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Polymorphism of Candida albicans is a major factor in the interaction with human dendritic cells
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International Journal of Medical Microbiology 295 (2005) 121–127 www.elsevier.de/ijmm
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Polymorphism of Candida albicans is a major factor in the interaction with human dendritic cells Oliver Kurzaia, Corinna Schmitta, Eva-B. Bro¨ckerb, Matthias Froscha, Annette Kolb-Ma¨urerb, a
Institute of Hygiene and Microbiology, University of Wu¨rzburg, Wu¨rzburg, Germany Department of Dermatology, University of Wu¨rzburg, Josef-Schneider-Str. 2, D-97080 Wu¨rzburg, Germany
b
Received 19 November 2004; received in revised form 24 January 2005; accepted 3 February 2005
Abstract Morphological plasticity of Candida albicans is a major virulence factor. Using pH-dependent dimorphism we show, that human dendritic cells (DC) recognize filamentous forms and blastoconidia of a virulent C. albicans isolate (strain SC5314). Heat inactivated and viable blastoconidia are rapidly phagocytosed by human DC. However, viable yeast cells start to filament inside the DC at later stages of infection, leading to penetration and loss of cellular integrity. The cytokine burst of human DC induced upon contact with Candida is dominated by the granulocyte-activating, chemotactic factor IL-8 and the proinflammatory mediator TNF-a: Blastoconidia induce markedly lower cytokine levels than filamentous forms. Whereas IL-8 secretion is mainly cell mass dependent, release of TNF-a; a major proinflammatory cytokine, is clearly dependent on the morphology of Candida. r 2005 Elsevier GmbH. All rights reserved. Keywords: Candida; Dendritic cell; Polymorphism; Cytokine; TNF-a
Introduction Although fungal infections caused by rare species of low pathogenic potential become more and more important, Candida albicans is still the major problem in medical mycology as a cause of infections in immunocompromised patients (Singh, 2001). Between 1980 and 1990 a doubling in the rate of nosocomial fungal infections has been documented in the United States, and C. albicans is now the fourth most commonly isolated pathogen from bloodstream infections (Singh, 2001). From an immunological point of Corresponding author. Tel.: +49 931 2012 6710; fax: +49 931 2012 6700. E-mail address: [email protected] (A. Kolb-Ma¨urer).
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view, the T-cell-mediated immunity is crucial for protection against invasive candidiasis (Romani, 1999). Extensive studies in the mouse model revealed that a Th1 response is protective in systemic candidiasis, whereas a Th2-balanced reaction is not (Romani, 1999). Antigen-presenting cells of the innate immune system are the essential mediators for the induction of Th1 CD4 T-cells. Dendritic cells (DC) are the most potent antigen-presenting cells, with an extraordinary capacity to initiate and direct an immune response (Banchereau and Steinman, 1998; Rescigno and Borrow, 2001; Kolb-Ma¨urer et al., 2003). These cells have been shown to play a major role in the regulation of a T-cell response towards either Th1 or Th2. This effect is likely to have a major impact on the outcome of infection in systemic candidiasis (Banchereau and Steinman, 1998; Liu, 2001; Claudia et al., 2002).
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Both hyphae and yeast cells of C. albicans are recognized by murine DC (Fe` d’Ostiani et al., 2000). However, whereas blastoconidia induced a protective Th1 response, hyphae inhibited IL-12 secretion and Th1 priming in these cells (Fe` d’Ostiani et al., 2000; Romani et al., 2002). Cytokine secretion of human monocytes was similarly found to depend on the morphotype (Chiani et al., 2000; Torosantucci et al., 2000). Filamentous forms of C. albicans induced significantly lower levels of chemotactic factors like MIP1-alpha, MIP1-beta, IL-8 and MCP-1, probably due to different levels of beta-1,6-glucan in the cell wall (Torosantucci et al., 2000). Furthermore, the differentiation of human monocytes into DC has been shown to be impaired after infection of the monocytes with C. albicans, and infected monocytes are unable to secrete IL-12, a major Th1 promoting cytokine (Torosantucci et al., 2004). Germ-tube-infected monocytes also exhibited functional deficits in this study. Human DC also recognize and phagocytose C. albicans (Rittig et al., 1998; Newman and Holly, 2001). This interaction leads to efficient processing of the fungi and induction of a Th1 promoting mature DC both for yeast and germ-tubes of C. albicans. However, most studies published so far about the influence of C. albicans morphology on the interaction with immune cells are hampered by the fact, that avirulent strains or mutants have been used to grow yeast forms, whereas virulent strains gave rise to the respective filamentous forms (Chiani et al., 2000; Fe` d’Ostiani et al., 2000; Liu et al., 2001; Phan et al., 2000) (problem reviewed in Rooney and Klein, 2002). Only recently, Romagnoli et al. (2004) have employed different culture conditions prior to the infection experiments for generation of blastoconidia and filaments of a single strain. In their study, the authors could not show a difference in IL12 secretion by DC pulsed with either hyphae, germ-tubes or blastoconidia of C. albicans (Romagnoli et al., 2004). In the study presented here, pH- and temperature-dependent dimorphism is used for assessing the influence of different morphotypes on the interaction of a virulent C. albicans strain with human DC. pH-dependent dimorphism is a major characteristic of C. albicans shared only by the closely related fungal pathogen Candida dubliniensis (Kurzai et al., 2000; reviewed in Kurzai et al., 2001). Signal transduction via the central transcriptional regulator RIM101 enables C. albicans to sense ambient pH and respond by morphological changes: 37 1C and neutral to alkaline pH promote filamentous growth, whereas 20 1C and acidic pH promote growth in the yeast form (Kurzai et al., 1999; El Barkani et al., 2000; Heinz et al., 2000). Using different culture conditions prior to infection, this characteristic of virulent C. albicans strains can be exploited to analyze the role of fungal morphology in the interaction with human DC.
Materials and methods Yeast strains and culture conditions C. albicans SC5314 (Gillum et al., 1984) and C. dubliniensis NCPF3949 (Sullivan et al., 1995) were grown on Sabouraud-Dextrose agar (SDA) at 37 1C overnight before infection experiments. For induction of pH-dependent dimorphism, both strains were incubated in M199 medium (Gibco BRL, Karlsruhe, Germany) (pH 4, 28 1C) and in M199 medium (pH 8, 37 1C). After 24 h fungi had obtained a filamentous morphotype at pH 8 and 37 1C, whereas fungi had remained in the yeast form at pH 4 and 28 1C. Prior to infection experiments, fungi were harvested and washed in RPMI to remove residual M199 medium. For experiments with inactivated fungi, yeasts or filamentous forms (germ-tubes and pseudohyphae) were resuspended in PBS and heated to 68 1C for 40 min. Complete inactivation was confirmed by plating an aliquot on SDA and incubation at 37 1C for 24 h.
Dendritic cells and infection experiments Immature DC were generated from peripheral blood monocytes of healthy human volunteers as previously described in detail (Kolb-Ma¨urer et al., 2001). For infection experiments, DC were harvested on day 7 and washed twice in RPMI 1640 with 2 mM L-glutamine. Cell density was then adjusted to 106 per ml and 500 ml of the cell suspension was transferred in one well of a 24well plate. Fungal cell density was adjusted using a hemocytometer to a multiplicity of infection (MOI) of 1, unless indicated otherwise. Aggregates formed by the filamentous forms of C. albicans and C. dubliniensis were separated by repeatedly aspirating the suspension with a 26G needle before counting. Purity of DC in FACS analyses was higher than 90%. The primary antibodies used were: CD1a (OKT6, Rockville, MD), a-HLA class II DR/DQ (9.3F10) (American Type Culture Collection, Manassas, VA), CD83 (clone HB15e, BD Pharmingen, Heidelberg, Germany), and CD86 (clone 2331(FUN-1), BD Pharmingen). The stained cells were analyzed on an EPICS XL-MCL (Coulter Immunotech Diagnostics, Krefeld, Germany).
Microscopy For microscopy, sterile glass coverslips were placed in the respective well of a 24-well microtitre plate prior to addition of the immature DC. Infection was performed exactly as described earlier. After the indicated time, glass coverslips were removed and washed in pre-warmed PBS. For light microscopy, specimens were fixed in methanol for 5 min and stained with Giemsa.
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For transmission electron microscopy (TEM), cells were washed, fixed in 2.5% glutaraldehyde, post-fixed in 2% osmium tetroxide and stained with 0.5% uranyl acetate. Cells were dehydrated in graded alcohols and embedded in Lowicryl K4M 812 overnight. Electron microscopy was performed with Zeiss EM900 and EM10 microscopes. FITC labeling of heat-inactivated fungi was achieved by incubating the fungal cells in a FITC/PBS solution (0.01 mg/ml) for 30 min and subsequent washing (4 ) in PBS prior to infection. Quenching of extracellular fungi was achieved by adding 1 mg/ml trypan blue at the end of the incubation period for 15 min at room temperature as described previously (Newman and Holly, 2001). For immunofluorescence, infection experiments were preformed on sterile glass coverslips essentially as described above. Cells were fixed in 3.7% formaldehyde (15 min at room temperature) and subsequently permeabilized with 1% Triton X-100 (15 min, room temperature). For staining of C. albicans, polyclonal antiserum (BioDesign International, obtained from Dunn Laboratories, Asbach, Germany; concentration 1:1000) was used. The actin cytoskeleton was stained using ALEXA-Phalloidin (Mobitec, Go¨ttingen, Germany) according to the manufacturer’s instructions for 15 min. Stained specimens were mounted in fluoprep (BioMerieux, Nu¨rtingen, Germany) and examined using a Zeiss LSM 510 confocal microscope.
Cytokine assays Supernatants of infection experiments were collected at the indicated time and immediately stored at 80 1C. Cytokine concentrations in the supernatants were measured with the Luminex technology (obtained from BioSource, Nivelles, Belgium) using the human inflammatory five-plex kit (GM-CSF, TNF-a; IL-6, IL-8, IL1b) and beads for IL-12p40, IL-10 and IFN-g (BioSource). Supernatants were diluted 1:10 in sterile water and 50 ml of the dilution were used in the assay according to the manufacturer’s instructions. Data were evaluated with the MasterPlex QT software (MiraiBio, Alameda, USA). Alternatively, standard ELISA assays were used to determine TNF-a; IL-6, IL-8, IL-1b; IFN-g (ImmunoKontact, AMS Biotech, Wiesbaden, Germany) and IL-10, IL-12p70 (Biocarta, Hamburg, Germany). Data gained with the Luminex technology correlated well with data from standard ELISA systems, as indicated by the manufacturer.
Results and discussion Localization of C. albicans during the infection of human DC was monitored microscopically. Heat-
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inactivated (65 1C, 30 min) blastoconidia of C. albicans were rapidly associated with DC and appeared to be localized mainly inside DC after 30–45 min. In contrast, heat-inactivated filaments of C. albicans mainly remained outside the cells after this period. To confirm phagocytosis of inactivated C. albicans blastoconidia, DC were infected with FITC-labeled yeasts (Fig. 1a). After 30 min of infection, fluorescence of extracellular yeast cells was quenched by addition of trypan blue and intracellular yeasts were visualized using immunofluorescence microscopy. At this time, the majority of yeast cells had been ingested by DC (Fig. 1a). To test whether viable C. albicans blastoconidia of a virulent strain were also phagocytosed rapidly, DC were infected with yeast cells of strain SC5314. Viable C. albicans blastoconidia were internalized by DC in the same period of time as observed for heat-inactivated yeasts (Fig. 1b). However, after 30–45 min the viable blastoconidia started to filament inside the cells and short germ-tubes could be observed microscopically (Fig. 1b). At a later time (90–120 min) after infection most C. albicans cells displayed a filamentous phenotype and were at least partially located extracellularly (Fig. 1b). To test whether filamentation during the infection could be induced by the medium alone, yeast cells of SC5314 were inoculated to RPMI 1640 medium at 37 1C (5% CO2). Filamentous forms were observed after 1 h, indicating that these conditions are sufficient to induce filamentation of C. albicans. Therefore unequivocal localization of the fungi was necessary. This was achieved by TEM. Phagocytosis as observed by TEM was of typical morphology and blastoconidia clearly resided inside the DC (Fig. 1c). Filamention of C. albicans resulted in penetration of one or even several DC 2 h post-infection. (Fig. 1c). In immunofluorescence microscopy, penetration of the filament through DC was found to induce significant reorganization of the actin cytoskeleton, leading to the formation of actin collars around the fungi, most likely resembling a protective mechanism of the DC (Fig. 1d). However, after prolonged incubation for 20 h no intact DCs could be observed, and C. albicans had totally converted to a filamentous morphology. Infection experiments using viable C. albicans filaments also resulted in complete destruction of the DC after prolonged incubation (20 h). Pseudohyphae, which could not be completely ingested by DC due to their size, penetrated DC and induced actin collars identical to those observed for intracellularly germinating yeast cells. Contact of human DC with either blastoconidia or germ-tubes of C. albicans resulted in phenotypic maturation of the DC as indicated by upregulation of CD83, CD86 and MHC II as has been shown previously by other authors (data not shown). Another important surrogate for DC maturation is secretion of cytokines. To assess this, concentrations of GM-CSF, TNF-a;
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Fig. 1. Morphology of DC–Candida interaction. (a) Heat-inactivated yeast cells of the virulent C. albicans strain SC5314 are rapidly ingested by human DC. Yeast cells were labeled with FITC, and fluorescence of extracellular fungi was quenched by addition of trypan blue. At 30 min post-infection, the majority of inactivated yeast cells have been phagocytosed. (b) Light microscopy (Giemsa, 1000 oil immersion). Viable yeast cells are mainly associated with DC after 30 min and appear to be phagocytosed at 60 min postinfection. Germination starts after 45–60 min. Two hours post-infection the majority of the fungi has formed germ-tubes, which penetrate the DC in the later course of infection. (c) In transmission electron microscopy, phagocytosis of viable C. albicans yeasts is of typical morphology. At later stages of infection yeast cells are clearly localized intracellularly. After 2 h of infection, germination of C. albicans leads to penetration of the DC and loss of cell integrity. (d) Filamentation of C. albicans results in massive reorganization of the actin cytoskeleton. Actin collars are formed around penetrating germ-tubes (arrows).
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IL-6, IL-8, IL-1b; IL-12p40, IL-10, and IFN-g in the supernatants were measured with the Luminex multiplex bead technology. The cytokine pattern was dominated by IL-8 and TNF-a in all experiments (Fig. 2a). In contrast, only low levels of IFN-g and IL-1b could be found. IL-6 and IL-12 levels were variable and strongly 35000
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Fig. 2. Cytokine pattern induced by yeast cells and filaments of C. albicans. (a) Cytokine concentrations in the 24-h supernatants induced by filamentous forms are higher than for yeast cells (MOI ¼ 1). Arithmetic means7standard deviation of three independent experiments are shown. IL-6 secretion was strongly donor dependent; in some experiments no IL-6 could be detected for blastoconidia and hyphae of C. albicans. (b) Influence of cell mass on secretion of IL-8. Infections were performed with blastoconidia at MOIs of 0.1, 1, 10 and 100 and with germ-tubes at MOI of 1. Arithmetic means7standard deviation of three independent experiments are shown. Secretion of IL-8 is largely dependent on the cell mass. (c) Secretion of TNF-alpha is dependent on the morphotype rather than cell mass. medium: cytokines secreted by uninfected DC. p-value was calculated using a two-tailed Student’s t-test.
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donor dependent. Generally, cytokine levels induced by Candida were significantly lower than those obtained with bacteria or bacterial lipopolysaccharide (KolbMa¨urer et al., 2001). To assess the impact of different morphotypes on cytokine secretion by human DC, cells were incubated with either yeasts or filamentous forms of C. albicans. In all experiments, IL-8 and TNF-a levels induced by filamentous forms were significantly higher than those triggered by the yeast forms when used at the same MOI (Fig. 2a). As filamentous morphotypes are larger in size than yeasts a pure mass-effect on cytokine secretion by DC had to be excluded. For that purpose DC were incubated with different MOIs of C. albicans yeast cells (0.1, 1, 10, 100) and cytokine levels were assayed in 24 h culture supernatants. IL-8 secretion induced by C. albicans blastoconidia was highly dependent on the MOI (Fig. 2b). In contrast, TNF-a levels increased only weekly when the MOI was raised from 0.1 to 100 and were far lower than those obtained with hyphae of the same strain at an MOI of 1 (Fig. 2c). Therefore TNF-a induction seems to be dependent on hyphae-specific factors, rather than cell mass. IL-12 is one of the major cytokines required to trigger a Th-1 response, which is protective in invasive Candida infections (Claudia et al., 2002). Hyphae, but not yeasts of C. albicans have been described to suppress IL-12 secretion by murine DC and human monocytes (Chiani et al., 2000; Fe` d’Ostiani et al., 2000). To test, whether this suppressive effect is also of importance for the interaction with human DC, IL-12p40 and IL-12p70 levels were measured after 24 h of incubation. Secretion of both forms was found to be highly donor dependent and no characteristic pattern could be defined for either blastoconidia or hyphae of C. albicans or C. dubliniensis. To test whether soluble factors are responsible for triggering the cytokine burst of human DC, supernatants were collected from 24-h C. albicans SC5314 yeasts and hyphae cultures. Cultures were centrifuged, supernatants were filtered (pore size 0.2 mm) and equilibrated to neutral pH. Cytokine secretion by human DC was measured after 24 h of incubation in the respective supernatants. No significant induction of IL-8, TNF-a; IL-6, IL-12p70, IFN-g or IL-1b could be found for any of the culture supernatants (data not shown). Candida dubliniensis is phylogenetically closely related to C. albicans. Both species share the ability to respond to environmental changes with a switch between yeast form and filamentous growth forms. However, the pathogenic potential of C. dubliniensis is lower than that of C. albicans. To test whether C. dubliniensis also induces a morphotype-dependent pattern of cytokine secretion, DC were infected with filamentous forms and yeasts of strain NCPF3949. Although the cytokine levels induced by C. dubliniensis were lower than those found with C. albicans, a similar difference between yeasts and filamentous forms could
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be observed (data not shown). In conclusion, human DC are capable of recognizing and phagocytosing C. albicans and C. dubliniensis. The cytokine response induced by Candida is dominated by IL-8 and TNF-a: In contrast to most bacteria, only low levels of IL-6 were induced (Kolb-Ma¨urer et al., 2001, 2003). This corresponds to the clinical experience that Candida septicemia is accompanied by a low IL-6 to IL-8 ratio, as well as to previous studies with other cell types (Steele and Fidel, 2002). In addition, some data suggest, that low IL-6 levels might favor clearance of C. albicans, whereas patients suffering from chronic Candida infections release high IL-6 levels (Lilic et al., 2003). Romani et al. described a suppressive effect of C. albicans hyphae on IL-12 secretion of murine DCs (Fe` d’Ostiani et al., 2000; Romani et al., 2002). Interestingly, we could not find a similar effect for human DC in our experiments. In contrast, levels of IL-12 secretion were largely donor dependent. Individual differences in the ability of inducing a protective immune response have been linked to defective IL-12 induction in cases of chronic mucocutaneous Candida infection (Lilic et al., 2003). Both IL-8 and TNF-a are induced to a much higher degree by filamentous forms than by blastoconidia when used at the same MOI. However, whereas this seems to merely depend on the cell mass in the case of IL-8, TNF-a induction is strongly dependent on the morphotype. Interestingly, cytokine levels induced by C. dubliniensis are considerably lower than for C. albicans – a fact that may be related to the lower pathogenic potential of the latter species. However, as only a single type strain of each species was tested in this study, further experiments are necessary to confirm this hypothesis, as intra-species variations of cytokine levels between single strains could also explain this observation. Most of the previous studies on the influence of fungal morphology in Candida–DC interaction have been hampered by the use of non-isogenic strains or by employing mutants which lack central transcriptional regulators (Rooney and Klein, 2002). By using pHdependent dimorphism for defining the morphotype of a single, virulent strain in vitro this handicap has been overcome in this study. The results obtained with filaments and yeast cells of a single virulent C. albicans isolate are compatible with the findings of Romagnoli et al. (2004), a recent study which used a different culture-based approach for generation of filaments and yeast cells of another fully virulent C. albicans strain. Taken together, these data show that morphological plasticity of C. albicans is of major importance in the interaction with human DC, but does most likely not affect the IL-12 secretion of human DC. Experimental application of environmentally regulated dimorphism will enable the use of isogenic strains for defining morphology-related effects in future.
Acknowledgements The authors are grateful to Prof. G. Krohne, C. Gehrig and D. Bunsen for help with electron microscopy, F. Hartmann for help with the luminex technology, J. Waschke for help with immunofluorescence microscopy, V. Hornich and J. O¨chsner for technical assistance, and M. Ma¨urer for critically reading the manuscript. This study was supported by a fellowship from the ‘‘Bayerischen Staatsministerium fu¨r Wissenschaft, Forschung und Kunst’’ to A. Kolb-Ma¨urer.
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International Journal of Medical Microbiology 295 (2005) 121–127 www.elsevier.de/ijmm
SHORT COMMUNICATION
Polymorphism of Candida albicans is a major factor in the interaction with human dendritic cells Oliver Kurzaia, Corinna Schmitta, Eva-B. Bro¨ckerb, Matthias Froscha, Annette Kolb-Ma¨urerb, a
Institute of Hygiene and Microbiology, University of Wu¨rzburg, Wu¨rzburg, Germany Department of Dermatology, University of Wu¨rzburg, Josef-Schneider-Str. 2, D-97080 Wu¨rzburg, Germany
b
Received 19 November 2004; received in revised form 24 January 2005; accepted 3 February 2005
Abstract Morphological plasticity of Candida albicans is a major virulence factor. Using pH-dependent dimorphism we show, that human dendritic cells (DC) recognize filamentous forms and blastoconidia of a virulent C. albicans isolate (strain SC5314). Heat inactivated and viable blastoconidia are rapidly phagocytosed by human DC. However, viable yeast cells start to filament inside the DC at later stages of infection, leading to penetration and loss of cellular integrity. The cytokine burst of human DC induced upon contact with Candida is dominated by the granulocyte-activating, chemotactic factor IL-8 and the proinflammatory mediator TNF-a: Blastoconidia induce markedly lower cytokine levels than filamentous forms. Whereas IL-8 secretion is mainly cell mass dependent, release of TNF-a; a major proinflammatory cytokine, is clearly dependent on the morphology of Candida. r 2005 Elsevier GmbH. All rights reserved. Keywords: Candida; Dendritic cell; Polymorphism; Cytokine; TNF-a
Introduction Although fungal infections caused by rare species of low pathogenic potential become more and more important, Candida albicans is still the major problem in medical mycology as a cause of infections in immunocompromised patients (Singh, 2001). Between 1980 and 1990 a doubling in the rate of nosocomial fungal infections has been documented in the United States, and C. albicans is now the fourth most commonly isolated pathogen from bloodstream infections (Singh, 2001). From an immunological point of Corresponding author. Tel.: +49 931 2012 6710; fax: +49 931 2012 6700. E-mail address: [email protected] (A. Kolb-Ma¨urer).
1438-4221/$ - see front matter r 2005 Elsevier GmbH. All rights reserved. doi:10.1016/j.ijmm.2005.02.003
view, the T-cell-mediated immunity is crucial for protection against invasive candidiasis (Romani, 1999). Extensive studies in the mouse model revealed that a Th1 response is protective in systemic candidiasis, whereas a Th2-balanced reaction is not (Romani, 1999). Antigen-presenting cells of the innate immune system are the essential mediators for the induction of Th1 CD4 T-cells. Dendritic cells (DC) are the most potent antigen-presenting cells, with an extraordinary capacity to initiate and direct an immune response (Banchereau and Steinman, 1998; Rescigno and Borrow, 2001; Kolb-Ma¨urer et al., 2003). These cells have been shown to play a major role in the regulation of a T-cell response towards either Th1 or Th2. This effect is likely to have a major impact on the outcome of infection in systemic candidiasis (Banchereau and Steinman, 1998; Liu, 2001; Claudia et al., 2002).
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Both hyphae and yeast cells of C. albicans are recognized by murine DC (Fe` d’Ostiani et al., 2000). However, whereas blastoconidia induced a protective Th1 response, hyphae inhibited IL-12 secretion and Th1 priming in these cells (Fe` d’Ostiani et al., 2000; Romani et al., 2002). Cytokine secretion of human monocytes was similarly found to depend on the morphotype (Chiani et al., 2000; Torosantucci et al., 2000). Filamentous forms of C. albicans induced significantly lower levels of chemotactic factors like MIP1-alpha, MIP1-beta, IL-8 and MCP-1, probably due to different levels of beta-1,6-glucan in the cell wall (Torosantucci et al., 2000). Furthermore, the differentiation of human monocytes into DC has been shown to be impaired after infection of the monocytes with C. albicans, and infected monocytes are unable to secrete IL-12, a major Th1 promoting cytokine (Torosantucci et al., 2004). Germ-tube-infected monocytes also exhibited functional deficits in this study. Human DC also recognize and phagocytose C. albicans (Rittig et al., 1998; Newman and Holly, 2001). This interaction leads to efficient processing of the fungi and induction of a Th1 promoting mature DC both for yeast and germ-tubes of C. albicans. However, most studies published so far about the influence of C. albicans morphology on the interaction with immune cells are hampered by the fact, that avirulent strains or mutants have been used to grow yeast forms, whereas virulent strains gave rise to the respective filamentous forms (Chiani et al., 2000; Fe` d’Ostiani et al., 2000; Liu et al., 2001; Phan et al., 2000) (problem reviewed in Rooney and Klein, 2002). Only recently, Romagnoli et al. (2004) have employed different culture conditions prior to the infection experiments for generation of blastoconidia and filaments of a single strain. In their study, the authors could not show a difference in IL12 secretion by DC pulsed with either hyphae, germ-tubes or blastoconidia of C. albicans (Romagnoli et al., 2004). In the study presented here, pH- and temperature-dependent dimorphism is used for assessing the influence of different morphotypes on the interaction of a virulent C. albicans strain with human DC. pH-dependent dimorphism is a major characteristic of C. albicans shared only by the closely related fungal pathogen Candida dubliniensis (Kurzai et al., 2000; reviewed in Kurzai et al., 2001). Signal transduction via the central transcriptional regulator RIM101 enables C. albicans to sense ambient pH and respond by morphological changes: 37 1C and neutral to alkaline pH promote filamentous growth, whereas 20 1C and acidic pH promote growth in the yeast form (Kurzai et al., 1999; El Barkani et al., 2000; Heinz et al., 2000). Using different culture conditions prior to infection, this characteristic of virulent C. albicans strains can be exploited to analyze the role of fungal morphology in the interaction with human DC.
Materials and methods Yeast strains and culture conditions C. albicans SC5314 (Gillum et al., 1984) and C. dubliniensis NCPF3949 (Sullivan et al., 1995) were grown on Sabouraud-Dextrose agar (SDA) at 37 1C overnight before infection experiments. For induction of pH-dependent dimorphism, both strains were incubated in M199 medium (Gibco BRL, Karlsruhe, Germany) (pH 4, 28 1C) and in M199 medium (pH 8, 37 1C). After 24 h fungi had obtained a filamentous morphotype at pH 8 and 37 1C, whereas fungi had remained in the yeast form at pH 4 and 28 1C. Prior to infection experiments, fungi were harvested and washed in RPMI to remove residual M199 medium. For experiments with inactivated fungi, yeasts or filamentous forms (germ-tubes and pseudohyphae) were resuspended in PBS and heated to 68 1C for 40 min. Complete inactivation was confirmed by plating an aliquot on SDA and incubation at 37 1C for 24 h.
Dendritic cells and infection experiments Immature DC were generated from peripheral blood monocytes of healthy human volunteers as previously described in detail (Kolb-Ma¨urer et al., 2001). For infection experiments, DC were harvested on day 7 and washed twice in RPMI 1640 with 2 mM L-glutamine. Cell density was then adjusted to 106 per ml and 500 ml of the cell suspension was transferred in one well of a 24well plate. Fungal cell density was adjusted using a hemocytometer to a multiplicity of infection (MOI) of 1, unless indicated otherwise. Aggregates formed by the filamentous forms of C. albicans and C. dubliniensis were separated by repeatedly aspirating the suspension with a 26G needle before counting. Purity of DC in FACS analyses was higher than 90%. The primary antibodies used were: CD1a (OKT6, Rockville, MD), a-HLA class II DR/DQ (9.3F10) (American Type Culture Collection, Manassas, VA), CD83 (clone HB15e, BD Pharmingen, Heidelberg, Germany), and CD86 (clone 2331(FUN-1), BD Pharmingen). The stained cells were analyzed on an EPICS XL-MCL (Coulter Immunotech Diagnostics, Krefeld, Germany).
Microscopy For microscopy, sterile glass coverslips were placed in the respective well of a 24-well microtitre plate prior to addition of the immature DC. Infection was performed exactly as described earlier. After the indicated time, glass coverslips were removed and washed in pre-warmed PBS. For light microscopy, specimens were fixed in methanol for 5 min and stained with Giemsa.
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For transmission electron microscopy (TEM), cells were washed, fixed in 2.5% glutaraldehyde, post-fixed in 2% osmium tetroxide and stained with 0.5% uranyl acetate. Cells were dehydrated in graded alcohols and embedded in Lowicryl K4M 812 overnight. Electron microscopy was performed with Zeiss EM900 and EM10 microscopes. FITC labeling of heat-inactivated fungi was achieved by incubating the fungal cells in a FITC/PBS solution (0.01 mg/ml) for 30 min and subsequent washing (4 ) in PBS prior to infection. Quenching of extracellular fungi was achieved by adding 1 mg/ml trypan blue at the end of the incubation period for 15 min at room temperature as described previously (Newman and Holly, 2001). For immunofluorescence, infection experiments were preformed on sterile glass coverslips essentially as described above. Cells were fixed in 3.7% formaldehyde (15 min at room temperature) and subsequently permeabilized with 1% Triton X-100 (15 min, room temperature). For staining of C. albicans, polyclonal antiserum (BioDesign International, obtained from Dunn Laboratories, Asbach, Germany; concentration 1:1000) was used. The actin cytoskeleton was stained using ALEXA-Phalloidin (Mobitec, Go¨ttingen, Germany) according to the manufacturer’s instructions for 15 min. Stained specimens were mounted in fluoprep (BioMerieux, Nu¨rtingen, Germany) and examined using a Zeiss LSM 510 confocal microscope.
Cytokine assays Supernatants of infection experiments were collected at the indicated time and immediately stored at 80 1C. Cytokine concentrations in the supernatants were measured with the Luminex technology (obtained from BioSource, Nivelles, Belgium) using the human inflammatory five-plex kit (GM-CSF, TNF-a; IL-6, IL-8, IL1b) and beads for IL-12p40, IL-10 and IFN-g (BioSource). Supernatants were diluted 1:10 in sterile water and 50 ml of the dilution were used in the assay according to the manufacturer’s instructions. Data were evaluated with the MasterPlex QT software (MiraiBio, Alameda, USA). Alternatively, standard ELISA assays were used to determine TNF-a; IL-6, IL-8, IL-1b; IFN-g (ImmunoKontact, AMS Biotech, Wiesbaden, Germany) and IL-10, IL-12p70 (Biocarta, Hamburg, Germany). Data gained with the Luminex technology correlated well with data from standard ELISA systems, as indicated by the manufacturer.
Results and discussion Localization of C. albicans during the infection of human DC was monitored microscopically. Heat-
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inactivated (65 1C, 30 min) blastoconidia of C. albicans were rapidly associated with DC and appeared to be localized mainly inside DC after 30–45 min. In contrast, heat-inactivated filaments of C. albicans mainly remained outside the cells after this period. To confirm phagocytosis of inactivated C. albicans blastoconidia, DC were infected with FITC-labeled yeasts (Fig. 1a). After 30 min of infection, fluorescence of extracellular yeast cells was quenched by addition of trypan blue and intracellular yeasts were visualized using immunofluorescence microscopy. At this time, the majority of yeast cells had been ingested by DC (Fig. 1a). To test whether viable C. albicans blastoconidia of a virulent strain were also phagocytosed rapidly, DC were infected with yeast cells of strain SC5314. Viable C. albicans blastoconidia were internalized by DC in the same period of time as observed for heat-inactivated yeasts (Fig. 1b). However, after 30–45 min the viable blastoconidia started to filament inside the cells and short germ-tubes could be observed microscopically (Fig. 1b). At a later time (90–120 min) after infection most C. albicans cells displayed a filamentous phenotype and were at least partially located extracellularly (Fig. 1b). To test whether filamentation during the infection could be induced by the medium alone, yeast cells of SC5314 were inoculated to RPMI 1640 medium at 37 1C (5% CO2). Filamentous forms were observed after 1 h, indicating that these conditions are sufficient to induce filamentation of C. albicans. Therefore unequivocal localization of the fungi was necessary. This was achieved by TEM. Phagocytosis as observed by TEM was of typical morphology and blastoconidia clearly resided inside the DC (Fig. 1c). Filamention of C. albicans resulted in penetration of one or even several DC 2 h post-infection. (Fig. 1c). In immunofluorescence microscopy, penetration of the filament through DC was found to induce significant reorganization of the actin cytoskeleton, leading to the formation of actin collars around the fungi, most likely resembling a protective mechanism of the DC (Fig. 1d). However, after prolonged incubation for 20 h no intact DCs could be observed, and C. albicans had totally converted to a filamentous morphology. Infection experiments using viable C. albicans filaments also resulted in complete destruction of the DC after prolonged incubation (20 h). Pseudohyphae, which could not be completely ingested by DC due to their size, penetrated DC and induced actin collars identical to those observed for intracellularly germinating yeast cells. Contact of human DC with either blastoconidia or germ-tubes of C. albicans resulted in phenotypic maturation of the DC as indicated by upregulation of CD83, CD86 and MHC II as has been shown previously by other authors (data not shown). Another important surrogate for DC maturation is secretion of cytokines. To assess this, concentrations of GM-CSF, TNF-a;
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Fig. 1. Morphology of DC–Candida interaction. (a) Heat-inactivated yeast cells of the virulent C. albicans strain SC5314 are rapidly ingested by human DC. Yeast cells were labeled with FITC, and fluorescence of extracellular fungi was quenched by addition of trypan blue. At 30 min post-infection, the majority of inactivated yeast cells have been phagocytosed. (b) Light microscopy (Giemsa, 1000 oil immersion). Viable yeast cells are mainly associated with DC after 30 min and appear to be phagocytosed at 60 min postinfection. Germination starts after 45–60 min. Two hours post-infection the majority of the fungi has formed germ-tubes, which penetrate the DC in the later course of infection. (c) In transmission electron microscopy, phagocytosis of viable C. albicans yeasts is of typical morphology. At later stages of infection yeast cells are clearly localized intracellularly. After 2 h of infection, germination of C. albicans leads to penetration of the DC and loss of cell integrity. (d) Filamentation of C. albicans results in massive reorganization of the actin cytoskeleton. Actin collars are formed around penetrating germ-tubes (arrows).
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IL-6, IL-8, IL-1b; IL-12p40, IL-10, and IFN-g in the supernatants were measured with the Luminex multiplex bead technology. The cytokine pattern was dominated by IL-8 and TNF-a in all experiments (Fig. 2a). In contrast, only low levels of IFN-g and IL-1b could be found. IL-6 and IL-12 levels were variable and strongly 35000
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Fig. 2. Cytokine pattern induced by yeast cells and filaments of C. albicans. (a) Cytokine concentrations in the 24-h supernatants induced by filamentous forms are higher than for yeast cells (MOI ¼ 1). Arithmetic means7standard deviation of three independent experiments are shown. IL-6 secretion was strongly donor dependent; in some experiments no IL-6 could be detected for blastoconidia and hyphae of C. albicans. (b) Influence of cell mass on secretion of IL-8. Infections were performed with blastoconidia at MOIs of 0.1, 1, 10 and 100 and with germ-tubes at MOI of 1. Arithmetic means7standard deviation of three independent experiments are shown. Secretion of IL-8 is largely dependent on the cell mass. (c) Secretion of TNF-alpha is dependent on the morphotype rather than cell mass. medium: cytokines secreted by uninfected DC. p-value was calculated using a two-tailed Student’s t-test.
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donor dependent. Generally, cytokine levels induced by Candida were significantly lower than those obtained with bacteria or bacterial lipopolysaccharide (KolbMa¨urer et al., 2001). To assess the impact of different morphotypes on cytokine secretion by human DC, cells were incubated with either yeasts or filamentous forms of C. albicans. In all experiments, IL-8 and TNF-a levels induced by filamentous forms were significantly higher than those triggered by the yeast forms when used at the same MOI (Fig. 2a). As filamentous morphotypes are larger in size than yeasts a pure mass-effect on cytokine secretion by DC had to be excluded. For that purpose DC were incubated with different MOIs of C. albicans yeast cells (0.1, 1, 10, 100) and cytokine levels were assayed in 24 h culture supernatants. IL-8 secretion induced by C. albicans blastoconidia was highly dependent on the MOI (Fig. 2b). In contrast, TNF-a levels increased only weekly when the MOI was raised from 0.1 to 100 and were far lower than those obtained with hyphae of the same strain at an MOI of 1 (Fig. 2c). Therefore TNF-a induction seems to be dependent on hyphae-specific factors, rather than cell mass. IL-12 is one of the major cytokines required to trigger a Th-1 response, which is protective in invasive Candida infections (Claudia et al., 2002). Hyphae, but not yeasts of C. albicans have been described to suppress IL-12 secretion by murine DC and human monocytes (Chiani et al., 2000; Fe` d’Ostiani et al., 2000). To test, whether this suppressive effect is also of importance for the interaction with human DC, IL-12p40 and IL-12p70 levels were measured after 24 h of incubation. Secretion of both forms was found to be highly donor dependent and no characteristic pattern could be defined for either blastoconidia or hyphae of C. albicans or C. dubliniensis. To test whether soluble factors are responsible for triggering the cytokine burst of human DC, supernatants were collected from 24-h C. albicans SC5314 yeasts and hyphae cultures. Cultures were centrifuged, supernatants were filtered (pore size 0.2 mm) and equilibrated to neutral pH. Cytokine secretion by human DC was measured after 24 h of incubation in the respective supernatants. No significant induction of IL-8, TNF-a; IL-6, IL-12p70, IFN-g or IL-1b could be found for any of the culture supernatants (data not shown). Candida dubliniensis is phylogenetically closely related to C. albicans. Both species share the ability to respond to environmental changes with a switch between yeast form and filamentous growth forms. However, the pathogenic potential of C. dubliniensis is lower than that of C. albicans. To test whether C. dubliniensis also induces a morphotype-dependent pattern of cytokine secretion, DC were infected with filamentous forms and yeasts of strain NCPF3949. Although the cytokine levels induced by C. dubliniensis were lower than those found with C. albicans, a similar difference between yeasts and filamentous forms could
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be observed (data not shown). In conclusion, human DC are capable of recognizing and phagocytosing C. albicans and C. dubliniensis. The cytokine response induced by Candida is dominated by IL-8 and TNF-a: In contrast to most bacteria, only low levels of IL-6 were induced (Kolb-Ma¨urer et al., 2001, 2003). This corresponds to the clinical experience that Candida septicemia is accompanied by a low IL-6 to IL-8 ratio, as well as to previous studies with other cell types (Steele and Fidel, 2002). In addition, some data suggest, that low IL-6 levels might favor clearance of C. albicans, whereas patients suffering from chronic Candida infections release high IL-6 levels (Lilic et al., 2003). Romani et al. described a suppressive effect of C. albicans hyphae on IL-12 secretion of murine DCs (Fe` d’Ostiani et al., 2000; Romani et al., 2002). Interestingly, we could not find a similar effect for human DC in our experiments. In contrast, levels of IL-12 secretion were largely donor dependent. Individual differences in the ability of inducing a protective immune response have been linked to defective IL-12 induction in cases of chronic mucocutaneous Candida infection (Lilic et al., 2003). Both IL-8 and TNF-a are induced to a much higher degree by filamentous forms than by blastoconidia when used at the same MOI. However, whereas this seems to merely depend on the cell mass in the case of IL-8, TNF-a induction is strongly dependent on the morphotype. Interestingly, cytokine levels induced by C. dubliniensis are considerably lower than for C. albicans – a fact that may be related to the lower pathogenic potential of the latter species. However, as only a single type strain of each species was tested in this study, further experiments are necessary to confirm this hypothesis, as intra-species variations of cytokine levels between single strains could also explain this observation. Most of the previous studies on the influence of fungal morphology in Candida–DC interaction have been hampered by the use of non-isogenic strains or by employing mutants which lack central transcriptional regulators (Rooney and Klein, 2002). By using pHdependent dimorphism for defining the morphotype of a single, virulent strain in vitro this handicap has been overcome in this study. The results obtained with filaments and yeast cells of a single virulent C. albicans isolate are compatible with the findings of Romagnoli et al. (2004), a recent study which used a different culture-based approach for generation of filaments and yeast cells of another fully virulent C. albicans strain. Taken together, these data show that morphological plasticity of C. albicans is of major importance in the interaction with human DC, but does most likely not affect the IL-12 secretion of human DC. Experimental application of environmentally regulated dimorphism will enable the use of isogenic strains for defining morphology-related effects in future.
Acknowledgements The authors are grateful to Prof. G. Krohne, C. Gehrig and D. Bunsen for help with electron microscopy, F. Hartmann for help with the luminex technology, J. Waschke for help with immunofluorescence microscopy, V. Hornich and J. O¨chsner for technical assistance, and M. Ma¨urer for critically reading the manuscript. This study was supported by a fellowship from the ‘‘Bayerischen Staatsministerium fu¨r Wissenschaft, Forschung und Kunst’’ to A. Kolb-Ma¨urer.
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