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Originally published online as doi:10.1189/jlb.0307160 on July 26, 2007

Published online before print July 26, 2007
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(Journal of Leukocyte Biology. 2007;82:1136-1142.)
© 2007 by Society for Leukocyte Biology

β-Glucan of Candida albicans cell wall causes the subversion of human monocyte differentiation into dendritic cells

Roberto Nisini*,1, Antonella Torosantucci*, Giulia Romagnoli{dagger}, Paola Chiani, Simona Donati{dagger}, Maria Cristina Gagliardi*, Raffaela Teloni*, Valeria Sargentini*, Sabrina Mariotti*, Egidio Iorio{dagger} and Antonio Cassone*

* Dipartimento di Malattie Infettive, Parassitarie e Immunomediate; and
{dagger} Dipartimento Biologia Cellulare e Neuroscienze. Istituto Superiore di Sanità, Rome, Italy

1 Correspondence: Dipartimento di Malattie Infettive, Parassitarie e Immunomediate, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Roma, Italy. E-mail: roberto.nisini{at}iss.it


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ABSTRACT
 
The functional consequences of treating human monocytes with purified and chemically characterized Candida albicans β-glucan—a major microbial pathogen associated molecular pattern—on their differentiation into dendritic cells (DC) were investigated. We show here that β-glucan-treated monocytes differentiated into mature DC (Glu-MoDC) with altered phenotype and functional behavior, similarly to DC derived from C. albicans germ-tubes-infected monocytes (Gt-MoDC). They failed to express CD1a and to up-regulate CD80 and DR molecules. Moreover, they produced IL-10 but not IL-12 and primed naive T cells without inducing their functional polarization into effector cells. Since C. albicans β-glucan is a mixture of both β-(1,3) and β-(1,6) glucan, we investigated their relative contribution by the use of non-Candida β-glucan structural analogs. We found that high molecular weight (MW) glucans β–(1,6) pustulan and β-(1,3) curdlan totally mimicked the effect of C. albicans β-glucan, while the low MW β-(1,3) glucan laminarin did not have any effect. Because β-glucan is a common constituent of all fungi and is abundantly released in vivo during systemic fungal infection, this novel effect of β-glucan has potential implications for host-parasite relationship in candidiasis and other mycoses. In particular, our data suggest that β-glucan could bias noninfected, bystander monocytes, thus aggravating the general immunodeficiency, predisposing to systemic fungal infection.

Key Words: fungi • antigen presentation/processing • β-glucan structure


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INTRODUCTION
 
Candida albicans is an opportunistic fungal pathogen commonly found in human gastrointestinal and female lower genital tracts. It is capable of colonizing, infecting, and persisting on mucosal surfaces, thus stimulating mucosal and systemic immune responses [1 ,2 ]. If the ability of C. albicans to establish a disseminated infection involves neutropenia as a major predisposing factor, its ability to persist in infected tissues or to behave as a commensal may primarily involve down-regulation of host cell-mediated adaptive immunity, and induction of tolerance [3 ]. These data argue for the importance of the innate and adaptive immunity in the control of C. albicans infections. On the other hand, the occurrence of chronic and/or recurrent diseases and, perhaps, the status of commensalism itself, indicate that C. albicans possesses a number of virulence traits, which allows the fungus to escape from eradicating host immunity. The unique ability of C. albicans to switch from a unicellular yeast (Y) into an hyphal form through a germ-tube (Gt) intermediate is a major one among such traits, conferring upon the fungus enhanced tissue-invasive and immunoevasive properties [4 5 6 ].

We have previously shown that both Y and Gt forms are equally capable of inducing maturation of monocyte-derived DC in vitro. Furthermore, both Y- and Gt-matured DC prime allogeneic naive T lymphocyte for a Th1 polarization [7 ]. Nonetheless, Y and Gt cells have different and profound effects on monocyte differentiation into DC [8 ]. In particular, we have shown that Y-phagocytosing monocytes did not differentiate into DC, but rather into macrophages, whereas Gt-phagocytosing monocytes differentiated into a subset of DC with an unusual phenotype and a reduced capacity to prime Th1 responses.

During Y to Gt transition, rather profound rearrangements of molecular patterns actually or potentially interacting with host cell surface receptors take place. On this basis, we have hypothesized that the different arrays of molecular patterns expressed on Y and Gt cells of C. albicans [9 ] would be responsible for the differential impact of the two fungal forms on the process of monocyte differentiation into DC. In this paper, we have focused on β-glucan, a molecular pattern strongly involved in activating and modulating innate immunity [10 ]. This compound proved to be likely responsible for the inability of monocytes infected by the Gt forms of C. albicans to differentiate into DC fully functional for T cell priming.


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MATERIALS AND METHODS
 
Reagents
Phytohemagglutinin (PHA) was obtained from Murex (Dartford, United Kingdom). Recombinant IL-2 was a kind gift from EuroCetus (Milano, Italy) GM-CSF (Leucomax) was purchased from Sandoz (Basel, Switzerland) and IL-4 from R&D systems (Minneapolis, MN, USA). Tritiated thymidine was purchased from Amersham (Little Chalfont, UK). Amphotericin B was purchased from Gibco BRL (Grand Island, NY, USA), PMA and ionomycin were from Sigma Chemical (St .Louis, MO, USA), brefeldin (Golgi Plug) was from BD PharMingen.

Media
RPMI 1640 (Euroclone Ltd, West York, UK) was used supplemented with 100 U/ml kanamycin, 1 mM L-glutamine, 1 mM sodium pyruvate, 1% nonessential amino acids (complete medium). Where indicated, 10% fetal calf serum (FCS, Hyclone, Logan, UT, USA) or 5% human autologous serum were added.

Fungal cells and antigens
The virulent, hyphal-conversion competent C. albicans strain BP, serotype A, from the established type collection of the Istituto Superiore di Sanità (Rome, Italy) was used throughout this study. Germ-tube forms were obtained by culturing yeast cells in germ-tubes growth-inducing medium, at 37°C, as described previously [11 ]. C. albicans MP-F2, a mannoprotein preparation enriched with the immunodominant MP65 antigen was purified as described by Gomez et al. [12 ,13 ].

Glucans
Microparticulate, alkali-acid insoluble C. albicans β-glucan (Glu) was obtained by repeated hot alkali and acid extraction of delipidized fungal cell walls, as described in previous reports [14 ,15 ]. The extraction procedure consisted in a 24-h treatment with NaOH 1% (wt/v) at 100°C, followed by extensive washing (to neutrality) of the insoluble residue, and a second 24-h extraction with 0.5 M acetic acid at 80°C. After repeated washings, the alkali-acid insoluble β-glucan pellet was lyophilized and stored at +4°C. Sterile, endotoxin-free water, labware, and reagents were used throughout the whole procedure. The β-(1, 6) glucan pustulan and the β–(1,3) curdlan were purchased from Calbiochem (La Jolla, CA, USA) and Wako Pure Chemicals (Osaka, Japan), respectively; laminarin, a low molecular weight (MW) β-(1, 3) glucan, from Sigma Chemical.

Analytical determinations
Total polysaccharide content in Glu was measured by the phenol-sulfuric acid assay of Dubois et al. using glucose as the standard [16 ]. Chitin was determined on the basis of the glucosamine content in HCl-hydrolyzed samples, as described previously [17 ,18 ], and monosaccharide composition was analyzed by standard gas-liquid chromatography techniques [15 ]. Endotoxin activity in Glu suspensions administered to monocytes was evaluated by the Limulus Amoebocyte Lysate (LAL) assay (sensitivity, {lambda} = 0.125 endotoxin units/ml, International PBI, Milan, Italy).

1H NMR analyses were performed on a Bruker AVANCE spectrometer operating at 9.4 T (32K complex data points acquisition, 128 FIDs averaging, with a 30° observe pulse, AQ = 4.05 s and 5 s repetition delay). Glu samples were solubilized in 6:1 (vol/vol) Me2So-d6:D2O and analyzed at 80°C, according to Kim et al. [19 ] In some NMR experiments, Glu was subjected to mild digestion by purified β-(1, 3)- or β-(1, 6)-endoglucanases [Zymoliase 100T, (Seikagaku, Tokyo, Japan) or recombinant β-(1, 6)-endoglucanase, respectively (Prozyme, San Leandro, CA, USA)], lyophilized and then analyzed as described above.

Differentiation into DC of Gt-infected, glucan-treated, or control monocytes
Peripheral blood mononuclear cells (PBMC) were purified from heparinized blood obtained by healthy donors who gave their informed consent (Blood Bank of University "La Sapienza," Roma, Italy) on a density gradient (Lymphoprep, Nycomed Pharma AS, Oslo, Norway) as described previously [20 ] and resuspended in PBS+10% FCS. Monocytes were positively sorted using anti-CD14 labeled magnetic beads (MACS, Miltenyi, Cologne, Germany), according to the manufacturer's instructions. Freshly isolated monocytes were resuspended in complete medium +10%FCS containing GM-CSF (50 U/ml), IL-4 (1000 U/ml) and amphotericin B (0.62 µg/ml) at 4x105 cell/ml and added to 6-well culture plate at 3 ml/well for 6 days (MoDC).

In parallel, monocytes were incubated with glucans (β glucan extracted from C. albicans, β-(1, 3) laminarin and curdlan or β-(1, 6) pustulan) at different concentrations (ranging from 100 to 10 µg/ml), or infected with single cell suspensions of C. albicans Gt (MOI = 3:1 fungus:monocyte) and then cultured in RPMI containing GM-CSF, IL-4, and amphotericin B for 6 days. In some conditions, LPS from E. coli (Sigma) at 0.1µg/ml was added in the last 18 h of culture to induce the maturation to Gt-infected, glucan-treated and control cells.

FACS analysis
Cells were stained with the following antibodies: anti-HLA class I, HLA class II, CD1a, CD14, CD40, CD80, CD83, CD86, and anti-chemokine receptor CCR5, CXCR4, and CCR7, all purchased from PharMingen (San Diego, CA, USA). A goat anti-mouse IgM (Southern Biotechnology Association, Birmingham, AL, USA) was used as secondary antibody in CCR7 staining.

Staining of intracellular cytokines in T cells was performed using phycoerythrin (PE)-conjugated rat anti-human IL-4 or IL-10 and fluorescein isothiocyanate (FITC)-conjugated mouse anti-human IFN{gamma} in PerCp-conjugated anti-CD3-positive cells (PharMingen) after fixation and permeabilization with Cytofix/Cytoperm (PharMingen), used according to manufacturer's instructions. Stained cells were analyzed by flow cytometry using a FACScan cytometer (Becton Dickinson, Mountain View, CA, USA) and CellQuest Software (Becton Dickinson). Propidium iodide (Sigma Chemical Co) was used to exclude dead cells.

Naive T cell priming
Decreasing numbers of immature DC (imDC), mature DC (DC+LPS), and DC derived from monocytes infected with Gt or glucan (Glu-MoDC and Gt-MoDC, respectively) were tested for their capacity to induce proliferation of 5x104 allogeneic CD4+ T cells/well purified by indirect magnetic sorting (Miltenyi Biotec) from cord blood mononuclear cells. The proliferative response was measured on day 6 by a 16-h pulse with 3H thymidine (3H-Thy) [21 ]. Some of the T cells were stimulated with 10–7 M PMA and 1µg/ml ionomycin for 5 h, with 2 µg/ml brefeldin A (Golgi Plug) being added during the last 2 hours and then analyzed by flow cytometry for their intracellular cytokine production.

Candida-specific T cell clones and antigen presentation assays
T cell clones (TCC) were derived from PBMC of a DRB1*1101 (DR5) normal donor as described previously [22 ]. Briefly, PBMC were purified from heparinized blood on a density gradient (Lymphoprep, Nycomed Pharma AS, Oslo, Norway) and resuspended in complete medium with the addition of 5% autologous serum, in the presence or absence of 5 µg/ml MP-F2. After 5 and 10 days, 10 and 100 U/ml of IL-2, respectively, were added to the cultures. After five additional days, cultures showing a significant cell growth were considered positive. Cells were counted and cloned by limiting dilution in the presence of 5x105 irradiated PBMC, 1 µg/ml PHA, and 100 U/ml of IL-2. After 10–15 days, growing cultures were expanded in medium containing 100 U/ml IL-2 and finally tested for MP-F2 specificity in a proliferation assay using irradiated autologous PBMC prepulsed or not with MP-F2 at 5 µg/ml. MP-F2-specific TCC were maintained and expanded in culture with 25- 35-day cycles of restimulation with PHA and irradiated PBMC [20 ]. To test the antigen presentation capacity of Glu-MoDC and Gt-MoDC, monocytes isolated from a DR5-positive healthy control were treated with β-glucan, infected with Gt or left untreated and cultured in GM-CSF and IL-4 as described above. After 5 days of culture, MP-F2 (20µg/ml) was added to part of DC derived from noninfected monocytes and to Glu-MoDC. Control DC, Glu-MoDC, and Gt-MoDC used as antigen-presenting cells (APC) were then washed and resuspended for accurate counting. Decreasing numbers (1x104 – 150 cell/well) of APC were plated into 96-well flat-bottomed plates together with TCC (3x104/wt), previously washed out of IL-2. Proliferation was measured as 3H-thymidine incorporation.

Cytokine determination
MoDC, Glu-MoDC, and Gt-MoDC were collected after 5 days of culture, washed, and resuspended with or without LPS (0.1 µg/ml). Supernatants were collected after o/n incubation and frozen until use. Cytokine secretion (IL-10, IL-12 p70, IL-6) was determined using commercially available kits (R&D), according to the manufacturer instructions and expressed in picograms per milliliter.

Statistical analysis
Data were analyzed using the SPSS program (SPSS Inc. Chicago, IL, USA). The statistical significance of the difference between groups of data with a normal distribution was determined by the ANOVA test with Student-Newman-Keulz posttests.


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RESULTS
 
Chemical characterization of Glucan
Crude chemical analysis of the glucan fraction of C. albicans used throughout this study demonstrated that this fraction was almost entirely composed (>97% wt/wt) of exoses and hexosamine, namely glucose accounted for >93% (wt/wt) of the preparation, which also contained a minor amount (3–4% wt/wt) of hexosamine, to constitute chitin, a fungal polysaccharide known to be covalently linked to β-glucan in C. albicans cell wall [23 ]. Mannan, a major component of the outer portion of yeast cell wall was <1% of the fraction, as inferred from mannose content in hydrolized glucan fraction. Protein content was negligible (<0.05% wt/wt) and endotoxin contamination was below the sensitivity threshold of our LAL assay (0.125 endotoxin units/ml). Overall, chemical analysis indicated that the glucan fraction quite entirely consisted of glucan polysaccharide, with a gross composition comparable to that reported by previous authors for the alkali-acid-insoluble β-glucan moiety of C. albicans cell wall [23 ]. According to the prevailing view, this cell wall moiety is made by β-(1,3)-linked glucopyransyl backbone(s) with β-(1,6)-linked side chains of varying length, which greatly influence the overall immunomodulatory properties of β-glucan itself [24 ]. To elucidate the structure of β-glucan(s) in our glucan fraction, 1H NMR analyses were carried out on materials dissolved under conditions preventing β-glucan aggregation, thus making it possible to separate peaks due to all individual 1H NMR signals in C1 position of different glucopyranosyl units (such as H1 protons of the reducing and nonreducing terminals as well as to the backbone (BC) residues). Distinct doublets corresponding to both β-D-(1->6)-linked (at 4.33 ppm) and β-D-(1->3)-linked glucopyranosyl chains (at 4.59 ppm), which practically coincided with those of the glucopyranosyl backbone of pustulan and laminarin, respectively, were detected by this analysis (not shown). The peak area ratio of the above signals was ~1:1, showing that the fraction contained about equal amounts of β-D-(1->3)- and β-D-(1->6) linked glucopyranosyl units. Moreover, analysis of the products of selective digestion of the fraction with β-(1,3) or β-(1,6) endoglycosidase confirmed the coexistence in the fraction of long β-D-(1->6) and β-D-(1->3) chains (E. Iorio et al., unpublished data).

C. albicans β-glucan subverts the differentiation of monocyte into DC
We previously demonstrated that the Y and Gt forms of C. albicans differently interfere with the process of monocyte differentiation into DC. Y-treated monocytes differentiated into macrophages (Y-MoM{phi}), while Gt-treated monocytes into subverted DC (Gt-MoDC) [8 ]. Thus, to test the possible role of β-glucan in the interfererence with human monocyte differentiation, we first analyzed the surface phenotype of DC derived from monocytes treated with the glucan fraction (chemically characterized as above) from C. albicans (Glu-MoDC). DC derived from untreated monocytes (MoDC) with or without LPS treatment at day 5 of culture, served as control. As shown in Fig. 1 Glu-MoDC population was CD14–, but expressed CD86 and CD83 without acquiring CD1a. In striking contrast, the expression of B7.1 (CD80) was markedly reduced, and the median intensity of fluorescence of MHC class II DR molecules was constantly lower than that of LPS treated, mature DC. Overall, the phenotype of Glu-MoDC closely resembled that observed in DC originated from Gt-phagocytosing monocytes (Gt-MoDC, Fig. 1 ), rather than Y-phagocytosing monocytes, which were shown to differentiate into CD14+ macrophage like cells (Y-MoM{phi},) [7 ].


Figure 1
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  		Figure 1. Monocytes treated with β-glucan isolated from Candida albicansdifferentiate into atypical mature dendritic cells (DC) with the same phenotype of DC derived from germ-tubes (Gt)-infected monocytes. Human monocytes from healthy donors were allowed to phagocytose C. albicans Gt forms or C. albicans β-glucan and then induced to differentiate into DC for 6 days in the presence of GM-CSF and IL-4 Gt-MoDC and Glu-MoDC, respectively. Analysis of the indicated surface molecules was performed by flow cytometry. Filled histograms represent LPS-treated Gt-MoDC and Glu-MoDC, while empty histograms represent nontreated cells. Isotypic controls are reported as histograms with dotted lines. Reported data are from one experiment representative of five independent experiments.

A further similarity between Gt-MoDC and Glu-MoDC was observed at the level of cytokine secretion. Both DC populations were unable to produce IL-12 even after LPS stimulation (Fig. 2 ), in sharp contrast with their "mature" pattern of membrane molecule expression, as defined by the CD83 and CD86 expression. Glu-MoDC spontaneously produced IL-6 and low amounts of IL-10, but the secretion of these cytokines was not significantly increased by LPS treatment (Fig. 2) .


Figure 2
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  		Figure 2. Glu-MoDC do not secrete IL-12. IL-12p70, L-10, and IL-6 secretion was analyzed in supernatants of Glu-MoDC. K: noninfected control DC; K+LPS: control DC stimulated overnight with LPS; Glu-MoDC+LPS: Glu-MoDC stimulated with LPS. The asterisk indicates values significantly different from control DC stimulated with LPS, calculated with ANOVA, and Student-Newman-Keuls post hoc test. Results are expressed as means ± SD of three independent experiments.

These data show that C. albicans β-glucan can mimic the effect of the whole Gt form of the fungus in altering monocyte differentiation into DC. Because C. albicans β-glucan is a highly branched polymer consisting of both β-(1,3) and β-(1,6) glucan moieties, we investigated whether a specific molecular configuration was responsible for its subversive effect. Because the two molecular types are covalently linked to one another in fungal β-glucan, making their separation particularly hard [25 ], we resorted to the use of chemical analogs, such as laminarin, a purified and a well-characterized low MW β-(1,3) glucan, the high MW β-(1,3) glucan curdlan or pustulan, a commercial preparation of lichen β-(1,6) glucan. As shown in Fig. 3 , treatment of monocytes with both pustulan and curdlan drastically inhibited acquisition of CD1a expression, while inducing monocyte differentiation into mature DC, as shown by the up-regulation of CD86. On the contrary, treatment with a large range of laminarin concentrations did not interfere with monocyte differentiation into DC. All together, these data suggest that the overall molecular configuration of the β-glucan and its degree of molecular complexity rather than the type of bonding (β-(1,3) or β-(1,6)) were the critical factors to mirror the effects of Gt. Whichever the molecular mechanism, these data indicate that Candida glucan may represent the major constituent of Gt involved in the subversion of monocyte differentiation into DC (see also below).


Figure 3
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  		Figure 3. Treatment with high MW glucans alters monocyte differentiation into DC. Monocytes were treated with both high MW glucans β-(1,6) pustulan and β-(1,3) curdlan or the low MW β-(1,3) laminarin and then induced to differentiate into DC (Pust-MoDC, Curd-MoDC, and Lam-MoDC, respectively). Expression of the indicated surface molecules was analyzed by flow cytometry. Numbers in dot plot panels indicate the percentage of positive cells. Numbers in histogram panels indicate the median of fluorescence intensity. One representative experiment of three performed is shown.

Antigen presentation capacity of Glu-MoDC and Gt-MoDC
We then examined whether and to what extent Glu-MoDC possessed antigen presentation capacity. As shown in Fig. 4A , Glu-MoDC were capable of inducing proliferation of naive CD4+ cord blood T cells (CB-T), and their capacity of alloantigen presentation overlapped that of previously described Gt-MoDC [8 ]. The T cell proliferation data show that Glu-MoDC, as well as Gt-MoDC, were more efficient (less APC required to obtain the same levels of T cell proliferation) and efficacious (judged from maximum level of T cell proliferation induced) than immature DC. However, efficiency of alloantigen presentation by LPS-matured DC was always higher than that showed by Glu-MoDC or Gt-MoDC. On the other hand, Gt-MoDC and Glu-MoDC pulsed with major C. albicans mannoprotein antigens (MP-F2) were shown to be capable of processing a complex antigen and presenting it to specific T cell clones (TCC), since they induced TCC proliferation with an efficiency comparable to that of control MP-F2 pulsed immature DC (Fig. 4B) . These data demonstrate that the subverted differentiation into DC caused by monocyte infection with Gt does not alter the processing and presentation of C. albicans antigens that occur during the differentiation period. Moreover, these data also suggest that glucan does not impair antigen uptake by subverted DC. All together, the implication here is that a functional impairment in the capacity of stimulating naive T cells is associated with the altered phenotype of Glu-MoDC or Gt-MoDC, without consequences on their ability to process and present antigen to memory T cells, such as TCC.


Figure 4
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  		Figure 4. Antigen presentation capacity of Gt-MoDC or Glu-MoDC. (A) Proliferative response of CD4+ naive T lymphocytes isolated from cord blood stimulated with allogeneic immature DCs (DC), LPS matured DC (DC+LPS) or with cells derived from C. albicans β-glucan treated- (Glu-MoDC) or germ tube-infected monocytes (Gt-MoDC). Control DCs, Gt-MoDC and Glu-MoDC were obtained culturing monocytes from the same healthy donor after 6 days of differentiation culture in the presence of GM-CSF and IL-4. Data are from one experiment representative of four. (B) Proliferative response of a CD4+ T cell clone specific for the C. albicans mannoprotein MP65 in response to decreasing number of autologous APC. Monocytes isolated from the same donor enrolled for TCC generation were infected with C. albicans-Gt, treated with β-glucan or left untreated and cultured for 5 days in GM-GSF and IL-4. Cells were then washed and resuspended in complete medium containing 10% FCS in the presence (DC+MP-F2 and Glu-MoDC+ MP-F2) or absence (DC no Ag and Gt-MoDC) of a purified fraction of C. albicans culture supernatant containing MP-65. After overnight incubation, APC were tested with 3x104 TCC/well. Data are from one experiment representative of two.

Functional polarization of naive T cells by Glu-MoDC
Previously, we showed that the capacity of priming naive T lymphocytes does not always correlate with the ability of an APC to induce functional polarization of responder T lymphocytes [7 ,26 ]. Thus, we analyzed cytokine production by alloreactive CB-T primed in vitro by Glu-MoDC. In comparison to CB-T lymphocytes stimulated with LPS-treated DCs, a significantly reduced number of CB-T lymphocytes stimulated with Glu-MoDC secreted IFN{gamma} (Fig. 5 ). Neither IL-4 nor IL-10-producing cells were observed upon stimulation of CB-T with Glu-MoDC (data not shown). Similar results were reproduced with Gt-MoDC (not shown). Overall, these data suggest that DC derived from monocytes that undergo differentiation in the presence of C. albicans Gt or β-glucan are inefficient in generating a strongly polarized Th1 immune response.


Figure 5
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  		Figure 5. Naive T cell primed by Glu-MoDC are not functionally polarized. Flow cytometric analysis of intracellular cytokine (IL-4 and IFN-{gamma}) accumulation in CD4+ naive T lymphocytes isolated from cord blood (CB-T) after coculture with allogeneic APC. The intracellular cytokine accumulation was detected in CD3-positive cells after a 6-days-culture with allogeneic noninfected DCs treated (DC+LPS) or not (imDC) with LPS in the last ~18 h of culture, or cells derived from Candida β-glucan treated monocytes (Glu-MoDC). Numbers report the percentage of cells in the relative quadrant. Data are from one experiment representative of two.


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DISCUSSION
 
In this study, we show that C. albicans β-glucan, composed of complex polymers of β-(1,3)- and β-(1,6)-linked molecular constituents, causes the differentiation of monocytes into a subset of DC with altered phenotype and function. The effect of C. albicans β-glucan could be similarly obtained by high MW curdlan, a β-(1,3) glucan and pustulan, a β-(1,6) glucan, but not by laminarin, a low MW, soluble β-(1,3) glucan, suggesting that molecular complexity is more relevant than type of glucan bonding for the observed effect. Interestingly, the phenotype and function of DC differentiated from C. albicans β-glucan-exposed monocytes (Glu-MoDC) are indistinguishable from those derived from monocytes infected with germ-tubes (Gt) of C. albicans (Gt-MoDC) and were clearly distinguishable from those infected by the yeast form of the fungus, as described previously [7 ], suggesting that β-glucan expressed on Gt cells may be responsible for Gt effect on monocytes.

The in vitro model that we have developed allows the analysis of the phenotypic and functional alterations of DC derived from monocytes infected with a given pathogen or processing its structural components. We suggest that this model may reproduce in vivo situations in which monocytes are recruited into infected, chronically inflamed sites, such as the oral or vaginal mucosa, or other body compartments altered by chronic candidiasis. In this case, monocyte differentiation into DC would occur in the presence of the causative microorganism or its products. This issue is of particular interest since monocytes sense microorganisms through an array of receptors, which transduce signals potentially conflicting with their differentiation program [27 ,28 ].

We previously showed that monocytes that had been exposed to Gt cells develop into mature DC, characterized by a peculiar phenotype and which are not able to prime a Th1 immune response [7 ]. Here, we show that Glu-MoDC share the same functional behavior and phenotype of Gt-MoDC. They lack CD1 molecule expression and present a reduced expression of CD80 and MHC class II molecules; moreover, they do not produce IL-12, nor do they release this cytokine in response to LPS stimulation. Remarkably, the inability to produce IL-12 is not associated with an inability to secrete other cytokines, such as IL-6 and IL-10, demonstrating that these cells are not functionally exhausted.

The most striking functional peculiarity of Glu-MoDC is their capacity of priming naive T cells associated with their noncompetence for T cell polarization. In particular, Glu-MoDC are able to expand T lymphocytes, which remain incapable of secreting IFN-{gamma}, a cytokine that plays a crucial role in anti-Candida defense [3 ,29 ]. Failure to induce polarization does not apparently depend on defective antigen presentation, since Glu-MoDc, as Gt-MoDCs, are perfectly able to stimulate a recall immune response. It may rather result from their unique combination of costimulatory molecule expression and cytokine secretion. This appears to be a distinctive feature of DC derived from pathogen-infected monocytes, as previously shown with mycobacteria-infected monocytes [26 ] (see below).

Thus, it can be hypothesized that interference with DC functions by β-glucan might attenuate Candida-specific Th1 immune responses and ultimately favor [30 ] persistence of the fungus on the host’s mucosa or in infectious foci. In this view, cell wall β-glucan could be regarded as a component of the immunoevasion array possessed by Candida albicans, and the recently described, anti-fungal anti-β-glucan antibodies [31 ] could play their protective role in vivo by hampering this immunoevasive potential.

It is of interest that this component, previously considered to be confined in the inner layers of fungal cell wall, has been recently found to be consistently exposed on the surface of germ-tube and hyphal, but not yeast, Candida cells [31 ,32 ]. This localization is fully compatible with the hypothesis that Gt-induced DC subversion is mostly mediated by β-glucan.

As reported in the literature and confirmed here for the specific preparation used in this investigation, C. albicans β-glucan is constituted by branched polysaccharide chains in both β-(1,6) or β-(1,3) configuration [25 ]. This raised the question of which one of the two configurations was mostly responsible of the observed interference with the DC differentiation process. Most previously reported immunological properties of β-glucans, such as their antitumoral and proinflammatory activities, are strongly dependent on configuration, degree of branching, and intermolecolar association [24 ]. We have here observed that the effects of C. albicans glucan can be mimicked by other high MW glucans with a defined β-(1,6) or β-(1,3) configuration, as pustulan and curdlan, but not by the low MW, β-(1,3) glucan laminarin. This finding suggests that, molecular size and a rather high degree of molecular complexity, including tertiary structure [33 ] is more important than a specific β-(1,6) or β-(1,3) configuration for glucan induction of the phenomenon here described. It is conceivable that high molecular weight glucans exert their effects acting as ligands for receptors present on monocytes, macrophages and DC and sensing microbial molecular patterns [34 ]. Further investigation is required to identify cellular receptors' involvement and the possible modulation of their signaling to counteract the subversive effect of glucans in monocyte differentiation. This investigation will also clarify whether other Gt components (i.e., mannan and its complexes with proteins) could participate in this phenomenon.

Of interest, phenotype and function of Gt-MoDC and Glu-MoDC are intriguingly similar to those of DC derived from monocytes infected with Mycobacterium tuberculosis (Mt-MoDC) [26 ,35 ]. This may imply that different microorganisms and their products engage the same membrane- or phagosome-associated PAMP receptors, leading to a predefined, alternative pathway of monocyte differentiation into DC. The identification of microbial constituents involved in this phenomenon may be instrumental for the identification of such cellular receptors. Studies are in progress to identify the role of signaling by receptors involved in the recognition of Candida albicans and β-glucan, namely Dectin-1 and CR3 [36 ].

Because nanogram concentrations of β-glucan can be found in serum of patients affected by severe fungal infections [37 ], it is reasonable that its concentration in sites of active fungal replication may reach sufficiently high levels to induce subversion in affluent monocytes undergoing DC differentiation in vivo. This might not only apply to Candida-infected, but also to bystander, noninfected monocytes, thus providing a possible, contributory explanation for the general immunosuppression observed in severe primary fungal infection, such as chronic mucocutaneous candidiasis [29 ]. In fact, our data provide some clues that fungal β-glucan, by affecting the process of monocyte differentiation into DC might contribute to the Candida albicans-driven weakening of T cell responses reported in early investigations. Recently, anti-β-glucan antibodies have been shown to mediate the antifungal protection induced by a glucan-protein conjugate vaccine [31 ]. Besides the demonstrated growth-inhibitory potential of these antibodies and their likely opsonic power [31 ], the data reported in this paper suggest that vaccine protection could also, in part, be dependent on antibody capacity to neutralize the subversion effect of fungal β-glucan on monocyte differentiation on DC and effective DC polarization.


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ACKNOWLEDGEMENTS
 
This paper was partially supported by "Progetto Italiano per la lotta contro l’AIDS," grant 50F/G, as well as the collaborative ISS-National Institutes of Health grant 5303.

Received March 14, 2007; revised June 20, 2007; accepted July 6, 2007.


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