Name: legionella pneumophila outer membrane vesicles: isolation and analysis of their pro-inflammatory potential on macrophages
Uploaded: 2017-02-22T13:00:00.000+00:00
Duration: 8 min 34 s
Description: Watch this Scientific Journal Video about Legionella pneumophila Outer Membrane Vesicles: Isolation and Analysis of Their Pro-inflammatory Potential on Macrophages at JoVE.com

我们感谢马库斯Schnare教授为我们提供了TLR2 - / -和TLR2 / 4 - / -小鼠和卡斯滕Kirschning教授博士TRIF / MyD88的- / -小鼠。这项工作的份资助的德国联邦联邦教育与研究部（E:生物miRSys - FKZ 0316175B，E:地中海CAPSYS - FKZ 01X1304E; http://www.bmbf.de/），德意志研究联合会（SFB / TR-84; http://www.sfb-tr84.de/）和Hessisches Ministerium献给Wissenschaft UND孔斯特（LOEWE医疗RNomics - FKZ 519/03 / 00.001-（0003）; http://www.proloewe.de/medicalrnomics）所有BS。

1.准备中型和琼脂平板<ol><li>准备1升肉汤培养基（YEB）的。溶解10克ACES和在900毫升的蒸馏水10g酵母提取物。将pH调节至6.9，用KOH（5N）。添加L-半胱氨酸（0.4克在10毫升的蒸馏水中）和10毫升的Fe的10毫升（NO 3）3 x9H 2 O（0.25克10毫升蒸馏水）。填充到1L蒸馏水，并过滤灭菌溶液（孔径:0.22微米）。保存在4℃。 </li><li>制备缓冲炭酵母提取物（BCYE）琼脂平板上。溶解10克ACES和在900毫升的蒸馏水10g酵母提取物。将pH调节至6.9，用KOH（5N）。加入琼脂15克和2.5克活性炭。填充到1升用蒸馏水和高压釜中。 <ol><li>添加L-半胱氨酸的10毫升（0.4克在10毫升的蒸馏水中）和10毫升的Fe（NO 3）3 x9H 2 O（0.25克10毫升的蒸馏水中，两个过滤灭菌通过0.22μm的孔）到冷却的BCYE（约50℃）。在4℃下倾板和存储。 </li></ol></li></ol> 2.培养嗜肺军团菌 <ol><li>上BCYE琼脂板上传播嗜肺军团菌菌株科比（野生型，WT）和在37°C孵育他们3天。接种10毫升YEB中的一个外径的0.3与来自预培养板嗜肺军团菌 600;孵育在6小时的旋转摇床（150rpm）下在37℃下的细菌。 </li><li>通过在血琼脂平板扩频100μL的悬浮液的验证液体培养的纯度。在37℃孵育过夜。 </li><li>剩余的液体培养中添加90毫升新鲜的YEB培养基，并孵育在旋转摇动器（37℃和150rpm下）到达的OD 3.0-3.5 600，其需要大约16-20小时。 </li></ol> 3.准备和量化嗜肺军团菌的OMV 注:执行以下所有离心步骤小号在无菌条件下，并在4℃。 <ol><li>离心培养液以4,000×g离心20分钟以沉淀细菌。将上清转移到新的离心管中，弃去细菌沉淀，并重复离心分离（4000×g离心20分钟）。重复此步骤一次。 </li><li>无菌过滤器的剩余的上清液两次（孔径:0.22微米）。传送无菌上清液超速离心管和超速离心机以100,000×g离心3小时。 </li><li>倒出上清液并丢弃它。重悬在无菌磷酸盐缓冲盐水（PBS）和超速离心（100,000×g离心3小时）的监查沉淀以除去污染性蛋白和LPS。 </li><li>弃上清，悬浮颗粒OMV在无菌PBS 500微升。条纹20微升在血琼脂平板并在BCYE琼脂板上以排除所制备的小泡的细菌污染。孵育3天（两个血琼脂平板过夜，并在BCYE琼脂板上在376; C）。 </li><li>量化从使用根据制造商的说明一个双金鸡宁酸测定法OMV制备得到的蛋白质量。 注:L的毫升100 菌培养物的浓度通常为1微克/微升。存放在-20°C的准备和量化的OMV。 </li></ol> 4.预巨噬细胞治疗<ol><li>制备THP-1细胞。 注:THP-1是从白血病患者来源的单核细胞系。 <ol><li>添加2×10 5 THP-1细胞每24个孔，并通过加入20nM的佛波醇12-肉豆蔻酸酯13-乙酸酯（PMA）为巨噬细胞样细胞区分。孵育在37℃下24小时。 </li><li>用新鲜培养基的500微升更换培养基并孵育另外24小时对THP-1细胞的最佳培养基组成的RPMI 1640高葡萄糖补充有10％胎牛血清的。 </li></ol></li><li>隔离的鼠骨髓衍生巨噬细胞（mBMDM），如在参见描述ENCE 20。 </li><li>治疗THP-1巨噬细胞或mBMDM用OMV的。 <ol><li>解冻在步骤3中制备的OMV的和根据它们的蛋白量（0.1，1和10微克/毫升）的人或鼠巨噬细胞添加它们。孵育OMV的巨噬细胞，在37℃下进行至少20小时。使用上清液ELISA或第5步前进。 </li></ol></li></ol> 5.感染巨噬细胞和评估细菌复制与菌落形成单位（CFU）分析<ol><li>从步骤2.1使用嗜肺 ，经预处理的THP-1细胞或mBMDM来自步骤4.3，和不进行预处理的巨噬细胞作为对照（2×10 5/24个孔）。不交换的媒介。 </li><li>感染THP-1细胞与嗜肺军团菌科比WT和mBMDM与嗜肺科比的鞭毛蛋白缺乏突变体（两者以感染复数为0.5（MOI）; 1×10 5 嗜肺 / 24孔）并孵育24和48小时，分别。准备博个嗜肺军团菌科比（WT或鞭毛蛋白缺乏突变体）如步骤2.1所述。 </li><li>裂解的细胞在通过加入皂苷其培养基（最终浓度:0.1％）和孵育在37℃下5分钟。 </li><li>通过移液重悬细菌和悬浮液转移到反应容器中。准备嗜肺军团菌含媒体无菌PBS系列稀释。 </li><li>条纹上BCYE琼脂板上所需的稀释液的50微升，并且在37°C孵育3天。 </li><li>视觉计算形成的殖民地。计算CFU <img alt="式（1）" src="/files/ftp_upload/55146/55146eq1.jpg" />规格化的CFU计数结果为不进行预处理，但感染的巨噬细胞，其被设置为100％。 </li></ol>

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Novartis.com Available from: <a target="_blank" href="https://www.novartis.com/news/media-releases/novartis-bexsero%C2%AE-vaccine-approved-fda-prevention-meningitis-b-leading-cause">https://www.novartis.com/news/media-releases/novartis-bexsero%C2%AE-vaccine-approved-fda-prevention-meningitis-b-leading-cause</a> (2016)</li></ol>

The authors have nothing to disclose.

细菌病原体和它们的靶细胞中的这些膜泡的影响的OMV目前正在深入研究。例如， 产气荚膜梭菌来源的OMV的诱导巨噬细胞分泌细胞因子，B淋巴细胞可以从莱姆病螺旋体的OMV被激活， 幽门螺杆菌 -released膜小泡可以在胃上皮细胞21，22，23采取行动。 嗜肺军团菌 ，细胞内病原体可诱导非典型肺炎的严重形式，也释放OMV的是能够激活肺上皮细胞和巨噬细胞16，19。在这里，我们提出了从液体培养嗜肺军团菌的OMV的小规模隔离研究的OMV在肺炎的潜在作用进行详细的协议。关键是在无菌康迪特工作离子和排除以获得纯嗜肺军团菌从其他细菌的污染来源的OMV制备。的OMV的隔离包括为了防止所得到的OMV粒料与嗜肺的污染，即使这降低了OMV产量，因为最大的OMV由该过滤步骤丢失通过0.22μm的微孔过滤步骤。 此外，我们测试了人类和小鼠巨噬细胞的隔离囊泡和感染细胞与嗜肺军团菌的响应更接近的军团菌肺炎，那里的OMV是由细菌外15日发布的LCV里面的情况。根据在孵育24小时后的体外人类巨噬细胞的感染的自由OMV量所采用的OMV剂量已被估计（在参考20描述）。对其他受体细胞的刺激或在体内实验中，其他OMV剂量可能是必要的，并且必须被建立。的嗜肺军团菌的OMV的效果分析表示前进到由雅格和施泰纳特24中描述的协议。 在这里，PMA分化的THP-1细胞作为肺泡巨噬细胞模型，由于主要人力物力有限的可用性。在加入PMA的区分单核细胞THP-1细胞为巨噬细胞样细胞25。此外，它们是嗜肺军团菌一个知名模特细胞系研究26。除了这种人类单核细胞系中，使用mBMDM细胞。 mBMDM被广泛用于嗜肺军团菌 27，28，29的影响的研究接受。利用基因敲除针对不同的TLR或其他蛋白质的可能性，使他们学习OMV效果的宝贵工具。在ORD器来降低每个实验小鼠的量，mBMDM来代替由于巨噬细胞的局限性肺泡巨噬细胞。关键的实验可能需要进行验证肺泡巨噬细胞。 除超速离心的本文所述协议来净化OMV的，它是能够进行密度梯度离心，它包括在由Chutkan 等的协议 30。这可以提高所得OMV制备的纯度，降低共纯化的蛋白聚集体，鞭毛蛋白，和LPS的量。所得到的OMV制备物的纯度可以通过透射电子显微镜或通过纳米粒子追踪分析的质量控制的补充步骤进行分析。这可以提供定量的另一种手段，超出这里提出的蛋白质测量程序。任选地，脂多糖浓度可以通过鲎变形细胞溶解物测试进行分析。如果OMV产量低，一经离心过滤器额外的浓缩步骤可以执行，这是不是在这里完成的。如果产量低于预期，OMV的被丢弃。 由于持续的努力阐明的OMV背后的生物学机制和功能的一部分，不同压力条件对OMV生产的影响可能进行测试。营养缺乏，改变培养温度，或暴露于有害物质可能对OMV分泌31产生影响。可能的应力条件在由Klimentova和Stulik 32的协议进行讨论。此外，可产生高血糖或hypovesiculating 嗜肺军团菌突变体。不同的OMV制剂然后可以在感染实验来分析与巨噬细胞，人肺组织外植体（在参考17中所述），或甚至在体内模型。另外的OMV在天然免疫信号中的作用，它们在细菌传播的影响力可以通过实验来解决。此外，各种先天免疫信号级联的影响可能通过使用鼠敲除细胞或CRISPR / Cas9击倒在人类细胞系的产生进行分析。在OMV的这个基本的研究将有助于新疫苗策略，已经存在由脑膜炎奈瑟菌 33传送乙脑的发展。 从上OMV分离和表征的协议开始，可以应用这对其它革兰氏阴性菌和其他宿主细胞;它只需要进行调整，以细菌的液体培养生长。通过Chutkan 等发表的协议。提供了来自大肠杆菌和绿脓杆菌 30产生的OMV的详细信息。培养不应以避免在裂解细菌的增加和污染蛋白和mem到达晚固定相branes。此外，用于宿主细胞的刺激OMV剂量需要根据OMV的本的过程中在体内感染的量来确定的，同时仍确保细胞毒性的低速率。以这种方式，的OMV的病理作用，它们对物种间的通信，以及宿主 - 病原体相互作用的影响可以被检查。 为了进一步研究嗜肺军团菌的OMV的肺炎中的作用，就需要有足够的产量和可比感染实验标准化OMV准备。该协议将有助于标准化分离程序，并监查研究扩展到其它的革兰氏阴性细菌和其他宿主细胞。此外，研究将在体外的知识，这可以用来延长实验在体内设置从详细受益。在未来，该协议可以扩展到从主生物材料的OMV，如血清或支气管LAV的隔离年龄液，以深入了解的OMV的生理条件下释放的成分。这将有助于确定OMV组成的关键参数，并了解在体外的OMV -生成的属性。

<html>细菌可以通过不同的机制1分泌致病因素。除了众所周知的分泌系统，革兰氏阴性细菌可以交换信息，并通过外膜囊泡（OMV的），这是小的，球体囊泡10-300纳米的粒径和具有双层膜结构传送的毒力因子。它们分泌的各种生长环境（液体培养，固体培养和生物膜），并在各生长阶段2，3。 OMV的是运输的重要手段（ 例如，蛋白质，粘附，毒素，和酶，以及对于LPS，该监查表面上找到）4。腔内货物免受蛋白水解降解，所以它能够在长距离行事，以及囊泡可在体液和远处器官5,6发现，"外部参照"&gt; 7，8。它们不仅可以识别和真核细胞9,10占用，但此外，它们能够促进细菌及其侵袭的结合到宿主细胞4。 嗜肺军团菌 （ 嗜肺军团菌 ）是一种革兰氏阴性细菌，可以释放的OMV。在人的肺，它主要感染肺泡巨噬细胞，尽管它的自然宿主是淡水变形虫11。一个嗜肺军团菌感染可引起军团病，肺炎12是一种严重的。在宿主细胞中它的块吞噬体 - 溶酶体融合。它还募集宿主细胞器，由此复制小生， 军团含液泡（LCV），形成13，14。溶酶体降解被抑制不仅效应蛋白TRAnslocation经由IV型分泌系统，也受到的OMV 15的释放。 的OMV的从细菌培养物中纯化需要分析其对受体细胞的效果。早期的研究集中于嗜肺军团菌的OMV的蛋白质含量和在囊泡上肺泡上皮细胞16，但与人肺组织移植后的研究的影响证明了嗜肺 OMV的由肺泡巨噬细胞17吸收。 作为OMV的本病原体相关分子模式（PAMP）和其他细菌抗原，它们可能对感染真核细胞的影响和调节宿主的免疫反应18。 嗜肺军团菌的OMV迅速与宿主细胞膜融合，而且，他们激活膜质TLR2 19。因为已知的是L. pneumophILA OMV的刺激巨噬细胞和上皮细胞的促炎方式16，图 17，我们分析的OMV对感染过程在人类和小鼠巨噬细胞的影响。 在这里，我们描述了嗜肺军团菌的培养的协议在液体培养通过差超速离心对分泌的OMV隔离并评估囊泡上真核宿主细胞的影响，无论是直接或以下的感染。

<table><tbody><tr><td>10 cm Petri dish</td><td>Sarstedt AG &amp;amp; Co KG (Nuembrecht, Germany)</td><td>82.1473</td><td></td></tr><tr><td>70 Ti rotor</td><td>Beckman Coulter Incorporation (California, USA)</td><td>337922</td><td></td></tr><tr><td>ACES</td><td>Carl Roth GmbH &amp;amp; Co KG (Karlsruhe, Germany)</td><td>9138.2</td><td></td></tr><tr><td>activated charcoal</td><td>Carl Roth GmbH &amp;amp; Co KG (Karlsruhe, Germany)</td><td>X865.2</td><td></td></tr><tr><td>agar-agar, Kobe I</td><td>Carl Roth GmbH &amp;amp; Co KG (Karlsruhe, Germany)</td><td>5210.2</td><td></td></tr><tr><td>Columbia agar with 5% sheep blood</td><td>Becton Dickinson GmbH (Heidelberg, Germany)</td><td>254005</td><td></td></tr><tr><td>cuvettes</td><td>Sarstedt AG &amp;amp; Co KG (Nuembrecht, Germany)</td><td>67.742</td><td></td></tr><tr><td>ELISA (human)</td><td>BD OptEIA&amp;trade;; Becton Dickinson GmbH (Heidelberg, Germany)</td><td>IL-8: 555244 IL-6: 550799</td><td></td></tr><tr><td>ELISA (murine)</td><td>DuoSet, R&amp;amp;D (Minneapolis, USA)</td><td>CXCL1: DY453-05</td><td></td></tr><tr><td>Fe(NO&lt;sub&gt;3&lt;/sub&gt;)&lt;sub&gt;3&lt;/sub&gt;x9H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Carl Roth GmbH &amp;amp; Co KG (Karlsruhe, Germany)</td><td>5632.1</td><td></td></tr><tr><td>Fetal calf serum (FCS)</td><td>Life Technologies GmbH (Darmstadt, Germany)</td><td>10270-106</td><td></td></tr><tr><td>Heracell 240i CO&lt;sub&gt;2&lt;/sub&gt; incubator</td><td>Thermo Fisher Scientific Germany BV &amp;amp; Co KG (Braunschweig, Germany)</td><td>40830469</td><td></td></tr><tr><td>Heraeus Multifuge X3R</td><td>Thermo Fisher Scientific Germany BV &amp;amp; Co KG (Braunschweig, Germany)</td><td>75004515</td><td></td></tr><tr><td>Inoculation loop&amp;nbsp;</td><td>Sarstedt AG &amp;amp; Co KG (Nuembrecht, Germany)</td><td>86.1567.010</td><td></td></tr><tr><td>KOH</td><td>Carl Roth GmbH &amp;amp; Co KG (Karlsruhe, Germany)</td><td>6751.1</td><td></td></tr><tr><td>&lt;em&gt;L. pneumophila&lt;/em&gt; Corby</td><td>---</td><td>---</td><td>kindly provided by Prof Dr Antje Flieger (RKI, Berlin, Germany)</td></tr><tr><td>&lt;em&gt;L. pneumophila&lt;/em&gt; Corby &amp;Delta;flaA</td><td>---</td><td>---</td><td>kindly provided by Prof Dr Klaus Heuner (RKI, Berlin, Germany)</td></tr><tr><td>L-cystein</td><td>Carl Roth GmbH &amp;amp; Co KG (Karlsruhe, Germany)</td><td></td><td></td></tr><tr><td>mBMDM</td><td>---</td><td>---</td><td>kindly provided by Prof Dr Markus Schnare (Philipps Univeristy Marburg, Marburg, Germany) and Prof Dr Carsten Kirschning (University Duisburg Essen, Essen, Germany)</td></tr><tr><td>PBS</td><td>Biochrom GmbH (Berlin, Germany)</td><td>L 1825</td><td></td></tr><tr><td>phorbol 12-myristate 13-acetate</td><td>Sigma-Aldrich Chemie GmbH (Taufkirchen, Germany)</td><td>P8139-1MG</td><td></td></tr><tr><td>rotating shaker (MaxQ 6000)</td><td>Thermo Fisher Scientific Germany BV &amp;amp; Co KG (Braunschweig, Germany)</td><td>SHKE6000</td><td></td></tr><tr><td>RPMI 1640 high glucose</td><td>Life Technologies GmbH (Darmstadt, Germany)</td><td>11875-093</td><td></td></tr><tr><td>saponin</td><td>Carl Roth GmbH &amp;amp; Co KG (Karlsruhe, Germany)</td><td>9622.1</td><td></td></tr><tr><td>Ultrospec 10</td><td>Biochrom Ltd (Cambridge, England)</td><td>80-2116-30</td><td></td></tr><tr><td>sterile filter (pore size: 0.22 &amp;micro;m)</td><td>Corning Incorporated (new York, USA)</td><td>431096</td><td></td></tr><tr><td>THP-1</td><td>Sigma-Aldrich Chemie GmbH (Taufkirchen, Germany)</td><td>88081201-1VL</td><td></td></tr><tr><td>Sorvall Discovery 100 SE</td><td>Thermo Fisher Scientific Germany BV &amp;amp; Co KG (Braunschweig, Germany)</td><td></td><td></td></tr><tr><td>yeast extract</td><td>Carl Roth GmbH &amp;amp; Co KG (Karlsruhe, Germany)</td><td>2363.2</td><td></td></tr><tr><td>Pierce BCA protein assay kit</td><td>Thermo Fisher Scientific Germany BV &amp;amp; Co KG (Braunschweig, Germany)</td><td>23225</td><td></td></tr></tbody></table>

legionella pneumophila outer membrane vesicles: isolation and analysis of their pro-inflammatory potential on macrophages

Bacteria are able to secrete a variety of molecules via various secretory systems. Besides the secretion of molecules into the extracellular space or directly into another cell, Gram-negative bacteria can also form outer membrane vesicles (OMVs). These membrane vesicles can deliver their cargo over long distances, and the cargo is protected from degradation by proteases and nucleases. 
Legionella pneumophila (L. pneumophila) is an intracellular, Gram-negative pathogen that causes a severe form of pneumonia. In humans, it infects alveolar macrophages, where it blocks lysosomal degradation and forms a specialized replication vacuole. Moreover, L. pneumophila produces OMVs under various growth conditions. To understand the role of OMVs in the infection process of human macrophages, we set up a protocol to purify bacterial membrane vesicles from liquid culture. The method is based on differential ultracentrifugation. The enriched OMVs were subsequently analyzed with regard to their protein and lipopolysaccharide (LPS) amount and were then used for the treatment of a human monocytic cell line or murine bone marrow-derived macrophages. The pro-inflammatory responses of those cells were analyzed by enzyme-linked immunosorbent assay. Furthermore, alterations in a subsequent infection were analyzed. To this end, the bacterial replication of L. pneumophila in macrophages was studied by colony-forming unit assays. 
Here, we describe a detailed protocol for the purification of L. pneumophila OMVs from liquid culture by ultracentrifugation and for the downstream analysis of their pro-inflammatory potential on macrophages.

实验装置准备嗜肺军团菌的OMV，并分析其对感染后图1描述了巨噬细胞的促炎性反应的影响。所制备的OMV的促炎症潜力可以在PMA分化的THP-1细胞，其在图2中所示进行分析。 THP-1细胞与IL-8和IL-6分泌的时间和剂量依赖性增加作出反应。此外，在嗜肺军团菌的OMV识别不同TLR的影响可以通过使用mBMDM从不同的遗传背景进行分析，如在图3中由CXCL1 ELISA分析。 mBMDM从OMV刺激后分泌CXCL1野生型小鼠，而mBMDM TLR2 / 4 - / -分泌显著少。研究嗜肺军团菌的OMV对细菌复制在THP-1巨噬细胞的影响，将细胞预温育与OMV的再另外感染与嗜肺军团菌 （ 图4 A）。 THP-1巨噬细胞的预刺激第一减少感染的24小时后的细菌复制，但是它会导致在CFU翻倍在稍后的时间点（48小时PI）的计数。感染的巨噬细胞的以下信号上的OMV识别Toll样受体（TLR）的影响，可以通过mBMDM进行评估，如在图4 B中。由十倍mBMDM来自WT动物OMV预孵育后细菌复制的增加，而TLR2 - / -和TRIF / MyD88的- / -细胞不显示这种增加嗜肺军团菌的复制。 <img alt="图1" src="/files/ftp_upload/55146/55146fig1.jpg" /> 图1:实验步骤。 （A）的 嗜肺军团菌科比WT从10厘米的BCYE琼脂板上被用于接种小液体培养（10毫升），其被转移到90毫升新鲜的YEB中6小时后网上平台。小体积也接种于血琼脂平板上，以检查纯度。细菌在37℃下培养，直到早期稳定期（OD 600 = 3.0-3.5）。 （B）中的液体培养物离心并无菌过滤以除去细菌。然后军团 -free上清液超离心以获得OMV粒料，其再悬浮在PBS中并超速离心一次。分离的囊泡再悬浮，检查纯度，以及该蛋白量进行定量。比例尺条为2.5厘米。 （C）的人或鼠巨噬细胞刺激与量化的OMV。细胞培养上清液可用于酶联免疫吸附，或巨噬细胞可以与嗜肺感染由CFU试验上10厘米BCYE琼脂板上以确定细菌复制。 <a href="http://ecsource.jove.com/files/ftp_upload/55146/55146fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> </一&gt; <img alt="图2" src="/files/ftp_upload/55146/55146fig2.jpg" /> 图2:由嗜肺军团菌的OMV THP-1细胞的促炎活性。 （A）的在此，单核细胞THP-1细胞系被用作肺泡巨噬细胞的模型。 PMA分化的THP-1细胞用24和48小时，分别增加剂量嗜肺军团菌的OMV（0.01-25微克/毫升）处理。用于IL-8的ELISA中无细胞上清液。三个独立实验±SEM的平均值被示出。 THP-1细胞的反应，以尽可能少为0.01微克/毫升嗜肺军团菌的OMV与显著的IL-8的分泌，这是时间和剂量依赖性。 （B）的 嗜肺军团菌的OMV（0.1-10微克/毫升）用于刺激PMA分化的THP-1细胞。上清液孵育24和48小时后收集，且所释放的IL-6通过ELISA法在上清液中测得的。三个独立实验±SEM的平均值被示出。 THP-1细胞分泌的显著量的IL-6，即使采用的OMV的最低剂量（0.1微克/毫升）。 IL-6的分泌随着OMV剂量和延长温育时间增加。统计:Mann-Whitney检验; * P &lt;0.05和** P &lt;0.01，相比于相应0微克/毫升的OMV。经许可后转载自参考20. <a href="http://ecsource.jove.com/files/ftp_upload/55146/55146fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/55146/55146fig3.jpg" /> 图3:巨噬细胞的促炎活性取决于TLR2 / 4。 mBMDM来自WT和TLR2 / 4 - / -小鼠用嗜肺军团菌的OMV（0.1或1微克/毫升）一起温育。 CXCL1分泌用ELISA 24和48小时后分别分析。该±三个独立实验的SEM平均值被示出。 mBMDM来自WT小鼠的回应嗜肺 OMV孵育后以剂量依赖性CXCL1分泌。 TLR2 / 4 - / -与WT相比mBMDM mBMDM分泌显著少CXCL1，这种分泌没有增加剂量依赖性。统计:Mann-Whitney检验; * P &lt;0.05，相比于相应0微克/毫升的OMV; ＃P &lt;0.05相比，同等对待WT样本。经许可后转载自参考20. <a href="http://ecsource.jove.com/files/ftp_upload/55146/55146fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/55146/55146fig4.jpg" /> 图4:在巨噬细胞嗜肺 OMV预孵育增加细菌复制。 （A）的分化的THP-1细胞预温育与OMV的（0.1,1或10μ克/毫升）或LPS / IFN-γ（200毫微克/毫升的每个）或不进行处理控制。预孵育（20小时）后，THP-1细胞分别感染嗜肺科比WT（MOI 0.5）2，24，和48小时，。 THP-1细胞通过加入皂苷裂解，将细菌铺在BCYE琼脂板上。 CFU的是各时间点后，0微克/毫升OMV相对计算。条形代表±三次独立实验，每次在技术重复进行SEM的平均值。有在细菌摄取（2小时后的感染（PI））相比，没有预先处理的细胞没有差异。在细菌复制改建分别24和48小时后测定。 LPS / IFN-γ预处理的THP-1细胞表现出细菌负荷24小时圆周率的降低这也观察剂量依赖性地为嗜肺军团菌的OMV预先处理的细胞。在稍后的时间点（48小时圆周率），经预处理的OMV THP-1细胞表现出嗜肺重增加了一倍褶皱，而LPS / IFN-γ预处理的巨噬细胞表现出细菌负荷的进一步的降低。统计:Mann-Whitney检验; * P &lt;0.05和** P &lt;0.01，相比于相应0微克/毫升的OMV。 （B）的 mBMDM来自具有不同遗传背景的小鼠（WT，TLR2 - / - ，和TRIF / MyD88的- / - ）中预温育用0.1微克/毫升嗜肺军团菌的OMV 20小时和然后感染了鞭毛蛋白嗜肺军团菌科比（MOI 0.5），48小时的缺失突变体。 mBMDM通过加入皂苷裂解，和军团菌接种在BCYE琼脂板上。在CFU被相对于0微克/毫升的OMV计算，由实线表示。条形代表平均值±±的三个独立的实验中，每个在重复进行扫描电镜。 mBMDM来自WT小鼠显示OMV预处理后的增加嗜肺军团菌的复制。 TLR2 - / -巨噬细胞表现出显著降低<em&gt;军团复制，这是堪比TRIF / MyD88的- / - mBMDM。统计:Mann-Whitney检验; P &lt;0.05相对于WT样品。经许可后转载自参考20. <a href="http://ecsource.jove.com/files/ftp_upload/55146/55146fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Legionella pneumophila Outer Membrane Vesicles: Isolation and Analysis of Their Pro-inflammatory Potential on Macrophages at JoVE.com

Legionella pneumophila Outer Membrane Vesicles: Isolation and Analysis of Their Pro-inflammatory Potential on Macrophages

这里，我们描述嗜肺军团菌 （ 嗜肺军团菌 ）从液体培养外膜囊泡（OMV的）的纯化。然后将这些纯化的囊泡用于巨噬细胞的治疗，以分析其促炎潜力。

嗜肺军团菌外膜囊泡:隔离及对巨噬细胞促炎潜力分析

Bacteria are able to secrete a variety of molecules via various secretory systems. Besides the secretion of molecules into the extracellular space or ...

legionella-pneumophila-outer-membrane-vesicles-isolation-analysis

Research

JoVE Journal

Immunology and Infection

Legionella pneumophila Outer Membrane Vesicles: Isolation and Analysis of Their Pro-inflammatory Potential on Macrophages

11.1K 次观看.  Philipps-University Marburg. 这里，我们描述嗜肺军团菌 （ 嗜肺军团菌 ）从液体培养外膜囊泡（OMV的）的纯化。然后将这些纯化的囊泡用于巨噬细胞的治疗，以分析其促炎潜力。 

视频: Legionella pneumophila Outer Membrane Vesicles: Isolation and Analysis of Their Pro-inflammatory Potential on Macrophages

Isolation Protocol of Mouse Monocyte-derived Dendritic Cells and Their Subsequent In Vitro Activation with Tumor Immune Complexes

Dendritic cells (DC) are heterogeneous cell populations that differ in their cell membrane markers, migration patterns and distribution, and in their antigen presentation and T cell activation capacities. Since most vaccinations of experimental tumor models require millions of DC, they are widely isolated from the bone marrow or spleen. However, these DC significantly differ from blood and tumor DC in their responses to immune complexes (IC), and presumably to other Syk-coupled lectin receptors. Importantly, given the sensitivity of DC to danger-associated molecules, the presence of endotoxins or antibodies that crosslink activation receptors in one of the isolating steps could result in the priming of DC and thus affect the parameters, or at least the dosage, required to activate them. Therefore, here we describe a detailed protocol for isolating MoDC from blood and tumors while avoiding their premature activation. In addition, a protocol is provided for MoDC activation with tumor IC, and their subsequent analyses.

Isolation Protocol of Mouse Monocyte-derived Dendritic Cells and Their Subsequent In Vitro Activation with Tumor Immune Complexes

Monocyte-derived DC (MoDC) can sense minor amounts of danger-associated molecules and are therefore easily primed. We provide a detailed protocol for the isolation of MoDC from blood and tumors and their activation with immune complexes while highlighting key precautions that should be considered in order to avoid their premature activation.

小鼠单核细胞衍生树突状细胞的分离及与肿瘤免疫配合物的后体外活化

Dendritic cells (DC) are heterogeneous cell populations that differ in their cell membrane markers, migration patterns and distribution, and in their ...

科研

癌症研究

Purification of Pathogen Vacuoles from Legionella-infected Phagocytes

The opportunistic pathogen Legionella pneumophila is an amoeba-resistant bacterium, which also replicates in alveolar macrophages thus causing the severe pneumonia "Legionnaires' disease"1. In protozoan and mammalian phagocytes, L. pneumophila employs a conserved mechanism to form a specific, replication-permissive compartment, the "Legionella-containing vacuole" (LCV). LCV formation requires the bacterial Icm/Dot type IV secretion system (T4SS), which translocates as many as 275 "effector" proteins into host cells. The effectors manipulate host proteins as well as lipids and communicate with secretory, endosomal and mitochondrial organelles2-4.
The formation of LCVs represents a complex, robust and redundant process, which is difficult to grasp in a reductionist manner. An integrative approach is required to comprehensively understand LCV formation, including a global analysis of pathogen-host factor interactions and their temporal and spatial dynamics. As a first step towards this goal, intact LCVs are purified and analyzed by proteomics and lipidomics.
The composition and formation of pathogen-containing vacuoles has been investigated by proteomic analysis using liquid chromatography or 2-D gel electrophoresis coupled to mass-spectrometry. Vacuoles isolated from either the social soil amoeba Dictyostelium discoideum or mammalian phagocytes harboured Leishmania5, Listeria6, Mycobacterium7, Rhodococcus8, Salmonella9 or Legionella spp.10. However, the purification protocols employed in these studies are time-consuming and tedious, as they require e.g. electron microscopy to analyse LCV morphology, integrity and purity. Additionally, these protocols do not exploit specific features of the pathogen vacuole for enrichment.
The method presented here overcomes these limitations by employing D. discoideum producing a fluorescent LCV marker and by targeting the bacterial effector protein SidC, which selectively anchors to the LCV membrane by binding to phosphatidylinositol 4-phosphate (PtdIns(4)P)3,11 . LCVs are enriched in a first step by immuno-magnetic separation using an affinity-purified primary antibody against SidC and a secondary antibody coupled to magnetic beads, followed in a second step by a classical Histodenz density gradient centrifugation12,13 (Fig. 1).
A proteome study of isolated LCVs from D. discoideum revealed more than 560 host cell proteins, including proteins associated with phagocytic vesicles, mitochondria, ER and Golgi, as well as several GTPases, which have not been implicated in LCV formation before13. LCVs enriched and purified with the protocol outlined here can be further analyzed by microscopy (immunofluorescence, electron microscopy), biochemical methods (Western blot) and proteomic or lipidomic approaches.

Purification of Pathogen Vacuoles from Legionella-infected Phagocytes

This article describes a method for the isolation and purification of intact Legionella-containing vacuoles (LCVs) from amoeba and macrophages. The two-step protocol comprises LCV enrichment by immuno-magnetic separation using an antibody against a bacterial LCV marker and further purification by density gradient centrifugation.

病原液泡的分离纯化<em&gt;军团</em&gt;感染吞噬细胞

The opportunistic pathogen Legionella pneumophila is an amoeba-resistant bacterium, which also replicates in alveolar macrophages thus causing the severe ...

免疫与感染

Proteomic Analysis of Human Macrophage Polarization Under a Low Oxygen Environment

Macrophages are innate immune cells involved in a number of physiological functions ranging from responses to infectious pathogens to tissue homeostasis. The various functions of these cells are related to their activation states, which is also called polarization. The precise molecular description of these various polarizations is a priority in the field of macrophage biology. It is currently acknowledged that a multidimensional approach is necessary to describe how polarization is controlled by environmental signals. In this report, we describe a protocol designed to obtain the proteomic signature of various polarizations in human macrophages. This protocol is based on a label-free quantification of macrophage protein expression obtained from in-gel fractionated and Lys C/trypsin-digested cellular lysis content. We also provide a protocol based on in-solution digestion and isoelectric focusing fractionation to use as an alternative. Because oxygen concentration is a relevant environmental parameter in tissues, we use this protocol to explore how atmospheric composition or a low oxygen environment affects the classification of macrophage polarization.

We present a protocol to obtain proteomic signatures of human macrophages and apply this to determination of the impact of a low oxygen environment on macrophage polarization.

低氧环境下人巨噬细胞极化的蛋白质组学分析

Macrophages are innate immune cells involved in a number of physiological functions ranging from responses to infectious pathogens to tissue homeostasis. ...

生物化学

Tractable Mammalian Cell Infections with Protozoan-primed Bacteria

Many intracellular bacterial pathogens use freshwater protozoans as a natural reservoir for proliferation in the environment. Legionella pneumophila, the causative agent of Legionnaires' pneumonia, gains a pathogenic advantage over in vitro cultured bacteria when first harvested from protozoan cells prior to infection of mammalian macrophages. This suggests that important virulence factors may not be properly expressed in vitro. We have developed a tractable system for priming L. pneumophila through its natural protozoan host Acanthamoeba castellanii prior to mammalian cell infection. The contribution of any virulence factor can be examined by comparing intracellular growth of a mutant strain to wild-type bacteria after protozoan priming. GFP-expressing wild-type and mutant L. pneumophila strains are used to infect protozoan monolayers in a priming step and allowed to reach late stages of intracellular growth. Fluorescent bacteria are then harvested from these infected cells and normalized by spectrophotometry to generate comparable numbers of bacteria for a subsequent infection into mammalian macrophages. For quantification, live bacteria are monitored after infection using fluorescence microscopy, flow cytometry, and by colony plating. This technique highlights and relies on the contribution of host cell-dependent gene expression by mimicking the environment that would be encountered in a natural acquisition route. This approach can be modified to accommodate any bacterium that uses an intermediary host as a means for gaining a pathogenic advantage.

This technique provides a method to harvest, normalize and quantify intracellular growth of bacterial pathogens that are pre-cultivated in natural protozoan host cells prior to infections of mammalian cells. This method can be modified to accommodate a wide variety of host cells for the priming stage as well as target cell types.

听话的哺乳动物细胞原生动物引发的细菌感染

Many intracellular bacterial pathogens use freshwater protozoans as a natural reservoir for proliferation in the environment. Legionella pneumophila, the ...

Use of Galleria mellonella as a Model Organism to Study Legionella pneumophila Infection

Legionella pneumophila, the causative agent of a severe pneumonia named Legionnaires&#39; disease, is an important human pathogen that infects and replicates within alveolar macrophages. Its virulence depends on the Dot/Icm type IV secretion system (T4SS), which is essential to establish a replication permissive vacuole known as the Legionella containing vacuole (LCV). L. pneumophila infection can be modeled in mice however most mouse strains are not permissive, leading to the search for novel infection models. We have recently shown that the larvae of the wax moth Galleria mellonella are suitable for investigation of L. pneumophila infection. G. mellonella is increasingly used as an infection model for human pathogens and a good correlation exists between virulence of several bacterial species in the insect and in mammalian models. A key component of the larvae&#39;s immune defenses are hemocytes, professional phagocytes, which take up and destroy invaders. L. pneumophila is able to infect, form a LCV and replicate within these cells. Here we demonstrate protocols for analyzing L. pneumophila virulence in the G. mellonella model, including how to grow infectious L. pneumophila, pretreat the larvae with inhibitors, infect the larvae and how to extract infected cells for quantification and immunofluorescence microscopy. We also describe how to quantify bacterial replication and fitness in competition assays. These approaches allow for the rapid screening of mutants to determine factors important in L. pneumophila virulence, describing a new tool to aid our understanding of this complex pathogen.

Use of Galleria mellonella as a Model Organism to Study Legionella pneumophila Infection

The larva of the wax moth Galleria mellonella was recently established as an in vivo model to study Legionella pneumophila infection. Here, we demonstrate fundamental techniques to characterize the pathogenesis of Legionella in the larvae, including inoculation, measurement of bacterial virulence and replication as well as extraction and analysis of infected hemocytes.

使用<em&gt;大蜡螟</em&gt;作为模式生物来研究<em&gt;嗜肺军团菌</em&gt;感染

Legionella pneumophila, the causative agent of a severe pneumonia named Legionnaires&#39; disease, is an important human pathogen that infects and ...

Isolation of Human Monocytes by Double Gradient Centrifugation and Their Differentiation to Macrophages in Teflon-coated Cell Culture Bags

Human macrophages are involved in a plethora of pathologic processes ranging from infectious diseases to cancer. Thus they pose a valuable tool to understand the underlying mechanisms of these diseases. We therefore present a straightforward protocol for the isolation of human monocytes from buffy coats, followed by a differentiation procedure which results in high macrophage yields. The technique relies mostly on commonly available lab equipment and thus provides a cost and time effective way to obtain large quantities of human macrophages. Briefly, buffy coats from healthy blood donors are subjected to a double density gradient centrifugation to harvest monocytes from the peripheral blood. These monocytes are then cultured in fluorinated ethylene propylene (FEP) Teflon-coated cell culture bags in the presence of macrophage colony-stimulating factor (M-CSF). The differentiated macrophages can be easily harvested and used for subsequent studies and functional assays. Important methods for quality control and validation of the isolation and differentiation steps will be highlighted within the protocol. In summary, the protocol described here enables scientists to routinely and reproducibly isolate human macrophages without the need for cost intensive tools. Furthermore, disease models can be studied in a syngeneic human system circumventing the use of murine macrophages.

We present a simple and efficient protocol for the generation of human macrophages. Buffy coats are processed by double density gradient centrifugation and isolated monocytes are then differentiated to macrophages in Teflon-coated cell culture bags. This maximizes macrophage yields and facilitates cell harvesting for subsequent experiments.

人类单核双梯度离心法和其分化为巨噬细胞的特富龙涂层的细胞培养袋隔离

Human macrophages are involved in a plethora of pathologic processes ranging from infectious diseases to cancer. Thus they pose a valuable tool to ...

Isolation of Murine Peritoneal Macrophages to Carry Out Gene Expression Analysis Upon Toll-like Receptors Stimulation

During infection and inflammation, circulating monocytes leave the bloodstream and migrate into tissues, where they differentiate into macrophages. Macrophages express surface Toll-like receptors (TLRs), which recognize molecular patterns conserved through evolution in a wide range of microorganisms. TLRs play a central role in macrophage activation which is usually associated with gene expression alteration. Macrophages are critical in many diseases and have emerged as attractive targets for therapy. In the following protocol, we describe a procedure to isolate murine peritoneal macrophages using Brewer&#8217;s thioglycollate medium. The latter will boost monocyte migration into the peritoneum, accordingly this will raise macrophage yield by 10-fold. Several studies have been carried out using bone marrow, spleen or peritoneal derived macrophages. However, peritoneal macrophages were shown to be more mature upon isolation and are more stable in their functionality and phenotype. Thus, macrophages isolated from murine peritoneal cavity present an important cell population that can serve in different immunological and metabolic studies. Once isolated, macrophages were stimulated with different TLR ligands and consequently&#160;gene expression was evaluated.

We describe here a simple protocol to isolate murine peritoneal macrophages. This procedure is followed by RNA extraction to carry out gene expression analysis upon Toll-like receptors stimulation.

小鼠腹腔巨噬细胞的分离，以贯彻基因表达分析后Toll样受体刺激

During infection and inflammation, circulating monocytes leave the bloodstream and migrate into tissues, where they differentiate into macrophages. ...

In Vitro Phagocytosis of Myelin Debris by Bone Marrow-Derived Macrophages

Bone marrow-derived macrophages (BMDMs) are mature leukocytes that serve a critical physiological role as professional phagocytes capable of clearing a variety of particles. Normally, BMDMs are restricted from the central nervous system (CNS), but following an injury, they can readily infiltrate. Once within the injured CNS tissue, BMDMs are the primary cell type responsible for the clearance of injury-derived cellular debris, including large quantities of lipid rich myelin debris. The neuropathological ramifications of BMDM infiltration and myelin debris phagocytosis within the CNS are complex and not well understood. The protocols described here, allow for the direct in vitro study of BMDMs in the context of CNS injury. We cover murine BMDM isolation and culture, myelin debris preparation, and assays to assess BMDM myelin debris phagocytosis. These techniques produce robust quantifiable results without the need for significant specialized equipment or materials, yet can be easily customized to meet the needs of researchers.

In Vitro Phagocytosis of Myelin Debris by Bone Marrow-Derived Macrophages

We present methods to assess the phagocytic capacity of primary murine bone marrow-derived macrophages using fluorescently labeled myelin debris and intracellular lipid droplet staining.

体外骨髓源性巨噬细胞对髓鞘碎片的吞噬

Bone marrow-derived macrophages (BMDMs) are mature leukocytes that serve a critical physiological role as professional phagocytes capable of clearing a ...

神经科学

Using a Bacterial Pathogen to Probe for Cellular and Organismic-level Host Responses

There are a variety of strategies bacterial pathogens employ to survive and proliferate once inside the eukaryotic cell. The so-called 'cytosolic' pathogens (Listeria monocytogenes, Shigella flexneri, Burkholderia pseudomallei, Francisella tularensis, and Rickettsia spp.) gain access to the infected cell cytosol by physically and enzymatically degrading the primary vacuolar membrane. Once in the cytosol, these pathogens both proliferate as well as generate sufficient mechanical forces to penetrate the plasma membrane of the host cell in order to infect new cells. Here, we show how this terminal step of the cellular infection cycle of L. monocytogenes (Lm) can be quantified by both colony-forming unit assays and flow cytometry and give examples of how both pathogen- and host-encoded factors impact this process. We also show a close correspondence of Lm infection dynamics of cultured cells infected in vitro and those of hepatic cells derived from mice infected in vivo. These function-based assays are relatively simple and can be readily scaled up for discovery-based high-throughput screens for modulators of eukaryotic cell function.

We describe both in vitro and in vivo infection assays that can be used to analyze the activities of host-encoding factors.

细菌病原体在细胞和有机水平宿主反应探针中的应用

There are a variety of strategies bacterial pathogens employ to survive and proliferate once inside the eukaryotic cell. The so-called 'cytosolic' ...

Flow Cytometry Analysis of Intracellular and Emergent Extracellular Listeria monocytogenes In Vitro

Source: Gayle, P. et al., <a href="https://app.jove.com/v/58775">Using a Bacterial Pathogen to Probe for Cellular and Organismic-level Host Responses.</a> J. Vis. Exp. (2019)
In this video, we quantify intracellular and extracellular Listeria monocytogenes, an intracellular pathogenic bacteria, following their infection of cultured macrophages in vitro using flow cytometry.

体外细胞内和新兴细胞外单核细胞增生李斯特菌的流式细胞术分析

Source: Gayle, P. et al., Using a Bacterial Pathogen to Probe for Cellular and Organismic-level Host Responses. J. Vis. Exp. (2019)
In this video, we ...