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Antimicrobial Agents and Chemotherapy, February 2008, p. 716-718, Vol. 52, No. 2
0066-4804/08/$08.00+0     doi:10.1128/AAC.00760-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Intranasal Granulocyte-Macrophage Colony-Stimulating Factor Reduces the Aspergillus Burden in an Immunosuppressed Murine Model of Pulmonary Aspergillosis{triangledown}

Gerardo Quezada,1 Nadezhda V. Koshkina,1 Patrick Zweidler-McKay,1 Zichao Zhou,1 Dimitrios P. Kontoyiannis,2 and Eugenie S. Kleinerman1*

The Children's Cancer Hospital, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Blvd., Houston, Texas 77030,1 Department of Infectious Diseases, Infection Control and Employee Health, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Blvd., Houston, Texas 770302

Received 12 June 2007/ Returned for modification 27 August 2007/ Accepted 26 October 2007


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ABSTRACT
 
We demonstrated that intranasal granulocyte-macrophage colony-stimulating factor given to immunosuppressed mice infected with pulmonary aspergillosis resulted in a sixfold reduction in the lung fungal burden compared to the result for saline-treated mice (P = 0.045). These data suggest that lung-targeted immunotherapy may be complementary to antifungal agents and may improve patient responses.


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TEXT
 
Invasive aspergillosis is a major cause of morbidity and mortality in heavily immunocompromised patients, such as those with hematologic malignancies and hematopoietic stem cell transplant recipients (9). There is a dire need for novel therapeutic strategies. Adjunct immunotherapy is an important step in this direction.

The efficacies of systemically administered granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor (GM-CSF) on Aspergillus have previously been studied in both in vitro and in vivo experiments (4, 14, 15, 16, 18). We conducted a pilot study to determine whether the intranasal delivery of GM-CSF may have therapeutic potential in a murine model of pulmonary aspergillosis.

Mice. Female Swiss Webster mice (Charles River Laboratories, Wilmington, MA) were used in quantitative PCR experiments. Female Swiss Webster mice (National Cancer Institute, Bethesda, MD) were used in the immunohistochemistry investigations.

Immunosuppression. Cyclophosphamide (Sigma-Aldrich, St. Louis, MO) dissolved in sterile saline (150 mg/kg of body weight per dose) was administered by intraperitoneal injection on days –4 and –1. Mice were neutropenic within 4 days of the first injection and for 4 days after the booster dose (7, 10, 11, 19). Cortisone acetate (Sigma-Aldrich, St. Louis, MO) dissolved (250 mg/kg/dose) in sterile saline was administered subcutaneously on day –1.

Isolate. Aspergillus fumigatus (AF293) spores were plated and incubated at 37°C for 72 h. They were harvested and then quantified using a hematocytometer, and viability was assessed through serial plating.

Infection. On day 0, the mice were rendered unconscious with isoflurane. A 20-µl droplet with 3 x 106 spores was delivered to the nares of each mouse. The mice showed signs of being sick within 48 h and were sacrificed no later than 96 h after infection (10, 11, 19). Individual lungs were harvested and stored at –20°C in cryovials.

Treatment. Escherichia coli-derived recombinant murine GM-CSF (ProSpec-Tany TechnoGene, Rehovot, Israel) was reconstituted per the manufacturer's recommendations and mixed in saline prior to administration. Daily intranasal treatments with either 10 ng of GM-CSF (treatment group) or saline (control group) were initiated within 6 h of infectious inoculation and repeated every 24 h until the mice were sacrificed. Three independent experiments were conducted.

Analysis. Pulmonary fungal burden was determined by real-time quantitative PCR analysis (3, 10, 12, 19). The lungs were homogenized with a mini bead beater homogenizer (BioSpec Products, Inc., Bartlesville, OK). DNA extraction and isolation were performed using Qiagen DNeasy kits. DNA yield per specimen was assessed spectrophotometrically.

DNA samples were analyzed using the iCycler iQ real-time PCR detection system (Bio-Rad Laboratories, Hercules, CA). Primers and a dual-labeled fluorescent hybridization probe were specific for the A. fumigatus 18S rRNA gene (GenBank accession number AB008401). The sequences of the primers and probe were 5'-ATGGCCGTTCTTAGTTGGTGGAGT-3' for the forward primer, 5'-AAATGCGGACCGGGCTATTTAAGG-3' for the reverse primer, and 5'-6-carboxyfluorescein-AATTGCGATAACGAACGAGACCTCGG-6-carboxytetramethylrhodamine-3' for the probe. The resulting primer product was 88 bp in length. Murine β-actin primers (forward, 5'-CTGAGAGGGAAATCGTGCG-3', and reverse, 5'-GGTGGTACCACCAGACAAC-3') were used as a loading control. β-Actin control samples were amplified in conjunction with the experimental samples. The threshold cycle of each sample, which identifies the cycle number during PCR when fluorescence exceeds a threshold value determined by the software, was then interpolated from an 8-point standard curve of threshold cycle values prepared by spiking uninfected mouse lungs with 101 to 108 A. fumigatus conidia (10, 12, 19). Results are reported as conidial equivalents. GraphPad InStat software, version 3.05, was used for the quantitative PCR data analysis. For a comparison of the conidial equivalent values, the Mann-Whitney test was used. A P value of <0.05 was considered statistically significant.

Immunohistochemistry was performed using the F4/80 rat monoclonal anti-murine macrophage antibody (AbD Serotec, Raleigh, NC) to identify activated and mature macrophages (2, 5, 6, 8).

Results and discussion. Both the immunocompetent and the immunosuppressed mice tolerated the 10-ng intranasal dose of GM-CSF without visible systemic toxicity. There was no difference in the daily weights (Fig. 1). Acute pulmonary aspergillosis infection was confirmed in the immunosuppressed mice by hematoxylin and eosin and Grocott's methenamine silver staining. At 48 and 72 h postintranasal GM-CSF, macrophage activation (F4/80 staining) was increased in both the uninfected immunocompetent and the immunosuppressed mice compared to the level in the saline-treated mice (Fig. 2A and B). There was no difference in F4/80 staining at 24, 48, and 72 h in either infected immunocompetent or immunosuppressed mice treated with either saline or GM-CSF. Germinating hyphae were visible by 48 h postinfection and widespread by 72 h. Mature activated macrophages were seen at both 48 and 72 h in the infected immunosuppressed mice treated with GM-CSF (Fig. 2C and D).


Figure 1
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  		FIG. 1. Mouse daily weights. To assess differences in fluid retention, individual mouse daily weights were obtained pre- and postintranasal treatment. There was no difference, regardless of immune status, in the average daily weights in either saline-treated or GM-CSF-treated mice. IS, immunosuppressed; IC, immunocompetent.


Figure 2
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  		FIG. 2. Immunohistochemistry with F4/80 staining. There was a visible increase in mature activated macrophages (brown staining) at 72 h postintranasal GM-CSF treatment (A) versus intranasal phosphate-buffered saline treatment (B) in uninfected immunosuppressed mice. Activated macrophages surrounding germinating hyphae in immunosuppressed mice at 48 (C) and 72 (D) h postinfection and treatment.

Quantitative PCR analysis demonstrated that immunosuppressed mice treated with intranasal GM-CSF had a sixfold reduction in the pulmonary fungal burden relative to the saline-treated control group. When plotted on a conidial-equivalent curve (Fig. 3), the difference between the two groups was found to be statistically significant (P = 0.045).


Figure 3
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  		FIG. 3. The therapeutic effect of intranasal GM-CSF on Aspergillus growth in vivo. Immunosuppressed mice with pulmonary aspergillosis were treated intranasally with either 10 ng of GM-CSF or saline. Daily treatments were continued every 24 h until the mice were sacrificed. Aspergillus conidial equivalents were determined by quantitative PCR as described in the text. The bars on the x axis represent the mean numbers of spores from the saline-treated group (n = 16; 1.41 ± 0.78) and the GM-CSF treatment group (n = 17; 0.42 ± 0.17). Results are shown as means ± standard errors (error bars). The difference in the fungal burden between the two groups was significant (P = 0.045; Mann-Whitney test).

We recognize that the mouse model used in these experiments does not ideally reproduce what is seen in the clinical setting. However, the finding that intranasal GM-CSF was successful in both increasing macrophage activation and reducing the fungal burden suggests the potential of this therapeutic approach whether it is used in a prophylactic manner or as part of a combination strategy with systemic antifungals. While there was no quantitative difference in activated macrophages in the infected immunosuppressed mice, the PCR data suggest an enhancement in the immune-mediated clearance of the fungal organism. In contrast to antifungals where resistance emerges, GM-CSF boosts the innate immune system broadly so it should not select for a resistant organism and might be beneficial against other emerging invasive molds. Our treatment approach in the clinical setting of pulmonary fungal infections would be by aerosol delivery of GM-CSF, since it is more feasible, practical, and reproducible than the intranasal approach. Aerosolized GM-CSF has been shown to be safe and well tolerated by patients in studies of metastatic sarcomas of the lungs and in pulmonary alveolar proteinosis (1, 13, 17, 20).

In summary, this pilot study indicates that GM-CSF administered intranasally may be a novel therapeutic approach for the prevention or treatment of pulmonary fungal infections and may augment the efficacies of antifungal agents.


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ACKNOWLEDGMENTS
 
We thank Georgios Chamilos, Gregory Lamaris, and Nathaniel Albert for their valuable advice with the pulmonary aspergillosis murine model. We also thank Lindsey DeLauter, Carol J. Oborn, and Nancy Gordon for their help and guidance with the immunohistochemistry.

Dimitrios P. Kontoyiannis has received research support and honoraria, but no financial support, from Schering-Plough, Pfizer, Astellas Pharma, Enzon Pharmaceuticals, and Merck & Co.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Pediatrics, 1515 Holcombe Blvd., Box 87, Houston, TX 77030. Phone: (713) 792-8110. Fax: (713) 792-0608. E-mail: ekleiner{at}mdanderson.org Back

{triangledown} Published ahead of print on 5 November 2007. Back


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Antimicrobial Agents and Chemotherapy, February 2008, p. 716-718, Vol. 52, No. 2
0066-4804/08/$08.00+0     doi:10.1128/AAC.00760-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.




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