Aspergillus fumigatus is a sporulating fungus found ubiquitously in the environment and is easily cleared from immunocompetent hosts. Invasive aspergillosis develops in immunocompromised patients, and is a leading cause of mortality in hematopoietic stem cell transplant recipients. CCR7 and its ligands, CCL19 and CCL21, are responsible for the migration of dendritic cells from sites of infection and inflammation to secondary lymphoid organs. To investigate the role of CCR7 during invasive aspergillosis, we used a well-characterized neutropenic murine model. During invasive aspergillosis, mice with a CCR7 deficiency in the hematopoietic compartment exhibited increased survival and less pulmonary injury compared with the appropriate wild-type control. Flow cytometric analysis of the chimeric mice revealed an increase in the number of dendritic cells present in the lungs of CCR7-deficient chimeras following infection with Aspergillus conidia. An adoptive transfer of dendritic cells into neutropenic mice provided a protective effect during invasive aspergillosis, which was further enhanced with the adoptive transfer of CCR7-deficient dendritic cells. Additionally, CCR7-deficient dendritic cells activated in vitro with Aspergillus conidia expressed higher TNF-α, CXCL10, and CXCL2 levels, indicating a more activated cellular response to the fungus. Our results suggest that the absence of CCR7 is protective during invasive aspergillosis in neutropenic mice. Collectively, these data demonstrate a potential deleterious role for CCR7 during primary immune responses directed against A. fumigatus.

Aspergillus fumigatus is a sporulating fungus present ubiquitously in the environment (1). Although most individuals are not affected by exposure to Aspergillus, the fungus can cause a broad spectrum of diseases, ranging from hypersensitivity reactions, such as allergic bronchopulmonary aspergillosis, to serious opportunisitic infections, including chronic pulmonary necrotizing aspergillosis and invasive aspergillosis (2). Invasive aspergillosis is a rapidly progressive disease, often originating in the pulmonary system, in which inhaled conidia from A. fumigatus germinate into hyphae and invade the lung parenchyma, leading to pneumonia and massive inflammation (3). This severe, usually fatal disease disproportionally affects immunocompromised individuals and is a leading cause of mortality in hematopoietic stem cell transplant recipients (4, 5, 6, 7). Neutropenia was initially described as the most significant risk factor for the development of invasive aspergillosis (8), but mounting evidence has shown the importance of several additional effector cell populations, most notably myeloid dendritic cells (DCs)3 (9, 10, 11).

DCs play a crucial role during the immune response to A. fumigatus. Studies have shown that DCs become activated by A. fumigatus conidia and hyphae, leading to DC maturation and production of inflammatory cytokines, such as TNF-α, IL-12, IL-6, and IL-10 (9). Mature DCs have been shown to clear both A. fumigatus conidia and hyphal elements, indicating their importance before, and following, the onset of invasive disease (12). DCs also participate in the adaptive immune response: when conidia-activated DCs were adoptively transferred, they activated IFN-γ-producing T cells, leading to fungal resistance.

In addition to effector cell production of cytokines in response to A. fumigatus, several chemokines and their receptors have been reported to play a significant role during invasive aspergillosis (10, 13, 14, 15). A key element of DC function is the ability to migrate to sites of inflammation and infection, which is mediated by CCR6 expressed on immature DCs (16, 17). A recent study has shown that CCR6−/− mice fail to recruit adequate DC numbers to the lung following conidia challenge, and they consequently had significantly enhanced morbidity and mortality compared with wild-type mice (15). Another study, using human DCs, showed that upon internalization of Aspergillus conidia, DCs significantly up-regulated their expression of CCR7, in addition to antifungal cytokines (18, 19).

In our studies, we were interested in the role of CCR7 on DCs during invasive aspergillosis. CCR7 is present on several cell subsets in addition to DCs, most notably T cells and B cells (20, 21, 22). CCR7 binds the ligands CCL19 and CCL21, which are expressed constitutively in secondary lymphoid organs (20, 23, 24, 25). CCR7 is up-regulated on DCs after exposure to a maturing stimulus, allowing DCs to migrate away from sites of inflammation to secondary lymphoid organs. Here, the fully mature DC, expressing costimulatory molecules, is able to prime an adaptive immune response (26).

Herein we address the role of CCR7 in a well-characterized murine model of invasive aspergillosis. We found that CCR7 deficiency (CCR7−/−) in the hematopoietic compartment markedly enhanced survival and fungal clearance following intratracheal (i.t.) administration of A. fumigatus conidia into neutropenic mice. This protective effect was associated with a significantly higher influx of DCs into the lungs of CCR7−/− bone marrow chimera mice than into the lungs of CCR7-sufficient chimeras. Additionally, it was found that CCR7−/− bone marrow-derived DCs (BMDCs) had a more activated cellular response to Aspergillus conidia than did wild-type BMDCs, and adoptive transfer of CCR7−/− BMDCs more effectively protected neutropenic wild-type mice from invasive aspergillosis than did wild-type BMDCs. Thus, CCR7 expression negatively regulates the innate response to Aspergillus conidia, thereby permitting the development of invasive pulmonary aspergillosis.

Wild-type female C57BL/6 mice (6–8 wk of age) were purchased from The Jackson Laboratory. CCR7−/− mice, generated on a C57BL/6 background by Dr. M. Lipp’s group (Max Delbrück Center for Molecular Medicine, Berlin, Germany), as previously described (27), were a kind gift from S. Lira (Mount Sinai School of Medicine, New York, NY), and breeding colonies of these mice were established and maintained in the University of Michigan Medical School Laboratory of Animal Medicine facility. For all experiments, mice were sex-matched and used between 8 and 10 wk of age. All animals were used in accordance with regulations mandated by the University Committee on Use and Care of Animals at the University of Michigan.

Wild-type mice were lethally irradiated with 1000 cGy using a cesium source. Within 12 h of irradiation, recipient mice were reconstituted with 5.0 × 106 whole bone marrow cells in PBS from wild-type or CCR7−/− mice via tail vein injection. Mice were considered fully chimeric 8–10 wk following bone marrow transplantation.

Mice were depleted of neutrophils with an i.p. injection of 100 μg of RB6–8C5 (anti-Gr-1) as previously described (28). One day after the induction of neutropenia, mice were anesthetized with a mixture of ketamine and xylazine and given an i.t. injection of 6.0 × 106A. fumigatus conidia suspended in 30 μl of 0.1% Tween 80 in PBS. The highly virulent A. fumigatus strain 13073 (American Type Culture Collection) was used to elicit a reproducible form of invasive aspergillosis in anti-Gr-1-treated mice (28).

Lungs were collected 2 days after conidial challenge from wild-type and CCR7−/− mice or wild-type and CCR7−/− chimeras. Whole lung samples for histologic analysis were excised, perfused with 10% formalin, and placed in fresh formalin for an additional 24–48 h. Routine histologic techniques were used to paraffin-embed this tissue, and 5-μm sections of whole lung were stained with H&E or with Gomori methenamine silver (GMS) stain to visualize A. fumigatus conidia and hyphal elements (black staining). Image capture was conducted with an Olympus BX40F microscope equipped with ×20/0.5 and ×100/1.3 objective lenses and a ×10 eyepiece (Olympus). Digital photographs were obtained with a Sony 3 CCD color video camera (model no. DXC-960MD), and IP Lab Spectrum software was used for image acquisition (Scanalytics).

Total RNA was isolated from homogenized mouse lungs or BMDCs with the TRIzol reagent (Invitrogen). Approximately 5 μg of purified RNA was reverse transcribed to yield cDNA. CCR7, CCL19, IL-12p35, IL-17a, TNF-α, CXCL10, CXCL2, CCL5, IL-10, MHC II, CD86, CD40, TLR2, TLR4, and dectin-1 gene expression was analyzed by real-time quantitative RT-PCR using an ABI Prism 7500 sequence detection system (Applied Biosystems). For quantitative TaqMan analysis of CCL21, primers were custom made as previously described (29). GAPDH was analyzed as an internal control, and gene expression was normalized to GAPDH. Fold changes in gene expression levels were calculated by comparison of the gene expression in unchallenged samples or mice, which were assigned an arbitrary value of 1. Additional analysis was performed by assigning wild-type challenged mice or BMDCs a value of 1 and comparing this with CCR7−/− challenged samples.

Whole lung cytokine and chemokine levels from wild-type or CCR7−/− chimeras were measured using a Bio-Plex bead-based cytokine assay purchased from Invitrogen or Bio-Rad and analyzed on the Bio-Rad Bio-Plex 200 system according to the manufacturer’s protocol.

Whole lung samples were minced using surgical scissors and incubated in RPMI 1640 supplemented with 5% FCS, type IV collagenase (Sigma-Aldrich), and DNase for 45 min. Cells were flushed through a nylon mesh filter and washed with FACS buffer, consisting of Ca2+- and Mg2+-free PBS with 0.1% azide, 1% BSA, and 5 mM EDTA as described (30). Before surface staining with labeled Abs, nonspecific binding was blocked by incubating cells with purified rat anti-mouse CD16/CD32 (FcγIII/II receptor) mAb from eBioscience. Flow cytometry analysis was performed with a Beckman Coulter Cytomics FC 500, and data were analyzed using FlowJo 8.2 software.

Chitin, a major constituent of the hyphal cell wall, is absent in mammalian tissue and thus can be used as a method to determine the fungal burden in the lungs of infected mice. The chitin assay was performed as described (10).

Bone marrow cells were flushed from the femurs and tibiae of wild-type or CCR7−/− mice with sterile media. A single-cell suspension was obtained by filtering bone marrow cells through a 70-μm nylon mesh filter. Bone marrow cells were plated at a concentration of 3.0 × 106 cells/10 ml in RPMI 1640 media containing 20 ng/ml murine GM-CSF (R&D Systems). Media was replenished at 3 days and BMDCs were purified at day 6 using anti-CD11c magnetic beads (Miltenyi Biotec). Cells were replated in fresh media, without GM-CSF, and incubated with live A. fumigatus conidia at a 1:5 ratio. Using the TRIzol reagent, RNA was extracted from the BMDCs after 2 h of culture with conidia to prevent fungal overgrowth and to prevent the use of antifungals, which can affect DC function and maturation (18). To determine T cell stimulatory capacity and BMDC surface expression of CD86, wild-type and CCR7−/− BMDCs were cultured with conidia at a 1:5 ratio for 2 h, at which time amphotericin B was added to all cultures. At 24 h, BMDCs were analyzed by flow cytometry for CD86 expression or were used to stimulate BALB/c T cells in an allogeneic MLR. At 72 h, T cell proliferation was measured by [3H]thymidine incorporation.

In vitro-derived wild-type or CCR7−/− BMDCs were cultured as described, and in some experiments were labeled with CFSE. BMDCs (1.0 × 106) were injected 6 h before conidia challenge or were coinjected with conidia (6.0 × 106 to 107) into wild-type mice 1 day after administration of 100 μg of anti-Gr-1. One set of mice was euthanized 24 h after the injection, and lungs were analyzed for CFSE expression. The remaining mice were sacrificed at 48 h for lung histology, protein, and chitin analysis.

All results are expressed as the means ± SE representative of two or more experiments. A Student’s t test or ANOVA was used to determine statistical significance between wild-type and CCR7−/− mice, chimeras, or BMDCs. Survival rates were expressed as percentages, and a log-rank test (χ2 test) was used to detect differences in mouse survival. Values of p < 0.05 were considered statistically significant.

During the past decade it has become increasingly clear that chemokines and their receptors play an important role in the antifungal response during invasive aspergillosis (10, 13, 15, 28, 31). To investigate whether CCR7 is involved in host defense to A. fumigatus, wild-type mice were first rendered neutropenic by the administration of 100 μg of anti-Gr-1 ∼24 h before an i.t. challenge with 6.0 × 106 conidia (Fig. 1, A and B). Lungs from infected mice were examined 48 h after infection by flow cytometry. DC numbers were significantly increased in the lungs of challenged mice 2 days after infection (Fig. 1,C). CCR7 expression is not limited to DCs but is highly expressed on activated DCs during invasive aspergillosis (Fig. 1,D). Lungs of the infected mice also had increased gene expression of CCR7 and its ligand CCL19 at 48 h postinfection (Fig. 1 E). Interestingly, gene expression of CCL21a and CCL21b, the other ligands for CCR7, are dramatically down-regulated in the lungs of infected mice. Taken together, these results suggest that CCR7 and its ligands play an important role in the immune response to A. fumigatus.

FIGURE 1.

Whole lung analysis of neutropenic mice following conidia challenge. A, Experimental scheme in which wild-type C57BL/6 mice were administered of 100 μg of anti-Gr-1 Ab 24 h before an i.t. challenge with 6.0 × 106 conidia. Forty-eight hours after fungal challenge, lungs were harvested for flow cytometry and real-time PCR analysis. B, Gr-1 expression in wild-type mice before (left) and after (right) the administration of anti-Gr-1 Ab. C, DC numbers, based on CD11c and CD11b expression, in the lungs of infected mice 48 h after conidia challenge, and DC surface expression of CCR7 (D). E, Real-time analysis of CCR7, CCL19, and CCL21a/b gene expression 48 h after conidia challenge. ∗, p < 0.05 between DC numbers in the lungs before and after conidia challenge.

FIGURE 1.

Whole lung analysis of neutropenic mice following conidia challenge. A, Experimental scheme in which wild-type C57BL/6 mice were administered of 100 μg of anti-Gr-1 Ab 24 h before an i.t. challenge with 6.0 × 106 conidia. Forty-eight hours after fungal challenge, lungs were harvested for flow cytometry and real-time PCR analysis. B, Gr-1 expression in wild-type mice before (left) and after (right) the administration of anti-Gr-1 Ab. C, DC numbers, based on CD11c and CD11b expression, in the lungs of infected mice 48 h after conidia challenge, and DC surface expression of CCR7 (D). E, Real-time analysis of CCR7, CCL19, and CCL21a/b gene expression 48 h after conidia challenge. ∗, p < 0.05 between DC numbers in the lungs before and after conidia challenge.

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To examine the effect of CCR7 during invasive pulmonary aspergillosis, we infected neutropenic wild-type and CCR7−/− mice with 6.0 × 106 conidia. CCR7−/− mice were less susceptible to death following fungal challenge when compared with their wild-type counterparts (Fig. 2,A). It was observed that the lungs of uninfected CCR7−/− mice had significant cellular infiltrate, which included the formation of lymphoid aggregates (Fig. 2,B, i and ii). Although there was substantial leukocytic infiltration in both wild-type and CCR7−/− mice 48 h following conidial challenge, the CCR7−/− mice demonstrated a decrease in pulmonary fungal elements as determined by GMS staining (Fig. 2,B, iii–vi). Using real-time PCR, the gene expression of whole lung cytokine levels was determined in uninfected mice and in mice 48 h after A. fumigatus infection (Fig. 2, C and D). Uninfected CCR7−/− mice had elevated levels of IL-12p35, TNF-α, IL-17a, CXCL10, and IL-10, when compared with unchallenged wild-type mice. Additionally, CCR7−/− mice had significantly higher gene expression of IFN-γ before infection (data not shown). Interestingly, 48 h following conidial challenge, CCR7−/− mice had lower expression of IL-12p35, TNF-α, ΙL-17a, IFN-γ, and IL-10 RNA than did wild-type mice (Fig. 2 D). Taken together, these data indicate that CCR7−/− mice are protected from invasive aspergillosis and have a regulated inflammatory response due to the accelerated clearance of A. fumigatus.

FIGURE 2.

Conidia challenge in neutropenic wild-type and CCR7−/− mice. Twenty-four hours after the administration of anti-Gr-1 Ab, both groups of mice were given an i.t. challenge with 6.0 × 106 conidia. A, Survival curve of wild-type and CCR7−/− mice following conidia challenge. B, Forty-eight hours after fungal administration, lungs were removed for histologic analysis. Results from wild-type mice are shown in the left panels and results from CCR7−/− mice are shown in the right panels. iiv, H & E staining of uninfected (i and ii) and infected (iii and iv) lungs at 48 h after conidia challenge. v and vi, GMS-stained lung at 48 h after fungal infection. C and D, Real-time PCR analysis of whole lung cytokine and chemokine levels before (C) and following (D) conidial challenge. The fold change represents CCR7−/− gene expression over wild-type gene expression. The black line represents the level of wild-type expression for each gene tested. Original magnifications were ×10. ∗, p < 0.05 when comparing survival of wild-type and CCR7−/− mice after conidial challenge.

FIGURE 2.

Conidia challenge in neutropenic wild-type and CCR7−/− mice. Twenty-four hours after the administration of anti-Gr-1 Ab, both groups of mice were given an i.t. challenge with 6.0 × 106 conidia. A, Survival curve of wild-type and CCR7−/− mice following conidia challenge. B, Forty-eight hours after fungal administration, lungs were removed for histologic analysis. Results from wild-type mice are shown in the left panels and results from CCR7−/− mice are shown in the right panels. iiv, H & E staining of uninfected (i and ii) and infected (iii and iv) lungs at 48 h after conidia challenge. v and vi, GMS-stained lung at 48 h after fungal infection. C and D, Real-time PCR analysis of whole lung cytokine and chemokine levels before (C) and following (D) conidial challenge. The fold change represents CCR7−/− gene expression over wild-type gene expression. The black line represents the level of wild-type expression for each gene tested. Original magnifications were ×10. ∗, p < 0.05 when comparing survival of wild-type and CCR7−/− mice after conidial challenge.

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Previous studies, and work in our laboratory, have demonstrated that CCR7−/− mice exhibit anatomical abnormalities, such as alterations in secondary lymphoid organs, and generalized autoimmunity (27, 32). Given this feature of CCR7−/− mice, we elected to make bone marrow chimeras to examine the effect of CCR7 solely on the hematopoietic system during invasive aspergillosis. CCR7−/− chimeras had significantly lower mortality (10%) than did their wild-type counterparts (66%) at day 5 postinfection with A. fumigatus (Fig. 3,A). Analysis of whole lung chitin levels revealed that CCR7−/− chimeric mice had a significantly lower fungal burden compared with wild-type chimeras (Fig. 3,B). Consistent with these data, GMS staining of whole lung sections showed significant fungal growth and hyphal formation in the wild-type chimera lung, which was widely absent in the CCR7−/− chimeras (Fig. 3, C–H). The lungs of uninfected CCR7−/− chimeras exhibited some lymphoid aggregates made up predominantly of B cells and T cells as well as additional cell infiltrate, which was not observed in the wild-type chimeras (Fig. 3, C and D). Interestingly, this infiltrate was markedly less than that observed in naive CCR7−/− mice of the same age. Two days after conidia challenge, H&E staining showed significant cell infiltrate in the lungs of both mice, but to a greater extent in the wild-type chimeras, when compared with CCR7−/− chimeras and to the unchallenged controls (Fig. 3, C–F). This cellular infiltrate corresponded to a massive inflammatory response to A. fumigatus. Despite the influx of cells in both groups of mice, wild-type chimeras were unable to control the growth of the fungus, while the knockout animals did not develop invasive aspergillosis (Fig. 3, G and H). These data clearly demonstrate that neutropenic CCR7−/− chimeric mice are protected from invasive aspergillosis.

FIGURE 3.

Survival, lung chitin content, and histology of wild-type and CCR7−/− bone marrow chimeras challenged with A. fumigatus conidia. Wild-type or CCR7−/− chimeric mice were given 100 μg of anti-Gr-1 Ab 24 h before an i.t. challenge with 6.0 × 106 conidia and followed for survival or analyzed at 48 h. A, Survival of wild-type and CCR7−/− chimeras; n = 10 for wild-type mice and n = 12 for CCR7−/− mice. B, Lung chitin content determined 48 h after the onset of infection. C–H, Histologic analysis of lung tissue 2 days after conidia challenge. H & E staining of uninfected lungs (C and D) or 48 h after conidia challenge (E and F). G and H, Representative GMS-stained sections from wild-type (G) or CCR7−/− chimeras (H) following conidial challenge; fungal elements are stained in black. Original magnification was ×20 for H & E-stained sections and ×40 for GMS sections. ∗, p < 0.05 when comparing survival and chitin in wild-type and CCR7−/− chimeras.

FIGURE 3.

Survival, lung chitin content, and histology of wild-type and CCR7−/− bone marrow chimeras challenged with A. fumigatus conidia. Wild-type or CCR7−/− chimeric mice were given 100 μg of anti-Gr-1 Ab 24 h before an i.t. challenge with 6.0 × 106 conidia and followed for survival or analyzed at 48 h. A, Survival of wild-type and CCR7−/− chimeras; n = 10 for wild-type mice and n = 12 for CCR7−/− mice. B, Lung chitin content determined 48 h after the onset of infection. C–H, Histologic analysis of lung tissue 2 days after conidia challenge. H & E staining of uninfected lungs (C and D) or 48 h after conidia challenge (E and F). G and H, Representative GMS-stained sections from wild-type (G) or CCR7−/− chimeras (H) following conidial challenge; fungal elements are stained in black. Original magnification was ×20 for H & E-stained sections and ×40 for GMS sections. ∗, p < 0.05 when comparing survival and chitin in wild-type and CCR7−/− chimeras.

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Since treatment of mice with anti-Gr-1 eliminates neutrophils, a major effector cell type in the aspergillus-infected lung, we examined whole lung samples by flow cytometery to identify the cells providing the protective effect observed in CCR7−/− chimeras. We saw a dramatic increase in myeloid DCs and immature myeloid cells (monocytes and recruited macrophages) at 48 h after aspergillus challenge in both the wild-type and CCR7−/− groups (Fig. 4). Conversely, the numbers of T cells, B cells, and NK cells were decreased 2 days after conidia challenge. While immature myeloid cell numbers were elevated in both groups, flow cytometric data revealed higher percentages of myeloid DCs in the lungs of CCR7−/− chimeric mice, which, in combination with higher absolute number of cells in the lungs of the knockout animals, resulted in a significantly greater number of DCs present (Fig. 4). It has been previously reported that DCs provide protection against invasive aspergillosis and that mice lacking adequate numbers of DCs are susceptible to disease (15). Therefore, antifungal responses in CCR7−/− chimeras appear to be the result of an enhanced accumulation of DCs.

FIGURE 4.

Leukocyte differentials in the lung at 48 h after A. fumigatus challenge in wild-type and CCR7−/− bone marrow chimeras. The left lung lobe from wild-type or CCR7−/− chimeras was analyzed by flow cytometery 48 h after fungal challenge. Myeloid DCs were CD11chigh, CD11bhigh cells (as shown in the dot plots). Monocytes/macrophages were CD11b+, F4/80+, and CD11c. NK cells were NK1.1+, T cells were CD3+ and CD4+ or CD8+, and B cells were CD19+. ∗, p < 0.05 when comparing DCs in wild-type and CCR7−/− mice on day 2 following infection.

FIGURE 4.

Leukocyte differentials in the lung at 48 h after A. fumigatus challenge in wild-type and CCR7−/− bone marrow chimeras. The left lung lobe from wild-type or CCR7−/− chimeras was analyzed by flow cytometery 48 h after fungal challenge. Myeloid DCs were CD11chigh, CD11bhigh cells (as shown in the dot plots). Monocytes/macrophages were CD11b+, F4/80+, and CD11c. NK cells were NK1.1+, T cells were CD3+ and CD4+ or CD8+, and B cells were CD19+. ∗, p < 0.05 when comparing DCs in wild-type and CCR7−/− mice on day 2 following infection.

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Inflammation is required for an appropriate antifungal immune response, but it has been reported that an uncontrolled inflammatory response is detrimental to the host (33, 34). We found that protein levels were significantly higher in both groups of mice 2 days after infection when compared with naive controls (Fig. 5). Unlike wild-type and CCR7−/− mice, there is no significant difference in the baseline cytokine expression between uninfected wild-type chimeras and CCR7−/− chimeras (Figs. 2,C and 5). The reduction of proinflammatory cytokine expression in uninfected CCR7−/− chimeras compared with CCR7−/− mice may be due to the reduction in lymphoid aggregates in the lung (Figs. 2,Bii and 3 D). Similarly to infected wild-type mice, conidial challenge in wild-type chimeras results in significantly higher expression of TNF-α, CCL2, CCL5, and CCL3, but lower levels of CXCL9 and CXCL10, when compared with CCR7−/− chimeras. While CCR7−/− chimeras have lower levels of inflammatory mediators, both groups produce the same amount of the antiinflammatory cytokine IL-10. Taken together, these findings suggest that the inflammatory response in the lung of CCR7−/− chimeric mice is appropriately regulated before and during infection, leading to the rapid clearance of A. fumigatus.

FIGURE 5.

Whole lung protein levels 48 h after conidial challenge in wild-type and CCR7−/− bone marrow chimeras. Cytokine and chemokine levels were determined using a Bio-Plex multiplex assay; all values are represented as pg/ml. ∗, p < 0.05 when comparing wild-type and CCR7−/− chimeras following conidia challenge.

FIGURE 5.

Whole lung protein levels 48 h after conidial challenge in wild-type and CCR7−/− bone marrow chimeras. Cytokine and chemokine levels were determined using a Bio-Plex multiplex assay; all values are represented as pg/ml. ∗, p < 0.05 when comparing wild-type and CCR7−/− chimeras following conidia challenge.

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Survival, histological, and fungal growth differences between wild-type and CCR7−/− chimeras appear to be the result of the accumulation of DCs in the lungs of the knockout mice. However, these differences could also be the consequence of differential activation and maturation by CCR7−/− DCs, or due to a difference in fungal recognition by these chemokine receptor-deficient DCs. To determine whether differences exist between wild-type and CCR7−/− BMDCs, bone marrow from both mice was grown in vitro for 8 days with GM-CSF and the resulting BMDCs were challenged with Aspergillus conidia. Two hours after fungal challenge, CCR7−/− and wild-type BMDCs had very similar gene expression of the costimulatory molecules MHC II, CD86, and CD40 (Fig. 6,A). When RNA was analyzed for expression of antifungal, proinflammatory cytokines at 2 h, CCR7−/− BMDCs showed higher levels of TNF-α, CXCL10, CCL2, and CXCL2 and similar levels of CCL5, when compared with wild-type BMDCs (Fig. 6,B). Given that it has been shown that a TLR2/dectin-1 complex and TLR4 recognize A. fumigatus (35, 36, 37), we examined the expression of these receptors on wild-type and CCR7−/− BMDCs. We found significantly more TLR2 gene expression present 2 h after conidia challenge, but decreased levels of dectin-1 in the CCR7−/− BMDCs. TLR4 levels were similar between the two groups (Fig. 6,C). When we cultured our cells for 24 h in the presence of amphotericin B, we observed that CCR7−/− DCs had higher expression of CD86 than did wild-type DCs (Fig. 6,D). Additionally, CCR7−/− DCs appeared to be functionally more mature, as they were able to stimulate allogeneic T cell proliferation in a MLR better than wild-type DCs (Fig. 6 E). Collectively, these data indicate that CCR7−/− DCs might provide protection via a more activated and mature phenotype, characterized by the higher expression of costimulatory molecules, and by the production of greater amounts of proinflammatory cytokines compared with their wild-type counterparts.

FIGURE 6.

Phenotypic and functional comparison of wild-type and CCR7−/− BMDCs. A–C, BMDCs were cocultured in a 1:5 ratio with conidia for 2 h before analysis by real-time PCR. Data are fold change of CCR7−/− BMDC gene expression over uninfected wild-type BMDC transcript expression. D and E, BMDCs were cultured for 24 h in a 1:5 ratio with conidia; amphotericin B was added to culture after 2 h. D, Relative expression of CD86 on wild-type (gray histogram) and CCR7−/− (solid black line) BMDCs (control is black histogram). E, Twenty-four hours after culture with conidia or LPS or in media, BALB/c T cells were added at a 10:1 ratio and allowed to proliferate for 72 h before the addition of tritiated thymidine ([3H]). cpm represents T cell proliferation. ∗, p < 0.05, which compares wild-type or CCR7−/− BMDCs.

FIGURE 6.

Phenotypic and functional comparison of wild-type and CCR7−/− BMDCs. A–C, BMDCs were cocultured in a 1:5 ratio with conidia for 2 h before analysis by real-time PCR. Data are fold change of CCR7−/− BMDC gene expression over uninfected wild-type BMDC transcript expression. D and E, BMDCs were cultured for 24 h in a 1:5 ratio with conidia; amphotericin B was added to culture after 2 h. D, Relative expression of CD86 on wild-type (gray histogram) and CCR7−/− (solid black line) BMDCs (control is black histogram). E, Twenty-four hours after culture with conidia or LPS or in media, BALB/c T cells were added at a 10:1 ratio and allowed to proliferate for 72 h before the addition of tritiated thymidine ([3H]). cpm represents T cell proliferation. ∗, p < 0.05, which compares wild-type or CCR7−/− BMDCs.

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DCs appear to be critically important in the host immune response to invasive aspergillosis in our model. To determine whether CCR7 deficiency on DCs alone was adequate to provide protection against A. fumigatus, we labeled 1.0 × 106 wild-type or CCR7−/− BMDCs (Fig. 7,A) with CFSE and coinjected them i.t. into neutropenic wild-type mice with 6.0 × 106 conidia. Twenty-four hours after the transfer, the lungs were analyzed for CFSE expression by flow cytometry. We found that mice receiving CCR7−/− BMDCs had significantly higher numbers of CFSE-positive cells than did mice receiving wild-type BMDCs (Fig. 7,B). Upon histological analysis, at 48 h, recipients of wild-type and CCR7−/− BMDCs both showed a decrease in fungal growth when compared with wild-type mice or chimeras without an adoptive transfer of BMDCs (Figs. 7, C and D, 2,Bv, and 3,G). Although fungal burden was diminished in both BMDC adoptive transfer groups compared with wild-type mice not receiving BMDCs, the histology indicates that mice receiving wild-type BMDCs had more fungal growth overall, especially in the air spaces. Additionally, protein data showed a similar trend to the wild-type and CCR7−/− chimeras infected with aspergillus (Fig. 7,E). TNF-α and CCL5 were statistically higher in the mice receiving wild-type BMDCs than in mice receiving CCR7−/− BMDCs. Since the adoptive transfer of BMDCs significantly decreased the fungal burden in both groups of mice, we increased the conidia inocula from 6.0 × 106 to 107spores to examine differences in lung chitin levels (Fig. 7 F). At 107 conidia, mice in both groups were susceptible to fungal growth, but mice receiving wild-type BMDCs had an increase in fungal burden. Collectively, these data indicate that CCR7−/− BMDCs provided better protection from invasive aspergillosis compared with wild-type BMDCs.

FIGURE 7.

Adoptive transfer of wild-type and CCR7−/− BMDCs into neutropenic C57BL/6 mice. A, Percentage of CD11c+ BMDCs used in the adoptive transfer, following in vitro culture and positive selection using magnetic beads. Black histogram indicates unstained, the gray histogram indicates wild-type BMDCs, and the solid black line indicates CCR7−/− BMDCs. B, BMDCs were labeled with CFSE and coinjected with 6.0 × 106 conidia. Data represent the numbers of labeled DCs present in the lungs at 24 h after injection of conidia. C and D, Lung histology 48 h after coinjection of BMDCs and conidia. Wild-type mice were given an adoptive transfer of 1.0 × 106 wild-type (C) or CCR7−/− (D) BMDCs 6 h before 6.0 × 106 conidia. Black arrows indicate fungal growth. E, Whole lung protein levels from the mice in C and D. F, Lung chitin levels 48 h following coinjection of 1.0 × 106 BMDCs and 107 conidia. WT indicates an adoptive transfer of wild-type DCs into a wild-type mouse, and CCR7−/− indicates CCR7−/− DCs transferred into a wild-type mouse. ∗, p < 0.05 between wild-type and CCR7−/− groups.

FIGURE 7.

Adoptive transfer of wild-type and CCR7−/− BMDCs into neutropenic C57BL/6 mice. A, Percentage of CD11c+ BMDCs used in the adoptive transfer, following in vitro culture and positive selection using magnetic beads. Black histogram indicates unstained, the gray histogram indicates wild-type BMDCs, and the solid black line indicates CCR7−/− BMDCs. B, BMDCs were labeled with CFSE and coinjected with 6.0 × 106 conidia. Data represent the numbers of labeled DCs present in the lungs at 24 h after injection of conidia. C and D, Lung histology 48 h after coinjection of BMDCs and conidia. Wild-type mice were given an adoptive transfer of 1.0 × 106 wild-type (C) or CCR7−/− (D) BMDCs 6 h before 6.0 × 106 conidia. Black arrows indicate fungal growth. E, Whole lung protein levels from the mice in C and D. F, Lung chitin levels 48 h following coinjection of 1.0 × 106 BMDCs and 107 conidia. WT indicates an adoptive transfer of wild-type DCs into a wild-type mouse, and CCR7−/− indicates CCR7−/− DCs transferred into a wild-type mouse. ∗, p < 0.05 between wild-type and CCR7−/− groups.

Close modal

Invasive aspergillosis represents a significant challenge in the clinic and remains a leading cause of death for immunocompromised individuals. While the highest incidence of disease is observed among bone marrow transplant recipients, invasive aspergillosis is also on the rise in solid organ transplant patients as well as in cancer and AIDS patients (4, 5, 6, 38, 39, 40). The present study investigated the role of the chemokine receptor CCR7 in a well-established neutropenic murine model of invasive aspergillosis. We found that CCR7−/− mice and CCR7−/− chimeric mice had a distinct survival advantage, as well as decreased fungal burden in the lungs, after challenge with A. fumigatus conidia when compared with their wild-type counterparts. This was an unexpected observation given that we found a significant up-regulation of CCR7 on DCs, in addition to increased levels of CCL19, in wild-type mice challenged with A. fumigatus conidia, which suggested the importance of this chemokine-chemokine receptor interaction during fungal pathogenesis. Our data thus suggest that CCR7 induction on DCs during invasive pulmonary aspergillosis promotes a pathological response to A. fumigatus.

DCs are an important effector cell during invasive aspergillosis. The Mehrad group showed that CCR6-deficient mice were significantly more susceptible to invasive aspergillosis than were CCR6-sufficient mice (15). CCR6 and its ligand, CCL20, are responsible for the migration of immature DCs to sights of inflammation, including the lung (16, 17, 41, 42). Consequently, DCs lacking the expression of CCR6 were unable to efficiently traffic into the infected lung, thus leaving a neutropenic host susceptible to invasive aspergillosis. As originally reported by Dieu et al. in human DCs, when DCs encounter a maturing stimulus, such as A. fumigatus, they down-regulate their expression of CCR6 and up-regulate their expression of CCR7 (43). DC up-regulation of CCR7 expression permits trafficking away from sites of infection and inflammation to secondary lymphoid organs, as the cells migrate in response to the chemokines CCL19 and CCL21 (20, 23, 24, 25).

Based on these observations, we hypothesized that CCR7−/− DCs, with functional CCR6 expression, would be able to efficiently traffic to the lungs of infected mice; once there, however, the DCs would accumulate as they would be unable to respond to the ligands, CCL19 and CCL21, and effectively traffic out of the lung to the draining lymph node. Indeed, our results showed that 48 h after infection with A. fumigatus conidia, there was a significant increase in the number of DCs in the CCR7−/− chimeras when compared with wild-type chimeric mice. We had similar findings in an adoptive transfer experiment where CCR7−/− or wild-type BMDCs are injected along with conidia into the lungs of wild-type neutropenic mice. Here we saw many-fold higher numbers of CCR7−/− BMDCs than wild-type BMDCs in the lungs of the neutropenic mice. These data support the hypothesis that CCR7−/− DCs remain in the lungs of infected animals, where they ultimately provide protection against invasive aspergillosis.

New evidence suggests that an improperly regulated inflammatory response in the lung during invasive aspergillosis is detrimental to the survival of the host (33, 34). When TNF-α, an important antifungal cytokine, was neutralized in a murine model of invasive aspergillosis, there was a significant increase in mortality associated with increased fungal burden (44). Consequently, significant levels of TNF-α are required for the clearance of Aspergillus, but dangerously high levels of TNF-α are implicated in severe inflammatory conditions such as sepsis (44, 45, 46, 47). Thus, a balance between pro- and antiinflammatory signals is required for appropriate fungal clearance, without excessive tissue injury. In our model, we found significantly higher production of TNF-α as well as the proinflammatory mediators CCL2, CCL5, and CCL3 in wild-type chimeras. Conversely, CCR7−/− chimeric mice produced significantly higher CXCL10, a chemokine recently shown to render the host susceptible to invasive aspergillosis if inappropriately expressed (31). Additionally, both wild-type and CCR7−/− chimeras showed similar expression of the potent antiinflammatory cytokine IL-10 after conidia challenge. Taken together, our results suggest that the cytokine milieu demonstrated by CCR7−/− chimeras may create a more adequate balance between pro- and antiinflammatory cytokines, which is required for appropriate fungal clearance. Alternatively, the CCR7−/− chimeras may simply have less inflammation, as these animals showed significantly less fungal growth in their lungs. The resulting decrease in fungal burden eliminated the need for a prolonged acute inflammatory response, ultimately leading to lower cytokine levels in the CCR7−/− mice.

While the accumulation of DCs in the lungs of CCR7−/− chimeras is likely playing an important role in the clearance of the fungus, it is possible that differences in DC phenotype or function also play a pivotal role during invasive aspergillosis. Previous reports have shown that human DCs exposed to A. fumigatus conidia in vitro up-regulate their expression of costimulatory molecules and their production of proinflammatory cytokines (18, 19). In the present study, we observed a trend indicating greater expression of the costimulatory molecules MHC II and CD86 on CCR7−/− BMDCs following conidia challenge. Additionally, CCR7−/− BMDCs had statistically higher gene expression of TNF-α, CXCL10, CCL2, and CXCL2 compared with wild-type BMDCs. Due to the importance of proinflammatory cytokines in the antifungal response, the protective phenotype provided by CCR7−/− DCs might derive not only from their accumulation in the lung, but also from their more robust cytokine response against A. fumigatus. A complication of these in vitro experiments is the 2 h time point, chosen to prevent the use of antifungal reagents, which have been shown to mature BMDCs in vitro (18). When amphotericin B was used in the MLR, we observed maturation of our media control BMDCs, leading to significant T cell proliferation. Importantly, T cell expansion was always greater when CCR7−/− BMDCs were used as T cell stimulators, regardless of the maturing stimulus used. Collectively, these data indicate that CCR7−/− BMDCs have a more activated and mature phenotype than do wild-type BMDCs during challenge with A. fumigatus conidia, which may augment the protective phenotype seen in vivo.

In summary, invasive aspergillosis is a severe and often fatal disease primarily affecting immunocompromised individuals. New therapies are required for the effective prevention and treatment of this opportunistic fungal infection. The present study demonstrates a deleterious role for CCR7 in a murine model of experimental invasive aspergillosis, indicating that selective targeting of CCR7 might provide a new course of therapies for invasive fungal diseases. More study is required to fully understand the beneficial mechanism of CCR7 deficiency during invasive aspergillosis, and to determine whether this receptor is clinically relevant to human disease.

We thank Ms. Holly Evanoff and Ms. Lisa Riggs for their outstanding technical assistance.

The authors have no financial conflict of interest.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1

This work was supported by National Institutes of Health Grant HL069865 (to C.M.H.) and Novartis Institutes for Biomedical Research.

3

Abbreviations used in this paper: DC, myeloid dendritic cell; BMDC, bone marrow-derived dendritic cell; GMS, Gomori methenamine silver; i.t., intratracheal(ly).

1
Mullins, J., R. Harvey, A. Seaton.
1976
. Sources and incidence of airborne Aspergillus fumigatus (Fres).
Clin. Allergy
6
:
209
-217.
2
Thompson, G. R., 3rd, T. F. Patterson.
2008
. Pulmonary aspergillosis.
Semin. Respir. Crit. Care Med.
29
:
103
-110.
3
Soubani, A. O., P. H. Chandrasekar.
2002
. The clinical spectrum of pulmonary aspergillosis.
Chest
121
:
1988
-1999.
4
Garcia-Vidal, C., A. Upton, K. A. Kirby, K. A. Marr.
2008
. Epidemiology of invasive mold infections in allogeneic stem cell transplant recipients: biological risk factors for infection according to time after transplantation.
Clin. Infect. Dis.
47
:
1041
-1050.
5
Jantunen, E., V. J. Anttila, T. Ruutu.
2002
. Aspergillus infections in allogeneic stem cell transplant recipients: have we made any progress?.
Bone Marrow Transplant.
30
:
925
-929.
6
Post, M. J., C. Lass-Floerl, G. Gastl, D. Nachbaur.
2007
. Invasive fungal infections in allogeneic and autologous stem cell transplant recipients: a single-center study of 166 transplanted patients.
Transpl. Infect. Dis.
9
:
189
-195.
7
Romani, L..
2004
. Immunity to fungal infections.
Nat. Rev. Immunol.
4
:
1
-23.
8
Gerson, S. L., G. H. Talbot, S. Hurwitz, B. L. Strom, E. J. Lusk, P. A. Cassileth.
1984
. Prolonged granulocytopenia: the major risk factor for invasive pulmonary aspergillosis in patients with acute leukemia.
Ann. Intern. Med.
100
:
345
-351.
9
Bozza, S., K. Perruccio, C. Montagnoli, R. Gaziano, S. Bellocchio, E. Burchielli, G. Nkwanyuo, L. Pitzurra, A. Velardi, L. Romani.
2003
. A dendritic cell vaccine against invasive aspergillosis in allogeneic hematopoietic transplantation.
Blood
102
:
3807
-3814.
10
Morrison, B. E., S. J. Park, J. M. Mooney, B. Mehrad.
2003
. Chemokine-mediated recruitment of NK cells is a critical host defense mechanism in invasive aspergillosis.
J. Clin. Invest.
112
:
1862
-1870.
11
Rivera, A., G. Ro, H. L. Van Epps, T. Simpson, I. Leiner, D. B. Sant'Angelo, E. G. Pamer.
2006
. Innate immune activation and CD4+ T cell priming during respiratory fungal infection.
Immunity
25
:
665
-675.
12
Bozza, S., R. Gaziano, A. Spreca, A. Bacci, C. Montagnoli, P. di Francesco, L. Romani.
2002
. Dendritic cells transport conidia and hyphae of Aspergillus fumigatus from the airways to the draining lymph nodes and initiate disparate Th responses to the fungus.
J. Immunol.
168
:
1362
-1371.
13
Carpenter, K. J., C. M. Hogaboam.
2005
. Immunosuppressive effects of CCL17 on pulmonary antifungal responses during pulmonary invasive aspergillosis.
Infect. Immun.
73
:
7198
-7207.
14
Mehrad, B., M. Wiekowski, B. E. Morrison, S. C. Chen, E. C. Coronel, D. J. Manfra, S. A. Lira.
2002
. Transient lung-specific expression of the chemokine KC improves outcome in invasive aspergillosis.
Am. J. Respir. Crit. Care Med.
166
:
1263
-1268.
15
Phadke, A. P., G. Akangire, S. J. Park, S. A. Lira, B. Mehrad.
2007
. The role of CC chemokine receptor 6 in host defense in a model of invasive pulmonary aspergillosis.
Am. J. Respir. Crit. Care Med.
175
:
1165
-1172.
16
Greaves, D. R., W. Wang, D. J. Dairaghi, M. C. Dieu, B. Saint-Vis, K. Franz-Bacon, D. Rossi, C. Caux, T. McClanahan, S. Gordon, et al
1997
. CCR6, a CC chemokine receptor that interacts with macrophage inflammatory protein 3α and is highly expressed in human dendritic cells.
J. Exp. Med.
186
:
837
-844.
17
Le Borgne, M., N. Etchart, A. Goubier, S. A. Lira, J. C. Sirard, N. van Rooijen, C. Caux, S. Ait-Yahia, A. Vicari, D. Kaiserlian, B. Dubois.
2006
. Dendritic cells rapidly recruited into epithelial tissues via CCR6/CCL20 are responsible for CD8+ T cell crosspriming in vivo.
Immunity
24
:
191
-201.
18
Gafa, V., R. Lande, M. C. Gagliardi, M. Severa, E. Giacomini, M. E. Remoli, R. Nisini, C. Ramoni, P. Di Francesco, D. Aldebert, et al
2006
. 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.
Infect. Immun.
74
:
1480
-1489.
19
Gafa, V., M. E. Remoli, E. Giacomini, M. C. Gagliardi, R. Lande, M. Severa, R. Grillot, E. M. Coccia.
2007
. In vitro infection of human dendritic cells by Aspergillus fumigatus conidia triggers the secretion of chemokines for neutrophil and Th1 lymphocyte recruitment.
Microbes Infect.
9
:
971
-980.
20
Saeki, H., A. M. Moore, M. J. Brown, S. T. Hwang.
1999
. Cutting edge: secondary lymphoid-tissue chemokine (SLC) and CC chemokine receptor 7 (CCR7) participate in the emigration pathway of mature dendritic cells from the skin to regional lymph nodes.
J. Immunol.
162
:
2472
-2475.
21
Sallusto, F., D. Lenig, R. Forster, M. Lipp, A. Lanzavecchia.
1999
. Two subsets of memory T lymphocytes with distinct homing potentials and effector functions.
Nature
401
:
708
-712.
22
Sanchez-Sanchez, N., L. Riol-Blanco, G. de la Rosa, A. Puig-Kroger, J. Garcia-Bordas, D. Martin, N. Longo, A. Cuadrado, C. Cabanas, A. L. Corbi, et al
2004
. Chemokine receptor CCR7 induces intracellular signaling that inhibits apoptosis of mature dendritic cells.
Blood
104
:
619
-625.
23
Gunn, M. D., K. Tangemann, C. Tam, J. G. Cyster, S. D. Rosen, L. T. Williams.
1998
. A chemokine expressed in lymphoid high endothelial venules promotes the adhesion and chemotaxis of naive T lymphocytes.
Proc. Natl. Acad. Sci. USA
95
:
258
-263.
24
Randolph, G. J., V. Angeli, M. A. Swartz.
2005
. Dendritic-cell trafficking to lymph nodes through lymphatic vessels.
Nat. Rev. Immunol.
5
:
617
-628.
25
Forster, R., A. C. Davalos-Misslitz, A. Rot.
2008
. CCR7 and its ligands: balancing immunity and tolerance.
Nat. Rev. Immunol.
8
:
362
-371.
26
Banchereau, J., R. M. Steinman.
1998
. Dendritic cells and the control of immunity.
Nature
392
:
245
-252.
27
Forster, R., A. Schubel, D. Breitfeld, E. Kremmer, I. Renner-Muller, E. Wolf, M. Lipp.
1999
. CCR7 coordinates the primary immune response by establishing functional microenvironments in secondary lymphoid organs.
Cell
99
:
23
-33.
28
Mehrad, B., R. M. Strieter, T. A. Moore, W. C. Tsai, S. A. Lira, T. J. Standiford.
1999
. CXC chemokine receptor-2 ligands are necessary components of neutrophil-mediated host defense in invasive pulmonary aspergillosis.
J. Immunol.
163
:
6086
-6094.
29
Chen, S. C., G. Vassileva, D. Kinsley, S. Holzmann, D. Manfra, M. T. Wiekowski, N. Romani, S. A. Lira.
2002
. Ectopic expression of the murine chemokines CCL21a and CCL21b induces the formation of lymph node-like structures in pancreas, but not skin, of transgenic mice.
J. Immunol.
168
:
1001
-1008.
30
Vermaelen, K., R. Pauwels.
2004
. Accurate and simple discrimination of mouse pulmonary dendritic cell and macrophage populations by flow cytometry: methodology and new insights.
Cytometry A
61
:
170
-177.
31
Mezger, M., M. Steffens, M. Beyer, C. Manger, J. Eberle, M. R. Toliat, T. F. Wienker, P. Ljungman, H. Hebart, H. J. Dornbusch, H. Einsele, J. Loeffler.
2008
. Polymorphisms in the chemokine (C-X-C motif) ligand 10 are associated with invasive aspergillosis after allogeneic stem-cell transplantation and influence CXCL10 expression in monocyte-derived dendritic cells.
Blood
111
:
534
-536.
32
Davalos-Misslitz, A. C., J. Rieckenberg, S. Willenzon, T. Worbs, E. Kremmer, G. Bernhardt, R. Forster.
2007
. Generalized multi-organ autoimmunity in CCR7-deficient mice.
Eur. J. Immunol.
37
:
613
-622.
33
Romani, L., F. Bistoni, K. Perruccio, C. Montagnoli, R. Gaziano, S. Bozza, P. Bonifazi, G. Bistoni, G. Rasi, A. Velardi, F. Fallarino, E. Garaci, P. Puccetti.
2006
. Thymosin α1 activates dendritic cell tryptophan catabolism and establishes a regulatory environment for balance of inflammation and tolerance.
Blood
108
:
2265
-2274.
34
Zelante, T., A. De Luca, P. Bonifazi, C. Montagnoli, S. Bozza, S. Moretti, M. L. Belladonna, C. Vacca, C. Conte, P. Mosci, et al
2007
. IL-23 and the Th17 pathway promote inflammation and impair antifungal immune resistance.
Eur. J. Immunol.
37
:
2695
-2706.
35
Mambula, S. S., K. Sau, P. Henneke, D. T. Golenbock, S. M. Levitz.
2002
. Toll-like receptor (TLR) signaling in response to Aspergillus fumigatus.
J. Biol. Chem.
277
:
39320
-39326.
36
Meier, A., C. J. Kirschning, T. Nikolaus, H. Wagner, J. Heesemann, F. Ebel.
2003
. Toll-like receptor (TLR) 2 and TLR4 are essential for Aspergillus-induced activation of murine macrophages.
Cell. Microbiol.
5
:
561
-570.
37
Steele, C., R. R. Rapaka, A. Metz, S. M. Pop, D. L. Williams, S. Gordon, J. K. Kolls, G. D. Brown.
2005
. The β-glucan receptor dectin-1 recognizes specific morphologies of Aspergillus fumigatus.
PLoS Pathog.
1
:
e42
38
Ali, R., F. Ozkalemkas, T. Ozcelik, V. Ozkocaman, A. Ozkan, S. Bayram, B. Ener, A. Ursavas, G. Ozal, A. Tunali.
2006
. Invasive pulmonary aspergillosis: role of early diagnosis and surgical treatment in patients with acute leukemia.
Ann. Clin. Microbiol. Antimicrob.
5
:
17
39
Morgan, J., K. A. Wannemuehler, K. A. Marr, S. Hadley, D. P. Kontoyiannis, T. J. Walsh, S. K. Fridkin, P. G. Pappas, D. W. Warnock.
2005
. Incidence of invasive aspergillosis following hematopoietic stem cell and solid organ transplantation: interim results of a prospective multicenter surveillance program.
Med. Mycol.
43
: (Suppl. 1):
S49
-S58.
40
Mylonakis, E., T. F. Barlam, T. Flanigan, J. D. Rich.
1998
. Pulmonary aspergillosis and invasive disease in AIDS: review of 342 cases.
Chest
114
:
251
-262.
41
Dieu-Nosjean, M. C., C. Massacrier, B. Homey, B. Vanbervliet, J. J. Pin, A. Vicari, S. Lebecque, C. Dezutter-Dambuyant, D. Schmitt, A. Zlotnik, C. Caux.
2000
. Macrophage inflammatory protein 3α is expressed at inflamed epithelial surfaces and is the most potent chemokine known in attracting Langerhans cell precursors.
J. Exp. Med.
192
:
705
-718.
42
Osterholzer, J. J., T. Ames, T. Polak, J. Sonstein, B. B. Moore, S. W. Chensue, G. B. Toews, J. L. Curtis.
2005
. CCR2 and CCR6, but not endothelial selectins, mediate the accumulation of immature dendritic cells within the lungs of mice in response to particulate antigen.
J. Immunol.
175
:
874
-883.
43
Dieu, M. C., B. Vanbervliet, A. Vicari, J. M. Bridon, E. Oldham, S. Ait-Yahia, F. Briere, A. Zlotnik, S. Lebecque, C. Caux.
1998
. Selective recruitment of immature and mature dendritic cells by distinct chemokines expressed in different anatomic sites.
J. Exp. Med.
188
:
373
-386.
44
Mehrad, B., R. M. Strieter, T. J. Standiford.
1999
. Role of TNF-α in pulmonary host defense in murine invasive aspergillosis.
J. Immunol.
162
:
1633
-1640.
45
Bone, R. C..
1991
. The pathogenesis of sepsis.
Ann. Intern. Med.
115
:
457
-469.
46
Michie, H. R., K. R. Manogue, D. R. Spriggs, A. Revhaug, S. O'Dwyer, C. A. Dinarello, A. Cerami, S. M. Wolff, D. W. Wilmore.
1988
. Detection of circulating tumor necrosis factor after endotoxin administration.
N. Engl. J. Med.
318
:
1481
-1486.
47
Nagai, H., J. Guo, H. Choi, V. Kurup.
1995
. Interferon-γ and tumor necrosis factor-alpha protect mice from invasive aspergillosis.
J. Infect. Dis.
172
:
1554
-1560.