Enhancement of anti-Aspergillus T helper type 1 response by interferon-β-conditioned dendritic cells

Abstract

Although data show the importance of type I interferons (IFNs) in the regulation of the innate and adaptive immunity elicited in response to viral, bacterial and parasitic infections, the functional activities of these cytokines during fungal infections are poorly understood. We examined here the impact of IFN-β on the response of human monocyte-derived dendritic cells (DCs) infected in vitro with Aspergillus fumigatus. Having found that A. fumigatus-infected DCs do not express IFN-β, we evaluated the effect of the exogenous addition of IFN-β on the maturation of human DCs induced by the infection with A. fumigatus conidia. Although the phagocytosis of the fungus was not affected by IFN-β treatment, the expression of CD86 and CD83 induced upon A. fumigatus challenge was enhanced in IFN-β-conditioned DCs, which also showed an increased expression of IL-27 and IL-12p70, members of IL-12 family. Through these modifications, IFN-β improved the capacity of DCs to promote an anti-Aspergillus T helper type 1 response, as evaluated by mixed leucocyte reaction, which plays a crucial role in the control of invasive aspergillosis. Our results identified a novel effect of IFN-β on anti-Aspergillus immune responses which, in turn, might open new perspectives on the use of IFN-β in immunotherapy for fungal infections aimed at enhancing the immunological functions of DCs.

Introduction

Aspergillus fumigatus (A. fumigatus) conidia are ubiquitous in the environment. Although the inhaled spores are normally eliminated without causing disease in an immunocompetent host, they can elicit invasive aspergillosis (IA), a rapidly progressive infection in patients who are severely immunosuppressed.

Efficient responses to the fungus require a complex network of immunological mechanisms. Together with alveolar macrophages and neutrophils, which constitute a primary line of innate cellular defence against A. fumigatus,^1,2 the crucial role of the adaptive immunity has been extensively demonstrated.³ Indeed, besides the well-characterized protective role of T helper type 1 (Th1) lymphocytes,^4–7 the newly described regulatory T cells and interleukin-17 (IL-17) -producing cells (Th17) represent important mediators of the inflammatory and anti-inflammatory host responses against A. fumigatus.⁸ However, dendritic cells (DCs) also play a fundamental function in initiating and modulating the specific immune responses upon recognition of A. fumigatus.^5,9,10 After internalization of A. fumigatus conidia, DCs mature and acquire the capacity to polarize naive T cells and, in turn, to promote a protective response.⁹ In keeping with these findings, in vivo results on the migration of lung DCs into lymphoid organs, where they drive an appropriate T-cell response to fungal antigens,¹¹ have brought DCs centre stage as promising targets for intervention in immunotherapy and fungal vaccine development.¹² In addition, it is important to consider several studies that have recently pointed to DCs and type I interferons (IFNs) as special players in the immune response tailored to combat tumours and infections.^13–15 Indeed, although the anti-microbial properties of these cytokines have not been fully characterized yet, type I IFNs represent important immunomodulators of the innate, as well as the adaptive, arm of the immune system. Type I IFN can promote the differentiation of human blood monocytes into DCs and contribute to their maturation.^16,17 This leads to the generation of DCs able to stimulate a primary human antibody response, a Th1 proliferation,¹⁸ and a cross-priming of CD8 T cells against viral antigens.¹⁹ In addition, one crucial outcome of type I IFN-induced effects is the ability to directly stimulate IFN-γ production in natural killer and T cells,^20–22 which in turn promotes the development of a cell-mediated immune response.

Based on these immunoregulatory properties, in this work the expression and the capacity of type I IFN, namely IFN-β, to modulate the T-cell polarizing capacity of A. fumigatus-infected DCs was investigated in an attempt to evaluate the effects induced by this cytokine on anti-fungal immunity. Although the phagocytosis of the fungus was not affected by IFN-β treatment, the maturation induced by A. fumigatus infection was enhanced in IFN-β-primed DCs, as evaluated by analysing the immunophenotype and the release of pro-inflammatory and regulatory cytokines. Accordingly, IFN-β endowed DCs with potent Th1 polarizing capacity because an enhanced IFN-γ production in T cells co-cultured with A. fumigatus-infected DCs was observed in the presence of IFN-β. These results indicate that IFN-β potentiates DC immunological functions following A. fumigatus infection, which suggests that IFN-β is a possible adjuvant to elicit an appropriate Th reactivity to A. fumigatus.

Materials and methods

Preparation of DCs

Dendritic cells were prepared as previously described.⁹ CD14⁺ monocytes were cultured with 25 ng/ml granulocyte–macrophage colony-stimulating factor (GM-CSF; Schering-Plough, Levallois Perret, France) and 1000 U/ml IL-4 (R&D Systems, Minneapolis, MN) for 5 days. On day 5, about 90% of the cells express CD1a⁺ and 95% express CD14⁻. The DCs were starved from IL-4 and GM-CSF for 20 hr before infection or treatments.

Antibodies and other reagents

Monoclonal antibodies specific for CD1a, CD14, CD38, CD40, CD83, CD86, HLA-DR, CD3 and CD4 as well as immunoglobulin G1 (IgG1), IgG2a and IgG2b (BD Bioscience PharMingen, San Diego, CA) were used as direct conjugates to fluorescein isothiocyanate (FITC) or phycoerythrin. Lipopolysaccharide (LPS) from Escherichia coli 0111:B4 (Sigma-Aldrich, St Louis, MO) was used at a concentration of 100 ng/ml to stimulate DC maturation and IFN-β expression. The IFN-β (Avonex®; Biogen Inc., Cambridge, MA) was used at 200 pm.

Aspergillus fumigatus preparation and infection

A wild-type clinical isolate of A. fumigatus (CBS 144 89) was grown on Sabouraud–chloramphenicol agar for 3 days, at 37°, as previously described.²³ Preparations of A. fumigatus were analysed for LPS contamination by the Limulus lysate assay (Biowhittaker, Verviers, Belgium) and were found to contain less than 10 pg/ml LPS. In all experiments, DCs were infected with live A. fumigatus conidia at a 1 : 1 ratio. Amphotericin B (0·75 μg/ml; Sigma-Aldrich) was added to the cell cultures to prevent fungal overgrowth 6 hr after infection when the internalization of A. fumigatus conidia was completed.⁹

For the adherence assay, A. fumigatus conidia were incubated with FITC at a final concentration of 3 mg/ml overnight at 4°, and then washed extensively with PBS. After a 6-hr incubation with FITC-labelled conidia (ratio 1 : 1), DCs were washed and the adherence was measured by flow cytometric analysis.

Flow cytometric analysis

The cells were incubated with purified monoclonal antibodies at 4° for 30 min. After washing, the cells were fixed with 2% paraformaldehyde before analysis on a FACScan using the cellquest software (BD Bioscience PharMingen). A total of 5000 cells were analysed per sample.

RNA isolation and real-time reverse transcription–polymerase chain reaction quantifications

RNA extraction, reverse transcription (RT) and real-time RT-polymerase chain reaction (PCR) assays were performed as previously described.²⁴ Sequences of the primer pairs used for glyceraldehyde 3-phosphate dehydrogenase (GaPDH), IFN-β, IL-12p35, IL-23p19 and IL-27p28 quantification were previously described.²⁴

Cytokine detection

Cytokine concentration in filtered supernatants was evaluated with the human inflammation cytometric bead array (CBA) [for IL-12p70, IL-10, tumour necrosis factor-α (TNF-α) and IL-6: BD Bioscience PharMingen] and enzyme-linked immunsorbent assay (ELISA; for IFN-β: PBL Biomedical Laboratories, Piscataway, NJ; for IL-23: eBioscience, San Diego, CA).

Mixed leucocyte reaction

Cord blood CD4⁺ T cells were purified by indirect magnetic sorting with CD4⁺ T Cell Isolation Kit (Miltenyi Biotec, Bergish Gladbach, Germany). The proliferative response was performed at various T-cells : DC ratios using a fixed number of T cells (3 × 10⁴) and evaluated after 5 days by measuring thymidine incorporation (0·5 μCi/well of [³H]thymidine; Amersham, Little Chalfont, UK). The results were expressed as mean counts per minute of triplicate cultures. Supernatant of T-cell cultures was harvested at day 6 after infection and IFN-γ and IL-4 concentrations were measured by ELISA kits (R&D Systems).

Statistical analysis

Statistical analysis was performed by non-parametric two-tailed Mann–Whitney U-test using the graphpad prism 4 software (GraphPad Inc., San Diego, CA).

Results

Aspergillus fumigatus-infected DCs do not express IFN-β

We and other authors previously observed that DCs could release IFN-β following viral and bacterial infection,^25–28 while no data have been published on the capacity of A. fumigatus to induce the expression of type I IFN in DCs. To investigate this aspect, DCs were infected with A. fumigatus and the expression of IFN-β was evaluated by real-time RT-PCR at various times after infection (2, 6 and 20 hr). To verify the capacity of DC culture to express IFN-β, a treatment with LPS was also included at each time-point. Interestingly, no induction of IFN-β expression was observed at early time-points, whereas only a slight increase was noted 20 hr after A. fumigatus infection (Fig. 1). The lack of IFN-β messenger RNA (mRNA) expression following A. fumigatus infection of DCs was confirmed by ELISA (data not shown). Conversely, IFN-β mRNA induction by LPS began 2 hr post-infection, the level remained elevated at 6 hr and declined rapidly as previously described.²⁵

Figure 1.

Lack of interferon-β (IFN-β) expression in Aspergillus fumigatus-infected dendritic cells (DCs). Total RNA was extracted at the indicated time-points following A. fumigatus conidia (Af) infection (1 : 1 ratio) and lipopolysaccharide (LPS) treatment (100 ng/ml). The expression of IFN-β was analysed by real-time reverse transcription–polymerase chain reaction (RT-PCR). The results are shown as a ratio to the GaPDH level and represent the mean ± SE of triplicate values. This is a representative real time RT-PCR experiment, which was repeated twice more with RNA extracted from different DC cultures.

Exogenous addition of IFN-β improves the maturation of A. fumigatus-infected DCs without affecting their capacity to uptake A. fumigatus conidia

After determining that A. fumigatus-infected DCs did not express IFN-β and knowing the potential immunoregulatory properties of this cytokine,¹⁶ we investigated whether the exogenous addition of IFN-β could modify DC responses to the fungal infection. Dendritic cells were pre-treated for 4 hr with IFN-β and then infected with A. fumigatus conidia for 24 hr. The immunophenotype of the DCs was evaluated by flow cytometry through the analysis of the molecules involved in T-cell activation, such as CD86, CD83, HLA-DR and CD38 (Fig. 2a). As previously shown, the treatment of immature DCs with IFN-β induced a selective increased expression of CD38 (an IFN-inducible marker) and CD86 but not of CD83.²⁴ Interestingly, while the IFN-β-induced expression of CD38 was not further increased upon A. fumigatus challenge, a strong effect of IFN-β was instead observed on CD83 and CD86 expression in A. fumigatus-infected cells. Conversely, the constitutive expression of HLA-DR was not significantly modified by A. fumigatus or IFN-β treatment.

Figure 2.

Influence of interferon-β (IFN-β) pre-treatment on the in vitro maturation of Aspergillus fumigatus-infected dendritic cells (DCs). (a) DCs were pre-treated for 4 hr with IFN-β (200 pm) before the infection with A. fumigatus (Af). After 24 hr, cells were stained for CD83, CD86, HLA-DR and CD38 and analysed by flow cytometry. The results are the mean of mean fluorescence intensity (MFI; subtracted from the isotype value) ± SE values from six different blood donors. (b) After a treatment with IFN-β (200 pm) or with lipopolysaccharide (LPS; 100 ng/ml) for 24 hr, DCs were incubated with fluorescein isothiocyanate (FITC) -labelled conidia at a ratio of 1 : 1 for 6 hr at 37°. The percentage of DCs with bound FITC-labelled A. fumigatus conidia (Af-FITC-positive DC) was measured by flow cytometric analysis. The results represent the mean ± SE of four separate experiments.

The phagocytosis of A. fumigatus conidia was then evaluated in DCs treated with IFN-β for 24 hr to check whether the IFN-β effect on DC maturation was the result of an enhanced capacity to uptake A. fumigatus. The ability of IFN-β-treated DCs to internalize A. fumigatus conidia was compared with LPS-matured DCs (100 ng/ml for 24 hr). The percentage of DCs that have fixed FITC-A. fumigatus conidia was analysed by flow cytometry at 6 hr post-infection. Treatment with IFN-β did not alter the capacity of DCs to internalize the pathogen whereas, as expected, LPS-matured DCs showed a reduced capacity to uptake A. fumigatus conidia. Indeed, 55% of control and IFN-β-treated DCs phagocytosed A. fumigatus but only 35% of LPS-stimulated DCs fixed the conidia (Fig. 2b).

Overall, our results indicated that IFN-β treatment did not modify the capacity of immature DCs to phagocytose A. fumigatus conidia although it strongly modulated the A. fumigatus-induced expression of co-stimulatory and maturation molecules. These results prompted us to further investigate the impact of IFN-β on the response of DCs to A. fumigatus infection.

Interferon-β modulates the A. fumigatus-stimulated expression of pro-inflammatory and regulatory cytokines

Cytokine production critically impacts the ability of DCs to activate and prime T cells. For this reason, we assessed IFN-β capacity to modulate the profile of cytokines released by A. fumigatus-infected DCs. To this end, supernatants were harvested from DCs stimulated for 24 hr with A. fumigatus with or without 4 hr IFN-β pre-treatment, and the release of IL-12p70, IL-23, IL-10, TNF-α and IL-6 was analysed (Fig. 3). The presence of IFN-β significantly increased the secretion of IL-12p70 and IL-6 by A. fumigatus-infected DCs but it did not modify the release of IL-10, IL-23 and TNF-α.

Figure 3.

Effects of interferon-β (IFN-β) pre-treatment on cytokine production by Aspergillus fumigatus-infected dendritic cells (DCs). DC were stimulated as described in Fig. 2 and cell culture supernatants were collected. The amounts of interleukin-12 p70 (IL-12p70), IL-23, IL-10, tumour necrosis factor-α (TNF-α), and IL-6 were measured in the supernatants by cytometric bead assay and enzyme-linked immunosorbent assay. The results represent the mean ± SE of four separate experiments performed with different donors.

To investigate whether the robust induction of IL-12p70 in IFN-β-primed DCs was determined by an increased transcription of the IL-12p35 subunit and to extend our study also to IL-27, a critical cytokine involved in regulating T cell differentiation and functions, we investigated in IFN-β-primed DC the expression of inducible subunits composing the IL-12 family members following A. fumigatus infection. Total RNA was extracted 20 hr after A. fumigatus infection with or without a 4-hr IFN-β priming and the transcripts encoding IL-12p35, IL-23p19, and IL-27p28 subunits were analysed by real-time RT-PCR (Fig. 4). The weak expression of IL-12p35 in A. fumigatus-infected cells was increased by IFN-β pre-treatment explaining the synergistic effect on IL-12p70 secretion observed by CBA analysis (compare Figs 3 and 4). Conversely, no effect of IFN-β treatment was observed on the expression of IL-23p19, confirming the ELISA results presented in Fig. 3. Interestingly, IFN-β pre-treatment induced a robust expression of IL-27p28, which was further increased in A. fumigatus-stimulated DCs. The lack of IL-27p28 expression, in DCs stimulated with A. fumigatus alone, correlated well with the incapacity of this fungus to stimulate IFN-β gene transcription (Fig. 1) and further highlighted the type I IFN-dependent expression of IL-27, as previously demonstrated.²⁴ All these data suggest that IFN-β can profoundly modify the response of DC to A. fumigatus infection acting on their ability to release pro-inflammatory and regulatory cytokines involved in orchestrating the Th response.

Figure 4.

Expression of interleukin-12 (IL-12) family members in interferon-β (IFN-β) pre-treated dendritic cells (DCs) upon stimulation with Aspergillus fumigatus. RNA was extracted from immature DCs, DCs treated for 20 hr with A. fumigatus (Af) with or without a 4-hr pre-treatment with IFN-β, or were stimulated with IFN-β alone. Real time reverse transcription–polymerase chain reaction was performed to measure the expression of IL-12p35, IL-23p19 and IL-27p28. The results are shown as a ratio to the GaPDH level and represent the mean ± SE of triplicate values. The results shown are from one of three experiments performed with different donors that yielded similar results.

Interferon-β pre-treatment strengthens the capacity of A. fumigatus-infected DCs to expand Th1-oriented CD4⁺ T cells

Next, T-cell proliferation and polarization were investigated by mixed leucocyte reaction to determine whether the effect induced by IFN-β on A. fumigatus-infected DC maturation resulted in an enhanced capacity in promoting the expansion of Th1-oriented CD4⁺ T cells. As shown in Fig. 5(a), A. fumigatus-infected DCs induced the proliferation of naive allogeneic cord blood CD4⁺ T cells, which was not significantly modified when infected DCs were primed with IFN-β. Interestingly, IFN-β priming of A. fumigatus-infected DCs highly enhanced the production of IFN-γ, as observed by the analysis of supernatants obtained from mixed leucocyte reaction cultures (Fig. 5b). Conversely, no induction of IL-4 was found when T cells were co-cultured with A. fumigatus-stimulated DCs in the presence or absence of IFN-β (data not shown).

Figure 5.

Immunoregulatory effect of interferon-β (IFN-β) on the priming of naive T-cell responses by Aspergillus fumigatus-infected dendritic cells (DCs). The DCs were either left untreated or were stimulated with IFN-β, or were infected for 24 hr with A. fumigatus (Af) with or without IFN-β pre-treatment. (a) A mixed leucocyte reaction assay was set up with DCs cultured at various cell numbers with 3 × 10⁴ purified allogeneic naive CD4⁺ T cells isolated from cord blood. The proliferative response was measured after 5 days and expressed as mean counts per minute (cpm). The results represent the mean ± SE of three separate experiments performed with different donors. (b) DCs were cultured at 1 : 10 ratio (stimulator : effector cells) with allogeneic naive T cells and after 5 days the supernatants were collected. IFN-γ production means ± SE were obtained in three independent experiments performed with different donors.

Discussion

Type I IFNs, originally identified for their ability to induce cellular resistance to viral infections, are key immunomodulators of the innate and adaptive immune responses.²⁹ By acting on DC differentiation and maturation, these cytokines can induce cross-priming of CD8 T cells¹⁹ and stimulate a Th1-oriented T-cell response.^21,22 Accordingly, our recent findings showed that IFN-β potentiates DC immunological functions following bacillus Calmette–Guérin infection, pointing to the importance of IFN-β in promoting a protective Th1 immune response against Mycobacterium tuberculosis.³⁰ Based on this evidence, the use of type I IFN constitutes a promising immunotherapy for infectious diseases.^13,15

Invasive aspergillosis is a serious opportunistic fungal infection in immunocompromised hosts. Advances in more potent and less toxic antifungal agents have reduced the mortality rate of IA and represent a promising area of research and development to cure invasive fungal infections. Moreover, novel strategies for immunotherapy and vaccine are also currently designed on the knowledge of the immunopathogenesis of fungal infections.³¹ Although clinical evidence points to a crucial role for the Th1 reactivity in the control of IA, more recently regulatory T cells and Th17 cells could display important functions in the scenario of the immune response against A. fumigatus.³² However, if the role of IL-17-producing T cells in protection versus pathology in fungal infections is still controversial,^33–35 it is generally accepted that a defective differentiation of regulatory T cells may cause an unacceptable level of tissue damage.³ Several studies in human and murine models have, however, confirmed the central role of IFN-γ released by interstitial lung lymphocytes in controlling IA through the stimulation of phagocytosis and intracellular antifungal killing mechanisms of neutrophils and macrophages.^4,36 In line with these data, an elegant demonstration of the importance of specific adjuvants in fungal vaccination development to selectively induce Th1-dominated immune responses was published by Romani et al.,³⁷ who demonstrated that thymosin-α1 stimulates the Th1-polarizing capacity of DC. CpG was also shown to act as a potent adjuvant for the vaccine-induced protection against the fungus by promoting a dominant Th1 response to Aspergillus antigens and allergens.^38,39 Given the capacity of CpG to stimulate the release of type I IFN by plasmacytoid DCs, it is tempting to speculate that these cytokines can act as immuno-adjuvants in fungal defence. In addition, the capacity of type I IFN to exert an anti-fungal activity by promoting natural killer cell activation in mice^40,41 suggests that these cytokines could represent a promising adjuvant able to reinforce also the innate defence mechanisms such as those involving natural killer cells.

Based on these data, we investigated whether IFN-β could also modulate the T-cell polarizing capacity of A. fumigatus-stimulated DCs, which fail to secrete this cytokine when challenged with A. fumigatus conidia. Interestingly, we observed that the exogenous addition of IFN-β to A. fumigatus-stimulated DCs reinforced the expression of CD86, CD83 and IL-12p70 and, in turn, the capacity to stimulate a Th1 response. Moreover, in the presence of IFN-β, DCs may also express IL-27, a key cytokine involved in controlling excessive inflammation by suppressing Th17 differentiation.⁴² In addition to that, in this scenario IFN-β could also limit the development of a Th17 inflammatory response acting directly on T cells as recently proposed in patients with multiple sclerosis undergoing IFN-β therapy.^43,44 Collectively these data identified a novel effect of IFN-β on the anti-Aspergillus immune response which, in turn, might open new perspectives on the use of IFN-β as a candidate adjuvant in immunotherapy for fungal infections aimed at potentiating DC immunological functions.

Glossary

Abbreviations:

CBA

cytometric bead array

DCs

dendritic cells

ELISA

enzyme-linked immunosorbent assay

FITC

fluorescein isothiocyanate

GaPDH

glyceraldehyde 3-phosphate dehydrogenase

GM-CSF

granulocyte–macrophage colony-stimulating factor

IA

invasive aspergillosis

IFN

interferon

IgG

immunoglobulin G

IL

interleukin

LPS

lipopolysaccharide

MFI

mean fluorescence intensity

RT-PCR

reverse transcription–polymerase chain reaction

Th1

T helper type 1

TNF

tumour necrosis factor

Disclosures

The authors declare no conflict of interest.