Reduced Nicotinamide Adenine Dinucleotide Phosphate Oxidase-Independent Resistance to Aspergillus fumigatus in Alveolar Macrophages This information is current as of September 27, 2021. E. Jean Cornish, Brady J. Hurtgen, Kate McInnerney, Nancy L. Burritt, Ross M. Taylor, James N. Jarvis, Shirley Y. Wang and James B. Burritt J Immunol 2008; 180:6854-6867; ; doi: 10.4049/jimmunol.180.10.6854 Downloaded from http://www.jimmunol.org/content/180/10/6854 References This article cites 80 articles, 29 of which you can access for free at: http://www.jimmunol.org/ http://www.jimmunol.org/content/180/10/6854.full#ref-list-1 Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists by guest on September 27, 2021 • Fast Publication! 4 weeks from acceptance to publication *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2008 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology Reduced Nicotinamide Adenine Dinucleotide Phosphate Oxidase-Independent Resistance to Aspergillus fumigatus in Alveolar Macrophages1 E. Jean Cornish,* Brady J. Hurtgen,* Kate McInnerney,* Nancy L. Burritt,* Ross M. Taylor,* James N. Jarvis,† Shirley Y. Wang,† and James B. Burritt2* The fungal pathogen Aspergillus fumigatus is responsible for increasing numbers of fatal infections in immune-compromised humans. Alveolar macrophages (AM) are important in the innate defense against aspergillosis, but little is known about their molecular responses to fungal conidia in vivo. We examined transcriptional changes and superoxide release by AM from C57BL/6 and gp91phox؊/؊ mice in response to conidia. Following introduction of conidia into the lung, microarray analysis of AM showed the transcripts most strongly up-regulated in vivo to encode chemokines and additional genes that play a critical role Downloaded from in neutrophil and monocyte recruitment, indicating that activation of phagocytes represents a critical early response of AM to fungal conidia. Of the 73 AM genes showing >2-fold changes, 8 were also increased in gp91phox؊/؊ mice by conidia and in C57BL/6 mice by polystyrene beads, suggesting a common innate response to particulate matter. Ingenuity analysis of the microarray data from C57BL/6 mice revealed immune cell signaling and gene expression as primary mechanisms of this response. Despite the well-established importance of phagocyte NADPH oxidase in resisting aspergillosis, we found no evidence of this mechanism in AM following introduction of conidia into the mouse lung using transcriptional, luminometry, http://www.jimmunol.org/ or NBT staining analysis. In support of these findings, we observed that AM from C57BL/6 and gp91phox؊/؊ mice inhibit conidial germination equally in vitro. Our results indicate that early transcription in mouse AM exposed to conidia in vivo targets neutrophil recruitment, and that NADPH oxidase-independent mechanisms in AM contribute to inhibition of conidial germination. The Journal of Immunology, 2008, 180: 6854–6867. spergillus fumigatus is the leading airborne fungal patho- AM are resident pulmonary phagocytes that respond early to gen in immune-compromised individuals, where it can inhaled A. fumigatus conidia. Numerous studies have examined the cause potentially fatal invasive pulmonary aspergillosis response of AM to A. fumigatus conidia, both in vivo and in vitro. A3 by guest on September 27, 2021 (IPA) (1–4). However, IPA is rare in individuals with a normal Despite the key role of AM in aspergillosis immunity, there is little inflammatory response, due primarily to innate immunity in which in vivo data that indicate how A. fumigatus conidia affect gene phagocytic leukocytes including alveolar macrophages (AM), expression in AM, limiting our understanding about the molecular polymorphonuclear neutrophils (PMN), and dendritic cells play an mechanisms by which AM respond to this fungus. In contrast to essential early role in the defense against aspergillosis (5). There- previous studies on in vitro transcriptional and functional changes fore, infections caused by A. fumigatus, which originate in the of peritoneal macrophages and AM following exposure to conidia airway of immune-compromised humans, arise because of a defect (6–9), the present study was designed to include transcriptional in one or more of the innate mechanisms of resistance that nor- responses of AM to conidia in mouse lungs, thus providing better mally protect from IPA. insight into the overall mechanism by which AM help resist in- fections by A. fumigatus in vivo. The generation of reactive oxygen species represents a well- *Department of Microbiology, Montana State University, Bozeman, MT 59717; and characterized antimicrobial mechanism used by phagocytic leu- † Oklahoma University Health Sciences Center, Oklahoma City, OK 73104 kocytes, and extensive clinical evidence implicates the super- Received for publication December 21, 2007. Accepted for publication March oxide-producing phagocyte NADPH oxidase in the resistance to 13, 2008. aspergillosis (10). Human patients with defects in this system The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance suffer from chronic granulomatous disease characterized by re- with 18 U.S.C. Section 1734 solely to indicate this fact. current infections, extensive granuloma formation, and fre- 1 This work was supported by National Institutes of Health (NIH) Award 1 R03 quently, IPA (11). Although it is generally accepted that AI057931-01 (to J.B.B.), American Heart Association Scientist Development Grant NADPH oxidase is necessary for killing A. fumigatus conidia 0630253N (to R.M.T.), and was also made possible by NIH Grant 1 P20 RR- 020185-01 from the National Center for Research Resources. by PMN (12, 13), there is conflicting information on the role of 2 Address correspondence and reprint requests to Dr. James B. Burritt, Department of this mechanism in AM-mediated resistance to IPA (12, 14–17). Microbiology, Montana State University, 109 Lewis Hall, Bozeman, MT 59717. E- Therefore, in addition to transcriptional studies on AM exposed mail address: [email protected] to A. fumigatus conidia, we have examined functional aspects of 3 Abbreviations used in this paper: IPA, invasive pulmonary aspergillosis; AM, al- AM for evidence of NADPH oxidase involvement following veolar macrophage; PMN, polymorphonuclear neutrophil; qRT-PCR, quantitative RT-PCR; BALF, bronchoalveolar lavage fluid; MCLA, 2-methyl-6-(4-methoxyphe- contact with conidia. nyl)imidazo[1,2-a] pyrazin-3(7H)-one; SOD, superoxide dismutase; HO, heme DNA microarray analysis is a powerful method for monitoring oxygenase. the effect of microbes on the global transcription of cellular gene Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00 products. Relevant to the present study, microarray analysis has www.jimmunol.org The Journal of Immunology 6855 previously been used to characterize changes in gene expression Materials and Methods associated with oxidative defense in phagocytes responding to Preparation of A. fumigatus conidia stimuli in vitro. For example, increased transcription of some A clinical isolate (no. 13073; American Type Culture Collection) of A. genes encoding oxidant scavengers was observed in human mono- fumigatus was grown on Sabouraud dextrose agar slants in 75-cm2 culture cytes in response to A. fumigatus conidia (6), as was increased flasks at 37°C for 5 days and conidia were collected in 0.1% Tween 20 in transcription of the gene encoding the gp91phox subunit of the HBSS (no. 10-547F; Cambrex Bio Science) by gentle rocking as described 6 NADPH oxidase in mouse macrophages when exposed to LPS (13). Conidia were then diluted in HBSS without Tween 20 to obtain either 10 or 107 conidia in 40 ␮l for intrapharyngeal administration. All in vivo inocu- (18). Additionally, the analysis of in vitro changes in transcription lations were performed using conidia harvested immediately before use. of human monocytes has provided valuable clues about innate re- sponses to A. fumigatus (6, 8). In those studies, the exposure of Animal handling and sample collection monocytes to conidia was found to induce genes involved in a All protocols involving mice were approved by the Institutional Review diverse array of cellular functions including leukocyte adhesion, Board of the Institutional Animal Care and Use Committee at Montana cell recruitment, endocytosis, phagocytosis, and oxidant stress re- State University. C57BL/6 male mice were obtained at 9–11 wk of age from Charles River Laboratories, kept in the Animal Resource Center at sponses. However, transcriptional differences between AM and Montana State University in microisolator cages, and given food and water monocytes (19, 20), and the influences on AM by other resident ad libitum. Breeder mice with a null allele corresponding