This work is supported by grant R01HL116826 to X. Zhao.

This protocol follows the guidelines of the Institutional Animal Care and Use Committee (IACUC) of Eastern Virginia Medical School.
1. Fluorescent Beads Phagocytosis
<ol>
	<li>Euthanize the mouse (C57BL/6J, 6 weeks old, female) by CO2 asphyxiation as per IACUC protocols for the ethical euthanasia of animals.</li>
	<li>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.</li>
	<li>Wet the mouse&#39;s throat, chest, and belly with 70% ethanol to disinfect and prevent the fur from sticking to the tools.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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&#160;into the trachea.</li>
	<li>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.&#160;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.</li>
	<li>Resuspend the pellet in 2 mL of Dulbecco&#39;s modified Eagle&#39;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 &#176;C in a humidified atmosphere.</li>
	<li>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 &#181;m in diameter, 50 beads/cell) and incubate for 1 h at 37 &#176;C in a humidified atmosphere.</li>
	<li>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). 
	&#8203;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.
	<ol>
		<li>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.</li>
	</ol>
	</li>
</ol>
2. Fc&#947;R- and CR-mediated Phagocytosis
<ol>
	<li>For the opsonization, incubate 2 x 108 sheep red blood cells (SRBCs) with 50 &#181;L of rabbit anti-SRBC-IgM or 50 &#181;L of rabbit anti-SRBC-IgG for 30 min at room temperature11.</li>
	<li>Incubate IgM-opsonized SRBCs with 50 &#181;L of C5-deficient (C5D) human serum for 30 min at 37 &#176;C to fix the complement fragments C3b and C3bi on IgM-coated SRBCs.</li>
	<li>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 &#181;L of 1 x 107/mL opsonized SRBCs to each well of MH-S cells and incubate for 1 h at 37 &#176;C. Wash unbound SRBCs very quickly (~1 min) with 100 &#181;L of ammonium chloride-potassium (ACK) lysis buffer.</li>
	<li>Lyse the cells with 0.1% SDS and add 50 &#181;L 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.</li>
	<li>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.</li>
</ol>
3. PRR-mediated Phagocytosis
<ol>
	<li>Follow steps 1.1-1.9 for the isolation and culturing of mouse primary alveolar macrophages.</li>
	<li>After 2 days, remove the media, wash the cells with 1 mL of PBS, and add 500 &#181;L of fresh media containing Alexa Fluor-488-conjugated zymosan-A bioparticles (100 particles/dish).</li>
	<li>Incubate for 1 h at 37 &#176;C. Stop the phagocytosis by adding 500 &#181;L of ice-cold PBS.</li>
	<li>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.</li>
	<li>Wash the cells extensively with PBS (1 mL at a time, for a total of five washes) and keep the cells in 500 &#181;L of PBS.</li>
	<li>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.</li>
</ol>
4. In Vivo Phagocytosis by Alveolar Macrophages
NOTE: Inoculate P. aeruginosa GFP on a nutrient agar plate and incubate the plate at 37&#176;C overnight. On the next day, inoculate the single colony to 2 mL of nutrient broth and grow the bacteria at 37&#176;C overnight.
<ol>
	<li>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.</li>
	<li>Lay the mouse on a flat board with a rubber band across the upper incisors and place it in a semirecumbent (45&#176;) position with the ventral surface and rostrum facing upward. Using curved forceps, partially retract the tongue. Using a microsprayer, intratracheally administer 50 &#181;L (5 x 106 colony-forming units [CFU])17 of P. aeruginosa GFP into the lungs of the anesthetized mice.</li>
	<li>After 1 h of infection, follow steps 1.1-1.7.</li>
	<li>Resuspend the cells in PBS and cytocentrifuge them (1,000 x g, 1 min at room temperature) onto a glass slide.</li>
	<li>Differentially stain the cytospin slides for alveolar macrophages, neutrophils, and lymphocytes, according to the manufacturer&#39;s instructions.</li>
	<li>Randomly select 100 AMs, count the AMs containing intracellular bacteria, and determine the percentage of phagocytosis.</li>
</ol>
5. In Vivo Bacteria Clearance Using P. aeruginosa
<ol>
	<li>Inoculate P. aeruginosa on a P. aeruginosa isolation agar plate and incubate the plate at 37&#176;C overnight. Inoculate the single colony to 2 mL of lysogeny broth (LB) and grow the bacteria at 37&#176;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.</li>
	<li>In trial 1, intratracheally inject a sublethal dose of ~2.5 x 105 CFU/mL P. aeruginosa into anesthetized wild-type (WT) and TRIM72KO mice, as stated in step 4.2. Measure the body weight daily for 6 days.</li>
	<li>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.</li>
	<li>In trial 3, using a different set of mice, inject WT and TRIM72KO mice with a lethal dose (3 x 107 CFU/mL) of P. aeruginosa and record the mortality within 2 days of injection.&#160;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.</li>
	<li>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.</li>
	<li>To test the lung bacterial burden, add 200 &#181;L 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 &#181;L of lung lysate on Pseudomonas isolation agar plates at 10-fold serial dilutions.</li>
	<li>Incubate the plates at 37 &#176;C for 24 h and count the bacterial colonies to determine the CFU per whole lung.</li>
</ol>
6. Statistical Analysis
<ol>
	<li>Use Student&#39;s t-test to determine the statistical significance of the difference between the two groups. Consider a difference statistically significant when p &#60; 0.05. All data are presented as means &#177; standard error of the mean (SEM).</li>
</ol>

<ol>
	<li>Hussell, T., Bell, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alveolar+macrophages:+plasticity+in+a+tissue-specific+context.">Alveolar macrophages: plasticity in a tissue-specific context.</a> Nature Reviews Immunology. 14, 81-93 (2014).</li><li>Belchamber, K. B. R., Donnelly, L. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macrophage+Dysfunction+in+Respiratory+Disease.">Macrophage Dysfunction in Respiratory Disease.</a> Results and Problems in Cell Differentiation. 62, 299-313 (2017).</li><li>Broug-Holub, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alveolar+macrophages+are+required+for+protective+pulmonary+defenses+in+murine+Klebsiella+pneumonia:+elimination+of+alveolar+macrophages+increases+neutrophil+recruitment+but+decreases+bacterial+clearance+and+survival.">Alveolar macrophages are required for protective pulmonary defenses in murine Klebsiella pneumonia: elimination of alveolar macrophages increases neutrophil recruitment but decreases bacterial clearance and survival.</a> Infection and Immunity. 65, 1139-1146 (1997).</li><li>Knapp, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alveolar+macrophages+have+a+protective+antiinflammatory+role+during+murine+pneumococcal+pneumonia.">Alveolar macrophages have a protective antiinflammatory role during murine pneumococcal pneumonia.</a> American Journal of Respiratory and Critical Care Medicine. 167, 171-179 (2003).</li><li>Bhatia, M., Zemans, R. L., Jeyaseelan, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+chemokines+in+the+pathogenesis+of+acute+lung+injury.">Role of chemokines in the pathogenesis of acute lung injury.</a> American Journal of Respiratory Cell and Molecular Biology. 46, 566-572 (2012).</li><li>Marriott, H. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interleukin-1beta+regulates+CXCL8+release+and+influences+disease+outcome+in+response+to+Streptococcus+pneumoniae,+defining+intercellular+cooperation+between+pulmonary+epithelial+cells+and+macrophages.">Interleukin-1beta regulates CXCL8 release and influences disease outcome in response to Streptococcus pneumoniae, defining intercellular cooperation between pulmonary epithelial cells and macrophages.</a> Infection and Immunity. 80, 1140-1149 (2012).</li><li>Greenlee-Wacker, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clearance+of+apoptotic+neutrophils+and+resolution+of+inflammation.">Clearance of apoptotic neutrophils and resolution of inflammation.</a> Immunological Reviews. 273, 357-370 (2016).</li><li>Haslett, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Granulocyte+apoptosis+and+its+role+in+the+resolution+and+control+of+lung+inflammation.">Granulocyte apoptosis and its role in the resolution and control of lung inflammation.</a> American Journal of Respiratory and Critical Care Medicine. 160, 5-11 (1999).</li><li>Cox, G., Crossley, J., Xing, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macrophage+engulfment+of+apoptotic+neutrophils+contributes+to+the+resolution+of+acute+pulmonary+inflammation+in+vivo.">Macrophage engulfment of apoptotic neutrophils contributes to the resolution of acute pulmonary inflammation in vivo.</a> American Journal of Respiratory Cell and Molecular Biology. 12, 232-237 (1995).</li><li>Groves, E., Dart, A. E., Covarelli, V., Caron, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mechanisms+of+phagocytic+uptake+in+mammalian+cells.">Molecular mechanisms of phagocytic uptake in mammalian cells.</a> Cellular and Molecular Life Sciences. 65, 1957-1976 (2008).</li><li>Mosser, D. M., Zhang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+Opsonic+Phagocytosis+via+Fc&#947;+Receptors+and+complement+receptors+on+macrophages.">Measuring Opsonic Phagocytosis via Fc&#947; Receptors and complement receptors on macrophages.</a> Current Protocols in Immunology. , (2011).</li><li>He, J. Q., Wiesmann, C., van Lookeren Campagne, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+role+of+macrophage+complement+receptor+CRIg+in+immune+clearance+and+inflammation.">A role of macrophage complement receptor CRIg in immune clearance and inflammation.</a> Molecular Immunology. 45, 4041-4047 (2008).</li><li>Nagre, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+Macrophage+Complement+Receptor+CRIg+by+TRIM72+Polarizes+Innate+Immunity+of+the+Lung.">Inhibition of Macrophage Complement Receptor CRIg by TRIM72 Polarizes Innate Immunity of the Lung.</a> American Journal of Respiratory Cell and Molecular Biology. 58 (6), 756-766 (2018).</li><li>Miksa, M., Komura, H., Wu, R., Shah, K. G., Wang, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Novel+Method+to+Determine+the+Engulfment+of+Apoptotic+Cells+by+Macrophages+using+pHrodo+Succinimidyl+Ester.">A Novel Method to Determine the Engulfment of Apoptotic Cells by Macrophages using pHrodo Succinimidyl Ester.</a> Journal of Immunological Methods. 342 (1-2), 71-77 (2009).</li><li>Su, H., Chen, H., Jen, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Severe+exercise+enhances+phagocytosis+by+murine+bronchoalveolar+macrophages.">Severe exercise enhances phagocytosis by murine bronchoalveolar macrophages.</a> Journal of Leukocyte Biology. 69, 75-80 (2001).</li><li>Amiel, E., Lovewell, R. R., O'Toole, G. A., Hogan, D. A., Berwin, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pseudomonas+aeruginosa.+evasion+of+phagocytosis+is+mediated+by+loss+of+swimming+motility+and+is+independent+of+flagellum+expression.">Pseudomonas aeruginosa. evasion of phagocytosis is mediated by loss of swimming motility and is independent of flagellum expression.</a> Infection and Immunity. 78, 2937-2945 (2010).</li><li>Giannoni, E., Sawa, T., Allen, L., Wiener-Kronish, J., Hawgood, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surfactant+Proteins+A+and+D+Enhance+Pulmonary+Clearance+of+Pseudomonas+aeruginosa.">Surfactant Proteins A and D Enhance Pulmonary Clearance of Pseudomonas aeruginosa.</a> American Journal of Respiratory Cell and Molecular Biology. 34, 704-710 (2006).</li></ol>

The authors have nothing to disclose.

While performing a gas exchange function, the lung persistently confronts foreign particles, pathogens, and allergens. AMs provide the first line of defense by virtue of their main function, namely phagocytosis. AMs also coordinate with other immune cells in destroying the pathogens and in the resolution of inflammation. Here, we described methods for specifically assessing phagocytosis by AMs isolated from the mouse lung. The protocol presented in this manuscript explains a detailed study of phagocytosis both in vivo an...

AMs are the major resident phagocytes in the alveoli at the resting stage and one of the major players of innate immune responses through the recognition and internalization of inhaled pathogens and foreign particles1,2. It has been reported that AMs are essential for the rapid clearance of many pulmonary pathogens such as P. aeruginosa and Klebsiella pneumonia3,4, so a deficiency in AM phagocytosis often results in respiratory infections, such as acute pneumonia, which cause higher mortality and morbidity rates.
AMs also initiate innate inflammatory responses in the lung by producing cytokines and chemokines such as TNF-&#945; and IL-1&#946;, which crosstalk with other cells of the alveolar environment to produce chemokines and recruit inflammatory neutrophils, monocytes, and adaptive immune cells in the lung5. For example, IL-1&#946; produced by AMs helps to prime the release of the neutrophil chemokine CXCL8 from epithelial cells6. Moreover, AMs contribute to the phagocytosis of apoptotic polymorphonuclear leukocytes (PMNs), failure of which leads to the sustained leakage of intracellular enzymes from PMNs to the surrounding tissue, resulting in tissue damage and prolonged inflammation7,8,9.
Phagocytosis by the AMs is mediated by a direct recognition of pathogen-associated molecular patterns at the pathogen surface by the pattern recognition receptors (PRRs) of the AMs or by the binding of opsonized pathogens with immune effector receptors of the AMs10. For the latter, AMs can recognize the targets opsonized with immunoglobulin (IgG) through their Fc&#947; receptors (Fc&#947;R) or the pathogens coated with complement fragments, C3b and C3bi, through their complement receptors (CR)11. Among complement receptors, the CR of the immunoglobulin superfamily (CRIg) is selectively expressed in tissue macrophages12, and a recent finding highlighted the role of the CRIg in AM phagocytosis in the context of P. aeruginosa pneumonia13.
Many original studies use methods to evaluate macrophage phagocytosis to describe the molecular mechanisms of macrophage function14,15. However, methods like in vivo phagocytosis require a precise quantification of phagocytosis. Here, we summarize a detailed methodology for both in vitro and in vivo phagocytosis using fluorescein isothiocyanate (FITC)-glass beads and P. aeruginosa green fluorescent protein (GFP), respectively. Further, we explain the method of differentiating among PRR-, CR-, and Fc&#947;R-mediated phagocytosis. Finally, we report a method to characterize bacterial clearance in mouse with respect to P. aeruginosa pneumonia.

<table><tbody><tr><td>18-G Needle</td><td>Nipro Medical&amp;nbsp;</td><td>CI+1832-2C</td><td>Molecular Biology grade</td></tr><tr><td>2,7-diaminofluorene (DAF)</td><td>Sigma-Aldrich</td><td>D17106</td><td>Molecular Biology grade</td></tr><tr><td>70% Ethanol</td><td>Decon Labs Inc.</td><td>18C27B</td><td>Analytical grade</td></tr><tr><td>96-well plate</td><td>Corning</td><td>3603</td><td>Cell Biology grade</td></tr><tr><td>ACK lysis buffer</td><td>Life Technologies</td><td>A10492</td><td>Molecular Biology grade</td></tr><tr><td>Alexa fluor-488 Zymosan-A-bioparticle</td><td>Thermofisher Scientific</td><td>Z23373</td><td>Molecular Biology grade</td></tr><tr><td>C5 deficient serum&amp;nbsp;</td><td>Sigma-Aldrich</td><td>C1163</td><td>Biochemical reagent</td></tr><tr><td>Centrifuge</td><td>Labnet International</td><td>C0160-R</td><td></td></tr><tr><td>Cytospin 4 Cytocentrifuge</td><td>Thermofisher Scientific</td><td>A78300101 Issue 11</td><td></td></tr><tr><td>DMEM Cell Culture Media</td><td>Gibco</td><td>11995-065</td><td>Cell Biology grade</td></tr><tr><td>FBS</td><td>Atlanta Biologicals</td><td>S11550</td><td>Cell Biology grade</td></tr><tr><td>Flow Cytometer</td><td>BD Biosciences</td><td>FACSCalibur</td><td></td></tr><tr><td>Flow Jo Software</td><td>FlowJo, LLC</td><td></td><td></td></tr><tr><td>Forceps</td><td>Dumont</td><td>0508-SS/45-PS-1</td><td>Suitable for laboratory animal dissection</td></tr><tr><td>FITC-carboxylated latex beads</td><td>Sigma-Aldrich</td><td>L4530</td><td>Cell Biology grade</td></tr><tr><td>GFP-&lt;em&gt;P. aeruginosa&lt;/em&gt;</td><td>ATCC</td><td>101045GFP</td><td>Suitable for&amp;nbsp; cell infection assays</td></tr><tr><td>Glass bottom dish</td><td>MatTek Corp.</td><td>P35G-0.170-14-C</td><td>Cell Biology grade</td></tr><tr><td>High-Pressure Syringe</td><td>Penn-Century</td><td>FMJ-250</td><td>Suitable for laboratory animal use</td></tr><tr><td>Homogenizer</td><td>Omni International</td><td>TH-01</td><td></td></tr><tr><td>Hydrogen peroxide</td><td>Sigma-Aldrich</td><td>H1009</td><td>Analytical grade</td></tr><tr><td>Inverted Fluorescence Microscope</td><td>Olympus</td><td>IX73</td><td></td></tr><tr><td>Ketamine Hydrochloride</td><td>Hospira</td><td>CA-2904</td><td>Pharmaceutical grade</td></tr><tr><td>Shandon Kwik-Diff Stains</td><td>Thermofisher Scientific</td><td>9990700</td><td>Cell Biology grade</td></tr><tr><td>LB Agar</td><td>Fisher Scientific</td><td>BP1425</td><td>Molecular Biology grade</td></tr><tr><td>LB Broth</td><td>Fisher Scientific</td><td>BP1427</td><td>Molecular Biology grade</td></tr><tr><td>MicroSprayer Aerosolizer</td><td>Penn-Century</td><td>IA-1C</td><td>Suitable for laboratory animal use</td></tr><tr><td>Paraformaldehyde</td><td>Sigma-Aldrich</td><td>P6148</td><td>Reagent grade</td></tr><tr><td>PBS</td><td>Gibco</td><td>20012-027</td><td>Cell Biology grade</td></tr><tr><td>rabbit anti-SRBC-IgG&amp;nbsp;</td><td>MP Biomedicals</td><td>55806</td><td>Suitable for immuno-assays</td></tr><tr><td>rabbit anti-SRBC-IgM&amp;nbsp;</td><td>Cedarline Laboratories</td><td>CL9000-M</td><td>Suitable for immuno-assays</td></tr><tr><td>Scissors</td><td>Miltex</td><td>5-2</td><td>Suitable for laboratory animal dissection</td></tr><tr><td>Small Animal Laryngoscope</td><td>Penn-Century</td><td>LS-2</td><td>Suitable for laboratory animal use</td></tr><tr><td>Sodium Dodecyl Sulfate (SDS)</td><td>BioRad</td><td>1610301</td><td>Analytical grade</td></tr><tr><td>Spring Scissors (Med)</td><td>Fine Science Tools</td><td>15012-12</td><td>Suitable for laboratory animal dissection</td></tr><tr><td>Spring Scissors (Small)</td><td>Fine Science Tools</td><td>91500-09</td><td>Suitable for laboratory animal dissection</td></tr><tr><td>sheep red blood cells (SRBCs)&amp;nbsp;</td><td>MP Biomedicals</td><td>55876</td><td>Washed, preserved SRBCs</td></tr><tr><td>Urea</td><td>Sigma-Aldrich</td><td>U5378</td><td>Molecular Biology grade</td></tr><tr><td>Xylazine&amp;nbsp;</td><td>Akorn Animal Health</td><td>59399-110-20</td><td>Pharmaceutical grade</td></tr></tbody></table>

alveolar macrophage phagocytosis and bacteria clearance in mice

Alveolar macrophages (AMs) guard the alveolar space of the lung. Phagocytosis by AMs plays a critical role in the defense against invading pathogens, the removal of dead cells or foreign particles, and in the resolution of inflammatory responses and tissue remodeling, processes that are mediated by various surface receptors of the AMs. Here, we report methods for the analysis of the phagocytic function of AMs using in vitro and in vivo assays and experimental strategies to differentiate between the pattern recognition receptor-, complement receptor-, and Fc gamma receptor-mediated phagocytosis. Finally, we discuss a method to establish and characterize a P. aeruginosa pneumonia model in mice to assess bacterial clearance in vivo. These assays represent the most common methods to evaluate AM functions and can also be used to study macrophage function and bacterial clearance in other organs.

We first performed the experiment to analyze phagocytosis by mouse primary AMs. Throughout all analyses, we compared AMs isolated from WT and TRIM72KO mice. As shown in Figure 1A, fluorescence microscopy revealed that phagocytosis of FITC-glass beads by mouse primary AMs occurs after 1 h of incubation. Figure 1B shows the analysis of phagocytosis by flow cytometry. The quantification o...

Watch this Scientific Journal Video about Alveolar Macrophage Phagocytosis and Bacteria Clearance in Mice at JoVE.com

Alveolar Macrophage Phagocytosis and Bacteria Clearance in Mice

Here we report common methods to analyze the phagocytic function of murine alveolar macrophages and bacterial clearance from the lung. These methods study in vitro phagocytosis of fluorescein isothiocyanate beads and in vivo phagocytosis of Pseudomonas aeruginosa Green Fluorescent Protein. We also describe a method for clearing P. aeruginosa in mice.

Alveolar macrophages (AMs) guard the alveolar space of the lung. Phagocytosis by AMs plays a critical role in the defense against invading pathogens, the ...

alveolar-macrophage-phagocytosis-and-bacteria-clearance-in-mice

Research

JoVE Journal

Immunology and Infection

11.1K Views.  Eastern Virginia Medical School. Here we report common methods to analyze the phagocytic function of murine alveolar macrophages and bacterial clearance from the lung. These methods study in vitro phagocytosis of fluorescein isothiocyanate beads and in vivo phagocytosis of Pseudomonas aeruginosa Green Fluorescent Protein. We also describe a method for clearing P. aeruginosa in mice.

Video: Alveolar Macrophage Phagocytosis and Bacteria Clearance in Mice

Harvesting Murine Alveolar Macrophages and Evaluating Cellular Activation Induced by Polyanhydride Nanoparticles

Biodegradable nanoparticles have emerged as a versatile platform for the design and implementation of new intranasal vaccines against respiratory infectious diseases. Specifically, polyanhydride nanoparticles composed of the aliphatic sebacic acid (SA), the aromatic 1,6-bis(p-carboxyphenoxy)hexane (CPH), or the amphiphilic 1,8-bis(p-carboxyphenoxy)-3,6-dioxaoctane (CPTEG) display unique bulk and surface erosion kinetics1,2 and can be exploited to slowly release functional biomolecules (e.g., protein antigens, immunoglobulins, etc.) in vivo3,4,5. These nanoparticles also possess intrinsic adjuvant activity, making them an excellent choice for a vaccine delivery platform6,7,8.
	In order to elucidate the mechanisms governing the activation of innate immunity following intranasal mucosal vaccination, one must evaluate the molecular and cellular responses of the antigen presenting cells (APCs) responsible for initiating immune responses. Dendritic cells are the principal APCs found in conducting airways, while alveolar macrophages (AM&#632;) predominate in the lung parenchyma9,10,11. AM&#632; are highly efficient in clearing the lungs of microbial pathogens and cell debris12,13. In addition, this cell type plays a valuable role in the transport of microbial antigens to the draining lymph nodes, which is an important first step in the initiation of an adaptive immune response9. AM&#632; also express elevated levels of innate pattern recognition and scavenger receptors, secrete pro-inflammatory mediators, and prime na&iuml;ve T cells12,14. A relatively pure population of AM&#632; (e.g., greater than 80%) can easily be obtained via lung lavage for study in the laboratory. Resident AM&#632; harvested from immune competent animals provide a representative phenotype of the macrophages that will encounter the particle-based vaccine in vivo. Herein, we describe the protocols used to harvest and culture AM&#632; from mice and examine the activation phenotype of the macrophages following treatment with polyanhydride nanoparticles in vitro.

Herein, we describe protocols for harvesting murine alveolar macrophages, which are resident innate immune cells in the lung, and examining their activation in response to co-culture with polyanhydride nanoparticles.

Biodegradable nanoparticles have emerged as a  versatile platform for the design and implementation of new intranasal vaccines  against respiratory ...

Bioengineering

Depletion and Reconstitution of Macrophages in Mice

Macrophages are critical players in the innate immune response to infectious challenge or injury, initiating the innate immune response and directing the acquired immune response. Macrophage dysfunction can lead to an inability to mount an appropriate immune response and as such, has been implicated in many disease processes, including inflammatory bowel diseases. Macrophages display polarized phenotypes that are broadly divided into two categories. Classically activated macrophages, activated by stimulation with IFN&gamma; or LPS, play an essential role in response to bacterial challenge whereas alternatively activated macrophages, activated by IL-4 or IL-13, participate in debris scavenging and tissue remodeling and have been implicated in the resolution phase of inflammation. During an inflammatory response in vivo, macrophages are found amid a complex mixture of infiltrating immune cells and may participate by exacerbating or resolving inflammation. To define the role of macrophages in situ in a whole animal model, it is necessary to examine the effect of depleting macrophages from the complex environment. To ask questions about the role of macrophage phenotype in situ, phenotypically defined polarized macrophages can be derived ex vivo, from bone marrow aspirates and added back to mice, with or without prior depletion of macrophages. In the protocol presented here clodronate-containing liposomes, versus PBS injected controls, were used to deplete colonic macrophages during dextran sodium sulfate (DSS)-induced colitis in mice. In addition, polarized macrophages were derived ex vivo and transferred to mice by intravenous injection. A caveat to this approach is that clodronate-containing liposomes deplete all professional phagocytes, including both dendritic cells and macrophages so to ensure the effect observed by depletion is macrophage-specific, reconstitution of phenotype by adoptive transfer of macrophages is necessary. Systemic macrophage depletion in mice can also be achieved by backcrossing mice onto a CD11b-DTR background, which is an excellent complementary approach. The advantage of clodronate-containing liposome-mediated depletion is that it does not require the time and expense involved in backcrossing mice and it can be used in mice regardless of the background of the mice (C57BL/6, BALB/c, or mixed background).

Macrophages play a central role in homeostasis and pathology in many tissues. The protocol presented here describes methods for depleting macrophages in vivo, deriving polarized macrophages from bone marrow aspirates, and adoptively transferring macrophages into mice. These techniques allow determination of the role that polarized macrophages play in health and disease.

Macrophages are critical players in the innate  immune response to infectious challenge or injury, initiating the innate immune  response and directing ...

Assessing Anti-fungal Activity of Isolated Alveolar Macrophages by Confocal Microscopy

The lung is an interface where host cells are routinely exposed to microbes and microbial products. Alveolar macrophages are the first-line phagocytic cells that encounter inhaled fungi and other microbes. Macrophages and other immune cells recognize Aspergillus motifs by pathogen recognition receptors and initiate downstream inflammatory responses. The phagocyte NADPH oxidase generates reactive oxygen intermediates (ROIs) and is critical for host defense. Although NADPH oxidase is critical for neutrophil-mediated host defense1-3, the importance of NADPH oxidase in macrophages is not well defined. The goal of this study was to delineate the specific role of NADPH oxidase in macrophages in mediating host defense against A. fumigatus. We found that NADPH oxidase in alveolar macrophages controls the growth of phagocytosed A. fumigatus spores4. Here, we describe a method for assessing the ability of mouse alveolar macrophages (AMs) to control the growth of phagocytosed Aspergillus spores (conidia). Alveolar macrophages are stained in vivo and ten days later isolated from mice by bronchoalveolar lavage (BAL). Macrophages are plated onto glass coverslips, then seeded with green fluorescent protein (GFP)-expressing A. fumigatus spores. At specified times, cells are fixed and the number of intact macrophages with phagocytosed spores is assessed by confocal microscopy.

A method to evaluate the ability of isolated mouse alveolar macrophages to control the growth of phagocytosed Aspergillus spores by confocal microscopy.

The lung is an interface where host cells are routinely exposed to microbes and microbial products. Alveolar macrophages are the first-line phagocytic ...

Studying Microbial Communities In Vivo: A Model of Host-mediated Interaction Between Candida Albicans and Pseudomonas Aeruginosa in the Airways

Studying host-pathogen interaction enables us to understand the underlying mechanisms of the pathogenicity during microbial infection. The prognosis of the host depends on the involvement of an adapted immune response against the pathogen1. Immune response is complex and results from interaction of the pathogens and several immune or non-immune cellular types2. In vitro studies cannot characterise these interactions and focus on cell-pathogen interactions. Moreover, in the airway3, particularly in patients with suppurative chronic lung disease or in mechanically ventilated patients, polymicrobial communities are present and complicate host-pathogen interaction. Pseudomonas aeruginosa and Candida albicans are both problem pathogens4, frequently isolated from tracheobronchial samples, and associated to severe infections, especially in intensive care unit5. Microbial interactions have been reported between these pathogens in vitro but the clinical impact of these interactions remains unclear6. To study the interactions between C. albicans and P. aeruginosa, a murine model of C. albicans airways colonization, followed by a P. aeruginosa-mediated acute lung infection was performed.

Studying Microbial Communities In Vivo: A Model of Host-mediated Interaction Between Candida Albicans and Pseudomonas Aeruginosa in the Airways

While in vitro study of host-pathogen interactions allow the characterization of specific immune responses, in vivo models are required to observe the effects of complex responses. Using Candida albicans exposure followed by Pseudomonas aeruginosa-mediated lung infection, we established a murine model of microbial interactions involved in ventilator-associated pneumonia pathogenicity.

Studying host-pathogen interaction enables us to understand the underlying mechanisms of the pathogenicity during microbial infection. The prognosis of ...

A Non-invasive and Technically Non-intensive Method for Induction and Phenotyping of Experimental Bacterial Pneumonia in Mice

Although community-acquired pneumonia remains a major public health problem, murine models of bacterial pneumonia have recently facilitated significant preclinical advances in our understanding of the underlying cellular and molecular pathogenesis. In vivo mouse models capture the integrated physiology and resilience of the host defense response in a manner not revealed by alternative, simplified ex vivo approaches. Several methods have been described in the literature for intrapulmonary inoculation of bacteria in mice, including aerosolization, intranasal delivery, peroral endotracheal cannulation under &#39;blind&#39; and visualized conditions, and transcutaneous endotracheal cannulation. All methods have relative merits and limitations. Herein, we describe in detail a non-invasive, technically non-intensive, inexpensive, and rapid method for intratracheal delivery of bacteria that involves aspiration (i.e., inhalation) by the mouse of an infectious inoculum pipetted into the oropharynx while under general anesthesia. This method can be used for pulmonary delivery of a wide variety of non-caustic biological and chemical agents, and is relatively easy to learn, even for laboratories with minimal prior experience with pulmonary procedures. In addition to describing the aspiration pneumonia method, we also provide step-by-step procedures for assaying the subsequent in vivo pulmonary innate immune response of the mouse, in particular, methods for quantifying bacterial clearance and the cellular immune response of the infected airway. This integrated and simple approach to pneumonia assessment allows for rapid and robust evaluation of the effect of genetic and environmental manipulations upon pulmonary innate immunity.

Several methods have been described in the literature for modeling bacterial pneumonia in mice. Herein, we describe a non-invasive, inexpensive, rapid method for inducing pneumonia via aspiration (i.e., inhalation) of a bacterial inoculum pipetted into the oropharynx. Downstream methods for assessment of the pulmonary innate immune response are also detailed.

Although community-acquired pneumonia remains a major public health problem, murine models of bacterial pneumonia have recently facilitated significant ...

Assessing Retinal Microglial Phagocytic Function In Vivo Using a Flow Cytometry-based Assay

Microglia are the tissue resident macrophages of the central nervous system (CNS) and they perform a variety of functions that support CNS homeostasis, including phagocytosis of damaged synapses or cells, debris, and/or invading pathogens. Impaired phagocytic function has been implicated in the pathogenesis of diseases such as Alzheimer's and age-related macular degeneration, where amyloid-&#946; plaque and drusen accumulate, respectively. Despite its importance, microglial phagocytosis has been challenging to assess in vivo. Here, we describe a simple, yet robust, technique for precisely monitoring and quantifying the in vivo phagocytic potential of retinal microglia. Previous methods have relied on immunohistochemical staining and imaging techniques. Our method uses flow cytometry to measure microglial uptake of fluorescently labeled particles after intravitreal delivery to the eye in live rodents. This method replaces conventional practices that involve laborious tissue sectioning, immunostaining, and imaging, allowing for more precise quantification of microglia phagocytic function in just under six hours. This procedure can also be adapted to test how various compounds alter microglial phagocytosis in physiological settings. While this technique was developed in the eye, its use is not limited to vision research.

Assessing Retinal Microglial Phagocytic Function In Vivo Using a Flow Cytometry-based Assay

Microglial phagocytosis is critical for the maintenance of tissue homeostasis and inadequate phagocytic function has been implicated in pathology. However, assessing microglia function in vivo is technically challenging. We have developed a simple but robust technique for precisely monitoring and quantifying the phagocytic potential of microglia in a physiological setting.

Microglia are the tissue resident macrophages of the central nervous system (CNS) and they perform a variety of functions that support CNS homeostasis, ...

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and their important role in lung development and function, as well as their lung-localized responses to infection and inflammation. To date, no unified method for identification, isolation, and handling of alveolar macrophages from humans and mice exists. Such a method is needed for studies on these important innate immune cells in various experimental settings. The method described here, which can be easily adopted by any laboratory, is a simplified approach to harvesting alveolar macrophages from bronchoalveolar lavage fluid or from lung tissue and maintaining them in vitro. Because alveolar macrophages primarily occur as adherent cells in the alveoli, the focus of this method is on dislodging them prior to harvest and identification. The lung is a highly vascularized organ, and various cell types of myeloid and lymphoid origin inhabit, interact, and are influenced by the lung microenvironment. By using the set of surface markers described here, researchers can easily and unambiguously distinguish alveolar macrophages from other leukocytes, and purify them for downstream applications. The culture method developed herein supports both human and mouse alveolar macrophages for in vitro growth, and is compatible with cellular and molecular studies.

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

This communication describes methodologies for isolation and culture of alveolar macrophages from humans and murine models for experimental purposes.

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and ...

Studying the Role of Alveolar Macrophages in Breast Cancer Metastasis

This paper describes the application of the syngeneic model of breast cancer (4T1) to the studies on a role of pulmonary alveolar macrophages in cancer metastasis. The 4T1 cells expressing GFP in combination with imaging and confocal microscopy are used to monitor tumor growth, track metastasizing tumor cells, and quantify the metastatic burden. These approaches are supplemented by digital histopathology that allows the automated and unbiased quantification of metastases. In this method the routinely prepared histological lung sections, which are stained with hematoxylin and eosin, are scanned and converted to the digital slides that are then analyzed by the self-trained pattern recognition software. In addition, we describe the flow cytometry approaches with the use of multiple cell surface markers to identify alveolar macrophages in the lungs. To determine impact of alveolar macrophages on metastases and antitumor immunity these cells are depleted with the clodronate-containing liposomes administrated intranasally to tumor-bearing mice. This approach leads to the specific and efficient depletion of this cell population as confirmed by flow cytometry. Tumor volumes and lung metastases are evaluated in mice depleted of alveolar macrophages, to determine the role of these cells in the metastatic progression of breast cancer.

Here we describe the model and approach to study functions of pulmonary alveolar macrophages in cancer metastasis. To demonstrate the role of these cells in metastasis, the syngeneic (4T1) model of breast cancer in conjunction with the depletion of alveolar macrophage with clodronate liposomes was used.

This paper describes the application of the syngeneic model of breast cancer (4T1) to the studies on a role of pulmonary alveolar macrophages in cancer ...

Medicine

In Vivo Assessment of Alveolar Macrophage Efferocytosis Following Ozone Exposure

Ozone (O3) is a criteria air pollutant that exacerbates and increases the incidence of chronic pulmonary diseases. O3 exposure is known to induce pulmonary inflammation, but little is known regarding how exposure alters processes important to the resolution of inflammation. Efferocytosis is a resolution process, whereby macrophages phagocytize apoptotic cells. The purpose of this protocol is to measure alveolar macrophage efferocytosis following O3-induced lung injury and inflammation. Several methods have been described for measuring efferocytosis; however, most require ex vivo manipulations. Described in detail here is a protocol to measure in vivo alveolar macrophage efferocytosis 24 h after O3 exposure, which avoids ex vivo manipulation of macrophages and serves as a simple technique that can be used to accurately represent perturbations in this resolution process. The protocol is a technically non-intensive and relatively inexpensive method that involves whole-body O3 inhalation followed by oropharyngeal aspiration of apoptotic cells (i.e., Jurkat T cells) while under general anesthesia. Alveolar macrophage efferocytosis is then measured by light microscopy evaluation of macrophages collected from bronchoalveolar (BAL) lavage. Efferocytosis is finally measured by calculating an efferocytic index. Collectively, the outlined methods quantify efferocytic activity in the lung in vivo while also serving to analyze the negative health effects of O3 or other inhaled insults.

This manuscript describes a protocol for determining whether exposure to ozone, a criteria air pollutant, impairs alveolar macrophage efferocytosis in vivo. This protocol utilizes commonly used reagents and techniques and can be adapted to multiple models of pulmonary injury to determine effects on alveolar macrophage efferocytosis.

Ozone (O3) is a criteria air pollutant that exacerbates and increases the incidence of chronic pulmonary diseases. O3 exposure is known to induce ...

Adoptive Transfer of IL-33-Stimulated Macrophages into Bleomycin-Induced Mouse Models to Study Their Effect on Idiopathic Pulmonary Fibrosis In Vivo

The inflammatory response caused by early lung injury is one of the important causes of the development of idiopathic pulmonary fibrosis (IPF), which is accompanied by the activation of inflammatory cells such as macrophages and neutrophils, as well as the release of inflammatory factors including TNF-&#945;, IL-1&#946;, and IL-6. Early inflammation caused by activated pulmonary interstitial macrophages (IMs) in response to IL-33 stimulation is known to play a vital role in the pathological process of IPF. This protocol describes the adoptive transfer of IMs stimulated by IL-33 into the lungs of mice to study IPF development. It involves the isolation and culture of primary IMs from host mouse lungs, followed by the adoptive transfer of stimulated IMs into the alveoli of bleomycin (BLM)-induced IPF recipient mice (which have been previously depleted of alveolar macrophages by treatment with clodronate liposomes), and the pathological evaluation of those mice. The representative results show that the adoptive transfer of IL-33-stimulated macrophages aggravates pulmonary fibrosis in mice, suggesting that the establishment of the macrophage adoptive transfer experiment is a good technical means to study IPF pathology.

Adoptive Transfer of IL-33-Stimulated Macrophages into Bleomycin-Induced Mouse Models to Study Their Effect on Idiopathic Pulmonary Fibrosis In Vivo

This protocol describes the isolation of pulmonary interstitial macrophages (IMs) and their adoptive transfer after IL-33 stimulation of the lung alveoli in a mouse model, which can facilitate the in vivo study of idiopathic pulmonary fibrosis (IPF).

The inflammatory response caused by early lung injury is one of the important causes of the development of idiopathic pulmonary fibrosis (IPF), which is ...

Alveolar Macrophage Phagocytosis and Bacteria Clearance in Mice

Summary

Explore More Videos

Harvesting Murine Alveolar Macrophages and Evaluating Cellular Activation Induced by Polyanhydride Nanoparticles

Depletion and Reconstitution of Macrophages in Mice

Assessing Anti-fungal Activity of Isolated Alveolar Macrophages by Confocal Microscopy

Studying Microbial Communities In Vivo: A Model of Host-mediated Interaction Between Candida Albicans and Pseudomonas Aeruginosa in the Airways

A Non-invasive and Technically Non-intensive Method for Induction and Phenotyping of Experimental Bacterial Pneumonia in Mice

Assessing Retinal Microglial Phagocytic Function In Vivo Using a Flow Cytometry-based Assay

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

Studying the Role of Alveolar Macrophages in Breast Cancer Metastasis

In Vivo Assessment of Alveolar Macrophage Efferocytosis Following Ozone Exposure

Adoptive Transfer of IL-33-Stimulated Macrophages into Bleomycin-Induced Mouse Models to Study Their Effect on Idiopathic Pulmonary Fibrosis In Vivo