| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
5
1 Department of Hematology and Oncology, Medizinische Klinik II, University of Würzburg, Würzburg, Germany
2 Department of Hematology and Oncology, Tübingen University Hospital, Tübingen, Germany
3 Interdisciplinary Centre for Clinical Research (IZKF) Microarray Facility, University of Tübingen, Tübingen, Germany
4 Institute for Medical Microbiology, Tübingen University Hospital, Tübingen, Germany
5 Department of Immunology, Institute for Cell Biology, University of Tübingen, Tübingen, Germany
6 Staufer-Klinik, Innere Medizin, Schwäbisch Gmünd, Germany
Correspondence
Juergen Loeffler
loeffler_j{at}klinik.uni-wuerzburg.de
Received May 8, 2008
Accepted September 18, 2008
Monocytes play a major role in the cellular defence against Aspergillus fumigatus in immunocompromised patients. To obtain a better understanding of the mechanisms involved in this interaction, phagocytosis and gene expression profiling of human monocytes was carried out after incubation with A. fumigatus resting, swollen and germinating conidia and hyphae (for 3, 6 and 9 h). The majority of monocytes phagocytosed up to three conidia during the first 3 h of incubation. Microarray analysis showed an increased expression level of immune-relevant genes, which was dependent on the germination state of the fungus and the incubation period. Among these genes, those encoding interleukin-8, macrophage inflammatory protein 3-
(CCL20) and monocyte chemotactic protein-1 (CCL2) were found to be potential key regulators involved in the A. fumigatus-induced immune response. In addition, A. fumigatus was found to be an inducer of the genes encoding urokinase type plasminogen activator (uPA), urokinase type plasminogen activator receptor (uPAR),plasminogen activator inhibitor (PAI), pentraxin-3 (PTX3) and intercellular adhesion molecule-1 (ICAM-1), which, in combination, may contribute to thrombosis and local lung tissue injury.
Abbreviations: IA, invasive aspergillosis; IL, interleukin; SLR, signal log2 ratio; TLR, Toll-like receptor; TNF, tumour necrosis factor.
These authors contributed equally to this work. 
 |
INTRODUCTION |
|---|
 TOP INTRODUCTION METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSReferences |
|---|
Activation of innate immune cells is crucial for successful control of IA. Phagocytes play an important role in the cellular defence against this fungus by attacking the fungus itself, as well as by the release of pro- and anti-inflammatory cytokines and chemokines (Liu & Pope, 2004).
Alveolar macrophages are the main pulmonary phagocytic cells and are thus the first line of defence against A. fumigatus. Inhaled A. fumigatus conidia can reach the alveoli where they germinate and subsequently invade blood vessels (Latge, 1999, 2001). At this stage of infection, Aspergillus germinates, growing small germ tubes (germinating conidia), followed by the generation of hyphal fragments. Immune cells of the peripheral blood, including polymorphonuclear neutrophils, dendritic cells and monocytes, attack A. fumigatus germlings and hyphae by phagocytosis, dragging or mere touching, thereby preventing further spread into other tissues (Behnsen et al., 2007). After internalization of germlings and upon direct cellular contact with hyphae, different immune-relevant genes are activated.
Antigen-presenting cells, such as monocytes, recognize pathogens using receptors including Toll-like receptors (TLRs) (Blander & Medzhitov, 2004; Wang et al., 2001), pentraxin-3 (PTX3) (Alles et al., 1994; Vouret-Craviari et al., 1997) and C-type lectins. PTX3 has been shown to bind microbial agents, including A. fumigatus conidia (Garlanda et al., 2002), to facilitate phagocytosis. Extensive analysis of the role of inflammatory cytokines in cellular host defence, such as tumour necrosis factor alpha (TNF-) (Frankenberger et al., 1996; Roilides et al., 1998), interleukin (IL)-6, IL-1 (Stordeur et al., 2003) and IL-8 has been carried out in other studies. Chemokines, such as CCL2 (monocyte chemotactic protein-1, MCP1) and CCL20 (MIP-3), have been shown to be significant contributors to chemotaxis (Allavena et al., 1994; Loetscher et al., 1994). The inflammatory response is accompanied by the regulation of a broad range of genes related to cell adhesion, apoptosis, transcription factors and heat-shock proteins.
In order to get a broader understanding of the genes involved in the host response to A. fumigatus, we analysed gene expression profiles in monocytes following incubation with A. fumigatus resting and germinating conidia, and ethanol-inactivated hyphae, in a time kinetic study. Analysis was performed by genome-wide expression profiling, as well as by real-time PCR and ELISA assays. In parallel, microscopic analysis of the phagocytosis of conidia was performed.
|
METHODS |
|---|
|
TOPINTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSReferences |
|---|
Preparation of A. fumigatus isolates. A. fumigatus isolated from a patient suffering from IA (Institute of Medical Microbiology, Tübingen University Hospital) was cultivated for 5–7 days on Sabouraud 2 % glucose agar (Merck) at room temperature. Conidia were harvested by washing the surface of the agar with PBS containing 0.01 % Tween (Merck), and were washed with RPMI 1640 and counted in a Neubauer counting chamber. Conidia were then resuspended in RPMI 1640 and a defined number of monocytes was added. After 3 h of incubation, conidia were mostly still in a resting state, whereas after 6 h, conidia were swollen and germination had started. After 9 h of incubation, the presence of germ tubes could be observed in all specimens.
A. fumigatus hyphae were prepared after culturing conidia for 18 h in 1 ml yeast nitrogen base (Merck), as reported by Denning (1996). Hyphae were inactivated in a 70 % ethanol/PBS (Merck) solution for 24 h at 4 °C, washed twice with PBS and stored at –80 °C until further use. The efficacy of inactivation was controlled by cultivating hyphae on Sabouraud 2 % glucose agar plates.
Phagocytosis assays. For phagocytosis assays, monocytes were labelled with a mouse anti-CD14 mAb, followed by goat anti-mouse Cy3-labelled antibody (both kindly provided by Christian Sinzger, Virology Institute, University of Tübingen) and incubated with germinating conidia at a ratio of 1 : 2 at 37 °C for 6 h. Two strategies were followed: (i) A. fumigatus conidia were harvested, washed and then stained with Fungi-Fluor dye (Polysciences) for 1 min, washed twice with RPMI 1640 and immediately incubated with the monocytes; or (ii) conidia were stained with Fungi-Fluor after 6 h of incubation with monocytes (using this approach, only non-phagocytosed conidia were stained). Phagocytosis rates were assessed by fluorescence microscopy.
Incubation assays and RNA isolation. For time-dependent gene expression analysis, monocytes from healthy donors (n=3) were incubated with different maturation forms of A. fumigatus: resting conidia, swollen and germinating conidia, and hyphae (for 3, 6 and 9 h). Briefly, 107 monocytes were incubated with 2x107 viable conidia or 106 hyphae at 37 °C and 5–10 % CO2.
RNA was isolated directly using an RNeasy mini kit and QiaShredder spin columns (Qiagen), according to the manufacturer's protocols. RNA concentration was measured by standard UV photometry (with a biophotometer; Eppendorf); an aliquot of RNA was taken for cDNA synthesis and the rest was frozen at –80 °C until further use.
Microarray analysis. For genome-wide expression analysis, microarray experiments were performed for every period of incubation and donor, using the HG-U133A array (Affymetrix). Briefly, double-stranded cDNA was synthesized from 5 µg total RNA with a SuperScript Choice kit (Invitrogen). cRNA was prepared by in vitro transcription (Enzo Biochemical) and labelled with biotin. Fragmentation followed by incubation at 94 °C for 35 min in the presence of 40 mM Tris/acetate (pH 8.1), 100 mM KOAc and 30 mM MgOAc. Hybridization was performed with 15 µg cRNA for 16 h at 45 °C. The arrays were then washed automatically, stained with streptavidin–phycoerythrin and scanned with a Genechip System confocal scanner (Agilent Technologies). Affymetrix Microarray Suite software (version 5.0), MicroDB, GCOS and the Data Mining Tool were used for analysis and evaluation of the arrays. Cluster analysis was performed using Genesis 1.3.0 (Institute for Bioinformatics), after determination of the mean values of the signals of the different incubation experiments. Functional analysis was performed using PathwayAssist software (Ariadne Genomics). For evaluation of up- or downregulated genes, as well as for cluster and pathway analysis, we set the baseline at signal log2 ratios (SLRs) of <–1 and >1.
Real-time RT-PCR. In order to validate stimulation of monocytes prior to genome-wide expression profiling and to confirm differentially expressed genes detected by the arrays, a variety of real-time RT-PCR assays was established on a LightCycler (Roche). cDNA was synthesized using an AMV First-Strand cDNA synthesis kit (Roche). Amplification and quantification were performed using a standard LightCycler protocol (Roche). Primer and probes were supplied by TIB MOLBIOL. External cDNA standards (from 106 to 103 copies) were used for quantification. Normalization of samples was performed using the LightCycler h-ALAS housekeeping gene set (Roche).
ELISA. To confirm differentially regulated genes at the protein level, quantitative determination of cytokine and chemokine concentrations (IL-8, CCL2 and CCL20) in the supernatants was performed using Quantikine immunoassay kits (R&D Systems).
|
RESULTS |
|---|
|
TOPINTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSReferences |
|---|
) and after (Fig. 1d, e, f) phagocytosis, phagocytosed and non-phagocytosed conidia could be differentiated. By counting monocytes (n=100) in three independent experiments, the following mean values were obtained: 40 monocytes phagocytosed two conidia, 23 phagocytosed three, 16 phagocytosed one, 10 phagocytosed four or more and 11 phagocytosed none. Phagocytosed conidia were found to swell over time (Fig. 1d), and monocytes changed their shape accordingly.
|
). Expression level varied depending on the incubation time and the morphological state of the fungus, with SLRs ranging from –3.7 to +4.7 for conidia, and from –4.1 to +3.5 for hyphae. Analysis revealed a wide range of differentially regulated immune-response genes including cytokines, chemokines and TLRs, as well as genes encoding signal-transduction and adhesion molecules.
|
), uptake of resting conidia did not induce expression of cytokines or chemokines as demonstrated by microarray (Table 2) and real-time PCR analysis (Fig. 2). In contrast, genes encoding adhesion and surface molecules were differentially regulated (Table 2). In contrast to resting conidia, hyphae induced the expression of genes encoding cytokines and chemokines such as TNF-, IL-1, IL-1β, IL-6, IL-10 and CCL7 by 3 h of incubation (SLRs of 1–1.8).
|
|
) after incubation with swollen and germinating conidia, but no differential expression after incubation with inactivated hyphae.
A. fumigatus germinating conidia and hyphae both stimulated cytokine and chemokine gene expression (Tables 2 and 3). Expression of the genes encoding IL-6, IL-8, IL-10 and IL-1β was upregulated up to 280-fold. In addition, various chemokines such as CCL2, CCL7 and CCL20 and members of the GRO family were found to be upregulated. Germinating conidia selectively upregulated expression of the genes encoding CSF1, TGF-β1, TNF- and CCL20.
|
). In addition, protein levels of CCL20, CCL2 and IL-8 were analysed and showed increased concentrations in culture supernatants after 6 and 9 h of incubation (Fig. 3).
|
).
|
Differential regulation of uPA, uPAR and PAI, as well as ICAM-1, encoding genes after incubation with germinating conidia and hyphae
We could demonstrate upregulation of the genes encoding urokinase type plasminogen activator (uPA; SLR 1.7) and intercellular adhesion molecule-1 (ICAM-1; SLR 1.4) by gene array analyses. Due to their potential involvement in local inflammation and haemorrhage during IA, we further analysed the expression of genes encoding ICAM-1, uPA, the uPA receptor (uPAR) and the uPA receptor inhibitor (PAI) by real-time PCR. Interestingly, only conidia led to a notable upregulation of the ICAM-1-encoding gene (23-fold after 3 h, 3-fold after 6 h, 3-fold after 9 h), whereas hyphae did not induce expression.
In parallel, conidia were able to augment levels of the genes encoding uPA, uPAR and PAI after 3, 6 and 9 h of co-cultivation (Fig. 5). Hyphae led to reduced gene expression for uPA, unchanged gene expression for uPAR and increased gene expression for PAI (Fig. 5). All analyses were performed with monocytes isolated from three to six independent blood donors.
|
|
DISCUSSION |
|---|
|
TOPINTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSReferences |
|---|
For a broader understanding of the defence strategies of peripheral monocytes against A. fumigatus, we compared data from interaction analyses of human monocytes with resting conidia (not present in physiopathological conditions), germlings and hyphae, by performing genome-wide expression profiling. Gene expression patterns were correlated with phagocytosis of conidia in a time-dependent manner.
The majority of monocytes were found to phagocytose up to three conidia within the first 3 h. The number of phagocytosed conidia remained unaltered after 6 h. However, conidia became swollen, and monocytes changed their shape accordingly. Conidia could partly escape phagocytosis, and non-phagocytosed conidia germinated to hyphae and overgrew the monocytes after 9 h.
Despite immediate phagocytosis of resting conidia, no differential regulation of genes encoding cytokines and chemokines could be observed. In contrast, hyphae induced a strong differential expression of immune-relevant genes after 3 h of incubation. Thus, we concluded that resting conidia are only weak stimuli for monocytes, whereas germinating conidia and hyphae are able to induce a strong immune response. Similar results have been shown previously for the interaction of endothelial cells with Pseudomonas aeruginosa (Grassme et al., 2003) where an inflammatory response could be detected in the later stages of phagocytosis only. Notably, preliminary experiments revealed that inactivation of A. fumigatus with 70 % ethanol did not significantly change the immune response of monocytes compared with living fungus.
Cortez et al. (2006) published their observations analysing the functional genomics of monocytes in response to A. fumigatus conidia. However, their data were limited to a maximum of 6 h of co-cultivation of conidia with monocytes, whereas our work was extended to 9 h (germinating conidia). As demonstrated, gene expression profiles changed significantly during the time-course experiments. Furthermore, we performed additional analysis of the interaction of monocytes with inactivated hyphae. Differential recognition of conidia and hyphae can skew downstream adaptive immune responses. This aspect is of special interest, even if the total number of regulated genes did not significantly vary between 6 and 9 h (418 genes vs 399 genes).
Recently, Rodland et al. (2008) presented microarray data exploring gene expression in human monocytes infected with A. fumigatus conidia. However, again, their study was limited to an incubation time of 6 h, using a mixed population of conidial morphotypes; they reported that, after 5 h of incubation, more than 50 % of the conidia were swollen, with diameters >3.3 µm.
Numerous genes involved in immune defence mechanisms were upregulated after 9 h of co-cultivation only. Among these were various chemokine genes, such as those encoding CCL7 and CCL20. CCL7 is involved in recruitment of macrophages to sites of inflammation. CCL20 shows activity towards neutrophils, monocytes and naive T lymphocytes and mobilizes intracellular calcium. In parallel, CCL20 was also found to be upregulated when A. fumigatus germinating conidia were co-cultivated with human dendritic cells (Mezger et al., 2008), but not when cells were co-cultivated with Streptococcus agalactiae (C. Beyrich, personal communication).
Expression of the genes encoding CCL7 and CCL20 is regulated by mediators of inflammation, such as TNF-. We observed upregulation of genes encoding TNF- and other proteins involved in the TNF- pathway, such as TNFSF3 and TNFSF15. TNF- is one of the key mediators involved in pro-inflammatory responses to bacteria and fungi. TNFSF15 is involved in endothelial-cell apoptosis via activation of the stress protein kinases SAPK/JNK and p38 MAPK, as well as caspase-3.
Interestingly, germinating conidia led to upregulation of expression of the CCL2-encoding gene (a chemokine that induces the recruitment of monocytes and neutrophils to infection sites) and, in parallel, to a downregulation of expression of the CCR2-encoding gene. Recent studies have described the significant role of CCR2 in clearance of A. fumigatus conidia from the airways of mice. CCR2–/– mice showed reduced neutrophil recruitment into the airways, as well as an increase in inflammation and subepithelial fibrosis (Blease et al., 2000). Lundien et al. (2002) demonstrated that CCR2 expression is inhibited in bronchial epithelial cells when incubated with LPS, a method that may be used by bacteria to promote cell infection. Similarly, A. fumigatus germinating conidia were able to inhibit expression of the CCR2-encoding gene in monocytes, whereas hyphae obviously lost the ability to inhibit this expression.
In parallel to the data of Cortez et al. (2006), we revealed strong upregulation of IL-8, which is another chemokine that enhances the phagocytosis of A. fumigatus by polymorphonuclear neutrophils. Indeed, induction of cytokine production in monocytes appeared to be strongly dependent on fungal elements, on the cell population and on the incubation time. In a recent study, Simitsopoulou et al.(2007) analysed the interaction of the THP-1 monocytic cell line with hyphal fragments of A. fumigatus for 20 h. In contrast to our findings, using freshly isolated monocytes from peripheral blood and shorter co-incubation periods (3–9 h), they described significant downregulation of genes encoding CCL2 and CCL20, whereas other genes, such as that encoding CCL5, showed similar results (upregulation) in both studies. The study of Simitsopoulou et al. (2007) also showed that the antifungal drug voriconazole strongly induced the expression of immunomodulatory genes in THP-1 cells, compared with hyphal fragments alone.
Activation of immune defence mechanisms by A. fumigatus is dependent on so-called pattern-recognition receptors, such as the mannose receptor, integrins and C-type lectins, as well as TLRs and PTX3. Although the interaction of TLR2 and TLR4 with A. fumigatus has been reported previously in numerous studies, we found no significant change in expression of genes encoding TLR2 and TLR4 during the 3, 6 and 9 h incubation periods. Changes in the expression of genes encoding TLR2 and TLR4 most likely occur following longer exposure to the organism, as also suggested by Cortez et al. (2006) in their studies with conidia only.
PTX3 is a lectin-like molecule that plays an essential role in antigen recognition and defence against conidia of A. fumigatus, which are opsonized by PTX3 (Garlanda et al., 2005). PTX3 production is induced by primary inflammatory signals and microbial moieties. Our data demonstrated that PTX3-encoding gene expression in monocytes was upregulated after 1 h of incubation by germinating conidia. Thus, PTX3 may act as a promoter of phagocytosis of conidia. These findings are in line with reports that describe the role of PTX3 in supporting phagocytosis of A. fumigatus conidia and in activating an antifungal cellular response (Garlanda et al., 2002, 2005; Gaziano et al., 2004).
Localized lung tissue damage and local thrombosis of the lung are major complications after allogenic stem-cell transplantation. They occur in 25–55 % of transplant patients and can account substantially for transplant-related mortality (Clark et al., 1999; Weiner et al., 1986). Our microarray data demonstrated that distinct mechanisms in monocytes, regulated by A. fumigatus, could be responsible for this observation. Augmented production of the chemokines CCL2, IL-8 and CXCL6 may contribute to morphological tissue damage in lungs and to local pulmonary haemorrhage. Furthermore, we found an increase in the expression of genes encoding uPA, uPAR and PAI after stimulation for 3 h with germinating conidia. uPA binds to its receptor (uPAR), resulting in the conversion of plasminogen to plasmin, which acts as a chemoattractant for monocytes (Syrovets et al., 1997). In this context, plasmin induces expression of inflammatory cytokines and chemokines at sites of inflammation (Burysek et al., 2002; Syrovets et al., 2001; Weide et al., 1996). Recently, it has also been demonstrated that plasminogen binds directly to A. fumigatus in a dose-dependent manner, influencing the pathogenesis of invasive fungal infections (Zaas et al., 2008).
In contrast, hyphae led to decreased gene expression for uPA and increased gene expression for PAI. Impaired levels of uPA and augmented levels of PAI result in progression of thrombosis and fibrosis, and in suppression of fibrinolysis. In parallel, higher expression of the PTX3-encoding gene after incubation with A. fumigatus hyphae may also increase coagulation, as PTX3 increases tissue factor expression in activated monocytes, demonstrating an additional link between inflammation and thrombogenesis (Napoleone et al., 2004). Additionally, we revealed increased expression levels of the gene encoding ICAM-1. ICAM-1 is a major adhesion molecule expressed on lymphocytes, and is also a critical accessory molecule for T cells. Increased levels of ICAM-1 result in oedema and haemorrhage. Furthermore, ICAM-1 leads to augmented infiltration of neutrophils and mast cells, which can be responsible for neutrophil-mediated local tissue injury (Westlin & Gimbrone, 1993; Yanaba et al., 2003). Taking these observations together, we presume that, among other factors, reactions triggered by monocytes may partly explain local thrombosis and local tissue damage during IA.
In summary, gene expression profiling of monocytes co-cultivated with A. fumigatus resting and swollen conidia and germlings, as well as hyphae, in a kinetic profile extends our knowledge of the host–pathogen interaction of this clinically relevant mould. Our data indicate that monocytes, along with other immune-effector cells, such as neutrophils and macrophages, play a major role in innate immune defence by inducing an inflammatory response to A. fumigatus conidia and hyphae. Production of different chemokines, adhesion molecules and immune receptors may contribute to local thrombosis and lung tissue injury.
|
ACKNOWLEDGEMENTS |
|---|
|
TOPINTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSReferences |
|---|
|
References |
|---|
|
TOPINTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSReferences |
|---|
B activation. Blood 97, 3941–3950.
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| INT J SYST EVOL MICROBIOL | J MED MICROBIOL | MICROBIOLOGY | J GEN VIROL | ALL SGM JOURNALS |
