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The Journal of Immunology, 2007, 179, 5999-6008
Copyright © 2007 by The American Association of Immunologists, Inc.
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Functional yet Balanced Reactivity to Candida albicans Requires TRIF, MyD88, and IDO-Dependent Inhibition of Rorc1

Antonella De Luca*, Claudia Montagnoli*, Teresa Zelante*, Pierluigi Bonifazi*, Silvia Bozza*, Silvia Moretti*, Carmen D’Angelo*, Carmine Vacca*, Louis Boon{dagger}, Francesco Bistoni*, Paolo Puccetti*, Francesca Fallarino* and Luigina Romani2,*

* Department of Experimental Medicine and Biochemical Sciences, University of Perugia, Perugia, Italy; and {dagger} Bioceros BV 3584 CM Utrecht, The Netherlands


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The ability of regulatory T (Treg) cells to inhibit aspects of innate and adaptive immunity is central to their protective function in fungal infections. In murine candidiasis, CD4+CD25+ Treg cells prevent excessive inflammation but enable fungal persistence in the gastrointestinal tract, which underlies the onset of durable antifungal protection. In this study, we show that fungal growth, inflammatory immunity, and tolerance to the fungus were all controlled by the coordinate activation of naturally occurring Treg cells, which limited early inflammation at the sites of infection, and pathogen-induced Treg cells (that regulated the expression of adaptive Th immunity in secondary lymphoid organs). Naturally occurring Treg cells required the TRIF pathway for migration to inflamed sites, where the MyD88 pathway would then restrain their suppressive function. Subsequent inflammatory Th1-type immunity was modulated by induced Treg cells, which required the TRIF pathway as well, and acted through activation of IDO in dendritic cells and Th17 cell antagonism. In vitro, using naive CD4+ cells from TRIF-deficient mice, tryptophan metabolites were capable of inducing the Foxp3-encoding gene transcriptionally and suppressing the gene encoding ROR{gamma}t, Th17 lineage specification factor. This is the first study to show that the same tryptophan catabolites can foster dendritic cell-supported generation of Foxp3+ cells and mediate, at the same time, inhibition of ROR{gamma}t-expressing T cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The balance between proinflammatory and anti-inflammatory signals is a prerequisite for successful host/pathogen interaction in fungal infections (1, 2). Although several cell types contribute to regulation of immune responses, via their production of IL-10 (3, 4, 5), regulatory T (Treg)3 cells with tolerogenic activity have been described in fungal infections of both mice (6, 7, 8, 9) and humans (10). The capacity of Treg cells to inhibit components of innate and adaptive immunity is pivotal in their regulatory function and has led to the concept of "protective tolerance," which implies that a host’s immune defense may be adequate for protection without necessarily eliminating fungal pathogens that would impair immune memory or cause an unacceptable level of tissue damage (2). By dampening Th1-dependent anticandidal immunity, mouse CD4+CD25+ Treg cells prevent eradication of the fungus from the gastrointestinal tract, but they enable the onset of memory immunity, which is contingent on fungal persistence. Thus, tolerogenic Treg cells are instrumental in permitting colonization and commensalism, and, at the same time, they allow for durable protection.

Besides ensuring self-tolerance, different types of Treg cells actively participate in immune responses (11). Naturally occurring Treg (nTreg) cells originate in the thymus and survive in the periphery as natural regulators, whereas inducible (or adaptive) Treg (iTreg) cells develop from conventional CD4+ T cells that are activated under conditions of impaired costimulatory signaling or are induced by deactivating cytokines and drugs (12). Recently, T cell activation in the presence of innate stimuli was found to divert iTreg generation to Th17 generation (13, 14, 15). Because the Th17 pathway is activated by Candida albicans (16, 17) and promotes susceptibility to infection (18), this further implicates Treg cells in fungal infections. However, the contribution of either type of Treg cell to an overall local T cell homeostasis and the dependency on specific TLRs have been unclear in candidiasis.

In the present study, we used mice genetically deficient for a specific pathway or TLR that were challenged intragastrically with C. albicans to assess the relative contributions of nTreg and iTreg cells to infection and their possible dependency on TLR signaling. We found that nTreg and iTreg cells are sequentially induced in infection and have non-overlapping, complementary roles. On examining the functional activity of CD4+CD25+ T cells recovered over time in infection, nTreg cells modulated the effector function and proinflammatory ability of polymorphonuclear neutrophils (PMNs) and thus exerted an early control over fungal growth and local inflammation. Later in time, however, Ag-specific iTreg cells were induced by the fungus that led to Th17 cell antagonism and protective Th immunity. nTreg cells required the TRIF pathway for migration to inflamed sites, where the MyD88 pathway restrained their suppressive activity. Subsequent inflammatory Th immunity was orchestrated by iTreg cells, induced by the TRIF pathway, that activated IDO in dendritic cells (DCs) and effectively opposed Th17 cell generation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

Female C57BL/6 mice, 8- to 10-wk-old, were purchased from Charles River Laboratories. Homozygous Tlr2–/–, Tlr3–/–, Tlr4–/–, Tlr9–/–, Myd88–/–, and Trif–/– mice on a C57BL/6 background were bred under specific, pathogen-free conditions at the Animal Facility of Perugia University. Five-week-old thymectomized C57BL/6 mice were also purchased from Charles River Laboratories. Experiments were performed according to the Italian Approved Animal Welfare Assurance A-3143-01.

C. albicans strains and infections

Isogenic strains of C. albicans, obtained by mutagenesis in vitro and capable (Vir–13) or not (Vir–3) of yeast-to-hypha transition as assessed by the germ-tube formation in vitro, were used (19, 20). 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, cells were harvested at the end of the exponential phase of growth. For gastrointestinal infection, 108 Candida cells of either isogenic strain were injected intragastrically and quantification of fungal growth was expressed as CFU per organ (mean ± SE). For histology, sections (3–4 µm) of paraffin-embedded tissues were stained with periodic acid-Schiff reagent and examined for histology. Treatments with 1 mg of anti-IL-10 (clone JES5.2A5), anti-CTLA-4 (clone 4F10), anti-TGF-beta (clone 1D11), anti-CD25 (clone PC61), or anti-beta-galactosidase (clone GL113) control mAb were done i.p., 5 and 24 h after the infection.

Purification of cells

Gr-1+CD11b+ PMNs, >98% pure on FACS analysis, were isolated from the peritoneal cavity of mice by magnetic-activating sorting using Ly-6G MicroBeads and MidiMacs (Miltenyi Biotec). CD4+CD25+ and CD4+CD25– cells were separated by magnetic cell sorting with MicroBeads and MidiMacs from mesenteric lymph nodes (MLNs) or the stomach. Stomach cells were isolated by enzymatic dissociation (700 µg/ml collagenase; Sigma-Aldrich) and 30 µg/ml DNase I (D5025; Sigma-Aldrich) followed by a discontinuous Percoll gradient (21). CD4+CD25–CD62Lh naive cells were separated from CD4+ T cells by magnetic-activated sorting. MLN DCs (>99% CD11c+ and <0.1% CD3+) were purified by magnetic-activated sorting using CD11c MicroBeads and MidiMacs. Bone marrow-derived DCs were obtained as described (22) by culturing bone marrow cells in Iscove’s modified medium, containing 10% filtered FCS, 50 µM 2-mercaptoethanol, 1 mM sodium pyruvate, 2 mM L-glutamine, 10 mM HEPES, 50 µg/ml gentamicin, and either 150 U/ml mouse rGM-CSF (Sigma-Aldrich) plus 75 U/ml rIL-4 (R&D Systems) for 7 days (GM-DCs) or 200 ng/ml FLT3-L for 9 days (FL-DCs). Final maturation was accomplished by the addition of 1 µg/ml LPS (for GM-DCs) or 2 µg/ml CpG (1668; for FL-DCs) for an additional 24 h.

Treg cell suppressive activity

PMNs were incubated at 37°C with live unopsonized yeasts (at a PMN/fungus ratio of 10:1) with and without T cells (at a PMN/Treg cell ratio of 2:1) or 20 µg/ml anti-IL-10, anti-CTLA-4, anti-TGF-beta or anti-beta-galactosidase control mAb for 60 min for candidacidal activity (8) or for 30 min for determination of Indo, NOS2 or NADPH oxidase p67phox mRNA expression by real-time RT-PCR. Preliminary time course experiment had shown that maximal NOS2 or NADPH p67 mRNA expression was observed at 30 min to decline thereafter, and no up-regulation of p47phox, gp91phox, or p22phox mRNA expression was observed in response to the fungus (data not shown). Purified CD4+CD25+ or CD4+CD25– cells from MLNs (at 14 days after infection) or naive CD4+CD25–CD62Lh (106/ml) cells were cultured in flat-bottom 96-well plates in the presence of 5 x 105 Candida-pulsed DCs for 5 days, before cytokine quantification in culture supernatants. Fungal overgrowth was prevented as described (23). Naive CD4+CD62Lh Th cells were restimulated with plate-bound anti-CD3{epsilon} (2 µg/ml), anti-CD28 (2 µg/ml), and 50 U/ml IL-2. In experiments involving the addition of tryptophan catabolites (that is, kynurenines), a mixture of 3-hydroxykynurenine, 3-hydroxyanthranilic acid, and quinolinic acid (Sigma-Aldrich), each at 10 µM, was added to the cultures as described (24). Purified CD4+CD25+ or CD4+CD25– cells were incubated either alone or in combination (at the optimal CD25+:CD25– ratio of 1:2) on anti-CD3{epsilon}-coated plates (clone 145–2C11; BD Pharmingen) in the presence of 1 µg/ml soluble anti-CD28 mAb (clone 37.51) (25) or with Candida-pulsed DCs for 72 or 120 h, respectively, before FACS analysis. Labeling with CFSE (Molecular Probes) was done as described (25).

Western blotting for IDO

Immunoblotting was performed with rabbit polyclonal IDO-specific Ab, as described (26). The positive control consisted of IDO-expressing MC24 transfectants and the negative control of mock-transfected MC22 cells.

Pulsing and adoptive transfer of DCs

MLN DCs were exposed to live unopsonized fungi or LPS from Escherichia coli (055:B5, 10 µg/ml) as described (8, 20). At 18 h of culture, cells were harvested for costimulatory molecule expression and IDO Western blotting, and supernatants were assessed for cytokine contents by ELISA. After 2 h of pulsing, DCs (5 x 105) were administered i.p. 1 and 2 wk before the infection. Mice were assessed for number of cytokine-producing MLN CD4+ cells 3 days later.

Flow cytometry

Staining was done as described (20). FITC-conjugated anti-mouse Abs were from R&D Systems (CCR9), BD Pharmingen (CD4, CD25, CD103, CD11b, CD11c, CD80, and CD86), Santa Cruz Biotechnology (CTLA-4), eBioscence (ICOS and Foxp3), and BioLegend (glucocorticoid-induced TNFR (GITR)). Propidium iodide was included at 1 µg/ml in the final wash after immunofluorescent staining to label dead cells. Cells were analyzed with a FACScan flow cytofluorometer (BD Biosciences) equipped with CELLQuest software. Control staining of cells with irrelevant Ab was used to obtain background fluorescence values. Intracellular staining for Foxp3 or CTLA-4 was performed by using specific staining kits. Data are expressed as a percentage of positive cells over total cells analyzed. Flow cytometry was used to determine the purity of CD4+ T cells (>98%) and the fractionated CD4+CD25– (>98%) or CD4+CD25+ (>82%).

Quantification of cytokines by real-time RT-PCR, ELISA, and ELISPOT assay

Real-time RT-PCR was performed using the iCycler iQ detection system (Bio-Rad) and SYBR Green chemistry (Finnzymes). Cells were lysed and total RNA was extracted using RNeasy mini kit (Qiagen) and was reverse transcribed with Sensiscript reverse transcriptase (Qiagen) according to the manufacturer’s directions. The PCR primers were as follows: forward primer 5'-CGGACTGAGAGGACACAGGTTAC-3' and 5'-ACACATACGCCATGGTGATGTAC-3' reverse primer for Indo; forward primer 5'-GGACGATCATCTGGGTCACATTGT-3' and 5'-GCCAGGGAACCGCTTATATG-3' reverse primer for Tbet; forward primer 5'-AGCAGTGTAATGTGGCCTAC-3' and 5'-GCACTTCTGCATGTAGACTG-3' reverse primer for Rorc; forward primer 5'-CTATCTGGGCAAGGCTACGGTT-3' and reverse primer 5'-CACAAAGCCAAACAATACGCG-3' for p67phox; forward primer 5'-CCTCCTCCACCCTACCAAGT-3' and reverse primer 5'-CACCCAAAGTGCTTCAGTCA-3' for NOS2; forward primer 5'-CGCAAAGACCTGTATGCCAAT-3' and reverse primer, 5'-GGGCTGTGATCTCCTTCTGC-3' for mouse {gamma}-actin-encoding gene. The PCR primers for Gata3 and Foxp3 were as described (8). Amplification efficiencies were validated and normalized against GAPDH. The thermal profile for SYBR Green real time PCR was at 95 °C for 3 min, followed by 40 cycles of denaturation for 30 s at 95 °C and an annealing/extension step of 30 s at 60 °C. Each data point was examined for integrity by analysis of the amplification plot. The mRNA-normalized data were expressed as relative cytokine mRNA in treated cells compared with that of mock-infected cells. Cytokine content was assessed by enzyme-linked immunosorbent assays (R&D Systems and, for IL-23, eBioscience) on supernatants of cultured cells. The detection limits of the assays were <30 for IL-23, <10 for IFN-{gamma}, <5 for IL-4, <3 for IL-10, <10 for IL-17, and <7 for IL-6. AID EliSpot assay kits (Amplimedical) were used on purified MLN CD4+ T cells cocultured with Candida-pulsed DCs for 3 days to enumerate cytokine-producing cells. Results were expressed as the mean number of cytokine-producing cells (±SE) per 104 cells, calculated using replicates of serial twofold dilutions of cells.

Statistical analysis

Student’s t test or ANOVA (ANOVA) and Bonferroni’s test were used to determine the statistical significance of differences in organ clearance and in vitro assays. Significance was defined as p < 0.05. The data reported were either from one representative experiment out of three independent experiments (FACS analysis and RT-PCR) or pooled from three to five experiments, otherwise. The in vivo groups consisted of 6–8 mice per group.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
MyD88–/– and Trif–/– mice show opposite patterns of anticandidal reactivity

We infected wild type (WT), Myd88–/–, and Trif–/– mice intragastrically with C. albicans and evaluated parameters of infection and local inflammatory pathology over the course of infection. Consistent with previous data (20), fungal growth was observed in the stomach and colon, but not kidneys, of WT mice early in infection. Both the qualitative and quantitative patterns of fungal growth were affected in Myd88–/– and Trif–/– mice. At 1 wk of infection, C. albicans growth was higher in the organs of Myd88–/– mice and declined thereafter. Growth was instead restrained in Trif–/– mice early in infection, but local outgrowth and peripheral dissemination were observed at later times (Fig. 1A). Severe signs of acanthosis, hyperkeratosis, and inflammatory cell (i.e., PMN) recruitment were found in the stomachs of Trif–/– mice, although these signs were not observed in Myd88–/– recipients (Fig. 1B).


Figure 1
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  		FIGURE 1. Pattern of susceptibility to candidiasis in Myd88–/– or Trif–/– mice. A, Fungal growth (CFU) in different organs at 1 and 2 wk after intragastric infection with 108 C. albicans Vir–13 yeasts. B, Periodic acid-Schiff-stained medial sections from the stomach of infected mice, 1 wk after infection. C, CFU in the stomach of TLR-deficient mice infected as above. In B, note the presence of abundant inflammatory cells (magnified in the inset) and signs of acanthosis and parakeratosis in the stomachs of Trif–/– or Tlr4–/– mice as opposed to the limited inflammatory reaction in C57BL6, Tlr3–/– and particularly Myd88–/– mice. Limited inflammatory cell recruitment (magnified in the inset) associated with prominent acanthosis or parakeratosis is seen in the stomachs of Tlr2–/– or Tlr9–/– mice, respectively. Bars indicate magnification. The results shown in A and C are pooled from four experiments, each consisting of 8 mice per group. Bars are SE. *, p < 0.05, CFU at 2 wk vs CFU at 1 wk. **, p < 0.05, Myd88–/–, Trif–/–, or TLR-deficient vs C57BL6 mice.

 
To identify the TLRs involved in those effects, we infected mice deficient in TLRs known to recruit the MyD88 (TLR2/TLR9), TRIF (TLR3), or both (TLR4) pathways. Neither Tlr2–/–, Tlr9–/–, nor Tlr3–/– mice mimicked the patterns of susceptibility and inflammation of Myd88–/– and Trif–/– mice. Fungal growth was restrained in Tlr9–/– mice but enhanced in Tlr2–/– mice and even more in Tlr3–/– hosts at 1 wk of infection, although all mice eventually cleared infection with no signs of overt pathology. In contrast, extensive fungal outgrowth and inflammation were all obvious in Tlr4–/– mice (Fig. 1, B and C). Thus TLR4 appeared to induce a protective response through nonredundant mechanisms of downstream signaling.

The sequential induction of nTreg and iTreg cells in infection is controlled by the TRIF pathway

The disparate patterns of response in Myd88–/– and Trif–/– mice may imply disparate controls of anticandidal reactivity via the two pathways. We asked whether, similar to that seen in mice with aspergillosis (8), Treg cells suppressing PMN effector function are present at sites of infection and how these cells relate to CD4+CD25+ Treg cells that inhibit antifungal Th1 immunity and are induced in MLNs or thymuses of mice with candidiasis (20). CD4+CD25+ T cells were purified from the stomach (at 1 wk) or MLNs (at 1 and 2 wk) of infected donors, including thymectomized mice, to be characterized for phenotypic markers. CD4+CD25+ T cells accumulated in the stomachs of infected WT and Myd88–/– but not Trif–/– or thymectomized mice (Fig. 2A), a finding suggesting that CD25+ T cells migrating to infected sites are bona fide nTreg cells. Phenotypic analysis showed that CD4+CD25+ T cells in the stomach had up-regulated expressions of Foxp3, CTLA-4, and GITR, but not ICOS, relative to uninfected controls (Fig. 2B). On staining for homing molecules defining the migratory capacity to the gut (27, 28), the sorted cells appeared to be CD103+ (Fig. 2B). CD4+CD25+ cells from MLNs, purified a week after the infection, appeared to be CD103+ and CCR9+ in WT but not in Trif–/– mice (Fig. 2C), a finding suggesting that failure to migrate may be an intrinsic defect of T cells under conditions of TRIF deficiency. Because these MLNs CD25+ T cells poorly expressed Foxp3 and CTLA-4 (data not shown), these findings suggested that nTreg cells up-regulated their surface markers once migrated to infected sites. Therefore, compartmentalization determines Treg cell functional activity in candidiasis, a process essentially controlled by the TRIF pathway.


Figure 2
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  		FIGURE 2. A, Detection of CD4+CD25+ T cells in the stomach or MLNs of C57BL/6, thymectomized, Myd88–/– or Trif–/– mice, uninfected (–) or infected intragastrically for 1 (stomach) or 2 (MLNs) wk (see also legend to Fig. 1). Five-week-old thymectomized mice were infected 3 wk after thymectomy. Numbers refer to percentages of positive cells in infected (black squares) or naive (gray squares) mice. B, Phenotypic analysis by flow cytometry of CD4+CD25+ T cells isolated from the stomachs or MLNs of WT mice infected as above. C, Phenotypic analysis of MLNs CD4+CD25+ T cells isolated from uninfected (–) or infected C57BL/6 or Trif–/–mice, a week after infection. In B and C, black and gray histograms indicate infected or naive mice, respectively. Black lines refer to control stained cells. Numbers indicate percentages of positive cells.

 
In line with previous findings (20), CD4+CD25+ T cells were expanded later in infection in MLNs of WT, Myd88–/– but not Trif–/– infected mice (Fig. 2A). They were also expanded in thymectomized mice, which suggests a relative thymus independence and confirms that peripheral conversion of CD4+CD25– cells into CD4+CD25+T cells does not require the thymus (29). MLN CD4+CD25+ T cells were Foxp3+CTLA-4+, CD103–, GITR+, and ICOS+ (Fig. 2B) and did not show up-regulation of CCR9 (data not shown). These data indicate that subsets of Treg cells, distinguishable by origin and phenotype, are sequentially induced in the course of infection through mechanisms involving a crucial participation of the TRIF pathway.

The anti-inflammatory activity of nTreg cells is modulated by the MyD88 pathway

PMNs play a central role in antifungal immunity and are down-regulated at sites of infection by the CTLA-4/IDO axis (26). To evaluate the suppressive activity of nTreg cells, PMNs from uninfected WT mice were assessed for candidacidal activity, respiratory burst (measured as NOS2 and NADPH-related p67 mRNA expression), and inflammatory state (Indo mRNA expression) in the presence of CD25+ T cells from the stomachs of WT or Myd88–/– mice. Preliminary assaying of NADPH enzymes had shown that p67 mRNA is selectively up-regulated by the fungus (data not shown). CD25+ but not CD25– T cells from infected, but not uninfected, WT mice suppressed killing and respiratory burst in PMNs (Fig. 3A). In contrast, the CD25+ T cells greatly potentiated IDO expression in PMNs (Fig. 3A) and decreased TNF-{alpha} production (data not shown). This suggested that nTreg cells exploit IDO to down-regulate the inflammatory state of PMNs, as previously observed (8, 26). nTreg cells were poorly suppressive of Ag-induced proliferation of the CD25– counterpart (Fig. 3B) and were not primed by Candida-pulsed DCs (unpublished observation). Therefore, nTreg cells at the sites of infection allow for fungal growth but oppose inflammation, and they exhibit limited suppressive activity on lymphocytes. Importantly, nTreg activities on PMN were enhanced in Myd88–/– mice (Fig. 3A), which suggests a role for TLR/MyD88 signaling in modulating nTreg cell function at inflamed sites.


Figure 3
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  		FIGURE 3. Modulation of anti-inflammatory activity of nTreg cells by the MyD88 pathway. Antifungal effector activity of PMNs (A) in the presence of CD4+CD25+ or CD4+CD25– cells obtained from the stomach of uninfected (–) or Vir–13-infected (+, for 1 wk) mice. Purified peritoneal PMNs were exposed to Candida yeasts in the presence of Treg cells for 60 min (for candidacidal activity), 30 min (for NADPH-related p67 or NOS2 mRNA expression by real-time RT-PCR) or 18 h (for Indo mRNA expression). B, CFSE-labeled CD4+CD25+ cells were incubated either alone (gray histograms) or in combination with CD4+CD25– cells (at an optimal CD25+:CD25– ratio of 1:2) (red lines) on Candida-pulsed DCs for 120 h, before FACS analysis. Black histograms, unstimulated CFSE-labeled cells. Numbers refer to mean channel fluorescence intensity. C, Candidacidal activity of PMNs exposed to CD4+CD25+ as above in the presence of 10 µg/ml of each mAb. Anti-beta-Gal, anti-beta-galactosidase control mAb. *, p < 0.05, unexposed vs Treg-exposed PMNs. **, p < 0.05, nTreg + anti-IL-10 or anti-CTLA-4 mAb vs Treg alone. D, Fungal growth in the stomach (1 wk after infection) of C57BL/6 mice with candidiasis, treated with 1 mg of each mAb, i.p., 5 and 24 h after infection. *, p < 0.05, treated vs untreated mice. **, p < 0.05, anti-IL-10 + anti-CTLA-4 mAb vs each mAb alone. Shown are results pooled from four experiments.

 
To directly correlate nTreg activity with susceptibility to infection, we determined whether blocking Treg cells would affect PMN killing and fungal growth. We examined the impact of TGF-beta, IL-10 or CTLA-4 blockade in vitro on nTreg-mediated control of PMNs and in vivo on fungal growth. As observed in aspergillosis (8), the suppressive function of nTreg cells on PMNs in vitro occurred through CTLA-4 and IL-10 but not TGF-beta (Fig. 3C). In vivo, fungal growth in the stomach, although somewhat decreased by CTLA-4 or IL-10 blockade when performed singularly, was greatly reduced by a combination of both, and it was similar to that observed with anti-CD25 treatment. TGF-beta blockade actually increased, rather than decreased, fungal growth in vivo (Fig. 3D). TNF-{alpha} increased in stomach homogenates-particularly after CD25+ T cell blockade (data not shown). These findings suggested that accumulation of nTreg cells at the sites of infection might control local fungal growth and inflammation. Therefore, the impaired nTreg cell migration to infected sites in Trif–/– mice and the opposite effect in Myd88–/– mice further substantiated the occurrence of disparate patterns of fungal growth and inflammation in Trif–/– and Myd88–/– mice with candidiasis.

Th/iTreg priming is subverted in Trif–/– mice

As CD4+CD25+ Treg cells expanded in MLNs of mice with candidiasis are known to modulate Th cell responses (20), we evaluated whether deregulated antifungal Th cell activation had occurred in WT, Myd88–/– or Trif–/– mice, at 2 wk of infection, a time when both local and peripheral dissemination could be observed in Trif–/– but not WT or Myd88–/– mice. We assessed the functional activity of Foxp3 mRNA expression in purified CD4+CD25+ T cells and the Th program of purified CD4+CD25– T cells by evaluating the Th1 (Tbet), Th2 (Gata3) or Th17 (ROR{gamma}t-encoding Rorc) lineage specification factors. Actual cytokine production by either subset was also evaluated in response to anti-CD3/anti-CD28 stimulation. CD4+CD25+ T cells from infected WT or Myd88–/– mice potently suppressed both Ag-specific and polyclonal proliferation of CD25– T cells, with concomitant inhibition of IFN-{gamma} production (data not shown), an effect observed neither in Trif–/– mice nor in uninfected controls (Fig. 4A). Inhibition was also seen with CD4+CD25+ T cells from thymectomized mice (data not shown). Inhibition was partially prevented by the presence of Abs to IL-10 or CTLA-4 but not TGF-beta (Fig. 4B), a finding suggestive of a common mechanism of action of Treg cells in infection. As no suppressive activity was observed on PMN functions (data not shown), these data suggested that phenotypically and functionally distinct Treg cells are induced over the course of candidiasis.


Figure 4
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  		FIGURE 4. Th/iTreg priming subversion in Trif–/– mice. A, CFSE-labeled CD4+CD25– cells, isolated from MLNs at 2 wk of intragastric infection with the Vir–13 C. albicans strain, were incubated either alone (black histograms) or in combination with CD4+CD25+ cells (red lines) on anti-CD3{epsilon}-coated plates in the presence of soluble anti-CD28 mAb or with Candida-pulsed DCs for 72 or 120 h, respectively, (gray histograms) before FACS analysis. Black histograms, unstimulated CFSE-labeled cells. Black lines, CD4+CD25+ cells from uninfected mice. Numbers refer to mean channel fluorescence intensity. B, CFSE-labeled CD4+CD25– were incubated with CD4+CD25+ cells on anti-CD3{epsilon}-coated plates and soluble anti-CD28 mAb (black lines) in the presence of 10 µg/ml of each mAb (red lines). Black and gray histograms are as in A. Numbers refer to mean channel fluorescence intensity. Results are from three experiments. Gene expression analysis (C) and cytokine production (B) in CD4+CD25– and CD4+CD25+ cells isolated from MLNs 2 wk after intragastric infection with the fungus (+). Purified CD4+CD25– cells were assessed for Tbet, Rorc, and Gata3 mRNA expression and the corresponding CD4+CD25+ cells for Foxp3 mRNA expression by RT-PCR. –, uninfected mice. D, Cytokine production by CD4+CD25– (IFN-{gamma}, IL-17, and IL-4) or CD4+CD25+ (IL-10) isolated from either uninfected (–) or infected (+) mice and stimulated with Candida-pulsed DCs for 5 days, before cytokines quantification in culture supernatants. Naive cells were restimulated with plate-bound anti-CD3 (2 µg/ml), anti-CD28 (2 µg/ml), and 50 U/ml IL-2. Cytokines (pg/ml) were assessed by ELISA. *, p < 0.05, infected vs uninfected. **, p < 0.05, Myd88–/– or Trif–/– cells vs C56BL/6 cells. Bars are SE. The results shown represent one representative experiment out of three (RT-PCR) or three independent experiments (cytokine production).

 
In terms of Th cell activation, Tbet, but neither Gata3 nor Rorc, mRNA expression was up-regulated in CD4+CD25– T cells from WT and, to a lesser extent, Myd88–/– mice (Fig. 4C). Because inhibition of Th1 cell proliferation by Myd88–/– CD25+ cells was not different from that of WT CD25+ cells, this finding confirmed the occurrence of a defective Th1 antifungal priming in the absence of MyD88 signaling (23, 30). The pattern of Th cell activation was completely different in Trif–/–CD25– cells. Tbet mRNA expression was poorly up-regulated in contrast to Gata3 and Rorc expressions (Fig. 4C), indicating that activation of Th2 and Th17 cells had occurred in those mice. Consistent with phenotype analysis, Foxp3 mRNA expression was up-regulated in CD25+ T cells from both WT and Myd88–/– mice, and much less in CD25+ cells from Trif–/– mice (Fig. 4C). The actual cytokine production by either T cell subset confirmed the release of IFN-{gamma} and IL-10 by CD25– and CD25+ cells, respectively, from both WT and Myd88–/– mice, with no IL-4 or IL-17. In contrast, the levels of IFN-{gamma} were low and those of IL-17, more than IL-4, were high in Trif–/–CD25– cells. Minimal production of IL-10 was observed with Trif–/–CD25+ T cells (Fig. 4D). Taken together, these data indicated that iTreg cells capable of suppressing antifungal Th1 priming are induced in candidiasis. These cells are expanded in Myd88–/– mice, in which reduced fungal clearance occurs, also due to defective Th1 priming. iTreg cells were deficient in Trif–/– mice, in which Th2/Th17, but not overt Th1, activation was observed, a finding consistent with the extensive fungal outgrowth and dissemination observed in those mice (31).

Trif–/– DCs prime for Th17 cell activation

During T cell activation, iTreg generation may shift to Th17 cell generation in the presence of innate stimuli (13, 14, 15). Among these, IL-6 has been found to be required for TGF-beta-dependent induction of Th17 cells (13), as well as for Th17 cell maintenance by IL-23 (32). We became interested in assessing the levels of IL-6 production under conditions of TRIF deficiency both in vivo (in MLNs from infected mice) and in vitro (using sorted DCs stimulated with Candida yeasts or hyphae, or LPS). We found that the levels of IL-6 in vivo were not different among WT, Myd88–/–, and Trif–/– mice (Fig. 5A). In vitro, however, the production of IL-6 in response to the fungus and LPS was observed in Trif–/– DCs. Importantly, IL-6 production by Trif–/– DCs was associated with increased IL-23 release particularly in response to yeasts and decreased IL-10 production, particularly in response to hyphae (Fig. 5B). Of interest, IL-10 production by Trif–/– DCs was decreased or increased by the respective addition or neutralization of IL-6 (data not shown). This might link IL-10 production to IL-6 signaling in DCs.


Figure 5
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  		FIGURE 5. Th17 cell priming by Trif–/– DCs. Cytokine production by MLNs from naive (–) or infected (for 2 wk) mice (A) or by CD11c+ DCs purified from MLNs of naive mice and stimulated with Vir–3 yeasts (Y), Vir–13 hyphae (H) or LPS (10 µg/ml) for 24 h in B. Naive MLNs were restimulated in vitro as in legend to Fig. 4. *, p < 0.05, Myd88–/– or Trif–/– vs C57BL6 MLNs and Trif–/– DCs vs C57BL/6 DCs. The results shown are pooled from four experiments in C. MLN CD4+CD62Lh naive Th cells from C57BL/6 or Trif–/– mice were cultured in the presence of the corresponding DCs either unpulsed (group 1) or pulsed with Candida (groups 2–5) for 5 days and restimulated as in legend to Fig. 4 before cytokine quantification in culture supernatants by ELISA. *, p < 0.05, groups 2 and 3 vs group 1; **, p < 0.05, groups 4 and 5 vs 3 and 2, respectively. D, Costimulatory and MHC class II Ag expression by MLN DCs purified from naive mice and pulsed in vitro with Candida hyphae for 18 h. Black and gray histograms indicate pulsed or unpulsed DCs, respectively. Black lines refer to control stained cells. E, Phenotypic analysis of bone marrow-derived DCs from C57BL/6 or Trif–/– mice grown on GM-CSF + IL-4 for 7 days (GM-DCs) or FLT3-L for 9 days (FL-DCs). In both D and E, numbers refer to percentages of positive cells. F, A total of 5 x 105 purified MLN CD11c+ DCs pulsed with Candida were adoptively transferred into recipient mice twice, a week apart, before intragastric infection with the fungus. Frequencies (mean ± SE per 104cells, by ELISPOT assay) of cytokine-producing cells in MLNs 3 days after infection. *, p < 0.05, mice receiving DCs vs mice not receiving DCs.

 
As a whole, these data indicated that under conditions of TRIF deficiency, the activation program of DCs in response to the fungus would predict an ability to activate Th17 rather than iTreg cells. This was indeed the case both in vitro and in vivo. In vitro, Trif–/– DCs induced IL-17 rather than IL-10 production by CD4+CD62Lh naive Th cells purified from Trif–/– mice, whereas WT DCs induced IL-10 and not IL-17 production by naive Th cells from WT mice (Fig. 5C). Interestingly, Trif–/– DCs also increased IL-17 production by naive WT cells, whereas WT DCs increased IL-10 production by Trif–/– T cells, suggesting a dysfunctional activity of the DC compartment in the absence of TRIF signaling.

To understand the level at which the dysfunctional DC activity might occur, we analyzed costimulatory molecule expression of MLN DCs upon pulsing with the fungus as well as DC subset development in bone marrow cultures from Trif–/– mice. Although both WT and Trif–/– DCs up-regulated costimulatory and MHC class II molecules upon pulsing with the fungus (Fig. 5D), the number of plasmacytoid DCs in bone marrow cultures (Fig. 5E) and MLNs (data not shown) was greatly reduced in Trif–/– mice, indicating that a defective developmental pathway in plasmacytoid DCs may contribute to the immunogenic potential of DCs in Trif–/– mice. In vivo, injection of Candida-pulsed Trif–/– DCs into WT mice increased the number of CD4+IL-17+ cells and decreased CD4+IL-10+ cells, whereas the opposite was observed after injection of Candida-pulsed WT DCs into Trif–/– mice (Fig. 5F). Accordingly, susceptibility to infection was increased in WT mice and decreased in Trif–/– mice (data not shown). These data suggested that Trif–/– DCs are sufficient to activate pathogenic Th17 cells in infection.

Failure to activate the tolerogenic IDO mechanism in Trif–/– mice

Although IL-6 and STA3 oppose Foxp3 expression and iTreg cell differentiation in cultured T cells (33), IL-6 is a potent inhibitor of IDO (34), an enzyme stringently required for induction of peripheral tolerance in candidiasis (26). Indeed, IDO+/IL-10+ DCs activate iTreg cells in candidiasis, whereas IDO blockade prevents iTreg cell generation (26). We assessed whether defective IDO activation would occur in Trif–/– mice. We assayed the levels of IDO protein expression in WT, Myd88–/–, and Trif–/– DCs following pulsing with the fungus in vitro as well as in MLNs from infected mice. IDO protein was detected in both WT and Myd88–/– DCs, but not in Trif–/– DCs pulsed with the fungus (Fig. 6A). Of interest, hyphae, but not yeasts, induced IDO protein expression. As tolerogenic potential in otherwise immunogenic DCs can be induced by exposure to tryptophan catabolites and this results in TGF-beta-dependent conversion of naive CD4+ cells into Foxp3+ Treg cells (24), CD4+CD62Lh naive Th cells were cocultured with Candida-pulsed, IDO-Trif–/– DCs in the presence of tryptophan catabolites and assessed for Foxp3 and Rorc mRNA expression as well as IL-10 and IL-17 production. Fig. 6B shows that under these conditions, the recovered T cells had an increased expression of Foxp3 mRNA and concomitant up-regulation of IL-10. Importantly, the Th17 lineage specification factor, Rorc, and related IL-17 production were both suppressed by kynurenins. This finding strongly supports the contention that an impaired IDO function is involved in defective tolerance and the induction of Th17 cell activity. Results obtained in vivo confirmed the above in vitro studies. IDO protein expression was not observed at earlier time points (data not shown), but it was incrementally induced in both WT and Myd88–/– but not Trif–/– mice at 1 wk of infection with Candida hyphae but not yeasts (Fig. 6C). IL-6 neutralization partially restored IDO protein expression in Trif–/– mice (data not shown). Thus the failure to activate IDO+ DCs combined with the subversion of IL-6/IL-10/IL-23 production may explain the shift from iTreg to Th17 induction in the absence of TRIF signaling.


Figure 6
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  		FIGURE 6. TRIF-dependent IDO activation in infection. A, IDO protein expression (Western blotting) in MLN CD11c+ DCs exposed or not (–) to live unopsonized Vir–3 yeasts or Vir–13 hyphae (at a DC/fungal ratio of 2:1) in vitro for 18 h. IDO expression was analyzed by immunoblotting with rabbit polyclonal IDO-specific Ab on whole cell lysates. The positive control consisted of IDO-expressing MC24 transfectants and the negative control of mock-transfected MC22 cells. B, MLN CD4+CD62Lh naive Th cells from Trif–/– mice were unstimulated (group 1), stimulated with plate bound anti-CD3 (2 µg/ml) and anti-CD28 (2 µg/ml) (groups 2 and 3) or cultured in the presence of the corresponding Vir–13-pulsed DCs (groups 4 and 5) for 5 days in the presence or not of kynurenins, a mixture of 3-hydroxykynurenine and 3-hydroxyanthranilic and quinolinic acids, each at 10 µM. Foxp3 and Rorc mRNA expression were done by RT-PCR and cytokine quantification in culture supernatants by ELISA. *, p < 0.05, groups 3 and 5 vs respective controls. C, Mice were infected intragastrically with Vir–3 yeasts or Vir–13 hyphae and assessed for IDO protein expression in MLNs 14 days later. Scanning densitometry was done on a Scion Image apparatus. The pixel density of IDO band was normalized against beta-tubulin. IDO expression was evaluated as in A.

 

   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
This study demonstrates that generation of protective immunity to C. albicans relies on the presence of functionally and phenotypically distinct Treg cell subsets that are sequentially induced in the course of infection through a process implicating distinct, nonredundant roles of MyD88 and TRIF pathways. Earlier studies have shown that Treg cells limiting antifungal Th1 immunity are induced in MLNs, and in the thymus as well, of mice with gastrointestinal candidiasis (20). Here we show that nTreg cells of thymic origin migrate to the sites of infection where, by inhibiting PMN effector function and sparing their inflammatory activity, they allow for fungal growth with limited inflammation by virtue of CTLA-4- and IL-10-dependent mechanisms. Thus, as observed in the Leishmania model (35), defective Treg cell accumulation to infected organs apparently increased resistance to candidiasis. However, an uncontrolled concomitant inflammation would later impair the development of protective Th immunity. Therefore, a tight control of both pathogen growth and host reactivity is needed in infection and nTreg cells might operate this control.

nTreg cells poorly accumulated at sites of infection in the absence of TRIF signaling, suggesting that TRIF crucially controls the migratory capacity of nTreg to sites of inflammation in the gut. As migration to inflamed sites is an essential component of nTreg suppressive activity (36), our findings reveal an unexpected role for TRIF in the orchestration of antimicrobial immune responses. TRIF seemed to affect the expression of molecules required for T cell homing to the gut. In preliminary experiments, adoptive transfer of nTreg cells from WT into Trif–/– mice ameliorated infection in the recipient hosts (data not shown), a finding suggesting that signals for local chemokine production required for nTreg cells recruitment may not be defective under conditions of TRIF deficiency.

In contrast to that seen in Leishmania major infection (37), site-restricted nTreg cells were not Ag-restricted but were sensitive to TLR modulation. Indeed, nTreg cell activity was heightened in Myd88–/– mice, suggesting that MyD88 signaling may promote inflammation by limiting the activity of nTreg cells at infection sites. The reduced ability to restrict fungal growth in both Tlr2–/– and Tlr3–/– mice without signs of exaggerated inflammatory response suggests a role for additional TLRs in controlling the quality of nTreg cell activity at the sites of infection. In this regard, a role for TLR2 in Treg cell function in disseminated candidiasis has been reported (38). As Tlr4–/– mice recapitulated the pattern of fungal growth and inflammatory pathology seen under conditions of MyD88 or TRIF deficiency, our results point to TLR4 as a fungal recognition pathway involved in both immunity and tolerance to the fungus.

Later in infection, persistence of the fungus with no obvious signs of inflammation, as observed in WT and Myd88–/– mice, was associated with the occurrence of Th1 and concomitant iTreg cell induction, as expected (20). Thus the presentation of fungal Ags in the context of limited danger signals likely results in successful activation of protective immunity to the fungus. Consistent with the notion that Th1 immunity to the fungus requires the MyD88 pathway (23), Th1 cell activity was reduced per se in Myd88–/– mice, thus contributing to a reduced fungal clearance in comparison to WT mice. Despite this impaired Th1 cell activation, iTreg cells were present, if not increased, in Myd88–/– mice, which points to a distinction between iTreg cells and suppressive Tbet+Foxp3– cells (39, 40). However, the fact that they were induced in thymectomized mice not only suggests a thymus independence, but it also indicates that iTreg cells take part in immune activation within secondary lymphoid organs.

One interesting observation in the present study was that failure to develop iTreg cells was associated with activation of Th2/Th17 cells as seen in Trif–/– mice, a finding consistent with the notion that iTreg cell induction may shift to Th17 cell generation in the presence of inflammatory stimuli (13). We have recently shown that, similar to Th2 activation (31), the Th17 pathway promotes inflammation and inhibits protective Th1 immunity in mice with candidiasis (18). Therefore, the importance of iTreg induction in candidiasis may go well beyond a mere control of the expression of Th1 immunity, to include functional Th17 antagonism. Because iTreg cells do not inhibit, and in fact seem to enhance, IL-17 production (41, 42), the suppressive effect of iTreg is likely contingent on the ability of these cells to block initial activation and Ag-induced T cell expansion.

Among inflammatory stimuli, IL-6 and other cytokines are known to divert iTreg generation to Th17 induction in the presence of TGF-beta (13). Trif–/– DCs produced IL-6 together with high levels of IL-23 and low levels of IL-10 in response to the fungus or TLR stimulation. Because of the ability of IL-6 to induce IL-23 responsiveness during Th17 differentiation (32), the coproduction of IL-6 and IL-23 under conditions of TRIF deficiency may favor Th17 activation. However, the ability of IL-6 to inhibit IDO, known to regulate IL-10 production by DCs and to be required for iTreg cell generation in candidiasis (26), would predict a direct inhibitory effect of IL-6 on iTreg cell induction, as already reported (32, 33). This might have occurred in Il6–/– mice, whose susceptibility to candidiasis was causally linked to an increased IL-10 production (43). The failure to activate iTreg cells under conditions of TRIF deficiency could be reverted by WT DCs with concomitant reduction of Th17 cell activation. In contrast, Trif–/– DCs inhibited iTreg cell development and favored Th17 induction in WT T cells. In vitro, the addition of tryptophan catabolites converted immunogenic Trif–/– DCs into tolerogenic cells fostering the generation of Foxp3- and IL-10-expressing cells, as already described (24). This finding strongly suggests that an impaired IDO function is involved in the defective tolerance of Trif–/– mice. In addition, this is the first time that kynurenins have been shown to mediate both induction of Foxp3 and transcriptional inhibition of Rorc in naive CD4+ T cells. These data further establish IDO as a truly immuno-regulatory mechanism in infection, controlling the balance between Th cell subsets and Treg cells (44).

The dysfunctional DC activity due to TRIF deficiency is of interest. Not only is the developmental pathway in plasmacytoid DCs defective, but, as ‘reverse signaling’ by Treg cells activates the suppressive pathway of tryptophan catabolism in DCs (45), it is also possible that a vicious circle occurs in the absence of TRIF signaling, whereby the defective number of tolerogenic DCs would combine with defective tolerogenic signals from iTreg cells.

Altogether, the current results suggest a scenario in which sensing of the fungus through both MyD88 and TRIF pathways mediates the induction of a state of protective tolerance, in which fungal persistence is maintained in the context of a poorly inflammatory environment. IDO, known to have a central role in the induction of Th1 immunity within a regulatory environment (22), appears to be involved in the TRIF-dependent tolerance to the fungus. In this regard, as iTreg cell induction did not occur under conditions of Th1 cell deficiency (our unpublished observations), and IFN-{gamma} is a potent IDO activator (46), this suggests the existence of an IFN-{gamma}/IDO-dependent pathway leading to sequential Th1/iTreg cell activation in infection. Interestingly, the fact that hyphae, more than yeasts, activate the TRIF-dependent expression of IDO ((26) and this study), further suggests that differential sensing of fungal morphotypes through distinct recognition receptors may promote distinct immune responses (47, 48). In addition, fungal hyphae, more than yeasts, may promote tolerance and thus contribute to commensalism and eventually to immunoevasion, as suggested (2). Because swollen Aspergillus conidia will promote inflammatory responses by subverting tolerance (2), our results confirm the central role of tolerance at the fungus/host interface and emphasize the role of tryptophan catabolism in the tolerant state (49). On a translational level, as Treg cell induction appears to be defective in patients with autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (50, 51), a disease associated with an often refractory chronic mucocutaneous candidiasis (CMC) (52), the results of the present study may provide new insights into the pathogenesis of chronic mucocutaneous candidiasis (CMC) with possible therapeutic implications.


   Acknowledgments
 
We thank Dr. Cristina Massi Benedetti for editorial support and Paolo Mosci for histology.


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


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

1 This work was supported by the National Research Project on Acquired Immunodeficiency Syndrome (AIDS) Contract No. 30G.28, Italy, and the Specific Targeted Research Project No. LSHM-CT-2005 (Contract No. 005223 (FP6)). Back

2 Address correspondence and reprint requests to Dr. Luigina Romani, Microbiology Section, Department of Experimental Medicine and Biochemical Sciences, University of Perugia, Via del Giochetto, Perugia, Italy. E-mail address: lromani{at}unipg.it Back

3 Abbreviations used in this paper: Treg cells, Regulatory T cells; nTreg, naturally-occurring Treg cells; iTreg cells, induced Treg cells; PMN, polymorphonuclear neutrophil; DC, dendritic cell; MLN, mesenteric lymph node; GITR, glucocorticoid-induced TNFR; WT, wild type. Back

Received for publication May 30, 2007. Accepted for publication August 24, 2007.


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 Materials and Methods
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