We thank Dr Jamil Jubrail for reading the manuscript. This work was supported by grants from CNRS, Inserm, Universit&#233; Paris Descartes, Agence Nationale de la Recherche (2011 BSV3 025 02), Fondation pour la Recherche M&#233;dicale (FRM DEQ20130326518 including a doctoral fellowship for GLB) and Agence Nationale de Recherche sur le SIDA et les h&#233;patites virales (ANRS, including a post-doctoral fellowship for CD) to FN. A. Dumas was supported by doctoral fellowships from Universit&#233; Paris Descartes and Sidaction.

プロトコルは、国内および国際的な法律や地方条例に厳密に従って行わなければなりません。研究目的のために献血をする彼らの同意を与えた健康なドナーからの血液は、機関が契約を締結していると輸血センターから得られています。ヒトの血液を使用した場合には、特別な保護がとられなければなりません。 HIV-1を用いた実験では現地の法律に従って、バイオセーフティーレベル3または2（BSL-3または2）実験室で行われなければなりません。 接着によって密度勾配遠心と選択によるヒト単球由来マクロファージ（hMDMs）の調製<ol><li>健康なドナー（9ミリリットル）からの新鮮な血液で起動します。 70 mlの最終容積を得ることが2+ の Ca 2+およびMgなしの滅菌1×リン酸緩衝生理食塩水（PBS）を用いて、新鮮な血液の全体積を希釈し、穏やかに（50ml×2回）コニカルチューブ（チューブにつき35 mL）に希釈された血液を追加、の15ミリリットルの上eutralは、非常に各チューブに既に​​溶液中に、高質量の親水性多糖分岐します。 </li><li>ブレーキなしで20℃で20分間、537×gでの血液の遠心分離は、両方の管。次に界面で濁ったセル環に ​​含まれる末梢血単核細胞（PBMC）を収集およびCa 2+及びMg 2+なしで、1×PBS 15 mlを含む新しい50mlチューブに移します。 </li><li>遠心分離機20℃で5分間、218×gで細胞およびCa 2+およびMgなしで1×PBSの45ミリリットル中にペレットを再懸濁2+。 </li><li> 20℃で5分間、218×gで遠心分離した細胞は、2+ の Ca 2+およびMgなしで1×PBS 10mlにペレットを再懸濁し、トリパンブルーで1/200の最終希釈に希釈することによって細胞をカウントします。 </li><li> 20℃で5分間、218×gで細胞を遠心し、1640培地は、2mMのL-グルタミンおよび100μg/ mlのpenicを補充したRPMI（ロズウェルパーク記念研究所）でペレットを再懸濁illin -ストレプトマイシン6ウェルプレートの各ウェルに培地2ml中、ウェルあたり7×10 6個のPBMCを有すること。 </li><li> 2時間、5％CO 2、37℃でプレートをインキュベートします。 2時間後、単球をプラスチックに付着しているでしょう。 </li><li>新たに単離した単球が、hMDMsに分化培地を吸引し、2mlでhMDM培地に交換できるようにする（RPMI 1640、10％ウシ胎児血清（FCS）、2mM L-グルタミンおよび1％ペニシリン - ストレプトマイシンをdecomplemented）を補充し組換えヒトマクロファージコロニー刺激因子（RHM-CSF）を10ng / mlの最終濃度で。 </li><li> 11日目（ 図1）、5％CO 2で37℃で細胞をインキュベートします。 </li><li> 11日目で培地を除去し、ウェルあたり冷たいhMDM培地2mlで2回洗浄します。次の冷1×PBS 1mlで各ウェルを2回洗浄します。 </li><li> （2 mMのethylenediaminetetraacetatic酸と冷たい1×PBSの1ミリリットルで、各ウェル1時間を洗って、完全に分化しhMDMsを切り離すにはEDTA）と4℃で15から60分間、ウェルあたり2 mMのEDTA冷1×PBSの2ミリリットルで細胞をインキュベートします。 注：細胞が存在して剥離する別の方法（ 例えば、トリプシンまたはトリプシン様活性）良い結果11を与えるかもしれません。 </li><li> （上下十分で静かにピペッティングすることにより完成）剥離した後、細胞を回収し、冷hMDM培地10mlを含む氷上で50mlチューブに入れ。 </li><li> 20℃で5分間、218×gで遠心分離した細胞は、冷たいhMDM培地10mlにペレットを再懸濁し、トリパンブルーで1/20に希釈することによって細胞をカウントします。 </li><li> 35ミリメートル顕微鏡グレードのガラスボトムディッシュあたり1×10 6 hMDMsで細胞をシードし、1日間、5％CO 2で37℃で培養します。 </li></ol> HIV-1ウイルスストックの2生産および定量注：M. SchindlからR5向性エンベロープを運ぶNLR4.3 HIV-1のGag-iGFP（緑色蛍光タンパク質）、ギフトER 10は、マクロファージを感染させるために、リアルタイムで感染細胞を見るために使用されます。 <ol><li>商業トランスフェクション試薬を用いて対応するプロウイルスDNAの6μgの持つヒト胚性腎臓293T細胞（2×10 6 100 mmディッシュ）のトランスフェクションによりウイルスストックを生成します。 </li><li>細胞計数のβ-ガラクトシダーゼ着色が続くウイルスストックの連続希釈液を用いて、（HIV-1 LTRの制御下でβ-ガラクトシダーゼ遺伝子を有する）HeLa細胞TZM-BL指標細胞を用いたウイルスストックの感染力を定量化12青色の細胞の。 </li></ol> HIV-1とhMDMs 3.感染<ol><li> hMDMs（1.13でプレートした細胞）に感染多重度（MOI）hMDM培地の1ミリリットル中に0.02から0.03でウイルスを追加します。ウェルはhMDM媒体の唯一の1ミリリットルを追加制御し、2日間（ 図1）、5％CO 2、37℃で皿をインキュベートします。 </li><li> 2日目にhMDM MEDで細胞を洗浄イウム3回と皿当たり新鮮hMDM培地の1ミリリットルを追加します。 6日目（ 図1）、5％CO 2で37℃で細胞をインキュベートします。 </li></ol>ヒツジ赤血球の4オプソニン<ol><li>皿当たり7×10 6 SRBCをの調製のために、4分間600×gで遠心分離すると、1×PBS中の0.1％ウシ血清アルブミン（BSA）を含む溶液100μlにSRBCを2回洗浄します。 </li><li> SRBCを5μlの当たりのサブ凝集濃度でウサギのIgG抗SRBCを含むPBS / BSA 0.1％、1×500μlの洗浄SRBCをを再懸濁し、室温で30分間回転させながらインキュベートします。 注：、抗SRBCをIgGのサブ凝集濃度を決定し、96ウェルプレート中の20μlの1/50から1/25600へのIgGの連続希釈物（13.1 mg / mlとでストック濃度）を調製しました。各ウェル中の20μlの2×10 6 SRBCを追加し、いくつかの時間の間、暗い部屋でプレートを置きます。サブ凝集濃度であります直前よく凝集抗体（IgG + SRBCを、ネットワークを形成する）とウェルの希釈。 </li><li>回転の後、4分間600×gでのIgGオプソニン化-SRBCをを遠心分離し、4分間、600×gの遠心分離で1×PBS / BSA 0.1％の100μlで洗います。 </li><li> 2mMのL-グルタミンおよび1％ペニシリン - ストレプトマイシン（1ミリリットル/皿）を補充した予め温めたフェノールレッドを含まないRPMI培地中のIgGオプソニン化-SRBCを再懸濁。 </li></ol> 5.ライブセルビデオ顕微鏡食作用アッセイ<ol><li>例えばCO 2は、加湿のために水でボトルを通過し、37℃で加熱室を備えた回転ディスクシステムのような共焦点イメージングシステムを使用してください。 </li><li>食作用アッセイの開始前に37℃で顕微鏡ステージを持つように、実験前に加熱室をオンにします。顕微鏡とコンピュータの電源を入れ、およびイメージングソフトウェアをロードします。 </li><li>このような走査速度、マニフィカ等の撮像設定を最適化イオン、解像度等が 60〜120分の間、毎分ごとにフィールドと、一つのフレーム画像に、少なくとも一つのセルを持っています。 注：ここでは、サンプルは63Xレンズと60分の間に1フレーム分毎に結像されます。 </li><li>顕微鏡ステージ上のイメージング皿を置きます。フィールドに一つだけの全体のHIV-1感染したマクロファージを見つけるために、フォーカスと位置を調整します。使用される撮像システムとプローブに基づいて適切な励起/発光の設定を使用します。ファゴソームを観察する明視野（BF）のチャンネルを含める（ 図1II）。透過光及び露光時間の割合を調整することにより、異なるチャネルの画像の外観を最適化します。 注：ここでは、NLR4.3 HIV-1のGag-iGFPは、レーザの20％（ 図1I）で長時間露光の50ミリ秒と491 nmのレーザーを使用して興奮していました。 </li><li>イメージング皿を外し、皿（ 図1）に7×10 6 SRBCを/ mlでSRBC懸濁液1mlを追加します。 </li><li> 500×gで遠心分離RTで2分間は、食作用を同期この遠心分離の終わりに時間を記録し、ステージに皿を返します。 </li><li>フォーカスを最適化し、（0.3ミクロンのステップ距離とセルの厚さを通して - 通常20面）のZスタックにGFPとBF画像を取り込む少なくとも1時間、毎分。使用される撮像システムのネイティブファイル形式でタイムラプスビデオを保存します。 注：ここでは、タイムラプス動画は、ネイティブ形式、* .STKファイルに保存されていました。 </li></ol>タイムラプスビデオの6.分析<ol><li>ビデオ編集ソフトの「レビュー多次元データ」ビデオ編集のために、ドロップダウンメニューをクリックして「アプリ」タブ上。 <ol><li>ファイルを開くには、「ディレクトリの選択」にし、「ベースファイルを選択」をクリックしてください。 「データ設定」ボックスで、（.nd形式）を分析し、「表示」をクリックして取得を選択します。データが列ANに時間を持つテーブルで表現されています行中のd Z-計画。 </li><li> Z-突起（ 図2、左パネル）に感染を表すために、「波長」ボックスに491 nmの波長を選択し、「すべての面」と「Z投影」タブをクリックします。 </li><li> （、時系列を分析する「波長」ボックスにBF波長を選択して、外部のSRBCを（ 図2、赤矢印）、内部SRBCを（ 図2、赤い円）と核を区別するために、Z軸上での最適な平面を選択するには図2、青丸）。 </li><li>ビデオモンタージュを保存するには、「選択[Xの]」タブで、次に「イメージのロード（S）」をクリックしてください。最後に、tifファイル形式で読み込んだ画像を保存し、ImageJソフトウェア上で次のそれらを開きます。 </li></ol></li><li>核の位置とBFチャネル（ 図3）で観察された異なるファゴソームを測定するためにImageJソフトウェア上のプラグイン「マニュアルトラッキング&quot;を使用してください。 <ol><li>ダウImageJのウェブサイトの「マニュアルトラッキング」プラグインをnload。 ImageJのでは、解析すべきプラグインと画像シーケンス（ 図3、ステップ1および2）を開きます。 </li><li>このような隣接するフレーム間の時間の量を表す「時間間隔」、および画素あたりの距離を表す「X / Yのキャリブレーション」（ 図3、ステップ3）などの設定を入力します。 注：6.45のx 6.45μmの2のピクセルサイズを持つ63Xズームとカメラが使用されているので、ここでは、各フレームと0.205ミクロンのX / Yのキャリブレーションの間に1分の時間間隔で画像シーケンスを保存します。 </li><li>追跡を開始するには、「トラックを追加」（ 図3、ステップ4）をクリックして、それが内在化される最初の時間でSRBCの中心をクリックしてください。次のフレームが自動的に表示されます。 </li><li>時間の間に位置が異なるために、すべてのフレームでSRBC中央をクリックし続けます（ 図3、赤いボックスのステップ6）。 <ol><li>その中心をクリックして、すべてのフレームでの地位を持っている核を追跡するために開始します。次回SRBCをの関数で異なるそれらの内在化（フレーム）で1ずつ（その中心をクリックして）ファゴソームを追跡します。 BFチャネルでSRBCを参照するには便宜上、トラッキング（ 図3、ステップ5）の間に「明るさとコントラスト」ウィンドウを使用します。 </li></ol></li><li>各SRBC追跡の間に、新しいトラックを持っている（ 図3、ステップ4）、「トラックを追加」をクリックしてください。追跡の数が第2の列に変更されます、結果テーブルの「トラックのn°」（ 図3、ステップ6）。 </li><li>分析の次のステップに進み、スプレッドシート内のデータを保存します。 </li></ol></li><li> （核とSRBCをの内在化後の最初の5分の間にファゴソームの速度に向かってSRBCを含有ファゴソームの走行距離を計算するためFiを提供して表計算ソフトを使用して、グレ4）。 <ol><li>スプレッドシートテーブルと新しいスプレッドシートファイルを開きます。この新たな次のパラメータファイルのみ、時間、x軸に移し、核およびすべてのSRBCを（ 図4A）のy座標。 </li><li> SRBCをのみそれらの座標と核の間の距離を計算するために、核とXY座標平面（ 図4B）でSRBCを検討してください。 注：2点間の距離は、それらを接続する経路の長さです。平面では、SRBCと核との間の距離は、ピタゴラスの定理により与えられます。 <ol><li> C（核とSRBCとの間の距離）を斜辺とAの長さを示し、B、他の2辺の長さを表す場合は、ピタゴラスの式のようにピタゴラスの定理を表現します： <img alt="式（1）" src="/files/ftp_upload/54568/54568eq1.jpg" /> 注：同時に、正規直交上、水平距離（X 核 -x SRBC）であり、垂直距離bは （SRBC -y Y 核 ）です。 </li><li>このように、核とSRBC（ 図4A、紫色のボックス）によるとの間の距離を計算します。 <img alt="式（2）" src="/files/ftp_upload/54568/54568eq2.jpg" /> 注：μm単位の距離を持っているのx / yのキャリブレーションによって（ピクセル単位）得られた距離値を乗算します。ここで、X / Yのキャリブレーションは0.205μmです。 </li><li>各時点で、核および初期距離（ 図4C）にSRBCとの間の距離を引きます。 </li><li>最初の5分間の時間に対する測定値をプロットします。プロットに線形トレンドライン（ 図5A）を適用し、SRBC内在化後の最初の5分の間に核に向かってファゴソーム速度を表す線形トレンドラインの傾きを決定します。 </li><li>の平均値と統計誤差を計算するためにデータを照合速度値と適切なソフトウェア（ 図5B）を使用して、適切な形式でそれらをプロットします。 </li></ol></li></ol></li></ol>

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E., Bucci, C., Vieira, O. V., Schroer, T. A., Grinstein, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phagosomes+fuse+with+late+endosomes+and/or+lysosomes+by+extension+of+membrane+protrusions+along+microtubules:+role+of+Rab7+and+RILP.">Phagosomes fuse with late endosomes and/or lysosomes by extension of membrane protrusions along microtubules: role of Rab7 and RILP.</a> Mol Cell Biol. 23, 6494-6506 (2003).</li><li>Toyohara, A., Inaba, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transport+of+phagosomes+in+mouse+peritoneal+macrophages.">Transport of phagosomes in mouse peritoneal macrophages.</a> J Cell Sci. 94 (Pt 1), 143-153 (1989).</li></ol>

The authors have nothing to disclose.

この技術は、いくつかの重要なステップがあります。感染の割合がドナー依存しているため、まず、HIV-1によるhMDMsの製造およびそれらの感染が重要です。注目すべきは、我々は潜在的に生体内でウイルスが遭遇したマクロファージの状態がよく、これまでに特徴付けされていないため、感染前に、in vitroで偏光されていないマクロファージを使用することを決定しました。我々?...

Macrophages play a major role in the innate immune system and in homeostasis. They are professional phagocytes that internalize and eliminate pathogens and debris by a process called phagocytosis 1,2. The phagosome, the closed compartment that forms after the engulfment of particulate material, undergoes a series of fusion and fission events with endocytic compartments, leading to a degradative compartment called the phagolysosome. This compartment has an acidic pH, due to the acquisition of proton-pumping v-ATPases, contains hydrolytic enzymes and is enriched in lysosomal-associated membrane proteins (LAMPs). The maturation of phagosomes is accompanied by their migration on microtubules 3,4 towards the cell center to reach a perinuclear location where lysosomes are accumulated.
Many pathogens have been reported to hijack phagosome maturation, including bacteria with intracellular lifestyles that modify the vacuolar environment where they reside 5. The Human Immunodeficiency Virus (HIV)-1 targets macrophages in addition to T cells. As macrophages are resistant to the cytopathic effects of the virus, unlike T cells, they can be considered as a reservoir for the virus. In addition, macrophages infected with HIV-1 show defective phagocytic functions and contribute to the emergence of opportunistic diseases. In particular, severe invasive non-typhoidal Salmonella disease caused by Salmonella Typhimurium ST313 has been prevalent for the last three decades in sub-Saharan African children or adults infected with HIV 6. It has been estimated that the risk of developing tuberculosis is more than 20 times greater in people living with HIV than among those without HIV infection. 
For all these reasons, it is important to better define the molecular mechanisms underlying the phagocytic defects in HIV-infected macrophages. We have shown that the uptake of particulate material, opsonized particles, bacteria or fungi, was inhibited in HIV-infected macrophages 7. Given that this inhibition is partial, we then set out to analyze the fate of the internalized phagosomes in HIV infected human macrophages 8. Because phagosome maturation is tightly connected with migration to the cell center and fusion with lysosomes, a defect in phagosomal maturation can be due to modifications of the trafficking modalities in the infected cell. The method described here uses IgG-opsonized Sheep Red Blood Cells (IgG-SRBCs) as a model to target receptor-mediated phagocytosis and in particular receptors for the Fc portion of immunoglobulins (FcR). These particles are easier to image in bright field (BF) than latex beads because extracellular and intracellular SRBCs show different refraction properties 9. To measure the velocity of phagosomes moving towards the nucleus in HIV-infected macrophages, we used a fluorescent virus 10 and set up a simple manual tracking method that is described here. The method does not require advanced programming and simply uses ImageJ. It is amenable to adherent cells and any type of particle or pathogen that can be visualized with bright field microscopy or with fluorescent imaging.

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			<td height="68" style="height:68px;width:212px;">Falcon 100mm TC-Treated Cell Culture Dish</td>
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			<td style="width:245px;">For viral production</td>
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			<td style="width:124px;">Thermo Fischer Scientific</td>
			<td style="width:111px;">Corning. Inc. 353934</td>
			<td style="width:245px;">For human monocyte-derived macrophages</td>
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		<tr height="68">
			<td height="68" style="height:68px;width:212px;">DPBS, no calcium, no magnesium</td>
			<td style="width:124px;">Thermo Fischer Scientific</td>
			<td style="width:111px;">14190-094</td>
			<td style="width:245px;">Room temperature</td>
		</tr>
		<tr height="68">
			<td height="68" style="height:68px;width:212px;">Dulbecco&#39;s Modified Eagle Medium (DMEM) 1X, liquid (High Glucose)</td>
			<td style="width:124px;">GIBCO, Molecular probes</td>
			<td style="width:111px;">31966-021</td>
			<td style="width:245px;">Conserved at 4&#176;C ; for HEK cells culture</td>
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		<tr height="68">
			<td height="68" style="height:68px;width:212px;">RPMI 1640 medium GLUTAMAX&#160; Supplement</td>
			<td style="width:124px;">Life technologies</td>
			<td style="width:111px;">61870-010</td>
			<td style="width:245px;">Conserved at 4&#176;C; for hMDMs culture</td>
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			<td height="68" style="height:68px;width:212px;">F&#339;tal Calf Serum (FCS)&#160;</td>
			<td style="width:124px;">Eurobio</td>
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			<td style="width:245px;">Conserved at -20&#176;C ; decomplemented</td>
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			<td height="68" style="height:68px;width:212px;">Penicillin-Streptomycin (10,000 U/mL)</td>
			<td style="width:124px;">Thermo Fischer Scientific</td>
			<td style="width:111px;">15140-122</td>
			<td style="width:245px;">Conserved at -20&#176;C ; for hMDMs culture</td>
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			<td height="68" style="height:68px;width:212px;">RPMI 1640 medium, no phenol red (10x500 ml)</td>
			<td style="width:124px;">Life technologies</td>
			<td style="width:111px;">11835-105</td>
			<td style="width:245px;">Warm in 37&#176;C water bath before use ; for phagocytosis assay</td>
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			<td height="68" style="height:68px;width:212px;">FuGENE6 Transfection Reagent</td>
			<td style="width:124px;">Promega</td>
			<td style="width:111px;">E2692</td>
			<td style="width:245px;">Conserved at 4&#176;C ; for viral production</td>
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		<tr height="68">
			<td height="68" style="height:68px;width:212px;">Sheep red blood cells (SRBCs)</td>
			<td style="width:124px;">Eurobio</td>
			<td style="width:111px;">DSGMTN00-0Q</td>
			<td style="width:245px;">Conserved in Alsever buffer at 4&#176;C before use</td>
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			<td height="68" style="height:68px;width:212px;">Anti-sheep red blood cells IgG</td>
			<td style="width:124px;">MP Biomedicals</td>
			<td style="width:111px;">55806</td>
			<td style="width:245px;">Conserved at 4&#176;C</td>
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			<td height="68" style="height:68px;width:212px;">Bovine Serum Albumin heat shock fraction, pH 7, &#8805;98%&#160;</td>
			<td style="width:124px;">Sigma</td>
			<td style="width:111px;">A7906</td>
			<td style="width:245px;">Conserved at -20&#176;C</td>
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			<td height="68" style="height:68px;width:212px;">Inverted microscope DMI600</td>
			<td style="width:124px;">Leica</td>
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			<td height="68" style="height:68px;width:212px;">Confocal Spinning Disk Unit&#160; CSU-X1M1</td>
			<td style="width:124px;">Yokogawa</td>
			<td style="width:111px;"></td>
			<td style="width:245px;"></td>
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			<td height="68" style="height:68px;width:212px;">491 nm 50mW laser</td>
			<td style="width:124px;">COBOLT CALYPSO</td>
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			<td style="width:245px;"></td>
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			<td height="68" style="height:68px;width:212px;">HCX PL APO CS Objectif</td>
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			<td style="width:245px;">Objective lens ; Magnification 100x ; Numerical aperture 1.40 ; Immersion oil</td>
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			<td height="68" style="height:68px;width:212px;">CoolSnap HQ2 (FireWire) Camera</td>
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			<td style="width:245px;">Pixel size 6.45 x 6.45 &#181;m ; Definition 1392 x 1040 ; Encoding the image in 14 Bit</td>
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			<td height="68" style="height:68px;width:212px;">Metamorph 7.7.5 software</td>
			<td style="width:124px;">Molecular Devices</td>
			<td style="width:111px;"></td>
			<td style="width:245px;">For the control of the microscope</td>
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phagosome migration and velocity measured in live primary human macrophages infected with hiv-1

Macrophages are phagocytic cells that play a major role at the crossroads between innate and specific immunity. They can be infected by the human immunodeficiency virus (HIV)-1 and because of their resistance to its cytopathic effects they can be considered to be persistent viral reservoirs. In addition, HIV-infected macrophages exhibit defective functions that contribute to the development of opportunistic diseases.
The exact mechanism by which HIV-1 impairs the phagocytic response of macrophages was unknown. We had previously shown that the uptake of various particulate material by macrophages was inhibited when they were infected with HIV-1. This inhibition was only partial and phagosomes did form within HIV-infected macrophages. Therefore, we focused on analyzing the fate of these phagosomes. Phagosome maturation is accompanied by migration of these compartments towards the cell center, where they fuse with lysosomes, generating phagolysosomes, responsible for degradation of the ingested material. We used IgG-opsonized Sheep Red Blood Cells as a target for phagocytosis. To measure the speed of centripetal movement of phagosomes in individual HIV-infected macrophages, we used a combination of bright field and fluorescence confocal microscopy. We established a method to calculate the distance of phagosomes towards the nucleus, and then to calculate the velocity of the phagosomes. HIV-infected cells were identified thanks to a GFP-expressing virus, but the method is applicable to non-infected cells or any type of infection or treatment.

HIV-1感染および非感染hMDMsによる食作用をのFcR媒介性は、モデル標的としてのIgGオプソニン化SRBCを（ 図1）を使用してここで説明されています。このプロトコルの重要なステップは、hMDMsの準備とHIV-1の感染です。確かに、分化したマクロファージの収量と品質は、ドナーの間で変化するだけでなく、10から40%の範囲の効率で感染率。これは破片としての代...

Watch this Scientific Journal Video about Phagosome Migration and Velocity Measured in Live Primary Human Macrophages Infected with HIV-1 at JoVE.com

Phagosome Migration and Velocity Measured in Live Primary Human Macrophages Infected with HIV-1

We describe a method to measure the velocity of phagosomes moving towards the cell center in living cells infected with or without the human immunodeficiency virus (HIV) type 1, using spinning disk confocal fluorescence microscopy to identify fluorescent infected cells and bright field microscopy to detect phagosomes.

HIV-1に感染したライブ初代ヒトマクロファージで測定ファゴソームの移行と速度

Macrophages are phagocytic cells that play a major role at the crossroads between innate and specific immunity. They can be infected by the human ...

phagosome-migration-velocity-measured-live-primary-human-macrophages

Research

JoVE Journal

Immunology and Infection

8.1K ビュー.  Inserm U1016, Institut Cochin, CNRS UMR 8104, Université Paris Descartes, Sorbonne Paris Cité. We describe a method to measure the velocity of phagosomes moving towards the cell center in living cells infected with or without the human immunodeficiency virus (HIV) type 1, using spinning disk confocal fluorescence microscopy to identify fluorescent infected cells and bright field microscopy to detect phagosomes. 

ビデオ: Phagosome Migration and Velocity Measured in Live Primary Human Macrophages Infected with HIV-1

Genotypic Inference of HIV-1 Tropism Using Population-based Sequencing of V3

Background: Prior to receiving a drug from CCR5-antagonist class in HIV therapy, a patient must undergo an HIV tropism test to confirm that his or her viral population uses the CCR5 coreceptor for cellular entry, and not an alternative coreceptor. One approach to tropism testing is to examine the sequence of the V3 region of the HIV envelope, which interacts with the coreceptor.
Methods: Viral RNA is extracted from blood plasma. The V3 region is amplified in triplicate with nested reverse transcriptase-PCR. The amplifications are then sequenced and analyzed using the software, RE_Call. Sequences are then submitted to a bioinformatic algorithm such as geno2pheno to infer viral tropism from the V3 region. Sequences are inferred to be non-R5 if their geno2pheno false positive rate falls below 5.75%. If any one of the three sequences from a sample is inferred to be non-R5, the patient is unlikely to respond to a CCR5-antagonist.

HIV tropism can be inferred from the V3 region of the viral envelope. V3 is PCR amplified in triplicate using nested RT-PCR, sequenced, and interpreted using bioinformatic software. Samples with with 1 or more sequence(s) with low g2P scores are classified as non-R5 virus.

V3の人口ベースのシーケンシングを用いたHIV - 1トロピズムの遺伝子型推論

Background: Prior to receiving a drug from CCR5-antagonist class in HIV therapy, a patient must undergo an HIV tropism test to confirm that his or her ...

免疫・感染学

Preparation and Use of HIV-1 Infected Primary CD4+ T-Cells as Target Cells in Natural Killer Cell Cytotoxic Assays

Natural killer (NK) cells are a vital component of the innate immune response to virus-infected cells. It is important to understand the ability of NK cells to recognize and lyse HIV-1 infected cells because identifying any aberrancy in NK cell function against HIV-infected cells could potentially lead to therapies that would enhance their cytolytic activity. There is a need to use HIV-infected primary T-cell blasts as target cells rather then infected-T-cell lines in the cytotoxicity assays. T-cell lines, even without infection, are quite susceptible to NK cell lysis. Furthermore, it is necessary to use autologous primary cells to prevent major histocompatibility complex class I mismatches between the target and effector cell that will result in lysis. Early studies evaluating NK cell cytolytic responses to primary HIV-infected cells failed to show significant killing of the infected cells 1,2. However, using HIV-1 infected primary T-cells as target cells in NK cell functional assays has been difficult due the presence of contaminating uninfected cells 3. This inconsistent infected cell to uninfected cell ratio will result in variation in NK cell killing between samples that may not be due to variability in donor NK cell function. Thus, it would be beneficial to work with a purified infected cell population in order to standardize the effector to target cell ratios between experiments 3,4. Here we demonstrate the isolation of a highly purified population of HIV-1 infected cells by taking advantage of HIV-1's ability to down-modulate CD4 on infected cells and the availability of commercial kits to remove dead or dying cells 3-6. The purified infected primary T-cell blasts can then be used as targets in either a degranulation or cytotoxic assay with purified NK cells as the effector population 5-7. Use of NK cells as effectors in a degranulation assay evaluates the ability of an NK cell to release the lytic contents of specialized lysosomes 8 called "cytolytic granules". By staining with a fluorochrome conjugated antibody against CD107a, a lysosomal membrane protein that becomes expressed on the NK cell surface when the cytolytic granules fuse to the plasma membrane, we can determine what percentage of NK cells degranulate in response to target cell recognition. Alternatively, NK cell lytic activity can be evaluated in a cytotoxic assay that allows for the determination of the percentage of target cells lysed by release of 51Cr from within the target cell in the presence of NK cells.

Cytotoxicity assays to measure natural killer cell lytic responses to HIV-infected cells is limited by the purity of the target cells. We demonstrate here the isolation of a highly purified population of HIV-1 infected primary T-cell blasts by taking advantage of HIV-1 s ability to down-modulate CD4.

ナチュラルキラー細胞傷害性アッセイにおけるHIV - 1感染プライマリCD4 + T細胞を標的細胞としての準備と使用

 Natural killer (NK) cells are a vital component of the innate immune response to virus-infected cells. It is important to understand the ability of NK ...

Amplifying and Quantifying HIV-1 RNA in HIV Infected Individuals with Viral Loads Below the Limit of Detection by Standard Clinical Assays

Amplifying viral genes and quantifying HIV-1 RNA in HIV-1 infected individuals with viral loads below the limit of detection by standard assays (below 50-75 copies/ml) is necessary to gain insight to viral dynamics and virus host interactions in patients who naturally control the infection and those who are on combination antiretroviral therapy (cART). 
Here we describe how to amplify viral genomes by single genome sequencing (the SGS protocol) 13, 19 and how to accurately quantify HIV-1 RNA in patients with low viral loads (the single-copy assay (SCA) protocol) 12, 20. 
The single-copy assay is a real-time PCR assay with sensitivity depending on the volume of plasma being assayed. If a single virus genome is detected in 7 ml of plasma, then the RNA copy number is reported to be 0.3 copies/ml. The assay has an internal control testing for the efficiency of RNA extraction, and controls for possible amplification from DNA or contamination. Patient samples are measured in triplicate.
The single-genome sequencing assay (SGS), now widely used and considered to be non-labor intensive 3, 7, 12, 14, 15,is a limiting dilution assay, in which endpoint diluted cDNA product is spread over a 96-well plate. According to a Poisson distribution, when less than 1/3 of the wells give product, there is an 80% chance of the PCR product being resultant of amplification from a single cDNA molecule. SGS has the advantage over cloning of not being subjected to resampling and not being biased by PCR-introduced recombination 19. However, the amplification success of SCA and SGS depend on primer design. Both assays were developed for HIV-1 subtype B, but can be adapted for other subtypes and other regions of the genome by changing primers, probes, and standards.

Quantifying levels of HIV-1 RNA in plasma and sequencing single HIV-1 genomes from individuals with viral loads below the limit of detection (50-75 copies/ml) is difficult. Here we describe how to extract and quantify plasma viral RNA using a real time PCR assay that reliably measures HIV-1 RNA down to 0.3 copies/ml and how to amplify viral genomes by single genome sequencing, from samples with very low viral loads.

標準的な臨床アッセイによる検出限界以下のウイルス量とHIV感染個体におけるHIV - 1 RNAを増幅して定量化

Amplifying viral genes and quantifying HIV-1 RNA in HIV-1 infected individuals with viral loads below the limit of detection by standard assays (below ...

In vitro Uncoating of HIV-1 Cores

The genome of the retroviruses is encased in a capsid surrounded by a lipid envelope. For lentiviruses, such as HIV-1, the conical capsid shell is composed of CA protein arranged as a lattice of hexagon. The capsid is closed by 7 pentamers at the broad end and 5 at the narrow end of the cone1, 2. Encased in this capsid shell is the viral ribonucleoprotein complex, and together they comprise the core.
Following fusion of the viral membrane with the target cell membrane, the HIV-1 is released into the cytoplasm. The capsid then disassembles releasing free CA in the soluble form3 in a process referred to as uncoating. The intracellular location and timing of HIV-1 uncoating are poorly understood. Single amino-acid substitutions in CA that alter the stability of the capsid also impair the ability of HIV-1 to infect cells4. This indicates that the stability of the capsid is critical for HIV-1 infection.
HIV-1 uncoating has been difficult to study due to lack of availability of sensitive and reliable assays for this process. Here we describe a quantitative method for studying uncoating in vitro using cores isolated from infectious HIV-1 particles. The approach involves isolation of cores by sedimentation of concentrated virions through a layer of detergent and into a linear sucrose gradient, in the cold. To quantify uncoating, the isolated cores are incubated at 37&deg;C for various timed intervals and subsequently pelleted by ultracentrifugation. The extent of uncoating is analyzed by quantifying the fraction of CA in the supernatant. This approach has been employed to analyze effects of viral mutations on HIV-1 capsid stability4, 5, 6. It should also be useful for studying the role of cellular factors in HIV-1 uncoating.

In vitro Uncoating of HIV-1 Cores

Uncoating is an essential step in the early phase of the HIV-1 life cycle and is defined as the disassembly of the capsid shell and the release of the viral ribonucleoprotein complex (vRNP). Here, we demonstrate techniques for isolating intact cores from HIV-1 virions and for quantifying their uncoating in vitro.

HIV - 1コアのin vitroでの脱殻で

The genome of the retroviruses is encased in a capsid surrounded by a lipid envelope. For lentiviruses, such as HIV-1, the conical capsid shell is ...

Imaging of HIV-1 Envelope-induced Virological Synapse and Signaling on Synthetic Lipid Bilayers

Human immunodeficiency virus type 1 (HIV-1) infection occurs most efficiently via cell to cell transmission2,10,11. This cell to cell transfer between CD4+ T cells involves the formation of a virological synapse (VS), which is an F-actin-dependent cell-cell junction formed upon the engagement of HIV-1 envelope gp120 on the infected cell with CD4 and the chemokine receptor (CKR) CCR5 or CXCR4 on the target cell 8. In addition to gp120 and its receptors, other membrane proteins, particularly the adhesion molecule LFA-1 and its ligands, the ICAM family, play a major role in VS formation and virus transmission as they are present on the surface of virus-infected donor cells and target cells, as well as on the envelope of HIV-1 virions1,4,5,6,7,13. VS formation is also accompanied by intracellular signaling events that are transduced as a result of gp120-engagement of its receptors. Indeed, we have recently showed that CD4+ T cell interaction with gp120 induces recruitment and phosphorylation of signaling molecules associated with the TCR signalosome including Lck, CD3&zeta;, ZAP70, LAT, SLP-76, Itk, and PLC&gamma;15.
In this article, we present a method to visualize supramolecular arrangement and membrane-proximal signaling events taking place during VS formation. We take advantage of the glass-supported planar bi-layer system as a reductionist model to represent the surface of HIV-infected cells bearing the viral envelope gp120 and the cellular adhesion molecule ICAM-1. The protocol describes general procedures for monitoring HIV-1 gp120-induced VS assembly and signal activation events that include i) bi-layer preparation and assembly in a flow cell, ii) injection of cells and immunofluorescence staining to detect intracellular signaling molecules on cells interacting with HIV-1 gp120 and ICAM-1 on bi-layers, iii) image acquisition by TIRF microscopy, and iv) data analysis. This system generates high-resolution images of VS interface beyond that achieved with the conventional cell-cell system as it allows detection of distinct clusters of individual molecular components of VS along with specific signaling molecules recruited to these sub-domains.

This article describes a method to visualize formation of an HIV-1 envelope-induced virological synapse on glass supported planar bilayers by total internal reflection fluorescence (TIRF) microscopy. The method can also be combined with immunofluorescence staining to detect activation and redistribution of signaling molecules that occur during HIV-1 envelope-induced virological synapse formation.

合成脂質二重膜上でHIV-1エンベロープ誘発性ウイルス学的シナプスとシグナリングのイメージング

Human immunodeficiency virus type 1 (HIV-1) infection occurs most efficiently via cell to cell transmission2,10,11. This cell to cell transfer between ...

An In vitro Co-infection Model to Study Plasmodium falciparum-HIV-1 Interactions in Human Primary Monocyte-derived Immune Cells

Plasmodium falciparum, the causative agent of the deadliest form of malaria, and human immunodeficiency virus type-1 (HIV-1) are among the most important health problems worldwide, being responsible for a total of 4 million deaths annually1. Due to their extensive overlap in developing regions, especially Sub-Saharan Africa, co-infections with malaria and HIV-1 are common, but the interplay between the two diseases is poorly understood. Epidemiological reports have suggested that malarial infection transiently enhances HIV-1 replication and increases HIV-1 viral load in co-infected individuals2,3. Because this viremia stays high for several weeks after treatment with antimalarials, this phenomenon could have an impact on disease progression and transmission.
The cellular immunological mechanisms behind these observations have been studied only scarcely. The few in vitro studies investigating the impact of malaria on HIV-1 have demonstrated that exposure to soluble malarial antigens can increase HIV-1 infection and reactivation in immune cells. However, these studies used whole cell extracts of P. falciparum schizont stage parasites and peripheral blood mononuclear cells (PBMC), making it hard to decipher which malarial component(s) was responsible for the observed effects and what the target host cells were4,5. Recent work has demonstrated that exposure of immature monocyte-derived dendritic cells to the malarial pigment hemozoin increased their ability to transfer HIV-1 to CD4+ T cells6,7, but that it decreased HIV-1 infection of macrophages8. To shed light on this complex process, a systematic analysis of the interactions between the malaria parasite and HIV-1 in different relevant human primary cell populations is critically needed.
Several techniques for investigating the impact of HIV-1 on the phagocytosis of micro-organisms and the effect of such pathogens on HIV-1 replication have been described. We here present a method to investigate the effects of P. falciparum-infected erythrocytes on the replication of HIV-1 in human primary monocyte-derived macrophages. The impact of parasite exposure on HIV-1 transcriptional/translational events is monitored by using single cycle pseudotyped viruses in which a luciferase reporter gene has replaced the Env gene while the effect on the quantity of virus released by the infected macrophages is determined by measuring the HIV-1 capsid protein p24 by ELISA in cell supernatants.

An In vitro Co-infection Model to Study Plasmodium falciparum-HIV-1 Interactions in Human Primary Monocyte-derived Immune Cells

We have developed an in vitro malaria-HIV-1 co-infection model to study the impact of Plasmodium falciparum on the HIV-1 replicative cycle in human primary monocyte-derived macrophages. This versatile system can easily be adapted to other primary cell types susceptible to HIV-1 infection.

不定冠詞<em&gt;のin vitro</em研究する&gt;共同感染モデル<em&gt;熱帯熱マラリア原虫（Plasmodium falciparum）</emヒトの第一次単球由来の免疫細胞に&gt;-HIV-1の相互作用

Plasmodium falciparum, the causative agent  of the deadliest form of malaria, and human immunodeficiency virus type-1 (HIV-1) are among the most important ...

New Tools to Expand Regulatory T Cells from HIV-1-infected Individuals

CD4+ Regulatory T cells (Tregs) are potent immune modulators and serve an important function in human immune homeostasis. Depletion of Tregs has led to measurable increases in antigen-specific T cell responses in vaccine settings for cancer and infectious pathogens. However, their role in HIV-1 immuno-pathogenesis remains controversial, as they could either serve to suppress deleterious HIV-1-associated immune activation and thus slow HIV-1 disease progression or alternatively suppress HIV-1-specific immunity and thereby promote virus spread. Understanding and modulating Treg function in the context of HIV-1 could lead to potential new strategies for immunotherapy or HIV vaccines. However, important open questions remain on their role in the context of HIV-1 infection, which needs to be carefully studied.
Representing roughly 5% of human CD4+ T cells in the peripheral blood, studying the Treg population has proven to be difficult, especially in HIV-1 infected individuals where HIV-1-associated CD4 T cell and with that Treg depletion occurs. The characterization of regulatory T cells in individuals with advanced HIV-1 disease or tissue samples, for which only very small biological samples can be obtained, is therefore extremely challenging. We propose a technical solution to overcome these limitations using isolation and expansion of Tregs from HIV-1-positive individuals.
Here we describe an easy and robust method to successfully expand Tregs isolated from HIV-1-infected individuals in vitro. Flow-sorted CD3+CD4+CD25+CD127low Tregs were stimulated with anti-CD3/anti-CD28 coated beads and cultured in the presence of IL-2. The expanded Tregs expressed high levels of FOXP3, CTLA4 and HELIOS compared to conventional T cells and were shown to be highly suppressive. Easier access to large numbers of Tregs will allow researchers to address important questions concerning their role in HIV-1 immunopathogenesis. We believe answering these questions may provide useful insight for the development of an effective HIV-1 vaccine.

CD4+ Regulatory T cells are potent immune-modulators and serve important functions in immune homeostasis. The paucity of these cells in peripheral blood makes functional studies challenging, specifically in the context of HIV-1-infection. We here describe a method to isolate and expand functional CD4+ Tregs from peripheral blood from HIV-1-infected individuals.

HIV-1に感染した個体からの制御性T細胞を増殖するための新しいツール

CD4+ Regulatory T cells (Tregs) are potent immune modulators and serve an important function in human immune homeostasis. Depletion of Tregs has led to ...

Study of Phagolysosome Biogenesis in Live Macrophages

Phagocytic cells play a major role in the innate immune system by removing and eliminating invading microorganisms in their phagosomes. Phagosome maturation is the complex and tightly regulated process during which a nascent phagosome undergoes drastic transformation through well-orchestrated interactions with various cellular organelles and compartments in the cytoplasm. This process, which is essential for the physiological function of phagocytic cells by endowing phagosomes with their lytic and bactericidal properties, culminates in fusion of phagosomes with lysosomes and biogenesis of phagolysosomes which is considered to be the last and critical stage of maturation for phagosomes. In this report, we describe a live cell imaging based method for qualitative and quantitative analysis of the dynamic process of lysosome to phagosome content delivery, which is a hallmark of phagolysosome biogenesis. This approach uses IgG-coated microbeads as a model for phagocytosis and fluorophore-conjugated dextran molecules as a luminal lysosomal cargo probe, in order to follow the dynamic delivery of lysosmal content to the phagosomes in real time in live macrophages using time-lapse imaging and confocal laser scanning microscopy. Here we describe in detail the background, the preparation steps and the step-by-step experimental setup to enable easy and precise deployment of this method in other labs. Our described method is simple, robust, and most importantly, can be easily adapted to study phagosomal interactions and maturation in different systems and under various experimental settings such as use of various phagocytic cells types, loss-of-function experiments, different probes, and phagocytic particles.

ライブマクロファージにおけるファゴリソソーム生合成の研究

Phagocytic cells play a major role in the innate immune system by removing and eliminating invading microorganisms in their phagosomes. Phagosome ...

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the protocols described here use freshly isolated human peripheral blood mononuclear cells (PBMCs) or plasmacytoid dendritic cells (pDCs) to sense infections in either T cell line (MT4) or heterologous primary CD4+ T cells. In order to ensure proper sensing, it is essential that PBMC are isolated immediately after blood collection and that optimal percentage of infected T cells are used. Furthermore, multi-parametric flow cytometric staining can be used to confirm that PBMC samples contain the different cell lineages at physiological ratios. A number of controls can also be included to evaluate viability and functionality of pDCs. These include, the presence of specific surface markers, assessing cellular responses to known agonist of Toll-Like Receptors (TLR) pathways, and confirming a lack of spontaneous type-I interferon (IFN) production. In this system, freshly isolated PBMCs or pDCs are co-cultured with HIV-1 infected cells in 96 well plates for 18-22 hr. Supernatants from these co-cultures are then used to determine the levels of bioactive type-I IFNs by monitoring the activation of the ISGF3 pathway in HEK-Blue IFN-&#945;/&#946; cells. Prior and during co-culture conditions, target cells can be subjected to flow cytometric analysis to determine a number of parameters, including the percentage of infected cells, levels of specific surface markers, and differential killing of infected cells. Although, these protocols were initially developed to follow type-I IFN production, they could potentially be used to study other imuno-modulatory molecules released from pDCs and to gain further insight into the molecular mechanisms governing HIV-1 innate sensing.

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

Unlike cell-free HIV-1 particles, infected CD4+ T cells are effectively sensed by plasmacytoid dendritic cells (pDCs). This manuscript describes a method where peripheral blood mononuclear cells (PBMCs) or isolated pDCs are co-cultured with HIV-1 infected T cells to evaluate innate sensing by pDCs as assessed by release of type-I IFN.

HIV-1感染CD4の生得センシングを評価<sup&gt; +</sup&gt;使用して形質細胞様樹状細胞によるT細胞<em&gt;エクスビボ</em&gt;共培養系。

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the ...

Measurement of In Vitro Integration Activity of HIV-1 Preintegration Complexes

HIV-1 envelope proteins engage cognate receptors on the target cell surface, which leads to viral-cell membrane fusion followed by the release of the viral capsid (CA) core into the cytoplasm. Subsequently, the viral Reverse Transcriptase (RT), as part of a namesake nucleoprotein complex termed the Reverse Transcription Complex (RTC), converts the viral single-stranded RNA genome into a double-stranded DNA copy (vDNA). This leads to the biogenesis of another nucleoprotein complex, termed the pre-integration complex (PIC), composed of the vDNA and associated virus proteins and host factors. The PIC-associated viral integrase (IN) orchestrates the integration of the vDNA into the host chromosomal DNA in a temporally and spatially regulated two-step process. First, the IN processes the 3&#39; ends of the vDNA in the cytoplasm and, second, after the PIC traffics to the nucleus, it mediates integration of the processed vDNA into the chromosomal DNA. The PICs isolated from target cells acutely infected with HIV-1 are functional in vitro, as they are competent to integrate the associated vDNA into an exogenously added heterologous target DNA. Such PIC-based in vitro integration assays have significantly contributed to delineating the mechanistic details of retroviral integration and to discovering IN inhibitors. In this report, we elaborate upon an updated HIV-1 PIC assay that employs a nested real-time quantitative Polymerase Chain Reaction (qPCR)-based strategy for measuring the in vitro integration activity of isolated native PICs.

Measurement of In Vitro Integration Activity of HIV-1 Preintegration Complexes

This report describes an in vitro assay for measuring the human immunodeficiency virus type 1 (HIV-1) preintegration complex (PIC)-associated integration activity in the cytoplasmic extracts of acutely infected cells. The integration of PIC-associated HIV-1 DNA into heterologous target DNA is quantified by using a nested real-time polymerase chain reaction strategy.

の測定<em&gt;インビトロ</em&gt; HIV-1プレインテグレーション複合体の統合活動

HIV-1 envelope proteins engage cognate receptors on the target cell surface, which leads to viral-cell membrane fusion followed by the release of the ...

HIV-1に感染したライブ初代ヒトマクロファージで測定ファゴソームの移行と速度

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