By mediating tryptophan catabolism, the enzyme indoleamine 2,3-dioxygenase (IDO) has a complex role in immunoregulation in infection, pregnancy, autoimmunity, transplantation, and neoplasia. We hypothesized that IDO might affect the outcome of the infection in mice infected with Candida albicans by virtue of its potent regulatory effects on inflammatory and T cell responses. IDO expression was examined in mice challenged with the fungus along with the consequences of its blockade by in vivo treatment with an enzyme inhibitor. We found that IDO activity was induced at sites of infection as well as in dendritic cells and effector neutrophils via IFN-γ- and CTLA-4-dependent mechanisms. IDO inhibition greatly exacerbated infection and associated inflammatory pathology as a result of deregulated innate and adaptive/regulatory immune responses. However, a role for tryptophan catabolism was also demonstrated in a fungus-autonomous fashion; its blockade in vitro promoted yeast-to-hyphal transition. These results provide novel mechanistic insights into complex events that, occurring at the fungus/pathogen interface, relate to the dynamics of host adaptation to the fungus. The production of IFN-γ may be squarely placed at this interface, where IDO activation probably exerts a fine control over fungal morphology as well as inflammatory and adaptive antifungal responses.

Protective immunity to the fungus Candida albicans is mediated by Ag-specific Th1 cells (1). Regulation is an absolute requirement for this fungus to ensure protective immunity in the absence of immune pathology, because the organism also behaves as a commensal of the human gastrointestinal and vaginal tracts (2). To limit the pathologic consequences of excessive inflammatory and cell-mediated reactions, the immune system resorts to a number of protective mechanisms. Early studies implicated a balance between protective Th1 and pathogenic Th2 responses as a major regulator of immunity to the fungus (3). Paradoxically, some Th2 cytokines, such as IL-4 (4), IL-10 (5), and TGF-β (6), were found to be required for the development and maintenance of Th1-mediated antifungal protection. In addition to the Th1/Th2 balance, it has recently been shown that the occurrence of long-lasting antifungal protection is dependent on the presence of CD4+CD25+ regulatory T cells (Treg)3 that, by negatively regulating anticandidal Th1 reactivity, limit exaggerated inflammatory responses (2). Although capable of efficiently restricting fungal growth, mice devoid of Treg were found to experience inflammatory pathology and to be incapable of resisting reinfection. Because the generation of CD4+ CD25+ Treg requires IL-10-producing, Candida-activated dendritic cells (DCs), this indicates a pivotal role for the innate immune system in determining the magnitude and quality of the subsequent adaptive immune response.

Recent studies have demonstrated a crucial role for tryptophan catabolism and kynurenine production in the induction of peripheral tolerance (7, 8). The enzyme indoleamine 2,3-dioxygenase (IDO) is an intracellular heme-containing enzyme that catalyzes the initial and rate-limiting step in tryptophan degradation along the so-called kynurenine pathway (9). IDO is widely expressed in a variety of human tissues as well as in macrophages and DCs and is induced in inflammatory states by IFN-γ and other proinflammatory cytokines. The production of IFN-γ and IDO induction represent important antimicrobial mechanisms (10). However, recent work has demonstrated a complex and crucial role for IDO in immunoregulation during infection, pregnancy, autoimmunity, transplantation, and neoplasia (8, 11). Local depletion of tryptophan and the production of proapoptotic kynurenines are among the mechanisms potentially responsible for the multiple activities observed after IDO induction, including effects on lymphocyte proliferation and survival (12). In this regard, it has recently been shown that IDO expression is regulated by environmental factors expressed by T cells in the form of ligands and cytokines (12, 13, 14).

Based on those previous results, we have hypothesized that IDO might contribute to disease course in mice with C. albicans infection, because it represents an important mechanism that regulates inflammatory and T cell responses. To explore this possibility, we assessed IDO functional expression in mice infected with C. albicans as well the consequences of its inhibition by treatment with a specific competitive inhibitor, 1-methyl-d,l-tryptophan (1-MT). We found that the production of IFN-γ and IDO activation are crucial elements at the host/pathogen interface, exerting a fine control over fungal morphology as well as inflammatory and adaptive antifungal responses.

Female BALB/c (H-2d) mice, 8–10 wk old, were purchased from Charles River Breeding Laboratories. Breeding pairs of homozygous B7-1-, B7-2- (2), and IFN-γ-deficient mice, raised on a BALB/c background (13), were bred under specific-pathogen free conditions at the breeding facilities of University of Perugia (Perugia, Italy). Procedures involving animals and their care were conducted in conformity with National and Perugia University animal care committee guidelines.

Isogenic strains of C. albicans, obtained by mutagenesis in vitro and capable (Vir13), or not (Vir3), of yeast-to-hyphal transition, as assessed by germ-tube formation in vitro, were used (14). Because the hyphal strain is capable of yeast-to-hyphal transition in vitro, whereas the yeast strain is not, the two strains were used as sources of hyphae and yeasts, respectively (15). For hyphae, cells were allowed to germinate by culture at 37°C in 5% CO2 for 2 h in RPMI 1640 medium (by that time, >98% of cells had germinated). For yeasts, the cells were harvested at the end of the exponential phase of growth, centrifuged, and resuspended in the above medium. The C. albicans highly virulent Vir13 (hereafter referred to as Candida hyphae) and the low virulence Vir3 (hereafter referred to as Candida yeasts) were i.v. injected (106 yeasts or 5 × 105 hyphae) for primary disseminated infection. For primary gastrointestinal (GI) infection, 108 hyphae were administered intragastrically (i.g.), and for secondary infection, mice with the GI infection were i.v. reinfected with 106 hyphae 14 days later (secondary infection). Quantification of fungal growth in the organs of infected mice was performed by plating serial dilutions of homogenized organs in Sabouraud dextrose agar (Difco), and the results (mean ± SE) were expressed as CFU per organ. Mice succumbing to fungal challenge were routinely necropsied for histopathological confirmation of candidiasis. Histology was performed on paraffin-embedded tissue sections (3–4 μm thick) stained with periodic acid-Schiff as previously described (2). 1-MT (Sigma-Aldrich), a known competitive inhibitor of IDO (16, 17), was dissolved in water and adjusted to pH 9.9 before use. Mice were given 1-MT in the drinking water (1 mg/ml) beginning the day of infection or reinfection and continuing until death. Candida yeasts and hyphae were exposed at 37°C in 5% CO2 in RPMI 1640 to 10 μM 1-MT with or without an excess of tryptophan (100 μM) for 2, 8, or 24 h before visualization of fungal morphology by light microscopy.

DCs were purified from Peyer patches (PP-DCs) by magnetic cell sorting with microbeads (Miltenyi Biotec) conjugated to hamster anti-mouse CD11c mAbs (clone N-418) as previously described (2). Consistent with previous reports (2), myeloid, lymphoid, and double-negative DCs were present in the PP-DCs on FACS analysis (data not shown). For cytokine determination, PP-DCs were incubated with yeasts or hyphae for 24 h at 37°C in the presence or the absence of 1-MT (10 μM/ml). CD11b+Gr-1+ polymorphonuclear neutrophils (PMNs) were positively selected with magnetic beads from the spleens of infected mice or the peritoneal cavities of uninfected mice 8 h after i.p. injection of 1 ml of endotoxin-free 10% thioglycolate solution. Endotoxin was depleted from all solutions with Detoxi-gel (Pierce). On FACS analysis, Gr-1+ PMNs were >98% pure and stained positively for the CD11b myeloid marker. CD4+ T cells were purified from mesenteric lymph nodes (MLN) as previously described (2).

For double staining, MLN cells were sequentially reacted with saturating amounts of FITC-conjugated anti-CD4 mAb and PE-conjugated anti-CD25 (PC61; BD Pharmingen). Before staining with the relevant mAb, cells were incubated at room temperature with 5 μg of anti-FcγR mAb (2.4G2; BD Pharmingen). Cells were analyzed with a FACScan flow cytofluorometer (BD Biosciences) equipped with LYSIS II software. Nonviable cells were excluded from analysis by standard procedures involving propidium iodide and narrow forward angle light scatter gating. Control staining of cells with irrelevant Ab was used to obtain background fluorescence values. Data are expressed as the percentage of positive cells over the total number of cells analyzed.

The levels of IL-4, IL-10, IL-12 p70, IL-6, and TNF-α in culture supernatants were determined by ELISA (R&D Systems). Cytokine titers were calculated by reference to standard curves constructed with known amounts of recombinant cytokines (BD Pharmingen). The detection limits (picograms per milliliter) of the assays were <3 for IL-4, <8 for IL-10, <16 for IL-12 p70, < 39 for IL-6, and <10 for TNF-α. IFN-γ- and IL-4-producing CD4+ T cells were enumerated by ELISPOT assay as previously described (15). Purified CD4+ T cells were cultured (105 cells/well) in complete medium (RPMI 1640 with 10% FCS, 50 mM 2-ME, and 50 mg/ml gentamicin sulfate) for 18 h in 96-well plates previously coated with rat anti-murine R4-6A-2 (for IFN-γ) and BVD4-1D11 (for IL-4) mAbs. Biotinylated AN-18.17.24 (for IFN-γ) and BVD6-24G2 (for IL-4) mAbs were used as the detecting reagents (BD Pharmingen). The enzyme used was avidin-alkaline phosphatase conjugate (Vector Laboratories), and the substrate was 5-bromo-4-chloro-3-indolyl phosphate-p-toluidine salt (Invitrogen Life Technologies). Results are expressed as the mean number of cytokine-producing cells (±SE) per 105 cells, calculated using replicates of serial 2-fold dilutions of cells. For intracellular staining, CD25+CD4+ T cells purified from the MLN were restimulated with PMA (1 mg/ml; Sigma-Aldrich) and ionomycin (50 ng/ml; Sigma-Aldrich) and stained for intracellular cytokines using Cytofix/Cytoperm kits (BD Pharmingen) as previously described (18). Cells were analyzed with a FACScan flow cytofluorometer equipped with LYSIS II software.

For phagocytosis, PMNs were incubated at 37°C with unopsonized Candida yeasts for 15 min, and the percentage of internalization was calculated on Giemsa-stained preparations (19). For fungicidal activity, PMNs were incubated with unopsonized yeasts and hyphae (at an effector to fungal cell ratio of 10:1) for 60 (conidia) or 120 (hyphae) min at 37°C. For the candidacidal assay, the percentage of CFU inhibition (mean ± SE) was determined as: percentage of colony formation inhibition = 100 − (CFU experimental group/CFU control cultures) × 100. To assess the damage to the hyphae, viability staining with the fluorescence probe FUN-1 (Molecular Probes Europe) was examined in a Fluorescence Microplate Reader (Titertek II; Flow Laboratories) at 485 nm excitation/620 nm emission (19). As controls, hyphae were incubated without cells and were treated with 96% ethanol. PMN production of reactive oxidant intermediates was determined by quantifying the release of superoxide anion (O2) in the culture supernatants (at 60 min for both yeasts and hyphae) through the measure of the superoxide dismutase-inhibitable reduction of cytochrome c (19). For O2, A550 was measured in a Microplate Reader (model 550; Bio-Rad), and the results were expressed as nanomoles of O2 per 106 cells. For apoptosis, PMNs were exposed to fungi for 5 and 24 h before suspension in 0.3 ml of hypotonic iodide solution (50 μg/ml propidium iodide (PI) in 0.1% sodium citrate plus 0.1% Triton X-100; Sigma-Aldrich) at 4°C in the dark for 1 h as previously described (12). The PI fluorescence of individual nuclei was measured by flow cytometry. The percentage of apoptotic nuclei (subdiploid DNA peak in the DNA fluorescence histogram) was calculated with specific FACScan research software (LYSIS II). 1-MT (10 μM, as suggested by initial experiments) was present throughout the assay. PMNs were also assessed after exposure in vitro to IFN-γ (200 U/ml) or CTLA-4-Ig (40 μg/ml) for 5 h, as suggested by initial experiments. CTLA-4-Ig consisted of a fusion protein between the extracellular domain of mouse CTLA-4 and the Fc portion of a mouse IgG3 Ab, as previously described (20). For experiments involving the fusion protein, control treatment consisted of native IgG3 or its Fc portion, produced as previously described (21).

IDO functional activity was measured in terms of the ability to metabolize tryptophan to kynurenine using 5 × 106 kidney or stomach cells. Kynurenine concentrations in the supernatants of 5-h cultures were measured by HPLC (12).

Protein assays (Bio-Rad) were performed on whole kidney or stomach cell lysates, PP-DCs, and PMNs from both uninfected and infected mice. PMNs from uninfected mice were also assessed after exposure in vitro to IFN-γ (200 U/ml) or CTLA-4-Ig (40 μg/ml) for 5 h. After SDS-PAGE resolution, immunoblotting was performed with rabbit polyclonal IDO-specific Ab, as previously described (22). Membranes were blocked in TBS containing 0, 05% Tween 20, 5% nonfat dried milk, and 1% BSA and were incubated sequentially with the Ab (1/3000) and HRP-conjugated anti-rabbit IgG (1/5000). The positive control consisted of IDO-expressing MC24 transfectants, and the negative control consisted of mock-transfected MC22 cells (22).

Total RNA, obtained by TRIzol extraction after mechanical disruption of fungal cells in liquid nitrogen, was reverse transcribed with Moloney murine leukemia virus-reverse transcriptase (Invitrogen Life Technologies). The PCR was performed in a thermal cycler (MasterCycler gradient; Eppendorf), cycling conditions were initial denaturation for 3 min at 95°C, followed by 30 cycles of 1 min at 95°C, 1 min at 50°C, and 20 s at 72°C, and a final extension for 10 min at 72°C. The sense (5′-ATC CCA AAT ATT GAA GAA TTC GAT G-3′) and antisense (5′-CTC TGC ACT GTT TGA GGA ATG G-3′) primers for IDO were designed on conserved regions of the genomic DNA alignment between Saccharomyces cerevisiae (NC_001142.3) BNA2 gene (23) and its C. albicans homologue (accession no. CA3435 of 〈http://genolist.pasteur.fr/Candida DB/genome.cgi?gene_detail+CA3435〉). The sequence analysis and primer design involved use of the DNASTAR software package. The sequence has 47% homology with that of S. cerevisiae and 31% homology with mammalian IDO. Sense (5′-GCATATCAATAAGCGGAGGAAAAG-3′) and antisense (5′-GCTCCGTGTTTCAAGACG-3′) primers for amplifying ∼610 bp of the large ribosomal subunit (RNA) gene (24) were used to normalize cDNA samples.

Student’s t test was used to determine the significance of differences in values between experimental groups (significance was defined as p < 0.05). Survival data were analyzed using the Mann-Whitney U test. The data reported were pooled from three to five experiments unless otherwise specified. The in vivo groups consisted of eight mice per group.

To assess IDO expression in candidiasis, mice were infected i.v. or i.g. with Candida, and IDO protein was detected by Western blot in kidneys and stomach, respectively. A 42-kDa band corresponding to IDO was observed in mice infected i.v. with yeasts or hyphae or i.g., as opposed to untreated mice (Fig. 1,A). Because IFN-γ is one major activating signal for IDO (8, 25) and IFN-γ expression, and protein production has been shown to occur early in the kidneys and stomach of mice infected i.v. or i.g. with Candida (26), we assessed IDO expression in IFN-γ-deficient mice and found no such expression after infection (Fig. 1,A). Immunostaining for IDO confirmed the increased presence of IDO in kidney epithelial cells after Candida i.v. infection, and PCR analysis confirmed that the transcriptional expression of IDO was significantly increased by infection (data not shown). IDO protein expression was also evaluated in PP-DCs and PMNs, because of the central role of these cells in the inductive and effector pathways of anticandidal immunity (27). IDO protein was increased in both types of cell after in vivo infection as well as in PMNs from untreated mice after exposure to IFN-γ in vitro (Fig. 1,B). To correlate protein expression with functional enzyme activity, we quantified kynurenine production in the kidneys and stomach of infected mice, because kynurenine levels are known to reflect IDO functional activity (28). Kynurenine production was higher in infected than in control mice (Fig. 1 C) and was undetectable in infected, IFN-γ-deficient mice (data not shown), suggesting that the increase in IDO expression was associated with enhanced functional activity. Therefore, there is a burst of IFN-γ-dependent IDO activation that occurs at the sites of infection as well as in DCs and PMNs.

FIGURE 1.

Increased IDO expression and functional activity in mice with candidiasis. BALB/c or IFN-γ-deficient (A) mice were infected with C. albicans yeasts or hyphae i.v. or i.g.; 2 days later, IDO protein expression was assessed by Western blotting on whole kidney or stomach lysates, respectively, on PP-DCs of i.g. infected mice, on splenic neutrophils (PMNs) from i.v. infected mice, or on peritoneal PMNs from uninfected mice after in vitro exposure to IFN-γ for 24 h. The positive control consisted of IDO-expressing MC24 transfectants, and the negative control consisted of mock-transfected MC22 cells. Kynurenines were measured in the supernatants of cells from kidney or stomach of infected mice 2 days after the infection. ∗, p < 0.05, infected vs uninfected (−) mice.

FIGURE 1.

Increased IDO expression and functional activity in mice with candidiasis. BALB/c or IFN-γ-deficient (A) mice were infected with C. albicans yeasts or hyphae i.v. or i.g.; 2 days later, IDO protein expression was assessed by Western blotting on whole kidney or stomach lysates, respectively, on PP-DCs of i.g. infected mice, on splenic neutrophils (PMNs) from i.v. infected mice, or on peritoneal PMNs from uninfected mice after in vitro exposure to IFN-γ for 24 h. The positive control consisted of IDO-expressing MC24 transfectants, and the negative control consisted of mock-transfected MC22 cells. Kynurenines were measured in the supernatants of cells from kidney or stomach of infected mice 2 days after the infection. ∗, p < 0.05, infected vs uninfected (−) mice.

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To assess the role of IDO in candidiasis, the course and outcome of primary disseminated infection with either Candida yeasts or hyphae as well as of GI infection were evaluated in mice with impaired IDO function resulting from treatment in vivo with 1-MT, which inhibits enzyme activity via tryptophan competition (16). Mice with GI infection were also assessed for resistance to reinfection at 2 wk of primary challenge. The animals were monitored for survival, fungal load, and histopathology. The results indicated that treatment with 1-MT reduced kynurenine production (data not shown) and at the same time drastically impaired resistance to infection. Not only did 1-MT treatment significantly decrease survival in mice infected i.v. with Candida hyphae, but it also induced mortality in animals that would otherwise survive challenge, such as mice infected with Candida yeasts i.v. or Candida hyphae i.g. or reinfected with Candida hyphae. The increased susceptibility to infection was associated with higher fungal burdens in the relevant target organs (Fig. 2) and with signs of dissemination to visceral organs in the GI infection (data not shown). Interestingly, treatment with 1-MT did not exacerbate the disease course if started 3 days after infection with Candida yeasts (data not shown), suggesting that IDO functional activity is tightly regulated during infection. The failure of 1-MT treatment to exacerbate infection in IFN-γ-deficient mice challenged with Candida (Fig. 2) also pointed to the IFN-γ dependency of IDO induction in the early control of infection. Treatment with 1-MT did not adversely affect survival in uninfected mice (data not shown). Histopathologic examination of the target organs revealed that treatment with 1-MT was associated with extensive fungal growth (extending to the kidney medullary) and the presence of numerous inflammatory abscesses in the cortex of kidneys in mice infected with Candida hyphae (Fig. 3,A). The inflammatory reaction was even more prominent in mice infected i.v. with Candida yeasts, in which fungal growth in the form of pseudohyphae was also observed (Fig. 3,B) as well as in the stomachs of mice infected i.g., in which numerous Candida hyphae were present in the keratinized layer. The hyphae penetrated the epithelial layer and were associated with signs of exaggerated inflammatory reactions and tissue destruction (Fig. 3 C). No signs of inflammatory cell recruitment or tissue destruction were observed in 1-MT-treated mice without infection (data not shown). Together, these results indicated that early induction of IDO may serve a protective role in Candida infections, such that its functional inhibition exacerbates fungal infection and pathology.

FIGURE 2.

IDO blockade impairs resistance to C. albicans infections. Mice were i.v. infected with C. albicans hyphae or yeasts (primary disseminated infection) or i.g. with hyphae (primary gastrointestinal infection) and were reinfected 2 wk later with Candida hyphae (secondary disseminated infection). Fungal growth (CFU) was evaluated in the kidneys or stomach 3 days after the primary infection or reinfection. Mice were given 1-MT in the drinking water (1 mg/ml) beginning the day of infection or reinfection and continuing until death. MST, median survival time (days). ▦, Wild-type mice with hyphal infection; ▪, wild-type mice with yeast infection; □, IFN-γ-deficient mice. ∗, p < 0.05, 1-MT-treated vs untreated mice.

FIGURE 2.

IDO blockade impairs resistance to C. albicans infections. Mice were i.v. infected with C. albicans hyphae or yeasts (primary disseminated infection) or i.g. with hyphae (primary gastrointestinal infection) and were reinfected 2 wk later with Candida hyphae (secondary disseminated infection). Fungal growth (CFU) was evaluated in the kidneys or stomach 3 days after the primary infection or reinfection. Mice were given 1-MT in the drinking water (1 mg/ml) beginning the day of infection or reinfection and continuing until death. MST, median survival time (days). ▦, Wild-type mice with hyphal infection; ▪, wild-type mice with yeast infection; □, IFN-γ-deficient mice. ∗, p < 0.05, 1-MT-treated vs untreated mice.

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FIGURE 3.

Pathologic analysis of organ tissues of mice with candidiasis. Periodic acid-Schiff-stained sections were prepared from the kidneys or stomach 3 days after the primary i.v. infection with Candida hyphae (A) or yeasts (B) or 7 days after i.g. infection (C), respectively. Treatment with 1-MT was performed as described in Fig. 2. Note the presence of more numerous inflammatory abscesses in the kidneys of 1-MT-treated mice associated with the presence of numerous fungal elements (magnified in the inset, A and B). Arrows indicate the presence of pseudohyphae in the kidneys of yeast-infected mice treated with 1-MT (B). Candida hyphae (arrows and magnified in the inset) were present in the keratinized layer and the epithelial layer and were associated with signs of exaggerated inflammatory reactions, such as parakeratosis, and tissue destruction in the stomach of 1-MT-treated mice (C).

FIGURE 3.

Pathologic analysis of organ tissues of mice with candidiasis. Periodic acid-Schiff-stained sections were prepared from the kidneys or stomach 3 days after the primary i.v. infection with Candida hyphae (A) or yeasts (B) or 7 days after i.g. infection (C), respectively. Treatment with 1-MT was performed as described in Fig. 2. Note the presence of more numerous inflammatory abscesses in the kidneys of 1-MT-treated mice associated with the presence of numerous fungal elements (magnified in the inset, A and B). Arrows indicate the presence of pseudohyphae in the kidneys of yeast-infected mice treated with 1-MT (B). Candida hyphae (arrows and magnified in the inset) were present in the keratinized layer and the epithelial layer and were associated with signs of exaggerated inflammatory reactions, such as parakeratosis, and tissue destruction in the stomach of 1-MT-treated mice (C).

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The occurrence of pseudohyphae in vivo in 1-MT-treated mice prompted us to evaluate whether 1-MT might have a direct effect on fungal morphology. To this purpose, the C. albicans variant strain, which is incapable of yeast-to-hyphal transition (14), as well as the parental strain were assessed for growth ability and morphology in vitro in the presence of 1-MT, either alone or together with tryptophan. The presence of 1-MT accelerated hyphal formation in the parental strain, as seen at 2 h of exposure; however, the effect was even more striking with the variant strain, whose growth as pseudohyphae and hyphae at 8 h, but maximally at 24 h, of exposure was greatly enhanced by 1-MT and was partially reversed by the addition of tryptophan. Because the tryptophan/kynurenine pathway has been implicated in the NAD+ metabolism of yeasts (29), we assessed the expression of IDO in fungal cells by RT-PCR. Because no IDO expression could be detected using primers for mammalian IDO (data not shown), we used fungus-specific primers. IDO expression was detected in both the yeast and hyphal forms of the fungus, and interestingly, it was inhibited by the presence of tryptophan and, to a lesser extent, 1-MT (Fig. 4). These data indicate that the tryptophan/kynurenine pathway of the fungus could be sensitive to the inhibitory activity of 1-MT, and this might impact on fungal morphology.

FIGURE 4.

IDO affects Candida albicans morphology. Candida hyphae and yeasts were exposed at 37°C in 5% CO2 in RPMI 1640 to 10 μM 1-MT with or without an excess of tryptophan (100 μM) for 2, 8, or 24 h before visualization of fungal morphology by light microscopy. IDO expression was assessed by RT-PCR on fungal cells using fungus-specific primers. cDNA levels were normalized against the RNA, large ribosomal subunit gene. Values are from one representative experiment of two performed.

FIGURE 4.

IDO affects Candida albicans morphology. Candida hyphae and yeasts were exposed at 37°C in 5% CO2 in RPMI 1640 to 10 μM 1-MT with or without an excess of tryptophan (100 μM) for 2, 8, or 24 h before visualization of fungal morphology by light microscopy. IDO expression was assessed by RT-PCR on fungal cells using fungus-specific primers. cDNA levels were normalized against the RNA, large ribosomal subunit gene. Values are from one representative experiment of two performed.

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Because IDO has regulatory effects on T cells (12) and is crucially involved in the accomplishment of antimicrobial effector functions (10), we determined the pattern of pro- and anti-inflammatory as well as Th1, Th2, and Treg cell activation in mice with primary disseminated or GI infections and in reinfection. Cytokine levels were determined locally in the kidneys in mice with primary or secondary disseminated infection and in the stomach of mice with GI infection. Increased production of TNF-α, IL-6, and IL-12 p70 was observed upon IDO blockade in each type of infection, although to different levels. In particular, TNF-α and IL-6 were not similarly increased after IDO blockade in mice with primary or secondary infection with hyphae despite a similar increase in fungal growth in the kidneys (Fig. 2). This suggests that the enhanced proinflammatory cytokine release is not obviously related to the fungal load. In contrast, IDO blockade also increased proinflammatory cytokine production and cell survival in cultures of splenocytes activated with mitogens (12) (data not shown), indicating that IDO inhibition per se is associated with augmentation of a certain cytokine profile. No significant differences were detected in the levels of IL-10 after treatment with 1-MT, except in the secondary infection (Fig. 5,A). However, on looking at the pattern of cytokine production by PP-DCs in response to the fungus, it was found that exposure to 1-MT significantly decreased the production of IL-10 and increased that of IL-12p70 and IL-6, particularly in response to hyphae (Fig. 5,B). Interestingly, production of IFN-γ was also observed in response to hyphae and was increased by exposure to 1-MT (Fig. 5 B).

FIGURE 5.

IDO blockade increases proinflammatory cytokine production in mice with candidiasis. Mice were infected and treated with 1-MT as described in Fig. 2. A, Cytokine content in kidney (primary and secondary infections) or stomach (GI infection) homogenates was determined by specific ELISA 3 days after infection and reinfection. B, Cytokine production by PP-DC upon exposure to yeasts and hyphae in vitro, with and without 1-MT. ∗, p < 0.05, 1-MT-treated vs untreated mice.

FIGURE 5.

IDO blockade increases proinflammatory cytokine production in mice with candidiasis. Mice were infected and treated with 1-MT as described in Fig. 2. A, Cytokine content in kidney (primary and secondary infections) or stomach (GI infection) homogenates was determined by specific ELISA 3 days after infection and reinfection. B, Cytokine production by PP-DC upon exposure to yeasts and hyphae in vitro, with and without 1-MT. ∗, p < 0.05, 1-MT-treated vs untreated mice.

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Because both IFN-γ-producing Th1 cells and IL-4-producing Th2 cells are activated in mice with GI infection (30), we determined the number of IFN-γ- and IL-4-producing CD4+ T cells in the MLNs of mice with GI infection. We found that the frequency of IFN-γ-producing cells was greatly increased, whereas that of IL-4-producing cells was decreased by IDO blockade (Fig. 6,A). Other results confirmed that Th1 cells are promoted and Th2 cells inhibited after IDO blockade, because the number of IFN-γ-producing cells was increased in the spleens of mice infected with yeasts and that of IL-4-producing cells was decreased in the spleens of mice infected with hyphae (Fig. 6,B). IL-4-producing cells were not detected in yeast-infected mice with or without 1-MT treatment, whereas the number of IFN-γ-producing cells was significantly increased in hypha-infected mice upon treatment with 1-MT (data not shown). To assess Treg activation, the number of CD4+CD25+ T cells producing IL-10 was determined in the MLNs of mice with GI infection, in which the occurrence of inhibitory Treg has been demonstrated (2, 31). We found that treatment with 1-MT decreased the number of CD4+ CD25+ T cells (Fig. 6,C), and this was associated with a decreased frequency of IL-10-producing CD4+ T cells (Fig. 6 D). Together, these results showed that IDO blockade favors the occurrence of an inflammatory state associated with the occurrence of Th1 reactivity, inflammatory pathology, and impairment of Treg activity in response to the fungus.

FIGURE 6.

IDO blockade increases Th1 cells and decreases Th2 and Treg cells in mice with candidiasis. Mice were infected and treated with 1-MT as described in Fig. 2. IFN-γ- and IL-4-producing cells were enumerated by ELISPOT assay on CD4+ T cells purified from the MLN (A) of mice with GI infection (7 days) or the spleens (B) of mice with the primary disseminated infection with Candida yeasts (IFN-γ) or Candida hyphae (IL-4) 6 days after the infection. Results are expressed as the mean number of cytokine-producing cells (±SE) per 105 cells. The number of CD25+CD4+ T cells (C) and the frequency of IL-10- and IFN-γ-producing cells (D) on purified CD4+ T cells from MLNs of mice with GI infection (7 days), treated or not with 1-MT, are shown. The numbers refer to the percentage of positive cells. Cells were analyzed with a FACScan flow cytofluorometer equipped with CellQuest software. Values are from one representative experiment of three performed. ∗, p < 0.05, 1-MT-treated vs untreated mice.

FIGURE 6.

IDO blockade increases Th1 cells and decreases Th2 and Treg cells in mice with candidiasis. Mice were infected and treated with 1-MT as described in Fig. 2. IFN-γ- and IL-4-producing cells were enumerated by ELISPOT assay on CD4+ T cells purified from the MLN (A) of mice with GI infection (7 days) or the spleens (B) of mice with the primary disseminated infection with Candida yeasts (IFN-γ) or Candida hyphae (IL-4) 6 days after the infection. Results are expressed as the mean number of cytokine-producing cells (±SE) per 105 cells. The number of CD25+CD4+ T cells (C) and the frequency of IL-10- and IFN-γ-producing cells (D) on purified CD4+ T cells from MLNs of mice with GI infection (7 days), treated or not with 1-MT, are shown. The numbers refer to the percentage of positive cells. Cells were analyzed with a FACScan flow cytofluorometer equipped with CellQuest software. Values are from one representative experiment of three performed. ∗, p < 0.05, 1-MT-treated vs untreated mice.

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The extensive fungal outgrowth observed in 1-MT-treated mice despite the occurrence of up-regulated Th1 reactivity prompted us to verify whether IDO blockade might have an effect on the antifungal effector functions of PMNs. Because the number of circulating PMNs was not affected by 1-MT treatment (data not shown), we assessed the phagocytic and killing capacity as well as the oxidant production by PMNs toward yeasts and hyphae of the fungus upon exposure to 1-MT in vitro. We also measured the effect of treatment on PMN apoptosis. The results show that both phagocytosis and killing of the fungus were impaired by treatment with 1-MT (Fig. 7,A), whereas both oxidant production (Fig. 7,B) and the number of apoptotic cells were increased (Fig. 7 C), particularly after exposure to hyphae. Therefore, these data suggest an important and previously unknown role for IDO in the overall expression of the antifungal effector state of PMNs.

FIGURE 7.

IDO blockade impairs PMN antifungal effector functions. PMNs from uninfected mice were exposed to Candida yeasts and hyphae in the presence of 10 μM 1-MT and assessed for phagocytosis and killing activities (A), O2 production (B), and apoptosis (C). The percentage of internalization was calculated on Giemsa-stained preparations after 15 min of PMN/Candida incubation. For fungicidal activity, PMNs were incubated with unopsonized yeasts and hyphae for 60 (yeasts) or 120 (hyphae) min, and the results are expressed as candidacidal activity and hyphal damage, as detailed in Materials and Methods. For superoxide anion (O2) quantification, the superoxide dismutase-inhibitable reduction of cytochrome c was performed in the culture supernatants (after 60-min incubation), and the results were expressed as nanomoles of O2 per 106 cells. For apoptosis, PMNs were exposed to yeasts and hyphae for the times indicated before assessment of apoptosis (PI staining) by flow cytometry. Values are the mean ± SE of samples taken from three independent experiments. ∗, p < 0.05, 1-MT-treated vs untreated mice.

FIGURE 7.

IDO blockade impairs PMN antifungal effector functions. PMNs from uninfected mice were exposed to Candida yeasts and hyphae in the presence of 10 μM 1-MT and assessed for phagocytosis and killing activities (A), O2 production (B), and apoptosis (C). The percentage of internalization was calculated on Giemsa-stained preparations after 15 min of PMN/Candida incubation. For fungicidal activity, PMNs were incubated with unopsonized yeasts and hyphae for 60 (yeasts) or 120 (hyphae) min, and the results are expressed as candidacidal activity and hyphal damage, as detailed in Materials and Methods. For superoxide anion (O2) quantification, the superoxide dismutase-inhibitable reduction of cytochrome c was performed in the culture supernatants (after 60-min incubation), and the results were expressed as nanomoles of O2 per 106 cells. For apoptosis, PMNs were exposed to yeasts and hyphae for the times indicated before assessment of apoptosis (PI staining) by flow cytometry. Values are the mean ± SE of samples taken from three independent experiments. ∗, p < 0.05, 1-MT-treated vs untreated mice.

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To gain insight into the mechanisms of IDO regulation in PMNs, we assessed whether, similar to what was observed in DCs (12), CTLA-4-Ig up-regulates IDO functional activity in PMNs. Because B7 receptor molecules are required for CTLA-4-Ig effects on DCs (20, 21, 22), we used PMNs from wild-type as well as B7-1- or B7-2-deficient mice. Peritoneal PMNs were pretreated with CTLA-4-Ig and, for comparison, IFN-γ before assessment of IDO expression and concomitant antifungal activity, such as the ability to kill yeasts and produce oxidants. FACS analysis revealed that binding of CTLA-4-Ig occurred on PMNs (data not shown). Similar to IFN-γ, CTLA-4-Ig induced IDO protein expression in both wild-type and B7-2-deficient mice, but not in B7-1-deficient mice (Fig. 8,A). Control IgG3 failed to induce IDO expression in any type of mouse (data not shown). In terms of antifungal effector activity, both treatments increased the candidacidal activity of and decreased oxidant production by either wild-type or B7-2-deficient PMNs, but not B7-1-deficient PMNs (Fig. 8, B and C). These data suggest that IDO functional activity in PMNs may be affected by IFN-γ and CTLA-4-Ig engagement of B7-1.

FIGURE 8.

CTLA-4-Ig increases IDO expression and function in PMNs. Peritoneal PMNs were exposed in vitro to IFN-γ (200 U/ml) or CTLA-4-Ig (40 μg/ml) for 5 h before being assessed for IDO protein expression by Western blotting (A), candidacidal activity (B), and O2 production (C) as described in Fig. 7. B7-1−/− or B7-2−/−, B7-1- or B7-2-deficient mice. Control treatment consisted of native IgG3. ∗, p < 0.05, treated vs control-treated (−) PMNs.

FIGURE 8.

CTLA-4-Ig increases IDO expression and function in PMNs. Peritoneal PMNs were exposed in vitro to IFN-γ (200 U/ml) or CTLA-4-Ig (40 μg/ml) for 5 h before being assessed for IDO protein expression by Western blotting (A), candidacidal activity (B), and O2 production (C) as described in Fig. 7. B7-1−/− or B7-2−/−, B7-1- or B7-2-deficient mice. Control treatment consisted of native IgG3. ∗, p < 0.05, treated vs control-treated (−) PMNs.

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This is the first report of the impact of IFN-γ/IDO-dependent tryptophan catabolism on a fungal infection. The study provides evidence for a central role of tryptophan catabolism in determining the outcome of mouse infection with C. albicans. The results showed that sustained activation of IDO occurred in the host concomitantly with infection, required IFN-γ, and was observed with both DCs and PMNs. Therefore, although IDO can be induced by IFN-γ-dependent and -independent mechanisms depending on the type of stimulus and tissue (31), our results point to the central role of IFN-γ in IDO activation in C. albicans infections.

The production of IFN-γ and IDO induction has been regarded as an important mechanism of antimicrobial resistance to intracellular organisms, such as parasites (32, 33, 34) and bacteria (35, 36, 37, 38). Our data are the first to provide evidence for an important and previously undescribed role for IDO in governing the antimicrobial state of PMNs. We found that the Candida growth inhibition activity of PMNs was reduced by IDO blockade in concomitance with increased oxidant generation and accelerated cell apoptosis, a finding compatible with the action of IDO as a free radical scavenger in this process (25). Therefore, more than a reduced tryptophan availability and an increased level of toxic metabolites, the growth restriction of IDO against Candida, a tryptophan prototrophic strain (14), appears to be the result of an action on the oxidant-antioxidant state of effector cells. Actually, a product of tryptophan catabolism, picolinic acid, has protective effects in mice with candidiasis (39). Similar to what was observed in DCs (20, 21, 22), IDO functional activity in PMNs was regulated by CTLA-4-Ig acting on B7-1 molecules, a finding that expands upon the importance of the B7-CTLA-4 coreceptor pathway in conditioning innate and adaptive immune responses (14).

Studies with Chlamidia spp have suggested that in the presence of IFN-γ, there exist unique host-parasite interactions that may contribute to persistent chlamidial infection (38). Because C. albicans is a commensal of the human gastrointestinal and genitourinary tracts, and IFN-γ is an important mediator of protective immunity to the fungus (40), persistence of the fungus in the human host needs to be accommodated in an environment rich in IFN-γ. Previous studies have shown that the production of IFN-γ occurs early, in response to both yeasts and hyphae, does not correlate with the occurrence of anticandidal Th1 responses, and is tightly regulated; signs of exaggerated inflammatory reaction are observed under conditions of both IFN-γ overproduction (41) and deficiency (26, 42, 43, 44). By regulating IDO activity, the present study suggests an additional, previously unexplored mechanism by which IFN-γ may affect host immune reactivity to the fungus. Both DCs and PMNs up-regulated IDO expression in infection, and as shown for DCs (45), IDO expression in PMNs was induced by IFN-γ. Therefore, both the inductive and effector pathways of antifungal immunity are affected by the IFN-γ/IDO-dependent pathway.

IDO expressing DCs are regarded as regulatory DCs specialized to cause Ag-specific deletional tolerance or otherwise negatively regulating responding T cells (17). IFN-γ is required for functional IDO enzymatic activity in DCs (17). We found that IDO expressing PP-DCs produce IL-10 in response to hyphae and IL-6/IL-12 in response to yeasts, a cytokine pattern that was completely reversed by IDO blockade. Consistent with the finding that PP-DCs producing IL-10 are absolutely required for the activation of CD4+ CD25+ Treg capable of negatively regulating the inflammatory response and antifungal Th1 immunity upon adoptive transfer in vivo (2), the number of IL-10-producing CD4+ CD25+ Treg was significantly decreased in the MLNs of mice with GI candidiasis after IDO blockade. Concomitantly, the number of CD4+ T cells producing IFN-γ increased, whereas that of cells producing IL-4 decreased. Because the adoptive transfer of CD4+ CD25+ Treg could partially reverse the inflammatory pathology associated with IDO blockade (data not shown), it appears that the activation of IL-10-producing CD4+ CD25+ T cells is one important mechanism through which the IFN-γ/IDO-dependent pathway may control the local inflammatory pathology and Th1 reactivity to the fungus. It is interesting to note that the same mechanism operates in the opposite direction, that is, Treg control of DCs, via CTLA-dependent regulation of IDO activity (22). In this regard, it is intriguing that both the occurrence of IL-10-producing DCs in candidiasis (2) and the modulatory activity of Treg on DCs (22, 46) are costimulation-dependent. Although the IDO-dependent effect on T cell subset regulation may also rely on the apoptotic activity of Th1 more than Th2 cells (12), whichever mechanism prevails, it will point to a major role for IDO in Th subset regulation in candidiasis.

In its ability to down-regulate the antifungal Th1 response in the GI tract, IDO behaves in a fashion similar to that described in mice with colitis, in which IDO expression correlates with the occurrence of local tolerogenic responses (47). In the basal state, the gut is the site of the highest levels of IDO expression (7), which is believed to be required to allow for microbial colonization and ingestion of dietary Ags without tissue-damaging inflammatory responses. The expression of IDO is indeed up-regulated in active inflammatory bowel disease (48).

The implication of IDO in immunoregulation in candidiasis may help to accommodate several, as yet unexplained findings. First, the selective expression of IDO in the gut may represent the missing tissue-dependent factor that conditions the ability of DCs to produce either IL-4 or IL-10 upon exposure to Candida hyphae, ultimately dictating the local pattern of both cytokine production and Th reactivity to the fungus (49). Second, the recovery of Candida from the GI tract as well as the detection of the underlying Th1 reactivity, including delayed-type hypersensitivity and lymphoproliferation, may fluctuate in healthy subjects and in infection (50). It is conceivable that changes in local IDO activity will affect the induction of Treg capable of opposing inflammatory pathology at the expense of fungal persistence. Alternatively, as a third possibility, high levels of IL-10 production, such as those seen in patients with chronic mucocutaneous candidiasis (51), may be a consequence of IDO activation by the fungus, impairing antifungal Th1 immunity and thus favoring persistent infection. In this regard, it has long been known that the ability of C. albicans to establish an infection involves multiple components of this fungal pathogen, but its ability to persist in host tissue may involve primarily the immunosuppressive property of a major cell wall glycoprotein, mannan (52, 53), which, interestingly, is a potent inducer of IL-10 production (51).

Finally, one novel finding in the present study relates to the detection of fungal IDO, which has 31% homology with mammalian IDO. Because the tryptophan/kynurenine pathway is involved in the NAD+ fungal metabolism under aerobic conditions (29), different enzymes of the kynurenine pathway have been characterized in S. cerevisiae (29, 54). However, to our knowledge, this is the first description of IDO expression in a fungus. IDO expression in C. albicans was inhibited in the presence of an excess of tryptophan, suggesting that IDO might be involved in tryptophan catabolism by the fungus. Furthermore, treatment with 1-MT promoted hyphal formation, a finding compatible with the dependency of fungal morphology on tryptophan auxotrophy (55). It is of interest that, contrary to IDO blockade, IFN-γ inhibited germ-tube formation in the fungus (56). Should the IFN-γ action on fungal cells be dependent on IDO, this would indicate that the fungal enzyme is highly responsive to signals from the mammalian host immune system. Consistent with this hypothesis, a recent review emphasized the role of IDO in evolution as “an example of the immune system ability to recycle effector mechanisms as it evolves” (11).

In summary, the results of this study provide a plausible mechanistic explanation of events occurring at the fungus/pathogen interface that encompass host adaptation to the fungus as well as failure to accomplish it (Fig. 9). The production of IFN-γ is squarely placed at the host/pathogen interface, where IDO activation may exert a fine control over fungal morphology, as well as the inductive and effector pathways of the immunological antifungal resistance.

FIGURE 9.

The central role of the IFN-γ/IDO-dependent pathway in C. albicans infection. The production of IFN-γ is squarely placed at the host/pathogen interface, where IDO activation may exert a fine control over fungal morphology as well as the inductive and effector pathways of the immunological antifungal resistance. Solid and dotted lines indicate positive and negative signals, respectively.

FIGURE 9.

The central role of the IFN-γ/IDO-dependent pathway in C. albicans infection. The production of IFN-γ is squarely placed at the host/pathogen interface, where IDO activation may exert a fine control over fungal morphology as well as the inductive and effector pathways of the immunological antifungal resistance. Solid and dotted lines indicate positive and negative signals, respectively.

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The authors have no financial conflict of interest.

We thank Dr. Lara Bellocchio for superb editorial assistance.

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 by the National Research Project on AIDS (Contract 50F.30).

3

Abbreviations used in this paper: Treg, regulatory T cell; DC, dendritic cell; GI, gastrointestinal infection; IDO, indoleamine 2,3-dioxygenase; i.g., intragastrically; MLN, mesenteric lymph node; 1-MT, 1-methyl-d,l-tryptophan; PI, propidium iodide; PMN, polymorphonuclear neutrophil; PP-DC, Peyer patch DC.

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