评估细菌病原体免疫反应的声吞噬杀灭试验
Summary
这种光吞噬杀灭方法用于比较吞噬免疫细胞根据不同的处理方法和条件对细菌做出反应和杀灭细菌的能力。传统上, 这种检测方法是评估针对视黄素细菌产生的抗体的效应功能的黄金标准。
Abstract
对宿主细菌定植的免疫反应的一个关键方面是吞噬。声嗜肺细胞杀伤试验 (OPKA) 是一种实验性方法, 其中吞噬细胞与细菌单位共同培养。免疫细胞会吞噬细胞, 以一种与互补有关的方式杀死细菌培养物。免疫介导的细胞杀伤效率取决于许多因素, 可以用来确定不同的细菌培养物在抵抗细胞死亡方面的比较。通过这种方式, 可以评估潜在的免疫疗法的有效性, 以适应特定的细菌菌株和/或血清型。在该协议中, 我们描述了一个简化的 OPKA, 利用基本的培养条件和细胞计数来确定细菌细胞的活力后, 共同培养的治疗条件和 HL-60 免疫细胞。该方法已成功地应用于多种肺炎球菌血清型、囊囊株和囊状菌株及其他细菌种类。该 OPKA 协议的优点是其简单、多功能 (因为此检测方法不限于作为视黄素的抗体处理), 以及最大限度地减少时间和试剂以评估基本实验组。
Introduction
光吞噬杀灭试验 (OPKA) 是将细菌结构或功能的改变与随后免疫反应和功能的变化联系起来的重要工具。因此, 它经常被用作一种补充检测方法, 以确定抗体治疗、候选疫苗、酶优化等基于免疫的疗效。虽然体内检测是确定细菌感染模型中有效清除或保护的必要条件, 但 OPKA 可用于评估细菌、免疫细胞和实验等最基本成分对细菌细胞死亡的免疫贡献治疗。以往的研究表明, opka 可以被修改和用于各种细菌和血清型, 包括肺炎链球菌1, 金黄色葡萄球菌 2, 铜绿假单胞菌3。此外, 这些优化的检测方法可用于评估不同的实验治疗方法, 包括酶使细菌更容易接触补充介导的免疫细胞4和抗体治疗以改善抗体的能力光圣化5。传统上, opka 检测已成功应用于基础和临床研究环境中, 作为由病原体特异性抗体6、7、8、9诱导的有力保护指标.
不同类型的免疫细胞可用于评估声超核细胞的杀伤。一个常用的吞噬种群是 HL-60 人白血病细胞系。该细胞系可作为培养中的灭活早幼粒细胞保存;然而, 他们可以通过不同的药物治疗区分成不同的激活状态10,11。用 N、n-二甲基甲酰胺处理 HL60 可将细胞系分化为具有较强吞噬活性的活化中性粒细胞11。虽然 HL-60 细胞已得到优化, 并经常用于这些吞噬检测 10, 其他原代多核白细胞可作为免疫臂的实验 12。
此外, 这些检测可以简化13或多路复用14 , 以查看要测试的细菌的多种抗生素耐药菌株。通过开发能够有效计算琼脂板15号单点细菌菌落形成单元 (Cfu) 的软件, 使多路复用方法更加可行.在这里, 我们描述了一种流线型的方法, 使用一种细菌菌株, hl-60 细胞, 小兔补充, 和血琼脂板。通过这种方法, 可以快速评估多种治疗方法, 以解决如何调节细菌感染的先天免疫反应的具体研究问题。
Protocol
Representative Results
Discussion
Opka 在评估疫苗6,8引起的抗体介导的免疫反应中发挥着至关重要的作用。这种简化的 OPKA 的主要意义是在需要测试的条件 (即抗体、酶处理等) 中的适应性。从这个意义上说, 虽然这种检测方法可以用来测试视黄素 (即抗体) 在吞噬中的贡献, 但也可以用来评估如何克服通常抑制吞噬途径的毒力因素 (即胶囊多糖)。最大限度地减少多路复用 OPKA 中通常使用的…
Disclosures
The authors have nothing to disclose.
Acknowledgements
我们感谢穆恩·纳姆博士 (伯明翰阿拉巴马大学) 在我们的实验室建立 OPKA 检测方面提供的宝贵协助。这项工作得到了国家卫生研究院1R1AI123383-01A1 至 FYA 的支持。
Materials
| Annexin V (APC conjugated) | BioLegend | 640919 | |
| anti-CD35, human (PE conjugated) | BioLegend | 333405 | |
| anti-CD71, human (PE conjugated) | BioLegend | 334105 | |
| bacterial strain to be used (ie, Streptococcus pneumoniae, WU2) | Bacterial Respiratory Reference Laboratory (Dr. Moon Nahm) | ||
| blood agar plates | Hardy Diagnostic | A10 | |
| Fetal Clone serum | HyClone | SH30080.03 | |
| glycerol | Sigma | G9012-1L | |
| HL-60 cells | ATCC | CCL-240 | |
| IgG Isotype Control (PE conjugated) | BioLegend | 400907 | |
| N,N-dimethylformamide (DMF) | Fisher Chemical | UN2265 | |
| propidium iodide | Sigma | P4864 | |
| RPMI media with L-glutamine | Corning | 10-040-CV |
References
- Romero-Steiner, S., et al. Standardization of an opsonophagocytic assay for the measurement of functional antibody activity against Streptococcus pneumoniae using differentiated HL-60 cells. Clinical and Diagnostic Laboratory Immunology. 4 (4), 415-422 (1997).
- Nanra, J. S., et al. Capsular polysaccharides are an important immune evasion mechanism for Staphylococcus aureus. Human Vaccines and Immunotherapeutics. 9 (3), 480-487 (2013).
- Ishibashi, K., Yamaguchi, O., Shiraiwa, Y., Ogihara, M., Shigeta, S. Combination therapy of Pseudomonas aeruginosa pyelonephritis in neutropenic mice with human antilipopolysaccharide monoclonal antibody and cefsulodin. Journal of Urology. 155 (6), 2094-2097 (1996).
- Middleton, D. R., Paschall, A. V., Duke, J. A., Avci, F. Y. Enzymatic Hydrolysis of Pneumococcal Capsular Polysaccharide Renders the Bacterium Vulnerable to Host Defense. Infection and Immunity. , (2018).
- Baker, C. J., Noya, F. J. Potential use of intravenous immune globulin for group B streptococcal infection. Reviews of Infectious Diseases. 12, 476-482 (1990).
- Tian, H., Weber, S., Thorkildson, P., Kozel, T. R., Pirofski, L. A. Efficacy of opsonic and nonopsonic serotype 3 pneumococcal capsular polysaccharide-specific monoclonal antibodies against intranasal challenge with Streptococcus pneumoniae in mice. Infection and Immunity. 77 (4), 1502-1513 (2009).
- Parameswarappa, S. G., et al. A Semi-synthetic Oligosaccharide Conjugate Vaccine Candidate Confers Protection against Streptococcus pneumoniae Serotype 3 Infection. Cell Chemical Biology. 23 (11), 1407-1416 (2016).
- Jackson, L., et al. Randomized clinical trial of a single versus a double dose of 13-valent pneumococcal conjugate vaccine in adults 55 through 74 years of age previously vaccinated with 23-valent pneumococcal polysaccharide vaccine. Vaccine. 36 (5), 606-614 (2018).
- Cywes-Bentley, C., et al. Antibody to a conserved antigenic target is protective against diverse prokaryotic and eukaryotic pathogens. Proceedings of the National Academy of Sciences of the USA. 110 (24), 2209-2218 (2013).
- Fleck, R. A., Romero-Steiner, S., Nahm, M. H. Use of HL-60 cell line to measure opsonic capacity of pneumococcal antibodies. Clinical and Diagnostic Laboratory Immunology. 12 (1), 19-27 (2005).
- Collins, S. J., Ruscetti, F. W., Gallagher, R. E., Gallo, R. C. Terminal differentiation of human promyelocytic leukemia cells induced by dimethyl sulfoxide and other polar compounds. Proceedings of the National Academy of Sciences of the USA. 75 (5), 2458-2462 (1978).
- Melnick, N., Rajam, G., Carlone, G. M., Sampson, J. S., Ades, E. W. Evaluation of a novel therapeutic approach to treating severe pneumococcal infection using a mouse model. Clinical and Vaccine Immunology. 16 (6), 806-810 (2009).
- Dwyer, M., Gadjeva, M. Opsonophagocytic assay. Methods in Molecular Biology. 1100, 373-379 (2014).
- Burton, R. L., Nahm, M. H. Development of a fourfold multiplexed opsonophagocytosis assay for pneumococcal antibodies against additional serotypes and discovery of serological subtypes in Streptococcus pneumoniae serotype 20. Clinical and Vaccine Immunololgy. 19 (6), 835-841 (2012).
- Clarke, M. L., et al. Low-cost, high-throughput, automated counting of bacterial colonies. Cytometry Part A. 77 (8), 790-797 (2010).
- Aanei, C. M., et al. Database-guided Flow-cytometry for Evaluation of Bone Marrow Myeloid Cell Maturation. Journal of Visualized Experiments. (141), e57867 (2018).
- Hirz, T., Dumontet, C. Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents. Journal of Visulaized Experiments. (109), e53846 (2016).
- Rieger, A. M., Nelson, K. L., Konowalchuk, J. D., Barreda, D. R. Modified annexin V/propidium iodide apoptosis assay for accurate assessment of cell death. Journal of Visualized Experiments. (50), 2597 (2011).
- Blair, O. C., Carbone, R., Sartorelli, A. C. Differentiation of HL-60 promyelocytic leukemia cells: simultaneous determination of phagocytic activity and cell cycle distribution by flow cytometry. Cytometry. 7 (2), 171-177 (1986).
- Middleton, D. R., et al. Identification and characterization of the Streptococcus pneumoniae type 3 capsule-specific glycoside hydrolase of Paenibacillus species 32352. Glycobiology. 28 (2), 90-99 (2018).
