Alveolar Macrophage Phagocytosis and Bacteria Clearance in Mice

Summary

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.

Abstract

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.

Introduction

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 particles^1,2. It has been reported that AMs are essential for the rapid clearance of many pulmonary pathogens such as P. aeruginosa and Klebsiella pneumonia^3,4, so a deficiency in AM phagocytosis often results in respiratory infections, such as acute pneumonia, which cause higher mortality and morbidity rates.

Protocol

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 disinf....

Results

We first performed the experiment to analyze phagocytosis by mouse primary AMs. Throughout all analyses, we compared AMs isolated from WT and TRIM72^KO 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.......

Discussion

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.......

Materials

List of materials used in this article

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