Introduction

Chronic mucocutaneous candidiasis (CMC) is a rare, complex, and heterogeneous group of syndromes characterized by persistent or recurrent infections of skin, nails, and mucosal tissues with the ubiquitous, opportunistic yeast Candida albicans (Kirkpatrick, 2001). The underlying defect may be a primary immune defect, as it is the case in the recently described subgroup "Autoimmune polyendocrinopathy candidiasis ectodermal dystrophy" (APECED) (Collins et al., 2006), or in a form of dominant inherited CMC with malfunction of the thyroid gland (Atkinson et al., 2001). However, the link between these mutations and the immune defect(s) remains unclear.

Even though a coordinated contribution of both innate and adaptive immunity is required in order to mount an effective host response against Candida, it seems that primarily a defect of the adaptive immune system leads to enhanced Candida infection. Patients lacking T cells due to a severe combined immunodeficiency often undergo chronic mucocutaneous candidiasis (IUIS scientific group, 1999). T-cell-knockout mice suffer from severe systemic candidiasis (Ashman et al., 1999); impaired T helper-1 (Th1) immune response leads to increased susceptibility to severe Candida infections (Mencacci et al., 2001), whereas reduction of IL-10 increases resistance against these infections (Tavares et al., 2000). The recently described T-cell subtype Th17, characterized by production of IL-17 and expression of CCR6 (Harrington, 2006; Singh et al., 2008), has been found, aside its involvement in autoimmune diseases and immunosurveillance, to be involved in host defense against Candida in mice and humans (Huang et al., 2004; Acosta-Rodriguez et al., 2007b; Bettelli et al., 2007).

Concerning CMC, we, and others, reported a heterogeneous, but not generally diminished, proliferative capacity of T cells from CMC patients (De Morales-Vasconcelos et al., 2001; Eyerich et al., 2007). A more critical parameter in the pathogenesis of CMC could be the cytokine secretion of T-cell subtypes rather than proliferation: recently, T-cell cytokine secretion has been the focus of numerous studies (Kobrynski et al., 1996; Lilic et al., 2003; Van der Graaf et al., 2003; Eyerich et al., 2007), describing an altered cytokine production with a reduced production of type-1 cytokines such as IFN-, IL-12, and IL-2, and increased secretion of IL-10 or IL-4.

The aim of this study was to elucidate a possible role for IL-17-producing T cells in the pathogenesis of CMC.

Results

Candida- and PHA-stimulated PBMCs from CMC patients transcribe and secrete less IL-17 and IL-22 compared with those in healthy controls

PBMCs of two CMC patients and two age- and sex-matched controls were stimulated with C. albicans. After 24 hours, total mRNA was isolated, reverse transcribed, and used for real-time PCR. In healthy (non-Candida infected) controls, IL-17F and IL-22 were significantly induced after 24 hours of stimulation with Candida or phytohemagglutinin (PHA), whereas CMC patients failed to upregulate the expression of these cytokines on mRNA level (Figure 1a), irrespective of the stimulus. IL-17A was not upregulated in CMC patients and healthy controls in the monitored time course (data not shown).

Figure 1.

PBMCs of CMC patients exhibit significantly reduced expression and release of IL-17 and IL-22 upon stimulation with Candida or PHA. Total mRNA of PBMCs was isolated 24 hours after stimulation with C. albicans or PHA, reverse transcribed, and analyzed by real-time PCR for IL-17F and IL-22 transcription (CMC patients, n=2; IC, non-Candida-infected control, n=2) (a). The culture supernatants of PBMCs stimulated for 72 hours with C. albicans, PHA or anti-CD3/anti-CD28 were analyzed by ELISA for protein content of IL-17 and IL-22 (CMC patients, n=4; IC, non-Candida-infected control, n=9; IC, Candida-infected control, n=6) (b). CD4+ or CD8+ T cells were incubated with autologous monocytes and Candida for 72 hours, and supernatants were analyzed for IL-17 and IL-22 by ELISA (CMC patients, n=2; IC, non-Candida-infected control, n=4) (c). Error bars indicate meanSD (IC, immunocompetent control; ^*P0.05; ^**P0.01; ^***P0.001).

Full figure and legend (66K)

The low transcription of IL-17 and IL-22 mRNA in CMC patients was reflected by the scarce and significantly lower secretion of IL-17 and IL-22 as compared with that in healthy controls as measured by ELISA after 72 hours of stimulation with PHA or Candida (Figure 1b). Notably, IL-17 and IL-22 release were also reduced in CMC patients when PBMCs were stimulated with anti-CD3/anti-CD28, pointing to a defect in the T-cell compartment rather than antigen-presentation or absent antigen-presenting cell-derived signals. Furthermore, PBMCs from immunocompetent patients with acute Candida infection exhibited not only significantly higher release of IL-17 upon stimulation with Candida compared with CMC patients, but also compared with healthy controls. This underlines the importance of IL-17 in defense against Candida.

Incubation of purified CD4+ and CD8+ T cells with autologous monocytes as antigen-presenting cells and Candida revealed that IL-17 and IL-22 are predominantly derived from CD4+ T cells in healthy controls, and conclusively, the disability of CMC patients to produce IL-17 and IL-22 is mainly attributed to CD4+ T cells (Figure 1c).

CMC patients exhibit reduced total number of IL-17-producing T cells, but normal amounts of CCR6+/CCR4+ T cells

The surface phenotype of IL-17-producing T cells is characterized by CCR4 and CCR6. Here we confirm these data showing that the majority of IL-17-producing cells expressed CCR6 (Figure 2a). In line with our data from ELISA, CMC patients showed an evanescent number of IL-17-producing (CCR6+) T cells after phorbol 12-myristate 13-acetate/ionomycin stimulation. However, no significant differences in the number of CCR4+, CCR6+ and CCR4/CCR6 double positive cells between CMC patients and healthy controls were observed (Figure 2b).

Figure 2.

The CCR6+ IL-17A+ cell population is strongly decreased in CMC patients, while the total number of CCR4+CCR6+ cells is not diminished. PBMCs of CMC patients and healthy controls were stained intracellularly after phorbol 12-myristate 13-acetate/ionomycin stimulation for IL-17 (a) (one representative experiment of three is shown) and analyzed for expression of the surface markers CCR4 and CCR6 (b) by flow cytometry (CMC patients, n=3 and healthy controls, n=4). Error bars indicate meanSD (IC, immunocompetent non-Candida-infected control).

Full figure and legend (27K)

PBMCs of CMC patients are able to secrete Th17-differentiating and Th17-maintaining cytokines

IL-1 and IL-6 are important for differentiation of human IL-17-producing T cells. We analyzed the expression of IL-1 and IL-6 at the transcriptional level by real-time PCR (Figure 3a). PBMCs of CMC patients stimulated with C. albicans for 24 hours tended to show higher mRNA expression of IL-1 and IL-6 compared those of with healthy controls. Transcriptional upregulation of these genes in CMC patients resulted in only slightly higher amounts of corresponding protein measured by ELISA in culture supernatants of Candida- or PHA-stimulated PBMCs after 72 hours (Figure 3b). IL-23, a cytokine important for maintenance of Th17 cells, was analyzed on mRNA level, revealing no differences in expression between patients and controls (data not shown).

Figure 3.

No significant differences in Th17-differentiating cytokine secretion between CMC patients and healthy volunteers. Total mRNA was isolated from Candida-stimulated PBMCs (CMC patients, n=2; IC, non-Candida-infected control, n=2) after 24 hours and analyzed for expression of IL-1 and IL-6 by real-time PCR (a). The supernatants were analyzed after 72 hours by ELISA (CMC patients, n=4; IC, non-Candida-infected control, n=9; IC, Candida-infected control, n=6) (b). There were no significant differences in secretion of IL-1 and IL-6 between patients and controls. Error bars indicate meanSD (IC, immunocompetent control).

Full figure and legend (45K)

Discussion

Recent data suggest that the inability to clear C. albicans in CMC patients is based upon a complex heterogeneity of immune defect(s), probably characteristic for various disease subgroups. In this study, we present four CMC patients deficient in the production of Th17-associated cytokines IL-17 and IL-22, whereas levels of the mediators important for differentiation (IL-6 and IL-1) and maintenance (IL-23) of the Th17 lineage were enhanced or not altered.

IL-17-producing T cells represent a recently described T-cell subset characterized by production of IL-17 and IL-22 (Liang et al., 2006; Bettelli et al., 2007). They are proinflammatory linkers between myeloid and lymphoid host defense that are able to help B cells, show low cytotoxicity, and poor regulative susceptibility to T regulatory cells (Annunziato et al., 2007).

First evidence for the importance of IL-17 in clearing Candida infections has been provided in the mouse system: IL-17A-receptor-knockout mice showed a dose-dependent, substantially reduced survival in a murine model of systemic candidiasis (Huang et al., 2004). Recent data revealed that infection with C. albicans leads to induction of murine IL-17-producing T cells (LeibundGut-Landmann et al., 2007). Furthermore, the hyphal form of C. albicans triggers IL-17 production of freshly isolated human CD4+ T cells of healthy donors in vitro (Acosta-Rodriguez et al., 2007b).

Our study affirms the importance of IL-17 producing-T cells in clearing Candida infections, because PBMCs of immunocompetent patients suffering from Candida infection showed significantly higher secretion of IL-17 and IL-22 after in vitro stimulation with C. albicans as compared with healthy (non-Candida-infected) donors. In concordance with previous reports (Annunziato et al., 2007; LeibundGut-Landmann et al., 2007; Singh et al., 2008), our data show that the source of IL-17 in PBMCs is almost exclusively limited to CD4+ CCR-6+ T cells. T-cell-receptor-specific stimulation of PBMCs and stimulation of isolated CD4+ T cells with autologous monocytes showed comparable amounts of secreted IL-17, indicating that IL-17 was predominantly derived from CD4+ T cells. Flow cytometry analysis of PBMCs revealed nearly all cells positive for IL-17 in intracellular staining to be positive also for CCR-6.

However, even though they had been chronically exposed to C. albicans, PBMCs from CMC patients secreted significantly lower amounts of IL-17 and IL-22 than PBMCs from healthy donors and patients with current Candida infection, after stimulation with C. albicans in vitro—both on the mRNA and on protein level. The underlying immune defect was not specific for the Candida stimulus, as mitogen stimulation (PHA) and T-cell-receptor-specific stimulation (anti-CD3/anti-CD28) also resulted in reduced secretion of IL-17 and IL-22 in CMC patients. This decrease was due to a strongly diminished total number of IL-17 producing T cells, as detected by surface CCR-6 and intracellular IL-17 staining of PBMCs by flow cytometry. The weaker secretion of IL-22 was less pronounced than that of IL-17, as compared with immunocompetent patients either infected or not infected with Candida. This could be explained by the fact that IL-22 production is not limited to Th17 cells, but is also produced by other activated T-cell subtypes (Xie et al., 2000; Conti et al., 2003).

The so far identified differentiation factors for human IL-17-producing T cells are IL-1 and IL-6 (Acosta-Rodriguez et al., 2007a), whereas IL-23 seems to be important for maintaining production of IL-17 and IL-22 in mouse (Chen et al., 2006; Veldhoen et al., 2006; Kreymborg et al., 2007), but to a lesser degree in human IL-17-producing T cells (Acosta-Rodriguez et al., 2007a, 2007b). In concordance with these studies, we observed strong enhancement of IL-1 and IL-6, but not of IL-23, after stimulation with Candida. The secretion of these cytokines in PBMCs of patients suffering from CMC was not diminished. In contrast, mRNA expression of IL-1 and IL-6 was induced much stronger in CMC patients, resulting in slightly higher release of proteins after 72 hours. This could indicate a defect in differentiation or survival of IL-17-producing T cells downstream of IL-1 and IL-6.

Concerning the mechanism of the candidicidal effects of IL-17-producing T cells, two possible pathways could be involved in clearing infection: on the one hand, there is the strong neutrophil-recruiting capacity of IL-17 via induction of IL-8 in human keratinocytes (Albanesi et al., 1999). On the other hand, IL-17 and IL-22 synergistically induce -defensins in human keratinocytes (Liang et al., 2006) that are able to kill C. albicans (Feng et al., 2005; Vylkova et al., 2007). Taken together, a decrease in the absolute number of IL-17-producing T cells and the resulting diminished stimulation of epithelial cells could explain why candidiasis is limited to the skin and mucosal membranes in CMC patients, and help to understand why such patients do not suffer from systemic candidiasis.

In summary, this study underlines the importance of IL-17-producing T cells for clearance of Candida infections. Furthermore, our data suggest that an impaired IL-17 and IL-22 response seems to be, at least in part, responsible for the pathogenesis of CMC. With its limited number of CMC patients, the power of this study is not high enough to draw general conclusions. Therefore, further studies with greater numbers of CMC patients will be required to investigate the immune response of IL-17-producing T cells in order to elucidate the pathomechanisms of CMC.

Materials And Methods

Patients

Four CMC patients (three female, one male; age 8–50 years, mean 31 years; all suffering from chronic Candida infections of the skin and esophagous since early childhood) were included into the study and compared with nine healthy, age and sex-matched volunteers. Furthermore, six patients suffering from an (at the time of investigation) untreated Candida infection (two paronychia, two oral, and two genital candidiasis) without any immune suppression were enrolled into the study. Before drawing blood, each participant provided informed consent. The study was approved by the ethical committee of the Technical University Munich and followed the Declaration of Helsinki Principles (41st World Medical Assembly, 1997).

Quantitative mRNA analysis

PBMCs of two CMC patients and two healthy volunteers were stimulated with Candida antigen (100 g ml^-1; Allergopharma, Hamburg, Germany), PHA (10 g ml^-1), or anti-CD3/antiCD28 antibodies, respectively, for 6 hours. Total mRNA was extracted using PeqGold RNA extraction buffer (Peqlab, Erlangen, Germany). RNA was reverse transcribed using oligo(dT) primers and avian myeloblastosis virus reverse transcriptase (Roche, Mannheim, Germany). PCRs were performed with the following primers: IL-17A (forward 5'-CTCGATTTCACATGCCTTCA-3'; reverse 5'-GAGGGGCCTTAATCTCCAAA-3'), IL-17F (forward 5'-AGTTGGAGAAGGTGCTGGTG-3'; reverse 5'-CCATCCGTGCAGGTCTTATT-3'), IL-22 (forward 5'-GAGGAATGTGCAAAAGCTGA-3'; reverse 5'-GCTTTGGGGCATCTAATTGT-3'), IL-23A (forward 5'-CAGTTCTGCTTGCAAAGGAT-3'; reverse 5'-ATCTGCTGAGTCTCCCAGTG-3'), IL-1 (forward 5'-TTCGACACATGGGATAACGA-3'; reverse 5'-TCTTTCAACACGCAGGACAG-3'), and IL-6 (forward 5'-ATGCAATAACCACCCCTGAC-3'; reverse 5'-GAGGTGCCCATGCTACATTT-3') (all from www.realtimeprimers.com) and SYBR green mastermix (Bio-Rad, Munich, Germany). PCRs were run on an ABI Prism 7000 Sequence Detection System (Applied Biosystems, Foster City, CA) using the following program: 10 minutes at 94 °C followed by 45 cycles of 15 seconds at 95 °C and 60 seconds at 58 °C. 18S RNA served as the housekeeping gene.

T-cell proliferation and cytokine secretion

PBMCs were separated as previously described (Eyerich et al., 2007) and stimulated for 60 hours either with 10 g ml^-1 PHA as a positive control or with 100 g ml^-1 C. albicans antigen (Allergopharma). After 60 hours, 100 l per well of supernatant was obtained and stored at -70 °C until further analysis by ELISA. Quantification of cytokines in the supernatants was performed by ELISA, according to the manufacturer's instructions (IL-17, IL-22; R&D Systems, Wiesbaden, Germany, and IL-1, IL-6; BD Biosciences, Heidelberg, Germany).

Additionally, CD4+ and CD8+ T cells were isolated from PBMCs of one CMC patient and a matched control, as previously described, and stimulated with mitogen or Candida in the presence of monocytes that served as antigen-presenting cells.

Intracellular cytokine staining

PBMCs from CMC patients and healthy controls were stimulated with phorbol 12-myristate 13-acetate (20 ng ml^-1) and ionomycin (1 ng ml^-1) (both from Sigma-Aldrich, Munich, Germany) for 6 hours and examined for intracellular IL-17 accumulation.

To prevent cytokine secretion, the stimulation was performed in the presence of Monensin (from the beginning) and brefeldin-A (10 g ml^-1; Sigma-Aldrich) was added for the final 4 hours. T cells were fixed (2% paraformaldehyde), permeabilized (0.5% saponin), and stained with phycoerythrin-conjugated anti-human IL-17 (eBioscience, San Diego, CA) antibody or isotype-matched control antibody, and analyzed by flow cytometry.

Statistical analysis

Statistical analysis was performed with the software program SPSS 14.0. All results were analyzed by Mann—Whitney U-test. Statistically significant differences between CMC patients and controls were defined as P<0.05.

Conflict of Interest

The authors state no conflict of interest.

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Acknowledgments

We thank Gaby Pleyl-Wisgickl for excellent technical assistance.