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In vitro Analysis for Macrophage Binding and Pro-inflammatory Responses to Candida albicans
巨噬细胞对白色念珠菌的结合和促炎反应的体外分析方法   

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Abstract

Macrophage recognition of Candida albicans (C. albicans) is facilitated by pattern recognition receptors that interact with the fungal pathogen associated molecular patterns (PAMPs). Dectin-1 is the major macrophage receptor that is known to recognize fungal Beta-glucans leading to induction of various immune responses. This receptor is also known to be required for in vivo protection against C. albicans (Taylor et al., 2007). We recently showed that the Dectin-1 mediated protection in vivo is strain-dependent, and that C. albicans can adapt to modulate immune recognition by Dectin-1 (Marakalala et al., 2013). In vitro analysis, however, showed a Dectin-1-dependent and pro-inflammatory responses against all strains tested. This protocol describes in detail the in vitro analysis used in the paper. In particular, methods involved in fluorescent labeling of live C. albicans, quantification of macrophage binding of the pathogen, and pro-inflammatory responses to yeast and hyphal forms of the fungi are described.

Keywords: Candida albicans(白色念珠菌), Dectin-1(树突状细胞相关性C型植物血凝素1), Beta-glucans(β-葡聚), Macrophages(巨噬细胞), Pro-inflammatory responses(炎性反应)

Materials and Reagents

  1. Candida albicans strains, SC5314 and ATCC18804
  2. Mice
    Note: 2x per WT or Dectin-1 KO mice on C57BL/6 background should give sufficient macrophages for a full 24-well plate experiment. Mice were age and sex matched.
  3. Thioglycollate-elicited macrophages
    Note: This method has been described in detail in Kerrigan et al. (2012).
  4. RPMI 1640 medium (Life Technologies, catalog number: 11875-093
  5. 70% ethanol
  6. Sabouraud Dextrose broth (Oxoid Limited, catalog number: CM0147 )
  7. Rhodamine Green-X (Life Technologies, InvitrogenTM, catalog number: R-6113 )
  8. Difco Thioglycollate Broth (BD Biosciences, catalog number: 225710 )
  9. Triton-X 100 (Sigma-Aldrich, catalog number: T8787 )
  10. OptEIA TNF kit (BD Biosciences, catalog number: 555268 )
  11. Zymosan (Life Technologies, InvitrogenTM, catalog number: Z2849 )
  12. Dulbecco’s Phosphate Buffered Saline (PBS) (Sigma-Aldrich, catalog number: D8662 )
  13. Fetal Bovine/Calf Serum (FCS) (Sigma-Aldrich, catalog number: F6178 )
  14. EDTA (Sigma-Aldrich, catalog number: E9884 )

Equipment

  1. Titer-Tek Fluoroskan II (Labsystems)
  2. Shaker
    Note: The shaker temperature was controlled at 30 °C and the speed was between 150 to 200 rpm.
  3. Centrifuge (Eppendorf, model: 5810R with swing bucket rotor S-4-72 )
  4. Light microscope
  5. Haemocytometer
  6. 24-well plates (BD Biosciences)
  7. 2 ml Eppendorf tube
  8. 96-well plates (black in color for fluorescence measurement) (BD Biosciences)
  9. Rubber back of syringe plunger
    Note: The make or brands for above equipment are not important for reproducibility.

Procedure

  1. Growth and Labeling of Candida albicans
    1. Start C. albicans cultures by inoculating frozen stocks into 5 ml Sabouraud Dextrose broth and incubate for 16 to 24 h at 30 °C with shaking at 200 rpm.
    2. Centrifuge the cells for 5 min at 3,000 rpm and wash three times in 10 ml Dulbecco’s PBS.
    3. Count the yeast cells under the microscope using a haemocytometer.
    4. Adjust the cell density to at least 3.2 x 106 yeast/ml, or as desirable in PBS and transfer into a 2 ml eppendorf tube.
    5. Add Rhodamine Green-X to a concentration of 200 μg/ml.
    6. Cover the tube with foil and incubate with gentle agitation at room temperature for 30 to 45 min.
    7. Centrifuge the labelled cells at 3,000 rpm for 5 min and resuspend in PBS.
    8. Wash the cells about ten times with 2 ml PBS or until free Rhodamine Green-X is removed.

  2. Macrophage binding and pro-inflammatory assays
    Notes:
    1.  Extraction of macrophages is done in a hood within animal facility.
    2. The treatment of macrophages with Candida albicans is performed in a hood in a tissue culture BSL2 area.
    3. The hood surface is sprayed with 70% ethanol to prevent sample contamination.
    1. Inject mice intraperitoneally with 1 ml of thioglycollate broth (Kerrigan et al., 2012). Gently hold the mouse to allow free space on the site of injection. Use a syringe to inject the thioglycollate broth in the lower quadrant of the abdomen; be careful not to prick vital organs such as the bladder or intestines.
    2. After four days, isolate peritoneal exudate cells by lavage with ice-cold 5 ml PBS containing 5 mM EDTA.
    3. Centrifuge the peritoneal cells at 1,000 rpm for 10 min and resuspend the pellet in 5 ml RPMI medium containing 10% (volume/volume) heat-inactivated FCS (by heating at 60 °C for 30 min).
      Note: All FCS used in the following steps are heat-inactivated.
    4. Count macrophages on a haemocytometer, dilute them to 5.0 x 105 cells/ml and seed them at a density of 2.5 x 105 cells/well in a 24-well plate with RPMI medium containing 10% FCS. Incubate the plate overnight at 37 °C (without any agitation).
    5. Aspirate wells and add 0.5 ml fresh RPMI plus 10% FCS to the macrophages that are attached to the wells.
    6. Add the Rhodamine Green-X-labelled C. albicans at the MOI (multiplicity of infection) of 5:1 or 10:1 to the macrophages.
    7. Fluorescein isothiocyanate–labelled zymosan can be used as a control at MOI of 25:1. Zymosan is made of Beta-glucan particles that are recognized by macrophage receptors and induce pro-inflammatory responses; Dectin-1 is a well-studied receptor for these particles (Brown et al., 2003).
      Notes:
      1. Zymosan is labelled with FITC according to manufacturer’s (Molecular Probe) instructions as described also in detail in Kerrigan et al. (2012).
      2. The expected results are that Zymosan will bind macrophage and induce proinflammatory responses in a Dectin-1 dependent manner.
    8. Incubate the plate on ice for 30 min to allow the particles to settle, and then a further 30 min incubation at 37 °C, 5% CO2.
    9. Wash the wells three times with 0.5 ml RPMI plus 10% FCS to remove the unbound particles.
    10. Add 500 μl of the RPMI plus 10% FCS to the wells and incubate at 37 °C for 3 h, 5% CO2.
    11. Aliquot 150 μl of the supernatant and store at -80 °C for later analysis of pro-inflammatory cytokine production.
    12. Wash the wells and lyse the cells by adding 150 μl 3% Triton X-100 (pH 7.5). Triton X-100 is diluted to 3% (v/v) in distilled water.
    13. Detach the cells using rubber back of syringe plunger and add 100 μl into black 96-well plates. Cell scraper can also be used.
    14. To quantify the binding of fungal particles to macrophages, measure the fluorescence at the excitation of 490 nm and the emission at 514 nm on a Titer-Tek Fluoroskan II. Expected results are as shown in Figure 4b in Marakalala et al. (2013).
    15. For pro-inflammatory responses, use ELISA (OptEIA TNF kit) to measure the TNF concentrations of the supernatant aliquots which had been stored at -80 °C.
      Note: Pro-inflammatory responses to hyphae can be measured similarly, except that macrophages are added directly to the wells that already contain live or heat-killed hyphae. See below for detailed description of hyphal induction.

  3. Pro-inflammatory responses to hyphae
    1. To induce hyphae formation, grow and quantify C. albicans as stated in steps A1-3.
    2. Incubate 2.5 x 106 yeast/well (cell density of about 3.2 x 106 yeast/ml or as per required amount) with RPMI medium for 3 h at 37 °C. View the cells under the microscope to confirm hyphae formation.
    3. Heat-kill the hyphae by incubating at 65 °C for 2 h, or use live hyphae if desired.
    4. Add 2.5 x 105 of thioglycollate-elicited macrophages (densisty is 5.0 x 105 cells/ml directly the hyphae-containing wells).
    5. Incubate the plates overnight at 37 °C, 5% CO2.
    6. Take the supernatant samples and store at -80 °C until needed for cytokine analysis.

Acknowledgments

This protocol was adapted from previous publications including (Marakalala et al., 2008; Brown et al., 2003; Kerrigan et al., 2012, and Taylor et al., 2007). I also acknowledge the Sydney Brenner Fellowship, National Research Foundation and Medical Research Council of South Africa for funding.

References

  1. Brown, G. D., Herre, J., Williams, D. L., Willment, J. A., Marshall, A. S. and Gordon, S. (2003). Dectin-1 mediates the biological effects of beta-glucans. J Exp Med 197(9): 1119-1124.
  2. Kerrigan, A. M., de Sousa Mda, G. and Brown, G. D. (2012). Simple assays for measuring innate interactions with fungi. Methods Mol Biol 845: 303-317.
  3. Marakalala, M. J., Vautier, S., Potrykus, J., Walker, L. A., Shepardson, K. M., Hopke, A., Mora-Montes, H. M., Kerrigan, A., Netea, M. G., Murray, G. I., Maccallum, D. M., Wheeler, R., Munro, C. A., Gow, N. A., Cramer, R. A., Brown, A. J. and Brown, G. D. (2013). Differential adaptation of Candida albicans in vivo modulates immune recognition by dectin-1. PLoS Pathog 9(4): e1003315.
  4. Taylor, P. R., Tsoni, S. V., Willment, J. A., Dennehy, K. M., Rosas, M., Findon, H., Haynes, K., Steele, C., Botto, M., Gordon, S. and Brown, G. D. (2007). Dectin-1 is required for beta-glucan recognition and control of fungal infection. Nat Immunol 8(1): 31-38.

简介

通过与真菌病原体相关分子模式(PAMP)相互作用的模式识别受体促进白色念珠菌(< em>白色念珠菌)的巨噬细胞识别。 Dectin-1是已知识别真菌β-葡聚糖导致各种免疫应答诱导的主要巨噬细胞受体。也已知这种受体对于体内抗em的保护是必需的。白色念珠菌(Taylor et al。,,2007)。我们最近显示Dectin-1介导的体内保护是应变依赖性的,而且C。白色念珠菌可以适应于调节Dectin-1的免疫识别(Marakalala等人,2013)。然而,体外分析显示针对所有测试的菌株的Dectin-1依赖性和促炎症反应。该协议详细描述了本文中使用的体外分析。特别地,涉及活的荧光标记的方法。白色念珠菌,对病原体的巨噬细胞结合的定量和对真菌的酵母和菌丝形式的促炎反应。

关键字:白色念珠菌, 树突状细胞相关性C型植物血凝素1, β-葡聚, 巨噬细胞, 炎性反应

材料和试剂

  1. 白色念珠菌菌株,SC5314和ATCC18804
  2. 小鼠
    注意:C57BL/6背景上的每只WT或Dectin-1 KO小鼠的2x应当为完整的24孔板实验提供足够的巨噬细胞。 小鼠年龄和性别匹配。
  3. 硫代乙醇酸酯引发的巨噬细胞
    注意:这种方法已经在Kerrigan等人 (2012)。
  4. RPMI 1640培养基(Life Technologies,目录号:11875-093)
  5. 70%乙醇
  6. Sabouraud葡萄糖肉汤(Oxoid Limited,目录号:CM0147)
  7. 罗丹明Green-X(Life Technologies,Invitrogen TM ,目录号:R-6113)
  8. Difco硫代乙醇酸液体培养基(BD Biosciences,目录号:225710)
  9. Triton-X 100(Sigma-Aldrich,目录号:T8787)
  10. OptEIA TNF试剂盒(BD Biosciences,目录号:555268)
  11. 酵母聚糖(Life Technologies,Invitrogen TM,目录号:Z2849)
  12. Dulbecco's磷酸盐缓冲盐水(PBS)(Sigma-Aldrich,目录号:D8662)
  13. 胎牛/小牛血清(FCS)(Sigma-Aldrich,目录号:F6178)
  14. EDTA(Sigma-Aldrich,目录号:E9884)

设备

  1. Titer-Tek Fluoroskan II(Labsystems)
  2. 振动器
    注意:振荡器温度控制在30℃,速度在150到200rpm之间。
  3. 离心机(Eppendorf,型号:5810R,带有旋转斗转子S-4-72)
  4. 光学显微镜
  5. 血细胞计数器
  6. 24孔板(BD Biosciences)
  7. 2 ml Eppendorf管
  8. 96孔板(用于荧光测量的黑色)(BD Biosciences)
  9. 注射器柱塞的橡胶背面
    注意:上述设备的品牌对于重现性不重要。

程序

  1. 白色念珠菌的生长和标记
    1. 开始 C。 白色念珠菌培养物接种冷冻储备液到5ml Sabouraud葡萄糖肉汤并在30℃下孵育16至24小时 以200rpm摇动
    2. 将细胞以3,000rpm离心5分钟,并在10ml Dulbecco's PBS中洗涤三次
    3. 在显微镜下使用血细胞计数器计数酵母细胞。
    4. 将细胞密度调节至至少3.2×10 6酵母/ml,或者在PBS中所需的并转移到2ml eppendorf管中。
    5. 加入Rhodamine Green-X至浓度为200μg/ml
    6. 用箔覆盖管,并在室温下温和搅拌孵育30至45分钟
    7. 将标记的细胞以3,000rpm离心5分钟,并重悬于PBS中
    8. 用2ml PBS洗涤细胞约10次,或直到除去游离的罗丹明绿-X。

  2. 巨噬细胞结合和促炎测定
    注意:
    1. 巨噬细胞的提取在动物设施内的罩中进行。
    2. 用白色念珠菌治疗巨噬细胞是在组织培养BSL2区域的罩中进行的。
    3. 用70%乙醇喷雾罩表面以防止样品污染。
    1. 用1ml巯基乙酸盐肉汤腹腔注射小鼠 (Kerrigan等人,2012)。 轻轻地握住鼠标以允许自由空间 注射部位。 使用注射器注射巯基乙酸盐培养液 在腹部的下象限; 小心不要刺伤重要 器官如膀胱或肠。
    2. 四天后,通过用冰冷的5ml含有5mM EDTA的PBS灌洗来分离腹膜渗出液细胞。
    3. 离心腹膜细胞在1,000 rpm 10分钟,并重悬 在含有10%(体积/体积) 热灭活的FCS(通过在60℃加热30分钟)。
      注意:以下步骤中使用的所有FCS都已被热灭活。
    4. 在血细胞计数器上计数巨噬细胞,将其稀释至5.0×10 5个细胞/ml,并将其以2.5×10 5个细胞/孔的密度接种在24孔 平板用含有10%FCS的RPMI培养基。 将板孵育过夜   在37℃(无任何搅拌)。
    5. 吸出孔,加入0.5 ml新鲜RPMI加10%FCS到附着在孔上的巨噬细胞。
    6. 将MOI(感染复数)为5:1或10:1的罗丹明绿-X-标记的白色念珠菌加入巨噬细胞。
    7. 荧光素异硫氰酸酯标记的酵母聚糖可用作对照 在MOI为25:1。 酵母聚糖由β-葡聚糖颗粒制成 被巨噬细胞受体识别并诱导促炎症 反应; Dectin-1是这些颗粒的受到很好研究的受体 (Brown等人,2003)。
      注意:
      1. 酵母聚糖用FITC标记 根据制造商(Molecular Probe)说明书进行 也在Kerrigan等人 (2012)。
      2. 预期的结果是 酵母聚糖将结合巨噬细胞并诱导炎症反应 Dectin-1依赖型。
    8. 孵育板在冰上30分钟   以允许颗粒沉降,然后再孵育30分钟 在37℃,5%CO 2下
    9. 用0.5ml RPMI加10%FCS洗涤孔三次,以除去未结合的颗粒
    10. 向孔中加入500μlRPMI加10%FCS,并在37℃下孵育3小时,5%CO 2。
    11. 等分150μl的上清液,并储存在-80℃,以便以后分析促炎细胞因子的产生
    12. 洗涤孔和裂解细胞,通过加入150μl3%Triton X-100(pH   7.5)。 将Triton X-100在蒸馏水中稀释至3%(v/v)
    13. 分离细胞使用注射器柱塞的橡胶背,并添加100微升 进入黑色96孔板。 也可以使用刮刀。
    14. 至 量化真菌颗粒与巨噬细胞的结合,测量 在490nm的激发下的荧光和在514nm的514nm的发射   Titer-Tek Fluoroskan II。 预期结果如图4b所示(Marakalala等人,2013年)。
    15. 对于促炎反应,使用ELISA(OptEIA   TNF试剂盒)以测量上清液等份的TNF浓度 其已储存在-80℃ 注意:促炎症反应 可以类似地测量菌丝,除了加入巨噬细胞 直接到已经含有活或热灭活菌丝的孔。 有关菌丝感应的详细说明,请参阅下文。

  3. 对菌丝的促炎反应
    1. 为了诱导菌丝形成,如步骤A1-3中所述,培养和定量白色念珠菌。
    2. 在37℃下用RPMI培养基孵育2.5×10 6个酵母/孔(细胞密度约3.2×10 6个酵母/ml或根据所需量)3小时 。 在显微镜下观察细胞以确认菌丝形成
    3. 热灭菌菌丝通过孵育在65°C 2小时,或使用活菌丝,如果需要。
    4. 加入2.5×10 5的巯基乙酸酯 - 引发的巨噬细胞(密度为5.0×10 5个细胞/ml,直接含有菌丝的孔)。
    5. 在37℃,5%CO 2下孵育平板过夜。
    6. 取上清液样品并储存于-80℃直到需要进行细胞因子分析

致谢

该方案改编自以前的出版物,包括(Marakalala等人,2008; Brown等人,2003; Kerrigan等人,2012年) ,和Taylor等人,2007)。 我也感谢悉尼布伦纳奖学金,国家研究基金会和南非医学研究委员会的资助。

参考文献

  1. Brown,G.D.,Herre,J.,Williams,D.L.,Willment,J.A.,Marshall,A.S.and Gordon,S。(2003)。 Dectin-1介导β-葡聚糖的生物学作用。 Med 197(9):1119-1124。
  2. Kerrigan,A.M.,de Sousa Mda,G.and Brown,G.D。(2012)。 测量与真菌的先天相互作用的简单测定法 em> 845:303-317。
  3. Marakalala,MJ,Vautier,S.,Potrykus,J.,Walker,LA,Shepardson,KM,Hopke,A.,Mora-Montes,HM,Kerrigan,A.,Netea,MG,Murray,GI,Maccallum, Wheeler,R.,Munro,CA,Gow,NA,Cramer,RA,Brown,AJand Brown,GD(2013)。 体内白色念珠菌的差异适应调节dectin-1的免疫识别 。 PLoS Pathog 9(4):e1003315。
  4. Taylor,PR,Tsoni,SV,Willment,JA,Dennehy,KM,Rosas,M.,Findon,H.,Haynes,K.,Steele,C.,Botto,M.,Gordon, 2007)。 Dectin-1是β-葡聚糖识别和控制真菌感染所必需的。 > Nat Immunol 8(1):31-38。
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Marakalala, M. J. (2014). In vitro Analysis for Macrophage Binding and Pro-inflammatory Responses to Candida albicans. Bio-protocol 4(9): e1123. DOI: 10.21769/BioProtoc.1123.
  2. Marakalala, M. J., Vautier, S., Potrykus, J., Walker, L. A., Shepardson, K. M., Hopke, A., Mora-Montes, H. M., Kerrigan, A., Netea, M. G., Murray, G. I., Maccallum, D. M., Wheeler, R., Munro, C. A., Gow, N. A., Cramer, R. A., Brown, A. J. and Brown, G. D. (2013). Differential adaptation of Candida albicans in vivo modulates immune recognition by dectin-1. PLoS Pathog 9(4): e1003315.
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