な培地（200μlのmを準備しますEDIA /ウェルの96ウェルプレートに加え、ピペッティングのために少し余分な）。 DMEM + FBS中に20ng / mlの最終濃度にLPSを希釈する。 </li><li> 96ウェルプレートから慎重に培地を吸引し、細胞にLPSを含む新鮮な培地を追加します。 </li><li>刺激後の様々な時点でLPSまたは他のPAMPへの応答を監視します。 6時間後には、IL-6またはTNFαなどのサイトカインのための強固な炎症性サイトカイン応答を生成するのに十分である。 </li></ol> 8.マクロファージにおいて誘導先天性免疫応答、遺伝子ノックダウン、および生存率のモニタリング<ol><li> LPS誘導性のサイトカイン産生を監視するために、96ウェル貯蔵プレートに細胞からの上清をピペット。サイトカイン産生をELISAによって測定することができる。 </li><li>上清を除去した後、フルオレセインジアセテート（生細胞における細胞エステラーゼによって切断されると、この化合物は蛍光性になる）を使用して、細胞生存率をモニターする。 Tを使用して、細胞の生存率を制御する遺伝子を標的とするsiRNAを特定彼のアプローチ。注意：適切に実行した場合のトランスフェクションプロセス自体が生存率にほとんど影響を持っている必要があります。 <ol><li> （：PBSで100最終希釈1）各ウェルにフルオレセインジアセテート（アセトン中5mg / mlストック）を追加します。 </li><li>一から五分間、室温でインキュベートした後、プレートリーダー（460 nm励起、515 nm発光）を用いて、細胞内の蛍光をモニターする。 </li><li>オプション：RIPAなどのような緩衝液を用いて細胞を溶解、ウェル内の蛍光が均一であることを確実にする。いくつかのプレートリーダーでウェルの小部分のみを監視するように構成されているように、これは、十分に均一な蛍光シグナルを提供する。細胞が均一にメッキされていない場合、フルオレセインジアセテートは、不正確な読み取り値を与えることができる。 </li></ol></li><li>細胞生存率を監視する代わりに、接着性細胞に由来するRNAを用いて定量PCRによりノックダウンsiRNAによる遺伝子をモニタリング。 <ol><li>上清を細胞から除去した後、広告を（上記8.1ステップ ）それらを直接溶解する接着細胞への（RNAの溶解および保存のために使用されている）からd RLT緩衝。 </li><li>このようにRNeasyミニキットとしてRNA調製キットを用いてRNAを単離し、そしてsiRNA処理を制御するために、遺伝子ノックダウンと比較して監視するために、遺伝子特異的プライマーを用いて定量PCRを使用する。正規化のためにβアクチンまたはGAPDHに対するプライマーを使用してください。 </li></ol></li><li>先天性免疫応答の他の側面を監視する別の代替方法として、FITC標識した大腸菌を添加することにより、マクロファージの貪食能をモニターキットを使用して付着細胞上に直接大腸菌粒子。ほんの数時間かかり、製造業者のプロトコール、続いて、プレートリーダーまたは顕微鏡を用いて貪食取り込みを監視します。 </li></ol>

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著者は、彼らが競合する経済的利益を持っていないことを宣言。この記事は、ロンザ社が主催した

多くの研究は、個々の遺伝子は、マウスのマクロファージにおけるsiRNAの標的とされている中で発表されている。脂質媒介トランスフェクションが個別にマクロファージ細胞株に対するsiRNAを送達するために使用されてきたが、これらの方法は、生存能力、限られた遺伝子ノックダウン、および遺伝子からの遺伝子の可変性に対する潜在的な影響に苦しんでいる。中·高スループット様式で遺?...

このプロトコルでは、siRNAを用いてマウスマクロファージ細胞株における遺伝子を抑制し、自然免疫応答におけるこれらの治療の効果をモニターするために効率的な方法を記載する。これらの手順は、中または高スループット様式でRNAiスクリーニングを可能にする、96ウェル形式で行われる。 感染に応答して、人間が直接の先天性免疫応答、より遅いが、より具体的な適応免疫応答をマウントする。この急速な自然免疫応答は、マクロファージ1を含む貪食自然免疫細胞の動員および活性化を伴う。古典的に活性化されたマクロファージは、急性炎症反応に関与し、抗菌タンパク質および化合物、サイトカイン及びケモカイン、および抗原を提示2,3を生成している。あるいは、活性化マクロファージは公差、組織修復を維持し、免疫を調節する役割を果たし、4-8創傷治癒 。理由の機能の彼らの広い配列のマクロファージは、アテローム性動脈硬化症、関節炎、および癌9を含む多数の疾患において役割を果たすことができる。したがって、マクロファージの研究では、病気の幅広い分野でいくつかの時間のための研究の重要な分野となっている。 先天性免疫応答におけるその重要性にもかかわらず、マクロファージはで動作するように細胞に挑戦することができます。特に、関連する毒性10,11なしマクロファージにおける脂質試薬を用いて効率的なトランスフェクションを得ることは困難である。 siRNAは効果的にマクロファージに送達される場合にもまた、RNAiを誘導遺伝子ノックダウンの頑健性は、多くの場合、かなり中程度であることができ、遺伝子に遺伝子を変化させることができる。 これらの技術的課題を克服するために、我々は、2つのマウスマクロファージ細胞株RAW264.7 17とJ774A.1 18でトランスフェクションし、ノックダウン技術12-16を最適化した。このアプローチは、トランスフェクションのためにAmaxa社のNucleofector 96ウェルシャトルシステムを使用しています。このシステム96ウェルフォーマット19で細胞をトランスフェクトするための特殊な試薬やエレクトロポレーションを組み合わせて使用します。トランスフェクションの後、我々は、細胞の回収および生存率を最大化するために、後続のsiRNAによる遺伝子ノックダウンを最大化するための方法を記載している。このアプローチの有用性を説明するために、リポ多糖（LPS）でこれらの細胞を刺激し、いくつかの炎症誘発性サイトカインの産生のレベルにおける先天性免疫応答をモニターし、これらのマクロファージ細胞株へのsiRNA送達のためのプロトコルを記述している。私たちはメンバー先天性免疫を調節するために、LPS及び他の病原体関連分子パターン（PAMP）を感知し、Toll様受容体（TLR）ファミリーをターゲットとしたサンプルデータを提供する。

<table border="0" cellpadding="0" cellspacing="0" >
	<tbody>
		<tr >
			<td >Amaxa nucleofector 96-well shuttle system</td>
			<td>Lonza</td>
			<td>AAM-1001S</td>
		<td></td></tr>
		<tr >
			<td >Amaxa SF cell line 96-well nucleofector kit</td>
			<td>Lonza</td>
			<td>V4SC-2096</td>
		<td></td></tr>
		<tr >
			<td >RAW264.7 mouse macrophage cell line</td>
			<td>ATCC</td>
			<td>TIB-71</td>
		<td></td></tr>
		<tr >
			<td >J774A.1 mouse macrophage cell line</td>
			<td>ATCC</td>
			<td>TIB-67</td>
		<td></td></tr>
		<tr >
			<td >siGenome smartpool siRNA</td>
			<td>Dharmacon</td>
			<td>varies depending on gene</td>
		<td></td></tr>
		<tr >
			<td >Non-targeting control siRNA pool</td>
			<td>Dharmacon</td>
			<td>D-001206-13-20</td>
		<td></td></tr>
		<tr >
			<td >Block-iT fluorescent oligo for electrooration</td>
			<td>Invitrogen</td>
			<td>13750062</td>
		<td></td></tr>
		<tr >
			<td >Ultrapure E. coli O111:B4 LPS</td>
			<td>List Biological Laboratories</td>
			<td>421</td>
		<td></td></tr>
		<tr >
			<td >DMEM, high glucose</td>
			<td>Invitrogen</td>
			<td>11995-065</td>
		<td></td></tr>
		<tr >
			<td >RPM1-1640</td>
			<td>Invitrogen</td>
			<td>11875-093</td>
		<td></td></tr>
		<tr >
			<td >Penicillin-Streptomycin Solution (Pen/Strep)</td>
			<td>Fisher</td>
			<td>SV30010</td>
		<td></td></tr>
		<tr >
			<td >0.25% Trypsin-EDTA</td>
			<td>Invitrogen</td>
			<td>25200072</td>
		<td></td></tr>
		<tr >
			<td >96 well tissue culture plates</td>
			<td>Fisher</td>
			<td>07-200-89 </td>
		<td></td></tr>
		<tr >
			<td >96 well round bottom sterile plates (not coated)</td>
			<td>Fisher</td>
			<td>07-200-745</td>
		<td></td></tr>
		<tr >
			<td >Mouse IL-6 Duoset ELISA kit</td>
			<td>R&#38;D Biosystems</td>
			<td>DY406</td>
		<td></td></tr>
		<tr >
			<td >Mouse TNFa Duoset ELISA kit</td>
			<td>R&#38;D Biosystems</td>
			<td>DY410</td>
		<td></td></tr>
		<tr >
			<td >Fluorescein diacetate</td>
			<td>Sigma-Aldrich</td>
			<td>F7378</td>
		<td></td></tr>
		<tr >
			<td >RLT Bufer</td>
			<td>Qiagen</td>
			<td>79216</td>
		<td></td></tr>
		<tr >
			<td >Rneasy mini kit</td>
			<td>Qiagen</td>
			<td>74134</td>
		<td></td></tr>
		<tr >
			<td >Vybrant Phagocytosis Assay Kit</td>
			<td>Invitrogen</td>
			<td>V-6694</td>
		<td></td></tr>
	</tbody>
</table>

using rna-interference to investigate the innate immune response in mouse macrophages

Macrophages are key phagocytic innate immune cells. When macrophages encounter a pathogen, they produce antimicrobial proteins and compounds to kill the pathogen, produce various cytokines and chemokines to recruit and stimulate other immune cells, and present antigens to stimulate the adaptive immune response. Thus, being able to efficiently manipulate macrophages with techniques such as RNA-interference (RNAi) is critical to our ability to investigate this important innate immune cell. However, macrophages can be technically challenging to transfect and can exhibit inefficient RNAi-induced gene knockdown. In this protocol, we describe methods to efficiently transfect two mouse macrophage cell lines (RAW264.7 and J774A.1) with siRNA using the Amaxa Nucleofector 96-well Shuttle System and describe procedures to maximize the effect of siRNA on gene knockdown. Moreover, the described methods are adapted to work in 96-well format, allowing for medium and high-throughput studies. To demonstrate the utility of this approach, we describe experiments that utilize RNAi to inhibit genes that regulate lipopolysaccharide (LPS)-induced cytokine production.

このアプローチを使用するトランスフェクションの効率を実証するために、我々は、フローサイトメーター（ 図1）を用いてFITC標識siRNAの取り込みを監視した。 先天性免疫応答をモニターするための我々のアプローチの有用性を説明するために、我々は、RAW264.7マクロファージ細胞株に知ら自然免疫調節遺伝子を標的とするsiRNAをトランスフェクトされたLPSで...

Watch this Scientific Journal Video about Using RNA-interference to Investigate the Innate Immune Response in Mouse Macrophages at JoVE.com

Using RNA-interference to Investigate the Innate Immune Response in Mouse Macrophages

In this protocol, we describe methods to efficiently transfect murine macrophage cell lines with siRNAs using the Amaxa Nucleofector 96-well Shuttle System, stimulate the macrophages with lipopolysaccharide, and monitor the effects on inflammatory cytokine production.

マウスマクロファージにおける先天性免疫応答を調査するために、RNA干渉を使用

Macrophages are key phagocytic innate immune cells.  When macrophages encounter a pathogen, they produce antimicrobial proteins and compounds to kill the ...

using-rna-interference-to-investigate-innate-immune-response-mouse

Research

JoVE Journal

Immunology and Infection

10.6K ビュー.  National Jewish Health and University of Colorado School of Medicine. In this protocol, we describe methods to efficiently transfect murine macrophage cell lines with siRNAs using the Amaxa Nucleofector 96-well Shuttle System, stimulate the macrophages with lipopolysaccharide, and monitor the effects on inflammatory cytokine production.

ビデオ: Using RNA-interference to Investigate the Innate Immune Response in Mouse Macrophages

RNA Interference in Ticks

Ticks are obligate hematophagous ectoparasites of wild and domestic animals and humans, and are considered to be second worldwide to mosquitoes as vectors of human diseases1 and the most important vectors affecting cattle industry worldwide2. Ticks are classified in the subclass Acari, order Parasitiformes, suborder Ixodida and are distributed worldwide from Arctic to tropical regions3. Despite efforts to control tick infestations, these ectoparasites remain a serious problem for human and animal health4,5.
RNA interference (RNAi)6 is a nucleic acid-based reverse genetic approach that involves disruption of gene expression in order to determine gene function or its effect on a metabolic pathway. Small interfering RNAs (siRNAs) are the effector molecules of the RNAi pathway that is initiated by double-stranded RNA (dsRNA) and results in a potent sequence-specific degradation of cytoplasmic mRNAs containing the same sequence as the dsRNA trigger7-9. Post-transcriptional gene silencing mechanisms initiated by dsRNA have been discovered in all eukaryotes studied thus far, and RNAi has been rapidly developed in a variety of organisms as a tool for functional genomics studies and other applications10.
RNAi has become the most widely used gene-silencing technique in ticks and other organisms where alternative approaches for genetic manipulation are not available or are unreliable5,11. The genetic characterization of ticks has been limited until the recent application of RNAi12,13. In the short time that RNAi has been available, it has proved to be a valuable tool for studying tick gene function, the characterization of the tick-pathogen interface and the screening and characterization of tick protective antigens14. Herein, a method for RNAi through injection of dsRNA into unfed ticks is described. It is likely that the knowledge gained from this experimental approach will contribute markedly to the understanding of basic biological systems and the development of vaccines to control tick infestations and prevent transmission of tick-borne pathogens15-19.

A method for RNA interference (RNAi) by injection of dsRNA into unfed ticks is described. RNAi is the most widely used gene-silencing technique in ticks where the use of other methods of genetic manipulation has been limited.

ダニのRNA干渉

Ticks are obligate hematophagous ectoparasites of wild and domestic animals and humans, and are considered to be second worldwide to mosquitoes as vectors ...

免疫・感染学

Seven Steps to Stellate Cells

Hepatic stellate cells are liver-resident cells of star-like morphology and are located in the space of Disse between liver sinusoidal endothelial cells and hepatocytes1,2. Stellate cells are derived from bone marrow precursors and store up to 80% of the total body vitamin A1, 2. Upon activation, stellate cells differentiate into myofibroblasts to produce extracellular matrix, thus contributing to liver fibrosis3. Based on their ability to contract, myofibroblastic stellate cells can regulate the vascular tone associated with portal hypertension4. Recently, we demonstrated that hepatic stellate cells are potent antigen presenting cells and can activate NKT cells as well as conventional T lymphocytes5. 

 Here we present a method for the efficient preparation of hepatic stellate cells from mouse liver. Due to their perisinusoidal localization, the isolation of hepatic stellate cells is a multi-step process. In order to render stellate cells accessible to isolation from the space of Disse, mouse livers are perfused in situ with the digestive enzymes Pronase E and Collagenase P. Following perfusion, the liver tissue is subjected to additional enzymatic treatment with Pronase E and Collagenase P in vitro. Subsequently, the method takes advantage of the massive amount of vitamin A-storing lipid droplets in hepatic stellate cells. This feature allows the separation of stellate cells from other hepatic cell types by centrifugation on an 8% Nycodenz gradient. The protocol described here yields a highly pure and homogenous population of stellate cells. Purity of preparations can be assessed by staining for the marker molecule glial fibrillary acidic protein (GFAP), prior to analysis by fluorescence microscopy or flow cytometry. Further, light microscopy reveals the unique appearance of star-shaped hepatic stellate cells that harbor high amounts of lipid droplets. 


 Taken together, we present a detailed protocol for the efficient isolation of hepatic stellate cells, including representative images of their morphological appearance and GFAP expression that help to define the stellate cell entity.

Here we describe a method for the isolation of hepatic stellate cells from mouse liver. For stellate cell purification, mouse livers are digested in situ and in vitro by pronase-collagenase treatment prior to density gradient centrifugation. This technique yields highly pure hepatic stellate cells.

細胞星状に7つのステップ

Hepatic stellate cells are liver-resident  cells of star-like morphology and are located in the space of Disse between  liver sinusoidal endothelial cells ...

An Introduction to Parasitic Wasps of Drosophila and the Antiparasite Immune Response

Most known parasitoid wasp species attack the larval or pupal stages of Drosophila. While Trichopria drosophilae infect the pupal stages of the host (Fig. 1A-C), females of the genus Leptopilina (Fig. 1D, 1F, 1G) and Ganaspis (Fig. 1E) attack the larval stages. We use these parasites to study the molecular basis of a biological arms race. Parasitic wasps have tremendous value as biocontrol agents. Most of them carry virulence and other factors that modify host physiology and immunity. Analysis of Drosophila wasps is providing insights into how species-specific interactions shape the genetic structures of natural communities. These studies also serve as a model for understanding the hosts' immune physiology and how coordinated immune reactions are thwarted by this class of parasites.
The larval/pupal cuticle serves as the first line of defense. The wasp ovipositor is a sharp needle-like structure that efficiently delivers eggs into the host hemocoel. Oviposition is followed by a wound healing reaction at the cuticle (Fig. 1C, arrowheads). Some wasps can insert two or more eggs into the same host, although the development of only one egg succeeds. Supernumerary eggs or developing larvae are eliminated by a process that is not yet understood. These wasps are therefore referred to as solitary parasitoids.
Depending on the fly strain and the wasp species, the wasp egg has one of two fates. It is either encapsulated, so that its development is blocked (host emerges; Fig. 2 left); or the wasp egg hatches, develops, molts, and grows into an adult (wasp emerges; Fig. 2 right). L. heterotoma is one of the best-studied species of Drosophila parasitic wasps. It is a "generalist," which means that it can utilize most Drosophila species as hosts1. L. heterotoma and L. victoriae are sister species and they produce virus-like particles that actively interfere with the encapsulation response2. Unlike L. heterotoma, L. boulardi is a specialist parasite and the range of Drosophila species it utilizes is relatively limited1. Strains of L. boulardi also produce virus-like particles3 although they differ significantly in their ability to succeed on D. melanogaster1. Some of these L. boulardi strains are difficult to grow on D. melanogaster1 as the fly host frequently succeeds in encapsulating their eggs. Thus, it is important to have the knowledge of both partners in specific experimental protocols.
In addition to barrier tissues (cuticle, gut and trachea), Drosophila larvae have systemic cellular and humoral immune responses that arise from functions of blood cells and the fat body, respectively. Oviposition by L. boulardi activates both immune arms1,4. Blood cells are found in circulation, in sessile populations under the segmented cuticle, and in the lymph gland. The lymph gland is a small hematopoietic organ on the dorsal side of the larva. Clusters of hematopoietic cells, called lobes, are arranged segmentally in pairs along the dorsal vessel that runs along the anterior-posterior axis of the animal (Fig. 3A). The fat body is a large multifunctional organ (Fig. 3B). It secretes antimicrobial peptides in response to microbial and metazoan infections.
Wasp infection activates immune signaling (Fig. 4)4. At the cellular level, it triggers division and differentiation of blood cells. In self defense, aggregates and capsules develop in the hemocoel of infected animals (Fig. 5)5,6. Activated blood cells migrate toward the wasp egg (or wasp larva) and begin to form a capsule around it (Fig. 5A-F). Some blood cells aggregate to form nodules (Fig. 5G-H). Careful analysis reveals that wasp infection induces the anterior-most lymph gland lobes to disperse at their peripheries (Fig. 6C, D).
We present representative data with Toll signal transduction pathway components Dorsal and Sp&auml;tzle (Figs. 4,5,7), and its target Drosomycin (Fig. 6), to illustrate how specific changes in the lymph gland and hemocoel can be studied after wasp infection. The dissection protocols described here also yield the wasp eggs (or developing stages of wasps) from the host hemolymph (Fig. 8).

An Introduction to Parasitic Wasps of Drosophila and the Antiparasite Immune Response

Parasitoid (parasitic) wasps constitute a major class of natural enemies of many insects including Drosophila melanogaster. We will introduce the techniques to propagate these parasites in Drosophila spp. and demonstrate how to analyze their effects on immune tissues of Drosophila larvae.

の寄生蜂の紹介<em&gt;ショウジョウバエ</em&gt;と抗寄生虫免疫応答

Most known parasitoid wasp species attack the larval or pupal stages of Drosophila. While Trichopria drosophilae infect the pupal stages of the host (Fig. ...

Intravital Imaging of the Mouse Thymus using 2-Photon Microscopy

Two-photon Microscopy (TPM) provides image acquisition in deep areas inside tissues and organs. In combination with the development of new stereotactic tools and surgical procedures, TPM becomes a powerful technique to identify "niches" inside organs and to document cellular "behaviors" in live animals. While intravital imaging provides information that best resembles the real cellular behavior inside the organ, it is both more laborious and technically demanding in terms of required equipment/procedures than alternative ex vivo imaging acquisition. Thus, we describe a surgical procedure and novel "stereotactic" organ holder that allows us to follow the movements of Foxp3+ cells within the thymus.
Foxp3 is the master regulator for the generation of regulatory T cells (Tregs). Moreover, these cells can be classified according to their origin: ie. thymus-differentiated Tregs are called "naturally-occurring Tregs" (nTregs), as opposed to peripherally-converted Tregs (pTregs). Although significant amount of research has been reported in the literature concerning the phenotype and physiology of these T cells, very little is known about their in vivo interactions with other cells. This deficiency may be due to the absence of techniques that would permit such observations. The protocol described in this paper provides a remedy for this situation.
Our protocol consists of using nude mice that lack an endogenous thymus since they have a punctual mutation in the DNA sequence that compromises the differentiation of some epithelial cells, including thymic epithelial cells. Nude mice were gamma-irradiated and reconstituted with bone marrows (BM) from Foxp3-KIgfp/gfp mice. After BM recovery (6 weeks), each animal received embryonic thymus transplantation inside the kidney capsule. After thymus acceptance (6 weeks), the animals were anesthetized; the kidney containing the transplanted thymus was exposed, fixed in our organ holder, and kept under physiological conditions for in vivo imaging by TPM. We have been using this approach to study the influence of drugs in the generation of regulatory T cells.

We have developed novel laboratory tools and protocols for intravital imaging acquisition of the thymus. Our technique should help in the identification of &#x201C;niches&#x201D; within the thymus where T cell development occurs.

2光子顕微鏡を用いてマウスの胸腺の生体内イメージング

Two-photon Microscopy (TPM) provides image acquisition in deep areas inside tissues and organs. In combination with the development of new stereotactic ...

Quantitative Measurement of the Immune Response and Sleep in Drosophila

A complex interaction between the immune response and host behavior has been described in a wide range of species. Excess sleep, in particular, is known to occur as a response to infection in mammals 1 and has also recently been described in Drosophila melanogaster2. It is generally accepted that sleep is beneficial to the host during an infection and that it is important for the maintenance of a robust immune system3,4. However, experimental evidence that supports this hypothesis is limited4, and the function of excess sleep during an immune response remains unclear. We have used a multidisciplinary approach to address this complex problem, and have conducted studies in the simple genetic model system, the fruitfly Drosophila melanogaster. We use a standard assay for measuring locomotor behavior and sleep in flies, and demonstrate how this assay is used to measure behavior in flies infected with a pathogenic strain of bacteria. This assay is also useful for monitoring the duration of survival in individual flies during an infection. Additional measures of immune function include the ability of flies to clear an infection and the activation of NF&kappa;B, a key transcription factor that is central to the innate immune response in Drosophila. Both survival outcome and bacterial clearance during infection together are indicators of resistance and tolerance to infection. Resistance refers to the ability of flies to clear an infection, while tolerance is defined as the ability of the host to limit damage from an infection and thereby survive despite high levels of pathogen within the system5. Real-time monitoring of NF&kappa;B activity during infection provides insight into a molecular mechanism of survival during infection. The use of Drosophila in these straightforward assays facilitates the genetic and molecular analyses of sleep and the immune response and how these two complex systems are reciprocally influenced.

Quantitative Measurement of the Immune Response and Sleep in Drosophila

To understand a link between the immune response and behavior, we describe a method to measure locomotor behavior in Drosophila during bacterial infection as well as the ability of flies to mount an immune response by monitoring survival, bacterial load, and real-time activity of a key regulator of innate immunity, NF&kappa;B.

免疫応答やスリープ中の定量的測定<em&gt;ショウジョウバエ</em

A complex interaction between the immune response and  host behavior has been described in a wide range of species. Excess sleep, in  particular, is known ...

Quantification of the Respiratory Burst Response as an Indicator of Innate Immune Health in Zebrafish

The phagocyte respiratory burst is part of the innate immune response to pathogen infection and involves the production of reactive oxygen species (ROS). ROS are toxic and function to kill phagocytized microorganisms. In vivo quantification of phagocyte-derived ROS provides information regarding an organism's ability to mount a robust innate immune response. Here we describe a protocol to quantify and compare ROS in whole zebrafish embryos upon chemical induction of the phagocyte respiratory burst. This method makes use of a non-fluorescent compound that becomes fluorescent upon oxidation by ROS. Individual zebrafish embryos are pipetted into the wells of a microplate and incubated in this fluorogenic substrate with or without a chemical inducer of the respiratory burst. Fluorescence in each well is quantified at desired time points using a microplate reader. Fluorescence readings are adjusted to eliminate background fluorescence and then compared using an unpaired t-test. This method allows for comparison of the respiratory burst potential of zebrafish embryos at different developmental stages and in response to experimental manipulations such as protein knockdown, overexpression, or treatment with pharmacological agents. This method can also be used to monitor the respiratory burst response in whole dissected kidneys or cell preparations from kidneys of adult zebrafish and some other fish species. We believe that the relative simplicity and adaptability of this protocol will complement existing protocols and will be of interest to researchers who seek to better understand the innate immune response.

The innate immune response protects organisms against pathogen infection. A critical component of the innate immune response, the phagocyte respiratory burst, generates reactive oxygen species that kill invading microorganisms. We describe a respiratory burst assay that quantifies reactive oxygen species produced when the innate immune response is chemically induced.

ゼブラフィッシュにおける自然免疫の健康の指標として呼吸バースト応答の定量化

The phagocyte respiratory burst is part of the innate immune response to  pathogen infection and involves the production of reactive oxygen species (ROS). ...

Dissecting Innate Immune Signaling in Viral Evasion of Cytokine Production

In response to a viral infection, the host innate immune response is activated to up-regulate gene expression and production of antiviral cytokines. Conversely, viruses have evolved intricate strategies to evade and exploit host immune signaling for survival and propagation. Viral immune evasion, entailing host defense and viral evasion, provides one of the most fascinating and dynamic interfaces to discern the host-virus interaction. These studies advance our understanding in innate immune regulation and pave our way to develop novel antiviral therapies.
Murine &#947;HV68 is a natural pathogen of murine rodents. &#947;HV68 infection of mice provides a tractable small animal model to examine the antiviral response to human KSHV and EBV of which perturbation of in vivo virus-host interactions is not applicable. Here we describe a protocol to determine the antiviral cytokine production. This protocol can be adapted to other viruses and signaling pathways.
Recently, we have discovered that &#947;HV68 hijacks MAVS and IKK&#946;, key innate immune signaling components downstream of the cytosolic RIG-I and MDA5, to abrogate NF&#922;B activation and antiviral cytokine production. Specifically, &#947;HV68 infection activates IKK&#946; and that activated IKK&#946; phosphorylates RelA to accelerate RelA degradation. As such, &#947;HV68 efficiently uncouples NF&#922;B activation from its upstream activated IKK&#946;, negating antiviral cytokine gene expression. This study elucidates an intricate strategy whereby the upstream innate immune activation is intercepted by a viral pathogen to nullify the immediate downstream transcriptional activation and evade antiviral cytokine production.

We describe a protocol to measure the antiviral cytokine production in mice infected with a model herpesvirus, murine gamma herpesvirus 68 (&#947;HV68) that is closely-related to human Kaposi&#8217;s sarcoma-associated herpesvirus (KSHV) and Epstein-Barr virus (EBV). Utilizing genetically modified mouse strains and mouse embryonic fibroblasts (MEFs), we assessed the antiviral cytokine production both in vivo and ex vivo. &#8220;Reconstituting&#8221; the expression of innate immune components in knockout embryonic fibroblasts by lentiviral transduction, we further pinpoint specific innate immune molecules and dissect the key signaling events that differentially regulate the antiviral cytokine production.

サイトカイン産生のウイルス回避における先天性免疫シグナルを解剖

In response to a viral infection, the host innate immune response is activated to up-regulate gene expression and production of antiviral cytokines. ...

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in mice. Recently, myeloid differentiation factor 88 (MyD88) signaling was shown to be important for initial T cell priming and memory T cell development during WNV NS4B-P38G mutant infection. In this study, two flow cytometry-based methods &#8211; an in vitro T cell priming assay and an intracellular cytokine staining (ICS) &#8211; were utilized to assess dendritic cells (DCs) and T cell functions. In the T cell priming assay, cell proliferation was analyzed by flow cytometry following co-culture of DCs from both groups of mice with carboxyfluorescein succinimidyl ester (CFSE) - labeled CD4+ T cells of OTII transgenic mice. This approach provided an accurate determination of the percentage of proliferating CD4+ T cells with significantly improved overall sensitivity than the traditional assays with radioactive reagents. A microcentrifuge tube system was used in both cell culture and cytokine staining procedures of the ICS protocol. Compared to the traditional tissue culture plate-based system, this modified procedure was easier to perform at biosafety level (BL) 3 facilities. Moreover, WNV- infected cells were treated with paraformaldehyde in both assays, which enabled further analysis outside BL3 facilities. Overall, these in vitro immunological assays can be used to efficiently assess cell-mediated immune responses during WNV infection.

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

Two flow cytometry-based methods &#8211; an in vitro T cell priming assay and intracellular cytokine staining were utilized to measure antigen presenting capacity of dendritic cells and antigen-specific T cell responses to a West Nile virus mutant infection in mice.

西ナイルウイルス変異株の感染に対するMyD88非介在細胞性免疫応答のインビトロ分析

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in ...

Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection

The aquatic pathogen, Streptococcus iniae, is responsible for over 100 million dollars in annual losses for the aquaculture industry and is capable of causing systemic disease in both fish and humans. A better understanding of S. iniae disease pathogenesis requires an appropriate model system. The genetic tractability and the optical transparency of the early developmental stages of zebrafish allow for the generation and non-invasive imaging of transgenic lines with fluorescently tagged immune cells. The adaptive immune system is not fully functional until several weeks post fertilization, but zebrafish larvae have a conserved vertebrate innate immune system with both neutrophils and macrophages. Thus, the generation of a larval infection model allows the study of the specific contribution of innate immunity in controlling S. iniae infection.
The site of microinjection will determine whether an infection is systemic or initially localized. Here, we present our protocols for otic vesicle injection of zebrafish aged 2-3 days post fertilization as well as our techniques for fluorescent confocal imaging of infection. A localized infection site allows observation of initial microbe invasion, recruitment of host cells and dissemination of infection. Our findings using the zebrafish larval model of S. iniae infection indicate that zebrafish can be used to examine the differing contributions of host neutrophils and macrophages in localized bacterial infections. In addition, we describe how photolabeling of immune cells can be used to track individual host cell fate during the course of infection.

Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection

Here, we present a protocol for the generation and imaging of a localized bacterial infection in the zebrafish otic vesicle.

のゼブラフィッシュ幼生モデルにおける先天性免疫応答の非侵襲的イメージング<em&gt;ストレプトコッカスイニエ</em&gt;感染

The aquatic pathogen, Streptococcus iniae, is responsible for over 100 million dollars in annual losses for the aquaculture industry and is capable of ...

Using Phylogenetic Analysis to Investigate Eukaryotic Gene Origin

Phylogenetic analysis uses nucleotide or amino acid sequences or other parameters, such as domain sequences and three-dimensional structure, to construct a tree to show the evolutionary relationship among different taxa (classification units) at the molecular level. Phylogenetic analysis can also be used to investigate domain relationships within an individual taxon, particularly for organisms that have undergone substantial change in morphology and physiology, but for which researchers lack fossil evidence due to the organisms&#39; long evolutionary history or scarcity of fossilization.
In this text, a detailed protocol is described for using the phylogenetic method, including amino acid sequence alignment using Clustal Omega, and subsequent phylogenetic tree construction using both Maximum Likelihood (ML) of Molecular Evolutionary Genetics Analysis (MEGA) and Bayesian Inference via MrBayes. To investigate the origin of eukaryotic Sugars Will Eventually be Exported Transporters (SWEET) genes, 228 SWEETs including 35 SWEET proteins from unicellular eukaryotes and 57 SemiSWEET proteins from prokaryotes were analyzed. Interestingly, SemiSWEETs were found in prokaryotes, but SWEETs were found in eukaryotes. Two phylogenetic trees constructed using theoretically distinct methods have consistently&#160;suggested that the first eukaryotic SWEET gene might stem from the fusion of a bacterial SemiSWEET gene and an archaeal SemiSWEET gene.&#160;It is worth noting that one should be cautious to draw a conclusion based only on phylogenetic analysis, although it is useful to explain the underlying relationship between different taxa, which is difficult or even impossible to discern through experimental means.

A method of constructing a phylogenetic tree based on sequence homology of SWEETs from eukaryotes and SemiSWEETs from prokaryotes is described. Phylogenetic analysis is a useful tool for explaining the evolutionary relatedness between homologous proteins or genes from different organism groups.

系統解析を使用して真核生物遺伝子の起源を調査するには

Phylogenetic analysis uses nucleotide or amino acid sequences or other parameters, such as domain sequences and three-dimensional structure, to construct ...