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Abstract
Introduction
Protocol
Results
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Disclosures
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Immunology and Infection

人外周血单核源性巨噬细胞内 基质木霉 分生孢子的活力测定

Published: October 20, 2023
doi:
10.3791/65231
, , , ,
1Laboratory of Immunobiology – Department of Biological Sciences,State University of Santa Cruz – UESC, 2Università Degli Studi di Genova

Summary

涉及巨噬细胞吞噬真菌分生孢子的技术被广泛用于评估对真菌免疫反应的调节的研究。本文的目的是提出一种评估分 生孢子木霉 刺激的人外周血单核来源巨噬细胞的吞噬作用和清除能力的方法。

Abstract

巨噬细胞是一道重要的防线，负责防止病原体在不同组织中的生长和定植。分生孢子吞噬作用是一个关键过程，可以研究巨噬细胞 – 病原体相互作用中涉及的细胞质和分子事件，以及确定内化分生孢子的死亡时间。涉及巨噬细胞吞噬真菌分生孢子的技术被广泛用于评估对真菌免疫反应的调节的研究。吞噬作用的逃避和吞噬体的逃逸是真菌毒力的机制。在这里，我们报告了可用于分析 T. stromaticum conidia 的吞噬作用、清除率和活力的方法，分生孢子是一种用作生物防治和生物肥料剂并能够诱导人类感染的真菌。该方案包括 1） 木霉 培养，2） 洗涤以获得分生孢子，3） 使用聚蔗糖溶液方法分离外周血单核细胞 （PBMC） 并将 PBMC 分化为巨噬细胞，4） 使用圆形玻璃盖玻片和着色的 体外 吞噬作用方法，以及 5） 清除测定以评估分生孢子吞噬后的分生孢子活力。综上所述，这些技术可用于测量巨噬细胞的真菌清除效率。

Introduction

木霉属（木霉属：木霉属，科：木霉科木霉属）由无处不在的腐生真菌组成，这些真菌是其他真菌物种的寄生虫，能够产生一系列商业上有用的酶1。这些真菌种类用于生产异源蛋白质2，生产纤维素3，乙醇，啤酒，葡萄酒和造纸4，在纺织工业5，食品工业6中，以及在农业中作为生物控制剂7,8。除了对这些真菌物种的工业兴趣外，人类感染数量的增加使一些木霉物种具有机会性病原体的地位9。

木霉属在培养物中生长迅速，最初有白色和棉质菌落，然后变成黄绿色至深绿色10.它们适应生活在广泛的pH值和温度条件下，机会主义物种能够在生理pH值和温度下生存，因此可以在不同的人体组织中定植11,12,13。重要的是，木霉属感染率的上升可能与毒力因素有关，而这些因素尚未得到很好的研究。此外，专注于了解针对机会性木霉菌种的免疫反应的研究仍然很少见。

在感染期间，巨噬细胞与中性粒细胞一起代表负责吞噬作用的防线，从而防止病原体在不同组织中的生长和定植。使用模式识别受体，如Toll样受体和C型凝集素受体，巨噬细胞吞噬真菌并将它们加工成吞噬溶酶体，从而促进呼吸爆发，促炎细胞因子的释放，以及吞噬微生物的破坏14。然而，吞噬作用的机制可以受到不同微生物策略的影响和逃避，例如真菌细胞的大小和形状;存在阻碍吞噬作用的胶囊;减少吞噬诱导受体的数量;细胞质中肌动蛋白纤维结构的重塑;阻碍伪足的形成;吞噬体或吞噬溶酶体在吞噬过程后逃逸14.

许多病原体，包括新型隐球菌，利用巨噬细胞作为在宿主体内生存、传播和诱导感染的生态位15。吞噬作用和清除测定用于评估对病原体的免疫反应，并鉴定用于逃避先天免疫系统的微生物策略15,16,17。这种类型的技术还可用于检查吞噬作用、延迟吞噬体酸化和氧化爆发的差异动力学，这些动力学导致真菌杀伤减少18。

可以使用不同的方法来评估吞噬作用、真菌存活和吞噬体成熟过程的逃避。这些包括荧光显微镜，用于观察吞噬作用、细胞位置和吞噬作用过程中产生的分子19;流式细胞术，其提供吞噬作用的定量数据，并用于评估该过程中涉及的不同标志物20,21;活体显微镜，用于评估微生物捕获和吞噬体成熟22;抗体介导的吞噬作用，用于评估病原体吞噬过程的特异性23;和其他24,25,26,27。

这里介绍的方案采用一种通用的、低成本的、直接的方法，使用光学显微镜和平板生长测定来评估真菌分生孢子的吞噬作用和杀伤作用。该方案将为读者提供使用暴露于 基质锥虫的人外周血单核来源的巨噬细胞进行吞噬作用和清除测定的分步说明。之所以使用PBMC，是因为分生 孢子木霉 被用作对抗植物病原体的生物防治剂和全球植物作物的生物肥料，并引起了几种人类感染，称为木霉病。除此之外，之前只有两篇研究分生 孢子 与人类免疫系统之间相互作用的工作，其中我们研究了中性粒细胞28 和巨噬细胞29中的自噬。本文首先展示了如何研究PBMC衍生的巨噬细胞对 T. stromaticum 分生孢子的吞噬作用，然后如何使用基于显微镜的简单技术评估被吞噬的分生孢子的活力。该方案可以进一步促进对巨噬细胞相关免疫反应或免疫系统调节相关机制的研究。

Protocol

伦理考虑和人类主体本研究中描述的所有人类实验均根据《赫尔辛基宣言》和巴西联邦法律进行，并经圣克鲁斯州立大学伦理委员会批准（项目识别代码：550.382/ 2014）。 从巴西巴伊亚州伊列乌斯市的健康志愿者那里收集人类外周血，这些志愿者没有暴露于与所研究真菌相关的职业活动。有健康医疗状况或使用药物的个人被排除在外。所有受试者在参与本研究?…

Representative Results

涉及巨噬细胞吞噬真菌分生孢子的技术被广泛用于评估对真菌免疫反应的调节的研究。我们利用 T. stromaticum 分生孢子的吞噬作用来评估吞噬作用后分生孢子的活力，因为吞噬作用的逃避和吞噬体的逃逸是真菌毒力的机制。研究人员在研究具有临床意义的物种时，应将这些技术作为首批检测之一。 <img alt="Figure 2" class="xfigimg" src="/files/ftp_up…

Discussion

对于几种真菌病原体，包括烟曲霉、隐球菌、白色念珠菌等，分生孢子或酵母吞噬作用是一个关键过程，可以研究巨噬细胞-病原体相互作用中的细胞质和分子事件，以及确定内化分生孢子的死亡时间14,39,40。吞噬作用是木霉-宿主相互作用的关键过程。木霉属调节多种吞噬途径，包括 Dectin-1 和 …

Disclosures

The authors have nothing to disclose.

Acknowledgements

这项工作得到了以下巴西金融机构的支持：巴伊亚州国家保护保护基金会（FAPESB），赠款RED0011/2012和RED008/2014。U.R.S.、J.O.C. 和 M.E.S.M. 分别感谢 Coordenação de Aperfeiçoamento de Pessoal de Nível Superior （CAPES） 和 FAPESB 颁发的奖学金。

Materials

15 mL centrifuge tubes Corning CLS431470 15 mL centrifuge tubes, polypropylene, conical bottom with lid, individually sterile
24-Well Flat Bottom Cell Culture Plate Kasvi K12-024 Made of polystyrene with alphanumeric identification; The Cell Culture Plate is DNase, RNase and pyrogen-free and free of cytotoxic substances; Sterilized by gamma radiation;
Cell culture CO2 incubator Sanyo 303082 A CO2 incubator serves to create and control conditions similar to a human body, thus allowing the in vitro growth and proliferation of different cell types.
Centrifuge Microtube (eppendorf type) 1.5 mL Capp 5101500 Made from polypropylene, with a cap attached to the tube for opening and closing with just one hand. It has a polished interior against protein adhesion and for sample visibility, being free of DNase, RNase and Pyrogens
Circular coverslip 15 mm Olen K5-0015 Circular coverslips are used for microscopy techniques in cell culture. Made of super transparent translucent glass; with thickness of 0.13 mm
Class II Type B2 (Total Exhaust) Biosafety Cabinets Esco Lifesciences group 2010274 Airstream Class II Type B2 Biosafety Cabinets (AB2) provide product, operator and environmental protection and are suitable for work with trace amounts of toxic chemicals and agents assigned to biological safety levels I, II or III. In a Class II Type B2 cabinet, all inflow and downflow air is exhausted after HEPA/ULPA filtration to the external environment without recirculation across the work surface.
Dextrose Potato Agar medium Merck 145 Potato Dextrose Agar is used in the cultivation and enumeration of yeasts and fungi
EDTA vacuum blood collection tube FirstLab FL5-1109L EDTA is the recommended anticoagulant for hematology routines as it is the best anticoagulant for preserving cell morphology.
Entellan Merck 1.07961  Fixative agent; Entellan is a waterless mounting medium for permanent mounting for microscopy.
Fetal Bovine Serum Gibco A2720801 Fetal bovine serum (FBS) is a universal growth supplement of cell and tissue culture media. FBS is a natural cocktail of most of the factors required for cell attachment, growth, and proliferation, effective for most types of human and animal (including insect) cells.
Flaticon  database of images
Glycerol Merck 24900988 The cryoprotectant agent glycerol is used for freezing cells and spores
Histopaque-1077 polysucrose solution
Image J  Image analysis software
Microscopy slides Precision 7105 Slide for Microscopy 26 x 76 mm Matte Lapped Thickness 1.0 to 1.2 mm. Made of special optical glass and packaged with silk paper divider with high quality transparency free of imperfections
Mini centrifuge Prism C1801 The Prism Mini Centrifuge was designed to be extremely compact with an exceptionally small footprint. Includes 2 interchangeable quick-release rotors that spin up to 6000 rpm. An electronic brake provides quick deceleration and the self-opening lid allows easy access to the sample, reducing handling time.
Neubauer chamber Kasvi K5-0011 The Neubauer Counting Chamber is used for counting cells or other suspended particles.
Panoptic fast  Laborclin 620529 Laborclin's  panoptic fast c is a kit for quick staining in hematology
Penicillin/Streptomycin Solution – 10,000U LGC- Biotechnology  BR3011001 antibiotic is used in order to avoid possible contamination by manipulation external to the laminar flow.
Petri dish 90 x 15 mm Smooth Cralplast 18248 Disposable Petri dish; Made of highly transparent polystyrene (PS); flat bottom; Smooth;Size: 90 x 15 mm.
Phosphate buffered saline (PBS) thermo fisher Scientific 10010001 PBS is a water-based saline solution with a simple formulation. It is isotonic and non-toxic to most cells. It includes sodium chloride and phosphate buffer and is formulated to prevent osmotic shock while maintaining the water balance of living cells.
Pipette Pasteur 3 mL Sterile Accumax AP-3-B-S STERILE ACCUMAX PASTEUR 3 ML PIPETTE with 3 mL capacity, made of transparent low-density polyethylene (LDPE) and individually sterile
Refrigerated Centrifuge Thermo Scientific TS-HM16R The Thermo Scientific Heraeus Megafuge 16R Refrigerated Centrifuge is a refrigerated centrifuge with the user-friendly control panel makes it easy to pre-set the speed, RCF value, running time, temperature, and running profile. The Megafuge 16R can reach maximum speeds of 15,200 RPM and maximum RCF of 25,830 x g.
RPMI-1640 Medium Merck MFCD00217820 HEPES Modification, with L-glutamine and 25 mM HEPES, without sodium bicarbonate, powder, suitable for cell culture
The single channel micropipettes Eppendorf Z683809 Single-channel micropipettes are used to accurately transfer and measure very small amounts of liquids.
Tip for Micropipettor Corning 4894 Capacity of 10 µL and 1,000 µL Autoclavable
Triocular inverted microscope LABOMED VU-7125500 It allows you to observe cells inside tubes and bottles, without having to open them, thus avoiding contamination problems.
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Tags

Keywords: TrichodermaMacrophagesPhagocytosisImmune SystemViability AssayConidial ClearanceBiocontrol AgentPathogenicityPeripheral Blood Mononuclear Cells (PBMCs)
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dos Santos, U. R., de Castro, J. O., Santos Matos, M. E., De Bonis, G., dos Santos, J. L. Viability Assay of Trichoderma stromaticum Conidia Inside Human Peripheral Blood Mononuclear-Derived Macrophages. J. Vis. Exp. (200), e65231, doi:10.3791/65231 (2023).
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