Pseudomonas aeruginosa is a BSL-2 level pathogen. Remember to follow all BSL-2 level safety practices when handling this organism. Here we will demonstrate primary cell isolation, in vitro phagocytosis, and phagocytic pathway analysis, and in vivo phagocytosis and bacterial clearance assessment in mice.
Successful intratracheal delivery requires practice to master. Make sure that the microsprayer is properly loaded, the needle is between the two vocal folds, and the plunger speed is properly controlled. Begin by securing the mouse in the supine position with the limbs spread on a dissection board covered with paper towels and hooking a string under the front teeth to retract the head so that the trachea is positioned straight and level.
Disinfect the mouse with 70%ethanol and use regular forceps to pull up the skin at the center line of the body. Use surgical scissors to cut the skin from the abdomen to the top of the throat. Use the blunt end of standard surgical scissors to carefully dissect the throat muscle and connective tissues.
Open the abdominal wall below the ribcage. Cut the diaphragm, and cut away the lower part of the ribcage to partially expose the lungs. Use spring scissors to expose the trachea and use the forceps to grasp a cartilage ring.
Use micro scissors to carefully make an approximately 1.5-millimeter incision on the ventral face of the trachea. Carefully string a short length of suture thread underneath the trachea and insert an 18 gauge cannula into the trachea. When the cannula is in place, gently lavage the lung three times with one milliliter of fresh PBS per wash, gently withdrawing the fluid into the syringe before reinfusing it back into the lung for three times in succession.
After each lavage, transfer the collected bronchoalveolar lavage fluid or BALF into a 15-milliliter conical tube for centrifugation of the total harvested fluid. Resuspend the pellet in one milliliter of fresh PBS for a second centrifugation and resuspend the washed alveolar macrophages into two milliliters of Dulbecco's Modified Eagles Medium or DMEM supplemented with 10%non-heat-inactivated fetal bovine serum. Then transfer the primary alveolar macrophages to a glass-bottom Petri dish for their two-day culture at 37 degrees Celsius in a cell incubator.
After 48 hours, wash the culture with one milliliter of PBS before feeding the culture with two milliliters of fresh medium. Next, add 50 two-micrometer diameter carboxylated latex FITC-conjugated beads per cell to the culture for a one-hour incubation at 37 degrees Celsius in a cell incubator. At the end of the incubation, wash the plate extensively five times with one milliliter of fresh PBS per wash to remove the extracellular beads and randomly image 100 cells to count the cells containing intracellular beads.
For an opsonization assay, incubate two times 10 to the eighth sheep red blood cells or SRBCs with 50 microliters of rabbit anti-SRBC immunoglobulin M or IgM for 30 minutes at room temperature. Then incubate the opsonized SRBCs with 50 microliters of C5-deficient human serum for 30 minutes at 37 degrees Celsius to fix the C3b and C3b inhibitor complement fragments on the IgM-coated SRBCs. Next, aspirate the media and add 100 microliters of one times 10 to the seven opsonized SRBCs per milliliter of medium to each well of a 96-well plate containing one times 10 to the fourth overnight-cultured murine macrophages per well.
Incubate the SRBC mouse macrophage coculture for one hour at 37 degrees Celsius and then remove the unbound SRBCs by washing with 100 microliters of ammonium chloride potassium lysis buffer for one minute. After the unbound SRBCs have been removed, rinse with 100 microliters of media. Lyse the remaining cells with 0.1%sodium dodecyl sulfate and treat the lysates with 50 microliters of 2, 7-diaminofluorene supplemented with 3%hydrogen peroxide and six molar urea.
Then measure the absorbance of the hemoglobin-catalyzed fluorene blue formation on a spectrophotometer at 620 nanometers. Use a standard curve at 620 nanometer absorbance values with known number of SRBCs to determine the number of opsonized SRBCs that are phagocytized. To assess pattern recognition receptor-mediated phagocytosis, wash two-day cultured mouse primary alveolar macrophages with one milliliter of PBS and treat the cells with 500 microliters of fresh medium containing 100 Alexa fluor-488 conjugated Zymosan-A-bioparticles.
After one hour at 37 degrees Celsius, arrest the phagocytosis with 500 microliters of ice-cold PBS and wash the cells extensively five times with one milliliter of PBS per wash. After the last wash, fix the cells with 4%paraformaldehyde for 10 minutes at room temperature and wash the cells five more times as demonstrated. After the last wash, cover the cells with 500 microliters of fresh PBS for imaging under differential interference contrast and fluorescent channel at 488 nanometers to quantify the number of Zymosan-A-bioparticle-containing alveolar macrophages.
For in vivo alveolar macrophage phagocytosis evaluation, confirm the appropriate level of sedation by lack of response to toe pinch in an anesthetized mouse and place the mouse on a flat board with a plastic wire under the upper incisors. Place the mouse in a semi-recumbent rostrum at 45 degree position with the ventral surface facing upward and use curved forceps to pull out and suppress the tongue. Then insert a microsprayer between the vocal folds to intratracheally administer 50 microliters of five times 10 to the six colony-forming units of P.aeruginosa GFP into the lungs of the anesthetized animal.
To confirm that the microsprayer needle is in the trachea, gently move the syringe and observe the vocal folds on either side of the needle. One hour after the infection, collect the lavage fluid as just demonstrated and pellet the alveolar macrophages by centrifugation. Resuspend one times 10 to the third cells in 100 microliters of fresh PBS for cytocentrifuge onto a glass slide.
Then differentially stain the cytospun slides for alveolar macrophages, neutrophils, and lymphocytes, according to the standard histochemical protocols. Randomly select 100 alveolar macrophages to quantify the percentage of cells that phagocytosed bacteria. In the first in vivo bacteria clearance trial, intratracheally inject a sublethal dose of approximately 2.5 to five times 10 the fifth colony-forming units per milliliter of P.aeruginosa into anesthetized wild type and mutant mice and measure the body weight of each animal every day for six days.
In the second trial, inject a new set of wild type and mutant mice with a lethal three times 10 to the seven colony-forming units per milliliter dose of P.aeruginosa and record the mortality within two days of an injection, collecting the whole lung tissue at the time of death or two days after the injection for quantification of the lung bacterial burden at peak infection. After harvesting the lungs, cut the lung samples into small pieces on ice and homogenize the lung fragments using an adjusted electric homogenizer setting. Then plate 100 microliters of homogenate onto Pseudomonas isolation agar plates in 10-fold serial dilutions.
Fluorescence microscopy reveals that mouse primary alveolar macrophage phagocytosis of FITC latex beads occurs after one hour of incubation, with TRIM72 knockout macrophages demonstrating a significantly higher phagocytic ability. Conversely, overexpression of TRIM72, a protein with unknown function in murine macrophage cells, results in a more than five-fold decrease in complement phagocytosis. Alexa fluor-488 conjugated Zymosan-A particles, however, are ingested in equal quantity by primary alveolar macrophages isolated from either wild type or knockout mice.
Differential staining of BALF harvested from wild type and knockout animals reveals a similar number of macrophages but a higher phagocytic ability for the macrophages harvested from knockout mice. Further, TRIM72 knockout mice demonstrate a quicker recovery of their body weights, maintain their survival, and exhibit a lower bacterial burden than do wild type animals after intratracheal P.aeruginosa administration. Using this technique, other phagocytic processes that are important for pneumonia such as neutrophil phagocytosis and the relative contribution of other phagocytes in bacteria clearance can be analyzed.
In combination with pharmacological inhibitors, adaptive transfer, and transgenic animals, this technique can help researchers to explore the molecular component of specific type of phagocytosis for the phagocytes of interest.
