Candida albicans and Candida parapsilosis Induce Different T-Cell Responses in Human Peripheral Blood Mononuclear Cells

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1 MAJOR ARTICLE Candida albicans and Candida parapsilosis Induce Different T-Cell Responses in Human Peripheral Blood Mononuclear Cells Adél Tóth, 1 Katalin Csonka, 1 Cor Jacobs, 2 Csaba Vágvölgyi, 1 Joshua D. Nosanchuk, 3 Mihai G. Netea, 2 and Attila Gácser 1 1 Department of Microbiology, University of Szeged, Hungary; 2 Department of Medicine, Radboud University Nijmegen Medical Center, the Netherlands; and 3 Department of Microbiology and Immunology, Albert Einstein College of Medicine, New York, New York Candida parapsilosis is the third most frequent cause of candidemia. Despite its clinical importance, little is known about the human immunological response to C. parapsilosis. In this study, we compared the cytokine responses evoked by Candida albicans and C. parapsilosis. C. parapsilosis stimulated human peripheral blood mononuclear cells (PBMCs) produced similar quantities of tumor necrosis factor α and interleukin 6 and slightly lower amounts of interleukin 1β, compared with C. albicans stimulated cells. PBMCs stimulated with C. parapsilosis displayed a skewed T-helper cell response, producing more interleukin 10 and less interferon γ than cells stimulated with C. albicans.notably,c. parapsilosis induced much less interleukin 17 and interleukin 22 production as compared to C. albicans. Inhibition of the 3 classical mitogen-activated protein kinases (p38 kinase, ERK, and JNK) revealed kinase-dependent differences in reductions in cytokine production by the 2 Candida species. Decreased cytokine production after inhibition of dectin 1 revealed that this receptor plays a major role in the recognition of both C. albicans and C. parapsilosis. These data improve understanding of the immune response triggered by C. parapsilosis,afirst step for the future design of immunotherapeutic strategies for these infections. Keywords. Candida parapsilosis; human PBMC; T cell response. Invasive candidiasis is a severe infection characterized by a high mortality rate, especially among immunocompromised individuals [1]. Although Candida albicans is the species most frequently isolated from Candida bloodstream infections, there has been a considerable increase in the incidence of infections due to Candida species other than C. albicans in recent years [2]. One of the emerging Candida species causing invasive infections is Candida parapsilosis [3], a fungal pathogen of growing importance because its incidence is much higher among young children (especially those Received 31 October 2012; accepted 12 February 2013; electronically published 9 May Presented in part: Annual Meeting of the Hungarian Society for Immunology, Hungary, October Correspondence: Attila Gácser, PhD, Department of Microbiology, University of Szeged, Közép fasor 52, H-6726 Szeged, Hungary (gacsera@gmail.com). The Journal of Infectious Diseases 2013;208:690 8 The Author Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please journals.permissions@oup.com. DOI: /infdis/jit188 aged <2 years), compared with adults [4]. However, despite its increasing importance as a pathogen, little is known about the recognition of C. parapsilosis by the immune system and the mechanisms of induction of host defense. Host defense against Candida infections is mediated by innate immune responses, especially by neutrophils and monocytes/macrophages, and by adaptive immune responses, especially by T lymphocytes. Before the discovery of the T-helper cell 17 (Th17) subset, differentiated CD4 + T-helper cells were divided into 2 subsets (the Th1 and Th2 lineages), and protection against fungal pathogens, including C. albicans, was associated with a Th1-biased response [5]. After the identification of the Th17 subset [6], the role of these cells in host defense against fungi has become increasingly evident. C. albicans induces a strong CD4 + Th17 response, and this has a crucial role during the clearance of the fungus [5]. The main cytokine produced by Th17 cells is interleukin 17 (IL-17). IL-17 induces the production of granulocyte colony-stimulating factor, antimicrobial 690 JID 2013:208 (15 August) Tóth et al

2 peptides (eg, human β defensin 2), and CXC chemokines, including CXCL8, which have a key role in the recruitment and activation of neutrophil granulocytes during inflammation [7]. Mice deficient in the expression of IL-17 receptor are more susceptible to C. albicans infection [8]. Additionally, defects of the IL-17 receptor or the incapacity to produce IL-17 in humans are associated with chronic mucocutaneous candidiasis [9 11]. The other signature cytokine of Th17 cells is interleukin 22 (IL-22), which is also involved in inflammatory responses, and induces the production of cytokines, chemokines, and defensins [7]. In recent years, there has been great progress in understanding immunity to C. albicans. However, despite the clinical importance of the host defense responses to C. parapsilosis, much less is known about these activities. In the present study, we aimed to investigate the capacity of C. parapsilosis to induce cytokine production, including the synthesis and release of T-helper cell cytokines such as interferon γ (IFN-γ), IL-17, and IL-22. MATERIALS AND METHODS Candida Strains The well-characterized Candida parapsilosis GA1 [21] and Candida albicans SC5314 wild-type strains were used throughout the study. Cells were grown overnight at 29 C in Sabouraud medium, harvested by centrifugation, washed twice with phosphate-buffered saline (PBS), and heat killed for 30 minutes at 100 C. Before stimulation experiments, cells were counted in a Bürker chamber and adjusted to the proper concentration. In all experiments, dead microorganisms were used because of differences in growth between strains, which would have impeded the proper comparison of the cytokines induced. Moreover, pilot experiments performed with live Candida organisms (10 5 or 10 6 cells) have shown an overgrowth of peripheral blood mononuclear cells (PBMCs) by the fungus at later time points, with host cell death, and an incapacity to assess T-helper cell derived cytokine production. Blood Donors Human primary PBMCs were isolated from buffy coats obtained from healthy blood donors at the Sanquin Blood Bank, Nijmegen, the Netherlands. The blood donors have given written approval for the use of their buffy coats for scientific purposes. Isolation and Stimulation of PBMCs Mononuclear cells were isolated by density centrifugation of PBS-diluted blood (dilution, 1:1) over a Ficoll-Paque PLUS gradient (GE Healthcare). PBMCs were washed 3 times with PBS and suspended in Roswell Park Memorial Institute (RPMI) 1640 culture medium, Dutch modification (Life Technologies, Paisley, UK), supplemented with 1% gentamicin, 1% pyruvate, and 1% L-glutamine. Cells were counted in a Coulter counter (Coulter Electronics), and the concentration was adjusted to cells/ml. For stimulation experiments, a 100- μl suspension of PBMCs in RPMI 1640 medium was added to round-bottomed 96-well plates (Greiner Bio-One) and incubated at 37 C for 24 hours, 48 hours, or 7 days with 100 μl of RPMI 1640 medium alone (negative control) or 100 μl of RPMI 1640 medium containing 10 4,10 5,or10 6 heatkilled C. albicans or C. parapsilosis. For the 7-day incubation, 10% pooled human serum was also added to the culture medium. In certain experiments, PBMCs were preincubated for 1 hour at 37 C with various inhibitors before being stimulated with Candida strains. After the incubation periods (24 hours for tumor necrosis factor α (TNF-α), interleukin 1β (IL-1β), and interleukin 6 (IL-6); 48 hours for interleukin 10 (IL-10) and IFN-γ; and 7 days for IL-17 and IL-22), the cell suspensions were centrifuged, and the supernatants were collected and stored at 20 C until assayed. Every stimulation experiment was performed in duplicate, and the supernatants were pooled before analysis by enzyme-linked immunosorbent assay (ELISA) kits. Reagents Laminarin (a specific inhibitor for dectin 1) was purchased from Sigma-Aldrich and used at a concentration of 50 µg/ml. s for MEK/ERK (U0126, Promega, WI), JNK (SP600125, A. G. Scientific, CA), and p38 kinase (SB , Sigma- Aldrich, MO) were used at concentrations of 10 µm, 20 µm, and 1 µm, respectively. Cytokine Assays The concentrations of targeted cytokines in cell culture supernatants were measured by commercial ELISA kits. IL-1β, TNFα, IL-17, and IL-22 were analyzed by an ELISA kit produced by R&D Systems (Abbingdon, United Kingdom), and IL-6, IFN-γ, and IL-10 were analyzed by a kit manufactured by Sanquin (Amsterdam, the Netherlands). All ELISAs were performed according to the manufacturers instructions. Intracellular Cytokine Staining and Flow Cytometry PBMCs were stimulated for 6 days with Candida strains (as described above) in the presence of 10% pooled human serum. After 6 days, cells were harvested by centrifugation and suspended in fresh cell culture medium containing 50 ng/ml PMA ( phorbol 12-myristate 13-acetate; Sigma), 1 mg/ml ionomycin (Sigma), and 1 µl/ml medium BD GolgiPlug (BD Biosciences), according to the manufacturers protocols. After 4 hours of incubation, cells were stained extracellularly, using an antibody to CD4 PECy7 (BD Biosciences). Subsequently, the cells were fixed and permeabilized with Cytofix/Cytoperm solution (BD Biosciences) and then stained intracellularly with FITC-labeled antibody to IFN-γ (ebiosciences) and AL647- labeled antibody against IL-17 (BD Biosciences). Samples were measured on a FACSCalibur, and data were analyzed using the CellQuest Pro software (BD Biosciences). Immune Response to Candida parapsilosis JID 2013:208 (15 August) 691

3 Statistical Analysis GraphPad Prism software, version 5.0, was used to perform statistical analysis. All experiments were performed at least twice. The data represent cumulative results of all experiments performed, and the number of donors is given as n in all figure legends. The differences between groups were analyzed by the Wilcoxon signed rank test or a 2-tailed paired t test (see figure legends) and were considered statistically significant at P values of <.05. RESULTS C. parapsilosis Induces Similar TNF-α and IL-6 Production but Slightly Lower Amounts of IL-1β in PBMCs, Compared With C. albicans Upon encountering a pathogen, a first step in the antifungal immune response is the release of inflammatory cytokines, such as TNF-α, IL-1β, and IL-6, by activated innate immune cells, especially neutrophil granulocytes, monocytes, and macrophages [12]. These mediators then contribute to the efficient phagocytosis and killing of pathogens and also take part in the initiation of the adaptive immune response. Since the growth rates of C. albicans and C. parapsilosis differ and only C. albicans forms hyphae in cell culture, we decided to use heat-killed yeast-phase organisms throughout our study. After 24 hours of incubation, both fungi induced cytokines in a dose-dependent manner (Figure 1). Use of 10 6 fungal cells resulted in secretion of similar amounts of TNF-α and IL-6 by PBMCs from 14 donors after stimulation with C. parapsilosis or C. albicans (mean TNF-α levels [±standard error of the mean {SEM}], 4563 ± 1797 pg/ml for C. parapsilosis and 4858 ± 1745 pg/ml for C. albicans; mean IL-6 levels [±SEM], 5515 ± 1573 pg/ml and 5464 ± 1414 pg/ml, respectively). However, C. parapsilosis induced slightly less IL-1β by PBMCs, compared with C. albicans (mean levels [±SEM], 3583 ± 2226 pg/ml vs 4300 ± 2297 pg/ml; Figure 1. Tumor necrosis factor α (TNF-α; A), interleukin 1β (IL-1β; B), and interleukin 6 (IL-6; C) production by peripheral blood mononuclear cells (PBMCs) after stimulation with heat-killed Candida albicans or Candida parapsilosis. A total of PBMCs were coincubated for 24 hours with either 10 5 or 10 6 yeast cells or RPMI 1640 medium (unstimulated control). Experiments were performed in duplicate, and cell culture supernatants were pooled before cytokine measurements. Data are mean ± standard error of the mean for 14 donors. Differences between groups were analyzed by the Wilcoxon signed rank test. *P <.05, **P <.01, ***P < JID 2013:208 (15 August) Tóth et al

4 Figure 2. Interferon γ (IFN-γ; A) and interleukin 10 (IL-10; B) production by peripheral blood mononuclear cells (PBMCs) after stimulation with heatkilled Candida albicans or Candida parapsilosis. A total of PBMCs were coincubated for 48 hours with either 10 5 or 10 6 yeast cells or RPMI 1640 medium (unstimulated control). Experiments were performed in duplicate, and cell culture supernatants were pooled before cytokine measurements. Data are mean ± standard error of the mean for 14 donors. Differences between groups were analyzed by the Wilcoxon signed rank test. *P <.05, **P <.01, ***P <.001. P <.001). We did not detect any significant difference between PBMCs from 14 donors after stimulation with 10 5 colonyforming units [CFU] of C. albicans or C. parapsilosis (mean TNF-α levels [±SEM], 1300 ± 773 pg for C. albicans and 1262 ± 832 for C. parapsilosis; mean IL-1β levels [±SEM], 1107 ± 667 pg/ml and 998 ± 717 pg/ml, respectively; mean IL-6 levels [±SEM], 1949 ± 1381 and 1791 ± 1460 pg/ml, respectively). C. albicans and C. parapsilosis Induce Different T-Helper Cell Responses In Vitro CD4 + T-helper cells play an essential role in antifungal host defense, and it is generally accepted that successful clearance of C. albicans infection requires a Th1- and Th17-dominated response, rather than a Th2-biased response [13]. Interestingly, we found that PBMCs produced 40% more IL-10 (mean levels [±SEM], ± pg/ml and ± pg/ml; P <.01; n = 14) and 74% less IFN-γ (mean levels [±SEM], ± pg/ml and ± pg/ml, respectively; P <.001; n = 14) when infected with 10 6 C. parapsilosis and C. albicans, respectively (Figure 2). Although this difference could only be seen using the higher (10 6 ) fungal cell count, it indicates a differential regulation of the Th1-Th2 balance during C. parapsilosis stimulation of the immune system. To investigate the role of the Th17 subset in immunity against C. parapsilosis, we analyzed the production of IL-17 and IL-22 after stimulating PBMCs with C. albicans or C. parapsilosis for 7 days (Figure 3). C. parapsilosis induced significantly lower IL-17 production as compared to C. albicans at yeast cell counts of both 10 5 (555.8 ± pg/ml vs 1280 ± pg/ml; P <.001; n = 14) and 10 6 (684.9 ± pg/ml vs 1456 ± 1068 pg/ml; P <.01;n=14).ThelevelofIL-22wasalsolowerinC. parapsilosis stimulated samples (457.3 ± and 2562 ± 2297 pg/ml for fungal cell counts of 10 5 and 10 6, respectively), compared with C. albicans stimulated samples (1037 ± pg/ml and 5567 ± 3187 pg/ml, respectively; P <.001 for both comparisons). Furthermore, when we repeated the stimulation experiments by using a lower number (10 4 ) of yeast cells, we found that, although C. albicans was able to induce considerable amounts of both IL-17 and IL-22 even at this low dose (851.8 ± 1120 pg/ml and ± 1108 pg/ml, respectively; n = 6), infection of PBMCs with 10 4 C. parapsilosis resulted in cytokine levels that barely reached the detection limit (57.79 ± pg/ml [P <.05] and ± pg/ml [P =.125], respectively; n=6). C. parapsilosis Induces Lower Th17 Differentiation, Compared With C. albicans To investigate whether the cause of decreased IL-17 production by C. parapsilosis stimulated PBMCs was due to a lower level of Th17 differentiation, we determined the number of IL-17 producing cells by means of flow cytometry after intracellular cytokine staining (Figure 4). PBMCs were incubated for 6 days with 10 5 C. albicans, 10 5 C. parapsilosis, or medium alone in the presence of 10% pooled human serum. Labeling of CD4 + cells for intracellular staining for IL-17 revealed a lower number of IL-17 producing cells in the CD4 + T-helper cell population after stimulation with C. parapsilosis, compared with C. albicans (3.1% ± 1.6% vs 6.1% ± 3.3%; P =.058; n = 5). Additionally, intracellular staining for IL-17 and IFN-γ showed that the number of cells producing both IL-17 and IFN-γ was also lower following stimulation with C. parapsilosis, compared with C. albicans (0.5% ± 0.2% vs 1.0% ± 0.3%; P <.05; n = 5). Immune Response to Candida parapsilosis JID 2013:208 (15 August) 693

5 Figure 3. Interleukin 17 (IL-17; A) and interleukin 22 (IL-22; B) production by peripheral blood mononuclear cells (PBMCs) stimulated with heat-killed Candida albicans or Candida parapsilosis. A total of PBMCs were coincubated for 7 days with 10 4,10 5,or10 6 yeast cells or RPMI 1640 medium (unstimulated control) in the presence of 10% pooled human serum. Every stimulation experiment was performed in duplicate, and cell culture supernatants were pooled before cytokine measurements. Data are mean ± standard error of the mean for at least 6 donors. Differences between groups were analyzed by the Wilcoxon signed rank test. *P <.05, **P <.01, ***P <.001. Inhibition of the Classical Mitogen-Activated Protein (MAP) Kinases p38 Kinase, ERK, or JNK in PBMCs Inhibits Cytokine Production Induced by C. albicans or C. parapsilosis MAP kinases are known to participate in signaling downstream of both Toll-like receptors and several C-type lectin receptors (such as dectin 1 and dectin 2), the 2 major receptor families playing a role in the immune recognition of C. albicans [14 16]. To investigate whether the difference in immune response induced by C. albicans and C. parapsilosis can be determined at the level of signal transduction, we inhibited the classical MAP kinases p38 kinase, ERK, and JNK before analyzing cytokine production by PBMCs. In these experiments, we preincubated the PBMCs for 1 hour with specific kinase inhibitors before adding the Candida strains to the samples. Blocking of p38 kinase, ERK, or JNK before stimulating PBMCs with C. albicans or C. parapsilosis resulted in different degrees of reduction in the levels of TNF-α, IL-1β, IL-6, IFN-γ, and IL-10 (Table 1). After p38 kinase inhibition, the decrease in the levels of TNF-α, IL-1β, and IL-6 was significantly greater in C. parapsilosis stimulated samples (mean percentage [±SD] of cytokine concentrations in control, 51.35% ± 13.03%, 11.36% ± 1.00%, and 35.71% ± 24.71%,respectively), compared with C. albicans stimulated cells (71.42% ± 12.24%, 16.06% ± 3.54%, and 61.76% ± 24.11%, respectively; P <.05). When inhibiting ERK, we also observed significant decreases in TNF-α, IL-1β, and IL-6 levels in C. parapsilosis stimulated samples (mean percentage [±SD] of cytokine concentrations in control, 30.29% ± 14.05%, 7.42% ± 2.44%, and 5.71% ± 8.61%, respectively), compared with C. albicans treated samples (37.16% ± 11.51%, 9.14% ± 3.26%, and 12.67% ± 9.63%, respectively; P <.05 for all comparisons). Inhibition of JNK resulted in a greater reduction in the levels of secreted TNF-α, IL-1β, and IL-6 in C. albicans stimulated samples (mean percentage [±SD] of cytokine concentrations in control, 34.63% ± 20.14%, 29.70% ± 23.94%, and 52.49% ± 43.63%, respectively), compared with C. parapsilosis treated samples (43.48% ± 21.53%, 34.60% ± 26.58%, and 61.72% ± 44.21%, respectively; P <.05 for comparisons of TNFα and IL-1β levels; P = not significant for comparison of IL-6 levels). Blockade of each of the tested kinases resulted in a greater decrease in IFN-γ levels following stimulation with C. albicans, compared with C. parapsilosis (mean percentage [±SD] of cytokine concentrations in control, 22.71% ± 11.37% vs 31.89% ± 22.96% for p38 kinase [P = not significant], 7.44% ± 6.78% vs 14.03% ± 11.02% for ERK [P <.05], and 7.76% ± 6.44% vs 15.20% ± 9.97% for JNK [P <.05]). The production of IL-10 was also reduced in these experiments, but the decrease was similar between samples stimulated with either Candida species. Therefore, we conclude that, after the recognition of C. albicans and C. parapsilosis by PBMCs, p38 kinase, ERK, and JNK all play a role in signal transduction, but their relative contribution to the resulting cytokine response differs slightly for these 2 species. Dectin 1 Is Involved in the Immune Recognition of C. parapsilosis Since dectin 1 is one of the most important receptors involved in the recognition of C. albicans [12], we wanted to know whether it plays a role in the immune sensing of C. parapsilosis, as well. To investigate this, we performed the same stimulation experiments after preincubating PBMCs with laminarin, a specific inhibitor of dectin 1. We found that inhibition of dectin 1 largely decreased the amount of secreted cytokines in response 694 JID 2013:208 (15 August) Tóth et al

6 Figure 4. Flow cytometric analysis of interleukin 17 (IL-17) and interferon γ (IFN-γ) producing CD4 + peripheral blood mononuclear cell (PBMC) populations after stimulation with heat-killed Candida albicans or Candida parapsilosis. A total of PBMCs were coincubated for 6 days with 10 5 yeast cells or RPMI 1640 medium (unstimulated control) in the presence of 10% pooled human serum. Every stimulation experiment was performed in duplicate, and cells were pooled before flow cytometric analysis. PBMCs were stained extracellularly using an anti-cd4 PECy7 antibody. Subsequently, the cells were fixed, permeabilized, and stained intracellularly with anti IFN-γ FITC and anti IL-17 AL647 antibodies. One representative picture of intracellular cytokine staining is shown in lower scatter plots, and data are summarized in panel A and B. Data are mean ± standard error of the mean for 5 donors. Differences between groups were analyzed by a paired t test. *P <.05, **P <.01, ***P <.001. to stimulation with either C. albicans or C. parapsilosis, indicating that this receptor plays an important role in the immune recognition of both species (Table 2). The reduction in the levels of TNF-α, IL-1β, IL-6, and IL-10 following dectin 1 inhibition was even larger, although nonsignificantly so (P <.05), only for IL-6 in C. parapsilosis stimulated PBMCs (mean percentage [±SD] of cytokine concentrations in control, 25.37% ± 19.01%, 25.85% ± 18.52%, 35.57% ± 20.06%, and 37.34% ± 35.32%, respectively), compared with C. albicans stimulated PBMCs (37.43% ± 28.71%, 38.23% ± 29.86%, 48.52% ± 21.87%, and 53.02% ± 35.91%, respectively). DISCUSSION In this study, we investigated and compared the capacity of C. albicans and C. parapsilosis to stimulate cytokine production by human PBMCs. First, we examined the production of several inflammatory cytokines after stimulating PBMCs with C. albicans and C. parapsilosis. Although C. parapsilosis is known to be less virulent [3] than C. albicans in certain patient populations, they induced similar levels of TNF-α and IL-6. However, PBMCs produced 17% less IL-1β when exposed to C. parapsilosis, compared with C. albicans. In contrast, C. albicans induced significantly higher amounts of IFN-γ and lower amounts of IL-10 as compared to C. parapsilosis. IFN-γ is known to be produced mainly by Th1 lymphocytes and to play a very important role in antifungal immunity through the activation of neutrophil granulocytes and macrophages [12]. In contrast, IL-10 is a typical antiinflammatory cytokine that promotes a Th2-dominated response. Therefore, our results indicate that while C. albicans induces a strong Th1-biased response, in agreement with previous studies [5], C. parapsilosis induces stronger Th2 cellular responses. In addition to assessing the role of Th1 and Th2 subsets, we also assessed the intensity of Th17 differentiation after stimulating PBMCs with either of the 2 Candida species, as the Th17 cell subset is acknowledged to play a significant role in antifungal immunity [5]. The 2 main effector cytokines produced by Th17 cells are IL-17 and IL-22, which induce the recruitment and activation of neutrophil granulocytes and the production of defensins by epithelial cells. The levels of secreted IL-17 and IL-22 were consistently lower in C. parapsilosis stimulated Immune Response to Candida parapsilosis JID 2013:208 (15 August) 695

7 696 JID 2013:208 (15 August) Tóth et al Effects of Mitogen-Activated Protein Kinase Inhibition on Cytokine Production by Peripheral Blood Mononuclear Cells Stimulated With 105 Candida albicans or Candida para- Table 1. psilosis Cytokine Production, % of Value, Mean ± SD Cytokine (Candida + vehicle) C. albicans + p38 Kinase C. parapsilosis + p38 Kinase Donors, No. C. albicans + p38 Kinase vs C. parapsilosis + p38 Kinase vs C. albicans + p38 Kinase vs C. parapsilosis + p38 Kinase TNF-α ± ± < IL-1β ± ± <.001 < IL ± ± IFN-γ ± ± < IL ± ± (Candida + vehicle) C. albicans + ERK C. parapsilosis + ERK C. albicans + ERK vs P C. parapsilosis + ERK vs C. albicans + ERK vs C. parapsilosis + ERK TNF-α ± ± <.001 < IL-1β ± ± <.001 < IL ± ± <.001 < IFN-γ ± ± <.001 < IL ± ± <.001 < (Candida + vehicle) C. albicans + JNK C. parapsilosis + JNK C. albicans + JNK vs C. parapsilosis + JNK vs C. albicans + JNK vs C. parapsilosis + JNK TNF-α ± ± < IL-1β ± ± < IL ± ± IFN-γ ± ± <.001 < IL ± ± Abbreviations: IFN-γ, interferon γ; IL-1β, interleukin 1β; IL-6, interleukin 6; IL-10, interleukin 10; TNF-α, tumor necrosis factor α.

8 Table 2. Cytokine Production by Peripheral Blood Mononuclear Cells Stimulated With Candida albicans or Candida parapsilosis in the Presence of Laminarin, a Dectin 1 Cytokine Production, % of Value, Mean ± SD P C. albicans + Dectin 1 vs C. parapsilosis + Dectin 1 C. parapsilosis + Dectin 1 vs C. albicans + Dectin 1 vs C. parapsilosis + Dectin 1 Donors, No. C. albicans + Dectin 1 (Candida + Vehicle) Cytokine TNF-α ± ± <.001 < IL-1β ± ± <.001 < IL ± ± <.001 < IFN-γ ± ± <.001 < IL ± ± Abbreviations: IFN-γ, interferon γ; IL-1β, interleukin 1β; IL-6, interleukin 6; IL-10, interleukin 10; TNF-α, tumor necrosis factor α. PBMCs, regardless of the concentration of stimulus used. Using intracellular cytokine staining and flow cytometry, we confirmed that the decreased production of IL-17 and IL-22 was in line with a lower number of IL-17 producing cells in the CD4 + cell population. In the light of this finding, the decreased production of IL-1β in C. parapsilosis stimulated samples is understandable, since this cytokine has been shown to be necessary for Th17 differentiation [17]. Considering that IL-6 is also important for Th17 differentiation [17], one might expect a difference in the level of secreted IL-6, as well. However, as stated, we found similar IL-6 production in both C. albicans and C. parapsilosis stimulated PBMCs. This means that, although IL-6 might be important during differentiation, it does not account for the different Th17 responses evoked by C. albicans and C. parapsilosis. Additionally, we found that the number of cells producing both IL-17 and IFN-γ was also lower in C. parapsilosis stimulated samples, although they constituted only a few percent of cells in the CD4 + cell population in both conditions. These special cells, termed Th17/Th1, have only recently been described [18], and their function during fungal infections needs further investigation. To study the signal transduction pathways following the recognition of C. albicans and C. parapsilosis, we compared the cytokine production by PBMCs after adding specific kinase inhibitors to the stimulation experiments. Inhibition experiments showed differential involvement of the MAP kinases p38, ERK, and JNK in the induction of cytokines following immune sensing of C. albicans and C. parapsilosis, demonstrating specific use of kinase pathways by these 2 Candida species. Finally, we wanted to determine whether the β-glucan receptor dectin 1 plays a role in the recognition of C. parapsilosis. Our results show that dectin 1 is indeed an important receptor for the immune sensing of this species, since inhibition of dectin 1 during the stimulation experiments resulted in significantly decreased cytokine levels. Taken together, our study contributes to the better understanding of the immune response against the emerging pathogen C. parapsilosis. Our results show that C. parapsilosis and C. albicans induce significantly different IFN-γ, IL-10, IL-17, and IL-22 production in vitro in human PBMCs. Although the underlying molecular mechanisms of the induction of these cytokines remain to be clarified, these findings may partly explain the different virulence of the 2 species and the increased susceptibility of certain populations to C. parapsilosis infection. The different cytokine-inducing capacity of the 2 Candida species may also have therapeutic consequences. Recombinant cytokines, such as rifn-γ, have already been successfully used in combination with antifungal drugs in invasive fungal infections [19], and they are promising therapeutics for invasive candidiasis [20]. However, their beneficial effects on health may vary when used against different Candida species. Thus, our Immune Response to Candida parapsilosis JID 2013:208 (15 August) 697

9 findings can contribute to the development of better antifungal therapies. Notes Financial support. This work was supported by the Federation of European Microbiological Societies (research fellowship to A. T.), OTKA (NF and ERA-Net Pathogenomics NN to A. G.), EMBO (installation grant 1813 to A. G.), the Hungarian Scientific Research Fund (NF and ERA-Net Pathogenomics NN to A. T. and A. G.), the National Institutes of Health (grant AI52733 to J. N.), the Irma T. Hirschl/Monique Weill-Caulier Trust (research award to J. N.), and the Netherlands Organization for Scientific Research (Vici grant to M. G. N.). Potential conflicts of interest. All authors: No reported conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed. References 1. Pfaller MA, Diekema DJ. Epidemiology of invasive candidiasis: a persistent public health problem. Clin Microbiol Rev 2007; 20: Pfaller MA, Diekema DJ, Gibbs DL, et al. Results from the ARTEMIS DISK Global Antifungal Surveillance Study, 1997 to 2007: a 10.5-year analysis of susceptibilities of Candida Species to fluconazole and voriconazole as determined by CLSI standardized disk diffusion. J Clin Microbiol 2010; 48: Trofa D, Gacser A, Nosanchuk JD. Candida parapsilosis, an emerging fungal pathogen. Clin Microbiol Rev 2008; 21: Dotis J, Prasad PA, Zaoutis T, Roilides E. Epidemiology, risk factors and outcome of Candida parapsilosis bloodstream infection in children. Pediatr Infect Dis J 2012; 31: Hernandez-Santos N, Gaffen SL. Th17 cells in immunity to Candida albicans. Cell Host Microbe 2012; 11: Harrington LE, Hatton RD, Mangan PR, et al. Interleukin 17-producing CD4+ effector T cells develop via a lineage distinct from the T helper type 1 and 2 lineages. Nat Immunol 2005; 6: Ouyang W, Kolls JK, Zheng Y. The biological functions of T helper 17 cell effector cytokines in inflammation. Immunity 2008; 28: Huang W, Na L, Fidel PL, Schwarzenberger P. Requirement of interleukin-17a for systemic anti-candida albicans host defense in mice. J Infect Dis 2004; 190: Puel A, Cypowyj S, Bustamante J, et al. Chronic mucocutaneous candidiasis in humans with inborn errors of interleukin-17 immunity. Science 2011; 332: van de Veerdonk FL, Plantinga TS, Hoischen A, et al. STAT1 mutations in autosomal dominant chronic mucocutaneous candidiasis. N Engl J Med 2011; 365: Liu L, Okada S, Kong XF, et al. Gain-of-function human STAT1 mutations impair IL-17 immunity and underlie chronic mucocutaneous candidiasis. J Exp Med 2011; 208: Netea MG, Gow NA, Munro CA, et al. Immune sensing of Candida albicans requires cooperative recognition of mannans and glucans by lectin and Toll-like receptors. J Clin Invest 2006; 116: van de Veerdonk FL, Netea MG. T-cell subsets and antifungal host defenses. Curr Fungal Infect Rep 2010; 4: Kerrigan AM, Brown GD. Syk-coupled C-type lectins in immunity. Trends Immunol 2011; 32: Netea MG, Brown GD, Kullberg BJ, Gow NA. An integrated model of the recognition of Candida albicans by the innate immune system. Nat Rev Microbiol 2008; 6: Kawai T, Akira S. TLR signaling. Cell Death Differ 2006; 13: Acosta-Rodriguez EV, Napolitani G, Lanzavecchia A, Sallusto F. Interleukins 1beta and 6 but not transforming growth factor-beta are essential for the differentiation of interleukin 17-producing human T helper cells. Nat Immunol 2007; 8: Annunziato F, Cosmi L, Santarlasci V, et al. Phenotypic and functional features of human Th17 cells. J Exp Med 2007; 204: Armstrong-James D, Teo IA, Shrivastava S, et al. Exogenous interferongamma immunotherapy for invasive fungal infections in kidney transplant patients. Am J Transplant 2010; 10: van de Veerdonk FL, Kullberg BJ, Netea MG. Adjunctive immunotherapy with recombinant cytokines for the treatment of disseminated candidiasis. Clin Microbiol Infect 2012; 18: Gacser A, Salomon S, Schafer W. Direct transformation of a clinical isolate of Candida parapsilosis using a dominant selection marker. FEMS Microbiol Lett 2005; 245: JID 2013:208 (15 August) Tóth et al