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Infection and Immunity, March 2006, p. 1480-1489, Vol. 74, No. 3
0019-9567/06/$08.00+0     doi:10.1128/IAI.74.3.1480-1489.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Human Dendritic Cells following Aspergillus fumigatus Infection Express the CCR7 Receptor and a Differential Pattern of Interleukin-12 (IL-12), IL-23, and IL-27 Cytokines, Which Lead to a Th1 Response

Valérie Gafa,1,2 Roberto Lande,1 Maria Cristina Gagliardi,1 Martina Severa,1 Elena Giacomini,1 Maria Elena Remoli,1 Roberto Nisini,1 Carlo Ramoni,3 Paolo Di Francesco,4 Delphine Aldebert,2 Renée Grillot,2 and Eliana M. Coccia1*

Department of Infectious, Parasitic, and Immuno-Mediated Diseases,1 Department of Cell Biology and Neuroscience, Istituto Superiore di Sanità, 00161 Rome,3 Department of Experimental Medicine and Biochemical Sciences, University of Rome Tor Vergata, Rome, Italy,4 Laboratoire Interactions Cellulaires Parasites-Hote, Faculté de Médecine-Pharmacie, Université Joseph Fourier, Grenoble I, La Tronche, France2

Received 22 October 2005/ Accepted 30 November 2005


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ABSTRACT
 
Aspergillus fumigatus is the most prevalent airborne fungal pathogen and causes fatal invasive aspergillosis in immunocompromised patients. Given the essential role of dendritic cells (DC) in initiating and regulating immune responses, we investigated the impact of A. fumigatus conidial infection on human DC. A. fumigatus conidia were rapidly internalized and induced the release of tumor necrosis factor alpha within the first 8 h. After A. fumigatus infection, the majority of DC underwent full maturation, although CCR7 expression was observed only in DC that had internalized the conidia. Additionally, the analysis of regulatory cytokines showed that infected DC simultaneously produced interleukin-12p70 (IL-12p70) and significant amounts of IL-10. IL-10 neutralization was not able to further increase IL-12p70 production from infected DC. Whereas the central role of IL-12 in the generation of Th1 cells has long been appreciated, recently two other members of the IL-12 family, IL-23 and IL-27, were reported to play important roles in the regulation of gamma interferon (IFN-{gamma}) production from naïve and memory T cells. A. fumigatus-infected DC were also able to express high levels of IL-23p19 and low levels of IL-27p28 at later stages of infection. According to this expression pattern, A. fumigatus-infected DC were able to prime IFN-{gamma} production of naïve T cells. Thus, this study on the expression of the new IL-12 family members controlling the Th1 response sheds light on a novel aspect of the contribution of DC to anti-Aspergillus immunity.


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INTRODUCTION
 
Aspergillus fumigatus is a saprophytic fungus responsible for approximately 90% of human cases of aspergillosis. It causes a usually fatal invasive aspergillosis (IA) in immunocompromised hosts (13). The airborne conidia of A. fumigatus present in the atmosphere are small enough (2 to 3 µm in diameter) to be continuously inhaled and to reach the human lung alveoli, where they are captured, ingested, and killed by resident phagocytes in immunocompetent individuals (20). Otherwise, in immunocompromised patients, the conidia can overcome the immune defense mechanisms, germinating into mycelia that invade the lung. The major recognized risk factors for IA are defects in phagocytic function (25), corticosteroid-induced suppression of macrophage conidiocidal activity (30, 43), and long-lasting neutropenia (15). Numerous studies have shown that innate immune mechanisms play a key role in the defense against A. fumigatus. In particular, alveolar macrophages and neutrophils are the main cells involved in the rapid killing of A. fumigatus through oxidative and nonoxidative mechanisms (33, 50).

Although the importance of the innate response has been well described both in vitro and in vivo, a recent observation showed that healthy individuals or patients with clinical evidence of IA and disease regression in antifungal therapy featured positive lymphoproliferation and a high level of gamma interferon (IFN-{gamma}) (19). Correlated to these results, studies with the murine model of IA showed that resistance to infection was associated with the production of IFN-{gamma}, tumor necrosis factor-{alpha} (TNF-{alpha}), and interleukin-12 (IL-12), whereas a dominant release of Th2 cytokines by interstitial lung lymphocytes was correlated with the development of disease (8, 9). The fact that the Th response may modify the outcome of IA indirectly indicates a key role for human dendritic cells (DC) in the modulation of the immune response against A. fumigatus. Indeed, at the site of primary infection, DC constitute an integral part of the innate immune system, recognizing the pathogen and secreting inflammatory cytokines, such as TNF-{alpha}, IL-6, and IL-1 (38). After interacting with the pathogen, DC mature and migrate into the lymphoid organs, where they interact with T cells, transmitting information on the type of infection encountered and inducing a T-cell response through a coordinated stimulation via T-cell receptor engagement, costimulatory molecules, and cytokine production (37).

IL-12p70, type I IFN, and IL-18 are well-documented examples of Th1-promoting cytokines (12). Recently other factors, belonging to the IL-12 family, were reported to play a key role in promoting a Th1 response. In particular, IL-23 acts primarily on effector T cells, prolonging and sustaining their IFN-{gamma} production (46) and stimulating the proliferation of memory T cells (27, 52), whereas IL-27 has a strong effect, especially on naïve Th cells inducing the early production of IFN-{gamma} (32).

The interaction of human DC with A. fumigatus and its consequence for the immune defense against this pathogen have been studied previously (4, 18, 41, 44). However in the present study, we investigated the impact of A. fumigatus infection on human DC maturation, focusing on the expression of CCR7 and novel members of the IL-12 family. Interestingly, we observed that the majority of DC underwent full maturation, although CCR7 expression was observed only in DC that had internalized the conidia. In addition, analysis of inflammatory and immunoregulatory cytokines was performed in order to determine the capacity of DC to elicit an anti-Aspergillus immune response. In particular, our results with regard to the IL-12, IL-23, and IL-27 expression profiles provide new insights into the mechanisms promoting the anti-Aspergillus Th1 response.


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MATERIALS AND METHODS
 
Generation of DC. DC were prepared as previously described (16). Peripheral blood mononuclear cells (PBMC) were isolated from healthy voluntary blood donors (Blood Bank of University "La Sapienza," Rome, Italy). Monocytes were purified by positive sorting using anti-CD14-conjugated magnetic microbeads (Miltenyi, Bergisch Gladbech, Germany). DC were generated by culturing monocytes in 6-well tissue culture plates (Costar Corporation, Cambridge, MA) with 25 ng/ml granulocyte-macrophage colony-stimulating factor (GM-CSF; Schering-Plough, Levallois Perret, France) and 1,000 U/ml of IL-4 (R&D Systems, Minneapolis, Minn.) for 5 days at 0.5 x 106 cells/ml in RPMI 1640 (Biowhittaker Europe, Verviers, Belgium) supplemented with 2 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, and 15% fetal calf serum (Biowhittaker Europe). On day 5 the cells were 70 to 90% CD1a+ and 95% CD14–. Then the culture medium containing IL-4 and GM-CSF was replaced with culture medium alone 20 h before infection or treatments.

Abs and other reagents. Monoclonal antibodies (MAbs) specific for CD86, CD83, CD1a, CD14, and CCR7, as well as immunoglobulin G1 (IgG1), IgG2a, and IgG2b (BD Bioscience PharMingen, San Diego, CA) were used as pure antibodies (Abs) or as direct conjugates to fluorescein isothiocyanate (FITC). FITC-conjugated goat anti-mouse IgG F(ab')2 was used as a secondary Ab when necessary. Lipopolysaccharide (LPS) from Escherichia coli 0111:B4 (Sigma-Aldrich, St. Louis, MO) was used at a concentration of 100 ng/ml to induce DC maturation. An anti-human IL-10 Ab and normal goat IgG (R&D Systems) were used at a concentration of 0.2 µg/ml.

Flow cytometric analysis. Cells (105) were aliquoted into tubes and washed once in phosphate-buffered saline (PBS) containing 2% fetal calf serum. The cells were incubated with purified MAbs at 4°C for 30 min. After a wash, the cells were fixed with 2% paraformaldehyde before analysis on a FACScan using CellQuest software (BD Bioscience PharMingen).

For the adherence assay, A. fumigatus conidia were first incubated with FITC at a final concentration of 3 µg/ml overnight at 4°C and then washed extensively with PBS. DC were challenged with FITC-labeled conidia (ratio, 1:1) and were incubated for 30 min, 2 h, or 6 h at 37°C. After a wash, adherence was measured by flow cytometric analysis.

Cell viability was assessed by propidium iodide (PI; Sigma-Aldrich) fluorescence. DC were washed with PBS and then incubated with PI (final concentration, 50 µg/ml) for 10 min at 4°C. The percentage of live cells (PI– cells) was evaluated by cytometric analysis.

Microorganism, culture conditions, and infection. The A. fumigatus strain referenced as CBS 144-89 (CBS, Utrecht, The Netherlands) was grown on Sabouraud-chloramphenicol agar for 3 days at 37°C. Conidia in the presence of sterile 0.1% Tween 20 in PBS were harvested by gentle shaking, washed, filtered, and suspended at a concentration of 108 conidia/ml. All A. fumigatus preparations were analyzed for LPS contamination by the Limulus lysate assay (Biowhittaker Europe) and were found to contain less than 10 pg/ml of LPS.

Amphotericin B (AB; Sigma-Aldrich) and voriconazole (VCZ; Molekula, La Tour du Pin, France) were added to the cell cultures 2 h after infection to prevent fungal overgrowth, enabling us to strictly study the interaction of conidia with DC. Dose-response experiments with AB and VCZ were carried out to identify the minimal dose able to inhibit the development of conidia into hyphae (data not shown). A dose of 0.5 µg/ml was considered the MIC of AB and VCZ.

After dilution in complete medium (50%, vol/vol), filtered supernatants were used to stimulate immature DC for 30 h.

Real-time PCR quantifications. Reverse transcriptions were performed as previously described (11). Quantitative PCR assays were performed at least in triplicate using Platinum Taq DNA polymerase (Invitrogen Life Technologies, Frederick, MD) and SYBR Green I (BioWhittaker Molecular Applications, Rockland, ME) on a LightCycler (Roche Diagnostics, Basel, Switzerland). Primer pairs for IL-p40, IL-12p35, IL-23p19, IL-27p28, and IL-27EBI3 have been described by Nagai et al.(26), those for IL-10 by Gibson et al. (17), and those for TNF-{alpha} by Overbergh et al. (28). All quantification data are presented as ratios to the level of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Only ratios with a standard error (SE) of 0.2 log unit (95% confidence limits) were considered for the determination of induction levels.

Cytokine detection. Supernatants from immature DC or from A. fumigatus-infected DC or LPS-treated DC cultures were harvested at the indicated times, filtered (0.2-µm filters), and stored at –80°C. IL-6, IL-10, IL-12p70, and TNF-{alpha} were measured with the human inflammation cytometric bead array (CBA) (BD Bioscience PharMingen). An IL-23-specific enzyme-linked immunosorbent assay (ELISA) kit was obtained from Bender MedSystems, Inc. (Burlingame, CA), and the assay was conducted according to the manufacturer's instructions.

CLSM analysis. Immature DC or DC treated with FITC-labeled conidia were analyzed by confocal laser scanning microscopy (CLSM) after 6 or 30 h at 37°C. After two washes with PBS, cells were labeled with MAbs against CD83, CCR7, and actin (30 min at 4°C) washed and incubated with an Alexa Fluor 594-conjugated goat anti-mouse IgG (Fab')2 fragment (Molecular Probes, Eugene, OR) as a secondary Ab. Where specified, cells were stained with FITC-conjugated anti-CD1a. After repeated washing with 1% bovine serum albumin (Sigma-Aldrich) in PBS, fixation was carried out at 4°C for 30 min with 3% paraformaldehyde, followed by permeabilization with 0.5% Triton X-100 for 10 min at room temperature. Cells were then seeded on a microscope slide with the Prolong reagent (Molecular Probes). CLSM observation was carried out on a Leica TCS SP2 apparatus, equipped with an argon laser (excitation wavelength, 488 nm) and an argon-krypton laser (excitation wavelength, 594 nm). Image acquisition and processing were performed by using the multicolor LCS (Leica Lasertechnik GmbH, Heidelberg, Germany) and Photoshop CS (Adobe Systems, Mountain View, CA) software. Several cells were analyzed for each labeling condition, and representative section results are shown.

Mixed leukocyte reaction (MLR). Cord blood CD4+ T cells were purified by indirect magnetic sorting with a CD4+ T-cell isolation kit (Miltenyi). Immature DC, A. fumigatus-infected DC, or LPS-treated DC were resuspended in their respective filtered supernatants. The proliferative response was assessed at various T-cell/DC ratios, using a fixed number of T cells (3 x 104), and was evaluated after 7 days by measuring thymidine incorporation (0.5 µCi/well of [3H]thymidine; Amersham, Little Chalfont, Buckinghamshire, United Kingdom). No thymidine incorporation by A. fumigatus alone was observed. Some T cells were stimulated with 10–7 M phorbol 12-myristate 13-acetate and 1 µg/ml ionomycin (Sigma-Aldrich) for 5 h, and brefeldin A (Golgi-Plug; BD Bioscience PharMingen) was added during the last 2 h. These T cells were then analyzed by flow cytometry for their intracellular cytokine production.

Analysis of intracellular cytokine production. Cytokines within T cells were stained with phycoerythrin-conjugated mouse anti-human IL-4 and FITC-conjugated mouse anti-human IFN-{gamma} (BD Bioscience PharMingen) after fixation and permeabilization using Cytofix/Cytoperm (BD Bioscience PharMingen), according to the manufacturer's instructions. Stained cells were analyzed by flow cytometry using a FACScan cytometer equipped with CellQuest software (BD Bioscience PharMingen).

Statistical analysis. Data are expressed as means ± SEs. The statistical significance of differences was determined by the Student t test (a P value of <0.05 was considered significant).


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RESULTS
 
Infection of DC with A. fumigatus. Initial studies were performed to examine the adherence of conidia to DC by analyzing the percentage of DC with bound FITC-labeled A. fumigatus conidia by flow cytometry. Thirty percent of DC fixed A. fumigatus conidia within 30 min, and this percentage increased to 48% after 2 h of infection and remained constant at 6 h, with 52% (Fig. 1A), and thereafter (data not shown). Then CLSM was performed to discriminate between attached and internalized conidia. At 6 h postinfection, conidia were observed inside the cells. In fact, sections obtained from the same cell showed that FITC-labeled conidia were localized in the cytoplasm area (Fig. 1B).


Figure 1
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  		FIG. 1. Infection of DC with A. fumigatus. DC were incubated with FITC-labeled conidia at a ratio of 1:1 for 30 min, 2 h, or 6 h at 37°C. (A) The percentage of DC with bound FITC-labeled A. fumigatus conidia (Af-FITC positive DC) was measured by flow cytometric analysis. Results of one representative experiment out of four performed are shown. (B) Internalization of A. fumigatus conidia in DC was observed at 6 h by CLSM. DC were incubated with FITC-labeled conidia (yellow pseudocolor). Cells were labeled with an anti-actin Ab followed by an Alexa 594-conjugated goat anti-human secondary Ab (red). The samples were scanned through a confocal laser microscope. Three representative sections of one cell are shown. The same analysis was performed on 200 cells from two different DC cultures. The relative position of the section in the cell is represented by "z." Bar, 5 µm.

Analysis of DC maturation following A. fumigatus infection. To analyze the impact of A. fumigatus infection on DC maturation, DC were infected for 30 h with A. fumigatus conidia, and the expression of B7.2 (CD86) and CD83 on the cell surface was examined (Fig. 2A). This long incubation time required the addition of antifungal drugs to the cell culture. Two drugs, AB and VCZ, affecting different cellular targets were used to verify that the data obtained were not due to an indirect effect of the antifungal agents on the DC activity. For both antifungal agents, the lowest dose preventing A. fumigatus outgrowth was 0.5 µg/ml. Thirty hours after A. fumigatus infection, all DC showed enhanced expression of CD86 and CD83, whatever the antifungal agent, suggesting that A. fumigatus-infected DC are likely to act as efficient antigen-presenting cells (Fig. 2A). As expected, LPS-treated DC displayed a mature phenotype.


Figure 2
Figure 2
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  		FIG. 2. Analysis of maturation markers and cytokine production in DC stimulated by LPS or A. fumigatus. DC were exposed to either of the two stimuli for 30 h at 37°C in the presence or absence of AB and VCZ. (A) The expression of the markers was analyzed by flow cytometry. A total of 5,000 cells were analyzed per sample. Nonstimulated and stimulated cells were stained with a control Ab (empty histogram). Representative flow cytometry profiles of one experiment, which was repeated three more times, are shown. DC from a total of four different blood donors were used. Ctr, control. (B) Cytokine production following the stimulation of DC with LPS or A. fumigatus. Cells were either left untreated (Ctr), infected with A. fumigatus conidia, or stimulated with LPS. Cell culture supernatants were collected 30 h after DC stimulation. The cytokine levels were measured by CBA. Results are means ± SEs from four separate experiments. All cytokines tested were significantly induced: P < 0.05 for A. fumigatus versus Ctr and for LPS versus Ctr in the presence of AB or VCZ. (C) DC viability assay assessed by PI fluorescence. The percentage of living cells (PI– cells) was determined by flow cytometry. Results are means ± SEs from four separate experiments. *, P < 0.05 for A. fumigatus versus Ctr in the presence of VCZ.

Cell culture supernatants were harvested 30 h after A. fumigatus infection and analyzed by CBA for the production of inflammatory and regulatory cytokines. A. fumigatus and LPS induced significant secretion of TNF-{alpha}, IL-6, and the regulatory cytokines IL-12 and IL-10 (Fig. 2B). Although A. fumigatus-infected DC produced larger amounts of all cytokines in the presence of VCZ than in the presence of AB, the same pattern of cytokines was produced in the presence of the two drugs. The viability of A. fumigatus-infected DC was altered in the presence of VCZ but was not significantly affected by exposure to AB (Fig. 2C). We decided to perform further A. fumigatus assays in the presence of AB because of the cell mortality found in A. fumigatus-infected DC exposed to VCZ and because the patterns of cytokine expression in the presence of the two drugs were similar. Control experiments indicated that in our DC cultures, AB alone (0.5 µg/ml) did not modify the pattern of cytokine production.

Kinetics of TNF-{alpha} expression in A. fumigatus-infected DC. We showed above that although only 52% of DC had internalized A. fumigatus, all DC expressed CD86 and CD83 markers, suggesting that the release of soluble molecules could also be involved in the induction of DC maturation. Thus, we focused our study on TNF-{alpha} expression, known for its contribution to DC maturation. The kinetics of mRNA expression was assessed by real-time PCR by using total RNA extracted at various time points after A. fumigatus infection. TNF-{alpha} mRNA induction by A. fumigatus began 2 h postinfection, reached a maximum at 20 h, and declined rapidly (Fig. 3A). The kinetics of TNF-{alpha} secretion by A. fumigatus-infected DC, evaluated by CBA, confirmed the profile of TNF-{alpha} mRNA expression (Fig. 3B). Indeed, A. fumigatus induced significant levels of TNF-{alpha} in culture supernatants as early as 8 h after infection, peaking at 48 h and declining thereafter.


Figure 3
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  		FIG. 3. Kinetics of TNF-{alpha} expression in A. fumigatus-infected DC. (A) Total RNA was extracted at the indicated time points. The expression of TNF-{alpha} was analyzed by real-time PCR. Results are means ± SEs of triplicate values. This is a representative real-time PCR experiment, which was repeated two more times with RNA extracted from various DC cultures. (B) The cytokine level was measured by CBA. Results are means ± SEs from three separate experiments. At all time points, TNF-{alpha} production was significantly induced: P < 0.05 for A. fumigatus-infected versus uninfected cells (0 h). (C) Analysis of maturation markers in immature DC stimulated by A. fumigatus-infected DC supernatants (SN). Immature DC were treated for 30 h with supernatants of uninfected cells (control [Ctr]) or supernatants of cells infected by A. fumigatus for 24 h (Af). The expression of CD83 and CCR7 markers was analyzed by flow cytometry. Representative flow cytometric profiles of one experiment, which was repeated three more times, are shown. DC from a total of four different blood donors were used.

Immature DC were stimulated for 30 h with supernatants from control or infected cells in order to study the functional impact of TNF-{alpha} release on DC maturation. A clear induction of CD83 was observed following the addition of supernatants from A. fumigatus-infected DC. We also extended our analysis to CCR7, a chemokine receptor involved in the migration of mature DC into secondary lymphoid organs (42). Interestingly, no induction of CCR7 was found on DC stimulated with supernatants from A. fumigatus-infected DC (Fig. 3C).

CCR7 expression on A. fumigatus-infected DC. We performed both flow cytometry analysis and CLSM to investigate whether infection with A. fumigatus could stimulate CCR7 expression on DC. Interestingly, we observed by flow cytometric analysis that only 58% of A. fumigatus-infected DC expressed CCR7 (Fig. 4A). Moreover, CLSM analysis indicated that only DC that had internalized A. fumigatus conidia expressed CCR7, whereas CD83 was present in all DC, including those that did not phagocytose the conidia (Fig. 4B). These results, together with those presented in Fig. 3C, suggest that only DC invaded by A. fumigatus may acquire the capacity to migrate into secondary lymph nodes.


Figure 4
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  		FIG. 4. Analysis of CCR7 expression on A. fumigatus-infected DC. (A) DC were exposed to A. fumigatus or LPS for 30 h, and CCR7 expression was analyzed by flow cytometry. Nonstimulated (control [Ctr]) and stimulated cells were stained with a control Ab (empty histogram). Representative flow cytometry profiles of one experiment, which was repeated five more times, are shown. DC from a total of six different blood donors were used. (B) DC were incubated with FITC-labeled conidia (yellow pseudocolor) at a ratio of 1:1 and were labeled with MAbs against CD83, CCR7 (red), and CD1a (green). Representative images from one experiment, which was repeated three more times, are shown. The samples (100 cells) were scanned through a confocal laser microscope, and the images shown are the arithmetic sums of entire stacks of the cell monolayer.

Kinetics of IL-12p70 and IL-10 expression following A. fumigatus infection. Next, we analyzed the kinetics of IL-12 and IL-10 mRNA expression; these cytokines are differentially involved in the immunoregulation of both innate and adaptive responses. We performed real-time PCR to analyze the two subunits of IL-12p70, p35 and p40, as well as to analyze IL-10, by using total RNA extracted at various time points after A. fumigatus infection. The analysis of p40 and p35 mRNAs indicated that in A. fumigatus-infected DC, the expression of both subunits increased only from 6 to 20 h and decreased 48 h after infection (Fig. 5A). Similar kinetics was observed for IL-10 mRNA expression (Fig. 5B). Taken together, these results suggest that A. fumigatus-infected DC might release these two cytokines at later stages of infection.


Figure 5
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  		FIG. 5. Kinetics of IL-12p70 and IL-10 expression. (A and B) Kinetics of IL-12p40, IL-12p35, and IL-10 expression in A. fumigatus-infected DC. Total RNA was extracted at the indicated time points. The expression of IL-12p40, IL-12p35, and IL-10 was analyzed by real-time PCR. Results are means ± SEs of triplicate values. These are representative real-time PCR experiments, which were repeated two more times with RNA extracted from various DC cultures. (C) Analysis of IL-12p70 production from unstimulated (control [Ctr]) or LPS- or A. fumigatus-stimulated DC following IL-10 neutralization. An anti-human IL-10 Ab or the normal anti-goat IgG was added to LPS- or A. fumigatus-stimulated or Ctr DC cultures. After 30 h of stimulation, the levels of IL-12p70 released in the supernatants of the different mature DC were analyzed by CBA. Results are means ± SEs from three separate experiments. *, P < 0.05 for LPS plus the anti-human IL-10 Ab versus LPS alone.

Based on these kinetics results, we wondered if IL-12 expression could be affected by the simultaneous expression of IL-10, a potent inhibitor of IL-12 production (1, 55). Thus, we tested whether by blocking IL-10 activity, IL-12p70 expression could be further increased in A. fumigatus-infected DC (Fig. 5C). DC cultures were stimulated for 30 h with LPS or A. fumigatus in the presence of an anti-IL-10 Ab or a control Ab. Although IL-12 production by LPS-treated DC was significantly enhanced by addition of an IL-10 neutralizing Ab, no effect was observed in A. fumigatus-infected DC, indicating that IL-12 secretion following A. fumigatus infection was not limited by the presence of IL-10 (Fig. 5C).

Expression of the novel IL-12 family members in A. fumigatus-infected DC. Other DC-derived cytokines that promote the development of Th1 cells include two novel IL-12 family members, IL-23 and IL-27. IL-23 is a heterodimer composed of two subunits, p19 and p40, whereas IL-27 is composed of the p28 and EBI3 subunits. The mRNA contents of the p19, p28, and EBI3 subunits were evaluated by real-time PCR to determine whether A. fumigatus was able to stimulate the expression of these molecules in DC. Total RNA was extracted at various time points after A. fumigatus infection or LPS treatment. Strikingly, IL-23p19 mRNA reached a much higher level at 20 to 48 h in A. fumigatus-infected DC than in LPS-treated DC (Fig. 6A). The analysis was performed over 48 h to exclude the possibility that these observations could be due to disparities in the kinetics of cytokine expression. The expression of p19 mRNA was up-regulated rapidly within the first 2 h following LPS treatment, remaining high from 6 to 20 h and decreasing to baseline levels at 48 h, while in A. fumigatus-infected DC, p19 expression was observed at 20 h postinfection and was still increasing at 48 h. The findings of delayed IL-23p19 and IL-12p40 mRNA expression, shown in Fig. 6A and 5A, respectively, suggested that IL-23, like IL-12, could be released from DC at the late stages of infection. This hypothesis was confirmed by ELISA (Fig. 6B), which showed a significant release of IL-23 from A. fumigatus-infected DC 30 h after infection.


Figure 6
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  		FIG. 6. Expression of IL-23 and IL-27 in DC stimulated with LPS or A. fumigatus. (A and C) The mRNA expression of IL-23p19 (A) and of IL-27p28 and IL-27EBI3 (C) was analyzed by real-time PCR. Total RNA was extracted at the indicated time points. Results are means ± SEs of triplicate values. This is a representative real-time PCR experiment, which was repeated two more times with RNA extracted from various DC cultures. (B) The cytokine level of IL-23 was measured by ELISA 30 h after DC stimulation with LPS or A. fumigatus. Results are means ± SEs for three separate experiments. *, P < 0.05 for A. fumigatus versus the control (Ctr).

The expression levels of the two IL-27 subunits, p28 and EBI3, were then analyzed. The levels of EBI3 mRNA increased gradually after 6 h in DC treated with either of the two stimuli (Fig. 6C). The expression of EBI3 remained significant at 48 h, although it reached various levels in stimulated DC. We analyzed IL-27p28 mRNA expression to assess whether high levels of EIB3, induced by both stimuli, reflected IL-27 production. A clear up-regulation of IL-27p28 was evident only upon LPS treatment, while a slight increase was noted at 20 h after A. fumigatus infection (Fig. 6C), suggesting that A. fumigatus-infected DC might release a small amount of this cytokine relative to that released by LPS-stimulated DC.

Analysis of T-cell priming and polarization by A. fumigatus-infected DC. We studied T-cell proliferation and polarization by MLR to evaluate whether A. fumigatus-infected DC were able to prime Th1 cells. As shown in Fig. 7A, A. fumigatus-infected DC induced a clear proliferation of naïve allogeneic cord blood CD4+ T cells compared to control DC. LPS-matured DC were used as positive controls for the T-cell proliferative response.


Figure 7
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  		FIG. 7. Stimulation of naïve CD4+ T cells. (A) DC were either left untreated or stimulated for 24 h with LPS or A. fumigatus conidia. An MLR assay was then set up with irradiated DC cultured at various cell numbers with 5 x 104 purified allogeneic CD4+ CD45RA+ T cells from cord blood. The proliferative response was measured after 6 days and is expressed as mean counts per minute of triplicate cultures. Results are means ± SEs of three experiments. (B) Intracellular staining for IL-4 and IFN-{gamma} production by T cells activated in the MLR for which results are shown in panel A. The results shown are from one representative experiment out of three performed.

Flow cytometric analysis of intracellular IL-4 and IFN-{gamma} accumulation in proliferating T lymphocytes showed that A. fumigatus-infected DC stimulated a clear induction of IFN-{gamma}-producing T cells (Fig. 7B). Conversely, no induction of IL-4-expressing T cells was obtained with A. fumigatus-matured DC.


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DISCUSSION
 
Although IA is a serious opportunistic fungal infection, especially in patients with deep and long-lasting neutropenia, IA has more recently been reported to occur also in nonneutropenic patients after allogeneic stem cell transplantation or solid organ transplantation (53), suggesting that cell-mediated immunity plays an important role in protection against this pathogen. Similarly, an increased incidence of invasive fungal infection was observed in patients after allogeneic bone marrow transplantation compared to peripheral blood stem cell transplantation, in which faster T-cell reconstitution occurred, indicating a potential role of T cells in the control of fungal infection (19, 49). Based on this evidence, we investigated the effects of A. fumigatus infection on human DC, since these cells are present in the lungs and are considered important in the regulation of both innate and acquired immune responses (2, 31, 40). In particular, we focused our studies on expression of IL-23 and IL-27, the novel members of the IL-12 family, described as key factors together with IL-12 in the promotion and maintenance of the Th1 cell response (6, 12, 29) required for efficient resistance to A. fumigatus infection (40).

In our experimental model, the internalization of A. fumigatus conidia was complete 6 h after interaction with DC. DC expressed CD86 and CD83 and secreted proinflammatory and regulatory cytokines following A. fumigatus infection, confirming previous observations (3, 31, 44). Interestingly, some differences in the magnitude of cytokine secretion by A. fumigatus-infected DC were observed, depending on the antifungal agent used. In fact, A. fumigatus-infected DC released larger amounts of all cytokines tested in the presence of VCZ than in the presence of AB. Since addition of either of these two antifungal agents did not affect cytokine secretion from LPS-stimulated DC, we hypothesized that the action of VCZ and AB on A. fumigatus development might directly influence the signal controlling cytokine production by infected DC. AB is known to bind to existing ergosterol in the fungal membrane, while VCZ blocks ergosterol biosynthesis, affecting A. fumigatus growth in two different stages, as previously suggested by Ramani et al. (34). VCZ might allow the early stages of germination (swelling) and the release of intracellular fungal constituents that are able to stimulate cytokine production, whereas killing by AB might be more efficient. Early germination in the presence of VCZ could also explain the alteration of the viability of A. fumigatus-infected DC. One of the fungal toxic metabolites released from VCZ-treated conidia involved in the loss of host cell viability could be gliotoxin, known to be involved in the apoptotic process (47).

The phagocytosis of A. fumigatus resulted in a strong and rapid release of TNF-{alpha}. Besides its key contribution to the anti-Aspergillus innate response (9, 14, 23, 39), TNF-{alpha} represents an essential cytokine involved in DC maturation (42). Indeed, the TNF-{alpha} present in the supernatants from A. fumigatus-infected DC could determine the induction of a partial maturation of DC, characterized by the acquisition of CD83 and the absence of CCR7. Thus, these partially mature DC might remain at the site of infection to present A. fumigatus antigens to CD4 and CD8 memory T cells present in the lung (22, 36). Conversely, DC that have internalized the conidia undergo full maturation, acquiring CCR7, and in turn might migrate into the lymph nodes to prime naïve CD4 T cells. This is supported by a study performed with a murine model showing that pulmonary DC transport A. fumigatus conidia from the alveolar spaces to the draining lymph nodes (3).

Inflammatory mediators, produced early at the site of infection, are replaced at later time points by cytokines promoting T-cell polarization. Our analysis was focused on Th1 polarizing cytokines usually associated with resistance to A. fumigatus infection (7, 10, 19). IL-12 is considered the main IFN-{gamma}-inducing cytokine. The local and rapid release of IL-12 is generally responsible for IFN-{gamma} production by NK or effector CD4+ T cells, while in the lymph nodes, the late IL-12 secretion leads to IFN-{gamma} production from naïve Th cells and therefore strongly promotes a Th1 response (38, 51). Human DC infected by A. fumigatus conidia produced detectable levels of IL-12p70. This correlates with recent observations by Romani and colleagues showing a similar production of IL-12p70 from human myeloid DC infected by A. fumigatus conidia (41). Conversely, the apparent discrepancy with the results obtained by Serrano-Gomez et al., describing a lack of IL-12 production 18 h after A. fumigatus infection (44), could be explained by the delayed IL-12 secretion observed in our experimental model (30 h after infection). Thus, our results suggest that A. fumigatus-induced IL-12p70 is mainly released by mature DC after their migration into lymph nodes. Interestingly, the concomitant release of IL-10 did not limit IL-12 production by A. fumigatus-infected DC, while IL-10 neutralization was able to reinforce only IL-12 production by LPS-stimulated DC, as previously described (1, 55).

Recently, two other members of the IL-12 family, IL-23 and IL-27, were reported to play important roles in the regulation of IFN-{gamma} production from naïve and memory T cells (12, 29, 52). Our real-time PCR analysis of the expression of IL-23 and IL-27 subunits indicated that A. fumigatus-infected DC do not produce relevant amounts of IL-27. Conversely, at later time points, A. fumigatus-infected DC induced robust expression of IL-23p19 and IL-12/IL-23p40 mRNAs and the release of IL-23 protein.

The observations that A. fumigatus-infected DC express high levels of IL-23p19 and no IFN-ß (data not shown) may suggest that this pathogen could mainly trigger the TLR2 pathway, since a similar profile of expression was observed in human DC stimulated by TLR2 agonists (35). However, TLR2 signaling generally does not lead to the production of IL-12 p70 that we observed following A. fumigatus infection, suggesting that other pattern recognition receptors, including TLR4 and the mannose receptor, could be involved in this induction (5, 21, 24, 40, 54).

Interestingly, A. fumigatus-matured DC primed naïve T cells for an allogeneic response, inducing clear lymphoproliferation and Th1 polarization. Indeed, naïve T cells expanded by A. fumigatus-matured DC were able to secrete IFN-{gamma} but not IL-4. Since no expression of IL-27 was detected in A. fumigatus-infected-DC, the observed Th1 response could be determined by the secretion of IL-12p70, which acts at an early stage of naïve Th cell differentiation. Moreover, the significant production of IL-23 from A. fumigatus-infected DC could play an important role in the establishment and maintenance of a pool of Th1 memory cells specific for A. fumigatus in healthy individuals (6, 12, 27).

In immunocompromised patients with T memory cell depletion, the immune system is unable to induce an effective Th1 response to A. fumigatus. In these patients a rapid recovery of functional Th1 cells is mandatory in order to control fungal invasion and disease progression. Thus, our results concerning the ability of A. fumigatus to induce a differential pattern of the novel IL-12 family members might be instrumental in reinforcing the new DC-based therapeutic approaches (31, 41, 45, 48). To this end, successful immunization protocols should be aimed at (i) inducing an important IL-12/IL-27 production to promote a strong and prompt Th1 response and simultaneously (ii) exploiting the A. fumigatus-induced IL-23 production to accelerate the reconstitution of the T memory cell repertoire.


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ACKNOWLEDGMENTS
 
This work was supported by grants from the ISS-NIH Program (5303) and from AIDS Research (50F/G) to E.M.C. and R.N.

We thank Luigina Romani, Antonella Torosantucci, Jean-Paul Latgé, and Pierre-Emmanuel Colle for valuable discussion and critical reading of the manuscript. We are grateful to Eugenio Morassi for preparing drawings.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Infectious, Parasitic, and Immuno-Mediated Diseases, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome, Italy. Phone: 39-0649903638. Fax: 39-0649387115. E-mail: e.coccia{at}iss.it. Back

Editor: A. Casadevall


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Infection and Immunity, March 2006, p. 1480-1489, Vol. 74, No. 3
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