Toll-Like Receptor 2 Suppresses Immunity against Candida albicans through Induction of IL-10 and Regulatory T Cells

Fungal infections in general, and acute disseminated candidiasis in particular, are severe infections occurring mainly in immunocompromised hosts. Mortality associated with disseminated candidiasis has changed little despite the availability of new antifungal drugs (1, 2). Despite the importance of Candida albicans in human pathology, relatively little is known of the mechanisms through which this fungus is recognized by the immune cells and triggers the host defense. We have recently shown that the Toll-like receptors (TLR)⁴2 and TLR4 are important recognition receptors for Candida, and we have demonstrated increased susceptibility of TLR4-deficient mice to disseminated candidiasis (3). In human blood cells, TLR2-derived signals were shown to contribute to production of proinflammatory cytokines induced by C. albicans blastoconidia (3). This finding could imply that TLR2 is a receptor involved in anticandidal host defense. These data are sustained by the finding that TLR2 is involved in the recognition of zymosan (a cell wall particle of the yeast Saccharomyces), leading to proinflammatory cytokine production (4).

In murine experimental infection, such as Staphylococcus aureus sepsis or pneumococcal meningitis, absence of TLR2 has been accompanied by increased mortality, although the precise mechanisms of TLR2 involvement in host defense have not been identified (5, 6, 7). Cytokine release, activation of leukocyte migration, and direct antimicrobial action have been implicated as potential defense mechanisms triggered by TLRs (8).

Based on our initial in vitro data, we hypothesized that TLR2 knockout (TLR2^−/−) mice would display a reduced activation of innate immunity during infection with C. albicans, and would prove more susceptible to disseminated candidiasis. However, the results of the present study reveal that the absence of TLR2 leads to increased resistance to candidiasis. We demonstrate that this is associated with decreased release of anti-inflammatory, but not proinflammatory cytokines, improved leukocyte recruitment to the site of infection and candidacidal activity, and decreased numbers of CD4⁺CD25⁺ regulatory T cells (Treg).

TLR2^−/− mice and TLR2^+/+ control littermates (20–25 g, 6–8 wk old) were kindly provided by Tularik (San Francisco, CA). TLR4-deficient ScCr mice were from a local colony at Nijmegen University, and control TLR4-competent C57BL/10J mice were obtained from The Jackson Laboratory (Bar Harbor, ME). The mice were fed sterilized laboratory chow (Hope Farms, Woerden, The Netherlands) and water ad libitum. The experiments were approved by the ethics committee on animal experiments of Nijmegen University.

C. albicans UC 820, a strain well described earlier (9), has been used in all experiments. A lethal experimental model of disseminated candidiasis was used to assess mortality, in which TLR2^−/− and TLR2^+/+ mice were injected i.v. with C. albicans (either 5 × 10⁶ or 5 × 10⁵ CFU/mouse) in a 100 μl vol of sterile pyrogen-free PBS. Survival was assessed daily for 14 days.

For the assessment of fungal growth in the organs, a nonlethal experimental model of disseminated candidiasis was used, in which TLR2^−/− and TLR2^+/+ mice were injected i.v. with C. albicans (1 × 10⁵ CFU/mouse). In the nonlethal model, subgroups of five animals were killed on day 1 or 7 of infection. To assess the tissue outgrowth of the microorganisms on these days, the liver, the left kidney, and the brain of the sacrificed animals were removed aseptically, weighed, and homogenized in sterile saline in a tissue grinder. The number of viable Candida cells in the tissues was determined by plating serial dilutions on Sabouraud dextrose agar plates, as previously described (10). The CFU were counted after 24 h of incubation at 37°C, and expressed as log CFU/g tissue. From the same animals, the right kidneys were fixed in formaldehyde (4%) and embedded in paraffin, and serial sections were examined microscopically after staining with periodic acid Schiff and H&E.

To investigate the recruitment of PMN and monocytes/macrophages at the site of Candida infection, groups of five TLR2^−/− and TLR2^+/+ mice were injected i.p. with 10⁷ C. albicans organisms, in a volume of 100 μl. The experiments on leukocyte recruitment were performed using heat-killed C. albicans, and not the live microorganisms, to avoid the bias induced by the differential candidacidal function of the leukocytes in the knockout mice and the wild-type controls. In pilot experiments, the maximum PMN infiltration after i.p. injection of heat-killed Candida was found after 4 h, whereas the maximal monocyte/macrophage infiltration was found after 72 h. After 4 or 72 h, peritoneal cells from TLR2^−/− and TLR2^+/+ mice were collected in sterile saline containing 0.38% sodium citrate, and the total cell number was counted in a hemocytometer. The percentage and the absolute numbers of neutrophils and macrophages were determined in Giemsa-stained cytocentrifuge preparations.

Either resident peritoneal macrophages or exudate peritoneal PMN were obtained, and phagocytosis and killing were performed by a modification of a method described earlier (11). Exudate peritoneal phagocytes from groups of five TLR2^−/− and TLR2^+/+ mice were elicited by an i.p. injection of 10% proteose peptone. Cells were collected in separate sterile tubes by washing the peritoneal cavity with 4 ml of ice-cold PBS containing 50 U/ml heparin, 4 h (65 ± 11% PMN) or 72 h (84 ± 6% macrophages) after injection. Phagocytes were centrifuged (10 min; 3600 rpm; 2250 × g), counted in a hemocytometer, and resuspended in RPMI 1640 Dutch modification (with 20 mM HEPES, without glutamine; ICN Biomedicals, Eschwege, Germany) supplemented with 5% heat-inactivated FCS, 1% gentamicin, 1% l-glutamine, and 1% pyruvate. The processes of phagocytosis and intracellular killing were studied in an adherent monolayer of phagocytes, as previously described (12). The percentage of phagocytized microorganisms was defined as (1 − (number of uningested CFU/CFU at the start of incubation)) × 100.

Killing of C. albicans by phagocytes was assessed in the same monolayers (12). After removal of the nonphagocytized Candida blastoconidia, 200 μl of culture medium, consisting of Sabouraud in MEM (50% v/v), was added to the monolayers. After 3 h of incubation at 37°C in air and 5% CO₂, the wells were gently scraped with a plastic paddle and washed with 200 μl of distilled H₂O to achieve lysis of phagocytes. The percentage of yeast killed by the phagocytes was determined as follows: (1 − (CFU after incubation/number of phagocytized CFU)) × 100. Phagocyte-free incubations of blastoconidia were included as a control for yeast viability.

Extracellular killing of C. albicans hyphae was determined by a modification of the XTT (2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide) (Sigma-Aldrich, St. Louis, MO) dye assay, as described elsewhere (13).

Groups of five TLR2^−/− and TLR2^+/+ mice were killed, and resident peritoneal macrophages were harvested by injecting 4 ml of sterile PBS containing 0.38% sodium citrate (11). After centrifugation and washing, the cells were resuspended in RPMI 1640 containing 1 mM pyruvate, 2 mM l-glutamine, 100 μg/ml gentamicin, and 2% fresh mouse plasma (culture medium). Cells were cultured in 96-well microtiter plates (Greiner, Alphen a/d Rijn, The Netherlands) at 10⁵ cells/well, in a final volume of 200 μl. The cells were stimulated with either control medium or heat-killed (1 h, 100°C) C. albicans at 1 × 10⁷ CFU/ml. After 24 h of incubation at 37°C, the plates were centrifuged (500 × g, 10 min), and the supernatant was collected and stored at −80°C until cytokine assays were performed.

For the assessment of IFN-γ and IL-10 production capacity, primed spleen cells from mice on day 7 of infection with 1 × 10⁵ CFU/mouse of C. albicans were stimulated in vitro with heat-killed Candida. Spleen cells were obtained by gently squeezing spleens in a sterile 200-μm filter chamber. Microscopic examination of Giemsa-stained cytospin preparations showed that these cells consisted of 95% lymphocytes, 2% monocytes, and 3% granulocytes. The cells were washed and resuspended in RPMI 1640 and counted in a Bürker counting chamber, and the number was adjusted to 5 × 10⁶/ml. One milliliter of the cell suspension was stimulated with 1 × 10⁷ heat-killed C. albicans yeasts (E:T ratio, 2:1). Measurement of IFN-γ and IL-10 concentrations was performed in supernatants collected after 48 h of incubation at 37°C in 5% CO₂ in 24-well plates (Greiner).

IL-1α, IL-1β, and TNF-α were determined by specific RIAs (detection limit 20 pg/ml), as previously described (14). IFN-γ and IL-10 concentrations were measured by a commercial ELISA (BioSource International, Camarillo, CA; detection limit 16 pg/ml), according to the instructions of the manufacturer.

To assess the presence of Treg cells in the circulation of TLR2^−/− and TLR2^+/+ mice, 50 μl of blood was collected from the tail of 10 deficient and 10 control mice. Analysis of cell surface markers on blood lymphocytes was performed using a FACSCalibur (BD Biosciences, Alphen aan de Rijn, The Netherlands) and CellQuest software. Blood (50 μl) was collected in heparin-coated tubes, and erythrocytes were lysed using standard laboratory protocols. The remaining lymphocytes were washed and incubated with anti-CD25 FITC (clone 7D4; BD PharMingen, San Diego, CA) and anti-CD4 allophycocyanin (BD PharMingen) and subsequently analyzed. Data are represented as the percentage of CD4⁺CD25⁺ cells of the total CD4⁺ T cell population.

In addition to TLR2^−/− mice, we also investigated the role of TLR4 in the generation of Treg cells, by assessing the CD4⁺CD25⁺ T cell population in TLR4-deficient ScCr mice and control C57BL/10J mice.

Spleens were mashed into single cell suspensions using a nylon filter. CD4⁺ T cells were presorted by magnetic purification using anti-CD4 (L3T4) microbeads and positively separated on large separation columns (both from Miltenyi Biotec, Auburn, CA). CD4⁺CD25⁻ and CD4⁺CD25⁺ T cell subsets were obtained by flow cytometry purification of the presorted T cells: CD4 cells were stained with allophycocyanin-conjugated CD4 mAb (BD Biosciences) and FITC-conjugated CD25 (PC61; BD Biosciences). Cell sorting was performed on a Coulter Elite cell sorter (Corixa, Seattle, WA). The purity of each cell preparation was >95%. Purified CD4⁺ T cell subsets were subsequently cultured for 3 days in complete medium (Iscove’s IMDM, 9% FCS) supplemented with, if indicated, 0.5 μg/ml highly purified Escherichia coli LPS (Sigma-Aldrich), 10 μg/ml peptidoglycan (Sigma-Aldrich), or 5 Cetus U/ml IL-2. After 3 days of culture, all surviving cells were harvested using PBS/1 mM EDTA, stained for CD4 and CD25 using the aforementioned Abs, and subsequently analyzed on a flow cytometer. Data indicate the average number and SEM of all surviving CD4⁺ cells of triplicate wells.

The possible effect of Treg cells on the anticandidal host defense was investigated by depletion of Treg cell population using a protein G-purified rat anti-mouse CD25 (300 μg, clone PC61; American Type Culture Collection, Manassas, VA) administered by i.p. injection 10 days before infection with 1 × 10⁵ CFU/mouse C. albicans. Rat IgG was used as control in a separate group of mice. On days 1 and 7 of infection, Candida outgrowth in the organs of mice receiving anti-CD25 or control Abs was assessed, as described above.

The mortality in the two groups was compared by Kaplan-Meier logarithm of rank test. The differences in the other parameters between groups were analyzed by Mann-Whitney U test, and where appropriate by Kruskal-Wallis ANOVA test. The level of significance between groups was set at p < 0.05. All experiments were performed at least twice, and the data are presented as cumulative results of all experiments performed.

TLR2 is one of the cellular receptors engaged by C. albicans (3). To determine the role of TLR2 in the host defense against C. albicans, we infected TLR2^−/− mice with Candida and compared their susceptibility to infection with that in wild-type mice. Surprisingly, TLR2^−/− mice survived longer after injection of a lethal amount of C. albicans (5 × 10⁶ CFU/mouse) than control mice (p < 0.05, Fig. 1,A). All deaths occurred during the first 10 days of infection, and no mortality was registered thereafter. When the mice were injected with a lower inoculum of 5 × 10⁵ CFU/ml, no mortality was recorded in either group (data not shown). The target organ for C. albicans growth in murine candidiasis, the kidneys, showed no differences in fungal outgrowth between deficient and control mice on day 1. However, on day 7 of infection, TLR2^−/− mice were found to have a 100-fold decreased load of C. albicans in their kidneys (p < 0.01), compared with TLR2^+/+ mice (Fig. 1,B). A similar tendency, although not significant, was apparent for the outgrowth of C. albicans in the liver and brain (Fig. 1,B). Histology of the kidneys showed no differences in the inflammatory infiltrate and the degree of Candida growth on day 1 of infection. In contrast, on day 7, control TLR2^+/+ mice displayed invasive growth of large amounts of C. albicans, especially in the pyelum, compared with larger inflammatory infiltrates and fewer Candida organisms in the TLR2^−/− mice (Fig. 2).

To investigate the recruitment of PMN and macrophages to the site of a C. albicans infection, groups of TLR2^+/+ and TLR2^−/− mice were injected i.p. with 10⁷ heat-killed C. albicans microorganisms, and exudate peritoneal neutrophils or macrophages were harvested 4 and 72 h later, respectively. As shown in Fig. 3 A, there was significantly more influx of monocytes/macrophages into the peritoneal cavity of TLR2^−/− mice than in that of TLR2^+/+ mice (p < 0.05), whereas the recruitment of PMN did not differ between the groups.

Phagocytosis of C. albicans by PMN or macrophages of TLR2^−/− mice was similar to that of TLR2^+/+ mice (Fig. 3,B). Both the intracellular killing of Candida blastoconidia and the extracellular killing of Candida hyphae by macrophages of TLR2^−/− mice were better than by macrophages of control TLR2^+/+ mice (Fig. 3, C and D). In contrast, neutrophils of TLR2^+/+ and TLR2^−/− mice were equally potent to kill both conidia and hyphae of Candida (Fig. 3, C and D).

To investigate the role of TLR2 in the stimulation of cytokines by C. albicans, we stimulated peritoneal macrophages of TLR2^−/− and control TLR2^+/+ mice with heat-killed Candida blastospores in vitro. Cytokine production by unstimulated macrophages of both mouse strains was below the detection limit for all cytokines studied (data not shown). Candida-stimulated production of TNF, IL-1β, and IL-6 was only 20–30% reduced in macrophages isolated from TLR2^−/− mice compared with control TLR2^+/+ mice (p > 0.05, Fig. 4,A). In contrast, the synthesis of the anti-inflammatory cytokine IL-10 was significantly lower in TLR2^−/− macrophages stimulated with Candida blastospores than in controls (42 ± 16 vs 98 ± 22 pg/ml, p < 0.05). Moreover, the IL-10 production by spleen cells isolated from C. albicans-infected TLR2^−/− mice on day 7 of infection was only 25–30% of that by TLR2^+/+ spleen cells when stimulated with Candida, whereas IFN-γ release was 3-fold higher than in TLR2^+/+ splenocytes (Fig. 4 B).

Recent data have implicated a role for Treg cells in the immune response to C. albicans (15), and it has been suggested that IL-10-mediated signals are involved in the generation of Treg cells (15). In contrast to TLR4^−/− mice, which showed normal numbers of Treg cells, there was a 50% decrease of the Treg population in uninfected TLR2^−/− mice compared with their wild-type littermates (p < 0.05; Fig. 5 A). There were no differences either in the total leukocyte counts of TLR2^+/+ and TLR2^−/− mice, or in the absolute numbers of CD4⁺ T lymphocytes (21.1 vs 19.7% of the total lymphocyte count; in absolute numbers, 1234 ± 342 vs 1102 ± 298 cells/mm³). This resulted in significantly decreased numbers of Treg cells in the TLR2^−/− mice (TLR2^+/+ vs TLR2^−/− mice: 10.5 vs 5.4%; 129 ± 35 vs 64 ± 16 cells/mm³, p < 0.05).

To establish the link between TLR2 and the number of Treg cells, we analyzed T cell survival of purified CD4⁺CD25⁺ and CD4⁺CD25⁻ subsets from wild-type animals. Three days of culture in the presence of IL-2 resulted in a 2-fold increase in T cell survival as compared with the medium control for both CD25⁺ (Fig. 5,B), as well as CD25⁻ T cells (data not shown). Interestingly, culture of T cells in medium supplemented with the TLR2 ligand peptidoglycan also improved Treg cell survival (Fig. 5,B). Surprisingly, the addition of highly purified LPS did not have a significant effect, indicating that in this particular setting, stimulation through TLR2, but not TLR4, prolongs Treg cell survival in vitro (Fig. 5 B).

Treg cells are known to produce large quantities of IL-10 and to decrease cellular defense, and both mechanisms may decrease innate resistance to C. albicans. We tested this hypothesis by depletion of Treg cells with an anti-CD25 mAb. In previous experiments, anti-CD25 Ab administration resulted in the depletion of ∼90% of the CD4⁺, CD25⁺ T cell population. As resting conventional T cells (both CD4 and CD8 T cells) are CD25 negative, the majority of the depleted CD25-expressing T cells will have the suppressor phenotype. At this moment, CD25 is the best cell surface marker for Treg in naive mice. As shown in Fig. 5 C, depletion of Treg cells in normal TLR2^+/+ mice resulted in a 10-fold decrease of fungal outgrowth in the kidneys on day 7, but not day 1 of infection, showing the crucial role of Treg cells in suppressing host defense in normal TLR2^+/+ mice.

Our results show that the absence of TLR2-mediated signaling results in an increased resistance to disseminated candidiasis. Whereas production of TNF and IL-1 is normal, IL-10 synthesis is severely impaired in TLR2^−/− mice. The decreased production of IL-10 is associated with an increased production of IFN-γ, a diminished generation of Treg cells, and improved candidacidal function of macrophages. This implies that C. albicans evades host defense through TLR2-mediated signals.

We have recently demonstrated that recognition of the C. albicans cell wall Ags involves both TLR2 and TLR4, and the absence of TLR4 signals increases susceptibility to disseminated candidiasis (3). Which C. albicans cell wall components stimulate TLR2 and TLR4 is unknown as yet, although there are indications that a mannan component is recognized by TLR4 (16). Because blockade of TLR2 in human mononuclear cells resulted in a 40–50% decrease of the production of proinflammatory cytokines in vitro (3), our initial hypothesis was that a similar defect in TLR2^−/− mice would lead to an increased susceptibility to systemic C. albicans infection, as both TNF and IL-1 are central for an effective antifungal defense (17). Surprisingly, the production of TNF and IL-1 was only marginally impaired in TLR2^−/− mice, suggesting that either other TLRs or receptors other than TLR such as mannose receptor (18), β-glucan receptor (19), complement receptor 3 (20), or dendritic cell-specific intercellular adhesion molecule-grabbing nonintegrin (21) are also involved in Candida-induced proinflammatory cytokine release. Moreover, the slight decrease in the TNF and IL-1 release in TLR2^−/− mice did not have functional consequences, implying that the remaining production of these cytokines is sufficient for effective host defense. It is also important to mention the cooperation between TLR2 and the β-glucan receptor in recognition of C. albicans (22, 23), and it is tempting to speculate on the role of β-glucan receptors in the findings described in this work.

The increased resistance to disseminated candidiasis was accompanied by increased capacity of TLR2^−/− monocytes to migrate to the site of infection and to kill Candida, whereas these functions of neutrophils were similar in the control and TLR2-deficient mice. This differential effect of TLR2 deficiency on the function of macrophages and neutrophils is in line with the relatively late effect on the fungal outgrowth in the kidneys (day 7), as neutrophils are the main cell population involved in the anticandidal defense during the first days of infection, and macrophages in the later phase.

To investigate the mechanisms behind the enhanced function of macrophages in the TLR2^−/− mice, we hypothesized that TLR2-mediated signals induce a suppressive signal, which is absent in the knockout mice. IL-10 is known to have strong inhibitory effects on the defense against C. albicans infection, as treatment of mice with anti-IL-10 Abs induces protection (24), and IL-10^−/− mice display an increased resistance to disseminated candidiasis (25). Indeed, C. albicans-stimulated IL-10 production by either peritoneal macrophages from naive TLR2^−/− mice or spleen cells from Candida-primed TLR2^−/− mice was severely impaired. Moreover, this was accompanied by increased IFN-γ production, very likely secondary to the absence of IL-10. These data indicate that TLR2 mainly induces anti-inflammatory signals during invasive candidiasis, and this may represent a mechanism to evade host defense. This new paradigm is sustained by the findings of in vitro studies suggesting that TLR2 mediates signals prone to induce Th2-type responses (26), as well as a recent study showing that Yersinia enterocolitica uses TLR2-dependent IL-10 release as a mechanism of immunosuppression. In accordance with this observation, TLR2^−/− mice are more resistant to Y. enterocolitica infection (27). However, TLR2 seems to provide beneficial signals in other types of infection such as staphylococcal sepsis (5) or pneumococcal meningitis (6, 7), although the mechanisms responsible for the protection in these models are unclear.

In addition to its direct inhibitory effects on the function of macrophages and neutrophils, recent data suggest that IL-10 also plays a central role in the shape of adaptive immunity. It has been shown recently that dendritic cell-derived IL-10 is necessary for proper development of a subset of CD4⁺CD25⁺ T lymphocytes (Treg) involved in regulation of effector T lymphocytes (15); in turn, secretion of IL-10 and TGF-β by Treg cells is responsible for their effects on innate and adaptive immunity (28). Because we observed a strong defect in the release of IL-10 in the TLR2^−/− mice, we have investigated both the presence of Treg cells in their circulation and the role of these cells during disseminated candidiasis. In contrast to TLR4^−/− mice, which displayed a normal population of Treg cells, TLR2^−/− mice displayed a 50% decrease in the Treg cell population. In addition, TLR2 ligation by peptidoglycan enhanced survival of Treg cells, sustaining the hypothesis that TLR2-mediated signals are crucial for the homeostasis of Treg cells. We could further demonstrate a deleterious role of these cells during disseminated candidiasis by depletion of CD25⁺ cells in normal mice: this led to increased resistance to C. albicans infection, as shown by the 10-fold decrease of the fungal outgrowth in the kidneys.

The demonstration of decreased IL-10 production, a reduced Treg cell population in TLR2^−/− mice, and beneficial effects of Treg cell depletion on disseminated candidiasis has two important conceptual consequences. First, these data strongly suggest that TLR2-mediated signals, very likely through IL-10 production, are crucial for the generation of Treg cells. This hypothesis is sustained by recent studies demonstrating that IL-10 is needed for generation of Treg cells during a mucosal model of Candida infection (15). Along the same line, TLR2-mediated signals induced by schistosomal lyso-phosphatidylserine lead to the development of Treg cells (29). Based on our data and the latter studies, we propose that TLR2-mediated IL-10 release is a stimulatory signal for generation and function of Treg cells. Others have described a role for the TLR4/TLR9-mediated IL-6 production in rendering CD4⁺ cells unresponsive to the Treg-mediated immunosuppression (30). Thus, different TLRs may modulate the adaptive immune response through either stimulation or inhibition of Treg cell functions. Despite the immunosuppressive effects during disseminated candidiasis, Treg cells have protective effects in other infections such as experimental models of pneumococcal meningitis, in which mortality is induced by overwhelming inflammation (6). In addition, Treg cells have proved to be beneficial in mucosal and cutaneous infections, and are essential components of the memory-protective immunity to C. albicans (15) or Leishmania major infection (31). CD4⁺CD25⁺ Treg cells are thought to be crucial in maintaining resistance to reinfection through suppression of effector CD4⁺CD25⁻ T cells. Thereby, the latter cells are no longer able to eliminate commensal microorganisms from sites of colonization (31). Conversely, our results could imply that the effects of Treg cells are deleterious when a pathogen subsequently penetrates the mucosa and disseminates through the bloodstream.

In addition, our data provide an answer to the paradox between the concept that Th1 cytokines are beneficial to host defense, and the observation that nude mice and mice depleted of CD4⁺ cells are relatively resistant to disseminated candidiasis (32, 33, 34). Our finding that selective depletion of CD4⁺CD25⁺ Treg cells beneficially influences the outcome of disseminated candidiasis may explain the increased resistance of nude (no T cells) and CD4⁺-depleted mice (including CD4⁺CD25⁺ Treg) to Candida infection (32, 33, 34). Of note, it has been demonstrated that depletion of CD8⁺ cells has no effects on the susceptibility for infection (34).

In conclusion, TLR2^−/−-deficient mice are relatively resistant to a disseminated infection with C. albicans. This reduced susceptibility to candidiasis is associated with impaired IL-10 production and a decreased number of Treg cells in TLR2 knockout animals, accompanied by more IFN-γ production and effective antifungal properties of TLR2^−/− macrophages. Thus, C. albicans evades host defense through TLR2-derived signals, and this represents a novel pathogenetic mechanism in fungal infections.