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. Author manuscript; available in PMC: 2012 Sep 6.
Published in final edited form as: J Immunol. 2009 Apr 15;182(8):4938–4946. doi: 10.4049/jimmunol.0804250

Requisite role for the Dectin-1 beta-glucan receptor in pulmonary defense against Aspergillus fumigatus1

Jessica L Werner 1, Allison E Metz 1, Dawn Horn 1, Trenton R Schoeb 2, Matthew M Hewitt 3, Lisa M Schwiebert 3, Ines Faro-Trindade 4, Gordon D Brown 4, Chad Steele 1
PMCID: PMC3434356  EMSID: UKMS49853  PMID: 19342673

Abstract

Immune suppression increases the incidence of invasive fungal infections, particularly those caused by the opportunistic mold Aspergillus fumigatus. Previous investigations revealed that members of the Toll-like receptor (TLR) family are not absolutely required for host defense against A. fumigatus in non-immunosuppressed hosts, suggesting that other pattern recognition receptors (PRRs) are involved. We show here that naive mice (i.e. not pharmacologically immunosuppressed) lacking the beta-glucan receptor Dectin-1 (Dectin-1−/−) are more sensitive to intratracheal challenge with A. fumigatus than control mice, exhibiting >80% mortality within 5 days, ultimately attributed to a compromise in respiratory mechanics. In response to A. fumigatus challenge, Dectin-1−/− mice demonstrated impaired interleukin (IL)-α, IL-1β, tumor necrosis factor (TNF)-α, CCL3/macrophage inflammatory protein (MIP)-α, CCL4/MIP-1β and CXCL1/KC production, which resulted in insufficient lung neutrophil recruitment and uncontrolled A. fumigatus lung growth. Alveolar macrophages from Dectin-1−/− mice failed to produce proinflammatory mediators in response to A. fumigatus, whereas neutrophils from Dectin-1−/− mice had impaired reactive oxygen species production and impaired killing of A. fumigatus. We further show that IL-17 production in the lung after A. fumigatus challenge was Dectin-1 dependent and that neutralization of IL-17 significantly impaired A. fumigatus clearance. Collectively, these results support a requisite role for Dectin-1 in in vivo defense against A. fumigatus.

Introduction

Patients with a history of solid organ or hematopoietic cell transplantation are at high risk for developing invasive fungal infections (1). Among these, invasive pulmonary aspergillosis (IPA) caused by the opportunistic fungal pathogen Aspergillus fumigatus is associated with an exceptional mortality rate. Predisposition to IPA is associated with impairments in innate immunity, primarily neutropenia (2, 3). In patients with hematologic malignancies, infections caused by A. fumigatus have increased from 0.9% to 2.9% between 1989-2003 (4). However, data are beginning to uncover an increase in IPA in non-neutropenic ICU patients who do not have classic risk factors. Recent studies report an appreciable incidence of IPA in patients with COPD (5, 6). Additionally, a recent study indicated the development of IPA in patients that were not neutropenic and had underlying diseases other than hematologic cancer, highlighting an increasing role for A. fumigatus as a cause of hospital-acquired pneumonia in seemingly immunocompetent patients (7).

Upon inhalation of A. fumigatus conidia into the lungs, initial recognition is the responsibility of the alveolar macrophage. Organisms that escape the effector functions of alveolar macrophages are subsequently targeted by neutrophils. An early landmark study showed that A. fumigatus conidia were more efficiently handled by macrophages, whereas neutrophils were the most effective against A. fumigatus germinating conidia/hyphae (8). Recognition of A. fumigatus by myeloid cells is reported to involve TLRs (9) and Dectin-1 (10, 11, 12). We have previously reported that interruption of A. fumigatus recognition by the beta-glucan receptor Dectin-1 attenuated alveolar macrophage inflammatory responses to A. fumigatus in vitro (10). Dectin-1 is a 43 kDa, type II transmembrane receptor containing a single cytoplasmic immuno-receptor tyrosine activation motif and a single extracellular C-type lectin recognition domain and is the predominant receptor for beta-1,3 glucans (13). In both humans (14) and mice (15), Dectin-1 is highly expressed on macrophages, neutrophils and dendritic cells.

Significant effort has been placed on delineating the role of innate receptors, particularly TLRs, in host defense against A. fumigatus. In vitro studies have primarily focused on the role of TLR2 and TLR4, with most studies showing a more prominent role for TLR2 (9). However, we have previously reported that TLR2 deficient alveolar macrophages have an intact inflammatory response to A. fumigatus (10). In vivo, MyD88, TLR2 or TLR4 deficient mice (16) have each been reported to have higher susceptibility to A. fumigatus lung infection, but only in the setting of neutropenia, as non-immunosuppressed MyD88, TLR2 and TLR4 deficient mice do not succumb to infection (17). However, a subsequent study has reported delayed lung clearance of A. fumigatus in non-immunosuppressed MyD88 deficient mice (18). These results suggest that TLRs are essential in the absence of neutrophils, but not in an immunocompetent host, and further suggests that other PRRs, such as Dectin-1, may be more critical for innate defense against A. fumigatus. In our previous study, and confirmed by others (11, 12), Dectin-1 expressed by alveolar macrophages recognized unmasked beta-glucan moieties in A. fumigatus swollen and germinating conidia, which led to a potent inflammatory response (10). We further showed that an attempt to block Dectin-1 in vivo after A. fumigatus challenge using soluble Dectin-1 resulted in moderate reductions in inflammatory mediator levels and a 30% increase in A. fumigatus lung burden, suggesting that Dectin-1 may have a role in the control of this organism in vivo (10). Therefore, in this report, we determined the consequences of Dectin-1 deficiency on lung host defense against invasive fungal disease caused by A. fumigatus.

Materials and Methods

Mice

Male 129/SvEv mice, 6-8 weeks of age, were purchased from Taconic Farms Incorporated (Germantown, NY). Dectin-1−/− mice were generated on the 129/SvEv background as previously described (19) and bred at Taconic. All mice were maintained in a specific pathogen free environment in microisolator cages within an American Association for Laboratory Animal Science certified animal facility in the Lyons Harrison Research Building at the University of Alabama at Birmingham. Animal studies were reviewed and approved by the University of Alabama at Birmingham Institutional Animal Care and Use Committee (IACUC).

Preparation of A. fumigatus conidia

A. fumigatus isolate 13073 (ATCC, Manassas, VA) was maintained on potato dextrose agar for 5-7 days at 37°C. Conidia were harvested by washing the culture flask with 50 ml of sterile phosphate buffered saline supplemented with 0.1% Tween 20. The conidia were then passed through a sterile 40 μm nylon membrane to remove hyphal fragments and enumerated on a hemacytometer.

In vivo A. fumigatus challenge, tissue burden assessment and histology

Mice were lightly anesthetized with isoflurane and administered 5-7 × 107 A. fumigatus conidia in a volume of 50 μl intratracheally. For survival studies, mice were monitored every 6 hours post A. fumigatus challenge and sacrificed when appearing moribund. For lung fungal burden analysis, the left lungs were collected and homogenized in 1 ml of PBS. Total RNA was extracted from 0.1 ml of unclarified lung homogenate using the MasterPure™ yeast RNA purification kit (Epicentre Biotechnologies, Madison, WI), which includes a DNAse treatment step to eliminate genomic DNA as previously reported (20). Lung A. fumigatus burden was analyzed with real time PCR measurement of the A. fumigatus 18S rRNA (GenBank accession number AB008401) (21) and quantified using a standard curve of A. fumigatus conidia (101 – 109) as previously described (20). Specifically, total RNA was isolated using the MasterPure™ kit from serial 1:10 dilutions of A. fumigatus conidia beginning with 109 and real time PCR amplification of A. fumigatus 18S rRNA was performed on each dilution. As a validation of the real-time PCR method, heat-killed A. fumigatus did not yield a signal by real-time PCR and were unable to grow on potato dextrose agar plates. In addition, no amplification controls (i.e. no reverse transcriptase included in the cDNA reaction) yielded a signal of <0.001% by real-time PCR, indicating that the DNAse treatment step was efficient at eliminating contaminating A. fumigatus DNA. For lung neutrophil recruitment, mice were challenged with A. fumigatus and a bronchoalveolar lavage was performed 24 h thereafter as previously described (10). Lavage cells were enumerated on a hemacytometer and then stained with the neutrophil marker Gr-1. Flow cytometry was performed to quantify the percentage of Gr-1 positive cells. For lung histology, the left lungs were collected and fixed in 4% formalin. The fixed lungs were paraffin-embedded and then processed and stained by the Comparative Pathology Laboratory at the University of Alabama at Birmingham. Imaging was performed using a Nikon Eclipse 90i microscope and Nikon NIS-Elements imaging analysis software.

Pulmonary function assessment

A tracheostomy was performed on individual anesthetized A. fumigatus-exposed wild-type and Dectin-1−/− mice. Each animal was then attached to a computer controlled volume ventilator (flexiVent, SCIREQ Montreal, PQ, Canada). Regular breathing was set at 150 bpm, with volume and pressure controlled by the flexiVent system based on individual animal weights. Positive end-expiratory pressure (PEEP) was set to 2 cm H2O and measured during each breath stroke. Respiratory input impedance (Zrs) was measured using the Forced Oscillation Technique controlled by the flexiVent system (22). The Single-Compartment Model was used to describe whole lung resistance (R) and the Constant Phase Model was used to describe Newtonian resistance (RN). All measurements were collected at baseline and after a linear dose response with methacholine challenge (10 – 40 mg/ml).No significant differences were observed in baseline and methacholine-challenged RN or R between WT and Dectin-1−/− mice in the absence of infection (P values for RN were 0.936 and 0.171 for 0 and 40 mg/ml methacholine; P values for R were 0.601 and 0.26 for 0 and 40 mg/ml methacholine; n = 3 - 4 mice per group).

Alveolar macrophage isolation and culture

For alveolar macrophage isolation, wild-type or Dectin-1−/− mice were anesthetized with intra-peritoneal ketamize/xylazine and sacrificed by exsanguination. Thereafter, lungs were lavaged through an intratracheal catheter with pre-warmed (37°C) calcium and magnesium-free PBS supplemented with 0.6 mM EDTA. A total of 10 ml was used in each mouse in 0.5 ml increments with a 30 second dwell-time. The lavage fluids were pooled and centrifuged at 300 × g for 10 min, and the cells collected for A. fumigatus co-culture. Alveolar macrophages (2-3 × 105) were co-cultured with A. fumigatus conidia at a ratio of 1:1 for 24 h in a 96-well plate at 37°C, 5% CO2. Controls for spontaneous cytokine and chemokine production included alveolar macrophages cultured in medium alone. Thereafter, the contents of each well were collected and the supernatants stored at −80°C until Bio-Plex cytokine and chemokine analysis.

Neutrophil isolation and culture

Thioglycollate-elicited peritoneal exudate cells were collected after 14 h from at least two mice per group by peritoneal lavage with ice-cold RPMI medium plus 10% heat-inactivated FCS. Pooled cells were used as a source of inflammatory granulocytes (mainly neutrophils) and monocytes. Inflammatory leukocytes (2 × 106) were mixed with live A. fumigatus swollen conidia (1 × 107) which were kept for 60 min at 4°C to allow the cells to ‘settle’, before being transferred to an incubator at 37°C for a further 60 min. For analysis of hydrogen peroxide generation, inflammatory cells were ‘loaded’ with dihydrorhodamine 123 at a final concentration of 1 uM. After incubation with live A. fumigatus swollen conidia, the conversion of dihydrorhodamine 123 was assessed by flow cytometry and was expressed as mean fluorescent intensity (19). Cells ‘loaded’ with dihydrorhodamine 123, but not treated with conidia, were used to assess background hydrogen peroxide production. For A. fumigatus killing in vitro, inflammatory leukocytes (1 × 105) were co-cultured at a 1:1 effector to target ratio with live A. fumigatus swollen/germinating conidia (10) for 2-4 h followed by RNA isolation with the MasterPure™ yeast RNA purification kit as described above. Controls included A. fumigatus swollen/germinating conidia cultured in the absence of inflammatory leukocytes for 2-4 h. To generate a standard curve, total RNA was isolated from serial 1:10 dilutions of A. fumigatus swollen/germinating conidia beginning at 106 that were simultaneously cultured for 2-4 h in the cell killing assay and real time PCR amplification of A. fumigatus 18S rRNA was performed on each dilution.

Analysis of IL-17

Wild-type and Dectin-1−/− mice were challenged intratracheally with 7 × 107 A. fumigatus conidia and IL-17 levels assessed in lung homogenates at 24 h by Bio-Plex. For in vivo IL-17 neutralization, mice were challenged intratracheally with 7 × 107 A. fumigatus conidia in 50 μl and 6 h thereafter, mice were administered 50 μg of rat anti-mouse IL-17A (Clone 50104; R&D Systems) (23) or rat IgG2a isotype control antibody. Twenty-four or forty-eight hours after challenge, mice were sacrificed, the left lungs were collected and homogenized in 1 ml of PBS. Total RNA was extracted from 0.1 ml of unclarified lung homogenate using the MasterPure™ yeast RNA purification kit and lung A. fumigatus burden was analyzed with real time PCR measurement of the A. fumigatus 18S rRNA.

Cytokine and chemokine quantification

For lung homogenates, the right lung was homogenized in PBS supplemented with Complete Mini protease inhibitor tablets (Roche), clarified by centrifugation and stored at −80°C. Samples were analyzed for protein levels of a panel of cytokines and chemokines using Luminex-based Bio-Plex multiplex suspension protein array (Bio-Rad Laboratories) according to the manufacturer’s instructions as previously described (10). Concentrations of each cytokine and chemokine were determined using Bio-Plex Manager version 4.1.1 software. In specific experiments, levels of IL-23 in lung homogenates were quantified by ELISA (R&D Systems).

Statistics

Data were analyzed using GraphPad Prism Version 5.0 statistical software. Comparisons between groups when data were normally distributed were made with the Student’s t-test. Comparisons among nonparametric data were made with the Mann-Whitney test. Survival analysis was performed using an asymmetrical 95% confidence interval for the Gehan-Breslow-Wilcoxon and Mantel-Cox log-rank tests. Significance was accepted at a value of P < 0.05.

Results

Enhanced susceptibility of Dectin-1 deficient mice after challenge with A. fumigatus

We first examined whether Dectin-1 was required for defense against the opportunistic mold A. fumigatus. Intratracheal administration of 5 × 107 A. fumigatus conidia resulted in rapid mortality in Dectin-1−/− mice, with greater than 60% of the mice succumbing by 4 days post-challenge (Figure 1A). Increasing the inoculum to 7 × 107 resulted in 100% mortality in Dectin-1−/− mice by 4 days (Figure 1B). Real-time PCR measurement of A. fumigatus 18S rRNA in lung tissue, recently demonstrated as the most sensitive method for determination of lung fungal burden in experimental aspergillosis (24), showed >4-fold higher A. fumigatus burden in Dectin-1−/− mice at 24 and 48 h (Figure 1C). Analysis of kidney and liver tissue in these animals indicated little A. fumigatus burden (< 102 in either organ) and no difference between wild-type and Dectin-1−/− mice (data not shown). Thus, Dectin-1 deficiency results in enhanced A. fumigatus growth in the lungs, but does not predispose to systemic infection that might otherwise explain the increased mortality.

Figure 1. Enhanced susceptibility of Dectin-1 deficient mice after challenge with A. fumigatus.

Figure 1

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Wild-type and Dectin-1−/− mice were challenged intratracheally with (A) 5 × 107 conidia in a volume of 50 ul or (B) 7 × 107 conidia in a volume of 50 ul and survival was monitored for 5 days. The Figure illustrates cumulative results from two independent studies per inoculum dose. *** represents a P value of < 0.001 (log rank Mantel-Cox test). (C) Real-time PCR analysis of A. fumigatus 18S rRNA levels in the lungs of wild-type and Dectin-1−/− mice challenged intratracheally with 7 × 107 conidia and collected at 24 h and 48 h post-challenge. The Figures illustrates cumulative data from three independent studies (n = 5 mice/group for each study). Data are expressed as mean A. fumigatus 18S rRNA levels + SEM. * represents a P value of < 0.05 (Unpaired two-tailed Student’s t test).

Defective A. fumigatus recognition by Dectin-1 deficient neutrophils

Neutrophil deficiency/dysfunction is the hallmark predisposing factor for the development of invasive pulmonary aspergillosis (2, 3). As Dectin-1−/− mice demonstrated increased mortality and higher lung burden after A. fumigatus challenge, we questioned whether defects existed in neutrophil recruitment and/or neutrophil function. Wild-type and Dectin-1−/− mice were challenged with A. fumigatus (7 × 107) and neutrophil levels in bronchoalveolar fluid were assessed at 24 h. Results in Figure 2A show that a 50% reduction in neutrophil recruitment to the lungs was observed in A. fumigatus-challenged Dectin-1−/− mice. Previous studies have shown that mice deficient in NADPH oxidase (gp91phox deficient mice) are highly susceptible to infection with A. fumigatus (25), despite having no impairment in killing of A. fumigatus by alveolar macrophages (26). More recent studies have shown extensive A. fumigatus germination in neutrophil aggregates from gp91phox deficient mice, but negligible germination in alveolar macrophages (27), suggesting that oxidative responses by neutrophils are critical for the prevention of A. fumigatus germination. A correlate of this is the observation that thioglycollate-elicited peritoneal neutrophils from uninfected Dectin-1−/− mice failed to activate NADPH oxidase in response to live A. fumigatus swollen/germinating conidia in vitro (Figure 2B). The lack of ROS production further correlated with an inability of Dectin-1−/− neutrophils to kill A. fumigatus in vitro (Figure 2C). Thus, one possible mechanism of the susceptibility observed in Dectin-1−/− mice may be impaired neutrophil oxidative killing of A. fumigatus.

Figure 2. Defective A. fumigatus recognition by Dectin-1 deficient neutrophils.

Figure 2

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(A) Neutrophil levels in BAL fluid were assessed by flow cytometry of individual mice 24 h after intratracheal challenge with 7 × 107 conidia. The Figure illustrates data from one of two independent studies (n = 5 mice/group). Data are expressed as neutrophils/ml of BAL fluid + SEM. ** P < 0.01 (Unpaired two-tailed Student’s t test). (B) Neutrophils were isolated from the peritoneum of wild-type and Dectin-1−/− mice challenged with 3% thioglycollate for 14 h and co-cultured with live A. fumigatus swollen conidia for 2 h. ROS production was assessed using dihydrorhodamine 123 conversion. The Figure illustrates cumulative data in triplicate wells from one of two independent studies. Data are expressed as mean fluorescent intensity ± SEM. ** and *** represent a P value of < 0.01 and 0.001, respectively (Unpaired two-tailed Student’s t test). (C) Peritoneal neutrophils were isolated and co-cultured with live A. fumigatus swollen conidia at a cell to organism ratio of 1:1 for 2-4 h. Controls included live A. fumigatus swollen conidia cultured in medium alone for 2-4 h. Thereafter, RNA was isolated from wild-type, Dectin-1−/− and A. fumigatus cultures and A. fumigatus 18S rRNA levels were quantified by real-time PCR. The Figure illustrates cumulative results from two independent studies each containing triplicate wells for wild-type, Dectin-1−/− and A. fumigatus cultures. Data are expressed as mean A. fumigatus 18S rRNA levels + SEM. * represents a P value of < 0.05 comparing wild-type to Aspergillus; # represents a P value of < 0.05 comparing wild-type to Dectin-1−/− (Unpaired two-tailed Student’s t test).

Hematoxylin and eosin (H&E)-staining staining of lung tissue sections revealed a profound, intense inflammatory response in wild-type mice (Figure 3A, left; 48 h). In contrast, inflammatory cell recruitment was significantly lower in Dectin-1−/− mice, despite the increased growth of A. fumigatus growth (Figure 3B, left). Moreover, dramatic alveolar edema, capillary congestion and proteinacious exudate deposition in the alveolar space was observed in Dectin-1−/− mice (Figure 3B, left; 48 h). Grocott’s methenamine silver (GMS) staining of lung tissue at 48 h revealed considerable A. fumigatus germinating conidia and hyphal masses in Dectin-1−/− mice (Figure 3B, right). In contrast, fewer germinating conidia and relatively few hyphae were observed in wild-type mice (Figure 3A, right). Germinating A. fumigatus was observed in Dectin-1−/− mice in areas containing inflammatory cells such as neutrophils, albeit significantly fewer in number compared to wild-type mice, which supports the in vitro data demonstrating impaired responses to A. fumigatus by Dectin-1−/− neutrophils.

Figure 3. Histological evidence for A. fumigatus invasion and lung damage in Dectin-1 deficient mice.

Figure 3

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Representative H&E-stained (left) and GMS-stained (right) lung sections from (A) wild-type mice and (B) Dectin-1−/− mice challenged intratracheally with 7 × 107 conidia for 48 h. Original magnification of 200X. Scale bar in A, left image represents 100 μm.

Requirement of Dectin-1 for the inflammatory response to A. fumigatus in vivo and in vitro

We next investigated proinflammatory cytokine and chemokine levels in lung homogenates in order to better understand inflammatory responses occurring early after exposure. Results showed that A. fumigatus elicited a robust inflammatory response in the lungs of wild-type mice at 24 h after challenge, characterized by the production of IL-α, IL-1β and TNF-α (Figure 4A). Each of these was significantly reduced in the lungs of Dectin-1−/− mice. The response to A. fumigatus in wild-type mice was also associated with significant production of CCL3/MIP-α, CCL4/MIP-1β and CXCL1/KC, chemokines essential for neutrophil recruitment during A. fumigatus lung infection (28, 29) (Figure 4A). Again, Dectin-1−/− mice had significant reductions in these mediators in response to A. fumigatus. Thus, Dectin-1 is required for optimal lung proinflammatory cytokine and chemokine production after A. fumigatus challenge.

Figure 4. Requirement for Dectin-1 in the inflammatory response to A. fumigatus in vivo and in vitro.

Figure 4

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(A) Wild-type and Dectin-1−/− mice were challenged intratracheally with 7 × 107 conidia and 24 h after exposure, IL-α, IL-1β, TNF-α, CCL3/MIP-α, CCL4/MIP-1β and CXCL1/KC levels were quantified in lung homogenates by Bio-Plex. The Figure illustrates cumulative data from four independent studies (n = 5 mice/group for each study). Data are expressed as mean pg/ml + SEM. *, ** and *** represent a P value of < 0.05, 0.01 and 0.001, respectively (Unpaired two-tailed Student’s t test). (B) Alveolar macrophages were isolated via BAL from wild-type or Dectin-1−/− mice and cultured with live A. fumigatus conidia for 24 h at a macrophage to conidium ratio of 1:1. Cytokine/chemokine concentrations in co-culture supernatants were determined by Bio-Plex. Unstimulated levels have been subtracted from each column. The Figure illustrates cumulative results from four independent studies. Data are expressed as mean pg/ml + SEM. * and ** represents a P value of < 0.05 and 0.01, respectively (Unpaired two-tailed Student’s t test).

As alveolar macrophages are a critical source for proinflammatory cytokine and chemokine production in response to A. fumigatus (30), we isolated alveolar macrophages from uninfected wild-type and Dectin-1−/− mice and assessed cytokine and chemokine production in response to live A. fumigatus in vitro. A. fumigatus elicited robust production of IL-α, IL-1β, TNF-α, CCL3/MIP-α, CCL4/MIP-1β and CXCL1/KC from wild-type alveolar macrophages (Figure 4B). In stark contrast, Dectin-1−/− alveolar macrophages were virtually unresponsive to A. fumigatus, despite undergoing stimulation with A. fumigatus for 24 h. Similar results were observed when co-culturing A. fumigatus swollen conidia (10) with Dectin-1−/− alveolar macrophages for 6 h (data not shown). The hyporesponsiveness of Dectin-1−/− macrophages is specific to beta-glucan containing ligands/organisms as responses to ligands for TLR2 and TLR4 have been shown to be unaffected in Dectin-1−/− macrophages (19 and data not shown). These results suggest that alveolar macrophages are a likely source of proinflammatory cytokine and chemokine production during A. fumigatus infection in vivo.

Compromised lung function in A. fumigatus-challenged Dectin-1 deficient mice

Histological assessment of Dectin-1−/− mice indicated A. fumigatus overgrowth and invasiveness in conjunction with severe alveolar damage. We therefore assessed measurements of respiratory mechanics to determine whether histological changes observed in Dectin-1−/− mice after A. fumigatus challenge correlated with a worsening in pulmonary function. For this, mice were challenged with 4 × 107 A. fumigatus conidia to allow for enough survival through 3 days post-challenge and pulmonary function was determined using the flexiVent system. Measurement of airway resistance indicated a functional compromise in the airways of A. fumigatus-challenged Dectin-1−/− mice at baseline, which was amplified upon treatment with the bronchoconstrictor methacholine (Figure 5A). Whole lung resistance was also found to be elevated in A. fumigatus-challenged Dectin-1−/− mice (Figure 5B). No differences were observed in baseline and methacholine-challenged airway resistance or whole lung resistance between WT and Dectin-1−/− mice in the absence of infection (data not shown). Thus, A. fumigatus infection in Dectin-1−/− mice is associated with increases in lung resistance.

Figure 5. Compromised lung function in A. fumigatus-challenged Dectin-1 deficient mice.

Figure 5

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Wild-type and Dectin-1−/− mice were challenged intratracheally with 4 × 107 conidia and 72 h after exposure, respiratory mechanics were analyzed via mechanical ventilation using the flexiVent system. Pulmonary function measurements were (A) airway resistance (RN) and (B) whole lung resistance (R). The Figure illustrates cumulative data from two independent studies (n = 5 mice/group for each study. Data are expressed as mean airway or whole lung resistance ± SEM. * and *** P < 0.05 and < 0.001, respectively (Unpaired two-tailed Student’s t test).

IL-17 production in Dectin-1 deficient mice exposed to A. fumigatus

Recent studies employing mice deficient in CARD9, a component in the signaling cascade of many immunoreceptors (31), including Dectin-1 (32, 33), have implicated a role for CARD9/Dectin-1 in generating protective Th17 responses during fungal infection (33). However, recent studies suggest an immunopathogenic, rather than protective, role for Th17/IL-17 during A. fumigatus infection (23, 34). Since IL-17 production has yet to be specifically characterized in Dectin-1−/− mice, we assessed IL-17 levels in wild-type and Dectin-1−/− mice 24 h after A. fumigatus levels in lung homogenates. Results in Figure 6A show that Dectin-1−/− mice have lower IL-17 levels in the lungs 24 h after A. fumigatus challenge. IL-12p40 (Figure 6B), a subunit of IL-23 (35), and IL-23 (Figure 6C), which is required for Th17 development (35), and were also observed to be significantly lower in Dectin-1−/− mice. Thus, Dectin-1−/− mice have impaired Th17 responses during fungal infection, putatively as a result of impaired IL-23 production.

Figure 6. IL-17 levels in the lungs of Dectin-1 deficient mice exposed to A. fumigatus.

Figure 6

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Wild-type and Dectin-1−/− mice were challenged intratracheally with 7 × 107 conidia and 24 h after exposure, (A) IL-17, (B) IL-12p40 and (C) IL-23 levels were quantified in lung homogenates. The Figure illustrates cumulative data from three independent studies (n = 5 mice per group per time point). Data are expressed as mean pg/ml + SEM. * and ** represent a P value of < 0.05 and 0.01, respectively (Unpaired two-tailed Student’s t test).

Neutralization of IL-17 impairs early A. fumigatus lung clearance

Neutralizing IL-17 in vivo has been shown to augment clearance of A. fumigatus in both normal and p47phox deficient mice (34). Our data indicates a requirement for Dectin-1 in IL-17 production early after A. fumigatus challenge (Figure 6A). We speculate that the lower levels of IL-17 in Dectin-1−/− mice early after challenge, in conjunction with the lower levels of other proinflammatory mediators (Figure 4A), may allow A. fumigatus infection to thrive. To address the role of IL-17 early, we sought to determine whether neutralizing IL-17 affected the development of A. fumigatus lung infection in wild-type mice. For this, we challenged mice with 7 × 107 A. fumigatus conidia and 6 h post-challenge, administered 50 μg of rat anti-mouse IL-17 antibody intratracheally (23). At 24 h post-challenge, mice were sacrificed and assessed for A. fumigatus lung burden. Treatment of mice with neutralizing antibodies significantly lowered IL-17 levels in the lungs at 24 h (Figure 7A) and resulted in a dramatic increase in A. fumigatus lung burden (Figure 7B). Similar results were also observed at 48 h (Figures 7C and 7D) Thus, IL-17 is involved in clearance of A. fumigatus from the lung.

Figure 7. Neutralization of IL-17 impairs early A. fumigatus lung clearance.

Figure 7

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Wild-type and Dectin-1−/− mice were challenged intratracheally with 7 × 107 conidia. Six hours after challenge, mice were administered 50 μg of rat anti-mouse IL-17 or rat IgG2A antibodies intratracheally. IL-17 levels were quantified in lung homogenates (A) 24 h and (C) 48 h after challenge by Bio-Plex. The Figure illustrates cumulative data from two independent studies (n = 5 mice per group per time point). Data are expressed as mean pg/ml + SEM. * and ** represents a P value of < 0.05 and 0.01, respectively (Unpaired two-tailed Student’s t test). Real-time PCR analysis of A. fumigatus 18S rRNA levels in the lungs of wild-type and Dectin-1−/− mice administered anti-IL-17 or isotype control antibodies (B) 24 h and (D) 48 h after challenge. The Figure illustrates cumulative data from two independent studies (n = 5 mice per group per time point). Data are expressed as mean A. fumigatus 18S rRNA + SEM. * represents a P value of < 0.05 (Unpaired two-tailed Student’s t test).

Discussion

When exposed to Pneumocystis carinii, mice deficient in Dectin-1 (Dectin-1−/−) show an early impairment in lung clearance, yet clear the organism similar to wild-type mice (36). Furthermore, Dectin-1−/− mice are not more susceptible to lung infection with the fungal organism Cryptococcus neoformans (37). These results may likely be explained by the beta-glucan content of these organisms, as P. carinii cysts have a much higher beta-glucan content than the more numerous trophozoites (38, 39) and C. neoformans not only possesses a large capsule, but has much lower beta-1,3-glucan content than most Dectin-1 dependent fungal organisms (37). Nevertheless, since these studies suggest a moderate role for Dectin-1 in lung defense against fungal pathogens, we questioned whether Dectin-1 was required for defense against a fungal pathogen acknowledged for its invasiveness, A. fumigatus. We found that mice deficient in Dectin-1 rapidly succumbed to invasive pulmonary disease caused by A. fumigatus as a result of functional defects in specific cell populations universally acknowledged to be required for clearing the organism from the lung. Dectin-1−/− alveolar macrophages had compromised production of proinflammatory cytokines and chemokines that were essential for neutrophil mobilization to the lung. Neutrophils in Dectin-1−/− mice were recruited to the lung in lower numbers and displayed defective anti-fungal defenses in vitro, which likely promoted invasion of A. fumigatus leading to dramatic changes in lung architecture and subsequently impaired lung function.

The phenotype of Dectin-1−/− mice challenged with A. fumigatus was dramatic, with these mice having 80-100% mortality within 5 days after exposure. One potential caveat of our study is employing high inocula of A. fumigatus to achieve mortality differences in Dectin-1−/− mice. Our mortality experiments were performed with inocula of 5-7 × 107, based on published reports employing inoculums at this level when assessing mortality and fungal clearance in animals that are not dually immunosuppressed with cyclophosphamide and cortisone. For example, several publications assessing the role of PRRs, such as TLRs or pentraxin 3, in defense against A. fumigatus administered inocula of 4-6 × 107 A. fumigatus conidia (16) (40) and even as high as 2 × 108 (41). It is important to note that our experiments were not conducted in the presence of immunosuppressive drugs (cyclophosphamide, cortisone acetate etc.) or during transient neutropenia (i.e. antibody-mediated depletion of neutrophils). Some studies employing a variety of genetically deficient mice have reported dramatic mortality phenotypes after A. fumigatus challenge under immunosuppressive conditions, however, our position was that induction of immunosuppression prior to infection may exaggerate the role of Dectin-1. A surprising observation in our studies was that despite these inocula, Dectin-1−/− mice did not have significant extra-pulmonary dissemination of A. fumigatus. These results suggest that the rapid mortality of Dectin-1−/− mice was primarily associated with events occurring in the lung. Assessing pulmonary function supports this hypothesis as Dectin-1−/− mice suffering from A. fumigatus demonstrated significant increases in airway and whole lung resistance. These increases were specifically associated with A. fumigatus infection, as no differences were observed between WT and Dectin-1−/− mice in the absence of infection (data not shown). The significance of this correlates with clinical observations as studies have reported that individuals who are diagnosed with IPA often initially present with dyspnea (42, 43). Thus, uncontrolled A. fumigatus lung infection in Dectin-1−/− mice results in dramatic structural changes in lung architecture, which impairs respiratory mechanics. Furthermore, our data strongly support a fundamental role for Dectin-1 in lung clearance of A. fumigatus in immunocompetent mice.

The necessity for Dectin-1-mediated recognition of A. fumigatus was understandable when analyzing the responses of alveolar macrophages and neutrophils. Alveolar macrophages are considered first-line defenders against inhaled A. fumigatus and are tasked with providing the initial wave of inflammatory mediator production and inflammatory signaling (30). Alveolar macrophages from Dectin-1−/− mice were not responsive to stimulation with A. fumigatus, producing approximately 15% of the IL-α, IL-1β, TNF-α, CCL3/MIP-α, CCL4/MIP-1β and CXCL1/KC levels elicited by A. fumigatus from wild-type alveolar macrophages. We further note that proinflammatory mediators produced by alveolar macrophages in a Dectin-1 dependent manner were also significantly lower in the lungs of Dectin-1−/− mice challenged with A. fumigatus. Previous studies investigating these cytokines and chemokines have provided critical insight into their role in lung defense against A. fumigatus. Neutralization of TNF-α has been shown to blunt lung neutrophil recruitment during A. fumigatus infection resulting in delayed fungal clearance and increased mortality (44). Mice lacking CCR1, the receptor for CCL3/MIP-α, are more susceptible to A. fumigatus lung infection, also through impaired neutrophil recruitment (45) as are mice administered CCL3/MIP-α neutralizing antibodies (29). Similarly, neutralization of CXCL1/KC also results in severe susceptibility to A. fumigatus infection (28). IL-1 is an understudied cytokine in lung defense against A. fumigatus, however ongoing studies in our laboratory indicate that mice deficient in both IL-α and IL-1β are more susceptible to A. fumigatus (unpublished data). Therefore, since mediators such as TNF-α, CCL3/MIP-α and CXCL1/KC are involved in neutrophil recruitment to the lung during A. fumigatus infection (28, 44, 45) and Dectin-1 is essential for these mediators to be produced at optimal levels in vivo (Figure 4), we can predict that Dectin-1 is involved in neutrophil mobilization to the lungs during fungal infection.

Neutrophil deficiency or dysfunction is the hallmark predisposing factor for developing IPA (46). Neutrophils kill A. fumigatus through a variety of different mechanisms, although the most well studied include ROS (25), MPO (47) and lactoferrin (48). Of these, ROS, specifically superoxide, is the most acclaimed for being indispensable for the killing of A. fumigatus by neutrophils. Indeed, NADPH oxidase deficiency in humans is uniquely associated with the development of IPA (49). Moreover, mice deficient in NADPH oxidase are arguably the most susceptible mouse strain for the development for experimental IPA (25, 26). We found that the oxidative burst by neutrophils in response to A. fumigatus swollen conidia, a morphological state which we have previously shown to have beta-glucans unmasked at the highest levels (10), was dependent on Dectin-1. The inability of Dectin-1−/− neutrophils to produce superoxide/hydrogen peroxide correlated with an inability to kill A. fumigatus, suggesting that beta-glucan recognition via Dectin-1 on neutrophils results in oxidative killing of A. fumigatus. Neutrophils possess additional non-oxidative antimicrobial mechanisms, such as serine proteases and antimicrobial peptides (50), therefore we can not eliminate the possibility that Dectin-1 is involved in the release of non-oxidative antimicrobial factors that also contribute to the killing of A. fumigatus observed here. Nevertheless, we propose that beta-glucan recognition via Dectin-1 is a central event in eliciting alveolar macrophage and neutrophil-mediated anti-fungal defense against A. fumigatus.

Recent studies have identified a major component of the Dectin-1 signaling pathway as CARD9 (32). Mice deficient in CARD9 have a similar phenotype as Dectin-1−/− mice after challenge with C. albicans (32) (19). First, our results partially support the finding of reduced IL-17 production in splenocyte cultures from C. albicans-challenged CARD9−/− mice (33), as Dectin-1−/− mice exposed to A. fumigatus for 24 h had lower levels of IL-17 protein in lung homogenates. We hypothesize this is due a dependency on Dectin-1 for IL-23 production, based on the impaired lung production of IL-23 in A. fumigatus challenged Dectin-1−/− mice. In fact, this is the first report to not only show impaired IL-17 production in the absence of Dectin-1, but also the first to specifically measure a defect in IL-23 production (rather than its surrogate marker, IL-12p40). With regards to the role of IL-17 in A. fumigatus host defense, IL-17 surprisingly appears to hamper neutrophil-mediated killing of A. fumigatus as well as in vivo clearance of the organism (23). Moreover, IL-17 appears to be a contributing factor to mortality observed in A. fumigatus exposed p47phox deficient mice and to a lesser extent, Tir8 deficient mice (51). In the case of p47phox deficient mice, deficient neutrophil function leads to high A. fumigatus burden in the lungs, which itself may promote enhanced antigen-driven Th17 develop and subsequent immunopathology (34). These data challenge the current dogma of IL-17 as an essential mediator for effective immune responses against microorganisms that cause infections at the mucosa (52). Furthermore, it is somewhat surprising that a pro-neutrophil cytokine such as IL-17 has a pronounced negative role against a pathogen, A. fumigatus, in which neutrophils are required for its elimination (2, 3) and neutrophil deficiency/dysfunction is the dominant predisposing factor for the incidence of disease (46). In our study, we were unable to show that neutralizing IL-17 in wild-type mice was beneficial for clearing A. fumigatus from the lungs, but rather observed that neutralizing IL-17 significantly worsened the infection. These results suggest that IL-17 is involved, at some level, in protective responses to A. fumigatus. Support for this comes from a recent study showing that A. fumigatus sensitized TLR9−/− mice challenged with swollen conidia had higher lung fungal burden 14 days post-challenge that correlated with lower Dectin-1 mRNA levels and lower IL-17 protein levels in the lung (53). Finally, clinical data, although limited, also suggest a protective role for Th17/IL-17 in defense against A. fumigatus. Studies have recently demonstrated that individuals with hyper-IgE syndrome (HIES, Job’s Syndrome), who have mutations in STAT3 and are unable to produce Th17 cells (54), are severely susceptible to a lung infection caused by A. fumigatus, most often when cavitary lung lesions are present (55). However, it remains to be determined whether A. fumigatus infection in HIES is the result of an inherent susceptibility of these patients, cavitary lung disease, or both.

In summary, we have identified a signaling pattern recognition receptor which is essential for host defense against A. fumigatus in an immunocompentent host. Moreover, we show a comprehensive role for Dectin-1 in multiple aspects of lung immunity against A. fumigatus including lung clearance, alveolar macrophage cytokine and chemokine production, neutrophil-mediated killing and in vivo lung inflammatory responses. We further provide experimental evidence for a protective role of IL-17 in host defense against A. fumigatus and that Dectin-1 mediated beta-glucan recognition is involved in the induction of Th17/IL-17 responses in the lung. We therefore feel these studies lay the foundation for further examination of Dectin-1 expression and function in individuals who are at-risk for IPA, which may provide valuable insight into the role of Dectin-1 in susceptibility to IPA in immunosuppressed patients.

Acknowledgements

The authors thank Dr. James Kreindler, University of Pennsylvania, and Dr. Lee Quinton, Boston University, for helpful discussion. G.D.B. is a Wellcome Trust Senior Research Fellow in Biomedical Science in South Africa.

Footnotes

1

This work was supported was supported by the Wellcome Trust (G.D.B.) and CANSA South Africa (G.D.B.) and grants from the Parker B. Francis Foundation (C.S.), the American Lung Association (C.S.) and PHS grant HL080317 (C.S.).

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