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

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.

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.

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.

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.

FIG. 3.

Kinetics of TNF-α expression in A. fumigatus-infected DC. (A) Total RNA was extracted at the indicated time points. The expression of TNF-α 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-α 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.

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.

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.

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).

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 × 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-γ 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.