野生型（ マーベリック+ / +）およびノックアウト（ マーベリック- / - ）マウスは、野生型（ マーベリック+ / +）およびノックアウト（ マーベリック- / - ）のMEFは、親切に博士Zhijian J·チェン（大学から提供されたテキサス南西医療センター）13。この出版物は、NIH（R01 CA134241とR01 DE021445）と米国がん協会（RSG-11から162-01-MPC）からの補助金によって資金を供給作業に基づいています。

倫理声明：すべての動物の作品は、国立衛生研究所の実験動物の管理と使用に関するガイドの推奨事項に厳密に従って下で行った。プロトコルは、南カリフォルニア大学の施設内動物管理使用委員会（IACUC）により承認された。 1。 γHV68感染マウスにおけるELISAによる定量的リアルタイムPCRおよび分泌によりサイトカイン遺伝子発現<ol><li>腹腔内キシラジンのketamine/0.12 Mgを1.5 mgの注射を経由して性別が一致する、6-8週齢のC57BL / 6（B6）同腹仔（8月12日マウス/群）を麻酔し、40μlのγHV68の40 PFUを鼻腔内接種（ドンと風水11で詳述感染症）。異なる時点で、感染後、頸椎脱臼、続いてCO 2吸入を使用して、マウスを安楽死させる。 </li><li> STER沿って左横のカットで胸を開きnumと鋭いハサミで肋骨を切断。肺組織を収集します。 </li><li> 500μlの1.0ミリメートルジルコニア/シリカビーズを含む滅菌1.5ミリリットルスクリューキャップチューブに肺組織（左葉）の半分を収集します。チューブに冷たい無血清DMEMの1ミリリットルを加え、30秒間ビーズ打つことによって肺組織を均質化する。 2分間氷上にチューブを冷却し、一度このプロセスを繰り返します。 </li><li>遠心管（15000 X 4°Cで10分間グラム）、上清を収集し、下記のようにサイトカイン産生を測定する。 </li><li>製造業者の指示に従って市販のサイトカインELISAキットを使用してサイトカインを測定します。 </li><li>別のビーズを含むチューブに肺組織（右葉）の残りの半分を収集し、1ミリリットルのTRIzolを追加。均質化し、製造者の指示に従って、ステップ1.3、抽出RNAで説明したように、上清を集める。 260および280 nmの吸光度の測定値を取ることによって、RNA濃度を決定します。 </li&gt; <li> 1μgの全RNAをcDNAを調製し、（総最終容量は20μlであった）製造者の指示に従って逆転写酵素。 DNアーゼとRNaseフリー水を用いてcDNAを50回希釈し、定量リアルタイムPCRを行う。 </li><li>定量リアルタイムPCR機器上のプログラムを実行します。各反応は、GAPDHの遺伝子特異的プライマーまたはコントロールプライマーの対の0.5μL（プライマーの作業濃度は、各プライマーのためのCE 500 nMの10μMであった）、SYBRマスターミックスおよび希薄化後の4μLの5μLが含まれていますcDNAを。抗ウイルスサイトカインのmRNA転写物の相対量を決定するためにΔΔCTを計算します。 </li></ol> 2。 ELISAおよび定量RT-PCRによるMEF細胞感染およびサイトカイン定量<ol><li> マーベリック+ / +およびマーベリックを育てる- / -細胞を播種する前に、サブコンフルエント（約80％）密度にMEF細胞。 </li><li> 12-WEにMEF細胞を分割うまく100,000細胞/ AT LLプレート（細胞密度は二日目に約70％になります）。サイトカイン誘導を同期して最大化する5月10日の感染多重度（MOI）で、三重でγHV68感染を実施しています。 </li><li>ウイルスの適切な量を含有する完全DMEM培地（各ウェル1ml）にγHV68懸濁液を調製する。 </li><li>培地を除去し、MEF細胞にγHV68含有懸濁液を追加します。 </li><li>組織培養インキュベーター中でプレートを置き、30分毎岩、インキュベーションの2時間の合計を可能にする。 </li><li> 、γHV68含有培地を外し、PBSで1回洗浄し、新鮮な完全DMEMと交換してください。 </li><li>種々の時点で1.5mlのエッペンドルフチュー​​ブに感染後、収穫媒体を指す。冷PBSで細胞を洗浄し、トリプシン消化に感染した細胞を収集します。 </li><li>ステップ1.5で説明したように培地中の抗ウイルスのサイトカインを測定します。 </li><li> RNAを抽出し、cDNAを調製し、抗ウイルス性サイトカイン遺伝子発現を決定するために、定量RT-PCRを行うことステップ1.5から1.7で説明したように。 </li></ol> 3。レンチウイルスの生産、感染症、安定した細胞株の生成<ol><li>トランスフェクションの一日前に、スプリット293T細胞、細胞は、トランスフェクションの時点で約40％コンフルエンシーに到達させる。 </li><li>パッケージングプラスミド（1.2のpVSV-Gのμgのと6μgのpDR8.9の）および7.2 pCDH-FLAG-MAVSまたはリン酸カルシウム沈殿によるpCDHコントロールのμgの293T細胞の10cmディッシュをトランスフェクト。 </li><li>新鮮な完全DMEMで6から8時間後、トランスフェクションで培地を交換してください。 </li><li> 72時間トランスフェクション後に、15分間4000×gでレンチウイルス、遠心分離を含む培地を収集し、0.22μmの膜でろ過する。レンチウイルス力価を増加させるために、我々は、4℃で2.5時間、110,000×gでウイルス含有培地を遠心分離慎重に興味のある少量の培地中のウイルスペレットを再懸濁します。 </li><li>シードマーベリック- / - MEF細胞や他のノックアウトMEF細胞1日befo〜30-40％の集密度で6 - ウェルプレートでの感染の再。 </li><li>を1mlレンチウイルス及び2mlのMEF細胞に感染を10μg/ mlの最終濃度までポリブレンを補充した新鮮な完全DMEM、。 </li><li> 30分間30℃で500×gで遠心分離し、プレートを組織培養インキュベーターにプレートを転送する。 </li><li>新鮮な完全DMEMで6時間感染後で培地を交換してください。 </li><li> 24時間の感染後に分割MEF細胞を、安定してMAVSを発現する細胞を選択するために48時間感染後に1μgの/ mlの最終濃度までピューロマイシンを追加する。 </li><li>ピューロ含有培地中でMEF細胞を維持し、対応する抗体を用いた免疫ブロッティングによってタンパク質の発現を確認します。 </li><li>プロトコール2に記載のように、細胞は、ウイルス感染、サイトカイン遺伝子発現および分泌実験のために使用することができる。 </li></ol>

<ol>
	<li>Akira, S., Uematsu, S., Takeuchi, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathogen+recognition+and+innate+immunity.">Pathogen recognition and innate immunity.</a> Cell. 124, 783-801 (2006).</li><li>Kato, H., Takahasi, K., Fujita, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RIG-I-like+receptors:+cytoplasmic+sensors+for+non-self+RNA.">RIG-I-like receptors: cytoplasmic sensors for non-self RNA.</a> Immunol. Rev. 243, 91-98 (2011).</li><li>Kawai, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IPS-1,+an+adaptor+triggering+RIG-I-+and+Mda5-mediated+type+I+interferon+induction.">IPS-1, an adaptor triggering RIG-I- and Mda5-mediated type I interferon induction.</a> Nat. Immunol. 6, 981-988 (2005).</li><li>Seth, R. B., Sun, L., Ea, C. K., Chen, Z. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+characterization+of+MAVS,+a+mitochondrial+antiviral+signaling+protein+that+activates+NF-kappaB+and+IRF+3.">Identification and characterization of MAVS, a mitochondrial antiviral signaling protein that activates NF-kappaB and IRF 3.</a> Cell. 122, 669-682 (2005).</li><li>Xu, L. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=VISA+is+an+adapter+protein+required+for+virus-triggered+IFN-beta+signaling.">VISA is an adapter protein required for virus-triggered IFN-beta signaling.</a> Mol. Cell. 19, 727-740 (2005).</li><li>Meylan, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardif+is+an+adaptor+protein+in+the+RIG-I+antiviral+pathway+and+is+targeted+by+hepatitis+C+virus.">Cardif is an adaptor protein in the RIG-I antiviral pathway and is targeted by hepatitis C virus.</a> Nature. 437, 1167-1172 (2005).</li><li>Speck, S. H., Virgin, H. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Host+and+viral+genetics+of+chronic+infection:+a+mouse+model+of+gamma-herpesvirus+pathogenesis.">Host and viral genetics of chronic infection: a mouse model of gamma-herpesvirus pathogenesis.</a> Curr. Opin. Microbiol. 2, 403-409 (1999).</li><li>Dong, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Murine+gamma-herpesvirus+68+hijacks+MAVS+and+IKKbeta+to+initiate+lytic+replication.">Murine gamma-herpesvirus 68 hijacks MAVS and IKKbeta to initiate lytic replication.</a> PLoS Pathog. , (2010).</li><li>Dong, X., Feng, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Murine+gamma+herpesvirus+68+hijacks+MAVS+and+IKKbeta+to+abrogate+NFkappaB+activation+and+antiviral+cytokine+production.">Murine gamma herpesvirus 68 hijacks MAVS and IKKbeta to abrogate NFkappaB activation and antiviral cytokine production.</a> PLoS Pathog. 7, (2011).</li><li>Dong, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Murine+gammaherpesvirus+68+evades+host+cytokine+production+via+replication+transactivator-induced+RelA+degradation.">Murine gammaherpesvirus 68 evades host cytokine production via replication transactivator-induced RelA degradation.</a> J. Virol. 86, 1930-1941 (2012).</li><li>Dong, X., Feng, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissecting+Host-virus+Interaction+in+Lytic+Replication+of+a+Model+Herpesvirus.">Dissecting Host-virus Interaction in Lytic Replication of a Model Herpesvirus.</a> J. Vis. Exp. , (2011).</li><li>He, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Receptor+interacting+protein+kinase-3+determines+cellular+necrotic+response+to+TNF-alpha.">Receptor interacting protein kinase-3 determines cellular necrotic response to TNF-alpha.</a> Cell. 137, 1100-1111 (2009).</li><li>Sun, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+specific+and+essential+role+of+MAVS+in+antiviral+innate+immune+responses.">The specific and essential role of MAVS in antiviral innate immune responses.</a> Immunity. 24, 633-642 (2006).</li></ol>

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ウイルス免疫回避は、ウイルス攻撃と宿主防御9をインターフェースする最もダイナミックで魅力的な相互作用の一つです。宿主生来の免疫成分は、シグナル伝達を効果的に開始され、忠実に伝達されるように構成されている。シグナル伝達カスケードの階層と規制の輪郭を描くことは自然免疫の抜群の話題です。ここでは、サイトカイン産生のウイルスの回避における自然免疫成分?...

最近の研究では、ホスト自然免疫応答を実装する際に、全体的なシグナル伝達カスケードを説明してきました。別個の区画内に存在する、パターン認識受容体（PRR）1シグナリング先天性免疫を誘発するために、多様な起源の病原体関連分子パターン（PAMP）を検出する。レチノイン酸誘導遺伝子I（RIG-I）とメラノーマ分化抗原5（MDA5）タンパク質は、特定の構造的特徴2でRNA種を認識し、細胞質ゾルセンサである。活性化されると、RIG-Iには、IKKiはとして知られ、その下流MAVS（また、IPS-1、VISA、およびCARDIFとして知られている）今度は、IKK（IKKαβγ）およびIKK関連キナーゼ（TBK1とIKKεを活性化し、アダプターと対話）3-6錯体。先天性免疫活性化キナーゼは、 例えば、IL6、TNF＆＃（その転写因子および阻害剤を含む、遺伝子発現の重要な調節因子をリン酸化し、宿主の抗ウイルス遺伝子の転写活性化を有効にする945;、CCL5、およびIFNβ）。これらのシグナル伝達カスケードは、感染の初期段階で病原体の伝播を制限する抗微生物状態を確立するための強力な内因性自然免疫応答を構成する。 ネズミγHV68は、人間の発癌KSHVおよびEBVに密接に関連している。従って、マウスのγHV68感染は、インビボ7におけるガンマヘルペスウイルス感染に対する宿主免疫応答を調べるために扱いやすい小動物モデルを提供する。 γHV68がウイルス感染を有効にするには、自然免疫のシグナル伝達をホストするハイジャックすることによりγHV68を使用して、私たちの研究室では、複雑な戦略を明らかにした。一方では、γHV68、複製ベーター（RTA）、γHV68複製8用のウイルス転写因子キーをリン酸化するために活性化IKKβを演出経由のウイルス転写活性化を促進するためのMAVS-IKKβ経路を活性化する。一方、IKKβ媒介リン酸化は劣化と任期のRelAをプライミングNFκB活性化9 inates。このように、γHV68感染を効果的に抗ウイルスサイトカインの産生を回避する。興味深いことに、γHV68の発現ライブラリーを用いたスクリーニングにより、RelAの分解を誘導し、NFκB活性化10を排除するE3リガーゼとして、RTAを同定した。これらの知見は、上流の免疫シグナルイベントは、究極の抗ウイルスサイトカイン産生を否定するγHV68によって利用されることにより、複雑な免疫回避戦略を明らかにします。 ここでは、in vivoおよびEX VIV Oの両方γHV68に感染したマウスにおける抗ウイルスサイトカイン産生を測定するためのプロトコルを記述します。プロトコルでは、我々はさらに、抗ウイルスサイトカイン産生を調節することで特定の自然免疫の分子の機能をピンポイントレンチウイルス形質導入によってノックアウト胚性線維芽細胞における自然免疫成分の「再構成」という表現を探る。このプロトコルは、簡単に他のヴィルに適合させることができるSESとシグナル伝達経路。

<table border="0" cellpadding="0" cellspacing="0" width="684">
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	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:251px;">Mouse CCL5 ELISA kit</td>
			<td style="width:211px;">R&#38;D systems</td>
			<td style="width:223px;"><a href="http://www.rndsystems.com/Products/DY478">DY478</a></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:251px;">1.0 mm Zirconia/Silica beads</td>
			<td style="width:211px;">BioSpec Products</td>
			<td style="width:223px;">11079110z</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:251px;">TRIzol</td>
			<td style="width:211px;">Invitrogen</td>
			<td style="width:223px;"><a href="http://products.invitrogen.com/ivgn/en/US/adirect/invitrogen?cmd=catProductDetail&amp;productID=15596018">15596-018</a></td>
		</tr>
		<tr height="79">
			<td height="98" rowspan="2" style="height:98px;width:251px;">SuperScript II Reverse Transcriptase</td>
			<td rowspan="2" style="width:211px;">Invitrogen</td>
			<td rowspan="2" style="width:223px;">18064-014</td>
		</tr>
		<tr height="19">
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"> </td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td colspan="2" height="21" style="height:21px;">CCL5 quantitative real-time PCR primers</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CCTGCTGCTTTGCCTACCTCTC</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">ACACACTTGGCGGTTCCTTCGA</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

dissecting innate immune signaling in viral evasion of cytokine production

ウイルス感染に応答して、宿主自然免疫応答は、抗ウイルス性サイトカインの遺伝子発現および産生をアップレギュレートに活性化される。逆に、ウイルスが生存と繁殖のために宿主の免疫シグナリングを回避し、悪用する複雑な戦略を進化させてきた。ウイルス免疫回避は、宿主防御およびウイルスの回避を伴う、宿主 - ウイルス相互作用を識別するための最も魅力的でダイナミックなインタフェースのいずれかを提供しています。これらの研究は、先天性免疫調節に我々の理解を進め、新たな抗ウイルス療法を開発する私たちの道を開く。 ネズミγHV68は、ネズミ、げっ歯類の自然な病原体である。マウスのγHV68感染は、in vivoでのウイルス-宿主相互作用の摂動が適用されない人間のKSHVおよびEBVに対する抗ウイルス応答を調べるために扱いやすい小型の動物モデルを提供する。ここでは、抗ウイルス性サイトカイン産生を決定するためのプロトコルを記載している。このプロトコルは、他のvirに適合させることができる使用しており、シグナル伝達経路。 最近では、NFκBの活性化​​および抗ウイルスサイトカイン産生を排除するために、γHV68がMAVSおよびIKKβ、細胞質ゾル、RIG-IとMDA5のダウンストリームキー自然免疫シグナル伝達成分をハイジャックすることを発見した。具体的には、γHV68感染は、IKKβを活性化し、活性化IKKβは、RelAの劣化を加速させるのRelAをリン酸化する。このように、γHV68を効率的に抗ウイルスサイトカイン遺伝子発現を否定、その上流活性化し、IKKβからNFκBの活性化を脱共役。本研究では、上流の先天性免疫活性化はすぐ下流の転写活性化を無効にし、抗ウイルス性サイトカイン産生を回避するためにウイルス病原体によって傍受される複雑な戦略を解明。

3つの代表的な数値はγHV68感染マーベリックの肺におけるサイトカイン産生を含め、ここに示されている+ / +およびマーベリック- / - / - - γHV68感染マーベリック + / +およびマーベリックのマウス、サイトカイン分泌および遺伝子発現レベルのMEF、およびγHV68感染マーベリックのサイトカインmRNAレベルを- / - MEFをMAVSで「再構成?...

Watch this Scientific Journal Video about Dissecting Innate Immune Signaling in Viral Evasion of Cytokine Production at JoVE.com

Dissecting Innate Immune Signaling in Viral Evasion of Cytokine Production

我々は、ヒトのカポジ肉腫関連ヘルペスウイルス（KSHV）、およびエプスタイン-バーウイルス（EBV）に密接に関連しているモデルヘルペスウイルス、マウスガンマヘルペスウイルス68（γHV68）に感染したマウスにおける抗ウイルスサイトカイン産生を測定するためのプロトコルを記載している。遺伝的に改変されたマウス系統およびマウス胚線維芽細胞（MEF）を利用して、我々は、in vivoおよびex vivoの両方の抗ウイルス性サイトカイン産生を評価した。レンチウイルス形質導入によるノックアウト胚性線維芽細胞における自然免疫成分の発現を「再構成」、我々はさらに、特定の自然免疫の分子を特定し、差別的な抗ウイルスサイトカイン産生を調節する重要なシグナル伝達事象を分析。

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. ...

dissecting-innate-immune-signaling-viral-evasion-cytokine

Research

JoVE Journal

Immunology and Infection

10.4K ビュー.  Keck School of Medicine, University of Southern California. 我々は、ヒトのカポジ肉腫関連ヘルペスウイルス（KSHV）、およびエプスタイン-バーウイルス（EBV）に密接に関連しているモデルヘルペスウイルス、マウスガンマヘルペスウイルス68（γHV68）に感染したマウスにおける抗ウイルスサイトカイン産生を測定するためのプロトコルを記載してい?...

ビデオ: Dissecting Innate Immune Signaling in Viral Evasion of Cytokine Production

Production and Titering of Recombinant Adeno-associated Viral Vectors

In recent years recombinant adeno-associated viral vectors (AAV) have become increasingly valuable for in vivo studies in animals, and are also currently being tested in human clinical trials. Wild-type AAV is a non-pathogenic member of the parvoviridae family and inherently replication-deficient. The broad transduction profile, low immune response as well as the strong and persistent transgene expression achieved with these vectors has made them a popular and versatile tool for in vitro and in vivo gene delivery. rAAVs can be easily and cheaply produced in the laboratory and, based on their favourable safety profile, are generally given a low safety classification. Here, we describe a method for the production and titering of chimeric rAAVs containing the capsid proteins of both AAV1 and AAV2. The use of these so-called chimeric vectors combines the benefits of both parental serotypes such as high titres stocks (AAV1) and purification by affinity chromatography (AAV2). These AAV serotypes are the best studied of all AAV serotypes, and individually have a broad infectivity pattern. The chimeric vectors described here should have the infectious properties of AAV1 and AAV2 and can thus be expected to infect a large range of tissues, including neurons, skeletal muscle, pancreas, kidney among others. The method described here uses heparin column purification, a method believed to give a higher viral titer and cleaner viral preparation than other purification methods, such as centrifugation through a caesium chloride gradient. Additionally, we describe how these vectors can be quickly and easily titered to give accurate reading of the number of infectious particles produced.

Recombinant adeno-associated virus (rAAVs) vectors are becoming increasingly valuable for in vivo studies in animals. We describe how rAAVs can be produced in the laboratory and how these vectors can be titered to give an accurate reading of the number of infectious particles produced.

組換えアデノ随伴ウイルスベクターの産生と力価測定

In recent years recombinant adeno-associated viral vectors (AAV) have become increasingly valuable for in vivo studies in animals, and are also currently ...

免疫・感染学

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). ...

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.

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 ...

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 ...

Dissecting Multi-protein Signaling Complexes by Bimolecular Complementation Affinity Purification (BiCAP)

The assembly of protein complexes is a central mechanism underlying the regulation of many cell signaling pathways. A major focus of biomedical research is deciphering how these dynamic protein complexes act to integrate signals from multiple sources in order to direct a specific biological response, and how this becomes deregulated in many disease settings. Despite the importance of this key biochemical mechanism, there is a lack of experimental techniques that can facilitate the specific and sensitive deconvolution of these multi-molecular signaling complexes.
Here this shortcoming is addressed through the combination of a protein complementation assay with a conformation-specific nanobody, which we have termed Bimolecular Complementation Affinity Purification (BiCAP). This novel technique facilitates the specific isolation and downstream proteomic characterization of any pair of interacting proteins, to the exclusion of un-complexed individual proteins and complexes formed with competing binding partners. 
The BiCAP technique is adaptable to a wide array of downstream experimental assays, and the high degree of specificity afforded by this technique allows more nuanced investigations into the mechanics of protein complex assembly than is currently possible using standard affinity purification techniques.

Dissecting Multi-protein Signaling Complexes by Bimolecular Complementation Affinity Purification BiCAP

This manuscript describes the protocol for Bimolecular Complementation Affinity Purification (BiCAP). This novel method facilitates the specific isolation and downstream proteomic characterization of any two interacting proteins, while excluding un-complexed individual proteins as well as complexes formed with competing binding partners.

解離性個体の親和性の浄化 (BiCAP) によって多蛋白質シグナリング複合体

The assembly of protein complexes is a central mechanism underlying the regulation of many cell signaling pathways. A major focus of biomedical research ...

Single-cell Analysis of Immunophenotype and Cytokine Production in Peripheral Whole Blood via Mass Cytometry

Cytokines play a pivotal role in the pathogenesis of autoimmune diseases. Hence, the measurement of cytokine levels has been the focus of multiple studies in an attempt to understand the precise mechanisms that lead to the breakdown of self-tolerance and subsequent autoimmunity. Approaches thus far have been based on the study of one specific aspect of the immune system (a single or few cell types or cytokines), and do not offer a global assessment of complex autoimmune disease. While patient sera-based studies have afforded important insights into autoimmunity, they do not provide the specific cellular source of the dysregulated cytokines detected. A comprehensive single-cell approach to evaluate cytokine production in multiple immune cell subsets, within the context of &#34;intrinsic&#34; patient-specific plasma circulating factors, is described here. This approach enables monitoring of the patient-specific immune phenotype (surface markers) and function (cytokines), either in its native &#34;intrinsic pathogenic&#34; disease state, or in the presence of therapeutic agents (in vivo or ex vivo).

Here we describe a single-cell proteomic approach to evaluate immune phenotypic and functional (intracellular cytokine induction) alterations in peripheral whole blood samples, analyzed via mass cytometry.

イムノフェノ タイプと大量のフローサイトメトリーによる末梢血中のサイトカイン産生の単一細胞解析

Cytokines play a pivotal role in the pathogenesis of autoimmune diseases. Hence, the measurement of cytokine levels has been the focus of multiple studies ...

A Mouse Model to Assess Innate Immune Response to Staphylococcus aureus Infection

Staphylococcus aureus (S. aureus)&#160;infections, including methicillin resistant stains, are an enormous burden on the healthcare system. With incidence rates of S. aureus infection climbing annually, there is a demand for additional research in its&#160;pathogenicity. Animal models of infectious disease advance our understanding of the host-pathogen response and lead to the development of effective therapeutics. Neutrophils play a primary role in the innate immune response that controls S. aureus infections by forming an abscess to wall off the infection and facilitate bacterial clearance; the number of neutrophils that infiltrate an S. aureus skin infection often correlates with disease outcome. LysM-EGFP mice, which possess the enhanced green fluorescent protein (EGFP) inserted in the Lysozyme M (LysM) promoter region (expressed primarily by neutrophils), when used in conjunction with in vivo whole animal fluorescence imaging (FLI) provide&#160;a means of quantifying neutrophil emigration noninvasively and longitudinally into wounded skin. When combined with a bioluminescent S. aureus strain and sequential in vivo whole animal bioluminescent imaging (BLI), it is possible to longitudinally monitor both the neutrophil recruitment dynamics and in vivo bacterial burden at the site of infection in anesthetized mice from onset of infection to resolution or death. Mice are more resistant to a number of virulence factors produced by S. aureus that facilitate effective colonization and infection in humans. Immunodeficient mice provide a more sensitive animal model to examine persistent S. aureus infections and the ability of therapeutics to boost innate immune responses. Herein, we characterize responses in LysM-EGFP mice that have been bred to MyD88-deficient mice (LysM-EGFP&#215;MyD88-/- mice) along with wild-type LysM-EGFP mice to investigate S. aureus skin wound infection. Multispectral simultaneous detection enabled study of neutrophil recruitment dynamics by using in vivo FLI, bacterial burden by using in vivo BLI, and wound healing longitudinally and noninvasively over time.

A Mouse Model to Assess Innate Immune Response to Staphylococcus aureus Infection

An approach is described for real-time detection of the innate immune response to cutaneous wounding and Staphylococcus aureus infection of mice. By comparing LysM-EGFP mice (which possess fluorescent neutrophils) with a LysM-EGFP crossbred immunodeficient mouse strain, we advance our understanding of infection and the development of approaches to combat infection.

マウスのモデルを評価する生得の免疫反応黄色ブドウ球菌感染症

Staphylococcus aureus (S. aureus)&#160;infections, including methicillin resistant stains, are an enormous burden on the healthcare system. With incidence ...

A Galleria mellonella Oral Administration Model to Study Commensal-Induced Innate Immune Responses

The investigation of the immunogenic potential of commensal bacteria on the host immune system is one essential component when studying intestinal host-microbe interactions. It is well established that different commensals exhibit a different potential to stimulate the host intestinal immune system. Such investigations involve vertebrate animals, especially rodents. Since increasing ethical concerns are linked with experiments involving vertebrates, there is a high demand for invertebrate replacements models. 
Here, we provide a Galleria mellonella oral administration model using commensal non-pathogenic bacteria and the possible assessment of the immunogenic potential of commensals on the G. mellonella immune system. We demonstrate that G. mellonella is a useful alternative invertebrate replacement model that allows the analysis of commensals with different immunogenic potential such as Bacteroides vulgatus and Escherichia coli. Interestingly, the bacteria exhibited no killing effect on the larvae, which is similar to mammals. The immune responses of G. mellonella were comparable with vertebrate innate immune responses and involve recognition of the bacteria and production of antimicrobial molecules. We propose that G. mellonella was able to restore previous microbiota balance, which is well known from healthy mammalian individuals. Although providing comparable innate immune responses in both G. mellonella and vertebrates, G. mellonella does not harbor an adaptive immune system. Since the investigated components of the innate immune system are evolutionary conserved, the model allows a prescreening and first analysis of bacterial immunogenic properties.

A Galleria mellonella Oral Administration Model to Study Commensal-Induced Innate Immune Responses

Here, we provide a detailed protocol for an oral administration model using Galleria mellonella larvae and how to characterize induced innate immune responses. Using this protocol, researchers without practical experience will be able to use the G. mellonella force-feeding method.

ハチミツガ口腔管理モデル Study Commensal-Induced 自然免疫応答

The investigation of the immunogenic potential of commensal bacteria on the host immune system is one essential component when studying intestinal ...

Isolation of Tonsillar Mononuclear Cells to Study Ex Vivo Innate Immune Responses in a Human Mucosal Lymphoid Tissue

Studying isolated cells from mucosa-associated lymphoid tissues (MALT) allows understanding of immune cells response in pathologies involving mucosal immunity, because they can model host-pathogen interactions in the tissue. While isolated cells derived from tissues were the first cell culture model, their use has been neglected because tissue can be hard to obtain. In the present protocol, we explain how to easily process and culture tonsillar mononuclear cells (TMCs) from healthy human tonsils to study innate immune responses upon activation, mimicking viral infection in mucosal tissues. Isolation of TMCs from the tonsils is quick, because the tonsils barely have any epithelium and yield up to billions of all major immune cell types. This method allows detection of cytokine production using several techniques, including immunoassays, qPCR, microscopy, flow cytometry, etc., similar to the use of peripheral mononuclear cells (PBMCs) from blood. Furthermore, TMCs show a higher sensitivity to drug testing than PBMCs, which needs to be considered for future toxicity assays. Thus, ex vivo TMCs cultures are an easy and accessible mucosal model.

In the present protocol, we explain how to easily process and culture tonsillar mononuclear cells from healthy humans undergoing partial surgical tonsillectomy to study innate immune responses upon activation, mimicking viral infection in mucosal tissues.

ヒト粘膜リンパ組織におけるEx Vivo自然免疫応答を研究する扁桃単核細胞の単核細胞の単離

Studying isolated cells from mucosa-associated lymphoid tissues (MALT) allows understanding of immune cells response in pathologies involving mucosal ...

Microfluidic Co-Culture Models for Dissecting the Immune Response in in vitro Tumor Microenvironments

Complex disease models demand cutting-edge tools able to deliver physiologically and pathologically relevant, actionable insights, and unveil otherwise invisible processes. Advanced cell assays closely mimicking in vivo scenery are establishing themselves as novel ways to visualize and measure the bidirectional tumor-host interplay influencing the progression of cancer. Here we describe two versatile protocols to recreate highly controllable 2D and 3D co-cultures in microdevices, mimicking the complexity of the tumor microenvironment (TME), under natural and therapy-induced immunosurveillance. In section 1, an experimental setting is provided to monitor crosstalk between adherent tumor cells and floating immune populations, by bright field time-lapse microscopy. As an applicative scenario, we analyze the effects of anti-cancer treatments, such as the so-called immunogenic cancer cell death inducers on the recruitment and activation of immune cells. In section 2, 3D tumor-immune microenvironments are assembled in a competitive layout. Differential immune infiltration is monitored by fluorescence snapshots up to 72 h, to evaluate combination therapeutic strategies. In both settings, image processing steps are illustrated to extract a plethora of immune cell parameters (e.g., immune cell migration and interaction, response to therapeutic agents). These simple and powerful methods can be further tailored to simulate the complexity of the TME encompassing the heterogeneity and plasticity of cancer, stromal and immune cells subtypes, as well as their reciprocal interactions as drivers of cancer evolution. The compliance of these rapidly evolving technologies with live-cell high-content imaging can lead to the generation of large informative datasets, bringing forth new challenges. Indeed, the triangle &#39;&#39;co-cultures/microscopy/advanced data analysis&#34; sets the path towards a precise problem parametrization that may assist tailor-made therapeutic protocols. We expect that future integration of cancer-immune on-a-chip with artificial intelligence for high-throughput processing will synergize a large step forward in leveraging the capabilities as predictive and preclinical tools for precision and personalized oncology.

In the age of immunotherapy and single-cell genomic profiling, cancer biology requires novel in vitro and computational tools for investigating the tumor-immune interface in a proper spatiotemporal context. We describe protocols to exploit tumor-immune microfluidic co-cultures in 2D and 3D settings, compatible with dynamic, multiparametric monitoring of cellular functions.

in vitro腫瘍微小環境における免疫応答を解剖するためのマイクロ流体共培養モデル

Complex disease models demand cutting-edge tools able to deliver physiologically and pathologically relevant, actionable insights, and unveil otherwise ...