Alveolar Macrophage Phagocytosis and Bacteria Clearance in Mice

This protocol follows the guidelines of the Institutional Animal Care and Use Committee (IACUC) of Eastern Virginia Medical School.

1. Fluorescent Beads Phagocytosis

1. Euthanize the mouse (C57BL/6J, 6 weeks old, female) by CO₂ asphyxiation as per IACUC protocols for the ethical euthanasia of animals.
2. Lay the mouse belly-up on a dissection board covered with paper towels. Pin its paws down with its limbs spread-eagle and hook a string under its front teeth to pull its head back so that the trachea is positioned straight and level.
3. Wet the mouse's throat, chest, and belly with 70% ethanol to disinfect and prevent the fur from sticking to the tools.
4. Using regular forceps, pull up the skin at the centerline of the body, and cut with surgical scissors up the centerline to the top of the throat.
5. Using the blunt end of standard surgical scissors, carefully move away the muscle and connective tissues on the throat and use spring scissors (microscissors) to expose the trachea.
6. Gripping a cartilage ring with the forceps, carefully make a small incision (~1.5 mm), using microscissors, on the ventricle face of the trachea and insert an 18 G cannula into the trachea.
7. Gently lavage 3 mL of phosphate-buffered saline (PBS), 1 mL at a time. Each time gently withdraw the fluid into the syringe and reinfuse it back into the lung, 3x in succession. After collecting the BALF, perform cervical dislocation to ensure euthanasia. Transfer the collected PBS (~2.8 mL), which is bronchoalveolar lavage fluid (BALF), to a tube, centrifuge at 1,000 x g for 10 min, and collect the pellet. Add 1 mL of fresh PBS to the tube and centrifuge at 1,000 x g for 10 min to wash the debris and collect the pelleted alveolar macrophages.
8. Resuspend the pellet in 2 mL of Dulbecco's modified Eagle's medium (DMEM) with 10% nonheat-inactivated fetal bovine serum (FBS) and culture primary alveolar macrophages in the same media for 2 days on a glass-bottom dish at 37 °C in a humidified atmosphere.
9. Aspirate the old media, wash it with 1 mL of PBS, and add 2 mL of fresh media. Add FITC beads (carboxylated latex beads, 2 µm in diameter, 50 beads/cell) and incubate for 1 h at 37 °C in a humidified atmosphere.
10. Wash extensively with PBS (1 mL at a time, for a total of five washes) to remove extracellular beads. Image 100 cells randomly and count the cells with intracellular beads (488 nm).
    ​NOTE: Phagocytic indexes are the number of ingested beads divided by the total number of macrophages; the percentage of phagocytic cells is the number of macrophages that ingest at least one bead divided by the total number of macrophages¹⁶.
    1. Alternatively, after a 1 h incubation with beads, wash the cells with 3 mL of PBS and process them for flow cytometry for the quantification of phagocytosis. Similarly, process AMs without beads as unstained cells or control cells. Calculate the percentage positivity and mean fluorescence intensity, using flow cytometry software, by selecting those options in the software.

2. FcγR- and CR-mediated Phagocytosis

1. For the opsonization, incubate 2 x 10⁸ sheep red blood cells (SRBCs) with 50 µL of rabbit anti-SRBC-IgM or 50 µL of rabbit anti-SRBC-IgG for 30 min at room temperature¹¹.
2. Incubate IgM-opsonized SRBCs with 50 µL of C5-deficient (C5D) human serum for 30 min at 37 °C to fix the complement fragments C3b and C3bi on IgM-coated SRBCs.
3. Seed murine macrophage cells (MH-S cells) (10,000 cells/well) in a 96-well plate and incubate overnight to get a ~70% confluence. Add 100 µL of 1 x 10⁷/mL opsonized SRBCs to each well of MH-S cells and incubate for 1 h at 37 °C. Wash unbound SRBCs very quickly (~1 min) with 100 µL of ammonium chloride-potassium (ACK) lysis buffer.
4. Lyse the cells with 0.1% SDS and add 50 µL of 2,7-diaminofluorene (DAF) containing 3% hydrogen peroxide and 6 M urea. Measure the absorbance of the hemoglobin-catalyzed fluorene blue formation at 620 nm.
5. Determine the number of SRBCs by using a standard curve at 620 nm absorbance values with a known number of SRBCs. Similarly, process MH-S cells incubated with nonopsonized SRBCs to use as negative controls.

3. PRR-mediated Phagocytosis

1. Follow steps 1.1-1.9 for the isolation and culturing of mouse primary alveolar macrophages.
2. After 2 days, remove the media, wash the cells with 1 mL of PBS, and add 500 µL of fresh media containing Alexa Fluor-488-conjugated zymosan-A bioparticles (100 particles/dish).
3. Incubate for 1 h at 37 °C. Stop the phagocytosis by adding 500 µL of ice-cold PBS.
4. Wash the cells extensively with PBS (1 mL at a time, for a total of five washes). Fix the cells with 4% paraformaldehyde for 10 min at room temperature.
5. Wash the cells extensively with PBS (1 mL at a time, for a total of five washes) and keep the cells in 500 µL of PBS.
6. Image the cells under differential interference contrast and a fluorescent channel at 488 nm. Count AMs containing zymosan-A bioparticles and determine the percentage of phagocytosis.

4. In Vivo Phagocytosis by Alveolar Macrophages

NOTE: Inoculate P. aeruginosa GFP on a nutrient agar plate and incubate the plate at 37°C overnight. On the next day, inoculate the single colony to 2 mL of nutrient broth and grow the bacteria at 37°C overnight.

1. The next day, anesthetize mice with an intraperitoneal administration of 100 mg/kg ketamine and 10 mg/kg xylazine. Confirm proper anesthetization via a lack of response to the toe pinch.
2. Lay the mouse on a flat board with a rubber band across the upper incisors and place it in a semirecumbent (45°) position with the ventral surface and rostrum facing upward. Using curved forceps, partially retract the tongue. Using a microsprayer, intratracheally administer 50 µL (5 x 10⁶ colony-forming units [CFU])¹⁷ of P. aeruginosa GFP into the lungs of the anesthetized mice.
3. After 1 h of infection, follow steps 1.1-1.7.
4. Resuspend the cells in PBS and cytocentrifuge them (1,000 x g, 1 min at room temperature) onto a glass slide.
5. Differentially stain the cytospin slides for alveolar macrophages, neutrophils, and lymphocytes, according to the manufacturer's instructions.
6. Randomly select 100 AMs, count the AMs containing intracellular bacteria, and determine the percentage of phagocytosis.

5. In Vivo Bacteria Clearance Using P. aeruginosa

1. Inoculate P. aeruginosa on a P. aeruginosa isolation agar plate and incubate the plate at 37°C overnight. Inoculate the single colony to 2 mL of lysogeny broth (LB) and grow the bacteria at 37°C overnight. Calculate the CFU, using the following formula.
   
   CFU/mL = (number of colonies x dilution factor)/volume of the culture plate.
   
   Dilute the culture with PBS to get the desired CFU/mL.
2. In trial 1, intratracheally inject a sublethal dose of ~2.5 x 10⁵ CFU/mL P. aeruginosa into anesthetized wild-type (WT) and TRIM72^KO mice, as stated in step 4.2. Measure the body weight daily for 6 days.
3. In trial 2, inject a second dose of P. aeruginosa (5 x 10⁵ CFU/mL) into the mice that survived in trial 1 and measure the body weight for 4 days.
4. In trial 3, using a different set of mice, inject WT and TRIM72^KO mice with a lethal dose (3 x 10⁷ CFU/mL) of P. aeruginosa and record the mortality within 2 days of injection. Animals showing signs of severe stress such as significant body weight loss, hunched back, anorexia, or dyspnea will be assessed by the attending vets. Untreatable animals will be sacrificed immediately.
5. Either at death or after euthanasia at day 2 after the injection, collect the whole-lung tissue for the quantification of the lung bacterial burden at peak infection.
6. To test the lung bacterial burden, add 200 µL of normal saline to the lung tissues and homogenize them, using a previously tested setting on an electronic homogenizer that completely disrupts the lung tissue without breaking bacteria. Adjust the total volume of the lung homogenate to 1 mL and plate 100 µL of lung lysate on Pseudomonas isolation agar plates at 10-fold serial dilutions.
7. Incubate the plates at 37 °C for 24 h and count the bacterial colonies to determine the CFU per whole lung.

6. Statistical Analysis

1. Use Student's t-test to determine the statistical significance of the difference between the two groups. Consider a difference statistically significant when p < 0.05. All data are presented as means ± standard error of the mean (SEM).