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The Journal of Immunology, 2006, 177: 4679-4687.
Copyright © 2006 by The American Association of Immunologists, Inc.

Specific Recognition of Candida albicans by Macrophages Requires Galectin-3 to Discriminate Saccharomyces cerevisiae and Needs Association with TLR2 for Signaling1

Thierry Jouault2,*, Miryam El Abed-El Behi*, María Martínez-Esparza§, Laetitia Breuilh{dagger}, Pierre-André Trinel*, Mathias Chamaillard{ddagger}, Francois Trottein{dagger} and Daniel Poulain*

* Institut National de la Santé et de la Recherche Médicale, Unité 799 and Université de Lille II, {dagger} Institut National de la Santé et de la Recherche Médicale, Unité 547, Institut Pasteur, and {ddagger} Institut National de la Santé et de la Recherche Médicale, Unité 795, Lille, France; and § Departamento de Bioquímica B e Inmunología, Facultad de Medicina, Universidad de Murcia, Murcia, Spain


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Stimulation of cells of the macrophage lineage is a crucial step in the sensing of yeasts by the immune system. Glycans present in both Candida albicans and Saccharomyces cerevisiae cell walls have been shown to act as ligands for different receptors leading to different stimulating pathways, some of which need receptor co-involvement. However, among these ligand-receptor couples, none has been shown to discriminate the pathogenic yeast C. albicans. We explored the role of galectin-3, which binds C. albicans beta-1,2 mannosides. These glycans are specifically and prominently expressed at the surface of C. albicans but not on S. cerevisiae. Using a mouse cell line and galectin-3-deleted cells from knockout mice, we demonstrated a specific enhancement of the cellular response to C. albicans compared with S. cerevisiae, which depended on galectin-3 expression. However, galectin-3 was not required for recognition and endocytosis of yeasts. In contrast, using PMA-induced differentiated THP-1, we observed that the presence of TLR2 was required for efficient uptake and endocytosis of both C. albicans and S. cerevisiae. TLR2 and galectin-3, which are expressed at the level of phagosomes containing C. albicans, were shown to be associated in differentiated macrophages after incubation with this sole species. These data suggest that macrophages differently sense C. albicans and S. cerevisiae through a mechanism involving TLR2 and galectin-3, which probably associate for binding of ligands expressing beta-1,2 mannosides specific to the C. albicans cell wall surface.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Host sensing of microbial exposure is conferred by the expression of extracellular and intracellular pathogen-recognition molecules (PRMs).3 Upon recognition of pathogen-associated molecular patterns (PAMPs) by PRMs, specific intracellular signaling pathways modulate essential host defense functions such as inflammation, cell death, and adaptive immunity. However, whereas the function of the membrane-bound PRMs known as TLRs has been studied extensively over the past few years (1), lectins have also been long established to confer an extracellular surveillance mechanism promoting phagocytosis and signaling (2, 3).

Candida albicans is one of the most prominent human pathogens, and also colonizes the mucocutaneous surfaces of the oral cavity, gastrointestinal tract and vagina. Given the high levels of morbidity and mortality induced by C. albicans in hospitalized patients, the pathogenic mechanisms of C. albicans have been investigated extensively (4). However, unlike Drosophila melanogaster, which provides a model for the sensing of indigenous and/or pathogenic fungi, the nature of the mammalian PRMs conferring discrimination between the large number of fungal species closely associated with different aspects of human life remains poorly understood.

The complex architecture of the yeast cell wall is composed of chitin, glucans, mannoproteins, mannan, and glycolipids. The protective and inflammatory response to fungal exposure is initiated by host recognition of specific PAMPs, which are expressed by pathogenic and/or symbiotic microorganisms (5). Notably, C. albicans and Saccharomyces cerevisiae can both lead to TLR-dependent proinflammatory cytokine production (6, 7). Alternatively, certain lectins, such as dectin-1, have been proposed to recognize surface sugars of microorganisms and to cooperate with TLRs for induction of a proinflammatory response (8). Notably, dectin-1 cooperates with TLR2 to confer cellular responsiveness to microbial beta-glucans (9, 10, 11). Association has also been demonstrated for SIGNR1, another lectin involved in yeast recognition (12, 13), which enhances TLR4/MD2-induced signaling by LPS (14). It therefore seems that lectins may act in specific recognition by TLRs, and enhance signals induced through these receptors.

Unlike beta-glucans and {alpha}-mannan, which are expressed by both nonpathogenic and pathogenic yeasts, host recognition of beta-1,2 mannosides predicts the host sensing of C. albicans (15). beta-1,2 mannosides are special types of glycans that are expressed by C. albicans and are associated with both mannan and phospholipomannan (PLM) (16), a glycolipid present in the cell wall (17) that binds to the macrophage membrane (18) and leads to TLR2-dependent NF-{kappa}B activation and TNF-{alpha} production (19). Purified beta-1,2 mannosides have been shown to elicit TNF-{alpha} (20) and eicosanoid (21) production. However, in contrast to other fungal mannosides, which react with C-lectins, such as the mannose receptor (22) or DC-SIGN/SIGNR1 (12), beta-1,2 mannosides bind to a host 32-kDa protein, identified as the S-lectin galectin-3 (23). In this study, we investigated the physiological role of galectin-3 in the immune response to C. albicans. Our results revealed that galectin-3, but not TLR2, is dispensable for the recognition and endocytosis of yeasts, but that galectin-3 is essential for TLR2-dependent cytokine production in response to C. albicans but not S. cerevisiae.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Reagents and Abs

All reagents were obtained from Sigma-Aldrich. The TLR2 agonist Pam3CSK4 was from EMC Microcollections. The anti-beta-1,2 mannoside mAb 5B2, a rat IgM, was developed in our laboratory (24). Anti-beta-1,3 glucan, a mouse monoclonal IgG, was provided from Biosupplies. Anti-human/mouse TLR2 Ab (clone TL2.5) was obtained from eBioscience. The human recombinant galectin-3 and polyclonal rabbit anti-human galectin-3 Abs were a gift from F. van den Brule, University of Liège (Liège, Belgium). mAbs to mouse or human galectin-3 (Novacastra Laboratories) were also used depending on the experiment. HRP-, FITC-, and PE-conjugated anti-mouse or anti-rabbit IgG or IgM were obtained from Southern Biotechnology Associates. Fluorescent phallotoxin (BODIPY FL phallacidin) was purchased from Molecular Probes (Interchim).

Cell lines

The mouse macrophage-like cell line J774 (ECCC 85011428) was derived from a tumor of a female BALB/c mouse. Adherent cells were cultured at 37°C in an atmosphere containing 5% CO2 in DMEM supplemented with 10% heat-inactivated FCS (Valbiotech), 5 mM L-glutamine, 100 µg/ml streptomycin, and 50 µg/ml penicillin (culture medium). Before use, cells were gently scraped off with a rubber policeman and, depending on the experiment, either plated into eight-well Lab-Tek tissue culture chambers (Nunc) or into 48-well tissue culture dishes at a concentration of 5 x 105 cells/well.

Human monocyte THP-1 cells (ATCC TIB 202) were maintained in RPMI 1640 medium supplemented with 10% heat-inactivated FCS. Cell differentiation was induced by treatment with different doses of PMA in culture medium. After 20 h incubation, the cells were washed and cultured for 12 h without PMA. With this treatment, cells acquired the morphologic characteristics of macrophages.

Isolation of peritoneal macrophages

The generation of galectin-3–/– mice by gene-targeting technology has been described elsewhere (25). As controls, age- and sex-matched wild-type (WT) littermate mice were used. Mice were maintained for us by Charles River Breeding Laboratories. Mouse peritoneal macrophages were obtained from 6- to 8-wk-old WT or galectin-3 knockout (KO) mice by injecting 2 ml of PBS containing 10% proteose peptone broth. After 4 days, the mice were killed and peritoneal macrophages were harvested by injecting 4 ml of sterile DMEM. After centrifugation and washing, the cells were resuspended in culture medium and cultured for 24 h in 75-cm2 culture flasks. For coculture experiments, adherent cells were recovered by gentle scraping and transferred into 96-well microtiter plates at 5 x 105 cells/well, in a final volume of 200 µl of culture medium.

Yeast culture

C. albicans (serotype A) and S. cerevisiae were maintained on Sabouraud dextrose agar at 4°C. Before the experiments, yeast cells were transferred onto fresh Sabouraud dextrose agar and incubated for 20 h at 37°C. Yeast cells were then recovered, washed with PBS, and transferred into DMEM. Three different strains of C. albicans (SC5314, the derived strain CAF and VW32, which was a strain isolated in our laboratory) were used. The S288C strain of S. cerevisiae was used as a reference. In some experiments, two additional strains derived from S288C were tested (S288C-1 and S288C-4).

Cytokine production by mammalian cells after stimulation by yeasts

Plated cells were incubated at 37°C with yeasts at a concentration of five yeasts per cell. Supernatants were collected after different periods of time and stored at –20°C until cytokine assays were performed. TNF-{alpha} concentrations in cell-free supernatants were measured with commercial ELISA kits according to the manufacturer’s instructions (R&D Systems). In some experiments, TNF-{alpha} production was also evaluated by RT-PCR (discussed below).

Microscopic examination and immunofluorescent staining

Yeasts were incubated with 100 µl of human recombinant galectin-3 in PBS for 2 h at 20°C. After washing, bound galectin-3 was revealed with a 1/100 dilution of rabbit polyclonal Ab specific for human galectin-3 previously adsorbed onto yeasts. After washing, 100 µl of a 1/100 dilution of FITC-conjugated goat anti-rabbit IgG in phosphate buffer was added for 1 h at 20°C. Slides were then washed and mounted for microscopic examination.

Expression of galectin-3 and TLR2 was evaluated on J774 and THP-1 cells previously plated into Lab-Tek wells in the absence of yeasts or after coculture of cells with yeasts for 2 h at 37°C. Cells were washed with warm DMEM, fixed and permeabilized with a mixture of 3.7% formaldehyde and 0.2% Triton X-100 in phosphate buffer at 20°C for 20 min. One hundred microliters of a 1/100 dilution of anti-galectin-3- or anti-TLR2-specific mAb was added for 2 h at 20°C. After five washes, 100 µl of a 1/100 dilution of FITC-conjugated goat anti-mouse IgG in phosphate buffer was added for 1 h at 20°C. In parallel, F-actin labeling was performed by incubating permeabilized cells with a 1/100 dilution of BODIPY FL phallacidin for 20 min at 20°C. Slides were then washed five times and mounted for microscopic examination.

Interaction of recombinant galectin-3 with C. albicans

Different concentrations of human recombinant galectin-3 in PBS were coated into 96-well culture plates for 1 h at 20°C followed by overnight incubation at 4°C. Plates were saturated with 5% BSA in PBS for 2 h at 20°C. After washing, 200 µl of yeast suspension was added to the wells and incubated for 2 h at 20°C. Unbound yeasts were discarded by washing with PBS containing 0.5% BSA and bound yeasts were revealed using the mAb EBCA1 directed against C. albicans mannan (26) and anti-mouse IgM FITC-conjugated Ab. Plates were read in a Fluorocount reader (Packard).

Flow cytometry analysis

The expression of beta-1,2 mannosides and beta-1,3 glucans on the yeast cell wall was studied by flow cytometry. For preparation of SC5314 heat-killed yeasts, the cells were incubated for 20 min at 90°C and washed once in PBS. For staining, 106 cells were washed with PBS containing 2% FCS and then incubated for 15 min with mAb 5B2 to beta-1,2 mannosides, diluted 1/200, or with a mAb to beta-1,3 glucans diluted 1/50. For Ab detection, cells were incubated for 15 min with FITC-labeled anti-rat IgM or PE-labeled anti-mouse IgG second Ab diluted 1/100. A negative control was performed by adding labeled second Ab at the same concentration. All processes were conducted at 4°C. After washing, cells were fixed in 0.4% paraformaldehyde and analyzed with a FACS.

Flow cytometry was performed with an EPICS XLMCL4 (Beckman Coulter) equipped with an argon ion laser with an excitation power of 15 mW at 488 nm. Forward and side light scatter were analyzed on linear scales, while green and red fluorescence intensity was determined on logarithmic scales. Analysis gates were set around debris and intact cells were set on a forward and side light scatter dot plot. The fluorescence histograms of 5000 cells were generated using gated data. Data acquisition and analysis were performed using WINMDI software (available from <http://facs.scripps.edu>). Mean fluorescence intensities were obtained by subtracting values for negative controls from the values given by each Ag.

Immunoprecipitation and Western blot analysis

For total extraction of proteins, macrophages (106 cells) were treated by boiling in 500 µl of 2x concentrated electrophoresis sample 1x buffer (125 mM Tris-HCl (pH 6.8), 2% SDS, 5% glycerol, 1% 2-ME, and bromphenol blue). Lysates were clarified by centrifugation for 10 min at 12,000 x g at 4°C. For immunoprecipitation, 107 cells were extracted with 500 µl of RIPA buffer (PBS containing 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, PMSF, aprotinin) for 30 min on ice. Lysates were collected and clarified by centrifugation for 10 min at 12,000 x g at 4°C. Supernatants (500 µl) were incubated with either anti-galectin-3 or anti-TLR2 Ab for 2 h at 4°C. Protein G-coupled agarose beads (10 µl) were added to each tube and incubated for 18 h at 4°C. Beads were washed 5 to 10 times with PBS. Precipitates were recovered by addition of 50 µl of sample buffer and boiled for 10 min at 100°C.

Extracted proteins or precipitates were separated by 10% SDS-PAGE before blotting onto a nitrocellulose membrane (Schleicher & Schuell) for 2 h at 200 mA in a semidry transfer system. After staining with 0.1% Ponceau S in 5% acetic acid to confirm equivalence of loading and transfer, the membrane was blocked by incubation with TNT buffer (10 mM Tris, 100 mM NaCl, and 0.1% Tween 20) containing 5% nonfat dry milk for 1 h at 20°C. Membranes were probed with anti-galectin-3 or anti-TLR2 Abs (diluted 1/1000) in TNT-5% BSA for 2 h at 20°C. After washing several times, the membranes were incubated for 1 h at 20°C with a 1/2000 dilution of HRP-conjugated anti-mouse IgG in TNT-5% BSA. After washing, the membrane was incubated with ECL detection reagents (SuperSignal Chemiluminescent substrate; Pierce) and exposed to Hyperfilm ECL.

RT-PCR

THP-1 cells (107) treated with different concentrations of PMA and incubated with or without C. albicans blastoconidia were washed with PBS and total RNA was extracted with an RNeasy Mini kit (Qiagen). The reverse transcription reaction was performed using Superscript II reverse transcriptase (Invitrogen Life Technologies) at 42°C for 60 min in the presence of 2 U of RNase inhibitor. Following reverse transcription, the enzyme was heat-inactivated at 99°C for 3 min. PCR was performed in an iCycler (Bio-Rad) with the primers designed using Primer 3 software from gene accession nos. NM002306, U88878, and AF043342 for human galectin-3, TLR2, and TNF-{alpha}, respectively. Control genes used were GAPDH or 28S rRNA. The following primers synthesized by Eurogentec were used for fragment amplification: galectin-3 302 bp, forward (353–373) 5'-GGCCACTGATTGTGCCTTAT-3' and reverse (654–674) 5'-TGCAACCTTGAAGTGGTCAG-3'; TLR2 311 bp, forward (2162–2182) 5'-ACTTCATTCCTGGCAAGTGG-3' and reverse (2472–2492) 5'-CGCAGCTCTCAGATTTACCC-3'; and TNF-{alpha} 335 bp, forward (419–439) 5'-AGCCCATGTTGTAGCAAACC-3' and reverse (753–773) 5'-GGAAGACCCCTCCCAGATAG-3'.

The PCR conditions used to amplify fragments were: 30 cycles at 94°C for 1 min, 60°C for 1 min, and 72°C for 45 s. Hot start at 94°C was performed using AmpliTaq Gold (Applied Biosystems). Products were analyzed by electrophoresis and staining with ethidium bromide. Semiquantification was performed after scanning with GelEval (FrogDance software).

Statistics

For cytokine determination, results of duplicate measurements (expressed as mean ± SD) from a representative experiment performed at least twice are shown. Significance of the data was evaluated by ANOVA. Other results were from three to five independent experiments.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
C. albicans, but not S. cerevisiae, expresses beta-1,2 mannosides

beta-1,2 mannosides have been previously shown to be essential for the binding of C. albicans to macrophages and to be recognized by a 32-kDa protein homologous to galectin-3 (15, 23). Using flow cytometry with specific mAbs, we determined and compared the yeasts surface expression of beta-1,3 glucans with beta-1,2 mannosides. Unlike beta-1,3 glucans, which were accessible only after heat treatment of C. albicans, beta-1,2 mannosides were constitutively expressed at the surface of both heat-killed and live Candida yeasts (Fig. 1). Furthermore, S. cerevisiae did not express beta-1,2 mannosides (Fig. 1), suggesting that galectin-3 might contribute to the sensing of C. albicans.


Figure 1
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  		FIGURE 1. Surface expression of beta-1,2 mannosides and beta-1,3 glucans by C. albicans and S. cerevisiae. Flow cytometry was performed with mAbs to {alpha}-mannoside or beta-1,2 mannosides and beta-1,3 glucans on live or heat-killed (HK) C. albicans strain SC5314 and compared with live S. cerevisiae strain S288C. The results presented are representative of three independent experiments. Gray-filled histogram represents negative control without primary Ab.

 
Galectin-3 binds C. albicans

To begin to understand the potential role of galectin-3 in the immune response to C. albicans, we conducted experiments to confirm the interaction of galectin-3 with C. albicans blastoconidia. C. albicans yeast cells were added to wells of microtiter plates coated with different concentrations of human recombinant galectin-3. Using an anti-mannan Ab, we revealed a dose-dependent binding of C. albicans to human recombinant galectin-3 (Fig. 2A). Furthermore, using a specific Ab to galectin-3, human recombinant galectin-3 was detected at the surface of C. albicans (Fig. 2B). Conversely, we failed to detect any binding of S. cerevisiae to human recombinant galectin-3 (data not shown).


Figure 2
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  		FIGURE 2. Binding of galectin-3 to C. albicans blastoconidia. A, Binding of yeasts to galectin-3 was evaluated by cyto-ELISA. After coating wells with different concentrations of galectin-3 (Gal-3), different amounts of C. albicans blastoconidia were added and bound yeasts were revealed with FITC-conjugated anti-C. albicans mannan mAb. Fluorescence of duplicate wells was measured in a fluorocount. B, Binding of human recombinant galectin-3 to blastoconidia was examined by indirect immunofluorescence using an anti-galectin-3 polyclonal Ab to reveal bound human recombinant galectin-3. Detail of a labeled yeast (inset) is shown.

 
Galectin-3 is localized in phagocytic cups and phagosomes containing C. albicans

To determine the localization of galectin-3 in macrophages engaged in phagocytosis of C. albicans blastoconidia, immunofluorescent microscopic analysis was performed using the mouse macrophage line J774. Galectin-3 expression was assessed in cells incubated with or without yeasts. As shown previously (27), entry of C. albicans into macrophages was associated with the condensation of actin filaments as revealed by the labeling of F-actin around ingested yeasts (Fig. 3, A and B). Parallel staining of galectin-3 revealed that after incubation with C. albicans, the lectin also localized around the yeasts (Fig. 3, C and D) either at the cell membrane at the level of phagocytic cups or within the cells (Fig. 3E).


Figure 3
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  		FIGURE 3. Localization of galectin-3 in J774 cells containing engulfed C. albicans blastoconidia. After coculture of J774 cells with yeasts at a yeast to cell ratio of 5:1, F-actin (A and B), and galectin-3 (C–E) distributions were determined using FITC-conjugated phallacidin and an anti-galectin-3 mAb, respectively. B and D, Corresponding phase-contrast images of fluorescence examination (A and C). E, Details of galectin-3 labeling show the arrows indicate phagocytic cups formed around the yeasts. Engulfed yeasts (*) are represented.

 
Higher amounts of TNF-{alpha} are produced after C. albicans stimulation of J774 cells

Because galectin-3 may play a role in the specific recognition of C. albicans compared with S. cerevisiae, the behavior of J774 cells toward both yeasts was examined, particularly whether the yeasts were recognized and engulfed by the cells. J774 cells were able to bind and endocytose both yeast species in a similar manner (Fig. 4A). The ability of the three strains of C. albicans and S. cerevisiae to stimulate macrophages was determined and compared (Fig. 4B). Unexpectedly, C. albicans induced a 2.5- to 3-fold higher TNF-{alpha} production compared with S. cerevisiae-stimulated cells, suggesting that C. albicans is somehow more efficient at triggering the proinflammatory response.


Figure 4
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  		FIGURE 4. Comparison of interaction of J774 cells with C. albicans and S. cerevisiae. A, J774 cells were incubated with either C. albicans strain SC5314 or S. cerevisiae strain S288C. After washing, cocultures were examined by phase-contrast microscopy. B, Stimulation of J774 cells by yeasts was evaluated by measuring the induction of TNF-{alpha} production after coculture of cells with three strains of either C. albicans or S. cerevisiae for different time points. Cytokine production was measured in cell-free supernatants by ELISA. Data are expressed as the mean ± SD of cytokine concentration and are from one representative among three experiments performed.

 
Galectin-3 deletion does not alter the ability of macrophages to recognize and endocytose C. albicans and S. cerevisiae

The role of galectin-3 in the enhancement of cell stimulation observed with C. albicans was then examined using macrophages isolated from either WT or galectin-3 KO mice. Absence of galectin-3 expression in macrophages from KO mice was first verified by Western blotting (Fig. 5A). The effect of deletion of the galectin-3 gene on the phagocytic capacity of macrophages toward the yeasts was then analyzed. As shown in Fig. 5B, both yeast strains were recognized by macrophages isolated from either WT or galectin-3 KO mice. This result shows that deletion of galectin-3 does not qualitatively alter the binding capacity of the macrophages toward yeasts.


Figure 5
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  		FIGURE 5. Galectin-3 is not necessary for binding and endocytosis of either C. albicans or S. cerevisiae blastoconidia. A, Expression of galectin-3 by peritoneal macrophages from galectin-3 KO or WT mice revealed by Western blotting using a mAb to galectin-3. B, Examination by phase-contrast microscopy of endocytosis of C. albicans and S. cerevisiae yeasts by macrophages from WT or galectin-3 (Gal-3) KO mice.

 
Specific induction of cytokines by C. albicans depends on galectin-3 expression by the cells

The effect of galectin-3 deficiency on the capacity of macrophages to respond to both C. albicans and S. cerevisiae was then determined. To this end, cells were incubated with either S. cerevisiae or C. albicans blastoconidia and the production of TNF-{alpha} was measured. With macrophages from WT mice, C. albicans induced a significantly (p < 0.05, n = 4) higher production of the cytokine (1424 ± 141 pg/ml) compared with that obtained with S. cerevisiae (352 ± 103 pg/ml). These results, obtained with primary macrophages from WT mice, confirm those obtained with J774 cells (as discussed). In contrast, when macrophages from galectin-3 KO mice were used in the experiment, no significant difference in TNF-{alpha} production was observed. In this case, the amount of cytokine obtained after incubation with C. albicans (366 ± 24 pg/ml) was similar to that observed with S. cerevisiae (241 ± 56 pg/ml). Similar results were observed whatever strain of C. albicans was used to stimulate the cells (Fig. 6B). To ascertain that this effect is not due to intrinsic defect of galectin-3-deficient cells to produce cytokines in response to TLR2, cells were stimulated with the specific TLR2 agonist Pam3CSK4. As observed in Fig. 6C, no differences were noticed between galectin-3-competent and galectin-3-deficient cells. Altogether, this indicates that the specific response of macrophages to C. albicans is due, at least in part, to galectin-3.


Figure 6
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  		FIGURE 6. Comparison of TNF-{alpha} production by macrophages from WT or galectin-3 (Gal-3) KO mice in response to C. albicans or S. cerevisiae. A, Macrophages from WT or galectin-3 KO mice were incubated with either C. albicans or S. cerevisiae at a yeast to cell ratio of 5:1. TNF-{alpha} production was measured in cell-free supernatants after 3 h. Data are the mean of four independent experiments ± SD. B, Production of TNF-{alpha} induced by two different strains of C. albicans (SC5314 and CAF). Results are expressed as the percentage of production induced by C. albicans, 100% being the level of cytokine obtained with S. cerevisiae. C, Production of TNF-{alpha} by cells from WT or galectin-3 KO mice in response to 500 ng of TLR2 agonist Pam3CSK4. Data are the mean of three independent experiments ± SD.

 
PMA differentiates THP-1 cells and induces galectin-3 and TLR2 expression

Human monocytic THP-1 cells, which are known to differentiate into macrophages after treatment with PMA, were used to examine the role of both receptors in the interaction of macrophages with yeasts. THP-1 cells were treated with different doses of PMA and maturation was followed according to adherence, morphology, and expression of galectin-3 and TLR2. Microscopic examination and fluorescence analysis (Fig. 7A) revealed that PMA treatment led to differentiation from a monocyte-like to a macrophage-like morphology and adherence. Immunofluorescence showed that both galectin-3 and TLR2 membrane expression was induced by PMA. Similarly, when galectin-3 gene expression was evaluated (Fig. 7B), a relationship between the amount of mRNA and the degree of cell maturation was observed, a maximum being obtained when cells had been treated with 10 ng/ml PMA. For TLR2, a lower dose of PMA was needed to obtain the maximum amount of mRNA. This increased significantly and reached a maximum with 1 ng/ml PMA. According to the results obtained by immunofluorescence, Western blotting of the corresponding proteins showed that TLR2 was absent and galectin-3 was present in undifferentiated cells (Fig. 7C). Expression of both proteins was increased after treatment of cells with 10 ng/ml PMA (Fig. 7C).


Figure 7
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  		FIGURE 7. Differential expression of galectin-3 and TLR2 by THP-1 cells according to their degree of maturation induced by PMA. A, Maturation of THP-1 cells was followed after treatment without (a, c, e, and g) or with (b, d, f, and h) 10 ng/ml PMA. A change in cell adherence to culture wells (a and b) and morphologic evolution of the cells after treatment (c and d) are shown. Influence of PMA treatment on surface expression of galectin-3 (e and f) or TLR2 (g and h) by cells, as revealed by immunofluorescence using specific mAbs, is shown. B, Determination by RT-PCR of galectin-3- and TLR2-specific mRNA expression by cells after treatment with PMA. Mitochondrial ribosomal protein 28 S mRNA (Ref) is indicated. C, Revelation after immunoprecipitation with specific mAbs in Western blotting using homologous Abs to galectin-3 and TLR2 protein in THP-1 cells treated with different doses of PMA.

 
Differentiated THP-1 cells express TLR2 associated with C. albicans-containing phagosomes and produce TNF-{alpha}

The effect of maturation on the ability of THP-1 cells to interact with C. albicans was investigated. Binding of blastoconidia to undifferentiated THP-1 cells was limited (Fig. 8A, a). In contrast, PMA-treated THP-1 cells (Fig. 8A, b) bound and endocytosed C. albicans in a manner that was similar to mouse primary macrophages (Fig. 5B) or J774 cells (Fig. 4A). With these cells, TLR2 could be localized at the level of engulfed yeasts (Fig. 8A, c and d). The ability of differentiated THP-1 cells, which express TLR2 and phagocytose yeasts, to respond to C. albicans was then investigated. In contrast to immature cells that do not produce TNF-{alpha} in response to C. albicans, after PMA treatment a high amount of mRNA was induced by yeasts in the cells (Fig. 8B).


Figure 8
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  		FIGURE 8. Effect of PMA treatment of THP-1 cells on endocytosis of C. albicans. A, Comparison of endocytic capability of cells untreated (a) or treated (b) with PMA toward C. albicans blastoconidia. Treated cells were incubated with C. albicans yeast cells and examined by phase-contrast microscopy. Localization of TLR2 in treated cells containing ingested C. albicans blastoconidia was performed after coculture by immunofluorescence with a specific mAb. Phase-contrast microcopy (c) and corresponding fluorescent staining (d) are shown. B, Determination by RT-PCR of TNF-{alpha} mRNA expression by cells incubated with C. albicans blastoconidia and treated with PMA is shown. GAPDH mRNA (Ref) is indicated.

 
TLR2 and galectin-3 associate when differentiated THP-1 cells are in contact with C. albicans

As suggested by our experiments, precipitation with their respective specific Abs demonstrated that both TLR2 and galectin-3 were increasingly present in macrophage extracts as a function of the PMA dose inducing THP-1 differentiation (Fig. 7). Whether an association of TLR2 and galectin-3 appeared in the presence of C. albicans was therefore explored through experiments based on coprecipitation with Abs to both proteins. When homologous Abs were used, it was possible to observe an increased expression of galectin-3 according to the dose of PMA used for treatment (Fig. 9, left). When an anti-TLR2 Ab was used for precipitation (Fig. 9, right), it was possible to reveal the presence of galectin-3 in the precipitates. This result depended on TLR2 expression by the cells. Absent in precipitates performed with untreated cells that did not express TLR2 (Fig. 9, right), galectin-3 was coprecipitated with TLR2, the amount of which depended on the dose of PMA used to differentiate the cells. Moreover, the lectin was not observed in precipitates in the absence of yeasts (data not shown).


Figure 9
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  		FIGURE 9. Galectin-3 and TLR2 coprecipitate in THP-1 cells after treatment with PMA and incubation with C. albicans blastoconidia. After treatment with different doses of PMA, THP-1 cells were incubated with C. albicans yeast cells. Proteins were extracted and incubated with either anti-galectin-3 or anti-TLR2 mAbs. Precipitates recovered on protein G-Sepharose beads were resolved by SDS-PAGE and transferred onto nitrocellulose. Presence of galectin-3 in the precipitates was then revealed by Western blotting with mAb to galectin-3.

 
Taken together with the previous results, these data strongly suggest that contact between the yeast cell wall and the cell membrane of differentiated macrophages specifically promotes the association of these two C. albicans interacting molecules.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Specific interaction between galectins and their glycoconjugate ligands are considered to be critical determinants in pathogen recognition (28). Galectin has been proposed as a molecule involved in "seeing strangers or announcing danger" (29). First identified as an Ag expressed on the surface of murine thioglycolate-elicited macrophages, galectin-3 has been implicated in some aspects of innate immunity. Interestingly, the level of galectin-3 expression in myeloid cells may be related to cell activity toward invading microbes. Expression is very low in monocytes, but increases in mature macrophages (30) and immature dendritic cells (31), two types of cell dedicated to recognition of pathogens at the level of tissue penetration. However, its transcription is down-modulated in mature dendritic cells (31) when endophagocytosis and sensing are accomplished and the cells migrate to the lymph nodes.

Present at the plasma membrane, galectin-3 is also localized within the cytoplasm and nucleus (32). Presence of galectin-3 has been demonstrated at the level of phagocytic cups and phagosomes (33), where it seems to play an important role in the regulation of phagolysosome maturation (34). An association with lysosomal membrane-associated glycoproteins has also been observed (35). Interestingly, although its physiological role remains unclear, galectin-3 promotes pathogen-macrophage interactions (36, 37, 38, 39, 40, 41, 42). For C. albicans, a recent proteomic study of infected cells showed that galectin-3 was one of the most regulated proteins whose expression was modulated during infection (43). However, although possible differential splicing has been observed leading to different proteins (44), the role of galectin-3 in signaling and recognition is still unclear because no transmembrane sequence or cytoplasmic tail has been demonstrated for this lectin (45).

Interaction of PAMPs with immune cells is now considered to be based on the assembly of a pluri-molecular complex that leads to signaling in the cells (2). This mechanism has been well documented for LPS, for which complex orchestration of multiple receptors, including TLR4 and CD14, has been demonstrated (46). Most of the pathogens interacting with galectin-3 also stimulate these cells through TLRs activation. This effect is the case for mycobacterial phosphatidylinositol mannosides, which have been shown to bind to galectin-3 (38) and to stimulate macrophages through TLR2 (47). By using either C. albicans or S. cerevisiae, we demonstrated that the interaction involving TLR2 and subsequent signaling leading to a proinflammatory response is not a trait specific to pathogens because both yeasts were able to stimulate cytokine production. However, although governed by identical signaling receptors (7), TNF-{alpha} induction was found to increase in the case of cells recognizing C. albicans. This finding suggests the involvement of another signal that would be associated with the specific recognition of this yeast. It has previously been shown that beta-1,2 mannosides, which are specific for C. albicans, participate in the recognition process of this yeast by interacting with galectin-3 (23). In this study, the use of recombinant galectin-3 allowed us to demonstrate direct binding of the lectin at the surface of live yeasts where beta-1,2 mannosides are expressed. This response contrasts with the reactivity of dectin-1 whose binding to live yeasts cells involving beta-glucans is limited at some stages of growth and can only be increased by heating of the yeasts (11, 48, 49) or antifungal drugs (50). Interaction of C. albicans with galectin-3 was also observed directly onto the cells when the cellular distribution of the lectin was examined after endocytosis of C. albicans. In this case, as already described in other systems (33), galectin-3 could be localized at the level of the phagocytic cups formed around the yeasts and also at the periphery of ingested yeasts.

Purified beta-1,2 mannosides stimulated macrophages to produce TNF-{alpha} (20, 21). Such activity involving TLR2 (19) has also been shown for PLM, a glycolipid expressing beta-1,2 mannosides (16) that binds to the macrophage membrane (18). The effect of deletion of galectin-3 on the cell response to yeasts was therefore investigated, hypothesizing that galectin-3 may associate with TLR2 for induction of specific stimulation by C. albicans. By using primary macrophages from galectin-3 KO mice, enhancement of the cell response toward C. albicans, which was observed with both macrophages from WT mice and the J774 cell line, was abrogated in cells that did not express galectin-3. In this case, C. albicans and S. cerevisiae blastoconidia induced similar cytokine production. This response could be explained by a lower capacity of the galectin-3-deficient cells to express TLRs or to recognize and/or endocytose the yeasts as suggested previously (34). However, comparison of cell response to specific agonist for TLRs revealed that both cell types expressed similarly TLR and that they presented identical capacity to produce cytokines. Moreover, endophagocytosis of both yeasts by macrophages from either WT or KO mice did not reveal any qualitative alteration at this step of the interaction. It therefore seems that two responses toward yeasts may exist. The first response, still stimulated in the absence of galectin-3, was induced by both yeast species; the second was induced specifically by C. albicans and depended on galectin-3 expression. This hypothesis, which agrees with results obtained in other systems (14), may explain for one part the observations obtained by Hobson et al. (51) who showed that disruption of the gene required for mannosyl phosphate transfer and, hence, the attachment of beta-1,2 mannose oligosaccharides to mannan, did not alter the interaction between C. albicans and macrophages. An important drawback from the conclusions raised in this study is that, beside the fraction attached to mannan by mannosyl phosphate, a large amount of beta-1,2 mannose oligosaccharides remained present at the cell wall surface, either attached to the mannan acid-stable part, the PLM, as well as to a large number of mannoproteins (52).

The level of galectin-3 has been shown to increase dramatically as monocytes differentiate into macrophages after culture in vitro or when cells were stimulated with phorbol esters (53). Among the different genes that are modulated in THP-1 cells in response to PMA, galectin-3 expression has been shown to be up-regulated by such treatment (30). We therefore took advantage of this model to explore the role of galectin-3 as a coreceptor for C. albicans recognition. Galectin-3 expression was revealed in cell extracts from undifferentiated cells and increased after PMA-induced differentiation. TLR2 protein was not observed in immature cells. Its expression increased according to the dose of PMA used for treatment. Interestingly, although no protein could be detected at the membrane or within cell extracts, a significant amount of the corresponding mRNA was apparent even in nontreated cells.

Although they expressed galectin-3, untreated THP-1 cells, which do not express TLR2, were not able to bind and endocytose C. albicans efficiently. Conversely, macrophages from galectin-3 KO mice phagocytosed the yeasts in a manner identical with macrophages from WT mice. After differentiation, THP-1 cells, which expressed both galectin-3 and TLR2, phagocytosed the yeasts. It therefore seems that galectin-3 is not critical for engulfment of yeasts and that endocytosis may be achieved only when the cells express TLR2. Although galectin-3 has been shown to be necessary for efficient IgG-mediated phagocytosis of erythrocytes by macrophages (34), these results show that the absence or presence of this lectin did not influence the binding and uptake of yeasts by the cells. In contrast, absence of TLR2 expression rendered the cells refractory to the yeasts, which confirms the results obtained by Blander and Medzhitov (54), who showed that phagocytosis of bacteria was impaired in the absence of TLR signaling. By using an anti-TLR2 Ab, it was possible to precipitate galectin-3 in extracts from cells that had been incubated with C. albicans. However, recovery of the lectin was dependent on the presence of TLR2 at the cell membrane because no galectin-3 was observed in precipitates obtained from undifferentiated cells that did not express TLR2 but nonetheless presented galectin-3. This observation suggests that galectin-3 may actually associate with TLR2 when both molecules are expressed at the membrane and after interaction with yeasts. Moreover, in the absence of TLR2 but presence of galectin-3 expression, which is the case of nondifferentiated THP-1 cells, no evidence of stimulation was obtained. In contrast, when both molecules were available, C. albicans induced a high level of TNF-{alpha} production. Previous results obtained with PLM (19, 55) or beta-1,2 mannosides (our unpublished data) on undifferentiated monocytes have already suggested the need for cell maturation to gain optimal induction of cytokines.

Evidence of the existence of complex assembly has been established for yeasts interacting with macrophages. Proinflammatory activity of fungal beta-glucans has been shown to depend on the cooperation between TLR2 and a dedicated coreceptor, dectin-1 (8), although recent findings showed that internalization of, and subsequent stimulation by, beta-glucan particles depended mainly on dectin-1 expression rather than on TLR2 (56). For mannan, both TLR4 and CD14 have been shown to be necessary for signaling (57). Recently, an association of SIGNR1, a C-type lectin that binds {alpha}-linked mannosides equivalent to DC-SIGN that has been shown to interact with C. albicans (12), with the TLR4/MD2 complex has been shown to enhance signaling induced by LPS (14). Thus, although signaling seems to require an association with TLRs, coreceptors, and in particular lectins may cooperate for specific recognition of PAMPs. It is clear that all components of the yeast cell wall are differently recognized by specific interaction with lectin receptors. This recognition is the case for beta-glucans, {alpha}-mannosides, and beta-mannosides. The behavior of the yeasts and of the interacting cells should depend on the glycan and lectin involved in this recognition. The fact that for C. albicans it is possible to show an interaction that involves the association of a peculiar lectin and specific mannosides may explain why sensing of C. albicans by macrophages differs from other yeasts.


   Acknowledgments
 
We thank Prof. F. Van den Brule for the precious gift of recombinant human galectin-3, and Jean-Pierre Kerckaert and Sabine Quief for help and technical assistance.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported in part by the European Community Feder Fund, Program Interreg IIIA. M.M.-E. was supported by the "Fundación Séneca", Murcia, Spain. Back

2 Address correspondence and reprint requests to Dr. Thierry Jouault, Institut National de la Santé et de la Recherche Médicale, Unité 799, Laboratoire de Mycologie Fondamentale et Appliquée, Université de Lille II, Faculté de Médecine H. Warembourg, Pôle Recherche, Place Verdun, 59037 Lille, France. E-mail address: tjouault{at}univ-lille2.fr Back

3 Abbreviations used in this paper: PRM, pathogen-recognition molecule; PAMP, pathogen-associated molecular pattern; PLM, phospholipomannan; KO, knockout; WT, wild type; DC-SIGN, dendritic cell-specific ICAM-3 grabbing nonintegrin; SIGNR1, a murine homolog of human DC-SIGN. Back

Received for publication December 7, 2005. Accepted for publication July 21, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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