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The Journal of Immunology, 2008, 180, 4022 -4031
Copyright © 2008 by The American Association of Immunologists, Inc.
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Lack of Toll IL-1R8 Exacerbates Th17 Cell Responses in Fungal Infection1

Silvia Bozza*, Teresa Zelante*, Silvia Moretti*, Pierluigi Bonifazi*, Antonella DeLuca*, Carmen D’Angelo*, Gloria Giovannini*, Cecilia Garlanda{dagger}, Louis Boon{ddagger}, Francesco Bistoni*, Paolo Puccetti*, Alberto Mantovani{dagger} and Luigina Romani2,*

* Department of Experimental Medicine and Biochemical Sciences, University of Perugia, Perugia, Italy, and Fondazione "Istituto di Ricovero e Cura per le Biotecnologie Trapiantologiche" I.B.i.T., Perugia, Italy; {dagger} Clinical Institute Humanitas, Istituti di Ricovero e Cura a Carattere Scientifico, Rozzano, Italy, and Institute of General Pathology, University of Milan, Milan, Italy; and {ddagger} Bioceros, Utrecht, The Netherlands


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
TLRs contribute to the inflammatory response in fungal infections. Although inflammation is an essential component of the protective response to fungi, its dysregulation may significantly worsen fungal diseases. In this study, we tested the hypothesis that Toll IL-1R8 (TIR8)/single Ig IL-1-related receptor, a member of the IL-1R family acting as a negative regulator of TLR/IL-1R signaling, affects TLR responses in fungal infections. Genetically engineered Tir8–/– mice were assessed for inflammatory and adaptive Th cell responses to Candida albicans and Aspergillus fumigatus. Inflammatory pathology and susceptibility to infection were higher in Tir8–/– mice and were causally linked to the activation of the Th17 pathway. IL-1R signaling was involved in Th17 cell activation by IL-6 and TGF-β in that limited inflammatory pathology and relative absence of Th17 cell activation were observed in IL-1RI–/– mice. These data demonstrate that TIR8 is required for host resistance to fungal infections and that it functions to negatively regulate IL-1-dependent activation of inflammatory Th17 responses. TIR8 may contribute toward fine-tuning the balance between protective immunity and immunopathology in infection.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The initial phase of host defense against microbes involves TLRs that function as primary sensors of microbial products and initiate innate immunity through activation of the transcription factor NF-{kappa}B and stress-activated protein kinases, leading to the expression of immune and proinflammatory genes (1). In this regard, TLR activation is a double-edged sword, and negative regulation of TLR signaling may be required to avoid detrimental and inappropriate inflammatory responses (2, 3), because the immune system needs to constantly balance activation by TLR signaling and inhibition by negative regulators.

Several negative regulators of TLR signaling have been identified over the past several years. The inhibitory response is achieved at multiple levels (2), which include production of soluble TLRs acting as decoy receptors, regulation of TLR expression, and sequestration of recruitment of adaptor molecules such as MyD88 and IL-1R-associated kinase by the transmembrane proteins IL-1RII, ST2, and the Toll IL-1R8 (TIR8)3 (also known as single Ig IL-1-related receptor (SIGIRR)) (4, 5, 6). TIR8 sequesters the formation of TLR signaling complexes and tunes the action of inflammatory cytokines/chemokines by inhibiting IL-1R and TLR4 signaling (6, 7, 8). Evidence suggests that TIR8 recruited at signaling receptor complexes may act as an intracellular molecular trap for components of the transduction cascade (TNFR-associated factor 6 and IL-1R-associated kinase 1) (6). Thus, TIR8 is a member of the TLR/IL-1R superfamily with unique anti-inflammatory properties.

Although TIR8 is normally expressed in various human (9) and murine (6, 7) tissues, high mRNA levels are found in kidney, gut, liver, and lung (10). Previous studies indicated that TIR8 is highly expressed in intestinal and colonic epithelial cells and is down-regulated in certain inflammatory conditions, suggesting that the inhibitory role of TIR8 signaling may play an important role in the regulation of gut mucosal immunity (7, 11). More recently, TIR8 has also been reported to negatively regulate the inflammatory response in infection in vivo (7, 12).

In fungal infections, despite extensive investigations on the role of selected TLRs in the elicitation of proinflammatory cytokines in vitro, little is known about the role of TLRs in contributing to the inflammatory pathology associated with infection. Although inflammation is an essential component of the protective response to fungi, its dysregulation may significantly worsen fungal diseases (13). Recent studies have shown that the Th17 pathway is activated by Candida albicans (14, 15), acts as a negative regulator of the Th1-mediated immune resistance to both C. albicans and Aspergillus fumigatus, and plays an inflammatory role previously attributed to uncontrolled Th1 cell responses (16). IL-23 acts as a molecular connection between uncontrolled fungal growth and inflammation, being produced by dendritic cells (DCs) in a TLR-dependent manner and counterregulating IL-12p70 production (16).

In this study, we investigated the function of TIR8/SIGIRR in the induction of inflammation and immunity in mice with candidiasis or aspergillosis, testing the hypothesis that TIR8/SIGIRR may be an important negative regulator of TLR-dependent activation of Th17 cells. Our data provide evidence that TIR8 is required for host resistance to fungal infection, by down-regulating IL-1 signaling-dependent activation of Th17 cell responses.


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

Eight - to 10-wk-old, Tir8-deficient mice (Tir8–/–) were raised on a 129/Sv and C57BL/6J mixed genetic background, and IL-1R1-deficient mice (hereafter indicated as IL-1R1–/–) were raised on a C57BL/6 background (7, 17). Wild-type mice included 129/Sv x C57BL/6J mice (Tir8+/+) or inbred C57BL/6 (IL-1R1+/+) mice obtained from Charles River Laboratories. Experiments were performed according to the Italian Approved Animal Welfare Assurance A-3143-01.

Fungi, infections, and treatments

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 (18, 19). 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 Vir–13 cells were injected intragastrically, and quantification of fungal growth was expressed as CFU/organ (mean ± SE), as described (18). For i.v. infection, mice received 5 x 105 Candida Vir–13 cells in 0.5 ml. The strain of A. fumigatus and culture conditions were as described (20). Mice received 2 x 107 resting Aspergillus conidia intranasally twice. Quantification of fungal growth was done by the chitin assay, and results are expressed as µg of glucosamine/organ. Fungi were suspended in endotoxin-free (Detoxi-gel; Pierce) solutions (<1.0 EU/ml, as determined by the Limulus amebocyte lysate method). For histology, tissues were excised and immediately fixed in formalin. Sections (3–4 µm) of paraffin-embedded tissues were stained with periodic acid-Schiff reagent and examined for histology (18, 20). Treatments with 200 µg/injection of p19-neutralizing rabbit IgG Abs (21), IL-17-neutralizing rat IgG2a mAb (mAb 421, clone 50104, noncross-reacting with IL-17E or IL-17F; R&D Systems, Space Import-Export), IL-12-neutralizing rat IgG1 mAbs (a 1:1 mixture of C15.6.7 and C15.1.2 mAbs, recognizing different epitopes of IL-12p40; BD Pharmingen), IFN-{gamma}-neutralizing rat IgG1 mAb (XMG.1.6; BD Pharmingen), and IL-6-neutralizing rat IgG1 mAb (MP5-20F3; eBioscience) were done i.p. on days 0 and 1 after the gastrointestinal infection. A total of 1 mg of purified mouse IgG1 anti-TGF-β 1,2,3 (clone 1D11) (22) was given i.p. on days 0 and 1 after infection. Treatments with 100 µg/injection of IL-17-neutralizing mAb or control rat IgG2a (MAB006, clone 5447; R&D Systems) were done in mice i.v. injected with 5 x 105 Candida Vir–13 cells, on days 0 and 1 after the infection. Anti-β-galactosidase (clone GL113; BD Pharmingen) rat IgG1 mAb was used as control in the gastrointestinal infection, because no differences were observed with each isotype control mAb. Blood smears were prepared and stained by the May-Grunewald-Giemsa method for differential counts.

Purification and culture of cells

Mesenteric lymph node (MLN) DCs (>99% CD11c+ and <0.1% CD3+) were purified by magnetic activated sorting using CD11c MicroBeads and MidiMacs (Miltenyi Biotec). DCs were exposed to live unopsonized yeasts or hyphae, as described (19, 23). At 18 h of culture, cells were harvested for costimulatory molecule expression and supernatants were assessed for cytokine contents by ELISA. Gr-1+ CD11b+ polymorphonuclear neutrophils (PMNs; >98% pure on FACS analysis) were positively selected with magnetic activated sorting using Ly-6G MicroBeads and MidiMacs (Miltenyi Biotec) from the peritoneal cavity of thioglycolate-treated mice. CD4+CD25+ and CD4+CD25–CD62Lh cells were purified from MLN or spleens by magnetic activated sorting (Miltenyi Biotec). Unfractionated MLN or thoracic lymph node (TLN) cells were cultured with live unopsonized fungi before cytokine determinations in culture supernatants 5 days later (18, 20). MLN CD4+CD25–CD62Lh T cells (106/ml) were cultured in flat-bottom 96-well plates in the presence of 5 x 105 Candida-pulsed DC for 5 days, with and without neutralizing Abs (10 µg/ml anti-IL-1β hamster IgG1, BD Pharmingen, or anti-IL-23 goat IgG, R&D Systems), before RT-PCR and cytokine quantification in culture supernatants by ELISA. Fungal overgrowth was prevented, as described (23). CD4+CD62Lh Th cells were restimulated with plate-bound anti-CD3{epsilon} (2 µg/ml) and anti-CD28 (2 µg/ml) and 50 U/ml IL-2, as described (22). Neither isotype control mAb added to the culture affected cytokine production (data not shown).

Flow cytometry

Staining was done, as described (18, 20). FITC-conjugated anti-mouse Abs were from BD Pharmingen (CD4, CD25, CD11b, CD11c, B220, CD80, and CD86) and eBioscence (Foxp3). 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 was performed by using a specific staining kit. Data are expressed as a percentage of positive cells over total cells analyzed. Flow cytometry was used to determine the purity of isolated cells.

Phagocytosis, antifungal effector activity, zymography, and myeloperoxidase (MPO) assays

Assays of PMN phagocytosis of unopsonized Candida yeasts or A. conidia and fungicidal activity were conducted, as described (17). Results are expressed as the percentage of CFU inhibition (mean ± SE). The expression of the NO synthase (NOS2)-encoding gene and Arg1 (coding for arginase 1) as well as matrix metalloproteinase 9 (MMP9)/MPO determinations were done after 60 min of fungal exposure. Gelatinolytic activity of MMP9 was determined by scanning densitometry in the 72-kDa area (24). MPO activity was determined by the oxidation of 3,3',5,5'-tetramethylbenzidine by H2O2, and light absorption was measured at a 655-nm wavelength. Units of MPO activity (per 10 min) were calculated from a standard curve that used the peroxidase (Sigma-Aldrich) enzyme as the standard enzyme. MPO data were expressed as U/106 cells, as described (24).

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 manufacturer’s directions. The PCR primers for genes encoding IL-12p35, IL-23p19, IL-12Rβ2, IL-23R, as well as Tbet, Rorc, Gata3, and Foxp3, were as described (16, 22). The PCR primers for Arg1 were as follows: forward primer, 5'-AGGAGAAGGCGTTTGCTTAG-3' and reverse primer, 5'-ACCATAAGCCAGGGACTGAC-3'. 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 ELISAs (R&D Systems; for IL-23, eBioscience) on tissue homogenates or supernatants of cultured cells. The detection limits (pg/ml) of the assays were <30 for IL-23, <10 for IFN-{gamma}, <5 for IL-4, <3 for IL-10, <10 for IL-17, <7 for IL-6, <6 for IL-1{alpha}, <3 for IL-1β, and <4 for TGF-β1. 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 (16, 22). Results were expressed as the mean number of cytokine-producing cells (±SE) per 104 cells, calculated using replicates of serial 2-fold dilutions of cells.

Statistical analysis

The double-tailed Student’s t test was used to compare the significance of differences between groups. Survival was calculated using Kaplan-Meier calculations. A value of p < 0.05 was considered significant. The data reported are either from one representative experiment of three independent experiments (FACS analysis) or pooled from three to five experiments otherwise. The in vivo groups consisted of 6–8 mice/group.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Tir8–/– mice are susceptible to candidiasis

To assess the susceptibility of Tir8–/– mice to C. albicans infection, mice were infected intragastrically or i.v. with the fungus and monitored for survival, fungal growth, and tissue pathology. Fig. 1 shows that Tir8–/– mice were highly susceptible to mucosal (A and C) and disseminated (B and D) infections, as judged by the decreased survival after either type of infection and the increased fungal burden in the stomach (A) or kidney (B), associated with a noticeable dissemination to visceral organs in both infections (data not shown). Histopathological examination of the stomach revealed no obvious differences between wild-type and Tir8–/– uninfected mice (data not shown). In the intragastric infection, however, a limited parakeratosis, acanthosis, and inflammatory reaction, characterized by infiltrates of mononuclear cells, were present in the stomach of Tir8+/+ mice. In contrast, numerous fungi were present in the keratinized layer in association with massive infiltrates of inflammatory cells, predominantly PMNs, acanthosis, and hyperkeratosis in the stomach of Tir8–/– mice (Fig. 1C). Similarly, in disseminated infection, few abscesses were present in the kidneys of Tir8+/+ mice, as opposed to numerous abscesses of mononuclear cells and PMNs in the kidneys of Tir8–/– mice (Fig. 1D). Circulating PMNs were also higher in Tir8–/– than Tir8+/+ mice in disseminated candidiasis (data not shown). These data indicate that TIR8 plays a crucial role in dampening the inflammatory host response to the fungus.


Figure 1
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  		FIGURE 1. Susceptibility of Tir8–/– mice to candidiasis. Tir8+/+ or Tir8–/– mice were injected either intragastrically (A and B) with 108 Vir–3 Candida or i.v. (C and D) with 5 x 105 Vir–3 Candida cells. Fungal growth (CFU) was evaluated 7 (A) or 3 (C) days after the infection. Shown are the results compiled from three experiments, each consisting of groups of six mice. Periodic acid-Schiff-stained medial sections of the stomach (B, 1 wk after infection) or kidneys (D, 3 days after infection) of mice with mucosal or disseminated disease, respectively. Note the presence of fungi (arrows) with signs of inflammatory cell recruitment and parakeratosis in the stomach of Tir8–/– mice as opposed to parakeratosis, but limited signs of inflammatory reaction in the stomach of Tir8+/+ mice. Few abscesses of mononuclear cells (arrows) were present in the kidney cortex from Tir8+/+ mice as opposed to diffuse infiltrates of inflammatory cells through the kidney parenchyma in Tir8–/– mice. Bars indicate magnification. Shown in insets are gross pathology specimens. Peritoneal polymorphonuclear neutrophils were assessed for fungicidal (E) and MPO (F) activity (assessed as described in Materials and Methods and confirmed by Western blotting (data not shown)), expression of NO synthase (NOS2 specific), and arginase (Arg1) mRNA (by real-time RT-PCR) and MMP9 production (by gelatin zimography) in PMNs either unexposed (–) or after the exposure (+) to Candida yeasts (effector to fungal cell ratio of 1:5), for 60 min. For the candidacidal activity, the values, expressed as percentage of killing, refer to percentage of CFU inhibition (mean ± SE). *, p < 0.05, Tir8–/– vs Tir8+/+ mice.

 
Alternative activation of PMNs in Tir8–/– mice

The finding that PMNs were abundantly recruited in the stomach of Tir8–/– mice in the presence of unimpaired fungal growth confirmed the finding that PMN recruitment is not affected in Tir8–/– mice (25) and led us to verify whether the antifungal effector and secretory functions of these cells were impaired. We evaluated phagocytosis, killing activity, respiratory burst, and the occurrence of metabolic pathways associated with IL-12 (NO synthase, NOS2-specific mRNA), IL-23 (arginase, Arg1 mRNA), or IL-17 (MMP9) activity on phagocytic cells (16, 22, 26). Although no differences were observed in the phagocytic capacity (data not shown), a defective killing activity (Fig. 1E), associated with an impairment of the respiratory burst (Fig. 1F), was found in PMN from Tir8–/– as compared with wild-type mice. Moreover, Tir8–/– PMNs failed to up-regulate NOS2-encoding gene expression in response to the fungus, as opposed to an up-regulated expression of Arg1 and high production of MMP9 (Fig. 1F). Therefore, PMNs from Tir8–/– mice have reduced antifungal effector functions and showed signatures of alternatively activated phagocytes (4, 27).

Increased inflammatory cytokine production and Th1/Th17 cell responses in Tir8–/– mice

It has recently been shown that the activation of inflammatory Th17 may accommodate the failure to control fungal growth in the face of an obvious inflammatory response (13, 16, 22). Because members of TLR/IL-1R superfamily are involved not only in innate, but also in Th adaptive immune responses after induction of inflammation (28), we assessed whether TIR8-modulated cytokines would associate with the adaptive immune response. We measured levels of proinflammatory/anti-inflammatory cytokines in the stomach or kidney homogenates of mice with mucosal or disseminated infection, respectively, the mRNA expression of IL-12β2R- and IL-23R-encoding genes, as well as of Tbet, Rorc, and Gata3 in CD4+ T cells, and the frequency of IFN-{gamma}-, IL-17-, IL-4-, or IL-10-producing CD4+ cells in MLNs 1 wk after mucosal infection. At sites of infections, the levels of proinflammatory cytokines (IL-12p70, IL-23, IL-6, and TNF-{alpha}) were higher, and those of IL-10/TGF-β lower, in the kidney (Fig. 2A) and stomach (Fig. 2B) homogenates of Tir8–/– mice. Levels of TGF-β could not be measured in the kidney homogenates of either type of mice upon infection, whereas levels of IL-10, similar to those of TGF-β, were greatly reduced in the stomach homogenates of Tir8–/– mice as compared with Tir8+/+ mice (data not shown). In terms of adaptive Th immunity, both the IL-12β2R/Tbet and IL-23R/Rorc mRNA expressions were higher in Tir8–/– than wild-type mice, whereas Gata3 mRNA expression was not increased in either genotype (Fig. 3C). The frequency of Th1 (IFN-{gamma}-producing) or Th17 (IL-17-producing) cells was also higher in Tir8–/– than Tir8+/+ mice, whereas IL-4-producing Th2 cells were not increased (Fig. 2D). Therefore, the high susceptibility of Tir8–/– mice to infection was associated with heightened Th1/Th17 cell responses with limited Th2 cell activation.


Figure 2
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  		FIGURE 2. Inflammatory cytokine production and Th1/Th2/Th17/Treg responses in Tir8–/– mice. Mice were infected (+) i.v. (A) or intragastrically (B–E) as in legend to Fig. 1, and evaluated for cytokine production in the kidney (A) or stomach (B) homogenates (3 days after the infection); Th1 (IL-12β2R-specific/Tbet), Th17 (IL-23R-specific/Rorc), and Th2 (Gata3) mRNA expression (C) and frequencies of IFN-{gamma}-, IL-170-, IL-4-, or IL-10-producing cells (D) in MLN CD4+ cells, 1 wk after the infection. E, Detection of and phenotypic analysis of purified CD4+CD25+ T cells in the MLNs of mice 1 wk after mucosal infection. Numbers refer to percentages of positive cells. Cytokines were measured by ELISA (pg/ml); mRNA expression was measured by real-time RT-PCR; the frequency of cytokine-producing cells by ELISPOT (values are the mean number of cytokine-producing cells (±SE) per 105 cells). Phenotypic analysis was done by flow cytometry. Shown are the results pooled from three experiments, each consisting of groups of six animals. –, Uninfected mice. *, p < 0.05, Tir8–/– vs Tir8+/+ mice.

 

Figure 3
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  		FIGURE 3. Effect of cytokine neutralization in Tir8+/+ or Tir8–/– mice with candidiasis. A, Mice were infected intragastrically, as in legend to Fig. 1, and treated with cytokine-neutralizing Abs on days 0 and 1 after the infection. Fungal growth (CFU) in the stomach and Tbet and Rorc mRNA expression in CD4+ T cells purified from MLNs were analyzed 1 wk after infection. B, Mice were infected intragastrically and treated with cytokine-neutralizing Abs as in A. mRNA expression was measured by real-time RT-PCR. None, Untreated mice. *, p < 0.05, Ab-treated vs untreated mice. Shown are results pooled from four experiments, each consisting of groups of four animals. C, Mice were infected i.v., as in legend to Fig. 1, and treated with IL-17-neutralizing mAbs or control IgG2a Ab on days 0 and 1 after the infection. CFU in the kidneys or brain, IL-17 production (by ELISA on kidney homogenates), and Tbet and Rorc mRNA expression in purified splenic CD4+CD25– T cells were analyzed 3 days after infection. Shown are results pooled from three experiments, each consisting of groups of six animals. *, p < 0.05, Ab-treated vs untreated mice.

 
IL-10-producing, CD4+CD25+ Foxp3+ regulatory T cells (Tregs) are induced in the MLNs of mice with mucosal candidiasis and are known to limit Th1 reactivity and to oppose Th17 cell development (18, 22). We looked for Tregs in MLNs of intragastrically infected mice and found that IL-10-producing cells were less in Tir8–/– than Tir8+/+ mice (Fig. 2D), and CD4+CD25+ Foxp3+ Tregs were not expanded in Tir8–/– mice (Fig. 2E). We concluded that Th1/Treg (IFN-{gamma}/IL-10-producing cell) activation occurred in wild-type mice, whereas Th1/Th17 cell activation occurred in Tir8–/– mice.

Neutralization of IL-23 or IL-17 increases resistance to infection

Although blockade of IL-23 or IL-17 increased antifungal resistance in vivo in mice with fungal infections (16), each cytokine was also found to mediate a degree of protection in condition of Th1 deficiency (16). The concomitant activation of both Th1 and Th17 responses in Tir8–/– mice with mucosal candidiasis appeared to provide a unique opportunity to dissect the relative contributions of both Th cell subsets in resistance and/or susceptibility to infection. Tir8–/– and Tir8+/+ mice were infected intragastrically with the fungus and concomitantly subjected to IL-12, IFN-{gamma}, IL-23, or IL-17 blockade by means of neutralizing Ab. In both genotypes, and particularly in Tir8–/– mice, blockade of IL-23 or IL-17 greatly reduced both the fungal growth and TNF-{alpha} production with concomitant increase of IL-10, a finding pointing to an effect on fungal growth, but also on local inflammatory state. The opposite results were obtained upon IL-12 or IFN-{gamma} neutralization, particularly in Tir8+/+ mice (Fig. 3A). Resistance to infection after IL-23 or IL-17 blockade correlated with an increased expression of Tbet and decreased Rorc in CD4+T cells from MLNs, and the opposite was true for IL-12 or IFN-{gamma} blockade (Fig. 3A). Therefore, whereas in condition of Th1 deficiency, Th17 may provide some protection in infection (16), when both components are present, protection is provided through the Th1 pathway and is opposed by the Th17 pathway.

Although levels of TGF-β did not increase in Tir8–/– mice in infection, the levels of IL-6 were greatly increased. Given the Th17-promoting activity of IL-6 when combined with TGF-β (29), we assessed the possible contribution of either IL-6 or TGF-β to Th17 cell development by evaluating the effect of IL-6 or TGF-β blockade on fungal growth and Th1/Th17 cell activation in mice with mucosal candidiasis. We found that treatment with IL-6- or TGF-β-neutralizing Ab increased fungal burden in Tir8+/+ mice with candidiasis and decreased Tbet, without significantly affecting Rorc in MLN CD4+ T cells. An opposite result was obtained in Tir8–/– mice, in which neutralization of IL-6 or TGF-β significantly reduced fungal growth in the stomach, and this was associated with reduced Rorc and increased Tbet expressions in MLN CD4+ T cells (Fig. 3B). Thus, consistent with previous data showing the association of IL-6 or TGF-β with the activation of protective Th1 responses (30), IL-6 or TGF-β contributes to Th1 immunity in Tir8+/+ mice, but to Th17 activation in Tir8–/– mice. These data suggest that the activation of Th17 cells by IL-6 and TGF-β is contingent upon additional environmental factors.

To assess whether IL-17 blockade also increases resistance to the disseminated infection, Tir8+/+ and Tir8–/– mice were i.v. infected with 5 x 105 Candida Vir–13 cells and concomitantly treated with IL-17-neutralizing mAb or isotype control Ab. As opposed to control Ab, IL-17 neutralization, although lowering the levels of IL-17 at the infection site, significantly increased resistance of mice to the infection, as revealed by the local (kidneys) reduced fungal growth and distal (brain) dissemination. The effect was greater in Tir8–/– than Tir8+/+ mice. Resistance to infection after IL-17 blockade correlated with a decreased expression of Rorc in splenic CD4+ T cells in Tir8–/– mice, and an increased expression of Tbet in Tir8+/+ mice (Fig. 3C). Interestingly, treatment with IL-17-neutralizing mAb greatly reduced the number of peripheral PMNs (data not shown), a finding that is consistent with the ability of IL-17 to recruit PMNs (31), and, at the same time, questions the role of emergency granulopoiesis in systemic candidiasis (32). Our data suggest that IL-17 contributes to the susceptibility to disseminated candidiasis in condition of TIR8 deficiency. However, because susceptibility to disseminated candidiasis is increased in IL-17AR-deficient mice (33), it is likely that the biological activity of IL-17 cannot be attributed solely to the presence and activation of IL-17AR, as suggested (34). In this regard, given that IL-17 signals through a heteromeric receptor complex (34), one potential explanation for the different results obtained in IL-17AR-deficient mice as opposed to those obtained upon IL-17A neutralization (this study) is that IL-17AR-deficient mice are deficient in both IL-17A and IL-17F. As a matter of fact, the IL-17 family has a complex role in regulating innate immune responses (31).

Dysfunctional DC activity in Tir8–/– mice

Because TIR8 is expressed by immature DCs and affects DC cytokine production (7), we assessed whether an altered DC activation program would occur in Tir8–/– mice in response to Candida. DCs from MLNs of naive mice were subjected to phenotypic analysis and assessed for activation and cytokine production after pulsing with Candida. Phenotypic analysis of purified CD11c+ cells revealed that the number of conventional CD11b+ DCs was higher, and that of B220+ DCs lower, in Tir8–/– than wild-type mice (data not shown). Nevertheless, DCs from either type of mice similarly up-regulated costimulatory molecule and MHC class II expression in response to the fungus in vitro (Fig. 4A) and showed a similar level of phagocytic activity (data not shown). In terms of cytokine production, however, and at variance with wild-type DCs, Tir8–/– DCs produced high levels of IL-12p70 and IL-23 in response to yeasts, low levels of IL-10 in response to hyphae, and high levels of IL-1{alpha} and IL-1β in response to both fungal morphotypes (Fig. 4B). Therefore, TIR8 affects the balance of pro- and anti-inflammatory cytokine production by DCs in response to the fungus and, importantly, greatly increases the production of both IL-1{alpha} and IL-1β.


Figure 4
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  		FIGURE 4. DC activity in Tir8–/– mice. CD11c+ DCs were purified from MLNs of uninfected Tir8+/+ or Tir8–/– mice and pulsed with unopsonized Candida yeasts (Y) or hyphae (H) before assessment of costimulatory and MHC class II Ag expression by FACS analysis (A) or cytokine production by ELISA (B), 18 h later. A, Green and black histograms, unpulsed vs yeast-pulsed DCs, respectively. Numbers refer to percentage of positive cells. Black lines refer to control staining of cells with irrelevant Ab. C, MLN CD4+CD62Lh naive Th cells from Tir8+/+ or Tir8–/– mice were stimulated with Candida-pulsed DCs for 5 days in the presence of 10 µg/ml each neutralizing mAb. Responder cells were restimulated with plate-bound anti-CD3 (2 µg/ml) and anti-CD28 (2 µg/ml) and 50 U/ml IL-2 before determination of IL-17 production (pg/ml) by ELISA and mRNA expression by RT-PCR. *, p < 0.05, Tir8–/– vs Tir8+/+ mice. Shown are results pooled from three experiments.

 
The ability of distinct DC subsets to produce directive cytokines in response to Candida is known to condition their ability to activate Th cell in vitro. IL-23-producing DCs are required and sufficient to activate IL-17-producing cells (16, 22). The above data suggest that under conditions of TIR8 deficiency, the activation program of DCs in response to the fungus would predict an ability to activate Th17 cells. Given the crucial role for IL-1 in the induction of IL-17-producing T cells (14, 35, 36), we assessed in this study whether IL-1 could contribute to the ability of Candida-pulsed DCs to prime CD4+CD62Lh naive cells for IL-17 production and Rorc mRNA expression in vitro in condition of TIR8 deficiency. To this purpose, IL-23- or IL-1β-neutralizing Abs were added to the cocultures of DCs and responder cells. Both the production of IL-17 and Rorc mRNA expression by TIR8–/– CD4+ cells were decreased in the presence of either type of Ab, an effect not observed in the presence of TIR8 (Fig. 3C). Instead, Tbet mRNA expression was increased upon IL-23 or IL-1β neutralization in the presence of TIR8 (data not shown). Thus, IL-1β, together with IL-23, contributes to Th17 cell activation in the absence of TIR8.

TIR8 inhibits IL-1 signaling in response to C. albicans

TIR8 is known to act as a decoy receptor for the formation of a signaling complex, which subsequently inhibits IL-1 and TLR4 signaling through NF-{kappa}B activation, and thereby fine-tunes IL-1- or TLR4-dependent inflammatory responses (4, 6, 33). Because Tlr4–/– mice are as susceptible as Tir8–/– mice to mucosal candidiasis with signs of exaggerated inflammatory reaction and PMN recruitment (17, 22), we thought it unlikely that an increased TLR4-dependent signaling could be mechanistically at work to explain the pathogenic inflammatory response observed in Tir8–/– mice. The availability of IL-1RI–/– mice prompted us to assess whether the observed Th17-dependent inflammatory response seen in condition of TIR8 deficiency could be due to an increased IL-1-dependent signaling. In this case, IL-1RI–/– mice would show limited signs of inflammatory cell recruitment and tissue pathology. Although IL-1RI–/– mice have been shown to be susceptible to candidiasis (17), the relative contribution of fungal outgrowth vs inflammatory pathology in infection is unknown. As already shown (17), IL-1 RI–/– mice showed an increased susceptibility to both mucosal (Fig. 5A) and disseminated (Fig. 5C) infections, both in terms of fungal growth in the target organs and dissemination to visceral and distant organs, such as the esophagus and kidneys in the mucosal infection and the brain in the disseminated infection (data not shown). In the disseminated infection, mice did not survive infection (Fig. 1C). At variance with what was observed in Tir8–/– mice, and in line with the notion that IL-1RI signaling is critical for host response to injury and infection (37), limited signs of inflammatory cell recruitment and tissue pathology were observed at sites of infections, such as the stomach (Fig. 5A) or the kidneys (Fig. 5B), where infiltrating cells were predominantly of mononuclear type. Consistent with the ability of IL-1β to recruit PMNs (38, 39), PMN count was significantly lower in the blood of IL-1RI–/– than IL-1RI+/+ mice with the disseminated infection (Fig. 5E). Moreover, IL-1RI–/– PMNs showed a remarkable impaired fungicidal activity, both from naive and infected mice (Fig. 5E), a finding pointing to the crucial role of IL-1RI signaling in PMN activity in response to the fungus.


Figure 5
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  		FIGURE 5. Susceptibility of IL-1RI–/– mice to candidiasis. Mice were injected either intragastrically (A and B) with 108 Vir–3 Candida or i.v. with 5 x 105 Vir–3 Candida cells (C and D). Fungal growth (CFU) was evaluated 7 (A) or 3 (C) days after infection. Shown are results pooled from three experiments, each consisting of groups of six mice. Pathological examination of the stomach (B, 1 wk after infection) or kidneys (D, 3 days after infection) of mice with mucosal or disseminated candidiasis, respectively. Periodic acid-Schiff-stained medial sections from the stomach showed signs of inflammatory cell recruitment associated with parakeratosis and acanthosis that were more prominent in IL-1RI+/+ than IL-1RI–/– mice. Similarly, in the kidneys, abscesses (arrows) were present in the parenchyma of IL-1RI+/+, but not IL-1RI+/+ mice. Bars, Magnification. E, PMN counts in the blood before and after the i.v. infection. Values are the mean ± SE of results from three different experiments. For killing activity of purified PMNs from naive (–) or 3-day-infected (+) mice, see legend to Fig. 3. F, Purified DCs from MLNs of naive mice were assessed for p19 or p35 mRNA (by real-time RT-PCR) before (–) or after (+) pulsing with live unopsonized Candida yeasts. G, Th1 (Tbet), Th17 (Rorc), and Th2 (Gata3) mRNA expression (RT-PCR) in CD4+ T cells purified from MLNs, 1 wk after the mucosal infection. *, p < 0.05, IL-1RI–/– vs IL-1RI+/+ mice.

 
On assessing parameters of adaptive Th immunity, MLN DCs from IL-1RI–/– mice showed a limited increased expression of p35- and p19-encoding genes upon exposure to fungal yeasts (Fig. 5C), and MLN CD4+ T cells from infected mice did not show up-regulated Tbet or Rorc expression. Interestingly, Gata3 expression was instead greatly up-regulated (Fig. 5D), and so was the number of IL-4-producing CD4+ T cells from MLNs of IL-1RI+/+ vs IL-1RI–/– mice with infection (from 3 up to 18 x 105 cells, respectively), a finding suggesting that Th2, rather than Th1/Th17, cell activation has occurred in these mice. As a matter of fact, IL-23 or IL-17 neutralization did not reverse the susceptibility to infection (data not shown). Together, these data suggest that TIR8 inhibits the IL-1 signaling pathway that has a key role in the induction of Th1/Th17 development in candidiasis. Moreover, the data also suggest that in the face of a comparable level of fungal outgrowth in the relevant target organs, the uncontrolled inflammatory response has a crucial pathogenic role in infection, as judged by the increased survival to the mucosal infection seen in concomitance with Th2 (IL-1RI–/– mice), but not Th17 (Tir8–/– mice) cell responses.

Tir8–/– mice are susceptible to aspergillosis

To understand whether TIR8 signaling may have an inhibitory role at sites other than the gut mucosa, we assessed whether TIR8 also negatively regulates Th17 responses in pulmonary aspergillosis. Tir8–/– mice were infected intranasally with A. conidia and assessed for susceptibility to infection in terms of survival, fungal growth, inflammatory tissue pathology, and Th1/Th17/Treg activation. Although both Tir8–/– and Tir8+/+ mice survived infection (Fig. 6A), a portion of Tir8–/– mice (30–40%) died within 1 wk of infection. Three days after infection, Tir8–/– mice showed a higher fungal growth associated with inflammatory PMN recruitment in the lungs as compared with Tir8+/+ mice (Fig. 6A). The number of PMNs was significantly greater in Tir8–/– mice than in Tir8+/+ mice, whereas the number of macrophages, F4/80– CD11c+DCs, CD4+, and CD8+ lymphocytes were not significantly different between Tir8–/– and Tir8+/+ mice (data not shown). In draining TLNs, activation of Th1/Th17, but not Foxp3+ Treg was observed in Tir8–/– mice and was associated with high levels of IFN-{gamma}/IL-17 production and low levels of IL-10 (Fig. 6C). Therefore, TIR8 also negatively regulates Th17 responses in pulmonary aspergillosis. At variance with what was observed in candidiasis, IL-1RI–/– mice were neither more susceptible to aspergillosis in terms of fungal growth or histopathology, nor did they show activation of IL-17+ CD4+ cells in the draining TLNs (data not shown).


Figure 6
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  		FIGURE 6. Susceptibility of Tir8–/– mice to aspergillosis. Mice were infected intranasally with 2 x 107 resting A. conidia and evaluated for A, survival, fungal growth (chitin content, microgram glucosamine/organ), and lung histology (periodic acid-Schiff staining at 3 days of infection), and B, cytokine production (ELISA) and mRNA expression (real-time RT-PCR) of purified CD4+ T cells from TLNs, 1 wk after the infection. Note the presence of a mild inflammatory pathology in Tir8+/+ mice, characterized by few infiltrates of inflammatory mononuclear cells scattered in an otherwise intact lung parenchyma as opposed to the massive infiltration of PMNs in the lung of Tir8–/– mice associated with numerous fungal hyphae (arrows). Shown in inset is gross pathology. Bars, Magnification. Shown are results pooled from three experiments. *, p < 0.05, Tir8–/– vs Tir8+/+ mice.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Excessive or inappropriate activation of immune cells and/or production of proinflammatory cytokines may lead to severe inflammatory diseases. We have recently shown that the Th17 pathway promotes inflammation, but inhibits the activation of protective Th1 responses in response to Candida and Aspergillus (16, 22). Thus, rigorous control of the inflammatory Th17 response is necessary to prevent immunopathology (13). In general, although the protective functions of TLRs and cytokine receptors in inflammation have been widely studied, less is known on how these pathways are negatively regulated (2). We show in this study that the TIR8/SIGIRR acts as a negative regulator of the Th17 pathway in fungal infections and is essentially required to fine-tune the inflammatory and adaptive immune response to C. albicans and A. fumigatus. Both Th1 and Th17 cell responses, with limited activation of either Th2 or Tregs, were heightened in Tir8–/– mice with either infection and were associated with susceptibility to challenge and inflammatory pathology. We could confirm in this study that blocking the Th17, but not the Th1 pathway limited inflammation and restored control of the infection in Tir8–/– mice, a finding clearly distinguishing the relative contribution of each subset in fungal infections.

It has been suggested that, in the presence of IL-6, TGF-β+ Tregs are diverted to inflammatory Th17 cells (29). We found in this study that the relative contribution of the IL-6/TGF-β-dependent axis to Th cell activation in candidiasis is contingent upon additional environmental factors, namely the IL-1RI signaling. The importance of IL-1 and IL-1RI signaling in Th17 and autoimmune T cell activation (14, 35, 36, 40, 41) has been extensively described. Consistent with previous data showing the association of either IL-6 (42) or TGF-β (30) with the activation of protective anticandidal Th1 responses, IL-6 or TGF-β contributes to Th1 immunity in Tir8+/+ mice, but to Th17 activation in condition of heightened IL-1RI signaling, such as in Tir8–/– mice. These data therefore reconcile the seemingly paradoxical protective effects exhibited by both IL-6 and TGF-β in candidiasis, despite their role in Th17 cell promotion.

The role of IL-6 in infection merits further consideration. IL-6, in addition to IL-1{alpha} and IL-1β, potently contributes to PMN recruitment and activation in candidiasis (43) as well as aspergillosis (44). As a matter of fact, PMNs were abundantly recruited at sites of infections in Tir8–/– mice. Interestingly, however, as opposed to wild-type PMNs, the arginine, but not the NO pathway was up-regulated in Tir8–/– PMNs with concomitant elevated MMP9 production and inhibition of the candidacidal and MPO activities. Thus, recruited PMNs showed a pattern of activation similar to that induced by IL-23 and IL-17 (16), resembling that of alternatively activated macrophages (27, 45). These data suggest that the effect of IL-6 on PMN activity may be also contingent upon the presence of IL-1RI signaling. Should this be the case, PMNs, alternatively activated by IL-6 in condition of up-regulated IL-1RI signaling, by perpetrating inflammation and concomitant defective fungal clearance, might contribute to a poor prognosis in fungal infections. As a matter of fact, despite the established role of neutrophils as a first line of defense against C. albicans (38, 39), unrestrained fungal growth and extensive tissue damage were both observed in experimental disseminated candidiasis in the face of emergency neutrophilia (32).

Studies have demonstrated that the activation of the Th17 pathway against fungi is under the strict control of TLRs, being positively regulated through the TLR2/MyD88 pathway and negatively regulated through the TLR4/Toll/IL-1 receptor-domain-containing adaptor protein-inducing IFN-β pathway (16, 22). Evidence suggests that TIR8/SIGIRR recruited at signaling receptor complexes may act as an intracellular decoy, trapping key components of the transduction cascade (4, 6). The increased responsiveness in terms of cytokine production, and increased severity of tissue inflammation in Tir8–/– mice is consistent with the view of this molecule as a molecular trap for components of the signaling cascade (4, 6). Both IL-1RI and TLR4 signaling are inhibited by TIR8/SIGIRR (6, 8). LPS stimulation leads to down-regulation of TIR8/SIGIRR expression in different mouse tissues (6), and SIGIRR/TIR8-deficient mice are more susceptible to the systemic toxicity of bacterial LPS (6, 7). Moreover, SIGIRR/TIR8-deficient mice show an enhanced inflammatory response to IL-1, specifically in the lung and colon (12, 25), and blocking IL-1β and TNF-{alpha} reverses susceptibility to Mycobacterium tuberculosis infection in SIGIRR/TIR8-deficient mice (25). We found that the production of both IL-1{alpha} and IL-1β was higher in DCs from Tir8–/– than Tir8+/+ mice and that Th17 cell activation did not occur in IL-1RI–/– mice. Tir8–/– DCs showed increased production of IL-12p70, IL-23, IL-1{alpha}, and IL-1β in response to Candida and a decreased production of IL-10, and were fully competent at up-regulating costimulatory and MHC class II Ag expression upon pulsing with the fungus. Because IL-1β significantly contributed to Th17 activation in the absence of TIR8, it can be concluded that the proinflammatory activity of Tir8–/– DCs includes the activation of Th17 cells, to which IL-1 signaling crucially contributes.

A number of studies has shown the important role of IL-1 and IL-1Rs in resistance to candidiasis (38, 39, 46). However, through different immune mechanisms, both IL-1{alpha} and IL-1β, produced through transcription and translation by the host (47), are involved in host defense against the fungus and contribute to the production of IFN-{gamma} (39). Consistent with the notion that both IL-1β and IL-1{alpha} are crucially involved in PMN recruitment and activation in response to the fungus (39) and MyD88 mediates PMN recruitment against Staphylococcus aureus initiated by IL-1R (48, 49), IL-1RI–/– mice were highly susceptible to both mucosal and disseminated candidiasis with unrestricted fungal growth and severe impairment or PMN recruitment and activation to the site of infection. Despite a similar degree of fungal growth in the target organs, at variance with Tir8–/– mice, IL-1RI–/– had less inflammatory pathology at sites of infections and survived the mucosal infection. This finding further points to the pathogenic role of inflammation in infection (13). As a matter of fact, neither the p35 nor the p19 mRNAs were up-regulated in IL-1RI–/– DCs in response to Candida, and neither Th1 nor Th17 cell activation occurred in infected IL-1RI–/– mice. As already shown by others (50), despite the critical role of IL-1/IL-1RI signaling for Th2 responses (51), Th2 cell activation was observed in IL-1RI–/– mice, a finding clearly indicating that the activation of antifungal Th2 cell associates with unrestricted fungal growth, but not immunological damage.

Although IL-1R is found ubiquitously on both resident cell and bone marrow-derived cells (47), IL-1RI signaling is not as crucial as in candidiasis in the initiation of the innate inflammatory response to Aspergillus, as already shown (17). These data suggest that TLR signaling inhibited by TIR8/SIGIRR may vary at different body sites. Further studies in search for ligands for TIR8/ SIGIRR, to be identified as yet, will help to clarify this issue.

In summary, our data provide evidence that TIR8 is required for host resistance against fungal infections and functions to negatively regulate the IL-1 signaling-dependent activation of Th17 activation. Our study also points to the infection-promoting role of a deregulated inflammation in fungal infections and highlights the relative contributions of the different adaptive Th cell subsets in the control of immunopathology. TIR8 proved to have a key role in immune regulation by providing a fine mechanism for the control of the balance between protective immune responses and immunopathology.


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


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


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

1 This study was supported by the National Research Project on AIDS, Contract 30G.28, "Opportunistic Infections and Tuberculosis," Italy, and by the Specific Targeted Research Project "EURAPS" (LSHM-CT-2005), Contract 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, 06126 Perugia, Italy. E-mail address: lromani{at}unipg.it Back

3 Abbreviations used in this paper: TIR8, Toll IL-1R8; DC, dendritic cell; MLN, mesenteric lymph node; MMP9, matrix metalloproteinase; MPO, myeloperoxidase; NOS2, NO synthase; PMN, polymorphonuclear neutrophil; SIGIRR, single Ig IL-1-related receptor; TLN, thoracic lymph node; Treg, regulatory T cell. Back

Received for publication September 13, 2007. Accepted for publication January 7, 2008.


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