This study is funded by Health Effects Institute Walter A. Rosenblith Award and NIEHS R01ES028829 (to K. M. G). We would like to thank Dr. Dianne Walters (Department of Physiology, ECU) for her assistance with obtaining representative images of alveolar macrophages.

All methods have been approved by the Institutional Animal Care and Use Committee (IACUC) of East Carolina University.
1. Ozone (O3) and filtered air exposures (Day 1)
<ol>
	<li>Place a maximum of 12 female C57BL/6J mice, 8-12 weeks old, in a steel cage (with 12 separate compartments) with wire mesh lids into an O3 exposure chamber.</li>
	<li>Place the thermometer in the exposure chamber with the cage to accurately record the temperature and humidity.</li>
	<li>Turn on the oxygen and ultraviolet (UV) light that is attached to the apparatus. 
	NOTE: Regulated airflow (&#62;30 air changes/h) with controlled temperature (22-23 &#176;C) and relative humidity (45%-50%) is obtained by the O3 apparatus. O3 is generated by the system in the exposure chamber by directing 100% oxygen through a UV light generator, then mixing with a filtered air supply.</li>
	<li>Adjust the O3 concentration to 1 ppm and regularly record O3 levels every 10 min for 3 h. Continuously monitor the temperature and humidity of chamber air, as is the O3 concentration with a UV light photometer. 
	NOTE: Filtered air exposures are performed in a similar apparatus, with only a filtered air supply flowing through the exposure chamber.</li>
	<li>Return the animals to their respective cages with bedding, food, and water ad libitum after 3 h of O3/filtered air exposure.</li>
</ol>
2. Preparation of Jurkat T cell line (Day 2)
NOTE: All procedures should be conducted in a class II biological safety cabinet.
<ol>
	<li>Culture Jurkat T cells in 24 mL of basal cell culture medium + 10% FBS + 5% penicillin/streptomycin at 37 &#176;C + 5% CO2 (Table of Material). Jurkat T cells are a suspension cell line that can be maintained through passaging 1:6-1:8 into pre-warmed culturing media every 3 days. Do not shake.</li>
	<li>To prepare apoptotic cells, grow them to 90% confluency in each flask (which takes 3-4 days to achieve after passaging). For this study, use cells from five T75 flasks to obtain the sufficient number of cells used in this protocol. 
	NOTE: A confluent flask contains about 20-24 million cells.</li>
	<li>Pipette up cells (which is the entire flask) from each flask (approximately 24 mL) and transfer cells to a sterile 50 mL conical tube using a serological pipette. Use multiple conical tubes for multiple flasks.</li>
	<li>Count cells by removing an 11 &#181;L aliquot of cells from the 50 mL conical tube and mix with 11 &#181;L of trypan blue stain and pipette 11 &#181;L onto hemocytometer slides.</li>
	<li>Insert slide into an automated cell counter and record the number of live cells to calculate the total cell count in each flask by multiplying the number of live cells by 24, as each flask contains 24 mL of media.</li>
	<li>Centrifuge the cell suspension at 271&#160;x g for 5 min at room temperature (RT) to pellet cells.</li>
	<li>Discard the supernatant by aspiration and resuspend the cell pellet in media to obtain 3.0 x 106 cells per mL.</li>
	<li>Aliquot 5 mL of cells in 100 mm x 20 mm tissue culture dishes (approximately nine dishes will be used; the total amount of cells in each dish should be ~15 x 106).</li>
	<li>Use one dish for control/unexposed, and the remaining dishes will be exposed to UV.</li>
	<li>Set the UV crosslinker to the correct energy level, press the energy button, and enter &#34;600&#34; using the number pad, which the machine will read as 600 &#181;J/cm2 x 100. 
	NOTE: UV crosslinker energy units is in &#181;J/cm2 x 100; therefore, to achieve 60 millijoules/cm2, convert units to match the UV crosslinker.</li>
	<li>Irradiate all dishes with cells, not including the control, at 60 millijoules (mJ)/cm2 using the UV crosslinker. Remove the top cover of the tissue culture dishes during UV exposure, as UV light will not penetrate the plastic cover.</li>
	<li>Incubate all the dishes in a cell culture incubator, including unexposed control, at 37 &#176;C at 5% CO2&#160;for 4 h.</li>
	<li>Confirm apoptosis by flow cytometry using an apoptosis assay detection kit containing annexin V and propidium iodide (PI) (markers for apoptosis and necrosis, respectively) after 4 h of incubation, per the manufacturer&#39;s instructions26,27. 
	NOTE: Irradiating Jurkat T cells in the UV crosslinker at an energy level of 600 &#181;J/cm2, following a 4 h incubation will lead to &#8805;75% of apoptotic (both early and late) cells having more early apoptotic phenotype than the late apoptotic phenotype. This makes it easier for alveolar macrophages to recognize them and engulf as their membranes are uncompromised, unlike late apoptotic cells, leading to a higher efferocytic index and more accurate imaging of alveolar macrophage efferocytosis in this study.
	<ol>
		<li>Pool 333 &#181;L (1 x 106 cells) of Jurkat T cells from several dishes (both &#34;no UV&#34; and &#34;UV-exposed&#34;) together to use for compensation analysis tubes.</li>
		<li>Aliquot 333 &#181;L of Jurkat T cells in an unstained, annexin V single stain, PI single stain, no UV control, and 600 &#181;J/cm2 UV-exposed labeled flow cytometry tubes.</li>
		<li>Centrifuge tubes at 188&#160;x g for 5 min at RT and decant the supernatant.</li>
		<li>Wash cells by resuspending in 500 &#181;L of cold, 1x phosphate-buffered saline (PBS).</li>
		<li>Centrifuge and pellet cells at 188&#160;x g for 5 min at RT. Discard the supernatant after centrifugation.</li>
		<li>Prepare 400 &#181;L of 1x binding buffer per flow tube by diluting 10x binding buffer with distilled water while cells are centrifuging.</li>
		<li>Prepare annexin V and PI incubation reagent (100 &#181;L per sample/tube) per the manufacturer&#39;s instructions.</li>
		<li>Decant the supernatant after centrifugation and gently resuspend all tubes in 400 &#181;L of 1x binding buffer, then add 100 &#181;L of annexin V incubation reagent to each sample tube. Lastly, add 100 &#181;L of annexin V single stain and PI single stain to their respective tubes, but do not add anything beyond the 1x binding buffer to the unstained tube.</li>
		<li>Incubate tubes in the dark for 15 min at RT.</li>
		<li>Centrifuge all cells at 188&#160;x g for 5 min at RT and decant supernatant.</li>
		<li>Resuspend cells in 400 &#181;L of 1x binding buffer, then analyze samples for apoptosis by flow cytometry. Collect at least 10,000 events per tube to allow accurate representation of staining.</li>
	</ol>
	</li>
	<li>Combine all the irradiated cells from dishes into a 50 mL conical tube and pellet cells by centrifugation at 271&#160;x g for 5 min at RT.</li>
	<li>Discard the supernatant from the tube by aspiration and resuspend cells in 24 mL of sterile phosphate buffered saline (PBS) and pellet cells by centrifugation at 271&#160;x g for 5 min at room temperature.</li>
	<li>Discard the supernatant from the tube by aspiration and resuspend cells in the amount of PBS used for dosing mice approved by IACUC. The dose used is between 5-10 x 106 cells/50 &#181;L per mouse; therefore, for 10 mice, resuspend in 500 &#181;L (number of cells in each dose varies depending on how many cells are cultured for irradiation). 
	NOTE: Makeup at least two additional doses to account for any liquid that may stick to the sides of the pipette tip resulting in the loss of cells.</li>
</ol>
3. Murine oropharyngeal instillation of apoptotic cells (Day 2)
<ol>
	<li>Prepare dosing inoculum of apoptotic cells using a P200 pipette prior to anesthetizing mice to expedite the procedure. As per the institutional guidelines, a volume of 50 &#181;L containing approximately 5-10 x 106 cells is utilized for oropharyngeal (o.p.) instillation to ensure best results.</li>
	<li>Anesthetize mice in a clear chamber with 2% isoflurane at a flow rate of 1 L/min or as per the institutional guidelines. Anesthetize one to two mice at a time; the number is determined by the comfort level of the experimenter. Observe the breathing pattern and confirm deep breaths are visible with 2&#8211;3 s counts between breaths. Check for the depth of anesthesia by the lack of response to the toe pinch.</li>
	<li>Position the mouse in a semi-recumbent supine position. Use a surgical string tied between pegs on a slanted acrylic sheet board to suspend by the maxillary incisors.</li>
	<li>Using a pair of blunt non-ridged forceps, lightly grab and pull the mouse tongue. Instill the apoptotic cells into the oral cavity with a P200 pipette. Dosing is successful when the mice make a crackling noise 1&#8211;2 s after giving the dose. 
	NOTE: Take care to avoid inducing trauma either to the tongue or oropharynx before the apoptotic cell instillation.</li>
	<li>With a gloved finger, gently block the nose until the mouse inhales while the tongue is retracted. Cover the nose until no liquid is visible in the oral cavity and the mouse has taken two or more inhalations. 
	NOTE: As mice are obligate nose breathers, covering the nose helps ensure that the mouse will inhale the apoptotic cells into the lungs.</li>
	<li>Remove the mouse from the inoculation board and return it to the cage to allow recovery from anesthesia. Place the mouse on its back to prevent bedding or debris from blocking the nares during the revovery.</li>
	<li>Wait 90 min after the mouse recovers from anesthesia to allow alveolar macrophages to engulf influx of apoptotic cells after all the mice have awoken from anesthesia. Typically, awakening after anesthesia will take 1&#8211;2 min, which should not affect the outcome/timing of instillation.</li>
</ol>
4. Bronchoalveolar lavage fluid collection and processing (Day 2)
<ol>
	<li>Euthanize each mouse per institutional guidelines 90 min after the mouse has woken up from anesthesia from dosing with apoptotic cells. Here, a lethal injection of ketamine and xylazine is used (90 mg/kg and 10 mg/kg, respectively) followed by excising the diaphragm. 
	NOTE: This time point allows sufficient time for alveolar macrophages to sense and engulf apoptotic cells38.</li>
	<li>Weigh all mice (g) on a scale and record weights. Use the body weight to calculate BAL volume (26.25 mL/kg body weight).</li>
	<li>Place mice on their backs and spray 70% ethanol to sterilize the chest and neck area.</li>
	<li>Make a 2&#8221; longitudinal cut just below the sternum along the entire ventral side with surgical scissors, and while holding the sternum with forceps, nick the diaphragm to allow the lungs to fall back into the chest cavity.</li>
	<li>Cut laterally along the sides of the rib cage to allow the lungs more room to expand when lavaging, then fold the chest cavity back with forceps.</li>
	<li>Make a 1&#34; vertical cut up along vasculature through the neck to expose the trachea.</li>
	<li>Use two forceps to pull muscle and tissue off the trachea and expose it. Avoid additional potential bleeding and cutting the trachea, since it is surrounded by vasculature, longitudinal muscles, and connective tissue.</li>
	<li>Use a needle to make a slit in the trachea (about one-quarter of the distance down from the head) and insert a cannula (18 G x 1.25&#34;) with a syringe pre-loaded with 1x PBS (26.25 mL/kg body weight, ~0.7-1.0 mL in an 8-10 week old female C57Bl/6J mouse) caudally into the trachea.</li>
	<li>Push volume of PBS into the lungs slowly to allow the lungs to inflate then, pull the volume back out into the syringe. Repeat this process a total of 3 times.</li>
	<li>Collect the pooled lavage fluid from each specific mouse in a 15 mL tube.</li>
	<li>Centrifuge the bronchoalveolar lavage at 610&#160;x g for 6 min at 4 &#176;C and collect supernatant into a 1.5 mL tube and freeze at -80 &#176;C. The pellet represents cells from the bronchoalveolar space.</li>
	<li>Remove residual red blood cells in collected BAL fluid by adding 1 mL of ACK RBC lysis buffer to the cell pellet, then vortex well and lyse for 1 min on ice. Afterwards, add 4 mL of PBS to stop the lysis reaction.</li>
	<li>Pellet cells by centrifugation at 610&#160;x g for 6 min at 4 &#176;C and aspirate the supernatant with a vacuum aspirator.</li>
	<li>Resuspend cells in 1 mL of 1x&#160;PBS + 10% FBS to each BAL sample tube. Count cells on a hemocytometer for the quantification of total airspace cells from each sample (no trypan blue). Centrifuge 120 &#181;L of each sample onto slides at 56&#160;x g for 3 min, using medium acceleration and a cytocentrifuge. Dry the slides overnight.</li>
</ol>
5. Calculation of alveolar macrophage efferocytic index (Day 3)
<ol>
	<li>Stain the slides with hematoxylin and eosin to allow for calculation of both efferocytic and differential cell counts, with at least 200 cells counted from each slide.</li>
	<li>View slides under a bright-field setting on a biological microscope (a 20x or 40x objective will work best).</li>
	<li>Calculate the efferocytic index based on the ratio of the number of alveolar macrophages that phagocytosed apoptotic Jurkat T cells to alveolar macrophages without apoptotic cell uptake out of a total 200 macrophages on a cell differential slide. Convert the ratio to a percentage for data input. Use the following equation:</li>
</ol>
<img src="/files/ftp_upload/60109/60109eq1.jpg" />

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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=Pulmonary+surfactant+protein+a+inhibits+macrophage+reactive+oxygen+intermediate+production+in+response+to+stimuli+by+reducing+NADPH+oxidase+activity.">Pulmonary surfactant protein a inhibits macrophage reactive oxygen intermediate production in response to stimuli by reducing NADPH oxidase activity.</a> Journal of Immunology. 172 (11), 6866-6874 (2004).</li><li>Silveyra, P., Floros, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+variant+associations+of+human+SP-A+and+SP-D+with+acute+and+chronic+lung+injury.">Genetic variant associations of human SP-A and SP-D with acute and chronic lung injury.</a> Frontiers in Bioscience. 17, 407-429 (2012).</li><li>Schagat, T. L., Wofford, J. A., Wright, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surfactant+protein+A+enhances+alveolar+macrophage+phagocytosis+of+apoptotic+neutrophils.">Surfactant protein A enhances alveolar macrophage phagocytosis of apoptotic neutrophils.</a> Journal of Immunology. 166 (4), 2727-2733 (2001).</li><li>Gomez Perdiguero, 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=Tissue-resident+macrophages+originate+from+yolk-sac-derived+erythro-myeloid+progenitors.">Tissue-resident macrophages originate from yolk-sac-derived erythro-myeloid progenitors.</a> Nature. 518 (7540), 547-551 (2015).</li><li>Nebbioso, A., 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=Time-resolved+analysis+of+DNA-protein+interactions+in+living+cells+by+UV+laser+pulses.">Time-resolved analysis of DNA-protein interactions in living cells by UV laser pulses.</a> Scientific Reports. 7 (1), 11725 (2017).</li><li>Novak, Z., 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=Efficacy+of+different+UV-emitting+light+sources+in+the+induction+of+T-cell+apoptosis.">Efficacy of different UV-emitting light sources in the induction of T-cell apoptosis.</a> Photochemistry and Photobiology. 79 (5), 434-439 (2004).</li><li>Park, Y. J., 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=PAI-1+inhibits+neutrophil+efferocytosis.">PAI-1 inhibits neutrophil efferocytosis.</a> Proceedings of the National Academy of Sciences of the United States of America. 105 (33), 11784-11789 (2008).</li><li>Kleeberger, S. R., Reddy, S., Zhang, L. Y., Jedlicka, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+susceptibility+to+ozone-induced+lung+hyperpermeability:+role+of+toll-like+receptor+4.">Genetic susceptibility to ozone-induced lung hyperpermeability: role of toll-like receptor 4.</a> American Journal of Respiratory Cell and Molecular Biology. 22 (5), 620-627 (2000).</li><li>Wesselkamper, S. C., Chen, L. C., Kleeberger, S. R., Gordon, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+variability+in+the+development+of+pulmonary+tolerance+to+inhaled+pollutants+in+inbred+mice.">Genetic variability in the development of pulmonary tolerance to inhaled pollutants in inbred mice.</a> American Journal of Physiology-Lung Cellular and Molecular Physiology. 281 (5), L1200-L1209 (2001).</li></ol>

The authors declare no conflicts of interest.

Efferocytosis is an anti-inflammatory process in which macrophages clear apoptotic cells and debris as well as produce multiple anti-inflammatory mediators9,10,11,12,16,18. Multiple models of efferocytosis have provided insight into how the macrophage is a critical cell in the resolution of inflammation6</su...

The lung is constantly exposed to environmental insults, including air particulates, viruses, bacteria, and oxidant gases that trigger pulmonary inflammation1,2,3. These insults can compromise gas exchange and induce irreversible tissue injury4,5. Alveolar macrophages, which constitute approximately 95% of the immune cells found in murine and human lungs at homeostasis, are critical regulators of pulmonary inflammation after environmental insults1,2,3,4,5. Alveolar macrophages are essential during the host defense by phagocytizing and eliminating pathogens. Recently, alveolar macrophages have been shown to promote tissue homeostasis and the resolution of inflammation through efferocytosis6,7. Efferocytosis is a phagocytic process in which macrophages engulf and eliminate apoptotic cells8,9,10. Efferocytosis also results in the production of mediators (i.e., IL-10, TGF-&#946;, PGE2, and nitric oxide) that further augment the process, resulting in the resolution of inflammation9,10,11,12,16,18. This process is necessary for preventing secondary necrosis and promoting tissue homeostasis12,13,14. Several studies have linked impaired efferocytosis with various chronic lung diseases, including asthma, chronic obstructive pulmonary disease, and idiopathic pulmonary fibrosis8,9,15,16,17.
O3 is a criteria air pollutant that exacerbates and increases the incidence of chronic pulmonary diseases19,20,21. O3 induces pulmonary inflammation and injury and is known to impair alveolar macrophage phagocytosis of bacterial pathogens22,23. However, it is unknown whether O3 impairs alveolar macrophage efferocytosis. Investigating O3-induced alterations in alveolar macrophage efferocytosis will provide potential insight into how exposure can lead to chronic pulmonary disease incidence and exacerbation. Described below is a simple method to evaluate alveolar macrophage efferocytosis in the lungs of female mice after acute O3 exposure.
The outlined method posseses several advantages over other efferocytosis protocols commonly used in the field by eliminating the use of costly fluorescent dyes, extensive flow cytometry measurements, and ex vivo manipulation of alveolar macrophages24,25. Additionally, this protocol measures alveolar macrophage efferocytosis in the context of the lung microenvironment, which can influence macrophage function.

<table><tbody><tr><td>Annexin V-FITC Kit</td><td>Trevigen</td><td>4830-250-K&amp;nbsp;</td><td>The TACS&amp;nbsp;Annexin V-FITC Kit allows rapid, specific, and quantitative identification of apoptosis in individual cells when using flow cytometry.</td></tr><tr><td>BCL2 Jurkat T Cells&amp;nbsp;</td><td>ATCC</td><td>ATCC CRL-2899</td><td>The BCL2 Jurkat cell line was derived by transfecting human Jurkat T cells with the pSFFV-neo mammalian expression vector containing the human BCL-2 ORF insert and a neomycin-resistant gene. Has been for models of measuring efferocytosis.&amp;nbsp;</td></tr><tr><td>Countess II Automated Cell Counter</td><td>Thermofisher</td><td>AMQAX1000</td><td>It is a benchtop assay platform equipped with state-of-the-art optics, full autofocus, and image analysis software for rapid assessment of cells in suspension. Very easy to use.</td></tr><tr><td>Cytospin 4 Cytocentrifuge</td><td>Thermofisher</td><td>A78300003</td><td>Provides economical thin-layer preparations from any liquid matrix, especially hypocellular fluids such as bronchoalveolar lavage fluid.</td></tr><tr><td>Fetal Bovine Serum, qualified, heat inactivated</td><td>Thermofisher</td><td>16140071</td><td>Provides Nutrients to cultured cells for them to grow. It is standard for cell culture.&amp;nbsp;</td></tr><tr><td>Kwik-Diff&amp;nbsp; Reagent 2, Eosin</td><td>Thermofisher</td><td>9990706</td><td>Eosin staining that stains cytoplasm.</td></tr><tr><td>Kwik-Diff Reagent 1, Fixative</td><td>Thermofisher</td><td>9990705</td><td>Fixes cells to be stained by H&amp;amp;E.</td></tr><tr><td>Kwik-Diff Reagent 3, Methylene Blue</td><td>Thermofisher</td><td>9990707</td><td>Methylene Blue staining that stains the nucleus.</td></tr><tr><td>Penicillin-Streptomycin</td><td>Sigma/Aldrich</td><td>P0781-100ML</td><td>Penicillin-Streptomycin is the most commonly used antibiotic solution for culture of mammalian cells. Additionally it is used to maintain sterile conditions during cell culture.</td></tr><tr><td>RPMI 1640 Medium, GlutaMAX Supplement&amp;nbsp;</td><td>Thermofisher</td><td>61870036</td><td>RPMI 1640 Medium (Roswell Park Memorial Institute 1640 Medium) was originally developed to culture human leukemic cells in suspension and as a monolayer. RPMI 1640 medium has since been found suitable for a variety of mammalian cells, including HeLa, Jurkat, MCF-7, PC12, PBMC, astrocytes, and carcinomas. Helps grow Jurkat T cells fast and efficiently.</td></tr><tr><td>Stratagene UV Stratalinker 1800 UV Crosslinker</td><td>Cambridge Scientific&amp;nbsp;</td><td>16659</td><td>The Stratalinker UV crosslinker is designed to induce apoptosis, crosslink DNA or RNA to nylon, nitrocellulose, or nylon-reinforced nitrocellulose membranes.</td></tr><tr><td>Teledyne T400 ultraviolet light photometer&amp;nbsp;</td><td>Teledyne API</td><td>T400</td><td>The Model T400 UV Absorption analyzer uses a system based on the Beer-Lambert law for measuring low ranges of ozone in ambient air.</td></tr><tr><td>Teledyne T703 Ozone calibrator</td><td>Teledyne API</td><td>T703</td><td>Provides feedback control of the UV lamp intensity, assuring stable ozone output.</td></tr></tbody></table>

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.

O3 exposure is known to induce pulmonary inflammation and injury, and efferocytosis is required to maintain tissue homeostasis. C57BL/6J female mice were exposed to filtered air (FA) or 1 ppm O3 for 3 h and necropsied 24 h post-exposure to examine pulmonary inflammation and injury. O3-exposed mice displayed a significant increase in macrophages and neutrophils in the airspace compared to the FA control group (Figure 1A...

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In Vivo Assessment of Alveolar Macrophage Efferocytosis Following Ozone Exposure

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 ...

in-vivo-assessment-alveolar-macrophage-efferocytosis-following-ozone

Research

JoVE Journal

Immunology and Infection

9.0K Views.  East Carolina University. 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.

Video: In Vivo Assessment of Alveolar Macrophage Efferocytosis Following Ozone Exposure

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

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 ...

Quantifying Carbon in Pulmonary Macrophages via Induced Sputum Analysis

Measuring Carbon Content in Airway Macrophages Exposed to Carbon-Containing Particulate Matters

Pulmonary macrophages exhibit a dose-dependent pattern in phagocytizing particles. Following engulfment, these macrophages are subsequently excreted with sputum, rendering macrophages and particles visible and quantifiable under light microscopy. Notably, elemental carbon within the mammalian body originates exclusively from external contaminants. Consequently, the carbon content in airway macrophages (CCAM) serves as a valid exposure biomarker, accurately estimating individual exposure to carbon-containing particulate matter (PM). This article delineates a protocol involving sputum collection, preservation, processing, slide preparation, and staining, as well as macrophage photo acquisition and analysis. After removing the macrophage nuclei, the proportion of cytoplasm area occupied by carbon particles (PCOC) was calculated to quantify carbon content in each macrophage. The results indicate an elevation in CCAM levels after exposure to carbon-containing PM. In summary, this non-invasive, precise, reliable, and standardized method enables the direct measurement of carbon particles within target cells and is utilized for large-scale quantification of individual CCAM through induced sputum.

This article describes a detailed experimental protocol for measuring the carbon content of airway macrophages with the aim of assessing the internal exposure dose at the level of individual particulate matter exposure.

Pulmonary macrophages exhibit a dose-dependent pattern in phagocytizing particles. Following engulfment, these macrophages are subsequently excreted with ...

Environment

Bronchoalveolar Lavage of Murine Lungs to Analyze Inflammatory Cell Infiltration

Bronchoalveolar Lavage (BAL) is an experimental procedure that is used to examine the cellular and acellular content of the lung lumen ex vivo to gain insight into an ongoing disease state.
Here, a simple and efficient method is described to perform BAL on murine lungs without the need of special tools or equipment. BAL fluid is isolated by inserting a catheter in the trachea of terminally anesthetized mice, through which a saline solution is instilled into the bronchioles. The instilled fluid is gently retracted to maximize BAL fluid retrieval and to minimize shearing forces. This technique allows the viability, function, and structure of cells within the airways and BAL fluid to be preserved.
Numerous techniques may be applied to gain further understanding of the disease state of the lung. Here, a commonly used technique for the identification and enumeration of different types of immune cells is described, where&#160;flow cytometry is combined with a select panel of fluorescently labeled cell surface-specific markers. The BAL procedure presented here can also be used to analyze infectious agents, fluid constituents, or inhaled particles within murine lungs.

The health status of the lung is reflected by the type and number of immune cells that are present in the bronchioles of the lung. We describe a bronchoalveolar lavage technique that allows the isolation and study of nonadherent cells and soluble factors from the lower respiratory tract of mice.

Bronchoalveolar Lavage (BAL) is an experimental procedure that is used to examine the cellular and acellular content of the lung lumen ex vivo to gain ...

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

Lung microRNA Profiling Across the Estrous Cycle in Ozone-exposed Mice

MicroRNA (miRNA) profiling has become of interest to researchers working in various research areas of biology and medicine. Current studies show a promising future of using miRNAs in the diagnosis and care of lung diseases. Here, we define a protocol for miRNA profiling to measure the relative abundance of a group of miRNAs predicted to regulate inflammatory genes in the lung tissue from of an ozone-induced airway inflammation mouse model. Because it has been shown that circulating sex hormone levels can affect the regulation of lung innate immunity in females, the purpose of this method is to describe an inflammatory miRNA profiling protocol in female mice, taking into consideration the estrous cycle stage of each animal at the time of ozone exposure. We also address applicable bioinformatics approaches to miRNA discovery and target identification methods using limma, an R/Bioconductor software, and functional analysis software to understand the biological context and pathways associated with differential miRNA expression.

Here we describe a method to assess lung expression of miRNAs that are predicted to regulate inflammatory genes using mice exposed to ozone or filtered air at different stages of the estrous cycle.

MicroRNA (miRNA) profiling has become of interest to researchers working in various research areas of biology and medicine. Current studies show a ...

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.

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 ...

Generation of a Chronic Obstructive Pulmonary Disease Model in Mice by Repeated Ozone Exposure

Chronic obstructive pulmonary disease (COPD) is characterized by persistent airflow limitation and lung parenchymal destruction. It has a very high incidence in aging populations. The current conventional therapies for COPD focus mainly on symptom-modifying drugs; thus, the development of new therapies is urgently needed. Qualified animal models of COPD could help to characterize the underlying mechanisms and can be used for new drug screening. Current COPD models, such as lipopolysaccharide (LPS) or the porcine pancreatic elastase (PPE)-induced emphysema model, generate COPD-like lesions in the lungs and airways but do not otherwise resemble the pathogenesis of human COPD. A cigarette smoke (CS)-induced model remains one of the most popular because it not only simulates COPD-like lesions in the respiratory system, but it is also based on one of the main hazardous materials that causes COPD in humans. However, the time-consuming and labor-intensive aspects of the CS-induced model dramatically limit its application in new drug screening. In this study, we successfully generated a new COPD model by exposing mice to high levels of ozone. This model demonstrated the following: 1) decreased forced expiratory volume 25, 50, and 75/forced vital capacity (FEV25/FVC, FEV50/FVC, and FEV75/FVC), indicating the deterioration of lung function; 2) enlarged lung alveoli, with lung parenchymal destruction; 3) reduced fatigue time and distance; and 4) increased inflammation. Taken together, these data demonstrate that the ozone exposure (OE) model is a reliable animal model that is similar to humans because ozone overexposure is one of the etiological factors of COPD. Additionally, it only took 6 - 8 weeks, based on our previous work, to create an OE model, whereas it requires 3 - 12 months to induce the cigarette smoke model, indicating that the OE model might be a good choice for COPD research.

This study describes the successful generation of a new chronic obstructive pulmonary disease (COPD) animal model by repeatedly exposing mice to high concentrations of ozone.

Chronic obstructive pulmonary disease (COPD) is characterized by persistent airflow limitation and lung parenchymal destruction. It has a very high ...

Visualizing Lung Cellular Adaptations during Combined Ozone and LPS Induced Murine Acute Lung Injury

Lungs are continually faced with direct and indirect insults in the form of sterile (particles or reactive toxins) and infectious (bacterial, viral or fungal) inflammatory conditions. An overwhelming host response may result in compromised respiration and acute lung injury, which is characterized by lung neutrophil recruitment as a result of the patho-logical host immune, coagulative and tissue remodeling response. Sensitive microscopic methods to visualize and quantify murine lung cellular adaptations, in response to low-dose (0.05 ppm) ozone, a potent environmental pollutant in combination with bacterial lipopolysaccharide, a TLR4 agonist, are crucial in order to understand the host inflammatory and repair mechanisms. We describe a comprehensive fluorescent microscopic analysis of various lung and systemic body compartments, namely the broncho-alveolar lavage fluid, lung vascular perfusate, left lung cryosections, and sternal bone marrow perfusate. We show damage of alveolar macrophages, neutrophils, lung parenchymal tissue, as well as bone marrow cells in correlation with a delayed (up to 36-72 h) immune response that is marked by discrete chemokine gradients in the analyzed compartments. In addition, we present lung extracellular matrix and cellular cytoskeletal interactions (actin, tubulin), mitochondrial and reactive oxygen species, anti-coagulative plasminogen, its anti-angiogenic peptide fragment angiostatin, the mitochondrial ATP synthase complex V subunits, &#945; and &#946;. These surrogate markers, when supplemented with adequate in vitro cell-based assays and in vivo animal imaging techniques such as intravital microscopy, can provide vital information towards understanding the lung response to novel immunomodulatory agents.

Combined ozone and bacterial endotoxin exposed mice show wide-spread cell death, including that of neutrophils. We observed cellular adaptations such as disruption of cytoskeletal lamellipodia, increased cellular expression of complex V ATP synthase subunit &#946; and angiostatin in broncho-alveolar lavage, suppression of the lung immune response and delayed neutrophil recruitment.

Lungs are continually faced with direct and indirect insults in the form of sterile (particles or reactive toxins) and infectious (bacterial, viral or ...

In Vivo Assessment of Alveolar Macrophage Efferocytosis Following Ozone Exposure

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

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

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Bronchoalveolar Lavage of Murine Lungs to Analyze Inflammatory Cell Infiltration

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

Studying the Role of Alveolar Macrophages in Breast Cancer Metastasis

Lung microRNA Profiling Across the Estrous Cycle in Ozone-exposed Mice

Alveolar Macrophage Phagocytosis and Bacteria Clearance in Mice

Generation of a Chronic Obstructive Pulmonary Disease Model in Mice by Repeated Ozone Exposure

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