Reduced Nicotinamide Adenine Dinucleotide Phosphate Oxidase-Independent Resistance to Aspergillus fumigatus in Alveolar Macrophages This information is current as of October 1, 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; ;

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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 in neutrophil and monocyte recruitment, indicating that activation of phagocytes represents a critical early response of AM Downloaded from 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, /or NBT staining analysis. In support of these findings, we observed that AM from C57BL/6 and gp91phox؊/؊ mice inhibit http://www.jimmunol.org 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 (IPA) (1–4). However, IPA is rare in individuals with a normal Despite the key role of AM in aspergillosis immunity, there is little by guest on October 1, 2021 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 to the X-linked and recruited cells in the lung, underscore the importance of ana- gp91phox component of the NADPH oxidase (B6.129S6-Cybbtm1Din) were lyzing the response of AM to conidia in vivo. There are some previously produced by backcrossing carrier females with C57BL/6 males for 13 generations. Breeding pairs of these mice were then obtained from published in vivo microarray data related to the effects of inflam- The Jackson Laboratory and reared in specific pathogen-free housing in the phoxϪ/Ϫ mation in AM (21), but to our knowledge, none relates specifically Animal Resource Center. Only male gp91 mice were used for mi- Downloaded from to the transcriptional responses to A. fumigatus in the lung. croarray, quantitative RT-PCR (qRT-PCR), and ELISA studies. They were The present study was undertaken to extend our previous ob- housed in microisolator cages in an environment of filtered air, given au- toclaved food ad libitum, and prophylactically treated with sulfamethox- servation that alveolar PMN both use and require NADPH oxidase azole-trimethoprim in their sterile, acidified drinking water. Three days for resistance to aspergillosis, and to more clearly define the early before use, their drinking water was changed to sterile acidified water Ϫ Ϫ in vivo responses of AM to A. fumigatus conidia, before conidial without antibiotics. Egr1 / mice generated on a C57BL/6 background were obtained from Taconic Farms. Homozygous female Egr1Ϫ/Ϫ mice

germination. Following the instillation of conidia into the lungs of tm1Jmi http://www.jimmunol.org/ phoxϪ/Ϫ 9–11 wk of age were used (no. 002013-M-F, B6.129-Egr1 N12) and C57BL/6 and gp91 mice, DNA microarray analysis was female Egrϩ/ϩ C57BL/6 mice were used for comparison in phagocyte used to evaluate both dose- and time-dependent changes in AM responses to conidia. For all inoculation studies, mice were briefly anes- gene transcription. Of key importance in the early innate AM re- thetized with isoflurane and inhaled 40 ␮l intrapharyngeally administered sponses to A. fumigatus conidia was up-regulation of genes for vehicle only (sterile HBSS) for mock inoculations, or vehicle containing either 106 or 107 freshly harvested conidia. In some experiments, 3-␮m PMN recruitment, previously associated with conidial display polystyrene microspheres (no. 17134; Polysciences) were used instead of of ␤-glucan (22, 23), augmenting the inflammatory response conidia to test for transcriptional changes to an alternative particle. At that protects normal animals from IPA. In our analyses, we indicated time points following in vivo incubation, mice were euthanized observed a group of genes commonly up-regulated by admin- with isoflurane and bronchoalveolar lavage fluid (BALF) was collected Ϫ Ϫ phox / through an 18-gauge angiocatheter needle (BD Biosciences) inserted into by guest on October 1, 2021 istration of conidia to C57BL/6 and gp91 mice, and by an incision in the exposed trachea as described (13). Lungs were perfused the administration of polystyrene beads to C57BL/6 mice, sug- with six 1-ml volumes of HBSS containing 3 mM EDTA, which were gesting an innate immune response to particulate agents. The pooled to produce a 6-ml BALF sample from each mouse. Two hundred current study examining in vivo responses of AM identifies a fifty microliters of BALF were used immediately for cytospin and Wright staining (to obtain leukocyte differentials and to verify pulmonary conidial focused and decisive immune response to A. fumigatus conidia delivery), and for cell counting by a hemocytometer. In certain experi- involving increased expression of the genes for TNF-␣ and ments, the levels of specific proteins were examined by ELISA on the PMN-recruiting chemokines to provide a protective inflamma- supernatants of BALF obtained from the first 2 ml of fluid following col- tory response. lection and storage at Ϫ80°C before analysis. Because the role of reactive oxygen species produced by AM in Isolation of mRNA from AM response to conidia remains a matter of debate, this study also Following the removal of samples of the BALF for cell count and differ- examined AM for superoxide production following exposure to A. ential, a pellet containing AM Ϯ conidia was obtained by centrifugation at fumigatus conidia for evidence of involvement of NADPH oxidase 150 ϫ g for 10 min at 4°C. The cell pellet was then resuspended in 450 ␮l in fungal killing. Our in vitro analysis of extracellular superoxide of RNA lysis tissue buffer from the RNeasy mini kit (no. 74904; Qiagen) production by luminometry indicated exposure of conidia to AM containing 1% 2-ME. The phagocytes were lysed by cycling the suspension through a pipette tip 10 times and vortexing for 20 s, then the sample was resulted in a reduction in measurable NADPH oxidase-dependent centrifuged at 20,800 ϫ g for 10 min at 4°C to pellet conidia (if present). superoxide generation, and that AM from C57BL/6 mice and those The supernatant samples without conidia were then processed according to from gp91phoxϪ/Ϫ mice lacking the NADPH oxidase were equally the RNeasy mini kit protocol. We have verified this procedure does not capable of suppressing conidial germination. Furthermore, we ob- extract fungal RNA as determined by RT-PCR with A. fumigatus-specific catalase A primers (forward primer: 5Ј-AAGACCTCCTCCAAGGGCAT- served that in vivo contact of conidia with AM did not lead to CATT; reverse primer: 5Ј-ACGGACTTTGTGGGCAAGTTCTTC). evidence of intracellular superoxide production in AM when Genomic DNA was removed from a final 45-␮l RNA eluate using a examined by NBT staining as it did in PMN. Taken together, TURBO DNA-free kit (no. 1907; Ambion) according to the manufacturer’s our results indicate the responses of AM to A. fumigatus conidia instructions. The resulting RNA was precipitated in 2.5 M lithium chloride according to the manufacturer’s protocol (no. 9480; Ambion) and stored include the production of soluble factors that lead to recruit- overnight at Ϫ20°C. Following this treatment, the RNA was pelleted by ment and activation of additional cell types of the innate im- centrifugation and washed three times with 70% ethanol. Following air mune system, but not activation of superoxide production by drying, the resulting RNA pellet was dissolved in 8–14 ␮l of nuclease-free the NADPH oxidase complex within AM themselves. The abil- water at 37°C, quantified using a Nano-Drop 1000 spectrophotometer and its quality was checked using an Agilent 2100 bioanalyzer. ity of AM to generate proteins that recruit PMN and prime their oxidative burst supports the hypothesis that superoxide produc- Analysis of transcriptional changes by microarray tion represents a crucial microbicidal mechanism used by PMN For microarray analysis, isolated RNA was amplified and biotin-labeled to prevent infections by A. fumigatus. using an Affymetrix two-cycle labeling and control reagent kit (no. 6856 AM RESISTANCE TO ASPERGILLOSIS

Table I. Primers for qRT-PCR

Gene Forward Primer Reverse Primer Amplicon bp

Tnf 5Ј-CCAACGGCATGGATCTCAAAGACA-3Ј 5Ј-TGAGATAGCAAATCGGCTGACGGT-3Ј 143 Egr1 5Ј-TTCCACAACAACAGGGAGACCTGA-3Ј 5Ј-TGGGTTTGATGAGCTGGGATTGGT-3Ј 186 Ereg 5Ј-TTCTGACATGGACGGCTACTGCTT-3Ј 5Ј-CTTTGCTCAAGGGTTGGTGAACAG-3Ј 143 Gapdh 5Ј-TCAACAGCAACTCCCACTCTTCCA-3Ј 5Ј-ACCCTGTTGCTGTAGCCGTATTCA-3Ј 115 Hmox1 5Ј-TAGCCCACTCCCTGTGTTTCCTTT-3Ј 5Ј-TGCTGGTTTCAAAGTTCAGGGCAC-3Ј 107

900494). The mouse genome array 430A 2.0 (no. 90499; Affymetrix) was ELISA used to analyze AM gene transcription following hybridization (Affymetrix ␣ ␤ Gene chip hybridization oven 640) and processing through a Gene Chip Analysis of TNF- , IL-1 , and CXCL2 was conducted on cell-free BALF Fluids Station 450. For these studies, each conidial and mock inoculation using the first 2 ml collected, according to the manufacturer’s protocol ␣ ␤ condition was replicated in quadruplicate for C57BL/6 mice, triplicate for (TNF- 88-7324-22 and IL-1 88-7013-22, eBioscience; CXCL2 MM200, gp91phoxϪ/Ϫ mice, and duplicate for C57BL/6 with polystyrene beads, gen- R&D Systems). erating 51 GeneChip experiments. Affymetrix GCOS software was used to Detection of pulmonary phagocyte superoxide production by convert raw scans to CEL and CHP data files; all of which were imported into GeneSpring GX7.3 (Agilent Technologies) for analysis. According to luminometry a recent examination of Affymetrix probe accuracy (24), sequence verified, Extracellular superoxide production by AM and LPS-elicited alveolar mismatch permissive chip definition files were imported into GeneSpring PMN was examined in white 96-well plates (no. EK-25075; E&K Scien- Downloaded from (from http://gbic.biol.rug.nl//supplementary/2006/probeverification/), and tific), using 2-methyl-6-(4-methoxyphenyl)-3,7-dihydroimidazol[1,2- present, marginal, and absent gene call information was extracted from the a]pyrazin-3-one (MCLA; no. M-23800, Molecular Probes-Invitrogen), a CHP files. Following robust multichip averaging normalization with me- luminometry reagent that has optimal sensitivity for detecting low-level dian polishing (25), CEL data was filtered for present gene calls and base- superoxide generation by phagocytes (35). For luminometry studies on line raw signal intensity of 100 in at least one replicate set equivalent (3 of AM, naive 10- to 12-wk-old C57BL/6 and gp91phoxϪ/Ϫ mice were sacri- 51 chips), an operation that trimmed the data being analyzed roughly in ficed and 8 ml of BALF was collected as described above. AM in the half (to 11,591 genes). The remaining genes were filtered by fold change BALF were counted, centrifuged at 300 ϫ g for 10 min at 4°C, and then 6 (2 in any comparison) and were then subjected to ANOVA with a Welch resuspended at 10 cells/ml in DMEM-10 (no. D5648; Sigma-Aldrich) http://www.jimmunol.org/ t test and Benjamini and Hochberg false discovery rate of 0.01. A hierar- containing 10% FCS and 4 mM L-glutamine. For superoxide production chical tree was generated using a Pearson’s correlation and an average assays, 105 freshly harvested AM in 100 ␮l of DMEM-10 were adhered to linkage clustering algorithm. Subsequent functional enrichment analysis wells of a 96-well microtiter plate in the absence or presence of conidia was conducted using DAVID software (www.DAVID.niaid.nih.gov) (26). (5:1 ratio of conidia:AM). Following 90- to 180-min incubation (data is Microarray data were deposited with Gene Expression Omnibus at the shown for 90-min incubation, but not different from 180-min incubation) in National Center for Biotechnology Information, and can be accessed a 37°C incubator with 5% CO2, the DMEM-10 was removed with rare through accession number GSE8997. nonadherent PMN and replaced with 100 ␮lof5␮M MCLA in 140 mM NaCl/well. In certain wells, phagocytes were also examined for oxidant Analysis of mRNA by qRT-PCR production for 30 min after exposure to 910 nM PMA (no. P-8139; Sigma-Aldrich).

To confirm results obtained in microarray studies, qRT-PCR was used to by guest on October 1, 2021 quantify transcription of a representative subset of AM genes significantly For examination of oxidant production in PMN, mice were first exposed altered following exposure to conidia and suspected to be involved in the to aerosolized LPS 12 h before BALF collection as described (13), pro- Ͼ resulting immune response. Thus, transcription of Ereg, Egr1, and Tnf was ducing BALF that contained 85% PMN (RBC were removed by hypo- examined relative to Gapdh (a constitutively expressed reference gene (27– tonic lysis). Following cell counting and Wright staining, PMN were re- 7 ␮ 5 30) at 2, 4, and 5 h after inoculation with 107 conidia or mock inoculation suspended at 10 cells/ml in HBSS, and then 10 l containing 10 PMN in male C57BL/6 and gp91phoxϪ/Ϫ mice. The Tnf transcript and protein was suspended in MCLA reagent described above, in the presence of 910 levels were of particular interest in the present study because of the reg- nM PMA. Data for superoxide liberation by both AM and PMN were ulatory role of TNF-␣ with respect to NADPH oxidase (31, 32). Due to collected using a Turner Biosystems GloRunner luminometer (1-s data possible effect on NADPH oxidase activity, transcription of Hmox1 was points collected each minute over a 30- to 120-min interval) with wells also examined by qRT-PCR, but only at4hinC57BL/6 mice and at 5 h blanked against representative wells containing 310 U/ml superoxide dis- in gp91phoxϪ/Ϫ mice. The gene-specific primer sets used in these analyses mutase (SOD; no. S-2515, Sigma-Aldrich). The average SOD-inhibitable Ϯ ϭ are shown in Table I. A Quanti-Tect SYBR Green RT-PCR kit (Qiagen) superoxide generation rate was then reported SEM for five mice (n 5). was used in combination with a Corbett Rotor Gene 3000 (no. 204243; The data were found to be reproducible with additional analyses performed Qiagen) to analyze transcription using 6 ng of purified total RNA per on 3 separate days. Ϯ sample. For these studies, the PCR amplification efficiencies were 1.01 Detection AM superoxide production by formazan deposition 0.04 for Gapdh, 1.07 Ϯ 0.07 for Ereg, 1.02 Ϯ 0.01 for Ereg1, 1.01 Ϯ 0.03 for Tnf, and 1.01 Ϯ 0.01 for Hmox1. In each experiment, controls using no Intracellular production of superoxide in AM and PMN was evaluated by template and no reverse transcriptase were included. In addition, melt exposure of cells to NBT (no. N-6876; Sigma-Aldrich), and microscopic curve analysis and gel electrophoresis were used to check product speci- examination for evidence of formazan deposition as previously described ficity. Analysis of qRT-PCR data was conducted by relative quantification (13, 17). Briefly, C57BL/6 or gp91phoxϪ/Ϫ mice were inoculated with 107 using REST software, which normalizes data and takes amplification effi- conidia as described above, then BALF was collected 4 or 12 h later and ciencies into account (27, 28, 33). exposed to 500 ␮M NBT for 30 min at 37°C. In certain experiments,

conidia were first preswollen in RPMI 1640 for8hat37°C in 5% CO2 then Molecular pathway analysis pelleted and resuspended in HBSS before inoculation of mice. For micros- From C57BL/6 mice inoculated with 107 conidia and AM collected at 4 h, copy, cytospins were then prepared and counterstained with safranin as networks of AM genes were constructed using Ingenuity analysis (www. described (13). In some cases, BALF phagocytes in contact with conidia ingenuity.com) as described (34). Briefly, microarray data (from Table II) were exposed to 910 nM PMA concurrently with NBT. containing gene identifiers and corresponding expression values were up- In vitro inhibition of A. fumigatus conidial germination by AM loaded to the Ingenuity program to probe the knowledge-based databases for information to suggest molecular participants involved in the response To compare the ability of AM from C57BL/6 and gp91phoxϪ/Ϫ mice to of AM to conidia. Only genes showing fold change values Ն2 were eval- inhibit conidial germination, BALF was collected from naive mice as de- uated, and only Ingenuity pathways with network scores of Ն15 were scribed above. AM were then pelleted at 150 ϫ g for 5 min at 4°C, re- considered further. We did not examine gp91phoxϪ/Ϫ data by Ingenuity suspended at 106 AM/ml in DMEM-10, and then introduced to wells in a analysis, because higher constitutive expression of inflammatory genes in 96-well tissue-culture plate at 105 cells/well. In this system, AM from gp91phoxϪ/Ϫ mice resulted in lower corresponding fold change values uninoculated mice remained Ͼ98% viable for 7 days (based on trypan blue which were needed to generate the networks. exclusion). Immediately after plating AM, conidia were then introduced The Journal of Immunology 6857

Table II. AM genes with roles in the immune response showing Ն2-fold changes (bolded) in transcription following in vivo exposure to conidia

C57BL/6 vs Mock gp91phoxϪ/Ϫ vs Mock

Common 2-h 106 2-h 107 4-h 106 4-h 107 2-h 106 2-h 107 4-h 106 4-h 107

Cxcl1 1.7 7.4 10.1 48.2 1.1 3.4 1.4 8.1 Cxcl2 3.5 14.6 20.7 39.8 1.3 3.7 1.5 4.3 Egr1 2.4 6.7 6.5 28.1 1.4 3.4 1.6 4.4 Ccl3 3.0 4.9 10.2 18.6 1.0 1.1 1.4 4.3 Il1b 1.4 2.1 15.4 15.8 0.9 1.0 0.8 1.9 1810011O10Rik 1.0 3.2 3.5 12.8 1.0 0.8 1.6 1.6 Socs3a 1.4 2.1 7.5 12.0 1.3 0.9 1.0 2.4 Nfkbiz 1.2 2.6 7.4 10.7 0.9 1.2 1.0 1.6 Ereg 1.4 3.8 7.7 9.8 1.0 1.7 1.3 3.3 Rgs1 2.1 6.1 3.1 7.5 0.9 1.4 2.1 2.4 Gdap10 1.1 1.7 1.7 7.2 1.1 1.4 1.1 1.4 Plk2 1.0 1.9 2.0 6.3 1.2 1.9 1.3 2.1 Clecsf9a 1.2 2.5 5.2 6.2 1.0 1.5 1.1 2.1 Ccrl2 1.2 2.2 4.0 6.1 0.9 1.0 1.2 1.9 Gadd45ba 1.1 1.6 3.4 5.3 1.2 1.3 1.1 1.6 Tnf 1.3 1.7 3.6 4.9 1.0 2.3 1.0 2.1 Maff 1.1 1.3 1.8 4.1 1.0 1.5 1.1 1.9 Downloaded from Atf3 1.4 2.8 2.2 3.7 1.2 1.5 1.1 1.8 Ifrd1 1.0 1.6 1.5 3.3 1.0 1.7 1.1 1.7 Hmox1 1.1 1.5 1.9 3.2 0.6 0.4 0.9 1.3 Il1a 1.0 1.4 3.0 3.1 0.9 1.2 1.1 2.1 Cflara 1.2 1.8 1.5 3.0 1.1 1.5 1.3 2.2 Ptp4a1 1.5 2.4 1.0 2.8 0.8 1.5 0.7 1.2

Ets2 1.5 2.6 1.9 2.7 1.1 1.4 1.2 1.5 http://www.jimmunol.org/ Zfp36 1.2 1.0 1.9 2.6 1.0 1.5 0.8 1.8 Becn1 0.9 0.8 1.0 2.6 0.9 0.6 0.9 1.2 Dhx9 1.0 1.3 0.6 2.6 0.8 2.0 1.1 2.2 Pde4b 1.1 1.7 1.5 2.5 0.9 1.3 1.2 1.2 Nfatc1 1.1 1.7 1.2 2.5 1.1 1.4 1.4 1.3 Ccl4 0.9 1.4 2.7 2.3 1.2 0.9 1.0 1.4 Gsr 1.2 1.8 0.9 2.2 0.9 1.8 1.0 1.2 Bcl3 1.1 1.8 3.1 2.2 1.1 1.4 0.7 1.4 Tde1 0.9 1.0 0.7 2.1 0.7 0.7 0.8 1.6 Dnaja1 1.3 2.4 1.2 2.1 0.9 1.1 1.0 1.0 Nck1 1.1 1.3 1.0 2.1 1.0 0.9 0.9 1.5 by guest on October 1, 2021 Slc20a1 0.9 1.4 1.2 2.1 1.2 1.6 1.1 1.4 Tob2 1.3 1.7 0.9 2.0 1.3 2.6 1.2 1.4 Icam1 1.0 1.3 1.8 2.0 1.1 1.3 1.0 1.3 Nfkbiaa 1.4 1.5 1.7 2.0 1.1 1.5 1.2 1.4 Txnrd1 1.2 1.7 1.3 2.0 1.0 1.2 1.2 1.9 Jun 1.6 2.4 1.8 2.0 1.1 1.3 1.1 1.0 Slc2a1 0.9 0.8 2.4 1.9 1.0 0.7 1.1 1.2 Tcf7l2 1.1 1.2 0.9 1.8 1.1 2.0 1.0 1.4 Abcc5 1.3 2.0 0.9 1.5 1.0 1.5 1.4 2.5 Fkbp2 0.9 0.7 1.0 1.4 0.9 0.5 1.0 1.0 Flt1 1.2 1.3 1.2 1.4 1.2 2.0 0.9 1.1 Calm3 0.7 0.6 1.0 1.0 1.2 0.5 0.8 1.0

a Average of two or more probe sets for gene on array. and incubated for 8 h (3:1 ratio, conidia:AM), during which time phago- evident in C57BL/6 mice, but was more frequently seen in cytosis of most conidia occurred. Cytospin mounts were then generated and gp91phoxϪ/Ϫ mice. At 6 h, 39.7 Ϯ 7.8% (n ϭ 3) of the cells in the Wright stained to determine the percentage of germinated intracellular and extracellular conidia by microscopy. BALF of C57BL/6 mice were PMN, so the transcription studies described below were not extended to samples collected beyond Results the 5 h time point. Of the total AM recovered in the BALF at 4 h Characterization of phagocytic cells in the mouse lung in after inoculation of C57BL/6 mice using 106 and 107 conidia, response to A. fumigatus 9.1 Ϯ 1.1 (n ϭ 8) and 21.1 Ϯ 2.2% (n ϭ 7) of AM were associated To better understand the in vivo engagement of A. fumigatus with conidia, respectively (Fig. 1A). In similar studies, the corre- phoxϪ/Ϫ Ϯ conidia by AM in a normal and susceptible mouse model, the sponding values at 4 h for gp91 mice were 7.5 2.6 and 7 BALF was examined at specific time points following instillation 21.3 Ϯ 1.5% (n ϭ 4), while the introduction of 10 polystyrene of conidia in C57BL/6 and gp91phoxϪ/Ϫ mice, respectively. At 2 beads (as a model particulate) resulted in 16.7 Ϯ 5% (n ϭ 3) of and 4 h after intrapharyngeal administration of conidia, PMN were AM associated with the beads. For each strain of mice, the largely absent in the BALF of both the C57BL/6 and gp91phoxϪ/Ϫ percentage of phagocytosing AM did not increase significantly strains, and in the rare cases when they were present, did not ex- ( p Ͼ 0.3) between 2 and5hateither dose of conidia (106 and ceed 4% of the total leukocyte number. At 5 h following admin- 107), suggesting few if any additional AM became involved in istration of 107 conidia, PMN recruitment was still generally not phagocytosis during that time. Further characterization of AM 6858 AM RESISTANCE TO ASPERGILLOSIS

When the microarray studies described above were conducted with AM following exposure to particulates, significant transcrip- A phox-/- C57Bl/6 gp91 tional changes were observed for cells isolated from both mouse phoxϪ/Ϫ 106 conidia strains. ANOVA analysis of data for C57BL/6 and gp91 7 30 10 conidia 30 mice after administration of A. fumigatus conidia or polystyrene 7 25 10 beads 25 beads indicated 73 gene probes showed transcriptional changes at Ͻ 20 20 least 2-fold ( p 0.01) when comparing inoculated animals to

15 15 their mock controls, in one or more of the experimental conditions

10 10 (data not shown). When these data were examined for conidia-

5 5 responsive genes, mouse strains tended to cluster together in 0 0 groups segregating by conidial dosage and timing (Fig. 2). Spe- 245 245cifically, AM from gp91phoxϪ/Ϫ mice showed overall higher con- stitutive gene expression and responded minimally to the 106 dose B of conidia at both 2 and 4 h, clustering with gp91phoxϪ/Ϫ time 0 8 10 and mock arrays. C57BL/6 mice also responded modestly to the 106 dose of conidia at 2 h; however, at 4 h, those from 106 dose 6 8 arrays clustered between those from 2 and 4 h following the 107 6 4 dose. Interestingly, AM from C57BL/6 mice 4 h after administered 4 107 polystyrene beads clustered most closely with 107 conidia at

2 Downloaded from 2 2 h. As administration of polystyrene beads to C57BL/6 mice elic- 0 0 ited less change than did conidia, it is possible that the type of 245 245 particle is important in the degree of up-regulation. A group of

Conidia/beads per AM %AM with conidia/beads %AM Conidia/beads per AM Time (h) eight genes was up-regulated at least 2-fold in C57BL/6 mice exposed to 107 polystyrene beads, and in both strains of mice FIGURE 1. In vivo AM-particulate engagement 2–5 h following intra- exposed to 107 conidia (Table III). The strongest transcriptional pharyngeal administration. A, Average percentage of AM containing A. changes in response to both conidia and polystyrene beads were http://www.jimmunol.org/ fumigatus conidia or polystyrene beads at the indicated time points in Ϫ Ϫ observed for genes encoding proteins important in PMN C57BL/6 (left panel) and gp91phox / mice (right panel). B, Average num- ber of conidia or polystyrene beads in AM involved in phagocytosis at time recruitment. points shown in A for the two mouse strains. Not all treatments were eval- Up-regulation of AM genes involved in PMN recruitment was uated at each time point, thus not all bars are shown. Replicates for conidia dependent on both conidial dose and duration of exposure in are n Ͼ 3, and for polystyrene beads n ϭ 3, with error bars indicating SEM. vivo After administration of 106 conidia to C57BL/6 mice, there were Ն2-fold ( p Ͻ 0.01) increases in transcription of Cxcl2 (MIP-2) at by guest on October 1, 2021 phagocytosis in the lung demonstrated that the average number of 2 h, and in Cxcl1 (KC), Cxcl2, and Il1b at 4 h (Table II). By particles within AM at the 4 h time point (after receiving 107 contrast in gp91phoxϪ/Ϫ mice receiving 106 conidia, no significant conidia) reached 5.3 Ϯ 0.17 (n ϭ 9) in C57BL/6 mice and 6.5 Ϯ increases in the genes encoding PMN recruiting molecules were Ϫ Ϫ 0.30 (n ϭ 4) in gp91phox / mice, while 4.7 Ϯ 0.88 (n ϭ 3) were observed at 2 and 4 h. After administration of 107 conidia, Cxcl1 observed in AM collected from C57BL/6 mice after inoculation and Cxcl2 were up-regulated in both strains at 2 h, while there was with 107 polystyrene beads (Fig. 1B). By comparison, an in vitro up-regulation of Il1b in C57BL/6 mice and Tnf in gp91phoxϪ/Ϫ study using A. fumigatus conidia and human monocytes, observed: mice. At 4 h after inoculation with 107 conidia, PMN were being 1) that approximately three times as many cells had engulfed recruited by Cxcl1, Cxcl2, Il1a, Il1b, and TNF-␣ in both mouse conidia at 4 h; 2) that the number of cells involved in phagocytosis strains (although Il1b in gp91phoxϪ/Ϫ mice minimally missed the was still increasing between 4 and 6 h; and 3) an average of only cutoff at a fold change of 1.9). The largest fold changes in the two conidia per phagocytosing AM at the 6-h time point (6). Such transcripts outlined above were observed at 4 h after 107 conidia in differences between those studies and our current data demonstrate C57BL/6 mice, when there were ϳ48- and 40-fold increases for the dependence of cell type and cellular environment on outcomes Cxcl1 and Cxcl2, respectively. In addition to these five chemokine when analyzing the response of phagocytic cells to A. fumigatus and cytokine genes involved in PMN recruitment, transcriptional conidia. changes in 42 additional genes with roles in the immune system were detected in our microarray analyses (Table II). Transcriptional analysis of AM in the presence and absence of A. fumigatus conidia Up-regulation of AM genes encoding oxidoreductases following To identify potential effector mechanisms used by AM to prevent exposure to conidia infection by A. fumigatus, AM were isolated from the lungs of both Due to the importance of a functional NADPH oxidase in resisting C57BL/6 and gp91phoxϪ/Ϫ mice (with or without conidia) for mi- aspergillosis, the list of AM oxidoreductase genes altered by ex- croarray analysis. In microarray studies comparing these two posure to conidia was evaluated. There was no up-regulation of mouse strains, the constitutive transcription in the absence of transcripts encoding cytosolic regulatory subunits of the NADPH conidia (time 0) was significantly different for many genes ( p Ͻ oxidase complex (p40phox, p47phox, p67phox, or Rac1/2) for any 0.05), demonstrating that elimination of functional NADPH oxi- condition, and similarly, there was no significant change in tran- dase has a marked effect on the basal AM transcriptome (data not scription of Cyba or Cybb (encoding the membrane-bound p22phox shown). This basic microarray characterization highlights the need and gp91phox subunits of flavocytochrome b-558, respectively). to account for basal transcriptional changes in studies using gene- We also observed no up-regulation of genes encoding the oxidant knockout mice, and is consistent with a broad role for superoxide scavengers superoxide dismutase or catalase under any conditions in signaling and regulation of gene transcription in AM (36, 37). following inoculation of either mouse strain with conidia. Genes The Journal of Immunology 6859

involved in cellular redox regulation that were altered included an increase in the transcript for heme oxygenase 1 (HO-1) HmoxI, marginal increases in the transcripts for glutathione reductase (Gsr) and thioredoxin reductase 1 (Txnrd1), and a down-regulation CXCl1 of the transcript for NADH dehydrogenase (ubiquinone) 1 ␤ sub- CXCl2 Egr1 complex, Ndufb7 (Table IV). AA408868 Ereg Ingenuity analysis of microarray data Plk2 1frd1 Ingenuity analysis was conducted on data from C57BL/6 mice to Slc20a1 explore relationships between AM genes involved in the response Il1b Socs3 to conidia. This analysis can identify relationships between genes Socs3 revealed by microarray analysis and overlay their corresponding Ccl4 expression levels (fold change), where the degree of up-regulation Bcl3 Tnf is indicated by the intensity of red color. None of the genes found Clecsf9 to be down-regulated by microarray analysis appeared in the net- Clecsf9 Gadd45b works. In the result of the network analysis, molecules or molec- Lcam1 ular complexes predicted by the program to participate in this re- Gadd45b Maff sponse, but not found significantly altered in microarray data, are 1810011010Rik included as well (symbols without color). Thus, Ingenuity analysis Downloaded from Cflar is capable not only of constructing associations of genes identified Cflar Cflar by microarray (including relative expression levels), but also of Txnrd1 predicting involvement of additional molecules not associated with Ccl3 Atf3 significant transcriptional changes. Rgs1 Using the Ingenuity search criteria described in Materials and Ccrl2 Methods, AM genes significantly altered in C57BL/6 mice 4 h

Il1a 7 http://www.jimmunol.org/ Ets2 after receiving 10 conidia segregated into three separate pathways Pde4b (Fig. 3) that differed in regard to the type of molecular response Nfkbia Gtpbp4 predicted by the program. Network A is involved primarily in im- Nfkbia mune response and contained many of the proinflammatory mol- Zfp36 ecules found up-regulated by microarray analysis. Network B in- Jun Hmox1 cluded responses involved in cell-to-cell signaling and cell Slc2a1 interaction. Molecules predicted to have the most complex neigh- Psmd10 ␣ ␤ Becn1 borhoods (hubs) in networks A and B were TNF- and IL-1 , both

Tde1 showing relationships with at least 20 other molecules in each of by guest on October 1, 2021 Nck1 the two networks. In network C, molecules (including NF-␬B, Gdap10 Nfatc1 p38MAPK, and AP-1) contributing primarily to early changes in Gsr gene expression are shown to form complex hubs, though none of Tob2 Tcf712 these three genes were identified by microarray data. Flt1 Ptp4a1 Verification of DNA microarray results by qRT-PCR Gopc Dnaja1 To better validate the microarray results reported in this study, Dhx9 transcriptional changes for several genes of particular interest Abcc5 Fkbp5 (Egr1, Ereg, Tnf, and Hmox1) were evaluated by qRT-PCR. All Fkbp2 qRT-PCR analysis was conducted on AM from mice treated as Grcc10 Mpdu1 indicated, but separate from those used for microarray analyses. Calm3 When the transcript levels of Egr1, Ereg, and Tnf were quantified 1110025Lo5Rik using qRT-PCR in the absence of conidia as well as 2, 4, and 5 h Mrpl23 7 Hist2h2aa1 after instillation of 10 conidia, increases measured for all three Ndufb7 genes determined by REST software (33) were found to parallel Ndufa8 Rab4b those observed by microarray analysis (Fig. 4). Furthermore, Rpl39 C57BL/6 mice showed more marked up-regulation than 0610010L17Rik gp91phoxϪ/Ϫ mice by qRT-PCR, which was consistent with our 2510027N19Rik 2610208E05Rik microarray data. qRT-PCR was also used to show a median fold Sdh1 increase of 5.4 for Hmox1 (SE 3.9–7.2) in C57BL/6 mice 4 h after Atp5k 7 Bloc1s1 inoculation with 10 conidia, which was similar to the 3.2-fold Fus increase found by microarray analysis. The Hmox1 transcript in AM from gp91phoxϪ/Ϫ mice showed a 1.3-fold increase at 4 h bl 0h gp 0hr bl 2h e6 bl 2h e7 bl 4h e6 bl 4h e7 gp 2h e6 gp 4h e6 gp 2h e7 gp 4h e7 bl 4h bead bl 4h mock gp 2h mock gp 4h mock bl 2h mock alone (Welch t test, Benjamini & Hochberg test correction, p Ͻ 0.01, fold FIGURE 2. Heat map showing in vivo differences in transcription of change Ն2). Replicates for conditions are: C57BL/6 mice receiving AM genes in C57BL/6 and gp91phoxϪ/Ϫ mice. The hierarchical tree reflects conidia (106 or 107) or mock, n ϭ 4; all gp91phoxϪ/Ϫ mice, n ϭ 3, C57BL/6 73 genes found significantly different by ANOVA. Data compare AM from mice receiving polystyrene beads, n ϭ 2. Abbreviations: gp, gp91phoxϪ/Ϫ; mice exposed to conidia with mock controls exposed to vehicle bl, C57BL/6; e6, 106 conidia; e7, 107 conidia; bead, 107 polystyrene beads. 6860 AM RESISTANCE TO ASPERGILLOSIS

Table III. Genes in AM up-regulated Ն2-fold at4hby107 A. fumigatus conidia and polystyrene beads

C57BL/6 C57BL/6 gp91phoxϪ/Ϫ Gene Name (Conidia) (Beads) (Conidia)

Cxcl2 Chemokine (cxc motif) ligand 1 39.8 13.4 4.3a Cxcl1 Chemokine (cxc motif) ligand 2 48.2 4.8 8.1 Egr1 Early growth response 1 28.1 4.3 4.4a Ereg Epiregulin 9.8 3.5 3.3 Clecsf9/Clec4e C-type lectin domain family 4, member e 6.2 2.9 2.1a Ccl3 Chemokine (cc motif) ligand 3 18.6 2.5 4.3a Plk2 Polo-like kinase 2 (Drosophila) 6.3 2.2 2.1 Socs3 Suppressor of cytokine signaling 3 12.0 2.3 2.4

a Constitutive expression was significantly greater in gp91phoxϪ/Ϫ mice than in C57BL/6 mice.

by microarray and a 1.8 (SE 1.8–2.4) fold increase by qRT- Functional analysis of the Egr1 gene product in response to PCR by 5 h. conidia 7 Validation of DNA microarray results by ELISA In light of the degree of up-regulation of Egr1 by 10 conidia in both C57BL/6 and gp91phoxϪ/Ϫ mice (28.1 and 4.4-fold increase, Downloaded from To examine the relationship between the observed transcription of respectively), and its connection with several other genes as pre- AM genes and levels of the protein product, three genes predicted dicted by Ingenuity analysis, responses of Egr1Ϫ/Ϫ mice (n ϭ 4) by Ingenuity analysis to play key roles and also known to encode and parental strain C57BL/6 mice (n ϭ 4) to conidia were com- ␣ ␤ proteins secreted into the BALF (TNF- , IL-1 , and CXCL2) pared. Administration of 107 conidia to Egr1Ϫ/Ϫ mice resulted in were chosen for analysis. CXCL2 protein concentrations in the 17.5 Ϯ 2.4% AM in the BALF with internalized conidia at 6 h

BALF of C57BL/6 mice were found to increase 22-fold by4hin http://www.jimmunol.org/ (compared with 22 Ϯ 0.6% in C57BL/6) ( p ϭ 0.16) and 6.5 Ϯ 0.6 mice receiving 107 conidia compared with those receiving sterile conidia/AM following phagocytosis (compared with 6.0 Ϯ 0.13% vehicle alone. In identically treated gp91phoxϪ/Ϫ mice, an increase in wild type) ( p ϭ 0.47). PMN recruitment was also not signifi- in CXCL2 protein of ϳ8-fold was found. Increases of CXCL2 Ϫ Ϫ cantly different between Egr1 / (1.6 ϫ 105 Ϯ 2.7 ϫ 104 PMN/ were found to be significant in both animal strains receiving lavage) and C57BL/6 mice (1.2 ϫ 105 Ϯ 1.5 ϫ 104 PMN/lavage), conidia relative to mock inoculated animals (n ϭ 5 p Ͻ 0.005), and and comparable levels of TNF-␣ were observed in the BALF of the increase in C57BL/6 mice was found to be significantly greater Ϫ/Ϫ Ϫ Ϫ both mouse strains (89 Ϯ 11.4 pg/ml for Egr1 mice and 124 Ϯ than in gp91phox / mice ( p ϭ 0.012). Following inoculation of 15.2 pg/ml for C57BL/6 mice, p ϭ 0.12). mice with 107 conidia, no changes in the concentrations of TNF-␣ or IL-1␤ protein in the BALF of C57BL/6 mice could be detected by guest on October 1, 2021 at 4 h, despite the increases in transcript levels observed in this Correlation of phagocyte superoxide production with the study. When these experiments were extended to 5 h, the concen- inhibition of conidial germination tration of TNF-␣ in the BALF of C57BL/6 mice was determined to be 7-fold higher in mice receiving conidia compared with those Our observation that TNF-␣ was increased at both the transcript phox mock-inoculated (n ϭ 4, p ϭ 0.012, Fig. 5). The TNF-␣ response and protein level indicated it could be up-regulating gp91 tran- was therefore slower in C57BL/6 mice following a dose of 107 scription and priming the respiratory burst in macrophages, as sug- conidia (this study), relative to what we previously observed in gested (31, 32). However, our microarray data did not identify BALB/c mice after receiving 3 ϫ 107 conidia (13). This apparent increased transcription in components of NADPH oxidase follow- delay in TNF-␣ production in C57BL/6 relative to BALB/c mice ing in vivo exposure to conidia, suggesting either that the NADPH may be linked to the slower PMN recruitment in C57BL/6 mice fol- oxidase components were preformed and activated following con- lowing conidial challenge (13). The conidia-treated gp91phoxϪ/Ϫ mice tact with conidia, or that they were not used in this response. Be- showed levels of TNF-␣ in the BALF similar to those measured in cause the molecular mechanisms by which phagocytes block ger- inoculated C57BL/6 mice ( p ϭ 0.3, Fig. 5). In contrast to results mination of A. fumigatus in the lung remain to be fully obtained with TNF-␣ and CXCL2, no measurable increase in the characterized, and to better describe innate response of AM to concentration of IL-1␤ was observed up to5hinthepresent study conidia, a functional role of superoxide production in this process (data not shown). was examined to complement transcriptional results of this study.

Table IV. Genes in AM which encode oxidoreductases showing Ն2-fold changes (bolded) in transcription following inhalation of A. fumigatus conidia

Fold Change

2-h 106 C57BL/6 4-h 106 Mice 2-h 107 4-h 107 2-h 106 gp91phoxϪ/Ϫ 4-h 106 Mice 2-h 107 4-h 107 Gene Conidia Conidia Conidia Conidia Conidia Conidia Conidia Conidia

Hmox1 1.5 1.5 1.9 3.2 0.6 0.4 0.9 1.3 Gsr 1.2 1.8 0.9 2.2 0.9 1.8 1.0 1.2 Txnrd1 1.2 1.7 1.3 2.0 1.0 1.2 1.2 1.9 Ndufb7 0.8 0.6 1.0 0.8 1.0 0.5 0.8 1.1 The Journal of Immunology 6861 A B CXCL1

CXCL2 Downloaded from http://www.jimmunol.org/

Legend: C cytokine ABbinding only enzyme ABinhibits by guest on October 1, 2021 ABacts on group or complex ABinhibits/acts on transcription regulator kinase ABtranslocates to direct interaction other indirect interaction transmembrane receptor

FIGURE 3. AM gene network analysis by Ingenuity. Analysis of microarray data by Ingenuity predicts three networks showing molecular interactions of AM genes from C57BL/6 mice 4 h after inoculation with 107 A. fumigatus conidia. Primary molecular responses identified by the analysis were: A, immune response; B, cell-to-cell signaling and interaction; and C, gene expression. Molecule symbols are marked with intensity of red color to indicate the relative degree of transcriptional up-regulation.

To measure extracellular superoxide production by AM from detect low-level NADPH oxidase activity. When exposed to 910 C57BL/6 and gp91phoxϪ/Ϫ mice following exposure to conidia, a nM PMA, AM from C57BL/6 but not gp91phoxϪ/Ϫ mice showed a sensitive MCLA-dependent luminometry method (35) was used to small but significant early increase in superoxide release ( p ϭ 6862 AM RESISTANCE TO ASPERGILLOSIS

Microarray qRT-PCR A C57Bl/6 AM Egr1 140 30 80 C57Bl/6 AM +conidia C57BL C57Bl/6 AM +PMA 25 gp91phox-/- 120 phox-/- 60 gp91 AM 20 15 40 100 10 20 80 5 0 0 60 2 4 Ereg 2 3 4 5 40 12 150

10 20 8 100 6 0 4 50 2 -20 0 10 20 30 Fold change 0 0 2 4 2 3 4 5 Tnf 5 40 B

5000 Downloaded from 4 30 3 20 C57Bl/6 PMN Relative Light Units (RLU) Relative 4000 2 gp91phox-/- PMN 10 1

0 0 3000 2 4 2 3 4 5

Time (h) after administration of conidia http://www.jimmunol.org/ 2000 FIGURE 4. Comparison of microarray and qRT-PCR transcriptional data for select AM genes following administration of 107 A. fumigatus conidia to C57BL/6 mice and gp91phoxϪ/Ϫ mice. Microarray data (left pan- 1000 els) show fold changes in transcription at 2 and 4 h, while qRT-PCR data (right panels) show median fold change examined at 2, 4, and 5 h. For ϭ 0 qRT-PCR, error bars indicate SE of median, n 4. 0 10 20 30 minutes by guest on October 1, 2021 0.014 at 3 min), which subsided within ϳ15 min and became in- FIGURE 6. Superoxide release from murine alveolar phagocytes de- distinguishable from C57BL/6 AM without PMA stimulation (Fig. tected by luminometry. All data represent the superoxide release from 105 6A). The relatively high concentration of 910 nM PMA was used phagocytes using MCLA-dependent luminometry, displayed in relative to elicit maximal response from phagocytes, but did not produce light units (RLU). A, AM collected from the BALF of naive C57BL/6 or phoxϪ/Ϫ measurable nonspecific effects in gp91phoxϪ/Ϫ cells that lack the gp91 mice were incubated in DMEM-10 for 90 min at 37°C in 5% CO and then examined for 30 min by luminometry. Adherent C57BL/6 functional NADPH oxidase. Surprisingly, when AM from 2 AM were examined alone, after stimulation by 910 nM PMA, and after C57BL/6 mice were exposed to a 5-fold excess of freshly har- incubation with a 5-fold excess (5 ϫ 105)ofA. fumigatus conidia. Signal vested conidia for 90 min at 37°C, the signal from superoxide of superoxide release from gp91phoxϪ/Ϫ AM alone is shown, and is not release was reduced, making it indistinguishable from that of AM distinguishable from that of gp91phoxϪ/Ϫ AM exposed to conidia or 910 nM phoxϪ/Ϫ isolated from gp91 mice. In these superoxide-production PMA (data not shown). B, PMN were examined by luminometry for com- parison to results obtained with AM under similar conditions. PMA-de- pendent superoxide release from LPS-recruited murine PMN is shown for C57BL/6 and gp91phoxϪ/Ϫ mice. Error bars indicate SEM (n ϭ 5) and 500 C57Bl/6-mock similar results were obtained in three additional experiments. phox-/- 400 gp91 -mock C57Bl/6-conidia phox-/- 300 gp91 -conidia assays, PMA-triggered alveolar PMN from C57BL/6 mice gener- 200 ated substantially higher levels of superoxide compared with C57BL/6-derived AM (Fig. 6B), while superoxide production was 100 phoxϪ/Ϫ

BALF protein(pg/ml) insignificant for PMN from gp91 mice. 0 Because functional and transcriptional analysis of AM respond- TNF-α CXCl2 ing to A. fumigatus conidia did not indicate obvious NADPH ox- idase involvement (in contrast to a report for murine peritoneal ␣ FIGURE 5. Comparison of TNF- and CXCL2 concentrations in macrophages responding to the Streptococcus pyogenes bacterium BALF from C57BL/6 and gp91phoxϪ/Ϫ mice after intrapharyngeal admin- (38)), we next examined AM from mice exposed to conidia in vivo istration of 107 A. fumigatus conidia. ELISA was used to measure the TNF-␣ and CXCL2 concentration in the first 2 ml of BALF collected from for evidence of intracellular superoxide production. To assess in- each mouse. For TNF-␣ measurements, BALF was collected 5 h after dose tracellular superoxide production, phagocytosing AM from phoxϪ/Ϫ (conidia or mock treatment) and BALF was collected 4 h after dose for C57BL/6 and gp91 mice collected in BALF 12 h after in- 6 CXCL2 measurements. Error bars indicate SEM (p Ͻ 0.05 for each strain stillation of 10 conidia were exposed to NBT and examined by of inoculated mice compared with uninoculated controls). microscopy. Despite the presence of functional NADPH oxidase in The Journal of Immunology 6863

ment in aggregates (Fig. 7A, panel 6). AM in the vicinity of the PMN shown in panel 6 (arrows) uniformly failed to produce formazan when in the presence of both conidia and PMA, which was consistent with our luminometry data shown in Fig. 6A. The in vivo studies outlined above comparing C57BL/6 and gp91phoxϪ/Ϫ mice suggest the phagocyte NADPH oxidase com- plex does not play a major role in the innate response of AM to A. fumigatus conidia, extending information we reported previously (13). However, it is possible that AM-independent influences in the mouse lung contribute to suppression of conidial germination inside AM. We therefore used an in vitro assay to determine whether a difference exists in the ability of AM from gp91phoxϪ/Ϫ and C57BL/6 mice to inhibit germination of phagocytosed conidia (Fig. 7B). Our results indicate that 8 h following exposure of AM to conidia in a tissue culture system (at which time conidia outside AM show abundant germination) inhibition of germination inside AM appeared equally effective for the two mouse strains, relative to conidia germinating outside the AM ( p Ͻ 0.0002). These results demonstrate that functional NADPH oxidase is not required by AM to prevent conidial germination under these in vitro assay Downloaded from conditions. This information suggests AM neither use nor require superoxide production by the NADPH oxidase to protect from as- pergillosis in the mouse lung.

Discussion http://www.jimmunol.org/ The goal of this study was to examine the responses of AM fol- lowing inoculation of mice with A. fumigatus conidia to better understand the innate resistance to IPA. To this end, we used a combination of in vivo and in vitro analyses to provide information that reflects the complexity of the innate response to A. fumigatus FIGURE 7. AM suppress conidial germination independently of conidia in the context of the lung. In vivo transcriptional changes NADPH oxidase activity. A, Following instillation of 107 A. fumigatus of AM responding to conidia were first determined by microarray conidia and in vivo incubation, BALF AM from C57BL/6 (panels 1 and 3) and Ingenuity analysis, with qRT-PCR verification of changes for phoxϪ/Ϫ by guest on October 1, 2021 or gp91 (panels 2 and 4) mice were found to contain conidia but no selected genes. TNF-␣ and CXCL2 were also correlated to protein evidence of NADPH oxidase-dependent formazan deposition. After instil- levels in the BALF. Of the 73 gene probes showing Ն2-fold lation of resting conidia and 12 h in vivo incubation, conidia were found to Ϫ Ϫ changes in transcription in AM from C57BL/6 and gp91phox / be ungerminated in both mouse strains (panels 1 and 2). Panels 3 and 4 show AM collected from mice 4 h after instillation of preswollen and mice responding to conidia (data not shown), 47 actual genes were germinating conidia. In contrast, BALF PMN from C57BL/6 mice were classified by DAVID Bioinformatics Resources 2007 Functional found to engage conidia with evidence of formazan deposition (panel 5), as Annotation as being involved in the immune response (Table II). did all BALF PMN (but not AM) from C57BL/6 mice after stimulation In addition, all significantly altered genes in C57BL/6 mice were with 910 nM PMA (panel 6, arrows show AM and arrowhead identifies examined by Ingenuity analysis to help predict key biochemical AM containing conidia). Scale bars represent 10 ␮m. B, In vitro suppres- networks involved in this early response phase. To our knowledge, sion of conidial germination following phagocytosis in AM from C57BL/6 the present study represents the first global analysis of transcrip- phoxϪ/Ϫ and gp91 mice. Resting conidia were combined with AM collected tional changes for AM in response to A. fumigatus conidia in from naive mice and incubated in vitro for8hat37°C in 5% CO . Ger- 2 the lung. mination percentages both inside and outside the AM were then determined Phagocytic leukocytes contain a multisubunit NADPH oxidase by microscopy following cytospin and Wright staining. Error bars indicate SEM (n Ն 5). complex that generates superoxide which serves as a precursor for a set of microbicidal reactive oxygen and nitrogen species. Be- cause superoxide has also been shown to regulate signal transduc- AM derived from C57BL/6 mice, no obvious difference could be tion cascades that alter gene transcription (37, 40), the responses of found in formazan deposition within AM obtained from the two AM to conidia in C57BL/6 mice (which are generally resistant to mouse strains engulfing resting conidia (Fig. 7A, panels 1 and 2). IPA) were compared with those in susceptible gp91phoxϪ/Ϫ mice In a previous study, oxidant production was observed when (12) by microarray analysis. In these studies, we observed strong A. fumigatus germlings were engaged by bone marrow derived differences in basal expression of numerous genes when compar- macrophages (39). We therefore inoculated mice with swollen ing C57BL/6 and gp91phoxϪ/Ϫ mice, even before exposure to and germinating conidia and found AM from C57BL/6 and conidia. These differences could be due in part to compensatory gp91phoxϪ/Ϫ mice 4 h after in vivo incubation to be indistinguish- responses in gp91phoxϪ/Ϫ mice, which lack metabolic regulatory able with regard to formazan deposition in the vicinity of engulfed mechanisms contributed by NADPH oxidase activity (41). In sup- developing conidia (Fig. 7A, panels 3 and 4). For comparison, port of this hypothesis, differences in constitutive gene transcrip- obvious formazan was found among PMN aggregates formed in tion have also been found in PMN from human patients with X- vivo in C57BL/6 mice in response to conidia (13) (Fig. 7A, panel linked chronic granulomatous disease compared with normal 5). Formazan deposition was also observed in PMN from C57BL/6 individuals (42). Recently, the altered inflammatory response of mice when exposed to 910 nM PMA, regardless of their involve- chronic granulomatous disease patients to pathogens including A. 6864 AM RESISTANCE TO ASPERGILLOSIS fumigatus has been linked to dysregulation of tryptophan metab- and CXCL2 protein in the BALF. Increased expression of Tnf was olism and alteration of T cell subsets (43). characterized in detail at both the transcript and protein level due Despite marked differences in constitutive expression of many to its predicted connection with other molecules in the response of genes in the C57BL/6 and gp91phoxϪ/Ϫ mouse strains used in the AM exposed to conidia, as well as its role in both regulation of present study, the innate response of the parental C57BL/6 strain gp91phox gene expression and priming the respiratory burst in mac- to conidia was conserved in a subset of genes in gp91phoxϪ/Ϫ mice. rophages and PMN (31). Increases in TNF-␣ protein production in However, a stronger overall transcriptional response to conidia response to A. fumigatus conidia have been reported in mouse and was observed in AM from C57BL/6 than in gp91phoxϪ/Ϫ mice. rat AM in vitro (46, 47, 57), and in mouse lung tissue (58). In- This difference does not appear to be due to greater phagocytosis creased levels of TNF-␣ have also been found in the BALF after of conidia by AM from C57BL/6 than gp91phoxϪ/Ϫ mice, as AM inhalation of A. fumigatus conidia (13, 44), but this TNF-␣ is prob- from the two strains were not significantly different with respect to ably not secreted only by AM (which represent the main source of the percentage of AM containing conidia. In fact, the average num- this cytokine in the lungs) but also by epithelial, mesenchymal and ber of conidia per AM was greater in gp91phoxϪ/Ϫ than in dendritic cells (59). In the present study using C57BL/6 mice, a C57BL/6 mice at both 2 and4h(p ϭ 0.0002 and 0.02, respec- delay was observed between increased transcription of Tnf and a tively). Approximately 40% of the genes up-regulated in response measurable increase in TNF-␣ protein, consistent with a similar to conidia in both strains of mice were also increased in C57BL/6 study using C57BL/6 mice (22). ELISA also revealed 22- and mice after administration of 107 polystyrene beads, suggesting 8-fold increases in CXCL2 protein in the BALF 4 h after exposure some of these changes in gene transcription occur as a general to 107 conidia, corresponding to 40- and 4-fold transcriptional up- response to inhaled particulates. regulation of the Cxcl2 gene in C57BL/6 and gp91phoxϪ/Ϫ mice, Previous experimental evidence has shown that phagocytic cells respectively. Downloaded from respond to A. fumigatus conidia by producing proinflammatory In contrast to results with TNF-␣ and CXCL2, we were unable chemokines and cytokines involved in PMN recruitment (6, 13, to detect significant changes in IL-1␤ concentration in the BALF 44–46). Our microarray data from C57BL/6 mice extend those up to 5 h after administration of conidia to C57BL/6 mice, al- studies by indicating the strongest increases in AM transcription though IL-1 protein secretion has been reported to increase fol- are in the chemokine genes Cxcl1 and Cxcl2. In the present mi- lowing prolonged exposure of murine AM to conidia (22, 60, 61). croarray study, increases in the transcripts for Tnf, Il1a, and Il1b These observations are in agreement with other reports showing http://www.jimmunol.org/ (genes that have additional roles in PMN recruitment (47, 48)) difficulty correlating IL-1␤ mRNA to protein levels (8, 44). In this were also observed. The above findings for AM exposed to conidia study, the lack of a corresponding increase in IL-1␤ protein con- in the mouse lung support the notion that early transcriptional centration in the BALF at 5 h despite more marked up-regulation changes observed in mouse AM represent a general proinflamma- of this gene transcript relative to that observed for TNF-␣ at4h tory response of mononuclear phagocytes that triggers PMN re- could indicate posttranscriptional regulation for expression of this cruitment. Exposure of AM to conidia in the lung also up-regulated gene. The difference in the results when comparing TNF-␣, IL-1␤, genes involved in monocyte recruitment, including Ccl3, which and CXCL2 (measured using comparable methods) emphasizes encodes Ccl3 (MIP-1␣), a chemoattractant for circulating mono- the fact that increased transcription may not necessarily be directly by guest on October 1, 2021 cytes (49, 50), and the chemokine receptor-like 2 gene (Ccrl2), linked with translation (62). which encodes a receptor for chemokines such as Ccl2 (51). In Previous studies have shown that some pathogens can overcome light of these results it is interesting to note that in BALB/c mice, natural resistance to infection by suppression of the host immune we previously found no evidence of a net increase in AM numbers response. The present study showed that exposure of both in the BALF until 24 h after intratracheal administration of 3 ϫ C57BL/6 and gp91phoxϪ/Ϫ mice to conidia caused up-regulation of 107 conidia, though PMN recruitment was evident within 3 h (13). two immune suppressive AM genes, Rgs1 and Socs3. The regula- Ingenuity analysis is a tool for comparing novel molecular data tor of G-protein signaling 1 (Rgs1) protein product has previously sets to a library of knowledge-based cellular and molecular re- been shown to play a role in deactivation of signaling through sponses. Through our examination of data from AM of C57BL/6 chemoattractant receptors (63), while the suppressor of cytokine mice, this method of data mining revealed particular complexity in signaling (Socs3) protein product has been shown to mediate neg- network neighborhoods for TNF-␣ and IL-1␤ (Fig. 3, networks ative feedback effects on IL-6 responses (64). It is important to A and B), supporting their importance in the inflammatory re- emphasize that the C57BL/6 mice used in our studies were not sponse to fungal pathogens including Aspergillus (8, 52, 53). specifically immune suppressed and have been shown to be gen- Our Ingenuity analysis also revealed molecular components un- erally resistant to aspergillosis. Therefore in animals with normal derrepresented in microarray data, or possibly those that did not immune status, anti-inflammatory effects induced by A. fumigatus participate as expected. For example, we did not observe increased conidia resulting from increased expression of Socs3 or Rgs1 transcription for p38 MAPK and NF-␬B in our microarray data, would be unlikely to lead to infection unless additional levels of though each was predicted by Ingenuity analysis to participate immune dysfunction (such as neutropenia) also existed. For this with several genes that were transcriptionally activated (Fig. 3, reason, responses in AM leading to anti-inflammatory effects may network C). Our Ingenuity analysis results support the importance be more likely to identify mechanisms by which the inflammatory of NF-␬B in aspergillosis immunity, as reduced nuclear NF-␬B response in the lung is attenuated to reduce tissue damage, rather translocation in macrophages was previously implicated in in- than those triggered by the fungus to overcome the immune re- creased susceptibility to infection following dexamethazone treat- sponse. It remains to be determined whether any host-induced anti- ment (54), and in sepsis (55). Our data are also consistent with a inflammatory responses triggered by conidia in AM are exploited role for both NF-␬B and p38 MAPK in dendritic cells following by the organism to cause disease. contact with A. fumigatus (56). Other than for Egr-1, the extent to Several lectin-like receptors on leukocytes have previously been which other hubs identified by Ingenuity analysis are important to reported to participate in binding of A. fumigatus conidia or hyphae aspergillosis immunity were not examined in the present study. (22, 39, 65). Our in vivo data from C57BL/6 mice indicated a The increased Tnf and Cxcl2 transcription observed by microar- 6.2-fold up-regulation of the transcript for Clecsf9 4 h after 107 ray was confirmed using ELISA by increased secretion of TNF-␣ conidia. Clecsf9 (Clec4e; macrophage-inducible C-type lectin The Journal of Immunology 6865

(Mincle)) encodes a group-II Ca2ϩ-dependent transmembrane lec- Hmox1 after contact with the bacterium, but showed both tran- tin with a role in inflammation and immunity (66), but has not been scriptional up-regulation and functional evidence of p47phox of the previously implicated in responses to conidia. In addition to being NADPH oxidase (38). up-regulated in AM by conidia, Clecsf9 was also up-regulated by Despite the requirement of a functional NADPH oxidase com- polystyrene beads, though to a lesser extent than by conidia. plex in humans for resistance to A. fumigatus, conflicting reports Additional novel findings in the present study were up-regula- exist concerning both the level of superoxide generated by AM and tion of Ereg and Egr1 in both strains of mice following exposure the role of superoxide in fungal killing by AM (12, 14–17). It is to conidia, and in C57BL/6 mice exposed to polystyrene beads. possible that differences in phagocyte types, animal strains, and Ereg encodes epiregulin, a transmembrane protein that is cleaved cell sources have contributed to contrasting interpretations in these by the metalloproteinase ADAM17, to form a soluble 5.5-kDa epi- studies. In the present study using a sensitive luminometry assay dermal growth factor receptor agonist that can exert a proliferative system, we identified low but significant NADPH oxidase activity or dedifferentiating autocrine effect in AM (67, 68). Previous stud- in adherent AM from C57BL/6 mice. We also observed this ad- ies have shown that Egr1, which encodes early growth response 1, herence-dependent superoxide production in AM incubated in 96- is up-regulated in human monocytes exposed to conidia in vitro well tissue culture plates using NBT to detect oxidant release (data (6), but not as strongly as we observed in murine AM isolated from not shown). The luminometry signal showing superoxide produced the lung. Early growth response-1 is a zinc-finger transcription by adherent AM was increased only slightly by exposure to 910 factor that binds enhancer elements of Tnf, Cxcl2, Ccl3, Gadd45, nM PMA (a concentration that did not elicit an increased signal in ␤ ␬ Ϫ Ϫ IL-1 , M-CSF, VEGF, NF- B, jun-D,c-myc, gene 475,c-myb, and AM from gp91phox / mice), which was strongly in contrast to the Egr1 (69–72), raising the possibility that some of the genes up-

behavior of PMN where robust superoxide generation was ob- Downloaded from regulated in AM by conidia may be responding to Egr1. Despite served in the presence of PMA. An almost 2-log difference be- the marked up-regulation of Egr1 by conidia observed in this study tween superoxide production by PMA-simulated AM relative to and suggested involvement of this gene by Ingenuity analysis, our that in PMN suggests that even small numbers of PMN that con- functional characterization of Egr1Ϫ/Ϫ mice (6 h after administra- 7 taminate AM samples could lead to a significant overestimation of tion of 10 conidia) indicated the lack of this gene product did not superoxide generation in AM. Thus, care was taken in the present significantly: 1) affect phagocytosis of conidia by AM; 2) influence

study to isolate AM samples before recruitment of PMN into the http://www.jimmunol.org/ the levels of TNF-␣ secretion into the BALF; or 3) delay PMN lung, and to remove nonadherent PMN from AM samples during recruitment into the lung. These results support the notion that a medium replacement before luminometry analysis. Using the lu- multiple levels of immune suppression are needed to predispose minometry assay system outlined above, we found no increase in to infections by A. fumigatus (73). Further studies are required superoxide generation in AM incubated 90–180 min with A. fu- to identify the potential role of Egr1 in the immune response to migatus conidia in vitro. In fact, the presence of a five-fold excess A. fumigatus. of conidia abolished all SOD-inhibitable NADPH oxidase activity Relevant to our analysis of superoxide production by AM in the in adherent AM. It is not known if this result is related to the present study, it is of interest to emphasize that while there was a reduced oxidant production from PMN following exposure to A. general lack of transcriptional change in genes involved in the fumigatus hyphal culture filtrates (79). Additional studies are by guest on October 1, 2021 generation and removal of oxidants (such as SOD, catalase, and therefore required to better understand the mechanism by which NADPH oxidase subunits), we measured the up-regulation of conidia influence the luminometry signal resulting from superox- Hmox1 (encoding HO-1) in C57BL/6 mice in response to A. fu- migatus conidia. HO-1 uses NADPH to convert heme to carbon ide produced by these phagocytes. monoxide and biliverdin, with both products showing anti- Importantly, we observed a lack of formazan deposition within inflammatory effects in cells (74, 75). Because both heme and AM following phagocytosis of conidia in vivo, which supports our NADPH are also required for function of gp91phox, it is possible hypothesis that AM do not use NADPH oxidase activity to neu- that HO-1 activity in AM competes with, and therefore suppresses tralize A. fumigatus conidia. The lack of obvious superoxide pro- NADPH oxidase activity. A previous study in RAW 264.7 mouse duction in AM containing conidia was in contrast to results ob- macrophages showed HO-1 indeed suppresses the activity of tained with PMN aggregates, where abundant formazan deposition NADPH oxidase through heme degradation and proteasome-de- is readily apparent in the vicinity of engaged conidia, and is of ␣ pendent degradation of gp91phox (76). In support of this view, we interest in light of increased production of TNF- (a known prim- did not observe an increase of Hmox1 in AM from gp91phoxϪ/Ϫ ing agent for superoxide production) by AM. It is possible that mice responding to conidia by either microarray or qRT-PCR anal- mechanisms in AM have evolved to suppress NADPH oxidase ysis. In fact, the strongest down-regulation of any immune func- activity in vivo, thus reducing tissue damage triggered by inhala- tion gene shown in Table II was for Hmox1, where it occurred in tion of airborne particulates. Because AM appear able to neutralize gp91phoxϪ/Ϫ mice 4 h following exposure to 106 conidia. These A. fumigatus conidia by NADPH oxidase-independent mecha- results raise the possibility that Hmox1 up-regulation is influenced nisms, it is unclear why AM do not appear capable of neutralizing by, and may in fact act to suppress a functional NADPH oxidase the conidial exposure that predisposes IPA in neutropenic or in AM. Further concerning the NADPH oxidase complex, the lack chronic granulomatous disease patients. Thus, additional studies of up-regulation observed for Cybb (encoding the gp91phox subunit are required to better determine which microbicidal mechanisms of flavocytochrome b558) following exposure to conidia is consis- are overcome in AM, PMN, or possibly nonphagocytic cells (80), tent with a report in human monocytes exposed to conidia (6), that enable conidia to germinate and lead to infections in immune although an increase in expression of this same gene was previ- suppressed individuals. ously observed in macrophages or macrophage cell lines in re- In summary, our results indicate that in murine AM exposed to sponse to LPS (18), Mycobacterium tuberculosis (77), Chlamydia A. fumigatus conidia in vivo, the most marked early transcriptional pneumoniae (78), and TNF-␣ (31). Our DNA microarray results changes occur in genes involved in PMN recruitment. This coop- differed in two respects when compared with an in vivo study on eration between AM and PMN in aspergillosis immunity appears peritoneal macrophages in response to S. pyogenes. Specifically, to be vital because both PMN and NADPH oxidase activity are the peritoneal macrophages did not show increased transcription of essential for preventing IPA (13, 81). As an apparent balance to 6866 AM RESISTANCE TO ASPERGILLOSIS this inflammatory response in AM, Socs3 and Rgs1 are also up- ences in the expression profiles of primary human monocytes, monocyte-derived regulated, presumably moderating the inflammatory response to macrophages, and alveolar macrophages. J. Leukocyte Biol. 81: 328–335. 20. Srivastava, M., S. Jung, J. Wilhelm, L. Fink, F. Buhling, T. Welte, R. M. Bohle, reduce damage to the lung. Of note, was also an observed paucity W. Seeger, J. Lohmeyer, and U. A. Maus. 2005. The inflammatory versus con- of conidia-induced transcription of AM genes encoding proteins stitutive trafficking of mononuclear phagocytes into the alveolar space of mice is associated with drastic changes in their gene expression profiles. J. Immunol. 175: associated with phagocytosis, pattern recognition receptors, oxi- 1884–1893. dant scavengers and specific microbicidal systems, which have 21. Woodruff, P. G., L. L. Koth, Y. H. Yang, M. W. Rodriguez, S. Favoreto, sometimes been described in leukocytes following in vitro expo- G. M. Dolganov, A. C. Paquet, and D. J. Erle. 2005. A distinctive alveolar macrophage activation state induced by cigarette smoking. Am. J. Respir. Crit. sure to conidia. We observed conserved transcriptional responses Care Med. 172: 1383–1392. phoxϪ/Ϫ related to innate immunity in AM of gp91 mice in response 22. Steele, C., R. R. Rapaka, A. Metz, S. M. Pop, D. L. Williams, S. Gordon, to conidia, despite marked differences in constitutive transcription J. K. Kolls, and G. D. Brown. 2005. The ␤-glucan receptor dectin-1 recognizes compared with control C57BL/6 mice. In addition, lack of func- specific morphologies of Aspergillus fumigatus. PLoS. Pathog. 1: e42. phoxϪ/Ϫ 23. Hohl, T. M., H. L. Van Epps, A. Rivera, L. A. Morgan, P. L. Chen, tional NADPH oxidase in gp91 mice exposed to conidia M. Feldmesser, and E. G. Pamer. 2005. Aspergillus fumigatus triggers inflam- did not significantly affect the phagocytic ability of AM or their matory responses by stage-specific ␤-glucan display. PLoS. Pathog. 1: e30. ability to inhibit germination of internalized conidia. The findings 24. Alberts, R., P. Terpstra, M. Hardonk, L. V. Bystrykh, H. G. de, R. Breitling, J. P. Nap, and R. C. Jansen. 2007. A verification protocol for the probe sequences in this study support the concept that a lack of superoxide produc- of Affymetrix genome arrays reveals high probe accuracy for studies in mouse, tion by the NADPH oxidase in PMN, rather than AM, is the key human and rat. BMC Bioinformatics 8: 132–142. defect that contributes susceptibility to IPA in chronic granuloma- 25. Bolstad, B. M., R. A. Irizarry, M. Astrand, and T. P. Speed. 2003. A comparison of normalization methods for high density oligonucleotide array data based on tous disease patients (10). variance and bias. Bioinformatics 19: 185–193. 26. Dennis, G., Jr., B. T. Sherman, D. A. Hosack, J. Yang, W. Gao, H. C. Lane, and R. A. Lempicki. 2003. DAVID: database for annotation, visualization, and inte- Downloaded from Disclosures grated discovery. Genome Biol. 4: 3. The authors have no financial conflict of interest. 27. Pfaffl, M. W. 2001. A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res. 29: 2002–2007. 28. Livak, K. J., and T. D. Schmittgen. 2001. Analysis of relative gene expression References data using real-time quantitative PCR and the 2Ϫ⌬⌬C(T) method. Methods 25: 1. Ampel, N. M. 1996. Emerging disease issues and fungal pathogens associated 402–408. with HIV infection. Emerg. Infect. Dis. 2: 109–116. 29. Radonic, A., S. Thulke, I. M. Mackay, O. Landt, W. Siegert, and A. Nitsche.

2. Maertens, J., M. Vrebos, and M. Boogaerts. 2001. Assessing risk factors for 2004. Guideline to reference gene selection for quantitative real-time PCR. Bio- http://www.jimmunol.org/ systemic fungal infections. Eur. J. Cancer Care 10: 56–62. chem. Biophys. Res. Commun. 313: 856–862. 3. Dykewicz, C. A. 2001. Hospital infection control in hematopoietic stem cell 30. Thellin, O., W. Zorzi, B. Lakaye, B. B. De, B. Coumans, G. Hennen, T. Grisar, transplant recipients. Emerg. Infect. Dis. 7: 263–267. A. Igout, and E. Heinen. 1999. Housekeeping genes as internal standards: use and 4. Walsh, T. J., and A. H. Groll. 1999. Emerging fungal pathogens: evolving chal- limits. J. Biotechnol. 75: 291–295. lenges to immunocompromised patients for the twenty-first century. Transpl. 31. Gauss, K. A., L. K. Nelson-Overton, D. W. Siemsen, Y. Gao, F. R. DeLeo, and Infect. Dis. 1: 247–261. M. T. Quinn. 2007. Role of NF-␬B in transcriptional regulation of the phagocyte 5. Latge, J. P. 2001. The pathobiology of Aspergillus fumigatus. Trends Microbiol. NADPH oxidase by tumor necrosis factor-␣. J. Leukocyte Biol. 82: 729–741. 9: 382–389. 32. Phillips, W. A., M. Croatto, and J. A. Hamilton. 1990. Priming the macrophage 6. Cortez, K. J., C. A. Lyman, S. Kottilil, H. S. Kim, E. Roilides, J. Yang, ␣ ␥ B. Fullmer, R. Lempicki, and T. J. Walsh. 2006. Functional genomics of innate respiratory burst with IL-4: enhancement with TNF- but inhibition by IFN- . host defense molecules in normal human monocytes in response to Aspergillus Immunology 70: 498–503. 33. Pfaffl, M. W., G. W. Horgan, and L. Dempfle. 2002. Relative expression software fumigatus. Infect. Immun. 74: 2353–2365. by guest on October 1, 2021 7. Brummer, E., J. H. Choi, and D. A. Stevens. 2005. Interaction between conidia, tool (REST) for group-wise comparison and statistical analysis of relative ex- lung macrophages, immunosuppressants, proinflammatory cytokines and tran- pression results in real-time PCR. Nucleic Acids Res. 30: e36. scriptional regulation. Med. Mycol. 43(Suppl. 1): S177–S179. 34. Calvano, S. E., W. Xiao, D. R. Richards, R. M. Felciano, H. V. Baker, R. J. Cho, 8. Simitsopoulou, M., E. Roilides, C. Likartsis, J. Ioannidis, A. Orfanou, R. O. Chen, B. H. Brownstein, J. P. Cobb, S. K. Tschoeke, et al. 2005. A network- F. Paliogianni, and T. J. Walsh. 2007. Expression of immunomodulatory genes in based analysis of systemic inflammation in humans. Nature 437: 1032–1037. human monocytes induced by voriconazole in the presence of Aspergillus fu- 35. Rinaldi, M., P. Moroni, M. J. Paape, and D. D. Bannerman. 2007. Evaluation of migatus. Antimicrob. Agents Chemother. 51: 1048–1054. assays for the measurement of bovine neutrophil reactive oxygen species. Vet. 9. Forman, H. J., H. Zhou, E. Gozal, and M. Torres. 1998. Modulation of the al- Immunol. Immunopathol. 115: 107–125. veolar macrophage superoxide production by protein phosphorylation. Environ. 36. Gwinn, M. R., and V. Vallyathan. 2006. Respiratory burst: role in signal trans- Health Perspect. 106(Suppl. 5): 1185–1190. duction in alveolar macrophages. J. Toxicol. Environ. Health B Crit. Rev. 9: 10. Johnston, R. B., Jr. 2001. Clinical aspects of chronic granulomatous disease. 27–39. Curr. Opin. Hematol. 8: 17–22. 37. Buetler, T. M., A. Krauskopf, and U. T. Ruegg. 2004. Role of superoxide as a 11. Almyroudis, N. G., S. M. Holland, and B. H. Segal. 2005. Invasive aspergillosis signaling molecule. News Physiol. Sci. 19: 120–123. in primary immunodeficiencies. Med. Mycol. 43(Suppl. 1): S247–S259. 38. Goldmann, O., M. von Kockritz-Blickwede, C. Holtje, G. S. Chhatwal, 12. Morgenstern, D. E., M. A. Gifford, L. L. Li, C. M. Doerschuk, and M. C. Dinauer. R. Geffers, and E. Medina. 2007. Transcriptome analysis of murine macrophages 1997. Absence of respiratory burst in X-linked chronic granulomatous disease in response to infection with Streptococcus pyogenes reveals an unusual activa- mice leads to abnormalities in both host defense and inflammatory response to tion program. Infect. Immun. 75: 4148–4157. Aspergillus fumigatus. J. Exp. Med. 185: 207–218. 39. Gersuk, G. M., D. M. Underhill, L. Zhu, and K. A. Marr. 2006. Dectin-1 and 13. Bonnett, C. R., E. J. Cornish, A. G. Harmsen, and J. B. Burritt. 2006. Early TLRs permit macrophages to distinguish between different Aspergillus fumigatus neutrophil recruitment and aggregation in the murine lung inhibit germination of cellular states. J. Immunol. 176: 3717–3724. Aspergillus fumigatus Conidia. Infect. Immun. 74: 6528–6539. 40. Finkel, T. 2000. Redox-dependent signal transduction. FEBS Lett. 476: 52–54. 14. Schaffner, A., H. Douglas, A. I. Braude, and C. E. Davis. 1983. Killing of As- pergillus spores depends on the anatomical source of the macrophage. Infect. 41. Quinn, M. T., M. C. Ammons, and F. R. DeLeo. 2006. The expanding role of Immun. 42: 1109–1115. NADPH oxidases in health and disease: no longer just agents of death and de- Clin. Sci. Lond. 15. Philippe, B., O. Ibrahim-Granet, M. C. Prevost, M. A. Gougerot-Pocidalo, struction. 111: 1–20. P. M. Sanchez, M. A. Van Der, and J. P. Latge. 2003. Killing of Aspergillus 42. Kobayashi, S. D., J. M. Voyich, K. R. Braughton, A. R. Whitney, W. M. Nauseef, fumigatus by alveolar macrophages is mediated by reactive oxidant intermedi- H. L. Malech, and F. R. DeLeo. 2004. Gene expression profiling provides insight ates. Infect. Immun. 71: 3034–3042. into the pathophysiology of chronic granulomatous disease. J. Immunol. 172: 16. Roilides, E., T. Sein, A. Holmes, S. Chanock, C. Blake, P. A. Pizzo, and 636–643. T. J. Walsh. 1995. Effects of macrophage colony-stimulating factor on antifungal 43. Romani, L., F. Fallarino, L. A. De, C. Montagnoli, C. D’Angelo, T. Zelante, activity of mononuclear phagocytes against Aspergillus fumigatus. J. Infect. Dis. C. Vacca, F. Bistoni, M. C. Fioretti, U. Grohmann, et al. 2008. Defective tryp- 172: 1028–1034. tophan catabolism underlies inflammation in mouse chronic granulomatous dis- 17. Nessa, K., L. Palmberg, U. Johard, P. Malmberg, C. Jarstrand, and P. Camner. ease. Nature 451: 211–215. 1997. Reaction of human alveolar macrophages to exposure to Aspergillus fu- 44. Duong, M., N. Ouellet, M. Simard, Y. Bergeron, M. Olivier, and M. G. Bergeron. migatus and inert particles. Environ. Res. 75: 141–148. 1998. Kinetic study of host defense and inflammatory response to Aspergillus 18. Wells, C. A., T. Ravasi, R. Sultana, K. Yagi, P. Carninci, H. Bono, G. Faulkner, fumigatus in steroid-induced immunosuppressed mice. J. Infect. Dis. 178: Y. Okazaki, J. Quackenbush, D. A. Hume, and P. A. Lyons. 2003. Continued 1472–1482. discovery of transcriptional units expressed in cells of the mouse mononuclear 45. Kim, H. S., E. H. Choi, J. Khan, E. Roilides, A. Francesconi, M. Kasai, T. Sein, phagocyte lineage. Genome Res. 13: 1360–1365. R. L. Schaufele, K. Sakurai, C. G. Son, et al. 2005. Expression of genes encoding 19. Li, J., D. K. Pritchard, X. Wang, D. R. Park, R. E. Bumgarner, S. M. Schwartz, innate host defense molecules in normal human monocytes in response to Can- and W. C. Liles. 2007. cDNA microarray analysis reveals fundamental differ- dida albicans. Infect. Immun. 73: 3714–3724. The Journal of Immunology 6867

46. Mehrad, B., R. M. Strieter, T. A. Moore, W. C. Tsai, S. A. Lira, and 64. Johnston, J. A., and J. J. O’Shea. 2003. Matching SOCS with function. Nat. T. J. Standiford. 1999. CXC chemokine receptor-2 ligands are necessary com- Immunol. 4: 507–509. ponents of neutrophil-mediated host defense in invasive pulmonary aspergillosis. 65. Kan, V. L., and J. E. Bennett. 1988. Lectin-like attachment sites on murine pul- J. Immunol. 163: 6086–6094. monary alveolar macrophages bind Aspergillus fumigatus conidia. J. Infect. Dis. 47. Mehrad, B., R. M. Strieter, and T. J. Standiford. 1999. Role of TNF-␣ in pul- 158: 407–414. monary host defense in murine invasive aspergillosis. J. Immunol. 162: 66. Marshall, A. S., and S. Gordon. 2004. Commentary: C-type lectins on the mac- 1633–1640. rophage cell surface-recent findings. Eur. J. Immunol. 34: 18–24. 48. Dinarello, C. A. 1996. Biologic basis for interleukin-1 in disease. Blood 87: 67. Shirakata, Y., T. Komurasaki, H. Toyoda, Y. Hanakawa, K. Yamasaki, 2095–2147. S. Tokumaru, K. Sayama, and K. Hashimoto. 2000. Epiregulin, a novel member 49. Haelens, A., A. Wuyts, P. Proost, S. Struyf, G. Opdenakker, and J. van Damme. of the epidermal growth factor family, is an autocrine growth factor in normal 1996. Leukocyte migration and activation by murine chemokines. Immunobiol- human keratinocytes. J. Biol. Chem. 275: 5748–5753. ogy 195: 499–521. 68. Takahashi, M., K. Hayashi, K. Yoshida, Y. Ohkawa, T. Komurasaki, 50. Sozzani, S., W. Luini, A. Borsatti, N. Polentarutti, D. Zhou, L. Piemonti, G. A. Kitabatake, A. Ogawa, W. Nishida, M. Yano, M. Monden, and K. Sobue. D’Amico, C. A. Power, T. N. Wells, M. Gobbi, et al. 1997. Receptor expression 2003. Epiregulin as a major autocrine/paracrine factor released from ERK- and and responsiveness of human dendritic cells to a defined set of CC and CXC p38MAPK-activated vascular smooth muscle cells. Circulation 108: 2524–2529. chemokines. J. Immunol. 159: 1993–2000. 69. Coleman, D. L., A. H. Bartiss, V. P. Sukhatme, J. Liu, and H. D. Rupprecht. 51. Shimada, T., M. Matsumoto, Y. Tatsumi, A. Kanamaru, and S. Akira. 1998. A 1992. Lipopolysaccharide induces Egr-1 mRNA and protein in murine peritoneal novel lipopolysaccharide inducible C-C chemokine receptor related gene in mu- macrophages. J. Immunol. 149: 3045–3051. rine macrophages. FEBS Lett. 425: 490–494. 70. Yan, S. F., J. Lu, L. Xu, Y. S. Zou, J. Tongers, W. Kisiel, N. Mackman, 52. Filler, S. G., M. R. Yeaman, and D. C. Sheppard. 2005. Tumor necrosis factor D. J. Pinsky, and D. M. Stern. 2000. Pulmonary expression of early growth inhibition and invasive fungal infections. Clin. Infect. Dis. 41(Suppl. 3): response-1: biphasic time course and effect of oxygen concentration. J. Appl. S208–S212. Physiol. 88: 2303–2309. 53. Shalit, I., D. Halperin, D. Haite, A. Levitov, J. Romano, N. Osherov, and 71. Kamimura, M., C. Viedt, A. Dalpke, M. E. Rosenfeld, N. Mackman, I. Fabian. 2006. Anti-inflammatory effects of moxifloxacin on IL-8: IL-1␤ and ␣ ␬ D. M. Cohen, E. Blessing, M. Preusch, C. M. Weber, J. Kreuzer, et al. 2005. TNF- secretion and NF B and MAP-kinase activation in human monocytes Interleukin-10 suppresses tissue factor expression in lipopolysaccharide-stimu- stimulated with Aspergillus fumigatus. J. Antimicrob. Chemother. 57: 230–235.

lated macrophages via inhibition of Egr-1 and a serum response element/MEK- Downloaded from 54. Choi, J. H., E. Brummer, Y. J. Kang, P. P. Jones, and D. A. Stevens. 2006. ERK1/2 pathway. Circ. Res. 97: 305–313. Inhibitor ␬B and nuclear factor ␬B in granulocyte-macrophage colony-stimulat- 72. Thyss, R., V. Virolle, V. Imbert, J. F. Peyron, D. Aberdam, and T. Virolle. 2005. ing factor antagonism of dexamethasone suppression of the macrophage response NF-␬B/Egr-1/Gadd45 are sequentially activated upon UVB irradiation to mediate to Aspergillus fumigatus conidia. J. Infect. Dis. 193: 1023–1028. epidermal cell death. EMBO J. 24: 128–137. 55. Benjamim, C. F., C. M. Hogaboam, N. W. Lukacs, and S. L. Kunkel. 2003. Septic 73. Zaas, A. K. 2006. Host genetics affect susceptibility to invasive aspergillosis. mice are susceptible to pulmonary aspergillosis. Am. J. Pathol. 163: 2605–2617. Med. Mycol. 44(Suppl.): 55–60. 56. Romani, L., F. Bistoni, R. Gaziano, S. Bozza, C. Montagnoli, K. Perruccio, L. Pitzurra, S. Bellocchio, A. Velardi, G. Rasi, et al. 2004. Thymosin ␣1 activates 74. Ryter, S. W., J. Alam, and A. M. Choi. 2006. Heme oxygenase-1/carbon mon- oxide: from basic science to therapeutic applications. Physiol. Rev. 86: 583–650. dendritic cells for antifungal Th1 resistance through Toll-like receptor signaling. http://www.jimmunol.org/ Blood 103: 4232–4239. 75. Fredenburgh, L. E., M. A. Perrella, and S. A. Mitsialis. 2007. The role of heme 57. Shahan, T. A., W. G. Sorenson, J. D. Paulauskis, R. Morey, and D. M. Lewis. oxygenase-1 in pulmonary disease. Am. J. Respir. Cell Mol. Biol. 36: 158–165. 1998. Concentration- and time-dependent upregulation and release of the cyto- 76. Taille, C., J. El-Benna, S. Lanone, M. C. Dang, E. Ogier-Denis, M. Aubier, and kines MIP-2. KC, TNF, and MIP-1␣ in rat alveolar macrophages by fungal spores J. Boczkowski. 2004. Induction of heme oxygenase-1 inhibits NAD(P)H oxidase implicated in airway inflammation. Am. J. Respir. Cell Mol. Biol. 18: 435–440. activity by down-regulating cytochrome b558 expression via the reduction of 58. Jiang, Y., D. I. Beller, G. Frendl, and D. T. Graves. 1992. Monocyte chemoat- heme availability. J. Biol. Chem. 279: 28681–28688. tractant protein-1 regulates adhesion molecule expression and cytokine produc- 77. Wang, J. P., S. E. Rought, J. Corbeil, and D. G. Guiney. 2003. Gene expression tion in human monocytes. J. Immunol. 148: 2423–2428. profiling detects patterns of human macrophage responses following Mycobac- 59. Wasylnka, J. A., and M. M. Moore. 2002. Uptake of Aspergillus fumigatus terium tuberculosis infection. FEMS Immunol. Med. Microbiol. 39: 163–172. conidia by phagocytic and nonphagocytic cells in vitro: quantitation using strains 78. Azenabor, A. A., S. Yang, G. Job, and O. O. Adedokun. 2005. Elicitation of expressing green fluorescent protein. Infect. Immun. 70: 3156–3163. reactive oxygen species in Chlamydia pneumoniae-stimulated macrophages: a 2ϩ 60. Taramelli, D., M. G. Malabarba, G. Sala, N. Basilico, and G. Cocuzza. 1996. Ca -dependent process involving simultaneous activation of NADPH oxidase by guest on October 1, 2021 Production of cytokines by alveolar and peritoneal macrophages stimulated by and cytochrome oxidase genes. Med. Microbiol. Immunol. 194: 91–103. Aspergillus fumigatus conidia or hyphae. J. Med. Vet. Mycol. 34: 49–56. 79. Sugui, J. A., J. Pardo, Y. C. Chang, A. Mullbacher, K. A. Zarember, 61. Schelenz, S., D. A. Smith, and G. J. Bancroft. 1999. Cytokine and chemokine E. M. Galvez, L. Brinster, P. Zerfas, J. I. Gallin, M. M. Simon, and responses following pulmonary challenge with Aspergillus fumigatus: obligatory K. J. Kwon-Chung. 2007. Role of laeA in the regulation of alb1, gliP, conidial role of TNF-␣ and GM-CSF in neutrophil recruitment. Med. Mycol. 37: 183–194. morphology and virulence in Aspergillus fumigatus. Eukaryot. Cell 1552–1561. 62. Gygi, S. P., Y. Rochon, B. R. Franza, and R. Aebersold. 1999. Correlation be- 80. Filler, S. G., and D. C. Sheppard. 2006. Fungal invasion of normally non-phago- tween protein and mRNA abundance in yeast. Mol. Cell. Biol. 19: 1720–1730. cytic host cells. PLoS. Pathog. 2: e129. 63. Denecke, B., A. Meyerdierks, and E. C. Bottger. 1999. RGS1 is expressed in 81. Stephens-Romero, S. D., A. J. Mednick, and M. Feldmesser. 2005. The patho- monocytes and acts as a GTPase-activating protein for G-protein-coupled che- genesis of fatal outcome in murine pulmonary aspergillosis depends on the neu- moattractant receptors. J. Biol. Chem. 274: 26860–26868. trophil depletion strategy. Infect. Immun. 73: 114–125.