Low Interleukin-17A Production in Response to Fungal Pathogens in Patients with Chronic Granulomatous Disease

Abstract

Patients with chronic granulomatous disease (CGD) cannot produce reactive oxygen species (ROS) due to a genetic defect in the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase system. Dysregulation of the l-tryptophan metabolism in mice with defects in NADPH oxidase, resulting in overproduction of interleukin (IL)-17, has been proposed to link ROS defects with hyperinflammation and susceptibility to pulmonary aspergillosis. In this study, we assessed the l-tryptophan metabolism and cytokine profiles in response to fungal pathogens in CGD patients. Peripheral blood mononuclear cells (PBMCs) from CGD patients showed increased production of IL-6, tumor necrosis factor-α, and interferon-γ upon stimulation with Aspergillus or Candida species, while IL-17A production was strikingly low compared with healthy controls. Indoleamine 2,3-dioxygenase expression was similar in PBMCs and neutrophils from CGD patients compared with healthy controls. Conversion of l-tryptophan to l-kynurenine, as measured by high-performance liquid chromatography, did not differ between CGD patients and healthy controls. Moreover, adding l-kynurenine to the cell cultures did not suppress fungal-induced IL-17A production. Although PBMCs of CGD patients produced more proinflammatory cytokines after stimulation, IL-17A production was strikingly low in response to fungal pathogens when compared with healthy controls. In addition, cells from CGD patients did not display a defective l-tryptophan metabolism.

Introduction

C hronic granulomatous disease (CGD) is a rare (incidence 1/250,000 live births) inherited immunodeficiency disorder due to a defect in any of the structural components of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. NADPH oxidase is a key player in innate host defense, and its activation results in production of superoxide anion and downstream reactive oxygen species (ROS). Clinically, CGD patients suffer from life-threatening bacterial and fungal infections and are characterized by abnormal inflammatory responses leading to granuloma formation. The precise “microbial” pathogenesis in CGD patients is unclear.

While the central role of ROS as direct antimicrobial effectors is still under discussion, ROS are also linked to key intracellular signaling events, such as the activation of transcription factors (Sen and Packer 1996; Sun and Oberley 1996), among which nuclear factor kappa-light-chain-enhancer of activated B cells (NFκβ) (Blackwell and others 1996; Rusyn and others 1998), induction of mitogenesis (Irani and others 1997; Joneson and Bar-Sagi 1998), and activation of proteases (Reeves and others 2002). ROS can also act as a substrate or cofactor for the catabolic indoleamine 2,3-dioxygenase enzyme (IDO) (Shimizu and others 1978; Hayaishi 1996; Thomas and Stocker 1999; Dang and others 2000). IDO is the rate-limiting enzyme in the tryptophan metabolism (Stone and Darlington 2002). Activation of IDO results in decreased levels of l-tryptophan, thus actively depriving invading microorganisms of an essential amino acid (Pfefferkorn 1984; Schmitz and others 1989; Hayaishi 1996). IDO is expressed in several tissues throughout the body and in immune cells like dendritic cells (DCs) (Mellor and Munn 2004), macrophages (Werner and others 1987; Carlin and others 1989), and eosinophils (Odemuyiwa and others 2004). IDO expression can be induced by mediators of immunity, such as interferon (IFN)-γ (Takikawa and others 1988; Krampera and others 2006) and tumor necrosis factor (TNF)-α (Fujigaki and others 2001). In addition, activation of tryptophan catabolism induces maternal immune tolerance (Munn and others 1998), indicating that IDO can regulate T-cell responses. IDO can suppress the Th1 response (Gurtner and others 2003; Kwidzinski and others 2005; Choi and others 2006), while in DC's IDO is able to induce a Th2 response (Xu and others 2008). In contrast, pulmonary IDO decreased Th2-driven experimental asthma (Hayashi and others 2004). The immunomodulatory functions of IDO have made it an important target of research in many inflammatory conditions.

The tryptophan metabolism has also been linked to Th17 responses. In a collagen-induced arthritis mice model, l-kynurenine dampened the Th17 response (Criado and others 2009). Furthermore, in a murine candidiasis model, retinoic acid receptor-related orphan receptor C transcription was decreased by the induction of IDO (De Luca and others 2007). In a recent article, Romani and others (2008) suggested that p47^phox-deficient mice that have a phenotype reminiscent of CGD have a defective tryptophan metabolism, leading to an increased interleukin (IL)-17 production. It has thus been proposed that the activation of IDO and subsequently conversion of l-tryptophan to l-kynurenine suppresses the Th17 response.

A robust Th17 response is necessary for an adequate defense against Candida (Huang and others 2004). Th17 cells are characterized by the production of IL-17A (Park and others 2005), which is important for attracting neutrophils to the tissues (Kolls and Linden 2004). The importance of IL-17A in antifungal host defense is reflected by the fact that IL-17A receptor antagonist-deficient mice have an increased susceptibility to disseminated candidiasis (Huang and others 2004). Furthermore, in a murine oropharyngeal candidiasis model, IL-17A- or IL-23p19-deficient animals showed increased severity of oropharyngeal candidiasis (Conti and others 2009). Also humans with chronic mucocuteneous candidiasis (CMC) and hyper-IgE syndrome (HIES) have decreased Th17 responses that are believed to be responsible for their susceptibility to fungal infections (Grimbacher and others 1999; Eyerich and others 2008; Ma and others 2008; Milner and others 2008; Minegishi and others 2009; van de Veerdonk and others 2011).

CGD is the primary immunodeficiency with the highest incidence of invasive fungal infections (Antachopoulos 2010), mainly due to Aspergillus spp. (lifetime incidence 25%–40%). Candida is commonly encountered in cases of fungal meningitis, fungemia, and lymphadenitis (Winkelstein and others 2000; van den Berg and others 2009; Antachopoulos 2010). It is currently not clear why patients with CGD are specifically susceptible to fungal infections, although infections with bacterial pathogens such as Staphylococcus aureus are also often encountered. The absence of ROS has been shown not to be the direct explanation of the increased susceptibility (Henriet and others 2011).

In the current study, the tryptophan metabolism and IL-17A response in human CGD patients were investigated. We demonstrate that CGD patients, in contrast to the p47^phox-deficient mice, are able to convert l-tryptophan to l-kynurenine, and that they have a lower IL-17A production in response to stimulation with the fungal pathogen Candida albicans, which might explain their incapacity to clear fungal infections.

Materials and Methods

Patients and healthy volunteers

Blood was collected from healthy volunteers who did not suffer from infectious or inflammatory diseases, and from 8 CGD patients, harboring homozygous mutations in the NCF1 gene (p47-phox) or CYBB gene (p91-phox). After informed consent was given, blood was collected by venipuncture into 10 mL ethylenediaminetetraacetic acid tubes (367525; BD). The clinical characteristics of all patients are presented in Table 1.

Table 1.

Clinical Characteristics of Chronic Granulomatous Disease Patients

Patient	Sex	Age	Disease history	Active infection at time of exp.?	Interferon-γ therapy at time of exp.?	Mutation
1	Male	38	Staphylococcus aureus, liver abscess, pneumonia (3 years)	No	No	NCF1 (p47-phox)
2	Male	26	Multiple S. aureus infections, including liver abscesses, and abscesses near the spine	No	No	NCF1 (p47-phox)
3	Male	29	S. aureus liver abscess (7 years), lung infection with exophiala dermatitidis (28 years)	No	No	NCF1 (p47-phox)
4	Male	20	Therapy-resistant acne	No	No	NCF1 (p47-phox)
5	Male	16	Aspergillus fumigatus infection (at age 5 years), no inflammatory complications	No	No	CYBB (gp91-phox)
6	Male	9	Mucormycosis (at age 10 months), no inflammatory complications	No	No	NCF1 (p47-phox)
7	Male	9	No history of invasive fungal disease, no inflammatory complications	No	No	NCF1 (p47-phox)
8	Male	19	Aspergillus nidulans infection (at age 5 years), A. fumigatus infection (8 years), no inflammatory complications	No	Yes	CYBB (gp91-phox)

Reagents

The following study materials were used: recombinant IFN-γ (Boehringer Ingelheim BV); l-kynurenine, l-tryptophan, 5-OH-tryptophan, serotonin, melatonin, niacin, and resveratrol were purchased from Sigma-Aldrich; αCD3αCD28 beads were prepared from a T-cell activation/expansion kit (130-091-441, MACS; Miltenyi Biotec); lipopolysaccharide (LPS) (Escherichia coli serotype 055:B5) was purchased from Sigma and an extra purification step was performed as described previously (Hirschfeld and others 2000). Purified LPS was tested in Toll-like receptor (TLR) 4-/- mice for the presence of contaminants and did not have any TLR4-independent activity (Sutmuller and others 2006). As culture medium, RPMI 1640 Dutch modifications (Sigma-Aldrich) was used, supplemented with 1% gentamicin, 1% l-glutamine, and 1% pyruvate (Life Technologies).

Microorganisms

C. albicans ATCC MYA-3573 (UC 820) (Lehrer and Cline 1969) was grown overnight in Sabouraud broth at 37°C; cells were harvested by centrifugation, washed twice, and resuspended in culture medium. C. albicans was heat killed for 1 h at 100°C; S. aureus (ATCC 25923) was heat killed for 30 min at 100°C; heat-killed conidia and hyphae of Aspergillus fumigatus (V4507) and Aspergillus nidulans (V44-46) were prepared as described previously (Chai and others 2009; Henriet and others 2011).

Human mononuclear cells and neutrophils

Peripheral blood mononuclear cells (PBMCs) and neutrophils were isolated from peripheral blood using density gradient centrifugation (800 g) of diluted blood (1 part blood to 1 part pyrogen-free saline) over Ficoll-Paque (Pharmacia Biotech) (Netea and others 2006). PBMCs were harvested, washed twice in phosphate buffered saline (PBS), and suspended in culture medium. To remove the erythrocytes from the neutrophils, the pellet was shocked at least twice with an ice-cold lysing reagent (2.75 g NH₄Cl + 0.25 g KHCO₃) for 15 min. Subsequently, cells were washed in PBS and resuspended in culture medium. The PBMCs and neutrophils were counted in a hemocytometer, and their concentration was adjusted to 5×10⁶ cells/mL.

About 5×10⁵ PBMCs or neutrophils in a total volume of 200 μL per well were incubated at 37°C in round-bottomed, 96-well plates (Greiner) with the different stimuli, as indicated in the figure legends. After 24 h, 48 h, or 7 days of incubation, supernatants were collected and stored at −20°C until assayed. When cells were cultured for 7 days, this was done in the presence of 10% human pooled serum.

Enzyme-linked immunosorbent assay

The concentration of IFN-γ, IL-6, IL-17A, and TNF-α was measured in cell culture supernatants using enzyme-linked immunosorbent assay (ELISA) (IL-17A and TNF-α: R&D Systems; IFN-γ and IL-6: Sanquin), according to the instructions of the manufacturer.

Real-time polymerase chain reaction

One million freshly isolated PBMCs or neutrophils were incubated with the various stimuli. After 24 h of incubation at 37°C, total ribonucleic acid (RNA) was extracted in 400 μL of TRIzol reagent (Invitrogen). Isolated RNA was being reverse transcribed into complementary DNA using oligo(dT) primers and MMLV reverse transcriptase. Polymerase chain reaction (PCR) was performed using a 7300 realtime PCR system (Applied Biosystems). The primer sequences for human IDO are as follows: 5-GGT-CAT-GGA-GAT-GTC-CGT-AA-3 (forward) and 5-ACC-AAT-AGA-GAG-ACC-AGG-AAG-AA-3 (reverse). β2M was used as a reference gene, for which the primers were 5-ATG-AGT-ATG-CCT-GCC-GTG-TG-3 (forward) and 5-CCA-AAT-GCG-GCA-TCT-TCA-AAC-3 (reverse). PCR conditions were as follows: 2 min at 50°C and 10 min at 95°C, followed by 40 cycles of PCR at 95°C for 15 seconds, and 60°C for 1 min. Data are expressed as fold increase compared with the unstimulated sample.

High-performance liquid chromatography

To compare the amount of l-tryptophan metabolized and the amount of l-kynurenine produced upon stimulation with fungal pathogens between CGD patients and healthy controls, cell culture supernatants were subjected to high-performance liquid chromatography (HPLC) analysis. HPLC was performed on a Spectra-SYSTEM autosampler and pump (Thermo Separation Products). Chromatographic separation was performed using an Inertsil 5 ODS-2 column (100 mm×3.0 ID; Varian Inc.). Absorbance was monitored with a diode-array detector (UV6000LP; Thermo Separation Products) at wavelength of 280 nm for tryptophan and 360 nm for kynurenine (Zucchelli and others 1997). The mobile phase for isocratic elution was made by dissolving 40 mM sodium acetate. The pH of the eluent was adjusted to pH 4.5 with a solution of 40 mM citric acid, and 2% acetonitrile of the total volume buffer was added. The continuous flow rate was 0.3 mL per minute (Krstulovic and others 1984). Data are presented as area under the curve.

Statistical analysis

Experiments were performed in duplicate, and supernatants were pooled. The differences between groups of 3 or more subjects were analyzed using the Mann–Whitney U test or the Wilcoxon signed rank test, for unpaired and paired data, respectively. The level of significance between groups was set at P<0.05 (*) and P<0.01 (**). Data are presented as mean±standard error of the mean.

Results

CGD patients have an increased production of proinflammatory cytokines in vitro

The cytokine profile of CGD patients upon stimulation with several stimuli was investigated. PBMCs of CGD patients produced more IL-6 and TNF-α upon stimulation with the specific TLR4 ligand LPS (Fig. 1). Similarly, heat-killed C. albicans induced more IL-6 and TNF-α production in PBMCs isolated from CGD patients (Fig. 1). In line with this, stimulation with live A. nidulans and A. fumigatus conidia, 2 commonly encountered species in CGD patients, resulted in a higher cytokine production by PBMCs of CGD patients compared with healthy controls (Fig. 1). Heat-killed Aspergillus conidia and hyphae showed to be poor stimulators of the proinflammatory cytokines.

FIG. 1.

CGD patients show increased production of proinflammatory cytokines upon fungal stimulation in vitro. PBMCs of healthy controls and CGD patients were stimulated for 24 h with Roswell Park Memorial Institute (RPMI) Medium, live and heat-killed (HK) Aspergillus nidulans (AN) and Aspergillus fumigatus (AF) conidia or hyphae, heat-killed Candida albicans (1×10⁶/mL), or LPS (10 ng/mL). IL-6 and TNF-α were measured in cell culture supernatants using ELISA. Data represent 5 CGD patients and 5 healthy controls from 3 different experiments. Data were analyzed using the Mann–Whitney U test (**P<0.01). Bars represent mean + SEM. CGD, chronic granulomatous disease; PBMCs, peripheral blood mononuclear cells; LPS, lipopolysaccharide; TNF-α, tumor necrosis factor-α; IL-6, interleukin-6; ELISA, enzyme-linked immunosorbent assay; SEM, standard error of the mean.

CGD patients have an intact L-tryptophan metabolism

Romani and others (2008) showed that p47^phox-deficient mice have a hyperinflammatory phenotype due to a defect in the conversion of l-tryptophan into l-kynurenine. Therefore we assessed whether the increased production of proinflammatory cytokines in patients with CGD could also be explained by an incapability to convert l-tryptophan into l-kynurenine. Cells isolated from CGD patients had a higher transcription of IDO upon stimulation with Candida and IFN-γ in both PBMCs and neutrophils, compared with healthy controls (Fig. 2A). Moreover, upon stimulation with Candida, both healthy controls and CGD patients were able to convert l-tryptophan into l-kynurenine (Fig. 2B). The conversion of l-tryptophan to l-kynurenine was independent of which fungal pathogen was used for stimulation (data not shown). This indicates that IDO functions normally in human CGD patients.

FIG. 2.

CGD patients do not have a defective l-tryptophan metabolism. (A) PBMCs and neutrophils of 2 healthy volunteers and of 3 CGD patients were stimulated for 48 h with heat-killed C. albicans (1×10⁶/mL) or IFN-γ (50 ng/mL). IDO expression was measured using qPCR. Bars represent mean + SEM. (B) PBMCs of healthy volunteers and CGD patients were cultured for 48 h in the presence of l-tryptophan (50 μg/mL), with or without heat-killed C. albicans (1×10⁶/mL). l-Tryptophan (285 nm; black lines and arrows) and l-kynurenine (360 nm; gray lines and arrows) were measured using HPLC. (C) PBMCs of healthy volunteers were stimulated for 7 days in the presence of 10% human pool serum with heat-killed C. albicans yeast (1×10⁶/mL), in the absence or presence of l-kynurenine (200 ng/mL, 1 μg/mL, or 10 μg/mL). IL-17 was measured in cell culture supernatants using ELISA. Experiments were performed in duplicate and cell culture supernatants were pooled. Data represent at least 7 healthy volunteers from at least 3 different experiments. Data were analyzed using the Wilcoxon signed rank test (*P<0.05). Bars represent mean + SEM. (D) PBMCs were stimulated for 7 days in the presence of 10% human pool serum with heat-killed C. albicans yeast (1×10⁶/mL), in the absence or presence of l-tryptophan (50 μg/mL), 5-OH-tryptophan (200 ng/mL), serotonin (1 μg/mL), melatonin (200 pg/mL), or resveratrol (10 μg/mL). IL-17A was measured in cell culture supernatants using ELISA. Experiments were performed in duplicate and cell culture supernatants were pooled. Data represent at least 5 healthy volunteers from at least 2 different experiments. Data were analyzed using the Wilcoxon signed rank test (**P<0.01). Bars represent mean + SEM. IDO, indoleamine 2,3-dioxygenase; qPCR, quantitative polymerase chain reaction; HPLC, high-performance liquid chromatography.

The tryptophan metabolite l-kynurenine has been suggested to be responsible for dampening the proinflammatory cytokine response, and has especially been associated with a lowering of IL-17A production by murine cells (Criado and others 2009; Desvignes and Ernst 2009). To determine whether l-kynurenine is able to influence the IL-17A response in human cells in vitro, we tested Candida-induced IL-17A production in healthy volunteers in the absence or presence of tryptophan metabolites. PBMCs from healthy controls were also stimulated with Candida in the absence or presence of increasing concentrations of l-kynurenine. There was no clear dose–response relationship between l-kynurenine and IL-17A (Fig. 2C). Furthermore, l-tryptophan significantly increased Candida-induced IL-17A production (Fig. 2D), while the other metabolites (5-OH-tryptophan, serotonin, melatonin, and resveratrol) did not influence Candida-induced IL-17 production.

CGD patients have a low IL-17A and high IFN-γ production upon stimulation with fungal pathogens

To investigate whether IL-17A production is affected in cells from CGD patients, PBMCs from CGD patients and healthy controls were stimulated for 7 days with αCD3αCD28-coated beads, LPS, different morphotypes of A. nidulans and A. fumigates, and heat-killed C. albicans. There was no difference in αCD3αCD28-induced IL-17 production between CGD patients and healthy controls (Fig. 3A). It is well established that especially in hyphal antifungal host defense, rapid and efficient recruitment of neutrophils is important (Bonnett and others 2006). Here we found that in response to A. nidulans hyphae and A. fumigates hyphae, the IL-17A production was relatively low in CGD patients although this was not significant (Fig. 3B). CGD patients produced more IFN-γ in response to all stimuli (Fig. 3B). PBMCs of CGD patients produced significantly less IL-17A upon stimulation with C. albicans (Fig. 3C).

FIG. 3.

CGD patients have a low IL-17A and high IFN-γ production upon stimulation with fungal pathogens. (A) PBMCs of healthy controls and CGD patients were stimulated for 7 days in the presence of 10% serum with RPMI or αCD3αCD28-coated beads. IL-17A was measured in cell culture supernatants using ELISA. Data represent 4 CGD patients and 5 healthy controls from 3 different experiments. (B) PBMCs of healthy controls and CGD patients were stimulated for 7 days in the presence of 10% serum with RPMI, heat-killed A. nidulans (AN) and A. fumigatus (AF) conidia or hyphae, or LPS (10 ng/mL). IL-17A and IFN-γ were measured in cell culture supernatants using ELISA. Data represent at least 5 CGD patients and 6 healthy controls from 4 different experiments. (C) PBMCs of healthy controls and CGD patients were stimulated for 7 days in the presence of 10% serum with RPMI or heat-killed C. albicans (1×10⁶/mL). IL-17A was measured in cell culture supernatants using ELISA. Data represent 8 CGD patients and 8 healthy controls from 7 different experiments. Data were analyzed using the Mann–Whitney U test (*P<0.05). Data are presented separately, and as mean + SEM.

Discussion

In the current study we demonstrate that human CGD patients produce markedly lower IL-17A concentrations upon stimulation with the fungal pathogen C. albicans compared with healthy controls, despite an overall higher proinflammatory cytokine production. CGD patients are able to convert l-tryptophan into l-kynurenine, and ROS are not crucial for the IDO activity, which is needed to facilitate this conversion in humans. Furthermore, l-kynurenine did not decrease Candida-induced IL-17A production. These data suggest that NADPH oxidase deficiency in humans is not associated with a defective tryptophan metabolism, but with a lower Th17 response against fungal pathogens that might explain their susceptibility to fungal infections.

Th17 responses have recently been described as a new T helper subset (McGeachy and Cua 2008). In recent years it has become apparent that the Th17 response is essential for antifungal host defense. The recurrent mucosal Candida infections in patients with primary immunodeficiency syndromes such as HIES and CMC have been specifically linked to a deficiency in their Th17 response (Eyerich and others 2008; Milner and others 2008; van de Veerdonk and others 2011).

The role of the Th17 response in the host defense against Aspergillus is controversial. On the one hand, IL-17 deficiency is associated with a higher susceptibility to Aspergillus infection in mice (Werner and others 2009; Rivera and others 2011). On the other hand, studies suggest that a high IL-17 production is linked to a higher susceptibility to Aspergillus infection due to detrimental effects caused by a hyperinflammatory response (Zelante and others 2007; Romani and others 2008). These observations do not need to be contradictory, and they might support the concept that there is an optimal window for IL-17 response in the host defense against Aspergillus.

The decreased IL-17 production in response to fungal PAMPs was rather unanticipated. We have previously shown that CGD patients have relatively high IL-1β production in response to fungal stimuli (van de Veerdonk and others 2010). IL-1β is a driver of Th17 responses (Chung and others, 2009) and therefore we predicted a high Th17 response. Furthermore, it has been reported that T-cells from CGD patients have increased IL-17 and IFN-γ production in response to mitogenic stimulation, because NADPH oxidase–deficient macrophages cannot sufficiently induce Tregs that are needed to control proinflammatory T helper responses (Kraaij and others 2010). Interestingly we observed no difference in αCD3αCD28-induced IL-17 production between CGD patients and healthy controls, indicating that the decreased IL-17 production observed in CGD patients is only present in the presence of a specific pathogen. This might explain why the rest of the phenotype of CGD patients is not similar to HIES or CMC.

A difference in proportion of γδ T-cells in PBMCs from CGD patients compared with healthy controls could be an explanation for the observed differences in IL-17A production (Roark and others 2008). Therefore we have determined the relative contribution of the γδ T-cells to IL-17A production in response to Candida in healthy volunteers. We found that a very low amount of γδ T-cells was IL-17 positive compared with CD4 T-cells (data not shown). These data suggest that the large proportion of IL-17 in response to C. albicans is from the CD4 T-cell population. Since CD4 lymphopenia is not described in CGD patients, it is unlikely that differential T-cell counts are responsible for the low IL-17A production in CGD patients.

It is interesting to observe that both p47^phox-deficient mice and patients with CGD are susceptible to Aspergillus infections, but the underlying mechanisms seem to differ. In mice it was suggested that the tryptophan metabolism was defective, which led to an increased level of IL-17 production that was detrimental for the mice. Here we confirm that in CGD patients the tryptophan metabolism is intact (De Ravin and others 2010), and moreover we observed that they had a lower IL-17 production in response to fungal stimulation. In addition, l-kynurenine did not decrease IL-17A production induced by C. albicans in PBMCs of healthy controls, indicating that in human primary cells l-kynurenine does not suppress the Th17 response. However, l-tryptophan itself could increase Candida-induced IL-17A production in healthy controls. In line with this, Veldhoen and others (2009) previously demonstrated that l-tryptophan is one of the aryl hydrocarbon receptor (AHR) agonists that can increase IL-17A production. The role of the tryptophan/kynurenine metabolism of the different human leucocytes was recently evaluated by De Ravin and others (2010). They compared cultured DCs, monocytes, and isolated PMNs. In line with our data obtained from monocytes, De Ravin and others (2010) found that highly purified PMN could indeed upregulate IDO expression after IFN-γ treatment.

In contrast to a low IL-17A production, we observed that the cells from CGD patients have an increased IFN-γ production compared with healthy controls in response to fungal stimuli. IFN-γ stimulates intracellular killing of C. albicans conidia by PMN in vitro (Djeu and others 1986; Kullberg and others 1993), and in a recent overview it has been reported that a large proportion of patients with defects in IL-12Rβ1 experience fungal infections (de Beaucoudrey and others 2010). However, both IFN-γ and IL-17 are required for an adequate antifungal host defense. Although there is no defect in IFN-γ production, the low IL-17A production might cause a sensitivity to fungal infections. CGD patients also have an increased susceptibility to S. aureus infection. Susceptibility to S. aureus has also been associated with a defective IL-17A response (van de Veerdonk and others 2009; Cho and others 2010), which is in line with the data presented here.

In conclusion, human CGD patients have a specific lower production of fungal pathogen-induced IL-17A production compared with healthy controls, while mitogenic stimulation of T-cells isolated from CGD patients does not result in an impaired IL-17 production. This might explain their inability to clear fungal infections. Furthermore, in contrast to a mouse CGD model, human CGD patients do not have a defect in their l-tryptophan metabolism to explain their susceptibility to fungal infections and their hyperinflammatory status.

Footnotes

Acknowledgments

M.G. Netea was supported by a Vici grant of the Netherlands Organization for Scientific Research (918.10.610). S.S.V. Henriet was supported by the ESPID-Wyeth Fellowship 2008–2010.

Author Disclosure Statement

No competing financial interests exist.

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