JoVE Journal > Immunology and Infection
Summary
Abstract
Protocol
Results
Discussion
Disclosures
Acknowledgements
Materials
References
Automatic Translation
Please note that all translations are automatically generated. Click here for the English version.
Immunology and Infection

肺炎链球菌在鼻内殖民期间炎症反应的特征

Published: January 17, 2014
doi:
10.3791/50490
, , ,
1Department of Pathology and Molecule Medicine,McMaster University

Summary

描述了 肺炎链球菌 的鼻咽结肠化以及随后对附体或招募细胞的提取。该技术涉及冲洗鼻咽和收集流体通过鼻孔，并适应各种读数，包括微分细胞定量和分析的mRNA表达 原位。

Abstract

肺炎链球菌的鼻咽结肠是侵入肺部或血液的先决条件。这种生物体能够殖民鼻咽的粘膜表面，在那里它可以居住，繁殖，并最终克服宿主防御入侵到宿主的其他组织。在通常的下呼吸道中发现感染会导致肺炎。或者，细菌可以传播到血液中，导致细菌血症，这与高死亡率2有关，否则直接导致肺炎球菌脑膜炎的发展。了解鼻咽结肠的动能和免疫反应是S.肺炎感染模型的一个重要方面。

我们的小鼠内殖民化模型改编自人类模型3， 并被多个研究小组用于鼻咽4-7宿主病原体反应的研究。在模型的第一部分，我们使用 肺炎 的临床分离建立一个自我限制的细菌殖民化，类似于人类成人的运输事件。此处详细说明的程序包括准备细菌接种，然后通过通过鼻内管理途径传递接种库建立殖民化事件。在稳定状态下，居民巨噬细胞是鼻咽的主要细胞类型。通常，在未受感染的小鼠8中，淋巴细胞很少，但粘膜结肠会导致低至高档炎症（取决于细菌物种和菌株的毒性），从而产生免疫反应，并随后招募宿主免疫细胞。这些细胞可以通过气管内含物的厕所进行分离，并与殖民化细菌的密度相关，以更好地了解感染的动能。

Protocol

在开始之前：除非另有说明，否则所有步骤均在生物危害 2 级 （BSL2） 生物安全柜 （BSC） 中完成。请确保在开始实验之前，您已获得适当的生物危害批准，以便根据机构指南使用传染性细菌病原体。此外，请确保您拥有进行事先准备的程序所需的所有材料和试剂。这些实验中使用的老鼠包括杰克逊实验室、查尔斯河或塔科尼奇的雌性C57BL/6小鼠，年龄在10-14周（尽管我们尚未发现鼻腔…

Representative Results

图 1 表示概述示意图，总结了协议的主要步骤。 图2-3 提供了本文所述协议所固有的微生物方法的可视化。 图4 表示小鼠进行鼻内殖民化的正确定位，而 图5 则描绘了与 S.肺炎 菌株P1547结肠的小鼠体重的典型变化。 图6-7 表示过程鼻腔熔洗部分的特定阶段，用于辅助可视化这两种技术。 图8-11 包括对从鼻腔清洗后从…

Discussion

在这项研究中，我们提出了使用 肺炎链球菌 临床分离菌株的小鼠的鼻内结肠的详细方法，以及随后针对细菌招募到鼻咽的免疫细胞的隔离和特征。我们演示了如何在营养丰富的介质中培养细菌，并用于在小鼠中建立殖民活动，最初仅限于鼻咽。然后，我们展示了如何通过使用罐装针头，在气管暴露、切口和鼻腔熔洗后分离出招募到鼻咽的免疫细胞类型。鼻腔熔岩样本可在PBS中采集，以分?…

Disclosures

The authors have nothing to disclose.

Acknowledgements

作者要感谢宾夕法尼亚大学的杰弗瑞·韦瑟博士赠送肺炎 链球菌临床菌株。这项工作由加拿大卫生研究所资助。简历由M.G.德格罗特奖学金和加拿大胸科学会的奖学金资助。这项工作由安大略肺脏协会和加拿大卫生研究所（CIHR）资助。鲍迪什实验室的工作部分得到了迈克尔·德格罗特传染病研究中心和麦克马斯特免疫学研究中心的支持。

Materials

Name of Reagent/Material Company Catalog Number
Anti-Mouse Ly6C FITC BD Pharmingen 553104
Anti-Mouse Ly6G PE BD Pharmingen
Anti-Mouse CD45.1 eFluor 450 eBioscience 48-0453-82
Anti-Mouse F4/80 Antigen APC eBioscience 17-4801-82
Anti-Mouse CD11c PerCP-Cy5.5 eBioscience 45-0114-82
Anti-Mouse CD11b PE-Cy7 eBioscience 25-0112-82
Anti-Mouse CD3 Alexa Fluor 700 eBioscience 56-0032-82
Anti-Mouse CD4 eFluor 605NC eBioscience 93-0041-42
Intramedic Polyethylene Tubing – PE20 Becton Dickinson 427406
BD 1ml Syringe Becton Dickinson 309659
BD 26G3/8 Intradermal Bevel Becton Dickinson 305110
Buffer RLT Lysis Buffer Qiagen 79216
Difco Tryptic Soy Agar Becton Dickinson 236950
Defibrinated Sheep Blood PML Microbiologicals A0404
RNAqueous-Micro Kit Ambion AM1931
M-MuLV Reverse Transcriptase New England Biolabs M0253L
GoTaq qPCR Master Mix Promega A6001
DOWNLOAD MATERIALS LIST

References

  1. Bogaert, D., de Groot, R., et al. Streptococcus pneumoniae colonisation: the key to pneumococcal disease. Lancet Infect. Dis. 4, 144-154 (2004).
  2. Kadioglu, A., Weiser, J. N., et al. The role of Streptococcus pneumoniae virulence factors in host respiratory colonization and disease. Nat. Rev. Microbiol. 6 (4), 288-301 (2008).
  3. McCool, T. L., Cate, T. R., et al. The immune response to pneumococcal proteins during experimental human carriage. J. Exp. Med. 195, 359-365 (2002).
  4. Nelson, A., Roche, A. M., et al. Capsule enhances pneumococcal colonisation by limiting mucus-mediated clearance. Infect. Immun. 75, 83-90 (2007).
  5. van Rossum, A., Lysenko, E., et al. Host and bacterial factors contributing to the clearance of colonisation by Streptococcus pneumoniae in a murine model. Infect. Immun. 73, 7718-7726 (2005).
  6. Barocchi, M. A., Ries, J., et al. A pneumococcal pilus influences virulence and host inflammatory responses. Proc. Natl. Acad. Sci. U.S.A. 103, 2857-2862 (2006).
  7. Malley, R., Henneke, P., et al. Recognition of pneumolysin by Toll-like receptor 4 confers resistance to pneumococcal infection. Proc. Natl. Acad. Sci. U.S.A. 100, 1966-1971 (2003).
  8. McCool, T. L., Weiser, J. N. Limited role of antibody in clearance of Streptococcus pneumoniae in a murine model of colonization. Infect. Immun. 72, 5807-5813 (2004).
  9. Gingles, N. A., et al. Role of genetic resistance in invasive pneumococcal infection: identification and study of susceptibility and resistance in inbred mouse strains. Infect. Immun. 69 (1), 426-434 (2001).
  10. Jeong, D., Jeong, E., et al. Difference in resistance to Streptococcus pneumoniae infection in mice. Lab Anim. Res. 27, 91-98 (2011).
  11. Wu, H. Y., Virolainen, A., et al. Establishment of a Streptococcus pneumoniae nasopharyngeal colonization model in adult mice. Microb. Pathog. 23, 127-137 (1997).
  12. Southam, D. S., Dolovich, M., et al. Distribution of intranasal instillations in mice: effects of volume, time, body position. Lung Physiol. 282, 833-839 (2002).
  13. Miller, M. A., Stabenow, J. M., et al. Visualization of Murine Intranasal Dosing Efficiency Using Luminescent Francisella tularensis: Effect of Instillation Volume and Form of Anesthesia. PLoS ONE. 7 (2), (2012).
  14. Briles, D. E., Novak, L. Nasal Colonization with Streptococcus pneumoniae includes subpopulations of surface and invasive pneumococci. Infect. Immun. 73 (10), 6945-6951 (2005).
  15. Wu, H. -. Y., Virolainen, A., et al. Establishment of a Streptococcus pneumoniae nasopharyngeal colonization model in adult mice. Microb. Pathog. 23, 127-137 (1997).
  16. Mo, Y., Wan, R., et al. Application of reverse transcription-PCR and real-time PCR in nanotoxicity research. Methods Mol. Biol. 926, 99-112 (2012).
  17. Kuper, C. F., Koornstra, P. J., et al. The role of nasopharyngeal lymphoid tissue. Trends Immunol. 13, 219-224 (1992).
  18. Zhang, Q., Leong, S. C., et al. Characterisation of regulatory T cells in nasal associated lymphoid tissue in children: relationships with pneumococcal colonization. PLoS Pathog. 7, (2011).
  19. Briles, D. E., Novak, L., et al. Nasal colonization with Streptococcus pneumoniae includes subpopulations of surface and invasive pneumococci. Infect. Immun. 73, 6945-6951 (2005).
  20. Weinberger, D. M., Trzcinski, K., et al. Pneumococcal capsular polysaccharide structure predicts serotype prevalence. PLoS Pathog. 5, (2009).
  21. Bryant, W. P., J, , et al. Which Pneumococcal Serogroups Cause the Most Invasive Disease: Implications for Conjugate Vaccine Formulation and Use, Part I.. Clin. Infect. Dis. 30, 100-121 (2000).
  22. Hausdorff, W. P., Feikin, D. R., et al. Epidemiological differences among pneumococcal serotypes. Lancet Infect. Dis. 5, 83-93 (2005).
  23. Brueggemann, A., Griffiths, D., et al. Clonal Relationships between Invasive and Carriage Streptococcus pneumoniae and Serotype and Clone Specific Differences in Invasive Disease Potential. J. Infect. Dis. 187, 1424-1432 (2003).
  24. Mohler, J., Azoulay-Dupis, E., et al. Streptococcus pneumoniae strain-dependent lung inflammatory responses in a murine model of pneumococcal pneumonia. Intensive Care Med. 29, 808-816 (2003).
  25. Wu, H. Y., Virolainen, A., Mathews, B., King, J., Russell, M. W., et al. Establishment of a Streptococcus pneumoniae nasopharyngeal colonization model in adult mice. Microb. Pathog. 23, 127-137 (1997).
  26. Zhang, Z., Clarke, T. B., et al. Cellular effectors mediating Th17-dependent clearance of pneumococcal colonization in mice. J. Clin. Invest. 119, 1899-1909 (2009).
  27. Parker, D., Martin, F. J., et al. Streptococcus pneumoniae DNA initiates type I interferon signaling in the respiratory tract. MBio. 2, (2011).
  28. Haya, D. L., Camilli, A. Large-scale identification of serotype 4 Streptococcus pneumoniae virulence factors. Mol. Microbiol. 45, 1389-1406 (2002).
  29. Nakamura, S., Favis, K. M., et al. Synergistic stimulation of type I interferons during influenza virus coinfection promotes Streptococcus pneumoniae colonization in mice. J. Clin. Invest. 121, 3657-3665 (2011).
  30. Kim, J. O., Weiser, J. N. Association of intrastrain phase variation in quantity of capsular polysaccharide and teichoic acid with the virulence of Streptococcus pneumoniae. J. Infect. Dis. 177, 368-377 (1998).
  31. Roche, A. M., King, S. J., et al. Live attenuated Streptococcus pneumoniae strains induce serotype-independent mucosal and systemic protection in mice. Infect. Immun. 75, 2469-2475 (2007).
  32. Cohen, J. M., Khandavalli, S., Camberlein, E., Hyams, C., Baxendale, H. E., Brown, J. S. Protective contributions against invasive Streptococcus pneumoniae pneumonia of antibody and Th17-Cell responses to nasopharyngeal colonisation. PLoS One. 6 (10), (2011).
  33. Cohen, J. M., Khandavalli, S., Camberlein, E., Hyams, C., Baxendale, H. E., Brown, J. S. Protective contributions against invasive Streptococcus pneumoniae pneumonia of antibody and Th17-Cell responses to nasopharyngeal colonisation. PLoS One. 6 (10), (2011).
  34. Richards, L., Ferreira, D. M., Miyaji, E. N., Andrew, P. W., Kadioglu, A. The immunising effect of pneumococcal nasopharyngeal colonisation; protection against future colonisation and fatal invasive disease. Immunobiology. , 215-251 (2010).
  35. Lanie, J. A., Ng, W. L., et al. Genome sequence of Avery’s virulent serotype 2 strain D39 of Streptococcus pneumoniae and comparison with that of unencapsulated laboratory strain R6. J. Bacteriol. 189, 38-51 (2007).
  36. Robertson, G. T., Ng, W. L., Foley, J., Gilmour, R., Winkler, M. E. Global transcriptional analysis of clpP mutations of type 2 Streptococcus pneumoniae and their effects on physiology and. 184, 3508-3520 (2002).
  37. Orihuela, C. J., Gao, G., et al. Tissue-specific contributions of pneumococcal virulence factors to pathogenesis. J. Infect. Dis. 190, 1661-1669 (2004).
  38. Orihuela, C. J., Gao, G., et al. Organ-specific models of Streptococcus pneumoniae disease. Scand. J. Infect. D. 35, 647-652 (2003).
  39. Swirski, F. K., Nahrendorf, M., et al. Identification of splenic reservoir monocytes and their deployment to inflammatory sites. Science. 325, 612-616 (2009).

Tags

Streptococcus PneumoniaeNasopharyngeal ColonizationIntranasalInflammatory ResponseImmune ResponseMouse ModelPneumoniaBacteremiaMeningitisResident MacrophagesLymphocytesBacterial Density

Play Video
PDF
DOI
DOWNLOAD MATERIALS LIST
Cite This Article
Puchta, A., Verschoor, C. P., Thurn, T., Bowdish, D. M. E. Characterization of Inflammatory Responses During Intranasal Colonization with Streptococcus pneumoniae. J. Vis. Exp. (83), e50490, doi:10.3791/50490 (2014).
Download Citation
Reprints and Permissions

View Video

✖

To prove you're not a robot, please enter the text in the image below

CAPTCHA Image
Play CAPTCHA Audio
Refresh Image