Characterization of Inflammatory Responses During Intranasal Colonization with Streptococcus pneumoniae

Before you begin: all steps are done in a Biohazard Level 2 (BSL2) Biological Safety Cabinet (BSC) unless otherwise stated. Please ensure that you have obtained the appropriate Biohazard Approval for use of infectious bacterial pathogens as per institutional guidelines prior to initiation of the experiments. Additionally, please ensure that you have all the materials and reagents necessary to conduct the procedure prepared beforehand. Mice used in these experiments have included female C57BL/6 mice from Jackson Laboratories, Charles River or Taconic and were 10-14 weeks of age (although we have not found any gender-dependent significant differences in kinetics of nasal colonization clearance or infection). All mice used in these experiments were bred and maintained under specific-pathogen free conditions, and were free of common viruses, (LCMV, MNV,MPV, reovirus ECTV, and other) bacteria (e.g. H. pylori) and parasites (e.g. pinworm, ectoparasites) by fecal sample testing as well as frequent anatomical assessment of sentinel mice cohoused within their facility rooms. When conducting these experiments, we recommend using control mice no younger than 10-12 weeks of age and no older than 6 months of age. Mice younger or older than this age range are more susceptible to longer nasopharyngeal carriage duration and increased likelihood of disseminating infection. Mouse background is another important consideration that may impact the outcomes of a colonization experiment, as several groups have demonstrated that mice of different genetic backgrounds have different susceptibilities to the S. pneumoniae D39 (serotype 2) strain^9,10. S. pneumoniae is not a naturally occurring murine pathogen and its only natural reservoir is the human nasopharynx. Transmission occurs via respiratory droplets, and as mice do not produce respiratory secretions, individual mice cannot transmit the bacterium to other mice, so there is no concern for mouse-to-mouse transmission¹¹. For a visual overview of the procedures described within this manuscript, please refer to Figure 1.

1. Preparation of S. pneumoniae Culture

1. Inoculate 5 ml of tryptic soy agar for the suspension growth of Streptococcus pneumoniae.
2. Culture under static conditions at 37 °C in 5% CO₂ until the bacterial inoculum reaches log phase growth with a corresponding liquid density of 10⁸ CFU/ml as determined by an odometer set to 600 nm. The exact reading corresponding to this CFU will differ depending on the specific selected bacterial strain; for most strains of S. pneumoniae this corresponds to an OD₆₀₀ range of 0.45-0.55. Typically, S. pneumoniae strains in liquid culture will grow to this density within 1.5-2.5 hr under the recommended conditions, with no need for subculturing. The culture should not be allowed to overgrow (beyond an OD reading of 0.75) as this represents the point at which the bacteria are no longer in log phase growth and are undergoing extensive autolysis.
3. Each mouse will be inoculated with approximately 10⁷ bacteria. Therefore, for every 9 mice to be colonized, pipette 1 ml of inoculum into an Eppendorf tube and spin at 15,000 x g for 1 min. A whitish pellet should be visible. Remove the supernatant, being careful not to disturb the pellet and resuspend the bacteria in 100 μl of phosphate buffered saline (PBS), thus increasing the concentration to 10⁹ CFU/ml. At this stage, the bacteria should remain viable, but will not readily replicate.
4. If using multiple aliquots, combine into one tube to control for slight inter-sample variations in bacterial density.
5. Keep bacteria on ice until ready for inoculation, for a maximum of 1 hr.
6. To obtain an exact bacterial count, perform log-wise serial dilution series starting with neat bacterial inoculum. Dilute serially 10-fold, adding 10 μl of to 90 μl of sterile PBS.
7. Plate out 3 drops of 10 μl samples of dilutions 10^-5 – 10^-9, plus a PBS-only contamination control, onto separately labeled sections of tryptic soy agar (TSA) plate supplemented with 5% sheep’s blood (Figure 2). Ensure that pipette tips are changed for each step diluting from a higher CFU concentration to a lower CFU concentration to avoid carrying over excess bacteria and increasing variability of results. Human blood agar (HBA) plates may also be used in lieu of TSA. Since many strains of S. pneumoniae are resistant to neomycin (from 5-20 μg/ml), this antibiotic may also be added to the agar medium of choice during the plate preparation phase. This facilitates enumeration as it eliminates nonresistant bacteria. The antibiotic susceptibility of each strain must be tested in advance to determine the optimal concentration of antibiotic to use for each bacterial strain.
8. Allow to dry for 15-30 min uncovered, then cover plates and place upside down in bacterial incubator set to 37 °C and 5% CO₂. Grow up bacterial colonies on plate for 24 hr.
9. Determine number of colony forming units and their corresponding concentration. Based on determinations of the OD₆₀₀ value, concentration should be within the range of 1-4 x 10⁹ CFU/ml. S. pneumoniae colonies should appear as small, circular colonies a yellowish-beige in color, with a small depression at the center giving them a donut-like appearance (Figure 3).

2. Murine Intranasal Colonization

1. Restrain mice by placing them into a mouse restrainer apparatus (a modified 50 ml Falcon tube with the tip cut off to create an aperture) securing them by the base of their body with thumb so that their noses just emerge out of tapered end of the restrainer apparatus (Figure 4). Use of this apparatus allows for immobilization of the mouse’s head and segregation of its nares in a manner that minimizes movement as well as impedes attempts from the animal to masticate the pipette tip, allowing for complete delivery of the inoculum. Alternatively, mice can be immobilized via scuffing at the neck and manual restraint. We do not recommend anesthesia of the animals prior to intranasal inoculation. Administrating the inoculum to animals under anesthesia results in some of the inoculum spreading to the lungs^12,13.
2. Using a P10 or P20 pipette, inoculate each mouse by depositing 10 μl of the prepared culture, distributing it evenly between both nares (allow inoculum to drip into nose by pulsing the inoculation gradually, taking time for mice to inhale inoculum). To achieve complete delivery of the inoculum, pause administration at any point the mouse begins to move its nose excessively. The entire inoculum may not be injected into the nares as the mice may expel some through the nose during exhalation; however, as the expelled amount tends to be minuscule, and the inoculum contains an extremely high amount of bacteria, this does not significantly affect the colonizing bacterial load. Additionally, the surface area available for colonization in the nasopharyngeal mucosa is limited and consequently we and others have found that the recommended 10⁷ dose is sufficient to obtain consistent levels of bacteria in all mice, resulting in minimal variability in initial amounts of colonizing bacteria^14,15 .
3. Weigh mice if utilizing weight indicators as part of your end-point monitoring. Monitor mice every 12-24 hr for clinical symptoms, including lethargy, ruffled fur and weight loss. Mice typically will not show symptoms of illness until 3-5 days post-colonization, and these will be preceded by weight loss which may average around 5% of total body weight a day. As the mice become increasingly ill they will assume hunched postures and show decreased activity and decreased responsiveness to stimulation, including handling. At this stage, illness is typically indicative of sepsis and/or pneumonia and will likely be terminal, although mice may be treated with 1 ml of subcutaneous saline daily to improve outcomes. Surviving mice should start showing improvement after day 7 post-colonization, as evidenced by stabilization of weight followed by weight gain, although different strains of S. pneumoniae may induce illness more quickly and result in progression of clinical symptoms along a different time-line. Please see Figure 5 for a representative result of weight tracked in mice colonized with the P1547 strain.

3. Nasal Lavage Sample Collection

Before beginning: prepare cannulated needles using 1 ml syringes capped with 26 3/8 G beveled needles. Cut 2.5 cm pieces of PE20 polyethylene tubing with an inner diameter 0.38 mm, ensuring that each end has a beveled tip. Using forceps, slide a 2.5 cm long piece of PE20 polyethylene tubing (inner diameter 0.38 mm) on to the needle tip, avoiding puncturing the tubing side. Cannulated needles can be kept in 70% ethanol until needed.

1. Euthanize experimental mice. As cervical dislocation can potentially damage the trachea, this method of euthanasia must be avoided. Our preferred method is isoflurane anesthesia followed by exsanguination, however, ensure you follow institutional guidelines when selecting mode of euthanasia.
2. Using 70% aqueous ethanol, sterilize the superoanterior fur of the animal, particularly the neck, taking care to prevent ethanol from accessing the nares.
3. Make a single longitudinal cut along the midline of the neck of the animal, and two horizontal cuts on either end, creating an opening to envision the trachea.
4. Carefully peel back skin to either side, revealing neck tissue beneath.
5. Trachea should be visible, surrounded by longitudinal muscles on either side. Carefully snip these to provide a clear view of the trachea itself, taking care not to sever the surrounding vasculature.
6. If the vasculature was cut and blood is present, prior to proceeding, allow bleeding to stop, and then cleanse the area several times by dispensing sterile PBS and using sterile gauze to gently soak up excess moisture in the area.
7. Once trachea is properly exposed, make a transverse, semilunar cut in the trachea about half-way up (Figure 6).
8. Draw up 1,000 μl of sterilized PBS into previously prepared cannulated needle.
9. Insert cannula into the trachea towards the nose, keeping the beveled edge pointing downwards for ease of insertion (Figure 7). Once needle is in place, rotate it 180°, and gently probe upwards until you feel light resistance.
10. Place Eppendorf designated for sample collection just beneath the nose of the mouse.
11. Test correct placement of needle by dispensing a minimal (~20 μl) amount of PBS lavage fluid – drop of fluid should form around the nares of the mouse; if this is the case, proceed to step 3.13).
12. If test PBS emerges directly out the mouth of the animal, pull the cannula back, and reposition by again gently probing forward until very slight resistance is felt – take care not to push cannula too far past this resistance, as you will move it past the nasal palate and through to the oral cavity.
13. Dispense contents of needle rapidly to help displace and collect maximal amount of cells – contents should flow out through the nares of the mouse and into collection tube. Place sample immediately on ice.
14. To collect samples for RNA analysis, repeat steps 3.8-3.13) using a cannulated needle containing 500 μl of RNA lysis buffer on the same mouse. This will allow the collection of lysate sample from the remaining cell populations, largely composed of the nasopharyngeal mucosal epithelium, as nonadherent cells should have been removed following the initial PBS lavage. Please note that the RNA lysis buffer will denude the epithelium and destroy surrounding tissue, so care must be taken to avoid contact with organs such as the lungs, if retention of these tissues is desired. Once collected, place sample in RNA lysis buffer directly on dry ice to snap-freeze. Once in lysis buffer, samples can be stored as per the manufacturer’s instructions, and are stable typically at -70 °C for several months.

4. Determination of Bacterial Load in the Nasopharynx

1. Quantitate bacteria by preparing a serial dilution series for each murine nasal lavage sample. In general, bacterial load can be expected to be between 0-10⁴ CFU, therefore conduct three 10-fold serial dilutions. Add 10 μl of the neat nasal lavage sample (100 CFU/ml) to the first tube to a concentration of 10^-1 CFU/ml. Vortex thoroughly.
2. Divide bacteriological plate into quadrants, and label quadrants each with a member of the dilution series (10⁰-10^-3 CFU/ml). Plate out 3 drops of 10 μl samples of the 3 dilutions and the neat sample on tryptic soy agar plates supplemented with 5% sheep’s blood, as in Figure 2.
3. Allow to dry for 15-30 min uncovered, then cover plates and place upside down in bacterial incubator with optimal conditions for bacterial growth (typically 37 °C and 5% CO₂).
4. Grow up bacterial colonies on plate for 18-24 hr.
5. Determine number of colonizing bacteria by averaging the colonies formed on plate for each dilution (Figure 3). Figure 8 demonstrates bacterial density during different timepoints, as determined by culture of nasal lavages, in mice colonized with 3 different strains of S. pneumoniae for up to 21 days.

5. Preparation of Samples for Flow Cytometery

Before beginning: Prepare mix of antibodies. For quantification of leukocyte populations, we recommend the following mix at the specified dilutions: PE-Ly6G (clone 1A8, 1 μg/ml), FITC-Ly6C (clone AL-21,1 μg/ml), eFluor 450-CD45 (clone 30-F11, 2.67 μg/ml), APC-F4/80 (clone PM8 RUO, 0.67 μg/ml), PerCP-Cy5.5-CD11c (clone N418 RUO, 0.5 μg/ml), PE-Cy7-CD11b (clone M1/70, 0.33 μg/ml), Alexa Fluor 700-CD3 (clone 1782, 4 μg/ml), eFluor 605NC-CD4 (clone GK1.5, 6.67 μg/ml). Please note that this mix is 2x concentration (see step 5.5). All antibodies should be diluted in FACs Wash buffer (0.5% fetal calf serum, 2mM EDTA, 0.1% sodium azide in PBS) which should also be prepared beforehand. In a mixture of isotype matched control antibodies, ideally from the same supplier as the labeled antibodies and at the same concentrations as the specific antibodies, should be prepared. The samples treated with the isotype control antibodies will function as the negative control. Any fluorescence observed in the samples treated with the isotype control antibodies should be considered background.

1. Prechill a centrifuge capable of spinning 1.5 ml Eppendorf tubes to 4 °C.
2. Centrifuge nasal lavage samples at 2,000 x g for 10 min at 4 °C. Carefully pipette out supernatant and reserve. Note: due to the small amount of cells within the nasopharynx, the cell pellet will not be visible unless there is undesired red blood cell contamination, which will bright red. If this is seen, sample should be discarded.
3. Resuspend sample in 50 μl of Fc?RIIb/CD16-2 (2.4G2) antibody (which binds Fc receptors and reduces nonspecific antibody binding) in FACs Wash Buffer at a concentration of 4 μg/ml.
4. Incubate sample on ice for 30 min.
5. Add 50 μl of preprepared 2x concentrated fluorescent antibody mix to sample. Set aside a representative sample from each experimental group to act as an isotype control. Add the isotype antibody mix to this sample in lieu of stain mix.
6. Incubate sample on ice for 1 hr.
7. Centrifuge samples at 2,000 x g for 10 min at 4 °C. Discard supernatant and resuspend in 200 μl of PBS.
8. Repeat step 5.7.
9. After the second wash, centrifuge samples again at 2,000 x g for 10 min at 4 °C.
10. Resuspend in either in PBS (if running sample immediately) or 2% paraformaldehyde (if running samples 1-3 days post-staining).
11. When conducting flow cytometry collect the maximum amount of events per sample or until the entire sample has been aspirated. In uninfected, healthy, young mice, this will be as little as 1,000-2,000 total events; During a bacterial colonization event, this number may increase more than 2-to-5-fold dependeing on disease status in animal and factors such as age and genetic background. Figure 9 shows representative flow cytometry results collected from a 3-laser Becton Dickenson LSRII flow cytometer using a Forward Scatter of 450 and Side Scatter of 300, although we recommend optimizing parameters for the specific flow cytometer you intend to use prior to sample collection. Note: if a sample contains even trace amounts of blood contamination, the total events collected will be significantly higher than expected and the sample should be discounted from analysis.

6. Quantitative PCR (qPCR) Analysis of Nasal Lavages

1. Thaw cell lysates from step 3.14 room temperature.
2. Follow the recommended protocol provided with the preferred RNA extraction of choice.
3. After completing RNA extraction procedure as instructed, quantify the amount of RNA using a spectrophotometer or electrophoresis based method (Figure 10). We routinely obtain between 975 and 3,250 ng total RNA per sample with a 260/280 nm ratio of >1.7 or an RNA integrity number (RIN) around, 8.1±0.13.
4. Transcribe cDNA using the M-MULV Reverse Transcriptase according to the manufacturer’s protocol with 1,000 ng of RNA (maximum 13 μl).
5. Dilute resulting cDNA samples 8x, and aliquot equally into 4 separate tubes for longterm storage at -20 or -80 °C.
6. To measure gene expression by qPCR, prepare 25 μl samples reactions in triplicate on ice or cold block containing: 12.5 μl of 2x qPCR master mix from qPCR kit of your choice, 0.25 μl reference dye, 2 μl of diluted cDNA (step 7.5), 1 μl of mixed forward and reverse primers (400 nM final), 9.25 μl of RNAse-DNAse free water. This protocol is an adaptation of previously published methods¹⁶.
7. In general we find that a two-step qPCR amplification ( 95 °C for 10 min followed by up to 40 cycles x [95 °C x 15 sec, 60 °C x 1 min]) is effective (Figure 11a); however, each primer pair must be optimized. Dissociation (melting) curves must be performed following amplification to ensure that no nonspecific amplification occurred. amplification (Figure 11b)
8. We routinely run standard curves for each gene analyzed as well as a standard calibrator (derived from lung or spleen homogenate) for each 96-well plate analyzed. Relative transcript amounts are obtained by first normalizing raw cycle threshold (Ct) values by the reference dye and transforming the resultant values through the respective standard curve. These relative amounts are subsequently normalized to the standard calibrator and a housekeeping gene, as applicable.