HIV-1陽性個体の血漿からエキソソームの単離

要約

Techniques describing a gradient procedure to separate exosomes from human immunodeficiency virus (HIV) particles are described. This procedure was used to isolate exosomes away from HIV particles in human plasma from HIV-infected individuals. The isolated exosomes were analyzed for cytokine/chemokine content.

要約

エキソソームは、様々な細胞型の両方から構成的刺激の際に放出された30〜100nmのナノメートルのサイズの範囲の小さな小胞です。これらは、生物学的流体の数に見出され、タンパク質、脂質、および核酸分子の多様性を運ぶことが知られています。もともと細胞破片のための貯水池より少しであると考えられ、生物学的プロセスを調節するエキソソームのと疾患における役割がますます高く評価されています。

いくつかの方法は、細胞培養培地および生物学的流体からエキソソームを単離するために記載されています。それらの小さなサイズ、低密度、差動超遠心分離及び/または限外ろ過のエキソソームの単離のために最も一般的に使用される技術です。しかし、HIV-1感染個体の血漿は、エキソソームと大きさと密度が類似しているHIVウイルス粒子、両方が含まれています。このように、ヒト血漿中のHIVウイルス粒子からのエキソソームの効率的な分離がされています挑戦。

この制限に対処するために、我々はCantin らから変更手続きを開発しました。ら、2008ヒト血漿中HIV粒子からエキソソームを精製します。イオジキサノール速度勾配は、HIV-1陽性個体の血漿中のHIV-1粒子からエキソソームを分離するために使用されました。ウイルス粒子は、p24をELISAにより同定しました。エキソソームは、エキソソームマーカーアセチルコリンエステラーゼ（AChE）およびCD9、CD63、およびCD45抗原に基づいて同定されました。我々の勾配手順は、ウイルス粒子の自由なエキソソーム調製物を得ました。ヒト血漿からエキソソームの効率的な精製は、血漿由来のエキソソームの内容を検討し、その免疫調節の可能性やその他の生物学的機能を調査することができました。

概要

HIV-1の流行は、世界中で重大な影響を与え続けています。 2013年の時点で、全世界で約35万人がHIVと共に生きているし、これらの210万は、新たに感染した個人の¹でした。予防戦略と抗レトロウイルス治療へのアクセスの増加は、HIVの全体的な買収を減らすのに役立っています。しかし、個々の集団は、依然として、HIV ¹の取得の上昇を経験しています。したがって、この流行に対処するための継続的な努力が必要とされています。

HIV疾患進行の最も強力な予測因子の一つは、慢性免疫活性化^{（CIA）2-10}です。 ; I型IFN ¹¹のI）連続樹状細胞の生産：Tリンパ球の表面に検出可能なサイトカインおよび高い発現マーカーの持続的に高いレベルによって定義され、CIAはに起因していますHIVタンパク質のTat、ネフとgp120の¹²で駆動される（ii）の直接免疫活性化。 （ハ腸管関連する免疫細胞⁶における細菌タンパク質の）転。しかし、HIV感染の慢性、全身性の免疫活性化の基礎となる正確なメカニズムは完全に解明されていません。

我々の研究グループなどは、HIV病原^15-18におけるエキソソームの役割を実証しました。当社グループは、Nefタンパク質がエキソソーム¹⁵に感染した細胞から分泌されることが決定しており、エキソソームネフ（exNef）はナノグラムレベル¹⁸でのHIV感染個体の血漿中に存在します。我々はまた、単球/マクロファージはexNef誘導アポトーシスに対して不応性であった。exNefにさらされたCD4 + T細胞は、CXCR4経路^19、20に依存して活性化誘導細胞死をもたらしたことを傍観者を示すが、変化した細胞機能やサイトカインの発現を示しました。最近、我々のグループは、HIV感染個体の血漿から単離したエキソソームは、炎症性サイトカインの様々なが含まれて示されています。フーrther、ナイーブ及び中央記憶CD4 +及びCD8 + T細胞上のCD38の発現を誘導したHIV感染患者からの血漿由来エキソソームに露出ナイーブ末梢血単核細胞。これはおそらく、バイスタンダー細胞の活性化^{21を介して}全身性炎症およびウイルス増殖に寄与し、エキソソームは、HIVの病因に重要な役割を果たすことを示唆しています。

HIVの病因におけるエキソソームの役割を調査するには、1つの課題は、HIV粒子から効率よく分離エキソソームエキソソームコンテンツを維持しながら、ならびにそれらの機能的な免疫調節能力に技術を開発しています。いくつかの方法は、細胞培養および生物学的流体^22,23からエキソソームを単離するために記載されています。サイズが小さいため、低密度（エキソソームは、1.15の密度で浮遊する- 1.19グラム/ ml）を、差動超遠心分離及び/または限外ろ過がエキソソームの単離²³のために最も一般的に使用される技術です。しかし、HIV感染細胞培養上清及び患者の血漿エキソソームおよびHIV-1ウイルス粒子の両方を含みます。エキソソームおよびHIV-1粒子は、サイズおよび密度の両方で非常に類似しています。あるいは、CD63、CD45、およびCD81のようなユニークなエキソソームのマーカーの発現を利用して、エキソソームは、免疫親和性捕捉法^{23を用いて、}単離されています。この手順は、エキソソームからウイルスを分離することができます。しかし、この手法の欠点は、培養中のエキソソームの免疫能力の評価を妨げる可能性精製エキソソームに対する抗体のタイト結合、です。

これらの制限に対処するために、我々は、イオジキサノール速度勾配を使用してHIVのCantinから修飾されたヒト血漿中の粒子および共同研究者²²からエキソソームを精製するための手順を開発しました。ウイルス粒子が高に偏析一方エキソソームは、イオジキサノール勾配の低密度/上側画分に分離することが見出されました密度/下分画。ウイルス粒子は、p24をELISAによって同定し、エキソソームは、エキソソームマーカーのAChE、CD9、CD63、およびCD45を用いて同定しました。収集上部低密度分画は、HIV-1のp24の汚染に対して陰性であったエキソソームを含んでいました。ヒト血漿中HIV粒子からエクソソームの効率的な精製および分離は、ヒト血漿、並びにそれらの免疫調節の可能性の調査とHIV-1病因におけるエキソソームの診断および予後値由来エキソソームの内容を正確に検査することができます。

プロトコル

A general diagram of the exosome isolation and purification procedure is provided in Figure 1. Whole blood was obtained from healthy volunteer donors and from HIV-positive individuals not receiving antiretroviral theraoy attending the Hope Clinic of Emory University and the Infectious Disease Program of Grady Health System in Atlanta, Georgia. This study was approved by the institutional review boards of Emory University and Morehouse School of Medicine. All persons participating in the study gave written and informed consent.

1. Preparation of Exosomes from Blood Plasma

1. Human Blood Collection and Processing
   1. Collect 10 ml of peripheral blood by venipuncture in EDTA blood collection tubes (containing 18 mg potassium EDTA), and gently invert five times to mix.
   2. Centrifuge the blood collection tubes at 1,000 x g for 20 min at RT to pellet blood cells. Use a sterile pipette to transfer the plasma fraction (4-5 ml) to a 25 ml conical tube. Dilute the plasma with 10 ml of 1 X PBS. Discard blood cells (red blood cells and white cells, also known as PBMC) in an appropriately marked container for biohazard waste.
      NOTE: In the case of uninfected donor blood samples, the PBMC can be reserved for further use (see procedure IV.2, below).
   3. Store the plasma samples at 4 ^oC for short term (2-3 days) or at -80 ^oC for longer term storage.
      NOTE: Bring any frozen plasma samples to 4 ^oC before further processing.
2. Preparation of Exosome Fraction from Plasma
   1. Centrifuge plasma at 10,000 x g for 30 min at 4 °C to remove cellular debris. Use a sterile serological pipette to transfer the cleared plasma supernatant to a clean 25 ml ultracentrifuge tube. Discard the pellet in the biohazard container.
   2. Centrifuge the cleared plasma at 100,000 x g for 2 hr at 4 °C to remove large vesicles. Remove the 100,000 x g supernatant carefully by pipetting, and discard in biohazard waste. Resuspend the 100,000 x g pellet in 1 ml of 1X PBS in a clean tube and incubate at RT for 30 min, swirling gently to dislodge and separate particles.
   3. Wash the suspended 100,000 x g exosome/virus pellet by adding 25 ml of PBS. Gently invert the tube five times to mix, then centrifuge again at 100,000 x g for 2 hr at 4 °C. Discard the PBS wash solution in the biohazard waste container.
   4. Resuspend the 100,000 x g pellet in 1 ml of 1 X PBS and incubate at RT for 30 min swirling gently to dislodge and dissolve the pellet.
      NOTE: If it is not possible to proceed directly to the iodixanol gradient step, store the pellet, containing exosomes and virus particles, at 4 °C for 1-2 days until the iodixanol gradient step.

2. Purification of Exosomes

1. Generate 6%-18% velocity gradients of iodixanol using a dual-chamber gradient former apparatus.
   1. Prepare 6% and 18% solutions of the iodixanol reagent, supplied as a 60% solution in water, by dilution in PBS. Pipet 5.5 ml of the 18% solution into the stirred chamber, and pipet 5.5 ml of the 6% solution into the reservoir chamber. Turn on the stirrer, open the stopcock, and allow each gradient to flow into a 14 ml ultracentrifuge tube.
      NOTE: Either use immediately or store prepared gradients O/N at 4 °C prior to use.
   2. Carefully layer 1 ml of the exosome/virus solution onto the top of each 11 ml gradient. Centrifuge gradients at 250,000 x g for 2 hr at 4 °C, using a SW40Ti swinging bucket rotor.
   3. Label twelve (12) 1.5 ml microcentrifuge tubes. Remove 1.0 ml from the top of the gradient and transfer to tube #1. Transfer the remaining 1ml fractions to tubes 2-12 in sequential order.
      NOTE: The topmost fraction will thus be #1, the bottom fraction, #12. Store the gradient fractions at 4 °C. The purified exosomes, which should be in the fractions 1-3 at the top of the gradient, are stable for 3-4 weeks when stored at 4 °C.

3. Exosome Characterization

1. Acetylcholinesterase (AChE) Activity Assay
   1. Prepare 100 mM substrate stock by mixing acetylthiocholine iodide 28.9 mg in 1 ml of 1X PBS. Store substrate stock at -20 °C up to 1 month.
   2. Prepare 10 mM color indicator stock by mixing benzoic acid 39.6mg and sodium bicarbonate 15 mg in 10 ml of 1X PBS. Store color indicator stock at 4 °C up to two weeks.
   3. Prepare assay reagent by mixing 1X PBS, substrate, and color indicator in a ratio of 100:2:5 (for example, 10 ml 1X PBS + 200 μl substrate + 500 μl color indicator).
   4. Transfer 50 μl from each 1.5 ml gradient fraction tube (labeled 1-12, from procedure III.3) to the wells of a 96-well microtiter plate. Prepare duplicate wells for each gradient sample.
   5. Prepare a set of standards by first making a 2000 μU/ml AChE stock in PBS. Make eleven (11) 2-fold serial dilutions of this stock and add 50 μl of each dilution to a single well of a microtiter plate, so that standard #1 = 2,000 μU/ml, #2 = 1,000 μU/ml, #3 = 500 μU/ml, etc. until #12 = 0.98 μU/ml. The twelve AchE standards will thus occupy a single row of a 96-well assay plate.
   6. Add 200 μl of assay reagent mixture to each well and incubate 20 min (in the dark) at RT to allow development of the colored reaction product. Measure AChE activity at a wavelength of 450 nm using a fluorescent microplate reader.
      NOTE: The percentage of starting material remaining after exosome purification is assessed by AchE assay of the unfractionated plasma.
2. Immunoblot Analysis
   1. Separate proteins in gradient fractions 1-12 (from procedure 2.1.3) by SDS-PAGE on 4-20% Tris-HCl precast gels at 100V x 1 hr. Transfer proteins in the gel to a nitrocellulose membrane using an electro-blotting transfer cell according to the manufacturer's instructions. Allow the transfers to blot for 12-16 hr (O/N) at 350V.
   2. Remove the membrane from the blotting apparatus and wash in Tris-Buffered Saline (TBS) for 5 min. Block with 5% nonfat milk in TTBS (TBS with 0.1% Tween 20) for 1 hr by shaking at RT.
   3. Incubate the membrane with primary antibodies specific for exosomes (CD9, CD45, CD63) or HIV-1 capsid protein (p24) in 5% nonfat milk with shaking at 4 °C for 12-16 hr (O/N). Dilutions of primary antibodies for immunoblots range from 1:2,000 -1:5,000.
   4. Wash the blot in TTBS for 20 min, followed by incubation with a 1: 2,000 dilution of horseradish peroxidase (HRP)-conjugated to anti-IgG (secondary antibody), in 5% nonfat milk for 1 hr at RT. Wash the blot 3 times with TTBS, 10 min per wash.
   5. Incubate the blot with luminol substrate reagent according to the manufacturer's directions, to generate a chemiluminescent signal from the HRP-labeled proteins Detect the chemiluminescent signal using an electronic imaging system with CCD camera.
   6. Save the images as TIFF files which can be viewed in Adobe Photoshop. Perform densitometry analysis of bands using ImageJ software (National Institutes of Health, Bethesda, MD).
3. Cytokine Assay
   1. Prior to the cytokine assay, disrupt immune complexes that are usually present in human plasma by acid dissociation and lyse the exosomes by detergent treatment. Assay both starting plasma samples and purified exosome preparations.
      1. To 100 μl of human plasma add 100 μl 0.33 N HCL and incubate at 37 °C for 1 hr. Add 100 μl 0.33 N NaOH to neutralize acid-treated plasma. Add Triton X-100 to both the neutralized plasma and purified exosome samples, to a final concentration of 1%, to cause lysis of exosomes.
   2. For this assay (a fluorescent bead-based procedure), use magnetic beads pre-coated with antibodies by the manufacturer. Use a custom-designed panel of anti-human cytokine antibody-coated beads; listed in Table 1. Vortex the antibody-coated magnetic beads from the cytokine assay kit for 30 sec and add 50 μl beads to each well of a 96-well plate to be used in the assay.
   3. Prepare a dilution series of standards in plasma standard buffer (both contained in the cytokine assay kit) as described in the instruction manual for the cytokine assay kit, to generate an 8-point standard curve.
   4. Add 150 μl of 1X wash buffer (from kit) to each well and wash the beads using a magnetic bead washer, as described in the manual. Add 25 μl plasma assay buffer (from kit) to each well. Add 25 μl of standards and samples to all wells used in the assay. Add 25 μl of plasma sample buffer to negative control wells. Seal and shake the plate at 700 rpm for 60 min at RT and incubate O/N at 4 °C.
   5. Add 150 μl of 1X wash buffer to each well and wash the plate (as in step 3.3.4, above). Add 25 μl of detection antibodies into each well, seal and shake plate at 700 rpm for 30 min at RT. Repeat the washing step.
   6. Add 50 µl of streptavidin solution (SAPE, from kit) into each well, seal and shake plate at 700 rpm for 30 min at RT. Repeat plate washes as in 3.3.4.
   7. Add 120 µl of reading buffer (from kit) into each well and shake on a laboratory tabletop shaker at 700 rpm for 5 min at RT to allow the fluorescent signal to develop. Read plate on the plate reader as per the manufacturer's instructions.
   8. In order to account for the amount of exosomes lost during the isolation procedure, use the following equation: [(original reading / plasma volume) x 66.6= adjusted cytokine reading].

4. Assay for Immunomodulatory Potential

1. Preparation of Culture Medium with Exosome-depleted Fetal Bovine Serum
   1. Centrifuge 500 ml of fetal bovine serum (FBS) at 100,000 x g for 20 hr at 4 °C to pellet contaminating exosomes and microvesicles. Carefully remove and save the exosome-depleted FBS supernatant. Discard the pellet in the biohazard waste.
   2. Combine 20% of the exosome-depleted FBS supernatant with 500 ml of Roswell Park Memorial Institute 1640 (RPMI 1640) medium. Filter the medium through a 0.45 µm membrane filter.
   3. Add streptomycin (100 U/ml), penicillin (100 U/ml), L-glutamine (2 mM), HEPES-buffered saline solution (10 µM), and IL-2 (20 U/ml) to the filtered medium.
2. Cell Culture and Exosome Exposure
   1. Expose exosomes to donor peripheral blood mononuclear cells (PBMC) obtained from healthy clinic volunteers. Suspend PBMC in prepared culture medium and count the cells using a cell/particle counter (according to the manufacturer's standard method).
      NOTE: The PBMC are obtained during the plasma preparation described above (step 1.2).
   2. Using prepared medium from procedure 4.1.3, co-culture 3.0 x 10⁶ (PBMC) with 1 μg/ml of pooled exosomes from three (3) HIV-1 seropositive or from three (3) HIV-1 seronegative individuals in a total volume of 1 ml in the wells of a 12-well culture plate (with lid). Incubate cultures for 48 hr at 37 °C.
   3. Prepare untreated PBMC cultures and cultures treated with Concanavalin A (Con A; 5 µg/ml) to serve as negative and positive controls, respectively, as described above (4.2.2). Incubate all PBMC cultures for 48 hr at 37 °C.
   4. Immediately prior to harvesting the cells, prepare dilutions of the following fluorochrome-conjugated monoclonal antibodies: Alexa Fluor 700-labeled anti-CD3 (1:400 dilution), allophycocyanin (APC)/cyanine 7 (Cy7)-labeled anti-CD4 (1:400), peridinin chlorophyll protein complex-labeled anti-CD4 (1:400), V450-labeled anti-CD8 (1:400), biotin-labeled anti-CD45RA (1:1,000), phycoerythrin (PE)/Cy7-labeled anti-CD62L (1:1,000), PE/cyanine 5 (Cy5)-labeled anti-CD38 (1:200), PE-Texas Red-labeled anti-streptavidin (1:2,000), and PE/Cy5-labeled mouse IgG1K isotype control (1:200).
   5. After the 48 hr incubation period, harvest the PBMC. Wash PBMC in PBS to remove exosomes and stain the cells by incubation for 1hr at 4 °C with individual fluorochrome-tagged antibodies. Analyze the stained PBMC by flow cytometry to quantify the expression of chemokines (as described ²¹).

結果

Exosomes are efficiently purified from HIV-1 positive human plasma. Isolated exosomes, identified by acetylcholinesterase (AChE) activity, segregated in lower density fractions 1-3 at the top of the iodixanol gradients, whereas virus particles, identified by HIV-1 antigen p24, segregated in the higher-density fractions (10-12, near the bottom). The presence of exosomes was further confirmed by immunoblot identification of exosome markers AChE, CD9, CD45, and CD63, and by ...

ディスカッション

Chronic immune activation (CIA) and CD4+ T cell depletion are two important hallmarks of HIV-1 infection. They have been established as predictors for pathogenesis, with CIA being the best predictor. However, the underlying mechanisms driving chronic systemic immune activation and CD4+ T cell decline still have not been fully elucidated. We and other labs have developed firm evidence that exosomes secreted from HIV-1 infected cells play a role in both hallmarks.

The continuing interest in both...

資料

Name	Company	Catalog Number	Comments
BD Vacutainer EDTA tubes (10ml)	Becton Dickinson	368589	pink top tubes
Lymphoprep Ficoll reagent	Cosmo Bio	AXS-1114545
Optiprep iodixanol reagent	Sigma	D1556
14ml ultracentrifuge tubes	Beckman Coulter	344060	ultraclear tubes
Gradient Former Model 485	BIO-RAD	165-4120
Acetylthiocholine iodide	Sigma	1480
Benzoic Acid	Sigma	D8130
Sodium Bicarbonate	Sigma	S5761
Acetyl Cholinesterase	Sigma	C3389
96-well clear microtiter plate	Medical Supply Partners	TR5003
SpectraMax 190 microplate reader	Molecular Devices	190	Fluorescent plate reader
Criterion Gel Electrophoresis Cell	BIO-RAD	165-6001
Transblot Gel	BIO-RAD	170-3910
Transfer Cell
Tris-HCl Criterion precast gels	BIO-RAD	567-1093
Anti-CD45 antibody	Abcam	Ab10558
CD63 Antibody (H-193)	Santa Cruz Biotech, Inc.	SC-15363
CD9 Antibody (H-110)	Santa Cruz Biotech, Inc.	SC-9148
Rabbit pAb p24 HIV-1	ImmunoDX, LLC	1303
Nitrocellulose membrane	BIO-RAD	G1472430
Tris Buffered Saline	BIO-RAD	170-6435
HRP-conjugated IgG (H+L) secondary antibody	Thermo Scientific	31460	Goat-Anti-Rabbit
HRP-conjugated IgG (H+L) secondary antibody	Thermo Scientific	31430	Goat-Anti-Mouse
Western Blotting Luminol Reagent	Santa Cruz Biotech, Inc.	SC-2048
GE LAS-4010 Imager	GE Healthcare	LAS-4010
Human Procarta Cytokine Immunoassay Kit	Affymetrix	N/A	Custom immunoassay panel
Bio-Plex 200 Immunobead Reader	BIO-RAD	171-000201
Coulter Z2 Particle Counter	Beckman Coulter	383552	Cell counter
Alexa Fluor 700-labeled anti-CD3	BD Bioscience (UCHT1)	300424
APC/Cy7-labeled anti-CD4	Biolegend (OKT4)	317418
PerCP-labeled anti-CD4	BD Bioscience (RPA-T8)	550631
V450-labeled anti-CD8	BD Bioscience (RPA-T8)	560347
Biotin-labeled anti-CD45RA	BD Bioscience (HI100)	555487
PE/Cy7-labeled anti-CD62L	Biolegend (DREG-56)	304822
PE/Cy5-labeled anti-CD38	Biolegend (HIT2)	303508
APC/Cy7-labeled anti-HLADR	Biolegend (L243)	307618
PE-Texas Red-labeled anti-streptavidin	BD Bioscience	551487
PE/Cy5-labeled mouse IgG1K	Biolegend (MOPC-21)	400116
APC/Cy7-labeled mouse IgG2aK	Biolegend (MOPC-173)	400229