Abstract

BackgroundInvasive aspergillosis (IA) is the most common cause of death associated with fungal infection in the developed world. Historically, susceptibility to IA has been associated with prolonged neutropenia; however, IA has now become a major problem in patients on calcineurin inhibitors and allogenic hematopoetic stem cell transplant patients following engraftment. These observations suggest complex cellular mechanisms govern immunity to IA

MethodsTo characterize the key early events that govern outcome from infection with Aspergillus fumigatus we performed a comparative immunochip microarray analysis of the pulmonary transcriptional response to IA between cyclophosphamide-treated mice and immunocompetent mice at 24 h after infection

ResultsWe demonstrate that death due to infection is associated with a failure to generate an incremental interferon-γ response, increased levels of interleukin-5 and interleukin-17a transcript, coordinated expression of a network of tumor necrosis factor–α-related genes, and increased levels of tumor necrosis factor–α. In contrast, clearance of infection is associated with increased expression of a number genes encoding proteins involved in innate pathogen clearance, as well as apoptosis and control of inflammation

ConclusionThis first organ-level immune response transcriptional analysis for IA has enabled us to gain new insights into the mechanisms that govern fungal immunity in the lung

The number of patients at risk of invasive mold infection has been increasing in parallel with an increasing number of new immunosuppressive therapies [1]. Consequently, invasive aspergillosis (IA) has supplanted invasive candidiasis as the most common fungal cause of death in modern health care settings [2]. This is partly due to the high mortality for IA, which ranges from 40% to 60% for pulmonary disease and approaches 90% for disseminated disease [3]. Historically, profound neutropenia has been a major risk factor for IA in patients with hematological malignancy; however, it is now recognized that, in the context of allogenic hematopoetic stem cell transplantation (HSCT), there is a bimodal distribution of cases, with up to 70% of patients developing IA late after transplantation, beyond the neutropenic period [4]. This observation is reiterated in patients who are taking calcineurin inhibitors, who develop IA in the absence of neutropenia. Because calcineurin inhibitors impair Th1-mediated responses, this suggests that Th1-mediated immunity is important for host immunity to IA [5]. This observation is further bolstered by mounting evidence that interferon (IFN)–γ, a Th1 signature cytokine, has utility as adjunctive therapy for invasive fungal diseases [6]. Gene knockout studies in murine models of IA further demonstrate the essential role of Th1-mediated immunity in host survival [7]

In this study, we used microarray immunochip analysis as an unbiased tool to characterize the early transcriptional pulmonary immune response to Aspergillus fumigatus for the first time, to our knowledge, in both immunocompetent mice and mice immunosuppressed with hydrocortisone and cyclophosphamide (an established and standardized model of IA) [8]. This approach enabled us to gain new insights into the key early innate immune responses to infection within the lung in both immunosuppressed and immunocompetent mice. We found that early stage infection in this model (leading ultimately to death) is associated with an inflammatory cell infiltrate at the site of infection, high tumor necrosis factor (TNF)–α levels, reduced levels of IFN-γ in the lung, failure to generate a Th1-polarized transcriptional program, and increased expression of a TNF-α–related network of genes, Il-5 and Il-17. In comparison, immunocompetent mice that rapidly cleared the infection within 24 h had higher levels of IFN-γ and lower levels of TNF-α protein in the lung and up-regulated a number of genes encoding proteins involved in innate processing of pathogens and control of inflammation. Transcriptional analysis of the pulmonary immune response in both immunocompetent and immunosuppressed mice has, therefore, enabled us to delineate the critical early events that may determine the outcome of infection with A. fumigatus

Materials and Methods

Murine fungal infectionsMurine infections were performed under UK Home Office Project Licence PPL/70/5361 and PPL70/6487 at Imperial College London in accordance with Home Office regulations. Groups of 6 out-bred male mice (strain CD1, 18–22 g; Harlan Ortech) were immunosuppressed with cyclophosphamide/hydrocortisone acetate, as described elsewhere [9]. Groups of 6 nonimmunosuppressed mice were infected in the same way. Mice were anesthetized by halothane inhalation and infected by intranasal instillation with 5×105A. fumigatus strain 237 conidia in 40 μL of normal saline for microarray and cytokine studies and from 5×105 to 1×107 conidia for survival studies, as described elsewhere [9]. Groups of 6 control mice were administered 40 μL of normal saline under the same conditions. Mice were culled by cervical dislocation at 8 and 24 h after infection (for microarray and cytokine studies) or at 20% weight loss (for survival curves). Pneumonectomy was performed and peripheral blood harvested. Lungs were instilled with 4% (w/v) formalin (Sigma) or TRIzol (Invitrogen) and snap frozen in liquid nitrogen

Isolation and preparation of labelled cDNA for immunochip analysisMouse lungs stored in TRIzol were homogenized at a ratio of 100 mg tissue to 1.5 ml of TRIzol. RNA was extracted and cleaned on RNeasy columns (Qiagen) in accordance with the manufacturer’s instructions. RNA concentration and quality were assessed by Nanodrop spectrophotometry (Nanodrop Technologies) and agarose gel electrophoresis. cDNA was generated by reverse transcription and amino-allyl labelling according to protocols from TIGR (http://www.tigr.org/tdb/microarray/protocolstigr.shtml) and dye incorporation estimated by Nanodrop spectrophotometry

Immunochip analysis of the pulmonary response to infection Mouse Immunology Oligo Set Arrays version 2 (consisting of 1104 genes encoding proteins either involved in the immune response or encoding immune-system–related transcription factors, as well as 1298 B cell clones) were kindly supplied by The Medical Research Council Rosalind Franklin Centre for Genomics Research (Hinxton). cDNA was hybridized to microarrays on the basis of standard protocols (http://www.tigr.org/tdb/microarray/protocolstigr.shtml). Microarray data were captured with a Genepix 4000b scanner (Axon Instruments) and were extracted with Genepix Pro software, version 3.0. Data were filtered and normalized with TIGR TM4 freeware (http://www.tm4.org/) by locally weighted scatter plot smoothing and standard deviation regularization. All microarray data were stored in a MIAME-compliant database (maxload2; COGEME) and submitted to Arrayexpress (E-MAXD-28)

Significance of fold changes in gene expression was calculated by 1-way significance analysis of microarrays for identification of significant changes in expression within experimental groups (when compared to control) and multivariate analysis of variance (MANOVA) for identification of changes in expression between immunosuppressed and immunocompetent infected animals. Differences between observed data were considered significant if the P or Q value was <.05 and the fold ratio was >1.7 standard deviations from the mean

Real-time Synergy Brands Incorporated (SYBR) green and Taqman reverse-transcription polymerase chain reaction (RT-PCR)A total of 1 μg of RNA was reverse-transcribed to cDNA with Superscript III Reverse Transcriptase (Invitrogen). Semi-quantitative PCR for TNF-α, IFN-γ, signal transducer and activator of transcription (STAT)–1α, interleukin (IL)–4, IL-12p40, IL-17A, and GATA-3 was performed on a Corbett Rotorgene 3000 (Corbett Life Sciences) using intron-spanning primers. The primer sequences used are available on request. Results were normalized to levels of the hypoxanthine-guanine phosphoribosyltransferase (HPRT) transcript and statistical significance was determined using repeated measures analysis of variance with appropriate Bonferroni corrections and significance (P<.05) reported, as appropriate

Cytokine protein expressionFrozen lungs were homogenized at 100 mg/mL in phosphate-buffered saline (PBS) and clarified by centrifugation at 2000 g for 10 min. Cytokine levels in lung homogenate supernatant were analyzed by Murine Th1/Th2 Cytokine Bead Array (BD Biosciences) in accordance with the manufacturer’s instructions. Statistical significance was determined using repeated measures analysis of variance, with Bonferroni correction, and statistical significance (P<.05) is reported where appropriate

Results

Immunochip analysis of the murine pulmonary response toA. fumigatusTo define outcomes from infections in our model of IA, we performed survival analysis, as well as histological examination and culture of lung homogenates at 24 h. More than 90% of immunosuppressed CD1 mice succumb to IA after intranasal instillation of 5×105A. fumigatus 237 conidia at 6 days after infection. In contrast, immunocompetent CD1 mice clear 5×105A. fumigatus 237 and up to 1×107A. fumigatus 237 conidia within 24 h after intranasal instillation. Immunocompetent mice exhibit 100% survival at 12 days. In immunosuppressed mice, inflammatory lesions could be identified in response to infection with A. fumigatus 237 conidia. In immunocompetent animals the lungs are hyperemic at 24 h, but there was no histological or microbiological evidence of infection or inflammatory infiltrates

To compare the pulmonary response to A. fumigatus infection in immunocompetent and immunosuppressed mice, we performed microarray analysis at 2 points: 8 and 24 h after infection. No significant differences were detectable between groups at 8 h by microarray, so this time-point will not be discussed further

To identify genes involved in the competent immune response to A. fumigatus the pulmonary transcriptional response to A. fumigatus infection in immunocompetent mice 24 h after infection was compared with than in immunocompetent, uninfected mice. A total of 18 genes had increased expression in the challenged immunocompetent mice in response to A. fumigatus infection (Table 1); these include 5 genes involved in inflammation and 7 involved in apoptosis and control of cellular differentiation. Analysis of pulmonary transcripts from A. fumigatus–challenged, immunosuppressed mice identified 42 genes with significantly increased expression, compared with unchallenged, immunosuppressed mice (Table 2). To further identify differences between immunocompetent A. fumigatus–infected mice and immunosuppressed A. fumigatus–infected mice, we performed MANOVA analysis between all 4 groups of mice. This approach identified 53 genes with significantly increased expression in infected, immunosuppressed mice, compared with infected, immunocompetent animals (Table 3)

Table 1

Genes with Increased Expression in Response to Aspergillus fumigatus Infection in Immunocompetent CD1 Mice versus Normal Saline–Inoculated, Nonimmunosuppressed Mice

Table 1

Genes with Increased Expression in Response to Aspergillus fumigatus Infection in Immunocompetent CD1 Mice versus Normal Saline–Inoculated, Nonimmunosuppressed Mice

Table 2

Genes with Increased Expression in Immunosuppressed, Aspergillus fumigatus–Infected CD1 Mice at 24 h after Infection, Compared with Normal Saline–Inoculated, Immunosuppressed Mice

Table 2

Genes with Increased Expression in Immunosuppressed, Aspergillus fumigatus–Infected CD1 Mice at 24 h after Infection, Compared with Normal Saline–Inoculated, Immunosuppressed Mice

Table 3

Genes with Increased Expression in Immunosuppressed, Aspergillus fumigatus–Infected CD1 Mice at 24 h after Infection, Compared with A. fumigatus–Infected, Nonimmunosuppressed Mice

Table 3

Genes with Increased Expression in Immunosuppressed, Aspergillus fumigatus–Infected CD1 Mice at 24 h after Infection, Compared with A. fumigatus–Infected, Nonimmunosuppressed Mice

Considering both groups of genes together, we identified 54 inflammatory genes with increased expression in response to IA; these included 8 genes encoding TNF-related proteins. In addition, the genes encoding IL-5 (a Th2 cytokine involved in B cell and eosinophil activation) and IL-17a (a Th17 inflammatory cytokine produced by T cells, NK cells, and iNKT cells) were both up-regulated. Furthermore, there was a striking increase (249-fold) in the expression of H2-dmb1

An additional 10 genes were identified encoding proteins with functions consistent with activation and expansion of CD4+ T cells. Furthermore, a number of genes with roles in lymphocyte proliferation were identified with increased expression

Real-time PCR analysis of cytokine responses toA. fumigatusintranasal infectionAn independent analysis was performed by real-time PCR for the genes encoding the Th1/2/17 cytokines TNF-α, IFN-γ, STAT-1α, IL-4, IL-12p40, IL-17A, and GATA-3, with intron-spanning primers. Expression level of these genes, after normalization to HRPT levels, was expressed as the ratio with respect to saline treated immunocompetent mice (Figure 1). Levels of ß-actin were analyzed as an extra control and showed no significant differences. Overall, there was a generalized reduction in cytokine expression in immunosuppressed mice versus immunocompetent mice

Figure 1

Cytokine expression in immunocompetent and immunosuppressed mice with pulmonary invasive aspergillosis. Reverse-transcription polymerase chain reaction analysis of murine lung cytokine levels was performed for tumor necrosis factor (TNF)–α, interferon (IFN)–γ, STAT-1α, interleukin (IL)–4, IL-12p40, IL-17a, and GATA-3 in immunocompetent and immunosuppressed mice 24 h after infection (intranasal) with 5×105Aspergillus fumigatus conidia. All cytokine levels were normalized to hypoxanthine-guanine phosphoribosyltransferase control levels and are expressed as a ratio compared with saline-inoculated immunocompetent mice. Levels for all cytokines are significantly reduced in immunosuppressed mice, compared with immunocompetent mice, before infection. In response to infection, Ifn-γ and Il-17a levels significantly increased in immunosuppressed mice but were still lower than in equivalent immunocompetent infected mice. Statistical significance (P<.05) was determined using repeated measures analysis of variance, with appropriate Bonferroni correction

Figure 1

Cytokine expression in immunocompetent and immunosuppressed mice with pulmonary invasive aspergillosis. Reverse-transcription polymerase chain reaction analysis of murine lung cytokine levels was performed for tumor necrosis factor (TNF)–α, interferon (IFN)–γ, STAT-1α, interleukin (IL)–4, IL-12p40, IL-17a, and GATA-3 in immunocompetent and immunosuppressed mice 24 h after infection (intranasal) with 5×105Aspergillus fumigatus conidia. All cytokine levels were normalized to hypoxanthine-guanine phosphoribosyltransferase control levels and are expressed as a ratio compared with saline-inoculated immunocompetent mice. Levels for all cytokines are significantly reduced in immunosuppressed mice, compared with immunocompetent mice, before infection. In response to infection, Ifn-γ and Il-17a levels significantly increased in immunosuppressed mice but were still lower than in equivalent immunocompetent infected mice. Statistical significance (P<.05) was determined using repeated measures analysis of variance, with appropriate Bonferroni correction

In immunocompetent mice, real-time RT-PCR demonstrated significant increases in Il-17a expression in response to infection, compared with saline controls. In immunosuppressed mice, there were significant increases in Ifn-γ and Il-17a expression in response to infection (Figure 1). Despite this, the absolute expression level for Ifn-γ in infected, immunosuppressed mice is still significantly lower than for infected, immunosuppressed mice. Pairwise comparison of immunocompetent, infected animals with immunosuppressed, infected animals demonstrated significantly reduced expression of Tnf-α, Ifn-γ, Il-4 and Il-12p40 associated with immunosuppression

Early infection in immunosuppressed mice is associated with a significant increase in TNF-α levels but no increase in IFN-γTo further characterize cytokine responses to A. fumigatus infection at a protein level, Murine Cytokine Bead Array (BD Biosciences) analysis was performed on whole-lung homogenates at 24 h after infection. Both immunosuppressed and immunocompetent mice demonstrated a significant increase in TNF-α in response to A. fumigatus challenge (P<.05) (Figure 2). Notably, basal TNF-α levels were reduced in immunosuppressed mice, compared with immunocompetent mice, but increased 15-fold in response to A. fumigatus inoculation (mean ± standard deviation, 947.62±108.41 pg/g). IFN-γ levels did not increase in immunosuppressed mice. There was a modest but significant increase in IL-5 protein levels in immunosuppressed mice. Levels of IL-4 and IL-2 did not significantly differ between infected and uninfected groups

Figure 2

Dramatic increase in tumor necrosis factor (TNF)–α levels and decrease in interferon (IFN)–γ levels in the lung in response to infection. The figure shows Murine Cytokine Bead Array analysis of whole-lung homogenates at 24 h after infection. Levels of TNF-α, IFN-γ, interleukin (IL)–5, IL-4, and IL-2 were determined in lung homogenates prepared from CD1 mice, with or without immunosuppression, killed 24 h after intranasal administration of saline or 5×105Aspergillus fumigatus conidia. Data represent the mean ± standard error of triplicate data from a minimum of 3 samples. Statistical significance (P<.05) was determined using repeated measures analysis of variance, with appropriate Bonferroni correction, and is indicated by →. There was a dramatic increase in the levels of TNF-α in infected, immunosuppressed mice, compared with equivalent immunocompetent mice. In contrast, IFN-γ levels appeared to decrease in immunosuppressed, infected mice and were significantly lower than in equivalent immunocompetent, infected mice

Figure 2

Dramatic increase in tumor necrosis factor (TNF)–α levels and decrease in interferon (IFN)–γ levels in the lung in response to infection. The figure shows Murine Cytokine Bead Array analysis of whole-lung homogenates at 24 h after infection. Levels of TNF-α, IFN-γ, interleukin (IL)–5, IL-4, and IL-2 were determined in lung homogenates prepared from CD1 mice, with or without immunosuppression, killed 24 h after intranasal administration of saline or 5×105Aspergillus fumigatus conidia. Data represent the mean ± standard error of triplicate data from a minimum of 3 samples. Statistical significance (P<.05) was determined using repeated measures analysis of variance, with appropriate Bonferroni correction, and is indicated by →. There was a dramatic increase in the levels of TNF-α in infected, immunosuppressed mice, compared with equivalent immunocompetent mice. In contrast, IFN-γ levels appeared to decrease in immunosuppressed, infected mice and were significantly lower than in equivalent immunocompetent, infected mice

Discussion

We present, to our knowledge, the first large-scale comparative analysis of the transcriptional response to A. fumigatus during the early stages of pulmonary infection between immunocompetent and immunosuppressed mice. This has enabled us to gain new insights into the key components of host immunity that govern resistance as well as susceptibility to IA. Although organ-level analysis does not account for the effects of differing lung cell populations on overall transcriptional profiles, it has enabled an overview of the immune response to infection not otherwise possible

First, we characterized the immune response to pulmonary infection with A. fumigatus in immunocompetent mice. At 24 h, we identified 18 genes with statistically significant increases in expression in response to A. fumigatus. Two core functional groups of genes were identified. Group 1 contained 7 genes encoding proteins with functions in control of cellular proliferation. IMAP38 is a poorly characterized macrophage protein involved in apoptosis of Th1 CD4 T cells and malarial immunity [10]. Bid3/Bim is a cytosolic protein that relays apoptotic signals to the mitochondrial death machinery [11]. Sim2 (single-minded 2) is a transcription factor essential for lung development that controls expression of matrix metalloproteinases [12]. Cbx8 is a polycomb protein that acts as a transcriptional repressor of cell proliferation [13]. CEBPB is a bZIP transcription factor that negatively regulates several cytokines. CCAAT/enhancer binding protein is a master regulator of macrophage differentiation [14], and both Pax2 and HoxD13 are transcription factors that act as key regulators of embryonic tissues [15, 16]. The finding that the genes encoding this group of proteins all demonstrated increased expression in the host lung during clearance of A. fumigatus infection strongly suggests that a key component of the normal response to infection is regulatory control of cellular proliferation and inflammation

A second group of genes encoding 5 innate inflammatory proteins were identified with increased expression in immunocompetent mice. These encode CD39, an ecto-pyrase critical in attenuating pulmonary neutrophil transmigration during lung injury [17]; CXCL15 lungkine, involved in lung neutrophil chemoattraction [18]; transforming growth factor-β interacting factor, a TGF-β transcriptional co-repressor that inhibits Smad-mediated transcriptional activation [19]; CD44, a cell-surface glycoprotein involved in lymphocyte activation [20]; and major basic protein 1, a secreted eosinophil granule proteglycan that causes phagocyte activation [21]. These proteins have roles in activating or regulating innate immune responses, providing further evidence that the competent immune response to A. fumigatus infection is primarily mediated by phagocytes within the lung. The high proportion of regulatory, apoptotic genes identified in this data set further suggests that tight control of cellular influx and activation at the site of infection limits inflammation and damage to the host. RT-PCR analysis further demonstrates that the competent immune response is characterized by inflammatory control, as there were no significant increases in expression of the genes encoding the pro-inflammatory cytokines STAT1, IFN-γ, TNF-α, IL-4, or IL-12. However, it is interesting to note that modest increases in Il-4 and Tnf-α were seen, which may have become significant had greater numbers of mice been used. Notably, there were significant increases in Il-17a expression. At a protein level, significant increases were only seen in TNF-α, although levels are still significantly lower than in infected, immunosuppressed mice

We then characterized the immune response to IA in neutropenic hydrocortisone/cyclophosphamide immunosuppressed mice that succumb to infection. At 24 h, we identified 54 genes with increased expression in response to infection by immunochip transcript profiling. Most strikingly, a group of 8 genes encoding proteins with roles in TNF-α signal transduction all demonstrated increased expression. These are TRAF2, a TNF receptor interacting protein which mediates macrophage differentiation [22]; TRAF-interacting protein, a regulator of TRAF2-mediated NF-κB activation [23]; RANK ligand, a TNF superfamily member involved in dendritic cell maturation [24]; RANK, a receptor activator of NF-κB that interacts with TRAF2 [25]; TNFSF12 (TWEAK), a protein with overlapping signalling functions with TNF-α involved in inflammation and remodelling [26]; Lck1, a T cell–activating tyrosine kinase [27]; ATF2, a TNF receptor–dependent transcription factor involved in p38 mitogen–activated protein-kinase dependent induction of TNF-α expression [28]; and glyceraldehyde-3-phosphate dehydrogenase, an inhibitor of TRAIL-mediated apoptosis [29]. Furthermore, there was a major increase in expression of the gene encoding H2-DMb1, an HLA class 2 molecule that plays a pivotal role in MHC II antigen presentation. This suggests that neutropenia leads to impairment of pathogen clearance, increased antigen presentation through MHC class II, and drives a TNF-α inflammatory response

In addition, a diverse group of genes encoding proteins with functions in lymphocyte development and immune activation were identified. These included HoxA7/9 (B cell development) [16], Pou2f2 (transcriptional activator of immunoglobulin expression) [30], Ciapin1 (cytokine-induced apoptosis inhibitor) [31], E2f1/2 (transcriptional activator, cellular proliferation) [32], CD33 (monocyte cell adhesion molecule) [33], Stra13 (positive regulator of T cell activation) [34], Ms4a4b (CD20 homolog, T cell differentiation) [35], transcription factor E3/EB (CD40 dependent CD4 T cell-dependent antibody production) [36], CXCR4 (lymphocyte chemotaxis) [37], Bap31 (MHC class 1 export) [38], CD22 (regulation of B cell signalling) [39], Ccl11 (Eotaxin 1, eosinophil recruitment) [40], CCR9 (mucosal lymphocyte recruitment) [41], C-src tyrosine kinase (T cell activation) [42], and FMS-like tyrosine kinase 4 (lymphatic development) [43]. This finding suggests that a key component of the early inflammatory response in IA is the activation of genes involved in lymphoproliferative responses. These observations indicate that, even at 24 h after infection, early components of adaptive immunity are being activated in pulmonary IA

Microarray analysis also identified the genes encoding IL-12 receptor β1, a component of the IL-12/IL-23 receptor complex [44], as up-regulated in mice that develop IA. However, IL-12p40 itself did not demonstrate increased expression in immunosuppressed mice with IA. Of note, it is IL-12 receptor β2 that is a critical determinant of Th1 differentiation, whereas β1 is constitutively expressed on naive T cells and is also involved in responses to the Th17 cytokine IL-23 [45]

Immunochip profiling also identified the genes encoding the Th2 cytokine IL-5 and the Th17 cytokine IL-17 as up-regulated in mice who fail to control infection. However, Il-5 was only identified when we compared the immune response to sham-infected mice, whereas Il-17A was identified in comparison with immunocompetent mice infected with A. fumigatus that clear infection. This finding demonstrates the utility of this comparative approach for delineating immunity to fungal infections and suggests that differential Il-17a expression may be a determinant of outcome from fungal infection. Interestingly, IL-5 is a key mediator of eosinophil activation. It is notable that Eotaxin 1 a chemokine centrally involved in eosinophil recruitment, is also up-regulated, suggesting that eosinophils are involved in the host response to IA [40]

Our study shows that susceptibility to IA is associated with high levels of TNF-α at the site of infection and with up-regulation of a network of TNF-α–related genes (Figure 2 and Tables 2 and 3). In addition, there appears to be a failure to increase IFN-γ levels within the lung (Figure 2). Interestingly, we recently found that a number of genes involved in gliotoxin synthesis are coordinately up-regulated during establishment of fungal infection [46]; this is relevant because gliotoxin has been shown to impair IFN-γ production [47]. Of note, this deficit only appears to be at the protein level, because there is a significant increase in IFN-γ transcript in these mice when assayed by real-time PCR, although this is from a very low baseline level

The kinetics of both cytokine and cellular responses to A. fumigatus infection in this model have been studied previously [48]. Cyclophosphamide-immunosuppressed mice, despite profound systemic leukopenia, develop increased pulmonary neutrophil, macrophage, and eosinophil influx 24 h after infection, but reduced lymphocyte counts [48]. This is associated with impaired ability of lung phagocytes to kill A. fumigatus conidia, slight reduction in bronchoalveolar lavage TNF-α level, and raised IL-10 level, in addition to dramatically reduced lymphocyte IFN-γ and IL-2 production. Kinetic studies suggest that lung TNF-α levels reach a peak at 24 h after infection, then rapidly fall away [49]. This finding may explain the discordant TNF-α mRNA levels (low) and protein levels (high) seen at 24 h in our model, suggesting down-regulation of TNF-α at this stage, although protein levels are still high

Murine gene knockout studies have demonstrated that IFN-γ deficiency leads to the generation of Th17-type immune responses, which, although nonsterilizing, may have a protective role in fungal infection [50]. This suggests that reduced IFN-γ levels within the lung may drive the development of Th17 responses. Real-time PCR confirms significant up-regulation of Il-17a in both immunosuppressed and immunocompetent mice inoculated with A. fumigatus. Notably, although absolute expression of Il-17a compared to HPRT appears higher in immunocompetent mice than in immunosuppressed mice, the fold increase in expression of Il-17a is 7.6 in immunosuppressed infected mice versus 4.1 in immunocompetent mice. Given this is a neutropenic model of IA, a possible source for IL-17A production is CD4 cells

In summary, in immunocompetent mice challenged with A. fumigatus we identified 18 genes with increased expression in the lung. Seven of these genes have roles in apoptosis and control of phagocyte differentiation. Five additional genes encoding cytokines with roles in phagocyte activation are also identified. Taken together, these observations suggest that clearance of infection in immunocompetent animals is effected through phagocytic innate immune responses in a tightly regulated process in which apoptotic mechanisms serve to limit host damage from inflammation

In neutropenic cyclophosphamide/hydrocortisone immunosuppressed mice, invasive infection is characterized by a marked inflammatory reaction, failure to increase IFN-γ protein levels, and high levels of TNF-α. Immunochip analysis demonstrates concerted up-regulation of a network of TNF-α–related genes and CD4 T cell markers in response to infection. However, analysis of key markers of Th1/Th2/Th17 cytokines does not point to a clearly polarized CD4 response. This is consistent with early-stage infection, when adaptive immune responses have not yet developed. However, it is interesting that there was significant up-regulation of Il-17a expression, in the context of a TNF-α– rather than IFN-γ–driven inflammatory response, and increased expression of the IL-12/23 receptor in the absence of significant increases in Il-12p40 expression. This observation is consistent with the development of a Th17 inflammatory response and adds to the increasing evidence that the tissue inflammation seen in IA is exacerbated by Th17-type responses [50]

Acknowledgments

K.H. sincerely thanks Graham Venn, Stuart McCorkell, Mark Searles, Karen Anderson, and all staff on Doulton Ward at St. Thomas’ Hospital for their skill and dedication in ensuring that he is still around to submit this paper

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Potential conflicts of interest: none reported
Presented in part: Advances Against Aspergillosis Meeting, February 2006, Athens, Greece
Financial support: This work was supported by grants from the BBSRC, CGD Research Trust, and the MRC
D.P.H.A.-J. and S.A.T. contributed equally to this work