巨噬细胞胆固醇消耗及其对的吞噬作用<em>新型隐球菌</em
Summary
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.
Abstract
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。
Introduction
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'专业'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。
Protocol
Representative Results
Discussion
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?…
Divulgaciones
The authors have nothing to disclose.
Acknowledgements
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。
Materials
| Name of Reagent/ Equipment | Company | Catalog Number | Comments/Description |
| Class II type A2 Biosafety Cabinet | Labconco | 3460009 | |
| J774A.1 cell line | ATCC | TIB-67 | Arrives Frozen. See ATCC instructions for culturing. |
| Dulbecco’s Modified Eagle Medium | Gibco | 11995-065 | Store at 4 °C and warm to 37 °C prior to use |
| HI Fetal Bovine Serum Performance Plus | Gibco | 10082-147 | Keep frozen at -20 °C and thaw before adding to DMEM |
| Penicillin-Streptomycin (10,000 U/mL) | Gibco | 15140-122 | Used to suplement DMEM |
| Isotemp Cell culture incubator | Fisher Scientific | Model # 3530 | |
| 96-Well culture dish | Corning Inc. Costar | 3595 | |
| 10x Phosphate Buffered Saline | Fisher Scientific BioReagents | BP3994 | Dilute to 1x and filter or autoclave prior to use. |
| Methyl-β-Cyclodextrin | Sigma Life Science | C4555-10G | Dissolve in 1x PBS to make solutions of 10mM and 30mM concentrations |
| Orbital Shaker | Labline | ||
| Amplex Red Cholesterol Assay Kit | Life Technologies Molecular Probes | A12216 | All reagents for Cholesterol Assay are contained within the kit. Follow Manufacturer instructions. |
| 96-Well Black Assay plate | Corning Inc. Costar | 3603 | |
| FilterMax microplate reader | Molecular Devices | Model F5 | |
| TLC Chamber | Sigma-Aldrich | Z126195-1EA | |
| Chloroform | Sigma-Aldrich | 650498-4L | |
| Methanol | Sigma-Aldrich | 34860-2L-R | |
| TLC Paper | Whatman | 3030917 | Cut down to size needed for TLC tank |
| Fume Hood | Any fume hood that complies with AIHA/ANSI Standards | ||
| 6-Well Plate | Corning Inc. Costar | 3506 | |
| Trypsin-EDTA | Gibco | 25300-054 | |
| Cell Scraper | Corning Inc. Costar | 3010 | |
| Hemocytometer | Hausser Scientific | 1490 | |
| Centrifuge | Beckman Coulter | Model Alegra x-30R | |
| Votex Mixer | Fisher Scientific | 12-812 | |
| Balance | Mettler Toledo | Model # MS104S Meaures down to .1 mg | |
| Glass Pasteur Pipette | Fisherbrand | 13-678-20A | |
| Cholesterol | Avanti Polar Lipids | 700000 | |
| SpeedVac Concentrator | Thermo Scientific | Model # SPD2010 | |
| Petroleum Ether | Fisher Scientific | E139-1 | |
| Diethyl Ether | Sigma-Aldrich | 309966 | |
| Acetic Acid | Sigma-Aldrich | 320099 | |
| TLC Silica Gel 60 with concentrating zone | Analytical Chromatograhy Millipore | 1.11845.0001 | |
| Iodine Chips | Sigma-Aldrich | 376558-50G | |
| Sulfuric Acid | Sigma-Aldrich | 320501 | |
| Manganese Chloride | Sigma-Aldrich | 244589 | |
| UVP EC3 Imaging System | Ultra-Violet Products Ltd. | Use the Vision Works LS software for densitometry analysis | |
| Glass Bottom Confocal Dish | MatTek | P35G-1.5-10C | www.glassbottomdishes.com |
| Cryptococcus neoformans (H99) | Obtained from Duke University Medical Center | ||
| YNB | BD | 239210 | See manufacturer for preparation instructions. Use a Glucose concentration of 20 g/L. |
| Lipopolysaccharide | Sigma | L4391-1MG | Dissolve in 1x PBS to make 1mg/mL stock. Store at -20 °C. |
| Interferon gamma | Sigma | I4777 | Dissolve in 1x PBS to make .1 mg/mL stock solution |
| Glucuronoxylomannan antibody (anti-GXM) | Gift from Arturo Casadevall's Lab concentration is 1.98 mg/mL | ||
| Giemsa | MP Biomedicals | 194591 | Dissolve .8 g of Giemsa in 25 mL of Glycerol and heat to 60 °C for 1 hour. Add 25 mL of methanol to the solution and allow to age at room temperature for at least 1 month. |
| Microscope | Zeiss | Observer.D1 microscope with AxioCam MRm for taking images |
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