Name: alveolar macrophage phagocytosis and bacteria clearance in mice
Uploaded: 2019-03-02T14:00:00
Description: Watch this Scientific Journal Video about Alveolar Macrophage Phagocytosis and Bacteria Clearance in Mice at JoVE.com

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...

<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. 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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. 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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. 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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.

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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 ...

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