Colonization of the murine nasopharynx with Streptococcus pneumoniae and the subsequent extraction of adherent or recruited cells is described. This technique involves flushing the nasopharynx and collection of the fluid through the nares and is adaptable for various readouts, including differential cell quantification and analysis of mRNA expression in situ.
Nasopharyngeal colonization by Streptococcus pneumoniae is a prerequisite to invasion to the lungs or bloodstream1. This organism is capable of colonizing the mucosal surface of the nasopharynx, where it can reside, multiply and eventually overcome host defences to invade to other tissues of the host. Establishment of an infection in the normally lower respiratory tract results in pneumonia. Alternatively, the bacteria can disseminate into the bloodstream causing bacteraemia, which is associated with high mortality rates2, or else lead directly to the development of pneumococcal meningitis. Understanding the kinetics of, and immune responses to, nasopharyngeal colonization is an important aspect of S. pneumoniae infection models.
Our mouse model of intranasal colonization is adapted from human models3 and has been used by multiple research groups in the study of host-pathogen responses in the nasopharynx4-7. In the first part of the model, we use a clinical isolate of S. pneumoniae to establish a self-limiting bacterial colonization that is similar to carriage events in human adults. The procedure detailed herein involves preparation of a bacterial inoculum, followed by the establishment of a colonization event through delivery of the inoculum via an intranasal route of administration. Resident macrophages are the predominant cell type in the nasopharynx during the steady state. Typically, there are few lymphocytes present in uninfected mice8, however mucosal colonization will lead to low- to high-grade inflammation (depending on the virulence of the bacterial species and strain) that will result in an immune response and the subsequent recruitment of host immune cells. These cells can be isolated by a lavage of the tracheal contents through the nares, and correlated to the density of colonization bacteria to better understand the kinetics of the infection.
In this study we presented detailed methods for the intranasal colonization of mice using a clinical isolate strain of Streptococcus pneumoniae and the subsequent isolation and characterization of the immune cells recruited to nasopharynx in response to the bacteria. We demonstrated how a bacterial inoculum can be cultured in nutrient-rich media and used to establish a colonization event in mice, which is initially restricted to the nasopharynx. We then showed how responding immune cell types that are recruited …
The authors have nothing to disclose.
The authors would like to thank Dr. Jeffery Weiser of the University of Pennsylvania for his gift of the clinical strains of Streptococcus pneumoniae. This work was funded by the Canadian Institutes for Health Research. CV was funded by a M. G. DeGroote fellowship and a fellowship from the Canadian Thoracic Society. This work was funded by the Ontario Lung Association and Canadian Institutes of Health Research (CIHR). Work in the Bowdish laboratory is supported in part by he Michael G. DeGroote Centre for Infectious Disease Research and the McMaster Immunology Research Centre.
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 |