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Keywords:

  • invasive aspergillosis;
  • innate immunity;
  • toll-like receptors;
  • dendritic cell activation

Summary

  1. Top of page
  2. Summary
  3. Material and methods
  4. Reagents, antibodies and plasmids
  5. Aspergillus fumigatus antigens
  6. Preparation and culture of human cells
  7. Generation of mouse DCs
  8. Phagocytosis
  9. Detection of human and mouse cytokines
  10. Quantifiable reverse transcription polymerase chain reaction (RT-PCR)
  11. Statistics
  12. Results
  13. Aspergillus fumigatus antigens activate human immature monocyte-derived DCs
  14. Aspergillus antigens stimulate effector functions of human neutrophils
  15. Aspergillus fumigatus mediates DC activation via TLR2 and TLR4
  16. Discussion
  17. Acknowledgments
  18. References

Invasive aspergillosis (IA) is a leading cause of mortality in haematological patients. Appropriate activation of the innate immune system is crucial for the successful clearance of IA. Therefore, we studied the Aspergillus fumigatus-mediated activation of human granulocytes and monocyte-derived immature dendritic cells (DCs), as well as murine bone marrow-derived DCs (BMDCs) from wild type, toll-like receptor (TLR)4-deficient, TLR2 knockout, and TLR2/TLR4 double deficient mice. Aspergillus fumigatus antigens induced the activation and maturation of immature DCs as characterized by CD83 expression, upregulation of major histocompatibility complex and co-stimulatory molecules. Moreover, fungal antigens enhanced the phagocytosis and production of interleukin (IL)-8 in granulocytes. The release of IL-12 by BMDCs in response to A. fumigatus antigens was dependent on the expression of TLR2, whereas the release of IL-6 was dependent on the expression of functional TLR4 molecules. The protein precipitate of A. fumigatus supernatant provided strong stimulation of DCs and granulocytes, indicating that a factor secreted by A. fumigatus might activate innate immune cells. In conclusion, A. fumigatus antigens induced the activation of DCs and granulocytes. Our results indicated that this activation was mediated via TLR2 and TLR4. Future studies are needed to assess the clinical impact of these findings in patients at high risk for IA.

Invasive aspergillosis (IA) has become a major cause of infection-related mortality in patients with haematological malignancies, especially after allogeneic stem cell transplantation (SCT) (Wald et al, 1997). Proven risk factors in human beings are defects in phagocyte function (Morgenstern et al, 1997), steroid-induced suppression of macrophage conidiocidal activity (Schaffner et al, 1982; Palmer et al, 1991), and chemotherapy-induced neutropenia (Gerson et al, 1984). Patients with a previous history of IA (Offner et al, 1998) and patients colonized in the lower respiratory tract without signs of tissue-invasive disease (Einsele et al, 1998) have been found to have an increased risk for recurrence of IA during a subsequent episode of neutropenia or immunosuppression (Offner et al, 1998). These observations implicate local cellular defects in the immune effector mechanisms as major predisposing factors of the host to IA (Schaffner et al, 1982; Romani, 1997; Cenci et al, 1998).

In Drosophila, toll participates in the defence against fungi by the induction of drosomycin secretion as an early form of an innate immune response (Lemaitre et al, 1996). In mammals, toll-like receptors (TLRs) are involved in the response to pathogens by the recognition of so-called pathogen-associated molecular patterns. These include lipopolysaccharide (LPS), peptidoglycans, lipoproteins and bacterial CpG-DNA, which are recognized by TLR4, TLR2 and TLR9 respectively (Aderem & Ulevitch, 2000; Hemmi et al, 2000).

Toll-like receptors have been demonstrated to induce tumour necrosis factor α (TNFα) release in murine peritoneal macrophages and transfected human cell lines after stimulation with different biological forms of Aspergillus fumigatus (Mambula et al, 2002) and preliminary data indicate a potential role of TLRs in the A. fumigatus-induced activation of human monocytes (Wang et al, 2001).

More recently, a critical role of T-helper (TH) cell responses in the control of IA has been reported in murine models (Clerici & Shearer, 1994; Cenci et al, 1997, 1998, 1999, 2000; Mehrad et al, 1999; Clemons et al, 2000) and in patients with haematological malignancies and allogeneic SCT recipients (Hebart et al, 2002). Dendritic cells (DCs) are recognized as the initiators of specific immune responses to pathogens (Banchereau & Steinman, 1998). In the murine model, pulmonary DCs were found to internalize Aspergillus conidia and hyphae, to undergo functional maturation upon migration to the draining lymph nodes and spleen and to induce T-cell priming of CD4+ T lymphocytes (Bozza et al, 2002). In vitro, DCs were found to restore Aspergillus-specific lymphoproliferation in haematological patients (Grazziutti et al, 2001).

In the current study, we assessed the A. fumigatus-induced activation of granulocytes and DCs.

Reagents, antibodies and plasmids

  1. Top of page
  2. Summary
  3. Material and methods
  4. Reagents, antibodies and plasmids
  5. Aspergillus fumigatus antigens
  6. Preparation and culture of human cells
  7. Generation of mouse DCs
  8. Phagocytosis
  9. Detection of human and mouse cytokines
  10. Quantifiable reverse transcription polymerase chain reaction (RT-PCR)
  11. Statistics
  12. Results
  13. Aspergillus fumigatus antigens activate human immature monocyte-derived DCs
  14. Aspergillus antigens stimulate effector functions of human neutrophils
  15. Aspergillus fumigatus mediates DC activation via TLR2 and TLR4
  16. Discussion
  17. Acknowledgments
  18. References

Lipopolysaccharide from Pseudomonas aeruginosa (kindly provided by T. Schröder, Tübingen, Germany) was used or LPS from Salmonella typhimurium was purchased from Sigma-Aldrich (Taufkirchen, Germany). Phosphothioate-stabilized CpG oligonucleotide 1668 (TCC-ATC-ACG-TTC-CTG-ATG-C) was purchased from TIB MOLBIOL (Berlin, Germany). Palmitoyl-3-Cys-Ser-(Lys)4 (Pam3Cys) was obtained from EMC microcollections GmbH (Tübingen, Germany).

The expression vectors for the N-terminus of human flag-tagged TLR2 and TLR4 were gifts from Tularik, Inc. (South San Francisco, CA, USA). The human MD2 expression vector was kindly provided by K. Miyake (Sage Medical School, Nabeshima, Japan). The luciferase reporter, driven by a synthetic enhancer harbouring six nuclear factor-κB binding consensus sites, was a gift from P. Baeuerle (München, Germany).

Cell staining was performed using fluorescein isothiocyanate- or phycoerythrin (PE)-conjugated mouse monoclonal antibodies against CD86, CD40, CD11b (purchased from Pharmingen, Hamburg, Germany); CD80, HLA-DR, CD14 (all purchased from Becton Dickinson, Heidelberg, Germany); CD66b or CD83 (all from Coulter-Immunotech Diagnostics, Hamburg, Germany), and CD1a (OKT6; Ortho Diagnostic Systems, Neckargemund, Germany), and mouse immunoglobulin (Ig)G isotype controls (Becton Dickinson). Samples were analysed on a FACScalibur (Becton Dickinson).

Aspergillus fumigatus antigens

  1. Top of page
  2. Summary
  3. Material and methods
  4. Reagents, antibodies and plasmids
  5. Aspergillus fumigatus antigens
  6. Preparation and culture of human cells
  7. Generation of mouse DCs
  8. Phagocytosis
  9. Detection of human and mouse cytokines
  10. Quantifiable reverse transcription polymerase chain reaction (RT-PCR)
  11. Statistics
  12. Results
  13. Aspergillus fumigatus antigens activate human immature monocyte-derived DCs
  14. Aspergillus antigens stimulate effector functions of human neutrophils
  15. Aspergillus fumigatus mediates DC activation via TLR2 and TLR4
  16. Discussion
  17. Acknowledgments
  18. References

Conidia of A. fumigatus (strain CBS 144-89) were inoculated in 150-ml Erlenmeyer flasks containing Sabouraud liquid medium [2% (w/v) glucose, 1% (w/v) mycopeptone]. Flasks were shaken for 24 h at 37°C and 200 rpm. Two-litre fermenters (LSL Biolafitte, Saint Germain en Laye, France) containing 1·2 l of Sabouraud medium were inoculated with the shaken flask cultures. The 18 h-culture conditions were as follows: inoculum 8% (v/v); temperature, 26°C; aeration, 50 l of air per minute; agitation, 500 rpm. The mycelial mat recovered by filtration was extensively washed with water. Mycelium was disrupted in a glass bead cell homogenizer in 50 mmol/l Tris–HCl buffer pH 7·5 and the water-soluble cellular extracts (EC SAB) were recovered after centrifugation. The ethanol precipitate (PP SAB) of A. fumigatus was prepared by precipitating the culture filtrate with 4 volumes of ethanol after 18 h of culture and stored at 4°C. Protein content was measured by the BioRad technique (Bio-Rad, Marne La Coquette, France) according to the manufacturer's instructions and estimated in milligram equivalent BSA per millilitre. EC SAB and PP SAB were tested endotoxin free (<0·1 endotoxin units/μg protein) by the limulus amebocyte lysate assay (BioWhittaker, Verviers, Belgium).

To generate hyphae, conidia (106 colony forming units/ml) were harvested after 3 d of culture on Sabouraud glucose agar, filtered through sterile gauze and then incubated in yeast nitrogen base for 18 h at 37°C, followed by a centrifugation for 10 min at 3000 g. Mycelia were washed twice and, for sterilization, incubated in ethanol-phosphate-buffered saline (ethanol-PBS; 70%) for 24 h at 4°C.

Preparation and culture of human cells

  1. Top of page
  2. Summary
  3. Material and methods
  4. Reagents, antibodies and plasmids
  5. Aspergillus fumigatus antigens
  6. Preparation and culture of human cells
  7. Generation of mouse DCs
  8. Phagocytosis
  9. Detection of human and mouse cytokines
  10. Quantifiable reverse transcription polymerase chain reaction (RT-PCR)
  11. Statistics
  12. Results
  13. Aspergillus fumigatus antigens activate human immature monocyte-derived DCs
  14. Aspergillus antigens stimulate effector functions of human neutrophils
  15. Aspergillus fumigatus mediates DC activation via TLR2 and TLR4
  16. Discussion
  17. Acknowledgments
  18. References

Polymorphonuclear neutrophils (PMNs) were separated from heparinized blood of healthy volunteer donors by dextran sedimentation using Polymorphprep (Nycomed, Oslo, Norway) according to the manufacturer's instructions and as described previously (Radsak et al, 2003). Contaminating red blood cells were removed by an in-house lysis buffer [150 mmol/l ammonium chloride, 1 mmol/l potassium bicarbonate, 0·1 mmol/l ethylene diamine tetra acetate (all from Sigma, St Louis, MO, USA) in distilled water, pH 7·3]. The purity of the cell preparation was assessed by flow cytometry with CD66b as marker for PMN. Usually, 95–98% of cells were CD66b positive. For stimulation of PMN, cells were suspended in a concentration of 2 × 106 cells/ml in Roswell Park Memorial Institute (RPMI) 1640 medium (PAN Biotech, Aidenbach, Germany) with 3% (v/v) heat inactivated (30 min at 56°C) fetal bovine serum (FBS; PAN Biotech) and cultivated at 37°C with 7·5% CO2 in air in the presence of various stimuli diluted in medium as indicated.

Human DCs were generated from heparinized blood of healthy donors as reported previously (Grigoleit et al, 2002). Briefly, peripheral blood mononuclear cells (PBMCs) were isolated using a Ficoll density gradient (LINARIS, Bettingen am Main, Germany), washed twice in sterile calcium- and magnesium-free Hanks’ balanced salt solution (GIBCO BRL, Karlsruhe, Germany) and re-suspended in RPMI 1640 medium supplemented with Glutamax-I, 25 mmol/l HEPES buffer (GIBCO BRL), 200 μg/ml Gentamicinsulphate (Refobacin® 80 mg; Merck, Darmstadt, Germany), and 10% fetal calf serum (FCS; Sigma). PBMCs were plated at a density of 10 × 106 cells/well for 2 h at 37°C and non-adherent cells were removed by washing with PBS. Adherent monocytes were cultured for 6 d in RP10 medium supplemented with 1000 IU/ml interleukin-4 (IL-4; R&D Systems, Minneapolis, MN, USA) and 100 ng/ml granulocyte-macrophage colony-stimulating factor (GM-CSF; Leukomax, Novartis Pharma GmbH, Nürnberg, Germany). The differentiation state of DCs was examined by flow cytometry.

Generation of mouse DCs

  1. Top of page
  2. Summary
  3. Material and methods
  4. Reagents, antibodies and plasmids
  5. Aspergillus fumigatus antigens
  6. Preparation and culture of human cells
  7. Generation of mouse DCs
  8. Phagocytosis
  9. Detection of human and mouse cytokines
  10. Quantifiable reverse transcription polymerase chain reaction (RT-PCR)
  11. Statistics
  12. Results
  13. Aspergillus fumigatus antigens activate human immature monocyte-derived DCs
  14. Aspergillus antigens stimulate effector functions of human neutrophils
  15. Aspergillus fumigatus mediates DC activation via TLR2 and TLR4
  16. Discussion
  17. Acknowledgments
  18. References

Mouse immature DCs were generated from bone marrow of C3H/HeN and C3H/HeJ (Tularic Inc., South San Fransisco, CA, USA). C3H/HeJ/TLR2−/− and TLR2−/− mice were generously provided by H. Wagner/K. Kirschning (Munich, Germany). For the generation of mouse bone marrow-derived DCs (BMDCs) Iscove's modified Dulbecco's medium (IMDM; BioWhittaker, Verviers, Belgium) was used supplemented with 2 mmol/l l-glutamine (GibcoBRL Life Technologies, Paisley, GB), 100 IU/ml penicillin/streptomycin (GibcoBRL), 10% FCS (PAA, Linz, Austria) and 200 U/ml GM-CSF (Peprotec, Rocky Hill, NJ, USA). Bone marrow cells were incubated in GM-CSF containing medium for 6–8 d and fresh medium with GM-CSF was replaced every 2 d. The obtained DCs were CD11c positive and CD14 negative.

Phagocytosis

  1. Top of page
  2. Summary
  3. Material and methods
  4. Reagents, antibodies and plasmids
  5. Aspergillus fumigatus antigens
  6. Preparation and culture of human cells
  7. Generation of mouse DCs
  8. Phagocytosis
  9. Detection of human and mouse cytokines
  10. Quantifiable reverse transcription polymerase chain reaction (RT-PCR)
  11. Statistics
  12. Results
  13. Aspergillus fumigatus antigens activate human immature monocyte-derived DCs
  14. Aspergillus antigens stimulate effector functions of human neutrophils
  15. Aspergillus fumigatus mediates DC activation via TLR2 and TLR4
  16. Discussion
  17. Acknowledgments
  18. References

For analysis of phagocytic activity, ingestion of PE-labelled polystyrene microspheres (diameter 1 μm, Fluoresbrite Plain Microspheres PCRed; Polysciences, Warrington, PA, USA) was evaluated as described previously (Lehmann et al, 2000; Radsak et al, 2003). Briefly, aliquots of 2 × 105 freshly purified PMN were incubated in the presence of stimuli as indicated and 5 × 106 microbeads [effector:target (E:T) ratio 1:25] for 60 min at 37°C, then kept on ice, washed twice in fluorescent-activated cell sorting (FACS) buffer and fixed in 1% paraformaldehyde in PBS. Analysis was performed by FACS.

Detection of human and mouse cytokines

  1. Top of page
  2. Summary
  3. Material and methods
  4. Reagents, antibodies and plasmids
  5. Aspergillus fumigatus antigens
  6. Preparation and culture of human cells
  7. Generation of mouse DCs
  8. Phagocytosis
  9. Detection of human and mouse cytokines
  10. Quantifiable reverse transcription polymerase chain reaction (RT-PCR)
  11. Statistics
  12. Results
  13. Aspergillus fumigatus antigens activate human immature monocyte-derived DCs
  14. Aspergillus antigens stimulate effector functions of human neutrophils
  15. Aspergillus fumigatus mediates DC activation via TLR2 and TLR4
  16. Discussion
  17. Acknowledgments
  18. References

For analysis of cytokines by enzyme-linked immunosorbent assay (ELISA), supernatants were derived from stimulation with purified human PMN or mouse DCs and frozen at −20°C until required.

Supernatants of PMN were analysed by ELISA for IL-8 (OptEIA from Pharmingen) according to the manufacturer's instructions. Supernatants derived from mouse DCs were analysed using a commercial IL-6 and IL-12 specific ELISA (BD Pharmingen) as described previously (Vabulas et al, 2002).

Quantifiable reverse transcription polymerase chain reaction (RT-PCR)

  1. Top of page
  2. Summary
  3. Material and methods
  4. Reagents, antibodies and plasmids
  5. Aspergillus fumigatus antigens
  6. Preparation and culture of human cells
  7. Generation of mouse DCs
  8. Phagocytosis
  9. Detection of human and mouse cytokines
  10. Quantifiable reverse transcription polymerase chain reaction (RT-PCR)
  11. Statistics
  12. Results
  13. Aspergillus fumigatus antigens activate human immature monocyte-derived DCs
  14. Aspergillus antigens stimulate effector functions of human neutrophils
  15. Aspergillus fumigatus mediates DC activation via TLR2 and TLR4
  16. Discussion
  17. Acknowledgments
  18. References

Total RNA was extracted using RNeasy spin columns (Qiagen, Hilden, Germany) followed by cDNA synthesis using the first strand cDNA synthesis kit based on avian myeloblastosis virus (AMV) reverse transcriptase (Roche, Mannheim, Germany). Quantifiable PCR assays were performed by real-time PCR with the LightCyclerTM instrument as reported previously (Loeffler et al, 2003). This technique is based on the fluorescence resonance energy transfer. The samples were quantified by defined external standards, ranging from 109 to 101 CFU/ml. Primers were specific for TNFα and IL-12 (5′-). The probes consisted of two parts: one part had been labelled at the 5′-end with the Light Cycler-Red 640 fluorophore (5′-), and the other at the 3′-end with fluorescein (5′-; Tibmolbiol, Berlin, Germany).

Aspergillus fumigatus antigens activate human immature monocyte-derived DCs

  1. Top of page
  2. Summary
  3. Material and methods
  4. Reagents, antibodies and plasmids
  5. Aspergillus fumigatus antigens
  6. Preparation and culture of human cells
  7. Generation of mouse DCs
  8. Phagocytosis
  9. Detection of human and mouse cytokines
  10. Quantifiable reverse transcription polymerase chain reaction (RT-PCR)
  11. Statistics
  12. Results
  13. Aspergillus fumigatus antigens activate human immature monocyte-derived DCs
  14. Aspergillus antigens stimulate effector functions of human neutrophils
  15. Aspergillus fumigatus mediates DC activation via TLR2 and TLR4
  16. Discussion
  17. Acknowledgments
  18. References

This study was intended to analyse the effects of A. fumigatus antigens on immature DCs. Therefore, monocyte-derived immature DCs were stimulated overnight with different antigen preparations from A. fumigatus including hyphae, a cellular extract (EC SAB) and the ethanol precipitate of the culture supernatant of A. fumigatus (PP SAB) and analysed for the surface expression of CD83, co-stimulatory and major histocompatibility complex (MHC) molecules as well as for the expression of IL-12p40 and TNFα by quantifiable RT-PCR. Aspergillus fumigatus antigens were found to activate immature DCs as demonstrated by an increased expression of co-stimulatory molecules and HLA-DR, and to induce maturation of immature DCs (Fig 1). Moreover, stimulation of immature DCs with A. fumigatus antigen preparations induced a strong expression of IL-12p40 (up to 1000-fold) and TNFα RNA (up to 100-fold) (data not shown). PP SAB was found to provide the strongest stimulation, but high input of hyphae yielded comparable result indicating that this effect might result from a higher concentration of TLR ligands in the PP SAB preparation. As PP SAB and EC SAB were found to stimulate immature DCs as effectively as Aspergillus hyphae, all further experiments were performed with EC SAB and PP SAB.

image

Figure 1. Aspergillus fumigatus hyphae induce activation and maturation of monocyte-derived human immature dendritic cells (DCs). The figure shows the result of one representative experiment. Overnight stimulation with A. fumigatus hyphae (one hyphae/DC) induces activation (upregulation of CD40, CD80, CD86, HLA-DR) and maturation (increased expression of CD83) of immature DCs. CD14 expression was unchanged. Isotype control, dotted line; negative control, thin line; A. fumigatus stimulated DCs, thick line.

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Aspergillus antigens stimulate effector functions of human neutrophils

  1. Top of page
  2. Summary
  3. Material and methods
  4. Reagents, antibodies and plasmids
  5. Aspergillus fumigatus antigens
  6. Preparation and culture of human cells
  7. Generation of mouse DCs
  8. Phagocytosis
  9. Detection of human and mouse cytokines
  10. Quantifiable reverse transcription polymerase chain reaction (RT-PCR)
  11. Statistics
  12. Results
  13. Aspergillus fumigatus antigens activate human immature monocyte-derived DCs
  14. Aspergillus antigens stimulate effector functions of human neutrophils
  15. Aspergillus fumigatus mediates DC activation via TLR2 and TLR4
  16. Discussion
  17. Acknowledgments
  18. References

Polymorphonuclear neutrophils play an important part in the first line of defence against microbial pathogens by contributing substantially to the innate host defence with their ability to rapidly extravasate into inflamed tissue and their employment of potent effector mechanisms, i.e. phagocytosis, production of reactive oxygen species, and release of mediators and antimicrobial substances (Van Furth et al, 1973; Lloyd & Oppenheim, 1992; Ben-Baruch et al, 1995; Greenberg & Grinstein, 2002).

Therefore, we studied whether PP SAB and EC SAB could also mediate activation of human PMN. In a first set of experiments, we investigated the ingestion of unopsonized fluorochrome-labelled polystyrene beads to measure for unspecific phagocytosis as described previously (Radsak et al, 2003). The phagocytic activity of PMN was markedly enhanced by PP SAB, but not by EC SAB (Table I). The activation was insensitive to the presence of polymyxin  B (not shown) indicating that the observed activation was not due to potential contaminations by bacterial endotoxins.

Table I. Aspergillus antigen PP SAB, but not EC SAB, enhances phagocytic activity of PMN.
StimulusFold dilutionMean (n = 8 experiments)SDP-value
  1. PMN, polymorphonuclear neutrophil; ns, not significant.

  2. Purified PMN (2 × 105) were incubated with the indicated stimuli in the presence of 5 × 106 fluorochrome-labelled microspheres as described in Materials and Methods. Cells were washed and fixed in 1% paraformaldehyde. Fluorescence intensity was evaluated by FACS. The summarized results of eight independent experiments with different donors are shown as mean with standard deviation (SD). Statistical analysis was performed by Mann–Witney U-test comparing the stimulated cells to the medium control.

Medium 267·0136·2 
PP SAB10935·0383·8<0·001
30744·3202·6<0·001
EC SAB10385·5334·5ns
30256·0215·0ns

In addition, Aspergillus antigens stimulated the release of IL-8 in purified PMN, as shown in Fig 2. In a similar way, Aspergillus antigens mediated degranulation in PMN, analysed by upregulation of surface expression of CD66b and Mac-1 (CD11b) indicating mobilization of specific and gelatinase granules (not shown).

image

Figure 2. Release of IL-8 by polymorphonuclear neutrophil (PMN) after stimulation with Aspergillus antigens. Freshly purified PMN (4 × 105) were incubated in the presence of the indicated Aspergillus antigens PP SAB (grey bars), EC SAB (open bars), or controls (medium alone or lipopolysaccharide 1 μg/ml; black bars). Culture supernatants were harvested after 6 h and assayed for IL-8 by ELISA. All samples were assayed in triplicates and are depicted as mean and standard deviation. The representative results from one of three independent experiments are shown.

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These results suggest that these Aspergillus antigens present potent stimuli for the activation of innate immune cells.

Aspergillus fumigatus mediates DC activation via TLR2 and TLR4

  1. Top of page
  2. Summary
  3. Material and methods
  4. Reagents, antibodies and plasmids
  5. Aspergillus fumigatus antigens
  6. Preparation and culture of human cells
  7. Generation of mouse DCs
  8. Phagocytosis
  9. Detection of human and mouse cytokines
  10. Quantifiable reverse transcription polymerase chain reaction (RT-PCR)
  11. Statistics
  12. Results
  13. Aspergillus fumigatus antigens activate human immature monocyte-derived DCs
  14. Aspergillus antigens stimulate effector functions of human neutrophils
  15. Aspergillus fumigatus mediates DC activation via TLR2 and TLR4
  16. Discussion
  17. Acknowledgments
  18. References

To analyse a potential role of TLR2 and TLR4 in the A. fumigatus antigen-mediated activation of innate immune cells, we investigated this interaction in detail in BMDCs from wild-type mice and mice lacking functional TLR2 or TLR4 molecules. In the first set of experiments, we analysed the secretion of the pro-inflammatory cytokines IL-12 and IL-6 in response to EC SAB and PP SAB or LPS, Pam3Cys and CpG-DNA as control stimuli. Upon stimulation BMDCs derived from TLR4 deficient mice and wild-type mice produced comparable amounts of IL-12 in response to EC SAB and PP SAB (Fig 3). In contrast, BMDCs from TLR2 knockout mice were still activated by PP SAB but not by EC SAB, suggesting that activation by EC SAB is strictly dependent on TLR2. In order to investigate this more closely, a second set of experiments was performed using also BMDCs from TLR2 and TLR4 double-deficient mice. As shown in Fig 4A, BMDCs from wild-type mice (C3H/HeN) and TLR4-deficient mice (C3H/HeJ) again gave a comparable IL-12 response to PP SAB and EC SAB (Fig 3). However, BMDCs from the TLR2/4 double-deficient mice did not respond to the PP SAB stimulus. As expected, BMDCs from TLR2/4 double-deficient mice did not respond to the TLR2-dependent IL-12 release induced by EC SAB. Analysing the production of IL-6 only showed a strong response to PP SAB in the wild-type BMDCs, but not to EC SAB (Fig 4B). Interestingly and unlike the production of IL-12, PP SAB-induced secretion of IL-6 was also affected in TLR4-deficient mice.

image

Figure 3. EC SAB-induced IL-12 production by DCs is TLR2 dependent. Bone marrow-derived dendritic cells (BMDCs) from the indicated mice strains were cultured in the presence of EC SAB (200-fold dilution), PP SAB (200-fold dilution), LPS (2·5 μg/ml), CpG ODN 1668 (2 μmol/l) or Pam3Cys (2 μg/ml). After 20 h IL-12 concentration in cell culture supernatant was measured by sandwich ELISA. BMDCs derived from C3H/HeN mice and C3H/HeJ mice produced comparable amounts of IL-12 in response to EC SAB and PP SAB. In contrast, TLR2 knockout mice-derived DCs responded to PP SAB but not to EC SAB regarding IL-12 production. The mean and standard deviation of triplicate wells is shown. The differences shown are statistically significant (P < 0·05 by two-way anova).

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image

Figure 4. Aspergillus fumigatus mediates dendritic cell (DC) activation via TLR2 and TLR4. Bone marrow-derived DCs (BMDCs) from the indicated mice strains were stimulated with EC SAB, PP SAB (indicated dilution), LPS (10 ng/ml), CpG ODN 1668 (5 μmol/l) or Pam3Cys (1 μg/ml). Cell culture supernatants were harvested after 20 h and analysed for IL-12 and IL-6 secretion by sandwich ELISA. (A) BMDCs from C3H/HeN mice and C3H/HeJ mice secreted comparable amounts of IL-12 in response to EC SAB and PP SAB. C3H/HeJ/TLR2−/− mice did not respond to either fungal preparation. (B) BMDCs from C3H/HeN produced significant amounts of IL-6 in response to PP SAB, but not to EC SAB. The IL-6 release upon stimulation with PP SAB was more dependent on TLR4 compared with the release of IL-12. The mean and standard deviation of triplicate wells is shown. The differences shown are statistically significant (P < 0·05 by two-way anova).

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These results clearly demonstrate that the activation of BMDCs by EC SAB is exclusively dependent on TLR2. The situation is more complex for PP SAB. In the case of IL-12 production induced by PP SAB, TLR2 and TLR4 can fully compensate for each other. However, IL-6 production in TLR4-deficient mice cannot be compensated by TLR2.

Discussion

  1. Top of page
  2. Summary
  3. Material and methods
  4. Reagents, antibodies and plasmids
  5. Aspergillus fumigatus antigens
  6. Preparation and culture of human cells
  7. Generation of mouse DCs
  8. Phagocytosis
  9. Detection of human and mouse cytokines
  10. Quantifiable reverse transcription polymerase chain reaction (RT-PCR)
  11. Statistics
  12. Results
  13. Aspergillus fumigatus antigens activate human immature monocyte-derived DCs
  14. Aspergillus antigens stimulate effector functions of human neutrophils
  15. Aspergillus fumigatus mediates DC activation via TLR2 and TLR4
  16. Discussion
  17. Acknowledgments
  18. References

In this report, we demonstrated that antigen preparations derived from A. fumigatus were potent activators of human immature monocyte-derived DCs, as demonstrated by the upregulation of MHC and co-stimulatory molecules, and an increased TNFα and IL-12p40 mRNA concentration. The presence of these pro-inflammatory cytokines can be expected to contribute to the development of a TH1-dominated immune response, which was found to be associated with control of IA. Moreover, CD83 was upregulated, indicating differentiation into mature DCs. In line with these results, the adoptive transfer of DCs pulsed with conidia and DCs transfected with conidial RNA were found to induce TH1-dependent antifungal resistance in allogeneic bone marrow transplanted mice (Bozza et al, 2003).

The precipitate of the culture supernatant (PP SAB) was found to induce the strongest stimulation of immature DCs in comparison with hyphae and the cellular extract of A. fumigatus (EC SAB). However, the high input of hyphae yielded comparable results, indicating that this effect might result from a higher concentration of TLR ligands in the PP SAB preparation. Whereas the cytokine release of DCs from TLR4- and TLR2-deficient mice was only slightly reduced upon stimulation with PP SAB, a complete blockade of the cytokine release was observed in DCs from TLR2 and TLR4 double-deficient mice (Fig 4). For signals mediated by PP SAB, TLR2 and TLR4 could fully compensate for each other if one was deficient, but deficiency of both, TLR2 and TLR4, could not be compensated for, demonstrating that TLRs are responsible for PP SAB-mediated cell activation. In contrast, signalling induced by the cellular extract of A. fumigatus EC SAB was found to be strictly TLR2 dependent, which is in line with data describing the TLR2 dependence of TNFα release in murine peritoneal macrophages after stimulation with Aspergillus conidia and hyphae (Mambula et al, 2002). More recently, A. fumigatus-induced activation of murine peritoneal macrophages has been described to be dependent on the expression of TLR2 and TLR4 (Meier et al, 2003).

Interestingly, PP SAB but not EC SAB was found to strongly induce IL-6 release in culture supernatants of murine immature DCs. This finding is of special interest as IL-6 release by DCs has recently been described as essential for blocking the suppressive effects of regulatory CD4+ CD25+ T cells on the initiation of pathogen-specific adaptive immune responses (Pasare & Medzhitov, 2003). The characterization of regulatory T cells has opened exciting opportunities to induce tolerance after transplantation and to prevent graft versus host disease after allogeneic SCT; however, these cells might induce potentially detrimental effects by down-regulation of immunity to infections and also to tumours (Wood & Sakaguchi, 2003). The characterization of the PP SAB-derived TLR ligand responsible for IL-6 release in DCs is thus of potential interest for the development of Aspergillus-directed immunotherapy protocols.

PP SAB-induced IL-6 release was dependent on the expression of functional TLR4 molecules whereas IL-12 release was TLR2 dependent. This suggests that various pattern recognition receptors might be involved in the complex interaction of microbes and DCs. Recently, the use of different TLRs to differentially induce the release of pro-inflammatory cytokines and chemokines has been described for the interaction of Candida albicans and DCs (Netea et al, 2002). Thus, differences in antigen preparations, cytokines analysed and cell types under study are likely to have a major impact on the results in the study of cell pathogen interactions and to explain differing results, as reported recently for TLR-usage in A. fumigatus-mediated cell activation (Wang et al, 2001; Mambula et al, 2002). Moreover, TLR4-mediated but not TLR2-mediated cytokine signals were found to be lost upon Aspergillus germination to hyphae indicating that phenotypic switching during germination might counteract host immune defence mechanisms (Netea et al, 2003).

Our findings obtained with mouse- and human-derived DCs are complemented by the experiments performed with human neutrophils. This cell type has been shown to express high levels of TLR2 and low levels of TLR4 on the surface (Kurt-Jones et al, 2002). In line with the results observed in DCs, PP SAB and EC SAB, although to a lesser extent, were found to augment neutrophil function as assessed by the release of IL-8, phagocytosis and degranulation. Thus, factors stimulated by A. fumigatus directly activate anti-Aspergillus effector functions of neutrophils and induce the release of IL-8 to attract further effector cells to the site of infection.

In conclusion, our results clearly indicate that A. fumigatus antigens induce the activation of immature DCs and granulocytes. According to our results, this activation is mediated via TLR2 and TLR4. Future studies are needed to assess the clinical impact of these findings in patients at high risk for IA, such as recipients of an allogeneic SCT.

References

  1. Top of page
  2. Summary
  3. Material and methods
  4. Reagents, antibodies and plasmids
  5. Aspergillus fumigatus antigens
  6. Preparation and culture of human cells
  7. Generation of mouse DCs
  8. Phagocytosis
  9. Detection of human and mouse cytokines
  10. Quantifiable reverse transcription polymerase chain reaction (RT-PCR)
  11. Statistics
  12. Results
  13. Aspergillus fumigatus antigens activate human immature monocyte-derived DCs
  14. Aspergillus antigens stimulate effector functions of human neutrophils
  15. Aspergillus fumigatus mediates DC activation via TLR2 and TLR4
  16. Discussion
  17. Acknowledgments
  18. References
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