Name: macrophage cholesterol depletion and its effect on the phagocytosis of cryptococcus neoformans
Uploaded: 2014-12-19T15:00:00.000+00:00
Duration: 11 min 7 s
Description: Watch this Scientific Journal Video about Macrophage Cholesterol Depletion and Its Effect on the Phagocytosis of Cryptococcus neoformans at JoVE.com

这项工作是由美国国立卫生研究院资助AI56168，AI71142，AI87541和AI100631到MDP支持。莫里吉奥德尔Poeta的是宝来惠康研究员在传染病。

J774A.1细胞MβCD1.胆固醇消耗<ol><li>在无菌生物安全柜，种子10 5 J774A.1巨噬细胞样细胞每孔在96孔细胞培养板中加入200μlDulbecco氏改良的Eagle培养基（DMEM）中的补充有10％胎牛血清（FBS）和1％的青霉素/链霉素（P / S）。在37℃和5％CO 2的O / N。 </li><li>从细胞单层除去介质并用1×磷酸盐缓冲盐水（PBS）中已被过滤或高压灭菌洗细胞两次。 </li><li>加入200微升MβCD溶液在所需浓度（10毫米或30毫米的PBS）或1x PBS作为对照，孵育30分钟，在37℃振荡。与市售试剂盒中取出上清液和储备在室温进行定量分析，立即过程如下。 </li><li>用1×PBS或无血清DMEM洗涤细胞两到三倍，并继续与感染或裂解细胞通过pipetti纳克的两到三倍，用去离子H 2 O代表的分析用薄层色谱或用的试剂盒。 注:按照胆固醇耗竭市售胆固醇定量试剂盒可以使用。详情请参阅材料部分。按照制造商的说明进行写入。 </li></ol> 2.观察胆固醇含量薄层色谱（TLC） <ol><li> （体积比1 65:30），用石油醚中的溶液洗TLC罐两次用丙酮和一次::乙醚乙酸。饱和罐用石油醚:乙醚:乙酸（65:30:1，体积比）溶液并离开O / N。 注:有机溶剂应始终在通风橱中使用，防止吸入蒸汽。手套和白大褂应该在任何时候都可以穿。乙酸是一种强酸，应谨慎使用。 </li><li>在无菌生物安全柜，种子10 6 J774A.1巨噬细胞样，每孔的细胞在6孔策LL培养板在5毫升温暖的DMEM补充有10％FBS和1％P / S的体积。孵育在37℃，5％的CO 2 O / N。 </li><li> 1.4，200微升，其中适用于占较大以及大小代1毫升 - 通过以下步骤1.2耗尽胆固醇的巨噬细胞。 </li><li>加入500μl胰蛋白酶EDTA至各孔中，孵育3分钟，在37℃，轻轻刮去细胞用细胞刮刀破碎。 </li><li>转移到一个离心管中，并添加另外500微升温暖的DMEM补充有10％FBS和1％P / S的。 </li><li>旋转细胞5分钟，在300×g离心并去除上清。 </li><li>增加一个额外的500微升温暖的DMEM补充有10％FBS和1％P / S的细胞沉淀，并通过上下抽吸小心重悬。 </li><li>去除10微升细胞，并在血细胞计数器计数细胞。正常化从每个样品中的细胞浓度和添加相等数目细胞的玻璃管。 注:在这一步骤中，可以选择组合的细胞来自相同的治疗组，以获得更集中的最终脂质提取物。 </li><li>离心机细胞在300 XG 5分钟在室温和删除介质。 </li><li>加入2毫升的甲醇和漩涡。然后，加入1毫升氯仿并涡旋。检查相状态，以确保在单相的解决方案。 注:管可存放在4°CO / N。 </li><li>离心在1700 xg离心在RT 10分钟，将上清转移到新的管</li><li>添加额外的1毫升氯仿接着1毫升卫生署2 O的涡旋两次30秒。离心在1700×g离心5分钟，在室温。 </li><li>权衡上的敏感平衡的玻璃管，并使用玻璃巴斯德吸管向下层相转移到已知重量的玻璃管中。 </li><li>干倒在离心蒸发器脂类至干（约2小时）。称取管干血脂，并计算干脂肪重量。 注:干脂质可以储存在-20℃，直至准备好执行TLC。 </li><li>稀干燥脂类在足够氯仿正常化脂质的浓度（通常为20 - 50微升）和负载20微升在硅胶TLC板上稀释的脂质。负载20胆固醇稀释在20μl的氯仿作为标准的微克。 </li><li>加入干薄层板上，以饱和TLC坦克，并允许溶剂可达1厘米迁移板边之前。从罐中取出TLC板，并让其干燥约5分钟。 </li><li>通过放置在碘蒸气罐，检查移民可视化脂类。删除并允许斑点褪色约10 - 引擎盖下15分钟。 注:碘是吸入危害。在通风橱中始终使用。 </li><li>通过组合60mL甲醇用60ml去离子H 2 O为4毫升硫酸，和630毫克的氯化锰制备用于中性脂质染色的溶液。 </li><li>小心缓慢地浸TLC板变成一个托盘中的中性脂肪染色解决方案，并删除不塌掉二氧化硅升艾尔。 注:中性脂质染色溶液可以重复使用好几次，因此它可以从托盘进行检索和放回瓶供以后使用。 </li><li>允许板引擎盖下干在室温，直到所有的泡沫已经消失了。加热TLC板上加热块设定为160℃和焦炭到所需的颜色。 注:光密度测定程序，如视觉作品LS可以用来量化烧焦频带进行比较的脂质样品。 </li></ol> 3.感染C.巨噬细胞新型隐球菌 （H99） <ol><li>在无菌生物安全柜，每孔在96孔细胞培养板在200μlDMEM中的种子10 5巨噬细胞样细胞在补充有10％FBS和1％P / S。孵育在37℃，5％CO 2，5％的CO 2 O / N。 注:感染，也可在玻璃完成有底为了便于成像的共焦碟子;所有金额保持不变。 </li><li> C.成长的文化新型隐球菌 （H99）接种10毫升YNB与从击打板获得的一个菌落，并与摇动孵育它O / N在30℃。 </li><li>洗和计数C.新型隐球菌 （H99）细胞。 <ol><li>离心机C.新型隐球菌 O / N培养在1700 XG为4℃10分钟。 </li><li>取出介质并丢弃。用5ml的1×PBS洗细胞。离心机在1700×g离心在4℃下10分钟。 </li><li>除去PBS中，洗涤用5ml过滤的1×PBS。离心机在1700×g离心在4℃下10分钟。重复此步骤2次以上。 </li><li>除去PBS，重悬在5毫升1X PBS的。 </li><li>使在PBS中系列稀释，得到1:500稀释洗涤培养物。 <ol><li>添加100微升原始样品至900微升的1×PBS，得到1:10稀释。 </li><li>加入100微升1时10稀释样品至900微升1×PBS中，得到1:100的稀释。 </li><li>加入200微升1:100稀释的样品到800微升1×PBS中，得到1:500 diluti上</li></ol></li><li>取10微升的1:500稀释并指望血球计算细胞的数目。 </li></ol></li><li>准备工作液激活巨噬细胞和opsonizing C.新型隐球菌。 <ol><li>加入10微升至990微升稀释LPS和IFNγ100倍的股票解决方案。 注:LPS和IFNγ用于增强巨噬细胞吞噬，但不是必须为吞噬作用。如果有兴趣的巨噬细胞的杀真菌活性，执行激活O / N在37℃下与在感染前晃动。 </li><li>每个样品结合7.5微升稀释毒素，1.25微升稀释IFNγ，1.25微升GXM抗体和C的量隐球菌培养，让1.25×10 5个细胞。带体积达到250微升乘以用DMEM样品补充有10％FBS和1％P / S的数量。 </li><li>涡旋孵育溶液中20分钟，在37℃振荡。 注意:胆固醇耗竭（步骤1.2 - 1.4）可以同时与调理步骤来完成。一定要善待之前，结合工作液的巨噬细胞，作为调理细胞应优化使用时间不超过20分钟以下步骤3.4.3孵化。 </li></ol></li><li>巨噬细胞感染<ol><li>巨噬细胞洗两次无血清DMEM，并添加200微升调理的C.隐球菌工作液到每个孔中。 </li><li>孵育2小时，在37℃。 </li></ol></li><li>修复和细胞染色<ol><li>删除媒体和DMEM洗涤细胞2次。 </li><li>空气干燥该细胞单层10分钟，并加入200微升冰冷的甲醇中以固定细胞。 </li><li>孵育15分钟，在RT和除去任何剩余的甲醇。 </li><li>加入200微升10×吉姆萨孵育5分钟，在室温。 </li><li>用去离子水和干O / N 3次盖帽关 - 洗2。 注:成像可以做到第二天或最多一周followi吴染色。 </li></ol></li><li>可视化和计数<ol><li>使用显微镜，计数每个数据点300细胞（如果有相同的治疗2计数150每孔），并注意感染的巨噬细胞的吞噬隐球菌细胞的数目和次数。 注:为了确保甚至每个条件采样每个数据点使用2片，选3每盘不重叠的区域和计数单位面积50个细胞。 </li><li>通过C的平均数乘以受感染的巨噬细胞的百分比计算吞噬指数每巨噬细胞隐球菌 。通过表达值作为1×PBS中的百分比处理的对照正常化吞噬指数。经过多次试验计算平均值和平均值的标准偏差，以确定在吞噬指数趋势。使用学生t检验，以确定显着性。 </li><li>就拿细胞的显微照片在1,000倍或400倍的放大倍率。 </li></ol></li></ol> 4.台盼蓝测定<ol><li>在无菌生物安全柜，种子10 6巨噬细胞样细胞，每孔在6孔细胞培养板中的体积5mL的Dulbecco补充有10％胎牛血清和1％青霉素/链霉素最小基本培养基（DMEM）中的。孵育在37℃，5％的CO 2 O / N。 </li><li> 1.4为200μl的在适用于占较大以及大小代2毫升 - 通过以下步骤1.2耗尽胆固醇的巨噬细胞。 注:一定要包括与1X PBS以及之前的任何处理被刮掉控制处理的对照。 </li><li>加入500微升的1×PBS的每个孔中，轻轻刮去细胞用细胞刮刀破碎。转移到一个离心管中，并轻轻吹打上下悬浮细胞。 </li><li>去除10微升细胞和染色用1微升4％台盼蓝。 </li><li>计数细胞在血细胞计数器，并使用以下等式计算存活率:％存活率= [1 - （蓝色细胞/总细胞）]×100标准化值，这是未经处理的控制。经过多次试验计算的平均值和标准偏差，以确定在存活力的趋势。使用学生t检验，以确定显着性。 </li></ol>

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K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydroxyurea+Treatment+Inhibits+Proliferation+Of+Cryptococcus+Neoformans+In+Mice.">Hydroxyurea Treatment Inhibits Proliferation Of Cryptococcus Neoformans In Mice.</a> Frontiers In Microbiology. 3, 187 (2012).</li><li>Chaka, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+Analysis+Of+Phagocytosis+And+Killing+Of+Cryptococcus+Neoformans+By+Human+Peripheral+Blood+Mononuclear+Cells+By+Flow+Cytometry.">Quantitative Analysis Of Phagocytosis And Killing Of Cryptococcus Neoformans By Human Peripheral Blood Mononuclear Cells By Flow Cytometry.</a> Clin. Diagn. Lab. Immunol. 2 (6), 753-759 (1995).</li></ol>

The authors have nothing to disclose.

在这个协议的工作是电镀时哺乳动物细胞和opsonizing C.获得准确的细胞计数是非常重要的新型隐球菌细胞。该试验间差异最小化，并保证准确的1:整个研究1目标效应比。这也是至关重要的协调胆固醇枯竭和感染的时间，以防止调理的酵母细胞或巨噬细胞处理细胞在RT中的过程之间的休息。漫长的等待时间可能会导致损失抗体的调理作用或贫胆固醇补给之前感染就可以开始。如果实验精?...

吞噬作用是由细胞外的实体是由宿主细胞内化的过程。它是在免疫系统中的武器库，以对抗病原体保卫一个关键武器，但该过程可以经常由病原体被颠覆，以允许内化和整个主体1传播。吞噬作用是由导致通过宿主细胞的细胞骨架的重排附着和吞噬几个信号传导事件介导的。职业"吞噬细胞能够识别和结合调理素的入侵病原体的表面上的信号用于附着和形成片状伪足的，其吞噬的病原体，并形成吞噬体2。其中所谓的&#39;专业&#39;吞噬细胞巨噬细胞是。巨噬细胞是高度特化的细胞即进行保护的功能包括寻找和消除致病剂，修复受损的组织，并介导的炎症，大多数的这些通过吞噬1,2的过程。 新生隐球菌是一个物种致病酵母导致称为隐球菌一个严重的疾病。 隐球菌孢子由主机吸入并导致肺部感染，通常是无症状的。据认为，曝光是极为普遍;对61名儿童从儿科传染病诊所在布朗克斯-黎巴嫩医院中心发现，所有的受访者有抗体的隐球菌多糖glucuronoxylomannan和其他研究的样本表明患病率在人类免疫缺陷病毒（HIV）感染和感染的成人3， 4。肺泡巨噬细胞是响应于肺部感染，并且在大多数情况下，成功地明确病原体的第一道防线。然而，在免疫受损的个体（ 例如，HIV和AIDS患者）的酵母是能够在巨噬细胞内生存。在这些的情况下，巨噬细胞可以作为利基病原体的复制，并且可以促进其传 ​​播到中枢神经系统（CNS），其中该疾病成为致命5 - 8。据认为，巨噬细胞可能甚至直接传递到酵母脑膜，帮助酵母通过"木马"的模式3,9穿过血脑屏障- 11。因此，为了理解吞噬的过程和影响它，特别是在隐球菌感染的因素是很重要的。 以前的工作在其它病原体系统指向由胆固醇作为具有重要作用，吞噬12发挥形成胆固醇和脂质筏- 15。胆固醇是最丰富的脂类物质在哺乳动物细胞中，并包括25 -哺乳动物细胞膜16的50％。已经发现，以在调节BIOP发挥作用膜通过改变其刚性物质环境的17性质。胆固醇和鞘脂在一起形成被称为脂筏在膜中的脂质微区。脂筏已经发现涉及在地层中的小窝，以及提供一个分离的结构域对某些类型的信号16 - 18。由于它们的尺寸小，它是很难研究脂质筏体内 。研究脂筏的作用的一个有用的方法是改变他们的选民。甲基β环糊精（MβCD）是已被发现从哺乳动物细胞膜耗尽胆固醇，通常用于研究脂质筏18的作用的化合物。 在这个协议中，我们提出了一个方法，以消耗从宿主细胞膜胆固醇并量化对宿主细胞的吞噬C的能力的耗尽的效果隐球菌体外 。此过程使用了细胞培养techniqUE的像细胞（J774A.1）作为模型感染的巨噬细胞的永生。胆固醇耗竭被完成通过暴露于MβCD，其具有特定的甾醇的尺寸的疏水性芯，并且能够充当胆固醇绘制出来的膜19的一个接收器。定量用市售试剂盒和定性使用改良的Bligh-Dyer的脂质提取，随后用薄层色谱法（TLC）20胆固醇耗竭进行测定。 23 -吞噬被感染与调理的酵母与干扰素γ和脂多糖的鸡尾酒激活巨噬细胞混合培养的细胞系测量隐球菌是使用glucuronoxylomannan（GXM）抗体21调理。染色和显微镜实验允许的细胞可视化和吞噬指数的计算，以评估细胞吞噬的程度。综上所述，该协议ðescribes集成脂质组合物具有的生理过程的改变的基本方法。

<table><tbody><tr><td>Class II type A2 Biosafety Cabinet</td><td>Labconco</td><td>3460009</td><td></td></tr><tr><td>J774A.1 cell line</td><td>ATCC</td><td>TIB-67</td><td>Arrives Frozen. See ATCC instructions for culturing.</td></tr><tr><td>Dulbecco&amp;rsquo;s Modified Eagle Medium</td><td>Gibco</td><td>11995-065</td><td>Store at 4 &amp;deg;C and warm to 37 &amp;deg;C prior to use</td></tr><tr><td>HI Fetal Bovine Serum Performance Plus</td><td>Gibco</td><td>10082-147</td><td>Keep frozen at -20 &amp;deg;C and thaw before adding to DMEM</td></tr><tr><td>Penicillin-Streptomycin (10,000 U/ml)</td><td>Gibco</td><td>15140-122</td><td>Used to suplement DMEM</td></tr><tr><td>Isotemp Cell culture incubator</td><td>Fisher Scientific</td><td></td><td>Model # 3530</td></tr><tr><td>96-Well culture dish</td><td>Corning Inc. Costar</td><td>3595</td><td></td></tr><tr><td>10x Phosphate Buffered Saline</td><td>Fisher Scientific BioReagents</td><td>BP3994</td><td>Dilute to 1x and filter or autoclave prior to use.</td></tr><tr><td>Methyl-&amp;beta;-cyclodextrin</td><td>Sigma Life Science</td><td>C4555-10G</td><td>Dissolve in 1x PBS to make solutions of 10 mM and 30 mM concentrations</td></tr><tr><td>Orbital shaker</td><td>Labline</td><td></td><td></td></tr><tr><td>Amplex Red Cholesterol Assay Kit</td><td>Life Technologies Molecular Probes</td><td>A12216</td><td>All reagents for Cholesterol Assay are contained within the kit. Follow Manufacturer instructions.</td></tr><tr><td>96-Well Black Assay plate</td><td>Corning Inc. Costar</td><td>3603</td><td></td></tr><tr><td>FilterMax microplate reader</td><td>Molecular Devices</td><td></td><td>Model F5</td></tr><tr><td>TLC chamber</td><td>Sigma-Aldrich</td><td>Z126195-1EA</td><td></td></tr><tr><td>Chloroform</td><td>Sigma-Aldrich</td><td>650498-4L</td><td></td></tr><tr><td>Methanol</td><td>Sigma-Aldrich</td><td>34860-2L-R</td><td></td></tr><tr><td>TLC Paper</td><td>Whatman</td><td>3030917</td><td>Cut down to size needed for TLC tank</td></tr><tr><td>Fume hood</td><td></td><td></td><td>Any fume hood that complies with AIHA/ANSI Standards</td></tr><tr><td>6-Well plate</td><td>Corning Inc. Costar</td><td>3506</td><td></td></tr><tr><td>Trypsin-EDTA</td><td>Gibco</td><td>25300-054</td><td></td></tr><tr><td>Cell scraper</td><td>Corning Inc. Costar</td><td>3010</td><td></td></tr><tr><td>Hemocytometer</td><td>Hausser Scientific</td><td>1490</td><td></td></tr><tr><td>Centrifuge</td><td>Beckman Coulter</td><td></td><td>Model Alegra x-30R</td></tr><tr><td>Votex mixer</td><td>Fisher Scientific</td><td>12-812</td><td></td></tr><tr><td>Balance</td><td>Mettler Toledo</td><td></td><td>Model # MS104S meaures down to 0.1 mg</td></tr><tr><td>Glass Pasteur pipette</td><td>Fisherbrand</td><td>13-678-20A</td><td></td></tr><tr><td>Cholesterol</td><td>Avanti Polar Lipids</td><td>700000</td><td></td></tr><tr><td>SpeedVac concentrator</td><td>Thermo Scientific</td><td></td><td>Model # SPD2010</td></tr><tr><td>Petroleum ether</td><td>Fisher Scientific</td><td>E139-1</td><td></td></tr><tr><td>Diethyl ether</td><td>Sigma-Aldrich</td><td>309966</td><td></td></tr><tr><td>Acetic acid</td><td>Sigma-Aldrich</td><td>320099</td><td></td></tr><tr><td>TLC Silica Gel 60 with concentrating zone</td><td>Analytical Chromatograhy Millipore</td><td>1.11845.0001</td><td></td></tr><tr><td>Iodine chips</td><td>Sigma-Aldrich</td><td>376558-50G</td><td></td></tr><tr><td>Sulfuric acid</td><td>Sigma-Aldrich</td><td>320501</td><td></td></tr><tr><td>Manganese chloride</td><td>Sigma-Aldrich</td><td>244589</td><td></td></tr><tr><td>UVP EC3 Imaging System</td><td>Ultra-Violet Products Ltd.</td><td></td><td>Use the Vision Works LS software for densitometry analysis</td></tr><tr><td>Glass bottom confocal dish</td><td>MatTek</td><td>P35G-1.5-10C</td><td>www.glassbottomdishes.com</td></tr><tr><td>Cryptococcus neoformans (H99)</td><td></td><td></td><td>Obtained from Duke University Medical Center</td></tr><tr><td>YNB</td><td>BD</td><td>239210</td><td>See manufacturer for preparation instructions. Use a glucose concentration of 20 g/L.</td></tr><tr><td>Lipopolysaccharide</td><td>Sigma</td><td>L4391-1MG</td><td>Dissolve in 1x PBS to make 1 mg/ml stock. Store at -20 &amp;deg;C.</td></tr><tr><td>Interferon gamma</td><td>Sigma</td><td>I4777</td><td>Dissolve in 1x PBS to make 0.1 mg/ml stock solution</td></tr><tr><td>Glucuronoxylomannan antibody (anti-GXM)</td><td></td><td></td><td>Gift from Arturo Casadevall&#039;s Lab concentration is 1.98 mg/ml</td></tr><tr><td>Giemsa</td><td>MP Biomedicals</td><td>194591</td><td>Dissolve 0.8 g of Giemsa in 25 ml of glycerol and heat to 60 &amp;deg;C for 1 hr. Add 25 ml of methanol to the solution and allow to age at room temperature for at least 1 month.</td></tr><tr><td>Microscope</td><td>Zeiss</td><td></td><td>Observer.D1 microscope with AxioCam MRm for taking images</td></tr></tbody></table>

macrophage cholesterol depletion and its effect on the phagocytosis of cryptococcus neoformans

隐球菌是一种威胁生命的感染引起的属隐球菌的致病性真菌。发生感染时吸入孢子，这是能够在肺部深处复制的。 隐球菌的巨噬细胞吞噬是，该疾病是能够传播到中枢神经系统以引起致死性脑膜脑炎的方法之一。因此， 隐球菌和巨噬细胞之间的关联的研究是非常重要的，以了解感染的进展。本研究描述了一个一步一步的协议，由C.研究巨噬细胞感染隐球菌体外 。使用该协议，主机固醇对宿主 - 病原体相互作用的作用进行了研究。不同浓度的甲基 - 环糊精（MCD）用于消耗来自鼠网肉瘤巨噬细胞样细胞系J774A.1胆固醇。胆固醇耗竭进行确认和定量同时使用市售availablË胆固醇定量试剂盒和薄层色谱法。用脂多糖（LPS）和干扰素γ（IFNγ）胆固醇耗竭细胞被激活，并感染抗体调理隐球菌野生型H99细胞以1的效应-目标:1的比例。 2小时温育与C之后感染的细胞进行了监测新型隐球菌及吞噬指数计算。胆固醇耗竭导致一显著降低的吞噬指数。所提出的协议，提供了一个方便的方法，以模仿感染过程的开始在实验室环境中，研究宿主脂质组合物对感染性的作用。

胆固醇消耗相比于1×PBS对照由以下中的Amplex红胆固醇测定试剂盒制造商的说明在协议的步骤1.3保留上清液的分析产生一个升高的浓度的胆固醇在MβCD处理过的样品。取决于细胞类型和MβCD浓度使用胆固醇耗竭可能会发生变化。对于J774用10mMMβCD处理后，观察到大约50％的损耗。耗尽可以使用从收集在步骤1.4（ 图1）的上清液和细胞裂解物获得的值来计算。 <p class="jove...

Watch this Scientific Journal Video about Macrophage Cholesterol Depletion and Its Effect on the Phagocytosis of Cryptococcus neoformans at JoVE.com

Macrophage Cholesterol Depletion and Its Effect on the Phagocytosis of Cryptococcus neoformans

In this article, a protocol for infection of macrophages with Cryptococcus neoformans is described. Also, a method for sterol depletion from the macrophages is explained. These protocols provide a guide to study fungal infections in vitro and examine the role of sterols in such infections.

巨噬细胞胆固醇消耗及其对的吞噬作用<em&gt;新型隐球菌</em

Cryptococcosis is a life-threatening infection caused by pathogenic fungi of the genus Cryptococcus. Infection occurs upon inhalation of spores, which are ...

macrophage-cholesterol-depletion-its-effect-on-phagocytosis

Research

JoVE Journal

Immunology and Infection

Macrophage Cholesterol Depletion and Its Effect on the Phagocytosis of Cryptococcus neoformans

12.6K 次观看.  Stony Brook University. In this article, a protocol for infection of macrophages with Cryptococcus neoformans is described. Also, a method for sterol depletion from the macrophages is explained. These protocols provide a guide to study fungal infections in vitro and examine the role of sterols in such infections. 

视频: Macrophage Cholesterol Depletion and Its Effect on the Phagocytosis of Cryptococcus neoformans

Depletion and Reconstitution of Macrophages in Mice

Macrophages are critical players in the innate immune response to infectious challenge or injury, initiating the innate immune response and directing the acquired immune response. Macrophage dysfunction can lead to an inability to mount an appropriate immune response and as such, has been implicated in many disease processes, including inflammatory bowel diseases. Macrophages display polarized phenotypes that are broadly divided into two categories. Classically activated macrophages, activated by stimulation with IFN&gamma; or LPS, play an essential role in response to bacterial challenge whereas alternatively activated macrophages, activated by IL-4 or IL-13, participate in debris scavenging and tissue remodeling and have been implicated in the resolution phase of inflammation. During an inflammatory response in vivo, macrophages are found amid a complex mixture of infiltrating immune cells and may participate by exacerbating or resolving inflammation. To define the role of macrophages in situ in a whole animal model, it is necessary to examine the effect of depleting macrophages from the complex environment. To ask questions about the role of macrophage phenotype in situ, phenotypically defined polarized macrophages can be derived ex vivo, from bone marrow aspirates and added back to mice, with or without prior depletion of macrophages. In the protocol presented here clodronate-containing liposomes, versus PBS injected controls, were used to deplete colonic macrophages during dextran sodium sulfate (DSS)-induced colitis in mice. In addition, polarized macrophages were derived ex vivo and transferred to mice by intravenous injection. A caveat to this approach is that clodronate-containing liposomes deplete all professional phagocytes, including both dendritic cells and macrophages so to ensure the effect observed by depletion is macrophage-specific, reconstitution of phenotype by adoptive transfer of macrophages is necessary. Systemic macrophage depletion in mice can also be achieved by backcrossing mice onto a CD11b-DTR background, which is an excellent complementary approach. The advantage of clodronate-containing liposome-mediated depletion is that it does not require the time and expense involved in backcrossing mice and it can be used in mice regardless of the background of the mice (C57BL/6, BALB/c, or mixed background).

Macrophages play a central role in homeostasis and pathology in many tissues. The protocol presented here describes methods for depleting macrophages in vivo, deriving polarized macrophages from bone marrow aspirates, and adoptively transferring macrophages into mice. These techniques allow determination of the role that polarized macrophages play in health and disease.

在小鼠体内的巨噬细胞的枯竭和重建

Macrophages are critical players in the innate  immune response to infectious challenge or injury, initiating the innate immune  response and directing ...

科研

免疫与感染

Live-cell Video Microscopy of Fungal Pathogen Phagocytosis

Phagocytic clearance of fungal pathogens, and microorganisms more generally, may be considered to consist of four distinct stages: (i) migration of phagocytes to the site where pathogens are located; (ii) recognition of pathogen-associated molecular patterns (PAMPs) through pattern recognition receptors (PRRs); (iii) engulfment of microorganisms bound to the phagocyte cell membrane, and (iv) processing of engulfed cells within maturing phagosomes and digestion of the ingested particle. Studies that assess phagocytosis in its entirety are informative1, 2, 3, 4, 5 but are limited in that they do not normally break the process down into migration, engulfment and phagosome maturation, which may be affected differentially. Furthermore, such studies assess uptake as a single event, rather than as a continuous dynamic process. We have recently developed advanced live-cell imaging technologies, and have combined these with genetic functional analysis of both pathogen and host cells to create a cross-disciplinary platform for the analysis of innate immune cell function and fungal pathogenesis. These studies have revealed novel aspects of phagocytosis that could only be observed using systematic temporal analysis of the molecular and cellular interactions between human phagocytes and fungal pathogens and infectious microorganisms more generally. For example, we have begun to define the following: (a) the components of the cell surface required for each stage of the process of recognition, engulfment and killing of fungal cells1, 6, 7, 8; (b) how surface geometry influences the efficiency of macrophage uptake and killing of yeast and hyphal cells7; and (c) how engulfment leads to alteration of the cell cycle and behavior of macrophages 9, 10.
In contrast to single time point snapshots, live-cell video microscopy enables a wide variety of host cells and pathogens to be studied as continuous sequences over lengthy time periods, providing spatial and temporal information on a broad range of dynamic processes, including cell migration, replication and vesicular trafficking. Here we describe in detail how to prepare host and fungal cells, and to conduct the video microscopy experiments. These methods can provide a user-guide for future studies with other phagocytes and microorganisms.

We describe methods for live-cell video microscopy of Candida albicans phagocytosis by macrophages. These methods enable stage-specific analysis of macrophage migration, recognition, engulfment and phagosome maturation and reveal novel aspects of phagocytosis.

活细胞视频显微镜病病原菌的吞噬作用

Phagocytic clearance of fungal pathogens, and microorganisms more generally, may be considered to consist of four distinct stages: (i) migration of ...

Assessing Anti-fungal Activity of Isolated Alveolar Macrophages by Confocal Microscopy

The lung is an interface where host cells are routinely exposed to microbes and microbial products. Alveolar macrophages are the first-line phagocytic cells that encounter inhaled fungi and other microbes. Macrophages and other immune cells recognize Aspergillus motifs by pathogen recognition receptors and initiate downstream inflammatory responses. The phagocyte NADPH oxidase generates reactive oxygen intermediates (ROIs) and is critical for host defense. Although NADPH oxidase is critical for neutrophil-mediated host defense1-3, the importance of NADPH oxidase in macrophages is not well defined. The goal of this study was to delineate the specific role of NADPH oxidase in macrophages in mediating host defense against A. fumigatus. We found that NADPH oxidase in alveolar macrophages controls the growth of phagocytosed A. fumigatus spores4. Here, we describe a method for assessing the ability of mouse alveolar macrophages (AMs) to control the growth of phagocytosed Aspergillus spores (conidia). Alveolar macrophages are stained in vivo and ten days later isolated from mice by bronchoalveolar lavage (BAL). Macrophages are plated onto glass coverslips, then seeded with green fluorescent protein (GFP)-expressing A. fumigatus spores. At specified times, cells are fixed and the number of intact macrophages with phagocytosed spores is assessed by confocal microscopy.

A method to evaluate the ability of isolated mouse alveolar macrophages to control the growth of phagocytosed Aspergillus spores by confocal microscopy.

评估隔离肺泡巨噬细胞的激光共聚焦显微镜抗真菌活性

The lung is an interface where host cells are routinely exposed to microbes and microbial products. Alveolar macrophages are the first-line phagocytic ...

Visualizing Non-lytic Exocytosis of Cryptococcus neoformans from Macrophages Using Digital Light Microscopy

Many aspects of the infection of macrophages by Cryptococcus neoformans have been extensively studied and well defined. However, one particular interaction that is not clearly understood is non-lytic exocytosis. In this process, yeast cells are released into the extracellular space by a poorly understood mechanism that leaves both the macrophage and Cn viable. Here, we describe how to follow a large number of individually infected macrophages for a 24 hr infection period by time-lapsed microscopy. Infected macrophages are housed in a heating chamber with a CO2 atmosphere attached to a microscope that provides the same conditions as a cell-culture incubator. Live digital microscopy can provide information about the dynamic interactions between a host and pathogen that is not available from static images. Being able to visualize each infected cell can provide clues as to how macrophages handle fungal infections, and vice versa. This technique is a powerful tool in studying the dynamics that are behind a complex phenomenon.

Visualizing Non-lytic Exocytosis of Cryptococcus neoformans from Macrophages Using Digital Light Microscopy

We describe how to visualize macrophage-C. neoformans (Cn) interactions in real time, with specific emphasis on the process of non-lytic exocytosis using digital light microscopy. Using this technique individually infected macrophages can be studied to ascertain various aspects of this phenomenon.

可视化的非溶胞胞吐作用<em&gt;新型隐球菌</em&gt;从巨噬细胞用数码光学显微镜

Many aspects of the infection of macrophages by Cryptococcus neoformans have been extensively studied and well defined. However, one particular ...

High Throughput Fluorometric Technique for Assessment of Macrophage Phagocytosis and Actin Polymerization

The goal of fluorometric analysis is to serve as an efficient, cost effective, high throughput method of analyzing phagocytosis and other cellular processes. This technique can be used on a variety of cell types, both adherent and non-adherent, to examine a variety of cellular properties. When studying phagocytosis, fluorometric technique utilizes phagocytic cell types such as macrophages, and fluorescently labeled opsonized particles whose fluorescence can be extinguished in the presence of trypan blue. Following plating of adherent macrophages in 96-well plates, fluorescent particles (green or red) are administered and cells are allowed to phagocytose for varied amounts of time. Following internalization of fluorescent particles, cells are washed with trypan blue, which facilitates extinction of fluorescent signal from bacteria which are not internalized, or are merely adhering to the cell surface. Following the trypan wash, cells are washed with PBS, fixed, and stained with DAPI (nuclear blue fluorescent label), which serves to label nuclei of cells. By a simple fluorometric quantification through plate reading of nuclear (blue) or particle (red/green) fluorescence we can examine the ratio of relative fluorescence units of green:blue and determine a phagocytic index indicative of amount of fluorescent bacteria internalized per cell. The duration of assay using a 96-well method and multichannel pipettes for washing, from end of phagocytosis to end of data acquisition, is less than 45 min. Flow cytometry could be used in a similar manner but the advantage of fluorometry is its high throughput, rapid method of assessment with minimal manipulation of samples and quick quantification of fluorescent intensity per cell. Similar strategies can be applied to non adherent cells, live labeled bacteria, actin polymerization, and essentially any process utilizing fluorescence. Therefore, fluorometry is a promising method for its low cost, high throughput capabilities in the study of cellular processes.

Here we present a protocol to quantify phagocytosis of fluorescent particles by adherent macrophage cell line using a fluorometric method. This method facilitates a high throughput quantification of particle internalization as well as the resulting actin polymerization.

高通量荧光技术巨噬细胞吞噬功能和肌动蛋白聚合的评估

The goal of fluorometric analysis is to serve as an efficient, cost effective, high throughput method of analyzing phagocytosis and other cellular ...

Culture of Macrophage Colony-stimulating Factor Differentiated Human Monocyte-derived Macrophages

A protocol is presented for cell culture of macrophage colony-stimulating factor (M-CSF) differentiated human monocyte-derived macrophages. For initiation of experiments, fresh or frozen monocytes are cultured in flasks for 1 week with M-CSF to induce their differentiation into macrophages. Then, the macrophages can be harvested and seeded into culture wells at required cell densities for carrying out experiments. The use of defined numbers of macrophages rather than defined numbers of monocytes to initiate macrophage cultures for experiments yields macrophage cultures in which the desired cell density can be more consistently attained. Use of cryopreserved monocytes reduces dependency on donor availability and produces more homogeneous macrophage cultures.

A protocol is presented for cell culture of macrophage colony-stimulating factor (M-CSF) differentiated human monocyte-derived macrophages. The protocol utilizes cryopreservation of monocytes coupled with their bulk differentiation into macrophages. Then harvested macrophages can then be seeded into culture wells at required cell densities for carrying out experiments.

巨噬细胞集落刺激因子分化的人单核细胞衍生的巨噬细胞的培养

A protocol is presented for cell culture of macrophage colony-stimulating factor (M-CSF) differentiated human monocyte-derived macrophages. For initiation ...

Methods to Study Lipid Alterations in Neutrophils and the Subsequent Formation of Neutrophil Extracellular Traps

Lipid analysis performed by high performance thin layer chromatography (HPTLC) is a relatively simple, cost-effective method of analyzing a broad range of lipids. The function of lipids (e.g., in host-pathogen interactions or host entry) has been reported to play a crucial role in cellular processes. Here, we show a method to determine lipid composition, with a focus on the cholesterol level of primary blood-derived neutrophils, by HPTLC in comparison to high performance liquid chromatography (HPLC). The aim was to investigate the role of lipid/cholesterol alterations in the formation of neutrophil extracellular traps (NETs). NET release is known as a host defense mechanism to prevent pathogens from spreading within the host. Therefore, blood-derived human neutrophils were treated with methyl-&#946;-cyclodextrin (M&#946;CD) to induce lipid alterations in the cells. Using HPTLC and HPLC, we have shown that M&#946;CD treatment of the cells leads to lipid alterations associated with a significant reduction in the cholesterol content of the cell. At the same time, M&#946;CD treatment of the neutrophils led to the formation of NETs, as shown by immunofluorescence microscopy. In summary, here we present a detailed method to study lipid alterations in neutrophils and the formation of NETs.

Lipids are known to play an important role in cellular functions. Here, we describe a method to determine the lipid composition of neutrophils, with emphasis on the cholesterol level, by using both HPTLC and HPLC to gain a better understanding of the underlying mechanisms of neutrophil extracellular trap formation.

方法来研究中性粒细胞脂质的改变和中性粒细胞胞外陷阱随后形成

Lipid analysis performed by high performance thin layer chromatography (HPTLC) is a relatively simple, cost-effective method of analyzing a broad range of ...

Experimental Analysis of Apoptotic Thymocyte Engulfment by Macrophages

Cell apoptosis is a natural process and plays a critical role in embryonic development, homeostatic regulation, immune tolerance induction, and resolution of inflammation. Accumulation of apoptotic debris in the body may trigger chronic inflammatory responses that lead to systemic autoimmune diseases over time. Impaired apoptotic cell clearance has been implicated in a variety of autoimmune diseases. Apoptotic clearance is a complex process rarely detected under physiological conditions. It involves abundant surface receptors and signaling molecules. Studying the process of apoptotic cell clearance provides insightful molecular mechanisms and subsequent biological responses, which may lead to the development of new therapeutics. Here, we describe protocols for the induction of apoptotic thymocytes, the preparation of peritoneal macrophages, and the analysis of apoptotic cell clearance by flow cytometry and microscopy. All cells will undergo apoptosis at a certain stage, and many residential and circulating cells can uptake apoptotic debris. Therefore, the protocol described here can be used in many applications to characterize apoptotic cell binding and ingestion by many other cell types.

Here, we present a protocol to prepare apoptotic thymocytes and peritoneal macrophages and analyze the efficiency of efferocytosis and the specific inhibitor-mediated blocking of apoptotic thymocytes engulfment. This protocol has a broad application in cell-mediated clearance of other particles including artificial beads and bacteria.

巨噬细胞吞噬胸腺细胞吞噬的实验分析

Cell apoptosis is a natural process and plays a critical role in embryonic development, homeostatic regulation, immune tolerance induction, and resolution ...

The Effect of Aspirin and Ibuprofen on the Phagocytic Ability of Macrophages

This video showcases a method to assess the impact of aspirin and ibuprofen, at their minimum inhibitory concentrations, on macrophage phagocytosis using pH-sensitive fluorescent dye-stained fungal cells. The drugs inhibit cyclooxygenase-2, decreasing prostaglandin formation and boosting macrophage phagocytosis, as observed through fluorescence measurements of pH-sensitive dye.

阿司匹林和布洛芬对巨噬细胞吞噬能力的影响

1. Safety considerations

	Personnel should use personal protective equipment (PPE).
	Handle the pathogen and macrophages in a Class II Biological safety ...

JoVE Encyclopedia of Experiments

Immunology

Immunodiagnostics

Cellular Immunodiagnostics

Effect of Aspirin and Ibuprofen on Phagocytic Ability of Macrophages

Begin with a culture of the murine macrophage cell line, grown in complete RPMI 1640 medium, as described in the text protocol.
Additionally, prepare a culture of Cryptococcus neoformans resuspended in PBS to the desired concentration, as mentioned in the text protocol.&#160;
To stain the cryptococcal cells with the phagocytosis stain, dispense 999 microliters of the cells into a microcentrifuge tube. Add 1 microliter of the stain to the tube. Incubate the cells for 1 hour at room temperature, in the dark, with slow agitation.
After incubation, wash the cells by first centrifugation. Then, discard the supernatant. Resuspend the cells with PBS and repeat the washing step two additional times.
Dispense 100 microliters of the Cryptococcus neoformans cell suspension into the wells of the 96-well plate containing the cultured macrophages.
To study the effect of the test drugs on macrophage phagocytosis, dispense 100 microliters of the desired test drug into appropriate wells of the 96-well plate as described in the text protocol.
Incubate the plate in a carbon dioxide incubator for 2 to 6 hours. At the end of the incubation, measure the fluorescence from the phagocytic stain.

巨噬细胞胆固醇消耗及其对的吞噬作用

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方法来研究中性粒细胞脂质的改变和中性粒细胞胞外陷阱随后形成

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阿司匹林和布洛芬对巨噬细胞吞噬能力的影响

阿司匹林和布洛芬对巨噬细胞吞噬能力的影响