This study was supported in part by an Indiana University School of Medicine Research Enhancement Grant and by NIH-NIAID 1R03AI122127-01. N.A. was partly supported during this period by a Careers in Immunology Fellowship from the American Association of Immunologists.

All animal procedures were approved by the Animal Care and Use Committee of Indiana State University, the host campus of Indiana University School of Medicine &#8212; Terre Haute.
1. Preparation of A. fumigatus Conidia for Infection
<ol>
	<li>While working in a well-ventilated fume hood, add 125 &#181;L of A. fumigatus 293 stock solution to 900 &#181;L cell culture grade water (CCGW). Then transfer the solution to malt extract agar (MEA) plate via pipette and spread evenly with an inoculating loop.</li>
	<li>Store the fungal plate in the dark at 24 &#176;C for at least 14 days and no more than 28 days.</li>
	<li>Harvest conidia using glass beads as previously described9.</li>
	<li>Pour 1.5 g of 0.5 mm glass beads onto the plate and gently tilt it back and forth until the beads are coated in order to extract conidia. Collect the bead/conidia mixture in a 15-mL conical tube. Suspend in 5 mL Dulbecco&#39;s phosphate-buffered saline (DPBS) and vortex.</li>
	<li>Count the conidia in a 50x dilution of supernatant in DPBS using a hemocytometer. Count the number of conidia in the area shown in Figure 1 and use Equation 1 to calculate the concentration. 
	#Conidia &#215; Dilution Factor &#215; 104=Conidia/mL (Equation 1) 
	NOTE: Additional 4 x 4 square corner areas may be counted, with the mean of these areas used as #Conidia in Equation 1.</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57155/57155fig1.jpg" /> 
Figure 1: Hemocytometer representative area used for conidia counting (box, arrow).
<ol start="6">
	<li>Dilute the conidia suspension with sterile saline (DPBS) to obtain the desired concentration, 1.0 x 108 conidia/mL for infection with 5 x 106 conidia/50 &#181;L. Prepare 50 &#181;L of conidia solution per &#39;n + 2&#39; mice to be infected to account for pipetting error. 
	NOTE: Standard fungus harvest and preparation/filtration may be alternatively used10. The glass bead method is best used to ensure that conidia samples with minimal small/soluble hyphal antigens are obtained for aspiration.</li>
</ol>
2. Immunosuppression and Infection of Murine Model
<ol>
	<li>Use 7-10-week-old mice. 
	NOTE: BALB/c, C57BL/6 (B6), eosinophil-deficient (&#916;dblGATA, BALB/c background), and &#947;&#948; T cell-deficient (TCR&#948;-/-, B6 background) mice were used for this study. For each experiment, age and gender-matched (both males and females) mice were used for each group.</li>
	<li>Deplete neutrophils via intraperitoneal (IP) injection of 0.1 mL (0.5 mg) anti-mouse-Ly-6G antibody in sterile saline at 45&#176; in the lateral lower right quadrant (For an experimental schedule, see Figure 2).</li>
	<li>24 h later, infect mice by aspiration with 5.0 x 106A. fumigatus conidia suspended in 50 &#181;L sterile saline (see steps 2.4-2.10) as previously described11. Following infection, inject a subset of animals with antifungal agent such as 5 mg/kg caspofungin diacetate in DPBS used in this study (Daily, see Figure 2).</li>
	<li>Anesthetize mice with 99.9% isoflurane using a veterinary anesthesia machine.
	<ol>
		<li>Place a paper towel on the floor of the induction chamber to catch feces and urine.</li>
		<li>Place the mouse in the induction chamber, secure the lid, and set the vaporizer to 5% isoflurane for 2 min and 30 s. 
		NOTE: Vet ointment is not used because the total time under anesthesia is less than 5 min.</li>
		<li>Reduce the vaporizer to half of the maximum setting for 30 s.</li>
	</ol>
	</li>
	<li>Confirm that the mouse is properly anesthetized by observing slowed breathing, lack of movement, and lack of response to toe-pinch stimulus.</li>
	<li>Remove the mouse from the induction chamber and place on a slant board. Place the mouse with its back against the board. Ensure that its upper incisors are gently restrained with a rubber band and the lower incisors are gently restrained with a metal wire.</li>
	<li>Carefully hold the tongue at full extension with small forceps and pipette 50 &#181;L of conidia/PBS suspension at the base of the tongue.</li>
	<li>Hold the tongue at full extension; listen for rapid breathing and the distinct clicking noise of aspiration. Hold for 20 s or until suspension is fully aspirated.</li>
	<li>Remove the mouse from the slant board and gently place in a cage for observation.</li>
	<li>Following aspiration, the mouse should regain consciousness within 1 min. Monitor the mouse until fully conscious and able to walk around the cage. The animals should be housed at 21 &#176;C and monitored twice daily until the lungs are harvested.</li>
	<li>Repeat the depletion of neutrophils (step 2.2) 24 h post-infection (Figure 2).</li>
</ol>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57155/57155fig2.jpg" /> 
Figure 2: Experimental schedule. This figure was modified from a previous publication8.
3. Harvest and Preservation of Mouse Lungs for Histological Analysis
<ol>
	<li>Deeply anesthetize the animal with a fatal dose of 0.1 ml of 390 mg/ml pentobarbital sodium solution IP.</li>
	<li>Confirm euthanasia by observing lack of spontaneous breathing and absence of response to limb stimulation with tweezers. Once euthanized, spray the carcass with 70% ethanol to sterilize.</li>
	<li>Place the carcass on a foam board covered with an absorbent pad. 
	NOTE: The carcass should be placed on its back with pins securing all four limbs to the foam board.</li>
	<li>Open the upper chest cavity using scissors with caution. Cut the rib cage away in order to expose the lungs and heart. Cut the inferior vena cava to allow for perfusion. Avoid cutting the blood vessels and puncturing organs.</li>
	<li>Slowly perfuse with 5 mL of cold DPBS in the right ventricle of the heart at a 45&#176; angle with a 25-G needle. Repeat the perfusion with 5 mL of 10% formalin.</li>
	<li>Carefully expose the trachea and detach the surrounding fat pads. Use caution and avoid puncturing the trachea. Ensure the excess connective tissue is removed in order to fully visualize the trachea before proceeding. Once the trachea is exposed, place forceps under the trachea to elevate it.</li>
	<li>Poke a small hole in the upper trachea using a 25-G needle. 
	NOTE: The hole should be positioned so that the entire cannula fits in the trachea. Use caution to avoid rupturing the trachea.</li>
	<li>Insert a cannula through the hole and towards the lungs. Secure the cannula with a surgical thread tied loosely around the trachea. 
	NOTE: If the trachea is ruptured, the cannula may be inserted past the trachea as far as possible in the airway.</li>
	<li>Using a syringe, inflate the lungs with 1 mL of 10% formalin buffer through the cannula. Ensure that the inflation of the lungs is visible. If not, adjust the catheter and repeat. Once the lungs are inflated, quickly remove the cannula with caution and tighten the thread securely to seal off the trachea. 
	NOTE: If the trachea is ruptured and the lungs cannot be inflated, the lungs may still be harvested but this may prevent proper fixation.</li>
	<li>Gently remove the trachea and lungs from the chest by carefully disconnecting the connective tissue of the trachea with forceps while gently pulling up on the thread. Place the lungs and trachea in a 50-mL conical tube with 15 mL of 10% formalin buffer. Place one end of the thread outside of the tube and seal the cap. Invert the tube to completely submerge the sample in fixative.</li>
	<li>Store the samples at room temperature for at least 24 h or until further processing. 
	CAUTION: Formalin is hazardous. Wear appropriate Personal Protective Equipment including goggles and a face mask. Work under a chemical hood when processing samples.</li>
</ol>
4. Harvest and Preservation of Mouse Lungs for DNA Fungal Burden
<ol>
	<li>Follow steps 3.1-3.5 as described above on a separate mouse.</li>
	<li>Slowly perfuse with 10 mL of cold DPBS in the right ventricle of the heart at a 45&#176; angle with a 25-G needle.</li>
	<li>Excise the lungs with scissors and place in labeled 1.5 mL tubes.</li>
	<li>Store the lungs at -80 &#176;C until further processing.</li>
	<li>Use standard techniques as described (embedding and sectioning) and according to manufacturer&#39;s protocols (see Table of Materials).</li>
</ol>
5. Microscopy and Imaging
<ol>
	<li>Open the imaging quantification program (see Table of Materials).</li>
	<li>Using a light microscope, obtain at least 4 distinct fields of the GMS stained slides (10X objective). Ensure that the images are representative of the infected airways within the lung with similar tissue density. 
	NOTE: If the density and distribution of hyphal foci appear markedly different between control and experimental groups, additional fields may be taken, or the GMS area of the whole lung section may be imaged (4X objective). Obtain images of the same areas of the lung (identified by equivalent tissue morphology) on a serial lung section stained with hematoxylin and eosin, if desired.</li>
	<li>Add scale bars to the images where necessary and save the images to be quantified. Select, Edit &#62; Add/Edit Calibration Marks &#62; Menu: choose line thickness, color and scale bar size &#62; click on the image for where the scale bar is to be placed. Close the Menu and choose, Edit &#62; Merge &#62; Merge Calibration Marks &#62; choose File Save As &#62; name the file and click on Save.</li>
</ol>
6. Using an Image Processing Program to Calculate the Fungal Burden (Figure 3)
<ol>
	<li>Open an image processing program. Select File &#62; folder with samples to quantify &#62; image (Figure 3A). Select Image &#62; Type &#62; convert to &#34;RGB Stack&#34; (Figure 3B).</li>
	<li>Use the right arrow key to select the second of the three given images (Figure 3B). Hit &#34;control + shift + T&#34; to bring up the &#34;Threshold&#34; menu settings adjustor (Figure 3C). Locate the original .tiff or .jpg image as a reference and open with an image viewing program (Figure 3C).</li>
	<li>Pull the top slider all the way to the left and adjust the bottom slider until the area selected (in red) is representative of the microscopy image (typically ranging from 130-160 as indicated to the right of the slider (Figure 3C).</li>
	<li>Once selected, press &#34;Set&#34; &#62; &#34;OK&#34; (Figure 3D). Select &#34;Analyze&#34; &#62; &#34;Set Measurements&#34; &#62; check &#34;Area, Area Fraction, Limit to Threshold, Display Label&#34; and leave the bottom settings (the drop-down menu and decimal places) as default &#62; &#34;OK&#34; (Figure 3D).</li>
	<li>Significant areas of white space or background staining may be excluded by selection of the appropriate tissue with an area or polygon tool.</li>
	<li>Finally select, &#34;Analyze&#34; &#62; &#34;Measure&#34; (a window with data should appear, or a tab on the windows taskbar will appear labeled, &#34;Results&#34;) (Figure 3E). Transfer the data to a data analysis program for graphing and statistical analysis.</li>
	<li>Enter the mean of the four area percentages for each lung sample. 5-8 samples are generally sufficient to detect significant differences between groups. If comparing two experimental groups, perform an unpaired student&#39;s t-test to determine significance.</li>
</ol>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57155/57155fig3.jpg" /> 
Figure 3: Representative screenshots for each step of GMS histological field quantification. (A) Select File &#62; folder with samples to quantify &#62; select image. (B) Select Image &#62; Type &#62; convert to &#34;RGB Stack&#34;. Use the right arrow key to select the second of the three given images. (C) Hit &#34;control + shift + T&#34; to bring up the &#34;Threshold&#34; menu settings adjustor. Locate the original .tiff or .jpg image as a reference and open with photo viewer program. Pull the top slider all the way left and adjust the bottom slider until the area selected (in red) is representative of the microscopy image (typically ranging from 130-160 as indicated to the right of the slider. (D) Once selected press &#34;Set&#34; &#62; &#34;OK&#34;. Select &#34;Analyze&#34; &#62; &#34;Set Measurements&#34; &#62; check &#34;Area, Area Fraction, Limit to Threshold, Display Label&#34; and leave the bottom settings (the drop-down menu and decimal places) as default &#62; &#34;OK&#34;. (E) Finally select &#34;Analyze&#34; &#62; &#34;Measure&#34; (a window with data should appear, or a tab on the windows taskbar will appear labeled &#34;Results&#34;). <a href="//cloudfront.jove.com/files/ftp_upload/57155/57155fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
7. DNA Fungal Burden
<ol>
	<li>Remove the lungs from -80 &#176;C and lyophilize for at least 4 h (overnight is optimal) or until completely dried by a freeze dry system.</li>
	<li>While the lungs are drying, prepare DNA extraction buffer (0.1 M NaCl, 10 mM EDTA pH 8.0, 10 mM Tris HCl pH 8.0, and 0.5% SDS).</li>
	<li>Place the prepared DNA extraction buffer in a 60 &#176;C water bath to pre-warm. Also, place phenol: chloroform: isoamyl alcohol (25:24:1) in a 50-mL conical tube and allow for separation.</li>
	<li>Place the lyophilized lungs in a 2-mL screw cap tube with 0.2 mL of glass beads. Using a bead beater, grind dry lung tissue for 15-30 s until it forms a fine powder.</li>
	<li>Add 0.8 mL warm DNA Extraction buffer to each tube and vortex well. Incubate each tube for 15 min in a 65 &#176;C bead bath and then vortex before incubating for an additional 15 min. After incubating, vortex and then centrifuge tubes at 25,000 x g for 10 min.</li>
	<li>Label 3 sets of 1.5 mL tubes for each of the samples and transfer the top layer of the supernatant into one set of tubes. Use caution while pipetting out the aqueous top layer to avoid disturbing the middle layer.</li>
	<li>Add equal volume of phenol:chloroform:isoamyl alcohol and mix well. Then centrifuge at 25,000 x g for 15 min before pipetting out the upper layer into the second set of tubes.</li>
	<li>Add equal volume of chloroform:isoamyl chloroform, mix well, and centrifuge at 25,000 x g for 10 min. Carefully pipette out the upper layer into the third set of tubes.</li>
	<li>Add 500 &#181;L of isopropanol and 50 &#181;L NaoAc (pH 5.2), mix well, and allow samples to incubate at room temperature for 10 min. Then centrifuge at 25,000 x g for 30 min and decant the supernatant.</li>
	<li>Wash the DNA pellet with 750 &#181;L of ice cold 70% ethanol and centrifuge for 5 min at 25,000 x g. Decant the ethanol and allow the pellets to air dry in the tubes in a fume hoods for 40 min.</li>
	<li>Resuspend the dry pellets in 300 &#181;L of TE buffer. 
	NOTE: The DNA may be stored at -20 &#176;C until quantified.</li>
	<li>Quantify DNA using a spectrophotometer and prepare 100 &#181;L of 0.2 &#181;g/&#181;L of each DNA sample for qPCR analysis.</li>
	<li>Perform a standard qPCR in triplicate as previously described with 1 &#181;g of DNA sample, fungal 18S rDNA primer, and probe sets with a modified probe quencher (5&#39;-/56-FAM/AGC CAG CGG/ZEN/CCC GCA AAT G/3IABkFQ/-3&#39;)12,13.</li>
	<li>Prepare a standard curve from A. fumigatus genomic DNA and calculate the concentration of fungal DNA in pg in each sample using the average reporter dye Ct values.</li>
	<li>Plot the pg/&#181;g of fungal DNA with standard error of the mean. Perform a student&#39;s t-test analysis to determine significance. 
	NOTE: As a less toxic alternative to the phenol-chloroform DNA extraction used herein, column based DNA extraction kits can be used.</li>
</ol>

<ol>
	<li>Hohl, T. M., Feldmesser, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aspergillus+fumigatus:+principles+of+pathogenesis+and+host+defense.">Aspergillus fumigatus: principles of pathogenesis and host defense.</a> Eukaryot Cell. 6 (11), 1953-1963 (2007).</li><li>Kwon-Chung, K. J., Sugui, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aspergillus+fumigatus--what+makes+the+species+a+ubiquitous+human+fungal+pathogen?.">Aspergillus fumigatus--what makes the species a ubiquitous human fungal pathogen?.</a> PLoS Pathog. 9 (12), e1003743 (2013).</li><li>Ostrosky-Zeichner, L., Casadevall, A., Galgiani, J. N., Odds, F. C., Rex, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+insight+into+the+antifungal+pipeline:+selected+new+molecules+and+beyond.">An insight into the antifungal pipeline: selected new molecules and beyond.</a> Nat Rev Drug Discov. 9 (9), 719-727 (2010).</li><li>Desoubeaux, G., Cray, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rodent+Models+of+Invasive+Aspergillosis+due+to+Aspergillus+fumigatus:+Still+a+Long+Path+toward+Standardization.">Rodent Models of Invasive Aspergillosis due to Aspergillus fumigatus: Still a Long Path toward Standardization.</a> Front Microbiol. 8, 841 (2017).</li><li>Hohl, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overview+of+vertebrate+animal+models+of+fungal+infection.">Overview of vertebrate animal models of fungal infection.</a> J Immunol Methods. 410, 100-112 (2014).</li><li>Sheppard, D. C., 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=Comparison+of+three+methodologies+for+the+determination+of+pulmonary+fungal+burden+in+experimental+murine+aspergillosis.">Comparison of three methodologies for the determination of pulmonary fungal burden in experimental murine aspergillosis.</a> Clin Microbiol Infect. 12 (4), 376-380 (2006).</li><li>Patterson, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+future+of+animal+models+of+invasive+aspergillosis.">The future of animal models of invasive aspergillosis.</a> Med Mycol. 43 Suppl 1, S115-S119 (2005).</li><li>Amarsaikhan, 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=Caspofungin+Increases+Fungal+Chitin+and+Eosinophil+and+gammadelta+T+Cell-Dependent+Pathology+in+Invasive+Aspergillosis.">Caspofungin Increases Fungal Chitin and Eosinophil and gammadelta T Cell-Dependent Pathology in Invasive Aspergillosis.</a> J Immunol. 199 (2), 624-632 (2017).</li><li>Templeton, S. P., Buskirk, A. D., Law, B., Green, B. J., Beezhold, D. H. <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+germination+in+murine+airway+CD8++T-cell+responses+to+Aspergillus+conidia.">Role of germination in murine airway CD8+ T-cell responses to Aspergillus conidia.</a> PLoS One. 6 (4), e18777 (2011).</li><li>Brunel, S. F., 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=Live+Imaging+of+Antifungal+Activity+by+Human+Primary+Neutrophils+and+Monocytes+in+Response+to+A.+fumigatus.">Live Imaging of Antifungal Activity by Human Primary Neutrophils and Monocytes in Response to A. fumigatus.</a> J Vis Exp. (122), (2017).</li><li>Rao, G. V., 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+a+technique+for+exposing+the+mouse+lung+to+particles+aspirated+from+the+pharynx.">Efficacy of a technique for exposing the mouse lung to particles aspirated from the pharynx.</a> J Toxicol Environ Health A. 66 (15), 1441-1452 (2003).</li><li>Bowman, J. C., 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=Quantitative+PCR+assay+to+measure+Aspergillus+fumigatus+burden+in+a+murine+model+of+disseminated+aspergillosis:+demonstration+of+efficacy+of+caspofungin+acetate.">Quantitative PCR assay to measure Aspergillus fumigatus burden in a murine model of disseminated aspergillosis: demonstration of efficacy of caspofungin acetate.</a> Antimicrob Agents Chemother. 45 (12), 3474-3481 (2001).</li><li>Li, H., 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=The+small+GTPase+RacA+mediates+intracellular+reactive+oxygen+species+production,+polarized+growth,+and+virulence+in+the+human+fungal+pathogen+Aspergillus+fumigatus.">The small GTPase RacA mediates intracellular reactive oxygen species production, polarized growth, and virulence in the human fungal pathogen Aspergillus fumigatus.</a> Eukaryot Cell. 10 (2), 174-186 (2011).</li><li>Sucher, A. J., Chahine, E. B., Balcer, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Echinocandins:+the+newest+class+of+antifungals.">Echinocandins: the newest class of antifungals.</a> Ann Pharmacother. 43 (10), 1647-1657 (2009).</li><li>Herrera, M. L., Vallor, A. C., Gelfond, J. A., Patterson, T. F., Wickes, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strain-dependent+variation+in+18S+ribosomal+DNA+Copy+numbers+in+Aspergillus+fumigatus.">Strain-dependent variation in 18S ribosomal DNA Copy numbers in Aspergillus fumigatus.</a> J Clin Microbiol. 47 (5), 1325-1332 (2009).</li><li>Kirkpatrick, W. R., Perea, S., Coco, B. J., Patterson, T. F. <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+caspofungin+alone+and+in+combination+with+voriconazole+in+a+Guinea+pig+model+of+invasive+aspergillosis.">Efficacy of caspofungin alone and in combination with voriconazole in a Guinea pig model of invasive aspergillosis.</a> Antimicrob Agents Chemother. 46 (8), 2564-2568 (2002).</li><li>Petraitiene, R., 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=Antifungal+efficacy+of+caspofungin+(MK-0991)+in+experimental+pulmonary+aspergillosis+in+persistently+neutropenic+rabbits:+pharmacokinetics,+drug+disposition,+and+relationship+to+galactomannan+antigenemia.">Antifungal efficacy of caspofungin (MK-0991) in experimental pulmonary aspergillosis in persistently neutropenic rabbits: pharmacokinetics, drug disposition, and relationship to galactomannan antigenemia.</a> Antimicrob Agents Chemother. 46 (1), 12-23 (2002).</li><li>Cowley, A. C., Thornton, D. J., Denning, D. W., Horsley, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aspergillosis+and+the+role+of+mucins+in+cystic+fibrosis.">Aspergillosis and the role of mucins in cystic fibrosis.</a> Pediatr Pulmonol. 52 (4), 548-555 (2017).</li><li>Taylor, M. 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=Detection+of+fungal+organisms+in+eosinophilic+mucin+using+a+fluorescein-labeled+chitin-specific+binding+protein.">Detection of fungal organisms in eosinophilic mucin using a fluorescein-labeled chitin-specific binding protein.</a> Otolaryngol Head Neck Surg. 127 (5), 377-383 (2002).</li></ol>

The authors have nothing to disclose.

The purpose of this article was to introduce a method for determination of lung fungal burden in mice with IA by utilizing GMS-stained lung histological sections for image analysis and quantification. In this study, treatment with the &#946;-glucan-synthesis-targeting antifungal drug caspofungin14 did not improve survival or fungal burden in neutropenic wild-type mice with IA8. However, in the absence of eosinophils or &#947;&#948; T cells, survival and fungal burden improved. The results of our study also demonstrated that comparable results may be obtained by GMS fungal burden in comparison to the widely-used fungal DNA qPCR method6.
There are several benefits to utilizing GMS fungal burden quantification. First, the process may utilize existing histological samples, thus potentially reducing the number of experiments required to determine significant differences. Second, in this study, less animals were required for GMS fungal burden quantification to achieve significant differences in comparison to qPCR of fungal DNA (Figure 6, Figure 7). Third, comparison of fungal burden in different isolates by qPCR may be affected by isolate-dependent differences in ribosomal DNA copy number15. In contrast, GMS quantification is not affected by copy number, as fungal burden is determined by relative levels of lung fungal growth. Thus, the use of GMS quantification for fungal burden reduces the use of vertebrate animals and does not require pre-determination of rDNA copy number. Finally, in addition to modifying fungal morphology, caspofungin therapy increases fungal fragmentation, and thus may artificially increase fungal burden when measured by isolation of colony forming units from lung homogenates12,16,17. Thus, GMS quantification of fungal burden avoids several limitations inherent with other commonly used methods.
However, the limitations of GMS quantification and/or this study are important to note. First, the authors assumed a comparable distribution of hyphal growth throughout the lungs of each experimental group, and thus used quantification from 4 representative 10x objective fields as a representative measurement for the fungal burden in the entire lung (Figure 6B). It is possible that in some instances the relative distribution, size, and density of hyphal foci would sufficiently differ so that the fungal burden would appear different with this method and equivalent by the qPCR method. However, our additional results with whole lung section quantification using the 4X objective fields showed similar, albeit less statistically significant, differences between groups (Figure 6C). The standard error of this quantification was increased with this strategy, likely due to decreased hyphal resolution with the 4X objective and increased background in suboptimal fields. Therefore, a representative quantification of fewer fields at higher magnification is preferred. Second, only a single, central section was used for each sample. It is possible, based on the fungal isolates or mice strains used, that some studies may result in an uneven distribution of hyphal growth. In those instances, additional sections throughout each paraffin block should be quantified to obtain a more representative burden. Third, in experiments that induce substantial production of mucins (i.e., quantifying airway fungal growth in allergic bronchopulmonary aspergillosis (ABPA)18 or cystic fibrosis (CF)18), GMS reactivity with polysaccharide-rich mucins19 could yield non-specific GMS+ results and thus skew some samples in favor of higher fungal burden. Since only the neutropenic model of IA was used in this study, it is possible that the use of other immune competent or suppressive models could results in less comparable results. Despite these caveats, GMS quantification provides a comparable technique to determine fungal burden, and its continued use in additional studies may further validate the utility of this method as consistent, reliable, and cost-effective.

IA is an opportunistic infection that may develop in susceptible individuals with congenital or acquired immune deficiencies due to immune suppressive therapy or chronic infection1,2. Primary infection often occurs in the lungs, although in some instances dissemination of Aspergillus fumigatus to the liver, kidneys, heart, and brain may occur, resulting in extensive tissue invasion of hyphae accompanied by severe disease and high rates of mortality1,2. Furthermore, the efficacy of existing pharmacotherapies is limited, and may be further weakened by the emergence of antifungal-resistant strains in the environment3. It is therefore important to understand the mechanisms of fungal virulence and host pathology that promote development or exacerbation of invasive fungal disease.
Murine models remain important for mechanistic IA studies, as they allow researchers to assess the roles of fungal virulence genes and host immune effectors for the establishment and growth of A. fumigatus in vivo4,5. Consequently, multiple strategies have been devised in order to effectively quantify or compare fungal burden in groups of experimental animals6,7. These strategies involve culture-based, biochemical, immunoassay, or qPCR methods, each with distinct advantages and disadvantages. Furthermore, each of these methods involve the dedication of a subset of animals in addition to those sacrificed for assessments of immune effector function, gene expression analysis, and comparative histopathology7. Thus, comprehensive IA studies often require significant numbers of research animals at a significant cost. Effective strategies that reduce experimental time, animal costs, and ethical considerations by utilizing animal tissues for multiple analyses are therefore extremely valuable7.
In this report, a method describing the quantification of GMS staining in histological sections for comparison of the relative fungal burden between experimental groups of mice with IA is introduced. Each step from fungal culture to infection, tissue harvest and processing, and image acquisition and data analysis, is described in detail. Fungal burdens obtained by GMS quantification were compared with qPCR in neutropenic models of IA, and in caspofungin-treated wild-type or eosinophil-deficient mice with IA8. The results show similarity with GMS quantification and qPCR of fungal DNA. This suggests that GMS quantification may be useful to researchers engaged in histological analyses as a supplemental or alternative method of comparison of relative fungal burden in mice with IA, and may ultimately reduce the cost and use of research animals in complex, mechanistic studies.

<table><tbody><tr><td>&lt;em&gt;Aspergillus fumigatus&lt;/em&gt; 293 Stock Solution</td><td>Fungal Genetics Stock Center</td><td>FGSC #A1100</td><td></td></tr><tr><td>HyPure Cell Culture Grade Water</td><td>Thermo Fisher Scientific</td><td>SH30529.03</td><td></td></tr><tr><td>Malt Extract</td><td>MP Biomedicals</td><td>2155315</td><td>White Powder</td></tr><tr><td>BD&amp;nbsp;BBL Acidicase Dehydrated Culture Media: Peptone</td><td>Fisher Scientific</td><td>L11843</td><td></td></tr><tr><td>Dextrose (D-Glucose) Anhydrous (Granular Powder/Certified ACS)</td><td>Fisher Scientific</td><td>D16-3</td><td></td></tr><tr><td>Fisher BioReagents Agar, Powder / Flakes, Fisher BioReagents</td><td>Fisher Scientific</td><td>BP1423-500</td><td></td></tr><tr><td>Auto Dry Cabinet</td><td>Shanghai Hasuc Instrument Manufacture Co.,LTD</td><td>HSFC160FD</td><td></td></tr><tr><td>1300 Series Class II, Type A2 Biological Safety Cabinet</td><td>Thermo Fisher Scientific</td><td>1375</td><td></td></tr><tr><td>0.5 mm Glass Beads</td><td>BioSpec Products</td><td>11079105</td><td></td></tr><tr><td>15 ml Conical Tubes</td><td>Thermo Fisher Scientific</td><td>339650</td><td></td></tr><tr><td>Hemacytometer</td><td>Fisher Scientific</td><td># 0267110</td><td></td></tr><tr><td>Leica Model DME Microscope</td><td>Leica</td><td>13595XXX</td><td></td></tr><tr><td>Dulbecco&#039;s Phosphate Buffered Saline</td><td>Sigma-Aldrich</td><td>D8662-500 ml</td><td></td></tr><tr><td>1.5 ml tubes</td><td>Fisher Scientific</td><td># 05-408-129</td><td></td></tr><tr><td>BD Precisionglide&amp;nbsp;syringe needles, gauge 27, L 1/2 in.</td><td>Sigma-Aldrich</td><td>Z192384</td><td></td></tr><tr><td>Anti-mouse-Ly-6G antibody</td><td>BioXCell</td><td>BP0075-1</td><td>Clone 1A8</td></tr><tr><td>Caspofungin diacetate</td><td>Sigma-Aldrich</td><td>SML0425</td><td></td></tr><tr><td>Isoflurane</td><td>Henry Schein Animal Health</td><td>1169567762</td><td></td></tr><tr><td>Non-Rebreathing Table Top Veterinary Anesthesia Machine</td><td>Supera Anesthesia Innovations</td><td>M3000</td><td></td></tr><tr><td>Pureline Oxygen Concentrator</td><td>Supera Anesthesia Innovations</td><td>OC8000</td><td></td></tr><tr><td>Slant Board Restraint</td><td>Indiana State University Facilities Management</td><td>Custom made</td><td></td></tr><tr><td>Gilson&amp;nbsp;PIPETMAN Classic 200 ml Pipets</td><td>Fisher Scientific</td><td>F123601G</td><td></td></tr><tr><td>Pentobarbital Sodium (Fatal-Plus)</td><td>Vortech Pharmaceuticals</td><td>0298-9373-68</td><td></td></tr><tr><td>General-Purpose Broad-Tipped Forceps</td><td>Fisher Scientific</td><td>10-300</td><td></td></tr><tr><td>Fisherbrand&amp;nbsp;Standard Dissecting Scissors</td><td>Fisher Scientific</td><td>08-951-20</td><td></td></tr><tr><td>Fisherbrand General-Purpose Curved Forceps</td><td>Fisher Scientific</td><td>10-275</td><td></td></tr><tr><td>Ethyl Alcohol-200 Proof</td><td>PHARMCO-AAPER</td><td>111000200</td><td></td></tr><tr><td>Fisherbrand Absorbent Underpads</td><td>Fisher Scientific</td><td>14-206-63</td><td></td></tr><tr><td>BD Precisionglide&amp;nbsp;syringe needles, gauge 25, L 1 in.</td><td>Fisher Scientific</td><td>Z192406</td><td></td></tr><tr><td>All-Plastic Norm-Ject Syringes</td><td>Fisher Scientific</td><td>14-817-30</td><td></td></tr><tr><td>IV CATH ANGIOCATH 22GX1GIN 50B</td><td>Fisher Scientific</td><td>NC9742754</td><td></td></tr><tr><td>Formalin Solution, neutral buffered, 10%</td><td>Sigma-Aldrich</td><td>HT501128-4L</td><td></td></tr><tr><td>50 ml Conical Tubes</td><td>Thermo Fisher Scientific</td><td>339652</td><td></td></tr><tr><td>Thermo Scientific Shandon Embedding Cassettes II in Tube Packs</td><td>Fisher Scientific</td><td>B1000729</td><td></td></tr><tr><td>Fisherfinest&amp;nbsp;Histoplast Paraffin Wax</td><td>Fisher Scientific</td><td>22-900-700</td><td></td></tr><tr><td>Disposable Base Molds</td><td>Fisher Scientific</td><td>22-363-554</td><td></td></tr><tr><td>Reichert Jung Histocut 820 Microtome</td><td>Labequip.com</td><td>31930</td><td></td></tr><tr><td>Water Bath</td><td>Precision Scientific</td><td>66630-23</td><td></td></tr><tr><td>CO2 Incubator</td><td>Fisher Scientific</td><td>116875H</td><td></td></tr><tr><td>Silver Stain (Modified GMS) Kit</td><td>Sigma-Aldrich</td><td>HT100A-1KT</td><td></td></tr><tr><td>Fast Green FCF</td><td>Fisher Scientific</td><td>AC410530250</td><td></td></tr><tr><td>Frosted Microscope Slides</td><td>Fisher Scientific</td><td>12-550-343</td><td></td></tr><tr><td>Fisherfinest Superslip Cover Glass</td><td>Fisher Scientific</td><td>12-545-89</td><td></td></tr><tr><td>Cytoseal XYL</td><td>Fisher Scientific</td><td>22-050-262</td><td></td></tr><tr><td>Olympus Provis AX70 Microscope</td><td>Olympus</td><td>OLYMPUS-AX70</td><td></td></tr><tr><td>U-PHOTO Universal Photo System</td><td>Olympus</td><td>OLYMPUS-U-PHOTO</td><td></td></tr><tr><td>U-MCB-2 MULTI CONTROL BOX</td><td>Olympus</td><td>OLYMPUS-U-MCB-2</td><td></td></tr><tr><td>U-PS POWER SUPPLY UNIT</td><td>Olympus</td><td>OLYMPUS-U-PS</td><td></td></tr><tr><td>FreeZone 1 Liter Benchtop Freeze Dry System</td><td>LABCONCO</td><td>7740020</td><td></td></tr><tr><td>Maxima C&amp;nbsp;Plus&amp;nbsp;Vacuum Pump</td><td>Fisher Scientific</td><td>01-257-80</td><td>Displacement- 6.1 cfm</td></tr><tr><td>1-Butanol</td><td>Fisher Scientific</td><td>A383-4</td><td></td></tr><tr><td>AMRESCO&amp;nbsp;PHENOL-CHLORFORM-OSOAMYL 100ML&amp;nbsp;DFS</td><td>Fisher Scientific</td><td>NC9573988</td><td></td></tr><tr><td>Free-Standing Microcentrifuge Tubes with Screw Caps</td><td>Fisher Scientific</td><td># 02-682-557</td><td></td></tr><tr><td>Mini-Beadbeater-24</td><td>BioSpec Products</td><td>112011</td><td></td></tr><tr><td>BioSpec ProductsSupplier Diversity Partner 2.3 MM ZIRCONIA BEADS</td><td>Fisher Scientific</td><td>NC0451999</td><td></td></tr><tr><td>Tris Base (White Crystals or Crystalline Powder/Molecular Biology)</td><td>Fisher Scientific</td><td>BP152-1</td><td></td></tr><tr><td>Hydrochloric Acid, Certified ACS Plus</td><td>Fisher Scientific</td><td>A144S</td><td></td></tr><tr><td>Ethylenediaminetetraacetic Acid, Disodium Salt Dihydrate</td><td>Fisher Scientific</td><td>S311</td><td></td></tr><tr><td>Sodium Dodecyl Sulfate (SDS), White Powder, Electrophoresis</td><td>Fisher Scientific</td><td>BP166</td><td></td></tr><tr><td>Chloroform/isoamyl alcohol 24:1</td><td>Fisher Scientific</td><td>AC327155000</td><td></td></tr><tr><td>Biotek Epoch Microplate Spectrophotometer</td><td>Fisher Scientific</td><td>11-120-570</td><td></td></tr><tr><td>Thermo Scientific&amp;trade;&amp;nbsp;ABsolute Blue qPCR Mixes</td><td>Fisher Scientific</td><td>AB4137A</td><td></td></tr><tr><td>Hybridization Probe, 5&amp;prime;-FAM-AGCCAGCGGCCCGCAAATG-TAMRA-3&amp;prime;</td><td>Integrated DNA Technologies</td><td>N/A</td><td></td></tr><tr><td>Sense Amplification Primer, 5&amp;prime;-GGCCCTTAAATAGCCCGGT-3&amp;prime;</td><td>Integrated DNA Technologies</td><td>N/A</td><td></td></tr><tr><td>Antisense Amplification Primer, 5&amp;prime;-TGAGCCGATAGTCCCCCTAA-3&amp;prime;</td><td>Integrated DNA Technologies</td><td>N/A</td><td></td></tr><tr><td>Applied Biosystems&amp;trade; MicroAmp&amp;trade; Optical 8-Tube Strip, 0.2mL</td><td>Fisher Scientific</td><td>43-165-67</td><td></td></tr><tr><td>Thermo Scientific&amp;nbsp;Domed and Flat PCR Cap Strips</td><td>Fisher Scientific</td><td>AB-0386</td><td></td></tr><tr><td>Mx3005P QPCR System, 110 Volt</td><td>Agilent</td><td>401443</td><td>Stratagene is now owned by Agilent</td></tr><tr><td>BALB/c mice</td><td>The Jackson Laboratory</td><td>000651</td><td></td></tr><tr><td>C57BL/6 (B6) mice</td><td>The Jackson Laboratory</td><td>000664</td><td></td></tr><tr><td>Eosinophil-deficient (&amp;Delta;dblGATA, BALB/c background) mice</td><td>The Jackson Laboratory</td><td>005653</td><td></td></tr><tr><td>&amp;gamma;&amp;delta; T cell-deficient (TCR&amp;delta;-/-, B6 background) mice</td><td>The Jackson Laboratory</td><td>002120</td><td></td></tr><tr><td>ImageJ Software</td><td>National Institutes of Health</td><td>N/A</td><td>https://imagej.nih.gov/ij/</td></tr><tr><td>Spot Advanced Software</td><td>Spot Imaging</td><td>SPOT53A</td><td>http://www.spotimaging.com/software/spot-advanced/</td></tr><tr><td>GraphPad Prism 6</td><td>GraphPad Software</td><td>N/A</td><td>https://www.graphpad.com/scientific-software/prism/</td></tr><tr><td>Fisherbrand Absorbent Underpads</td><td>Fisher Scientific</td><td>14-206-62</td><td></td></tr><tr><td>Step 4.5</td><td></td><td></td><td>http://www.bdbiosciences.com/sg/resources/protocols/paraffin_sections.jsp ; http://jove.unicartagenaproxy.elogim.com/science-education/5039 ; http://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/General_Information/1/ht100.pdf</td></tr></tbody></table>

histological quantification to determine lung fungal burden in experimental aspergillosis

The quantification of lung fungal burden is critical for the determination of the relative levels of immune protection and fungal virulence in mouse models of pulmonary fungal infection. Although multiple methods are used to assess fungal burden, quantitative polymerase chain reaction (qPCR) of fungal DNA has emerged as a technique with several advantages over previous culture-based methods. Currently, a comprehensive assessment of lung pathology, leukocyte recruitment, fungal burden, and gene expression in mice with invasive aspergillosis (IA) necessitates the use of a significant number of experimental and control animals. Here the quantification of lung histological staining to determine fungal burden using a reduced number of animals was examined in detail. Lung sections were stained to identify fungal structures with Gomori&#39;s modified methanamine silver (GMS) staining. Images were taken from the GMS-stained sections from 4 discrete fields of each formalin-fixed paraffin-embedded lung. The GMS stained areas within each image were quantified using an image analysis program, and from this quantification, the mean percentage of stained area was determined for each sample. Using this strategy, eosinophil-deficient mice exhibited decreased fungal burden and disease with caspofungin therapy, while wild-type mice with IA did not improve with caspofungin. Similarly, fungal burden in mice lacking &#947;&#948; T cells were also improved by caspofungin, as measured by qPCR and GMS quantification. GMS quantification is therefore introduced as a method for the determination of relative lung fungal burden that may ultimately reduce the quantity of experimental animals required for comprehensive studies of invasive aspergillosis.

Figure 4 includes a graph of survival of wild type or eosinophil-deficient mice with IA treated with caspofungin. The results show that eosinophil-deficient mice exhibited increased survival when compared to wild-type mice (50% mortality versus 100% mortality, respectively). Figure 5 shows representative GMS staining from neutropenic wild-type and eosinophil-deficient mice with IA treated or untreated with caspofungin. Caspofungin treatment resulted in relative fungal clearance in eosinophil-deficient, but not wild-type mice (right panels), while both groups that did not receive caspofungin were similar (left panels). Figure 6 shows the comparison of fungal burden in caspofungin-treated and control untreated wild-type or eosinophil-deficient mice, using both qPCR of fungal DNA (Figure 6A) and representative GMS quantification of 4 (10X objective) fields (Figure 6B). The results generated from both techniques show that caspofungin treatment results in the most significant fungal burden decrease between wild-type and eosinophil-deficient mice (Figure 6A). However, in untreated mice, only GMS quantification resulted in a significant decrease in mice lacking eosinophils (Figure 6B). When the mean area of whole-lung GMS-stained sections were calculated (4X objective), the differences were similar to the results obtained with 4 representative 10X fields, though with less statistical significance (Figure 6C). Figure 7 shows survival, images of representative GMS staining (4 10X fields), and quantification of fungal burden by both methods in &#947;&#948; T cell-deficient (TCR&#948;KO) mice that were treated with caspofungin in comparison to control, untreated mice. Caspofungin treatment improved survival (Figure 7A) and fungal burden (Figure 7B-D) in TCR&#948;KO mice. Similar to the results of Figure 6, the results of fungal burden as measured by qPCR (Figure 7B) and representative GMS quantification (Figure 7C) were comparable.
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57155/57155fig4.jpg" /> 
Figure 4: Increased survival in eosinophil-deficient (&#916;dblGATA) mice with IA after treatment with caspofungin. 15-30 mice/group. Summary of 3-6 experiments. **** p &#60; 0.0001. This figure was modified from a previous publication8. <a href="https://cloudflare.jove.com/files/ftp_upload/57155/57155fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/57155/57155fig5.jpg" /> 
Figure 5: Representative images of GMS-stained lung sections from wild-type, eosinophil deficient, and caspofungin-treated or control untreated mice with IA. Representative of 3-4 mice/group. Scale bar is equivalent to 100 &#181;m. This figure was modified from a previous publication8. <a href="https://cloudflare.jove.com/files/ftp_upload/57155/57155fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/57155/57155fig6.jpg" /> 
Figure 6: Fungal burden in wild-type, eosinophil-deficient mice with IA treated or control treated with caspofungin. (A) qPCR of fungal DNA. 15-30 mice/group, summary of 3-6 experiments. (B) GMS quantification of histological sections, mean% of GMS staining from 4 fields (10x objective). (C) GMS quantification, mean% of whole lung section (4X objective). B and C are a summary of 5 mice/group. * p &#60; 0.05. *** p &#60; 0.001. **** p &#60; 0.0001. This figure was generated using data that were reported in a previous publication8. <a href="https://cloudflare.jove.com/files/ftp_upload/57155/57155fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/57155/57155fig7.jpg" /> 
Figure 7: Decreased severity of IA in &#947;&#948; T cell-deficient mice after caspofungin treatment. (A) Survival. (B) qPCR of fungal DNA. (C) GMS quantification of histology. (D) Representative images of GMS-stained sections from &#947;&#948; T cells with IA, treated or untreated with caspofungin. A and B are a summary of two experiments with 7-10 mice/group. C and D are a summary of 4 mice/group. * p &#60; 0.05. ** p &#60; 0.01. *** p &#60; 0.001. This figure was modified from a previous publication8. <a href="https://cloudflare.jove.com/files/ftp_upload/57155/57155fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Histological Quantification to Determine Lung Fungal Burden in Experimental Aspergillosis at JoVE.com

Histological Quantification to Determine Lung Fungal Burden in Experimental Aspergillosis

Here we describe a protocol to determine pulmonary fungal burden in mice with invasive aspergillosis by quantification of Gomori&#39;s modified methanamine silver staining in histological sections. Use of this method resulted in comparable results with less animals compared to assessment of fungal burden by quantitative PCR of lung fungal DNA.

The quantification of lung fungal burden is critical for the determination of the relative levels of immune protection and fungal virulence in mouse ...

histological-quantification-to-determine-lung-fungal-burden

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

10.4K Views.  Indiana University School of Medicine - Terre Haute. Here we describe a protocol to determine pulmonary fungal burden in mice with invasive aspergillosis by quantification of Gomori's modified methanamine silver staining in histological sections. Use of this method resulted in comparable results with less animals compared to assessment of fungal burden by quantitative PCR of lung fungal DNA.

Video: Histological Quantification to Determine Lung Fungal Burden in Experimental Aspergillosis

Direct Observation of Phagocytosis and NET-formation by Neutrophils in Infected Lungs using 2-photon Microscopy

After the gastrointestinal tract, the lung is the second largest surface for interaction between the vertebrate body and the 
environment. Here, an effective gas exchange must be maintained, while at the same time avoiding infection by the multiple pathogens that are inhaled 
during normal breathing. To achieve this, a superb set of defense strategies combining humoral and cellular immune mechanisms exists. One of the most 
effective measures for acute defense of the lung is the recruitment of neutrophils, which either phagocytose the inhaled pathogens or kill them by 
releasing cytotoxic chemicals. A recent addition to the arsenal of neutrophils is their explosive release of extracellular DNA-NETs by which bacteria 
or fungi can be caught or inactivated even after the NET releasing cells have died. We present here a method that allows one to directly observe neutrophils, 
migrating within a recently infected lung, phagocytosing fungal pathogens as well as visualize the extensive NETs that they have produced throughout the 
infected tissue. The method describes the preparation of thick viable lung slices 7 hours after intratracheal infection of mice with conidia of the mold 
Aspergillus fumigatus and their examination by multicolor time-lapse 2-photon microscopy. This approach allows one to directly investigate antifungal 
defense in native lung tissue and thus opens a new avenue for the detailed investigation of pulmonary immunity.

We show, how to use 2-photon microscopy for the observation of the dynamics of neutrophil granulocytes in infected lungs while they phagocytose pathogens or produce neutrophil extracellular traps (NETs).

After the gastrointestinal tract, the lung is the second largest surface for interaction between the vertebrate body and the 
environment. Here, an ...

Quantification of Fungal Colonization, Sporogenesis, and Production of Mycotoxins Using Kernel Bioassays

The rotting of grains by seed-infecting fungi poses one of the greatest economic challenges to cereal production worldwide, not to mention serious risks to human and animal health. Among cereal production, maize is arguably the most affected crop, due to pathogen-induced losses in grain integrity and mycotoxin seed contamination. The two most prevalent and problematic mycotoxins for maize growers and food and feed processors are aflatoxin and fumonisin, produced by Aspergillus flavus and Fusarium verticillioides, respectively.
Recent studies in molecular plant-pathogen interactions have demonstrated promise in understanding specific mechanisms associated with plant responses to fungal infection and mycotoxin contamination1,2,3,4,5,6. Because many labs are using kernel assays to study plant-pathogen interactions, there is a need for a standardized method for quantifying different biological parameters, so results from different laboratories can be cross-interpreted. For a robust and reproducible means for quantitative analyses on seeds, we have developed in-lab kernel assays and subsequent methods to quantify fungal growth, biomass, and mycotoxin contamination. Four sterilized maize kernels are inoculated in glass vials with a fungal suspension (106) and incubated for a predetermined period. Sample vials are then selected for enumeration of conidia by hemocytometer, ergosterol-based biomass analysis by high performance liquid chromatography (HPLC), aflatoxin quantification using an AflaTest fluorometer method, and fumonisin quantification by HPLC.

The devastation of cereal crops by seed-infecting fungi has prompted numerous research efforts to better understand plant-pathogen interactions. To study seed-fungal interactions in a laboratory setting, we developed a robust method for the quantification of fungal reproduction, biomass, and mycotoxin contamination using kernel bioassays.

The rotting of grains by seed-infecting fungi poses one of the greatest economic challenges to cereal production worldwide, not to mention serious risks ...

Use of Image Cytometry for Quantification of Pathogenic Fungi in Association with Host Cells

Studies of the cellular pathogenesis mechanisms of pathogenic yeasts such as Candida albicans, Histoplasma capsulatum, and Cryptococcus neoformans commonly employ infection of mammalian hosts or host cells (i.e. macrophages) followed by yeast quantification using colony forming unit analysis or flow cytometry. While colony forming unit enumeration has been the most commonly used method in the field, this technique has disadvantages and limitations, including slow growth of some fungal species on solid media and low and/or variable plating efficiencies, which is of particular concern when comparing growth of wild-type and mutant strains. Flow cytometry can provide rapid quantitative information regarding yeast viability, however, adoption of flow cytometric detection for pathogenic yeasts has been limited for a number of practical reasons including its high cost and biosafety considerations. Here, we demonstrate an image-based cytometric methodology using the Cellometer Vision (Nexcelom Bioscience, LLC) for the quantification of viable pathogenic yeasts in co-culture with macrophages. Our studies focus on detection of two human fungal pathogens: Histoplasma capsulatum and Candida albicans. H. capsulatum colonizes alveolar macrophages by replicating within the macrophage phagosome, and here, we quantitatively assess the growth of H. capsulatum yeasts in RAW 264.7 macrophages using acridine orange/propidium iodide staining in combination with image cytometry. Our method faithfully recapitulates growth trends as measured by traditional colony forming unit enumeration, but with significantly increased sensitivity. Additionally, we directly assess infection of live macrophages with a GFP-expressing strain of C. albicans. Our methodology offers a rapid, accurate, and economical means for detection and quantification of important human fungal pathogens in association with host cells.

Here, we demonstrate how image cytometry can be used for quantification of pathogenic fungi in association with host cells in culture. This technique can be used as an alternative to CFU enumeration.

Studies of the cellular pathogenesis mechanisms of pathogenic yeasts such as Candida albicans, Histoplasma capsulatum, and Cryptococcus neoformans ...

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 ...

Measuring Phagocytosis of Aspergillus fumigatus Conidia by Human Leukocytes using Flow Cytometry

Invasive pulmonary infection by the mold Aspergillus fumigatus poses a great threat to immunocompromised patients. Inhaled fungal conidia (spores) are cleared from the human lung alveoli by being phagocytosed by innate monocytes and/or neutrophils. This protocol offers a fast and reliable measurement of phagocytosis by flow cytometry using fluorescein isothiocyanate (FITC)-labeled conidia for co-incubation with human leukocytes and subsequent counterstaining with an anti-FITC antibody to allow discrimination of internalized and cell-adherent conidia. Major advantages of this protocol are its rapidness, the possibility to combine the assay with cytometric analysis of other cell markers of interest, the simultaneous analysis of monocytes and neutrophils from a single sample and its applicability to other cell wall-bearing fungi or bacteria. Determination of percentages of phagocytosing leukocytes provides a means to microbiologists for evaluating virulence of a pathogen or for comparing pathogen wildtypes and mutants as well as to immunologists for investigating human leukocyte capabilities to combat pathogens.

Measuring Phagocytosis of Aspergillus fumigatus Conidia by Human Leukocytes using Flow Cytometry

This protocol provides a fast and reliable method to quantitatively measure phagocytosis of Aspergillus fumigatus conidia by human primary phagocytes using flow cytometry and to discriminate phagocytosis of conidia from mere adhesion to leukocytes.

Invasive pulmonary infection by the mold Aspergillus fumigatus poses a great threat to immunocompromised patients. Inhaled fungal conidia (spores) are ...

Infection of Zebrafish Larvae with Aspergillus Spores for Analysis of Host-Pathogen Interactions

Invasive aspergillosis (IA) is one of&#160;the most common fungal infections among immunocompromised individuals. Despite the availability of antifungal drugs, IA can cause &#62;50% mortality in infected immunocompromised patients. It is crucial to determine both host and pathogen factors that contribute to infection susceptibility and low survival rates in infected patients in order to develop novel therapeutics. Innate immune responses play a pivotal role in recognition and clearance of Aspergillus spores, though little is known about the exact cellular and molecular mechanisms. Reliable models are required to investigate detailed mechanistic interactions between the host and pathogen. The optical clarity and genetic tractability of zebrafish larvae make them an intriguing model&#160;to study host-pathogen interactions of multiple human bacterial and fungal infections in a live and intact host. This protocol describes a larval zebrafish Aspergillus infection model. First, Aspergillus spores are isolated and injected into the zebrafish hindbrain ventricle via microinjection. Then, chemical inhibitors such as immunosuppressive drugs are added directly to the larval water. Two methods to monitor the infection in injected larvae are described, including the 1) homogenization of larvae for colony forming unit (CFU) enumeration and 2) a repeated, daily live imaging setup. Overall, these techniques can be used to mechanistically analyze the progression of Aspergillus infection in vivo and can be applied to different host backgrounds and Aspergillus strains to interrogate host-pathogen interactions.

Infection of Zebrafish Larvae with Aspergillus Spores for Analysis of Host-Pathogen Interactions

This protocol describes an Aspergillus infection model in zebrafish larvae. Aspergillus spores are microinjected into the hindbrain of larvae, and chemical treatment is used to induce immunosuppression. Infection progression is monitored via a daily imaging setup to monitor fungal growth and immune responses as well as enumeration of live spores by colony forming unit plating.

Invasive aspergillosis (IA) is one of&#160;the most common fungal infections among immunocompromised individuals. Despite the availability of antifungal ...

Confocal Laser Scanning Microscopy-Based Quantitative Analysis of Aspergillus fumigatus Conidia Distribution in Whole-Mount Optically Cleared Mouse Lung

Aspergillus fumigatus conidia are airborne pathogens that can penetrate human airways. Immunocompetent people without allergies exhibit resistance and immunological tolerance, while in immunocompromised patients, conidia can colonize airways and cause severe invasive respiratory disorders. Various cells in different airway compartments are involved in the immune response that prevents fungal invasion; however, the spatio-temporal aspects of pathogen elimination are still not completely understood. Three-dimensional (3D) imaging of optically cleared whole-mount organs, particularly the lungs of experimental mice, permits detection of fluorescently labeled pathogens in the airways at different time points after infection. In the present study, we describe an experimental setup to perform a quantitative analysis of A. fumigatus conidia distribution in the airways. Using fluorescent confocal laser scanning microscopy (CLSM), we traced the location of fluorescently labeled&#160;conidia in the bronchial branches and the alveolar compartment 6 hours after oropharyngeal application to mice. The approach described here was previously used for detection of the precise pathogen location and identification of the pathogen-interacting cells at different phases of the immune response. The experimental setup can be used to estimate the kinetics of the pathogen elimination in different pathological conditions.

Confocal Laser Scanning Microscopy-Based Quantitative Analysis of Aspergillus fumigatus Conidia Distribution in Whole-Mount Optically Cleared Mouse Lung

We describe the method for quantitative analysis of the distribution of Aspergillus fumigatus conidia (3 &#181;m in size) in the airways of mice. The method also can be used for the analysis of microparticles and nanoparticle agglomerate distribution in the airways in various pathological condition models.

Aspergillus fumigatus conidia are airborne pathogens that can penetrate human airways. Immunocompetent people without allergies exhibit resistance and ...

Preserving Fungal Spore Localization in Lungs for Fluorescent Microscopy Analysis

Efficient Method for Imaging Murine Lungs that Preserves Spatial Dynamics of Fungal Spores in the Airways

Fungi infect humans when environmental spores are inhaled into the lungs. The lung is a heterogeneous organ. Conducting airways, including bronchi and bronchioles, branch until terminating in the alveolar airspace where gas exchange occurs. Infections originating in the bronchioles or alveoli elicit distinct host responses and disease manifestations. Therefore, understanding precisely where spores naturally localize in the lungs, particularly soon after infection, expands opportunities for investigation of host-pathogen interactions. Herein, we detail an in-situ analysis of lungs from mice infected with Coccidioides posadasii cts2/ard1/cts3&#916; arthroconidia. Conventional methods for histological preservation involve liquid inflation of the airways with a fixative solution, which displaces the natural location of aspirated fungal particles, pushing spores from proximal bronchioles to terminal airspaces.
Conversely, this method of air-inflation with blood vasculature perfusion-fixation preserves the physiologic position of fungal spores within the bronchioles. Moreover, we describe a simple approach to cryopreserving, embedding, and imaging lung specimens. We also share high-throughput computational techniques via the open-source QuPath program to analyze the spatial distribution of fungal spores within the lung. The method presented here is simple and quick, requires minimal equipment to perform, and can be easily adapted for use with many respiratory fungal infection models.

We present a method for a fungal pathogenesis model that preserves the natural positioning of fungal spores in the lung airways for analysis via fluorescent microscopy.

Fungi infect humans when environmental spores are inhaled into the lungs. The lung is a heterogeneous organ. Conducting airways, including bronchi and ...

Quantitative Analysis of Aspergillus nidulans Growth Rate using Live Microscopy and Open-Source Software

It is well established that colony growth of filamentous fungi, mostly dependent on changes in hyphae/mycelia apical growth rate, is macroscopically estimated on solidified media by comparing colony size. However, to quantitatively measure the growth rate of genetically different fungal strains or strains under different environmental/growth conditions (pH, temperature, carbon and nitrogen sources, antibiotics, etc.) is challenging. Thus, the pursuit of complementary approaches to quantify growth kinetics becomes mandatory in order to better understand fungal cell growth. Furthermore, it is well-known that filamentous fungi, including Aspergillus spp., have distinct modes of growth and differentiation under sub-aerial conditions on solid media or submerged cultures. Here, we detail a quantitative microscopic method for analyzing growth kinetics of&#160;the model fungus Aspergillus nidulans, using live imaging in both submerged cultures and solid media. We capture images, analyze, and quantify growth rates of different fungal strains in a reproducible and reliable manner using an open source, free software for bio-images (e.g., Fiji), in a way that does not require any prior image analysis expertise from the user.

Quantitative Analysis of Aspergillus nidulans Growth Rate using Live Microscopy and Open-Source Software

We present a label-free live imaging protocol using transmitted light microscopy techniques to capture images, analyze and quantify growth kinetics of the filamentous fungus A. nidulans in both submerged cultures and solid media. This protocol can be used in conjunction with fluorescence microscopy.

It is well established that colony growth of filamentous fungi, mostly dependent on changes in hyphae/mycelia apical growth rate, is macroscopically ...

Biology

A Technique to Quantify Fungal Infection in Zebrafish Larvae

Source: Thrikawala, S., et al. <a href="https://appjove.unicartagenaproxy.elogim.com/v/61165">Infection of Zebrafish Larvae with Aspergillus Spores for Analysis of Host-Pathogen Interactions.</a> J. Vis. Exp. (2020).
This video demonstrates a method to quantify fungal infection in zebrafish larvae. Fungal spores are microinjected into the larval hindbrain ventricle, and the larvae are incubated to allow infection development. Infected larvae are harvested at defined intervals, and infection progress is assessed by enumerating colony-forming units.

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. ...

Histological Quantification to Determine Lung Fungal Burden in Experimental Aspergillosis

Summary

Explore More Videos

Chapters in this video

Direct Observation of Phagocytosis and NET-formation by Neutrophils in Infected Lungs using 2-photon Microscopy

Quantification of Fungal Colonization, Sporogenesis, and Production of Mycotoxins Using Kernel Bioassays

Use of Image Cytometry for Quantification of Pathogenic Fungi in Association with Host Cells

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

Measuring Phagocytosis of Aspergillus fumigatus Conidia by Human Leukocytes using Flow Cytometry

Infection of Zebrafish Larvae with Aspergillus Spores for Analysis of Host-Pathogen Interactions

Confocal Laser Scanning Microscopy-Based Quantitative Analysis of Aspergillus fumigatus Conidia Distribution in Whole-Mount Optically Cleared Mouse Lung

Efficient Method for Imaging Murine Lungs that Preserves Spatial Dynamics of Fungal Spores in the Airways

Quantitative Analysis of Aspergillus nidulans Growth Rate using Live Microscopy and Open-Source Software

A Technique to Quantify Fungal Infection in Zebrafish Larvae