of February 7, 2016.This information is current as

Neutrophils with HumanCandida dubliniensis

and Candida albicansFungal Species Differential Interaction of the Two Related

Hennicke, Dagmar Barz and Mihály JózsiEliska Svobodová, Peter Staib, Josephine Losse, Florian

http://www.jimmunol.org/content/189/5/2502doi: 10.4049/jimmunol.1200185July 2012;

2012; 189:2502-2511; Prepublished online 30J Immunol 

MaterialSupplementary

5.DC1.htmlhttp://www.jimmunol.org/content/suppl/2012/07/30/jimmunol.120018

Referenceshttp://www.jimmunol.org/content/189/5/2502.full#ref-list-1

, 28 of which you can access for free at: cites 67 articlesThis article

Subscriptionshttp://jimmunol.org/subscriptions

is online at: The Journal of ImmunologyInformation about subscribing to

Permissionshttp://www.aai.org/ji/copyright.htmlSubmit copyright permission requests at:

Email Alertshttp://jimmunol.org/cgi/alerts/etocReceive free email-alerts when new articles cite this article. Sign up at:

Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists, Inc. All rights reserved.Copyright © 2012 by The American Association of9650 Rockville Pike, Bethesda, MD 20814-3994.The American Association of Immunologists, Inc.,

is published twice each month byThe Journal of Immunology

by guest on February 7, 2016http://w

ww

.jimm

unol.org/D

ownloaded from

by guest on February 7, 2016

http://ww

w.jim

munol.org/

Dow

nloaded from

The Journal of Immunology

Differential Interaction of the Two Related Fungal SpeciesCandida albicans and Candida dubliniensis with HumanNeutrophils

Eliska Svobodova,* Peter Staib,† Josephine Losse,* Florian Hennicke,† Dagmar Barz,‡ and

Mihaly Jozsi*

Candida albicans, the most common facultative human pathogenic fungus is of major medical importance, whereas the closely

related species Candida dubliniensis is less virulent and rarely causes life-threatening, systemic infections. Little is known,

however, about the reasons for this difference in pathogenicity, and especially on the interactions of C. dubliniensis with the

human immune system. Because innate immunity and, in particular, neutrophil granulocytes play a major role in host anti-

fungal defense, we studied the responses of human neutrophils to clinical isolates of both C. albicans and C. dubliniensis.

C. dubliniensis was found to support neutrophil migration and fungal cell uptake to a greater extent in comparison with

C. albicans, whereas inducing less neutrophil damage and extracellular trap formation. The production of antimicrobial reactive

oxygen species, myeloperoxidase, and lactoferrin, as well as the inflammatory chemokine IL-8 by neutrophils was increased

when stimulated with C. dubliniensis as compared with C. albicans. However, most of the analyzed macrophage-derived

inflammatory and regulatory cytokines and chemokines, such as IL-1a, IL-1b, IL-1ra, TNF-a, IL-10, G-CSF, and GM-CSF,

were less induced by C. dubliniensis. Similarly, the amounts of the antifungal immunity-related IL-17A produced by PBMCs

was significantly lower when challenged with C. dubliniensis than with C. albicans. These data indicate that C. dubliniensis

triggers stronger early neutrophil responses than C. albicans, thus providing insight into the differential virulence of these two

closely related fungal species, and suggest that this is, in part, due to their differential capacity to form hyphae. The Journal of

Immunology, 2012, 189: 2502–2511.

Fungi of the genus Candida are harmless commensalscoexisting with humans. In the immunocompromised host,however, Candida spp. can cause superficial and even life-

threatening, systemic infections. Although Candida albicans is themost common cause of candidiasis, the number of infectionscaused by nonalbicans species such as Candida glabrata, Candidaparapsilosis, and Candida dubliniensis, especially in HIV-infectedand AIDS patients, is quite high (1). C. dubliniensis was firstdiscovered in 1995 upon genetic characterization and is mainlyrecovered from oral candidosis of HIV+ patients (2). C. dubliniensisis the closest relative of C. albicans and is the only other Candidaspecies able to form true hyphae and chlamydospores (3).

Despite their similarity, C. dubliniensis appears to be less virulentin animal and mucosal infection models (4–7) according to its in-fection rate and the severity of disease. C. dubliniensis strains aremore rapidly cleared form the site of infection and are less able tocause disseminated infections. The phylogenetic properties of C.dubliniensis have been well described in the literature (2, 8). A DNAhybridization array (9) and lately comparison of both genomes (10)uncovered particular differences between C. dubliniensis and C.albicans in regard to putative virulence genes, including thoseencoding hyphae-associated factors such as specific secretedproteases or invasins. The ability of C. albicans to reversibly switchbetween yeast and filamentous growth forms has widely been docu-mented as an important virulence attribute of C. albicans, facilitatinghost invasion and colonization. C. dubliniensis also produces truehyphae, however, with a far lesser efficiency than C. albicans, whichputatively contributes to its comparatively poor virulence (6, 11).The control of fungal infection in the human host critically

depends on cells of the innate immunity. Neutrophil granulocytesare major phagocytic cells that are recruited early to the infectionsite, where they activate a large repertoire of antimicrobialmechanisms including phagocytosis, the generation of reactiveoxygen species (ROS), the release of granule enzymes and anti-microbial peptides, and the formation of neutrophil extracellulartraps (NETs) (12–14). Attracted monocytes possess less micro-bicidal capacity but are important in alerting cells of the innateand adaptive immune system, and give rise to macrophages, im-portant phagocytic and APCs. These innate immune cells recog-nize fungal pathogens via three major receptor families (i.e., theTLRs, the lectin receptors, and the NOD-like receptors), leadingto activation of signaling pathways that result in tightly regulatedand complementary immune responses (15, 16).

*Junior Research Group Cellular Immunobiology, Leibniz Institute for Natural Prod-uct Research and Infection Biology–Hans Knoll Institute, D-07745 Jena, Germany;†Junior Research Group Fundamental Molecular Biology of Pathogenic Fungi, Leib-niz Institute for Natural Product Research and Infection Biology–Hans Knoll Insti-tute, D-07745 Jena, Germany; and ‡Institute for Transfusion Medicine, UniversityHospital of Jena, D-07747 Jena, Germany

Received for publication January 13, 2012. Accepted for publication July 3, 2012.

This work was supported in part by the Deutsche Forschungsgemeinschaft (DFGGrant STA 1147/1-1 to P.S.).

Address correspondence and reprint requests to Dr. Mihaly Jozsi, Junior ResearchGroup Cellular Immunobiology, Leibniz Institute for Natural Product Research andInfection Biology–Hans Knoll Institute, Beutenbergstrasse 11a, D-07745 Jena, Germany.E-mail address: mihaly.jozsi@gmx.net

The online version of this article contains supplemental material.

Abbreviations used in this article: CFW, calcofluor white; DPBS, Dulbecco’sphosphate-buffered saline; LDH, lactate dehydrogenase; MDM, monocyte-derivedmacrophage; MOI, multiplicity of infection; MPO, myeloperoxidase; NET, neutro-phil extracellular trap; ROS, reactive oxygen species.

Copyright� 2012 by TheAmericanAssociation of Immunologists, Inc. 0022-1767/12/$16.00

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1200185

by guest on February 7, 2016http://w

ww

.jimm

unol.org/D

ownloaded from

To date, little is known about the Candida species-specificresponses of human innate immune cells. Because C. dubliniensishas a reduced ability to cause severe (i.e., systemic) infections,which are usually brought in context with C. albicans, one wouldexpect a better clearance of the former by host immune cells. Aprevious report found no difference in the phagocytosis, oxidativeburst, or killing induced by these two fungal species in wholeblood (17). Nevertheless, it has been shown that even though bothCandida species release a chemotactic factor for neutrophils,the chemotaxis toward C. dubliniensis is enhanced compared withC. albicans (18). Studies using epithelial cell infection mod-els demonstrated that C. dubliniensis causes minimal invasion(5, 19) and cytokine production (20, 21) compared with C.albicans, and that this effect is attributed to the ability of the latterto switch from yeast to hyphal morphology under cell cultureconditions (21).It is important to understand their interaction with the host

immune system in detail to decipher species-specific differences inthe pathogenicity of these two closely related Candida species.Therefore, we compared the activation of human neutrophil gran-ulocytes upon coincubation with C. albicans and C. dubliniensisin vitro, using a set of previously characterized clinical isolates.We found that C. dubliniensis strains enhanced neutrophil mi-gration and phagocytosis, as well as IL-8, ROS, and lactoferrinrelease, but caused less extracellular trap formation and neutrophildamage, in comparison with C. albicans strains. In addition, areduced ability of C. dubliniensis to stimulate the production ofIL-17 by mononuclear cells, a cytokine involved in neutrophilrecruitment and activation, was observed. These results demon-strating a differential interaction of the two Candida species withcells of the human innate immune system may, in part, explain thedifferences in their virulence.

Materials and MethodsCandida strains

The C. albicans and C. dubliniensis wild-type strains that were used arelisted in Table I. All strains were routinely maintained on yeast peptonedextrose agar (20 g peptone, 10 g yeast extract, 20 g glucose, 15 g agar perliter). For experiments, cells were grown in yeast peptone dextrose mediumat 30˚C with shaking for 16 h and washed twice in Dulbecco’s PBS(DPBS; Lonza, Wuppertal, Germany) before use. In case of C. albicans,hypha formation was induced in RPMI 1640 medium containing L-gluta-mine and 25 mM HEPES (Invitrogen, Karlsruhe, Germany) at 37˚C for 1h. For C. dubliniensis, hypha formation was induced in water containing10% FCS (PAA Laboratories, Colbe, Germany) at 37˚C for 1 h (11). Thehyphal morphology for both species was monitored microscopically toensure a similar hyphal percentage and length.

C. dubliniensis reporter strain construction

To visualize Candida cells during interaction with neutrophils, we alsoused GFP-labeled fungal cells. For this purpose, C. albicans and C. dub-liniensis strains were used that express the GFP gene from the constitu-tively active C. albicans and C. dubliniensis ADH1 promoter, respectively.The C. albicans reporter strain SCADH1G4A carrying a PCaADH1-GFPfusion in one of the ADH1 alleles of the wild-type strain SC5314 hadbeen described previously (26). Analogously, the C. dubliniensis strainCdADH1GFP1A, which harbors a PCdADH1-GFP fusion in one of theCdADH1 alleles of the wild-type strain Wu284, was constructed as follows.An ApaI-XhoI fragment with CdADH1 upstream sequences was amplifiedby PCR with the primers CdADH1-1 (59-ATATAGGGCCCCCGGGTT-CCGTCATGCAAGCAAGC-39) and CdADH1-2 (59-ATATACTCGAGG-TTTTTGTATTTGTATGACAATAACGTTTATTG-39). A PstI-SacII fragmentcontaining CdADH1 downstream sequences was obtained by PCR withthe primers CdADH1-3 (59-GCTGAACCAAACTGCAGTGAAGCTGAC-TTG-39) and CdADH1-4 (59-GTGTCTTTTCTGTTACCGCGGTAAGA-ACCTTTG-39). Genomic DNA from C. dubliniensis strain Wu284 wasused as a template for the PCR. The CdADH1 upstream and downstreamfragments were successively substituted for CaADH1 upstream and down-stream fragments in plasmid pADH1E1 (27) via the introduced restriction

sites, respectively, to result in pCdADH1E2. A SalI-BglII GFP frag-ment from pNIM1 (28) was cloned in the XhoI-BglII digested plasmidpCdADH1E2, resulting in pCdADH1GFP1. Flanked by CdADH1 upstreamand downstream sequences, pCdADH1GFP1 contains a cassette with theGFP gene under control of PCdADH1, followed by TCaACT1 terminationsequences, and the SAT1 marker for transformant selection after trans-formation. Transformation by electroporation of C. dubliniensis Wu284by use of the linear ApaI-SacII DNA cassette from pCdADH1GFP1,transformant selection on nourseothricin (Werner Bioagents, Jena, Ger-many), and Southern analysis of genomic DNA with ECL-labeled (GEHealthcare, Freiburg, Germany) upstream and downstream CdADH1probes for proving correct insertion of the GFP construct in the CdADH1locus of the resulting transformant CdADH1GFP1A were performed bystandard procedures as described previously (29).

Human cell culture and stimulation

Human neutrophil granulocytes were isolated from peripheral blood ofhealthy individuals as previously described (30). All blood donors gaveinformed consent. If not otherwise stated, neutrophils were cultured inserum-free X-VIVO 15 medium (Lonza) at a concentration of 2.5 3 106

cells/ml and cultivated at 37˚C in a humidified atmosphere containing5% CO2. If not stated otherwise, neutrophils were incubated withCandida cells at a multiplicity of infection (MOI) of 1. Culture super-natants were collected either after 1.5 h for ROS, myeloperoxidase(MPO), and lactoferrin measurement or after 20 h of coincubation forIL-8 measurement. All stimulations for each donor were performed induplicates.

PBMCs were isolated by Ficoll-Hypaque (GE Healthcare) densitygradient separation, and erythrocytes were lysed using a hypotonic saltsolution. PBMCs were washed with DPBS and cultured in X-VIVO 15medium containing 10% pooled normal human serum at a concentration of5 3 106 cells/ml. For stimulation experiments, PBMCs were incubated inmedium alone or in medium containing 13 104 live Candida cells/ml, andsupernatants were collected after 7 d for IL-17 measurement.

Monocytes were obtained from PBMCs by positive selection with CD14magnetic beads (Miltenyi Biotech, Bergisch Gladbach, Germany), fol-lowing the manufacturer’s instructions. In brief, PBMCs were resuspendedin MACS buffer (DPBS with 0.5% BSA and 2 mM EDTA) containingCD14 microbeads. After incubation for 30 min at 4˚C, cells were washedand resuspended in MACS buffer and loaded onto MiniMACS LS Sepa-ration Columns (Miltenyi Biotech). After washing out of nonlabeled cellswith MACS buffer, monocytes were eluted and the purity of CD14+

monocytes was determined by FACS.Monocyte-derived macrophages (MDMs) were obtained by culturing

isolated monocytes for 7 d in X-VIVO 15 medium supplemented with 10%heat-inactivated FCS, 2 mM ultraglutamine (Lonza), and 50 mg/ml gen-tamicin sulfate (Lonza), and by addition of 500 IU/ml GM-CSF (Immu-notools, Friesoythe, Germany) every 48 h. MDMs were resuspended at 23106 cells/ml in serum-free X-VIVO 15 medium and cultivated at 37˚C ina humidified atmosphere containing 5% CO2. Cells were infected withCandida at a MOI of 1 or 4, depending on experiment. Culture super-natants were collected 20 h later, and cytokine profiles were determined bycytokine array and/or ELISA.

Neutrophil migration assays

Migration assays were performed as described previously (30). Neutrophilswere labeled with calcein AM (Sigma-Aldrich, Taufkirchen, Germany),allowed to migrate toward Candida cells through a 3-mm-pore poly-carbonate membrane of a transwell system (Corning Life Sciences,Amsterdam, The Netherlands) for 60 min and were quantified in the lowerwell.

Neutrophil phagocytosis assay

Neutrophils (5 3 105) in X-VIVO 15 medium were added to coverslips,precoated with 0.01% poly-L-lysine, in 24-well plates and allowed to at-tach for 40 min at 37˚C. For some experiments, neutrophils were pretreatedwith 15 mg/ml Cytochalasin D (Sigma-Aldrich) for 30 min at 37˚C beforephagocytosis. Candida cells constitutively expressing GFP were added ata MOI of 2 and sedimented by centrifugation at 1400 rpm for 3 min. After40 min of coincubation at 37˚C and 5% CO2, plates were cooled on ice andnonphagocytosed Candida cells were stained with 10 mg/ml calcofluorwhite (CFW; fluorescent brightener 28; Sigma-Aldrich) for 20 min at roomtemperature. Cells were fixed with 4% paraformaldehyde (Carl Roth,Karlsruhe, Germany) for 15 min at 20˚C, coverslips were mounted on glassslides, and phagocytosis was analyzed using a 633 immersion oil objec-tive at room temperature with an LSM 710 confocal laser-scanning mi-

The Journal of Immunology 2503

by guest on February 7, 2016http://w

ww

.jimm

unol.org/D

ownloaded from

croscope running ZEN software (Carl Zeiss, Jena, Germany). At least fourof 3 3 3 microscopic fields (200 3 200 mm) were analyzed for thecolocalization of GFP and CFW staining using an argon laser witha wavelength of 488 nm and corresponding filters. The percentage ofphagocytosed cells was calculated from the GFP signal that does notcolocalize with the CFW signal.

Release of NETs

Neutrophils were attached to coverslip as described earlier for thephagocytosis assay. Afterward, 1 3 106 GFP-expressing yeast cells or 100nM PMA as a positive control were added. Fungal cells were sedimentedby centrifugation at 1400 rpm for 3 min. The coverslips were then incu-bated for 3 h at 37˚C in a humidified atmosphere containing 5% CO2.Extracellular DNA was stained with 20 mg/ml propidium iodide (Sigma-Aldrich) for 20 min at 37˚C. After washing gently with DPBS, the cov-erslips were placed on mounting solution on a glass slide, and NETs wereanalyzed using an LSM 710 confocal laser-scanning microscope (CarlZeiss). For quantification of NETs, neutrophils were coincubated withCandida cells in white 96-well plates. Released DNA was stained with 5mg/ml SYTOX Orange (Invitrogen), and the fluorescence was measured ina fluorescence reader (Tecan, Crailsheim, Germany) with emission andabsorption filters of 540 and 575 nm, respectively.

Neutrophil damage assay

Neutrophil granulocytes were seeded in a flat-bottom, 96-well plate ata concentration of 5 3 106 cells/ml. After addition of Candida cells ata MOI of 1, the plate was incubated for 20 h at 37˚C and 5% CO2. Forcontrol, neutrophils and Candida cells were incubated in medium only.The plate was equilibrated to 20˚C, supernatants were collected in a white96-well plate, and the lactate dehydrogenase (LDH) activity was analyzedusing a fluorescence-based CytoTox-ONE Homogeneous Membrane In-tegrity Assay kit (Promega, Mannheim, Germany) according to the man-ufacturer’s instructions. Experiments were performed in duplicates, andthe results are expressed as percentage of released LDH in the supernatantof lysed neutrophils.

Release of ROS, MPO, and lactoferrin

Neutrophils were coincubated in white 96-well plates (Greiner, Frick-enhausen, Germany) in 200 ml medium/well with yeasts of the various C.albicans and C. dubliniensis strains at a MOI of 1 in X-VIVO 15 me-dium at 37˚C. For some experiments, Candida cells were separated fromneutrophils through a 0.4-mm-pore membrane in a 24 well-plate trans-well system (Corning Life Sciences). ROS were measured after 90 minof coculture by incubating cells in X-VIVO 15 medium containing 10mM dihydrorhodamine (Sigma-Aldrich) for 15 min at 37˚C followedby lysis of neutrophils with DPBS supplemented with 1% Triton X-100.The fluorescence signal of the oxidized dihydrorhodamine was mea-sured in a fluorescence reader (Tecan) with excitation and emissionfilters of 485 and 538 nm. MPO and lactoferrin were detected using asandwich ELISA. In brief, 4 mg/ml anti-MPO or anti-lactoferrin mAb(HyTest, Turku, Finland) was immobilized on microtiter plates (Nunc,Wiesbaden, Germany) at 4˚C overnight. After blocking with 4% skimmedmilk for 2 h, supernatants of cocultures, taken after 90 min, were addedfor 1 h at room temperature. Lactoferrin and MPO were detected using50 ng/ml HRP-conjugated anti-lactoferrin IgG (antibodies-online, Aachen,Germany) and rabbit anti-MPO IgG (1:5000; HyCultec, Beutelsbach,Germany), followed by the corresponding secondary Ab. The ELISAwas developed using TMB PLUS substrate (Kem-En-Tec Diagnostics,Taastrup, Denmark), and the absorbance was measured at 450 nm.

Measurement of cytokine production

The production of IL-8 by human neutrophils was measured fromsupernatants of cocultures taken after 20 h using a commercial sandwichELISA kit (ImmunoTools). IL-1b and IL-17 were quantified from culturesupernatants of MDMs or PBMCs using commercial sandwich ELISAkits from ImmunoTools and eBioscience (Frankfurt, Germany), respec-tively. The amounts of other inflammation-related cytokines weremeasured after 24 h from the supernatants of Candida-macrophagecocultures incubated at a MOI of 1 in X-VIVO 15 medium usinga Proteome Profiler Human Cytokine Array Kit (Panel A; R&D Systems,Wiesbaden, Germany).

Statistical analysis

Statistical analysis of the results was carried out using an unpaired two-tailed Student t test. A p value #0.05 was considered significant.

ResultsHuman neutrophils migrate at a higher rate toward C.dubliniensis

Because both C. albicans and C. dubliniensis were shown to re-lease chemotactic factor(s) for neutrophils (18), we studied themigratory activity of neutrophils toward different strains of bothCandida species (Table I) in a transwell assay. All studied strainsof C. dubliniensis significantly enhanced the number of neutrophilsthat migrated through the porous membrane of the transwellsystem compared with the strains of C. albicans (Fig. 1). We didnot observe significant differences in the growth rate between thestudied strains in suspension cultures. During the 1-h incubationtime, the fungal cells did not multiply in the wells as monitored bymicroscopic analysis, and the C. albicans cells only started togerminate. Thus, the observed difference in supporting neutrophilmigration was not due to difference in fungal mass among thefungal species.

C. dubliniensis is rapidly phagocytosed by human neutrophils

To assess the phagocytosis of both Candida species, we coincu-bated blood-derived human neutrophils with GFP-labeled yeastcells (C. albicans strain SCADH1G4A and C. dubliniensis strainCdADH1GFP1A; for details, see Materials and Methods) at aMOI of 2. After 40 min, nonphagocytosed fungal cells werestained with CFW and the percentage of engulfed cells was de-termined microscopically. Under these conditions, ∼70% of the C.dubliniensis cells were internalized compared with only ∼20% ofC. albicans cells (Fig. 2). As a negative control, neutrophils werepretreated with Cytochalasin D before phagocytosis. Under thiscondition, almost no cells were internalized after the specifiedtime point (data not shown).

Reduced ability of C. dubliniensis to induce NETs

To analyze whether C. dubliniensis induces the production ofextracellular traps by human neutrophils, similar to C. albicans(14), we coincubated GFP-expressing yeast cells of both Candida

Table I. C. albicans and C. dubliniensis clinical isolates used in thisstudy

Strain Source Reference

C. albicansATCC10261 ATCC Reference strainATCC38696 ATCC Reference strainATCC44807 ATCC Reference strainATCC44808 ATCC Reference strainDSM1386 DSM Reference strainSC5314 F. Muhlschlegel 22WO-1 D. Soll 23MA13 Clinical isolate, Berlin 24Wu212 Clinical isolate, Wurzburg 24Wu277 Clinical isolate, Wurzburg 24C. dubliniensisH1 Clinical isolate, Berlin 25H12 Clinical isolate, Berlin 25VK9603-29 Clinical isolate, Berlin 25VK9603-34 Clinical isolate, Berlin 25VK9603-54 Clinical isolate, Berlin 25VK9603-63 Clinical isolate, Berlin 25Wu272 Clinical isolate, Wurzburg 25Wu284 Clinical isolate, Wurzburg 25Wu294 Clinical isolate, Wurzburg 25Wu361 Clinical isolate, Wurzburg 25Wu367 Clinical isolate, Wurzburg 25Wu370 Clinical isolate, Wurzburg 25

ATCC, American Type Culture Collection; DSM, German collection of micro-organisms.

2504 NEUTROPHIL RESPONSES TO C. ALBICANS AND C. DUBLINIENSIS

by guest on February 7, 2016http://w

ww

.jimm

unol.org/D

ownloaded from

species with neutrophils for 3 h at 37˚C at a MOI of 2. Confocallaser-scanning microscopy of cultures stained with propidium io-dide revealed that C. dubliniensis caused only little NET formation,whereas NETs were readily visible for neutrophils challenged withC. albicans (Fig. 3). Neutrophils incubated without fungi did notproduce NETs. As a positive control, NET formation was inducedusing PMA (data not shown). To quantify NET formation, weanalyzed the DNA content in the supernatants of cocultures ofCandida cells with neutrophils using SYTOX Orange. Neutrophilsefficiently formed NETs by 3 h after stimulation with C. albicansstrains. However, significantly less extracellular DNA was detectedfrom neutrophil cultures stimulated with C. dubliniensis strains(Fig. 4), confirming the microscopy data. We have repeated thisassay with fungal cells for which hypha formation was induced for1 h at 37˚C before addition to the neutrophils (Supplemental Fig. 1).C. dubliniensis strains again caused less NET formation comparedwith C. albicans strains (data not shown). Notably, after severalhours of coincubation with neutrophils, most C. dubliniensis cellsdid not maintain the hyphal morphology (Supplemental Fig. 2), asalso reported by Moyes et al. (21).

The ability of C. dubliniensis to damage neutrophils is reduced

One of the pathogenic attributes of C. albicans is the productionand elongation of hyphae, through which the pathogen is able to

damage and escape from host phagocytes (31). The ability of C.dubliniensis to switch from the yeast to the hyphal form is re-duced, especially in a nutrient-rich environment (11), which canbe found in the infection site in the human body. To test whetherC. dubliniensis can cause damage to human neutrophils to thesame extent as C. albicans, we determined the amounts of LDHreleased upon damage of the plasma membrane of the host cells,in culture supernatants after 20 h of coincubation of C. albicanscells (strains SC5314, ATCC38696, and ATCC44808) and C.dubliniensis cells (strains Wu272, Wu284, and H1) with neu-trophils. As a positive control, neutrophils were lysed, and asa negative control, LDH release was measured from neutrophilsincubated in medium only. Even though C. dubliniensis wasrapidly phagocytosed, the yeast cells were not able to switch intohyphae as efficiently as C. albicans, as observed microscopically(Supplemental Fig. 3), and caused only slight damage (∼20%) toneutrophils compared with the negative control representing deadneutrophils after 20 h (∼16%). In contrast, all C. albicans strainscaused more damage (30–50% after 20 h) to human neutrophils(Fig. 5). Similarly, C. dubliniensis strains caused less neutrophildamage compared with C. albicans strains when the fungal cellswere added after 1-h hypha induction (data not shown).

Increased release of antimicrobial substances and IL-8 byneutrophils stimulated with C. dubliniensis versus C. albicans

Antimicrobial compounds and cytokines produced by the innateimmune cells are important factors that affect the outcome offungal infections. We therefore analyzed the production and/orrelease of ROS, MPO, lactoferrin, and the proinflammatory cy-tokine IL-8 by human neutrophils coincubated with C. albicans orC. dubliniensis. Supernatants from neutrophils incubated in me-dium only or with PMA (potent neutrophil stimulator) were usedas a negative and positive control, respectively. All analyzedstrains of C. dubliniensis caused an increased release of ROS,MPO, lactoferrin, and IL-8 when compared with the strains of C.albicans (Table II).To analyze in more detail whether the morphology of theCandida

cells per se differentially influences this phenotype, hypha forma-tion was induced for C. albicans strains SC5314, ATCC38696,ATCC44808, and WO-1, and for C. dubliniensis strains H1,Wu272, Wu284, and VK9603-29 for 1 h at 37˚C before incubationwith human neutrophils (Supplemental Figs. 1, 2). These fungalcells caused an increased ROS and lactoferrin production comparedwith yeast cells; nevertheless, the elevation was more prominent inthe case of C. albicans than C. dubliniensis (Fig. 6).Further, we determined whether the stimulation of neutrophils,

and thus the induction of lactoferrin production, is dependent ona direct contact between Candida and neutrophils or is caused by

FIGURE 1. Enhanced neutrophil migration toward C. dubliniensis. Live

yeast cells were added to the lower chamber and calcein-labeled neutrophils

to the upper chamber of the transwell system. The fluorescence of neu-

trophils in the lower chamber was determined after 60 min of transmi-

gration. Transwells without yeasts were used as negative control. Results

shown are relative mean fluorescence intensities (MFI) 6 SD from at least

five experiments with different blood donors. The migration induced by

C. albicans strain SC5314 was set at 100%. The negative control was

subtracted, and mean value of the population of both species was derived

from values of all strains in this population. **p # 0.01, Student t test.

FIGURE 2. Phagocytosis of C. albicans and C. dubliniensis by human neutrophils. Blood-derived neutrophils were coincubated with GFP-expressing C.

albicans and C. dubliniensis cells for 40 min at a MOI of 2. Extracellular fungal cells were stained with CFW and analyzed at 3630 magnification in

a laser-scanning microscope. (A) Pictures represent overlay images of green (internalized fungal cells) and blue (nonphagocytosed fungal cells) fluores-

cence channels with bright field. Scale bar, 10 mm. (B) The percentage of internalized fungal cells was quantified microscopically. Results shown are

percentage of phagocytosed cells + SD from at least five experiments with different blood donors. ***p # 0.001, Student t test.

The Journal of Immunology 2505

by guest on February 7, 2016http://w

ww

.jimm

unol.org/D

ownloaded from

Candida products acting on neutrophils. For this purpose, wecompared the neutrophil response when coincubated with Can-dida cells directly and when separated by a porous membrane toallow exchange of medium and products. Whereas C. albicanscaused only a slightly elevated lactoferrin production upon directcontact compared with separated cells, the induction of lacto-ferrin production by C. dubliniensis was largely dependent on theCandida–neutrophil contact (Fig. 7).

C. dubliniensis stimulates less production of fungalimmunity-related cytokines

Because macrophages are important innate immunity cells thatrecruit additional cell types to the site of infection, we measuredchemokines and cytokines from MDMs after coincubation withboth Candida species for 24 h at a MOI of 1 using a commerciallyavailable inflammation array. C. dubliniensis and C. albicans in-duced several inflammatory molecules under these experimentalconditions in comparison with MDMs incubated without fungalcells (Fig. 8). Whereas some molecules were detected in similaramounts for both C. dubliniensis and C. albicans, such as C5/C5a,growth-regulated oncogene-a (also known as CXCL1), RANTES(CCL5), and MIP-1a (CCL3), a markedly reduced induction of G-CSF, GM-CSF, IL-1a, IL-1b, IL-1ra, IL-10, IL-16, macrophagemigration inhibitory factor, serine protease inhibitor 1, TNF-a,and soluble triggering receptor expressed on myeloid cells wasdetected when MDMs were exposed to C. dubliniensis. In con-trast, the chemoattractant IFN-g–induced protein 10 (also knownas CXCL10) was more strongly induced by the two testedC. dubliniensis strains (Wu272 and Wu284) compared withC. albicans SC5314 (Fig. 8).

Because the production of IL-17, an important cytokine in hostantifungal defense and a potent activator of neutrophils (32), is alsomediated through the action of the proinflammatory macrophage-derived IL-1b, we further analyzed the IL-1b production. MDMswere coincubated with yeast cells of C. albicans strains (SC5314and ATCC38696) and C. dubliniensis strains (Wu272 and Wu284)for 20 h at a MOI of 4, and IL-1b was measured from the culturesupernatants by ELISA. Both strains of C. albicans triggeredsignificantly higher IL-1b production than did the C. dubliniensisstrains (Fig. 9), which remained in yeast form, compared with thehypha-forming C. albicans (Supplemental Fig. 3).In addition, we have compared the amounts of IL-17A produced

by PBMCs upon stimulation with C. albicans and C. dubliniensis.PBMCs at a concentration of 5 3 106/ml were coincubated with13 104/ml yeast cells of C. albicans strains (SC5314, ATCC38696,ATCC44808, and WO-1) and C. dubliniensis strains (H1, Wu272,Wu284, and VK9603-29) for 7d, and IL-17A was measured fromthe supernatants by ELISA. C. albicans stimulated a higher IL-17A production in PBMCs compared with C. dubliniensis (Fig.10). This assay was also performed with fungal cells stimulatedfor 1 h at 37˚C to produce hyphae. Again, C. albicans cells causedmore IL-17A release than C. dubliniensis cells, and the levels ofIL-17A were increased for both fungal species compared with theyeast cells (Fig. 10).

DiscussionC. albicans is the most virulent species of the genus Candida, andcauses not only superficial but also life-threatening systemicinfections. C. albicans and C. dubliniensis are very closely relatedand share numerous phenotypes; intriguingly, however, systemic

FIGURE 3. Generation of NETs on stimulation

with C. albicans and C. dubliniensis. Neutrophils

were coincubated with GFP-expressing C. albicans

or C. dubliniensis cells (green fluorescence) for 3 h

at 37˚C. NETs were examined at 3630 mag-

nification using propidium iodide to stain the

DNA (red fluorescence). Images represent laser-

scanning microscopy fluorescence, bright field, and

overlay images. Scale bars, 10 mm. Images are

representative of four experiments.

FIGURE 4. C. dubliniensis causes less extracellular

trap formation by neutrophils. The release of NETs

was quantified after 3 h of coincubation with Candida

cells or PMA as a positive control. The fluorescence of

released DNA stained with SYTOX Orange was mea-

sured. C. dubliniensis strains (H1, Wu272, Wu284, and

VK9603-29; white bars) caused less NET release

than the C. albicans strains (SC5314, ATCC38696,

ATCC44808, and WO-1; black bars). Data represent

mean fluorescence intensity values (MFI) + SD of at

least five experiments performed in duplicates with

different cell donors. *p # 0.05, Student t test.

2506 NEUTROPHIL RESPONSES TO C. ALBICANS AND C. DUBLINIENSIS

by guest on February 7, 2016http://w

ww

.jimm

unol.org/D

ownloaded from

C. dubliniensis infections have rarely been observed (2), and thisspecies proved to be less virulent than C. albicans in various in-fection models (4–7). The differences in the interaction of bothfungal species with human immune cells remain largely unknown.Neutrophils play a major role in innate antifungal defense.Therefore, the understanding of neutrophil–Candida interaction isof particular importance. These observations instigated the com-parative analysis of the two species in the context of neutrophilinteraction in this study. By this approach, we unraveled that

C. dubliniensis species specifically induces increased migration,uptake, and antimicrobial effects, but less formation of NETs byneutrophils as compared with C. albicans, thereby providing newinsights into the differential virulence of the two species. Fur-thermore, our data suggest that this differential neutrophil inter-action is, in part, associated with the different capacity of the twoCandida species to form hyphae.Previous studies demonstrated that C. albicans releases a che-

motactic factor that can act through the formyl peptide receptor onneutrophils and induce migration (18, 33). Using the single-cellchemotaxis assay, Geiger et al. (18) showed that also C. dubliniensisreleases chemokinetic and chemotactic factors for human neu-trophils, and that the conditioned media of C. albicans resulted ina lower chemotactic index than did the conditioned supernatant ofC. dubliniensis. In agreement with this, we detected a highermigration of human neutrophils toward all tested isolates of C.dubliniensis compared with isolates of C. albicans (Fig. 1). Thechemoattractant of C. albicans has been identified as a smallpeptide (18, 33), but there is evidence for the involvement of otherfactors in the neutrophil migration toward C. albicans, such assoluble b-glucans (34). Although C. albicans hyphae induce ahigher neutrophil motility than the yeast form (35), in our assays,C. dubliniensis caused a higher migration rate of neutrophilsthan C. albicans (Fig. 1) despite its incapability of forming truehyphae under the tested cell culture conditions, suggesting thatthe chemoattractant produced by C. dubliniensis is not a hypha-specific product.Neutrophils also internalized C. dubliniensis cells more effi-

ciently than cells of C. albicans (Fig. 2). In a previous study,Peltroche-Llacsahuanga et al. (17) found no significant differencein the phagocytosis between different isolates of both fungal

FIGURE 5. Less neutrophil damage is caused by C. dubliniensis. The

concentration of LDH, as a measure of neutrophil damage, was determined

after 20 h of neutrophil-Candida coculture in the supernatants. C. dubliniensis

strains (H1, Wu272, and Wu284; white bars) caused less neutrophil damage

than did the C. albicans strains (SC5314, ATCC38696, and ATCC4408;

black bars). Results were normalized setting the lysis of all neutrophils to

100%. Data represent mean percentage values of relative fluorescence

intensities (% MFI) + SD of at least five experiments performed with

different cell donors. **p # 0.01, Student t test.

Table II. Increased release of ROS, MPO, lactoferrin, and IL-8 from neutrophils stimulated with C.dubliniensis compared with C. albicans

ROS MPO Lactoferrin IL-8

StrainDonorsTested % RFI t Test % Conc. t Test % Conc. t Test % Conc. t Test

C. albicansSC5314 8 100 100 100 100ATCC10261 8 95 6 11 101 6 39 82 6 38 100 6 22ATCC38696 3 93 6 3 45 6 6 * 34 6 2 * 107 6 1 *ATCC44807 3 106 6 3 92 6 13 77 6 19 89 6 5ATCC44808 8 120 6 20 119 6 22 164 6 78 114 6 17DMS1386 3 92 6 1 ** 69 6 9 * 69 6 12 * 88 6 3WO-1 8 66 6 22 80 6 39 108 6 76 104 6 21MA13 3 89 6 5 71 6 13 80 6 13 86 6 4Wu212 3 100 6 6 86 6 3 100 6 30 86 6 6Wu277 3 95 6 5 87 6 11 98 6 6 97 6 3C. dubliniensisH1 8 143 6 24 ** 192 6 26 ** 355 6 146 *** 196 6 25 *H12 8 157 6 23 ** 188 6 18 ** 377 6 168 *** 195 6 35 *Wu272 8 159 6 36 *** 194 6 32 ** 340 6 149 ** 165 6 30 *Wu284 8 157 6 35 ** 209 6 20 ** 387 6 195 ** 176 6 30 *Wu294 3 124 6 11 127 6 12 * 209 6 56 144 6 9 *Wu361 3 128 6 3 ** 133 6 18 * 220 6 47 134 6 4 *Wu367 3 135 6 5 * 140 6 11 * 273 6 38 ** 151 6 14 **Wu370 3 147 6 2 *** 137 6 3 * 249 6 30 ** 123 6 4 **VK9603-29 3 129 6 9 * 144 6 20 * 291 6 106 121 6 2 **VK9603-34 8 138 6 22 169 6 17 ** 309 6 128 *** 183 6 35 *VK9603-54 3 123 6 9 137 6 5 * 230 6 36 ** 129 6 8 *VK9603-63 3 115 6 7 123 6 4 178 6 21 * 116 6 7 *

Neutrophils were coincubated with live yeast cells of the indicated strains at a MOI of 1, or left untreated as a negativecontrol. ROS were measured intracellularly by detecting the fluorescence of ROS-converted fluorescent dye. MPO, lactoferrin,and IL-8 were measured from coculture supernatants by ELISA. Results shown are percentages of relative mean fluorescenceintensities (RFI; for ROS) or concentrations (Conc.; for MPO, lactoferrin, IL-8) 6 SD from at least three experiments withdifferent blood donors. Data were normalized to the value of C. albicans strain SC5314 (set to 100%) after the subtraction of thenegative control.

*p # 0.05, **p # 0.01, ***p # 0.001, Student t test.

The Journal of Immunology 2507

by guest on February 7, 2016http://w

ww

.jimm

unol.org/D

ownloaded from

species. This discrepancy is likely due to the use of whole hepa-rinized blood instead of purified human neutrophils. Phagocytosisof fungi by neutrophils involves complement receptor 3, FcRs, anddectin-1 (36–38). In our serum-free assays, the participation of acomplement-dependent uptake or an Ab-mediated uptake throughFcRs could be excluded. Dectin-1 is a major receptor for Candida(39, 40), recognizing b-glucan components of the fungal cell walland inducing phagocytosis, defense processes, and cytokine pro-duction by neutrophils, but its important role in yeast recognitionis contradictory (41). Further studies are needed to analyzewhether species-specific differences in the expression or avail-ability of b-glucan might explain a more efficient uptake of C.dubliniensis compared with C. albicans.The release of NETs is thought to be an immune defense strategy

that prevents the distribution of pathogens, trapping them intoa milieu of highly concentrated antimicrobial substances (42–44).NET formation is appreciated as a killing mechanism againstdifferent morphotypes of fungi (14, 45), because neutrophils thatare unable to produce NETs lack the ability to inhibit fungal in-fection efficiently (43, 46). However, the ability of NETs to di-rectly kill pathogens such as C. albicans is not unequivocallyestablished (47). Even though NETosis requires autophagy and theactivation of the respiratory burst machinery involving MPO andthe production of ROS (46, 48), our results show that despite thehigher MPO and ROS production by neutrophils stimulated withC. dubliniensis, these neutrophils do not undergo NETosis so ef-

ficiently as they do when in contact with C. albicans (Figs. 3, 4).Moreover, Yost et al. (49) demonstrated that ROS are necessarybut not sufficient for the induction of NETs, but the exact mech-anism of NETosis and all factors involved are yet unknown. Onemight speculate that because C. dubliniensis is more efficientlyphagocytosed than C. albicans, the formation of NETs would bedispensable; in contrast, C. albicans is also able to escape from theneutrophils by producing hyphae, and the large hyphae of C.albicans can be prevented from spreading when trapped in theNETs (14).Candida species can cause cell and tissue damage during in-

teraction with the host. Although C. dubliniensis is able to damagehost cells (6), our results show that isolates of C. dubliniensis donot cause as severe damage to human neutrophils in vitro as C.albicans strains (Fig. 5). These data are in line with earlier reportsshowing that the ability of C. dubliniensis to disseminate and tocause cell damage in vitro and in vivo is reduced (4–7). This re-duction in host cell damage is likely associated, in part, with thereduced hyphal formation of C. dubliniensis, because the hyphalmorphology serves the penetration of the host cells through me-chanical force of the hyphal tip (50). We have observed that underthe used cell culture conditions, C. dubliniensis strains were notable to efficiently form hyphae, and even after induction of hyphaformation, this morphology was not maintained over time (Sup-plemental Figs. 2, 3). Thus, both the ability to switch between thetwo morphological forms and to maintain hyphal growth seems tobe pivotal for Candida cells to adapt to different host niches andsurvive in the host (51). In addition, the hyphal morphology isassociated with the production of several virulence factors, such asproteases and adhesins, which contribute to the virulence in thehost (51). Thus, the observed differences between C. dubliniensishyphae compared with C. albicans hyphae in inflicting damage toneutrophils could be, in part, also due to differences in some of thevirulence factors between these fungal strains.Upon the encounter with the fungus, neutrophils use a set of

killing mechanisms and can further activate and recruit other im-mune cells by producing cytokines. Neutrophils produce micro-bicidal amounts of ROS such as superoxide, hydrogen peroxide,and hypochlorous acid, which damage fungi (13, 52). ROS pro-duction is catalyzed by the NADPH system and subsequently byMPO, a component of the azurophilic granules. In addition to theoxidative mechanisms, neutrophils can fight bacteria and fungithrough a set of antimicrobial substances such as lactoferrin,defensins, and cathelicidins. We found that in comparison withisolates of C. albicans, the studied C. dubliniensis isolates stim-ulated increased levels of ROS, MPO, and lactoferrin by human

FIGURE 6. Hyphae of C. albicans augment the release of ROS to a higher extent than hyphal cells of C. dubliniensis. Neutrophils were coincubated with

live yeast and hyphal cells at a MOI of 1, or left untreated as a negative control. ROS (A) were measured intracellularly by detecting the fluorescence of

ROS-converted fluorescent dye. Lactoferrin (B) was measured from coculture supernatants by ELISA. Results shown are percentages of relative mean

fluorescence intensities (ROS) or relative concentrations (lactoferrin) 6 SD from at least five experiments with different blood donors. Data were nor-

malized to the value of C. albicans strain SC5314 (set to 100%) after the subtraction of the negative control. *p# 0.05, **p# 0.01, Student t test. n.s., Not

significant.

FIGURE 7. Stimulation of lactoferrin release from neutrophils by C.

dubliniensis is dependent on the host cell–Candida contact. Neutrophils

were coincubated with live yeast cells at a MOI of 1 either in a direct

contact (black bars) or neutrophils were separated from the Candida cells

through a porous membrane (striped bars). Lactoferrin was measured from

coculture supernatants by ELISA. Results shown are lactoferrin concen-

trations, and data represent means + SD from three experiments with

different cell donors.

2508 NEUTROPHIL RESPONSES TO C. ALBICANS AND C. DUBLINIENSIS

by guest on February 7, 2016http://w

ww

.jimm

unol.org/D

ownloaded from

neutrophils (Table II). These data point to an early and morepronounced activation of neutrophil antifungal activity by C.dubliniensis. These observations are in agreement with the studyof Vilela et al. (7) where a relatively strong inflammatory reactionof mice infected with C. dubliniensis was seen compared withthose infected with C. albicans. Furthermore, our results indicatethat the strong stimulation of lactoferrin production in neutrophilsby C. dubliniensis requires the fungus–host cell contact (Fig. 7).This suggests that either C. dubliniensis more strongly activatesneutrophils, for example, because of surface-expressed molecularpatterns engaged with host cell receptors, or this species lacks certainfactor(s) that counteract or suppress the reaction of the host cells.We have also investigated the release of fungal immunity-related

cytokines upon encounter with both Candida species. IL-8 is re-leased by neutrophils and several other cell types in response tofungal or bacterial infection and triggers the mobilization of in-flammatory cells into the site of infection (52). Similar to theantimicrobial substances, the amount of IL-8 released from neu-trophils was also significantly higher when coincubated with C.dubliniensis than C. albicans. In addition, several differences inthe response of macrophages to the two related fungal specieswere observed. Most molecules detected with the used array wereless induced by C. dubliniensis, such as G-CSF, GM-CSF, IL-1a,IL-1b, IL-1ra, IL-10, IL-16, serine protease inhibitor-1, TNF-a,and soluble triggering receptor expressed on myeloid cell, butIFN-g–induced protein 10 was enhanced, indicating important

differences in the chemokine and cytokine pattern induced by thetwo Candida species. Because the production of several of thesecytokines and chemokines was shown to be induced by dectin-1– and/or dectin-2–mediated signaling (53, 54), the differentialcytokine and chemokine induction by C. albicans and C. dubliniensismay indicate their differential recognition via these host cellpattern recognition receptors. Similarly, it has been shown that themore invasive C. albicans induces considerably higher productionof IL-1a, IL-8 (20), G-CSF, GM-CSF, and IL-6 (21) in recon-stituted human epithelium than C. dubliniensis.Many studies have demonstrated that IL-17 is a key cytokine in

the host defense against C. albicans infection (55–58). This cy-tokine is mainly produced by Th17 cells, which develop in thepresence of IL-1b, IL-6, and TGF-b, whereas IL-23 is importantfor the expansion, maintenance, and function of Th17 cells (32).C. albicans induces IL-17 production dependent on macrophagemannose receptor, and this IL-17 production is enhanced by theTLR2/dectin-1 activation pathway (59). Because the hyphal formof C. albicans is recognized preferentially by TLR2 on mononu-clear cells, in contrast with the yeast cells recognized mainly byTLR4 (60), the morphological plasticity of Candida cells might bean important aspect in regulating IL-17 production. Furthermore,IL-1b is produced after the stimulation of the inflammasome (61),which also responds to the morphology shift of C. albicans and iscrucial in the secretion of IL-17. Indeed, it has been shown that C.albicans hyphae specifically induce inflammasome activation in

FIGURE 8. Differential macrophage cytokine response to C. dubliniensis and C. albicans. MDMs were coincubated with live yeast cells of C. albicans

(strain SC5314) and C. dubliniensis (strains Wu272 and Wu284) for 24 h at a MOI of 1, or left untreated as a negative control. Cytokine levels were

measured from coculture supernatants using a cytokine array. Results shown represent means from two experiments with different blood donors. Data were

normalized to the value of the arrays’ positive control (set to 100% after subtracting the background). GRO-a, Growth regulated oncogene-a; IP-10, IFN-g–

induced protein 10; MIF, macrophage migration inhibitory factor; Serpin-1, serine protease inhibitor-1; sTREM, soluble triggering receptor expressed on

myeloid cells.

FIGURE 9. C. dubliniensis triggers less IL-1b production by human

macrophages than C. albicans. MDMs were coincubated with live yeast

cells for 20 h at a MOI of 4, or left untreated as a negative control. IL-1b

was measured from coculture supernatants by ELISA. Results shown are

relative IL-1b concentrations + SD from at least five experiments with

different blood donors. Data were normalized to the value of C. albicans

strain SC5314 (set to 100%). **p # 0.01, Student t test.

FIGURE 10. PBMCs produce less IL-17A when stimulated with C.

dubliniensis compared with stimulation with C. albicans. PBMCs were

coincubated with live yeast and hyphal cells for 7 d at a MOI of 0.002, or

left untreated as a negative control. IL-17A was measured from coculture

supernatants by ELISA. Results shown are relative IL-17A concentrations

6 SD from at least six experiments with different blood donors. Data were

normalized to the value of C. albicans strain SC5314 (set to 100%). *p #

0.05, ***p # 0.001, Student t test. n.s., Not significant.

The Journal of Immunology 2509

by guest on February 7, 2016http://w

ww

.jimm

unol.org/D

ownloaded from

macrophages in a partly dectin-1–dependent manner, resulting inenhanced IL-1b secretion and induction of IL-17 production (53).In this article, we demonstrate that the production of IL-1b isreduced when MDMs are stimulated with C. dubliniensis incomparison with C. albicans (Fig. 9), supporting the importantrole of the morphological conversion. Furthermore, the release ofIL-17 from PBMCs is also reduced for C. dubliniensis whencompared with C. albicans (Fig. 10). These differences are likelydue to the fact that C. albicans readily formed hyphae under theused culture conditions, whereas C. dubliniensis did not. IL-17plays an important role in the recruitment of neutrophils (62).Although C. dubliniensis induced less IL-17A production in ourassays, measured after 7 d, it did enhance the migration of neu-trophils directly and the production of IL-8, which also supportsneutrophil migration. The induction of these immediate and earlyneutrophil responses, as well as a more efficient phagocytosis, andthe limited induction of later responses, including production ofinflammatory cytokines and IL-17, suggest that partly because ofits restricted capacity to form hyphae, C. dubliniensis poses lessdanger to the host organism than C. albicans, which is reflected inthe attenuated late host response.Key virulence factors of Candida include the ability to switch

between yeast and hypha morphology enabling the fungus in itsspreading and invasion of the human host (50, 63), the productionof enzymes such as secreted aspartyl proteases and phospholipases(64), and the expression of adhesins (65). Some of these virulencefactors, found in C. albicans, however, are lacking or are reducedin C. dubliniensis (9, 10, 66). Aside from C. albicans, C. dubliniensisis the only Candida species that has the capacity to produce truehyphae (3), but with far less efficiency and under fewer conditions.In our experiments, C. dubliniensis did not form filaments duringcoincubation with neutrophils in cell culture medium (Supple-mental Fig. 3).We and others previously showed that neutrophils respond more

strongly to C. albicans hyphae than to yeast cells (30, 35). Inagreement with this, we detected an enhanced production andrelease of ROS and lactoferrin for neutrophils coincubated withhyphal cells of various C. albicans isolates in comparison withyeast cells (Fig. 6). However, in case of the neutrophil challengewith both morphotypes of C. dubliniensis, the levels of releasedROS and lactoferrin remained almost the same. When coincu-bating neutrophils or PBMCs with fungal cells that were inducedfor 1 h to form hyphae, increased amounts of ROS, lactoferrin, andIL-17A were detected for both species. Nevertheless, hyphae-induced ROS, lactoferrin, and IL-17A production for isolatesof C. dubliniensis did not reach the levels of those induced byC. albicans hyphae. This could be because of the fact thatC. dubliniensis does not persist in the state of hyphal form whenintroduced to the nutrition-rich environment (21), as we also ob-served during coincubation with neutrophils (Supplemental Fig. 2).The lack of the filamentation in C. dubliniensis is supported

by expression of the NRG1 repressor and suppression of UME6induction in a nutrient-dependent manner (11, 67). Thus, fila-mentation of C. dubliniensis can be increased after deletion of theNRG1 gene, which leads to an improved invasion of human epi-thelium and survival in macrophage cocultures in vitro (67), or ina nutrient-poor environment (11). The role of hypha production byC. dubliniensis in the interaction with host immune cells could befurther studied using such genetically manipulated fungal cells,although the conclusions would be limited because of the multipleeffects of NRG1 in addition to influencing filamentous growth.In conclusion, our data indicate that, in comparison with C.

albicans, early responses such as neutrophil migration, phagocy-tosis, and the release of antimicrobial substances are increased

when encountering C. dubliniensis. Late responses, such as NETosisand the release of cytokines by mononuclear cells, including IL-1band IL-17A involved in antifungal immunity, are reduced, whichmight indicate a partly dispensable late response because of theearly neutrophil activation and fungal clearance. Future studiesshould identify the fungal and host factors underlying this differ-ential neutrophil response.

AcknowledgmentsWe thank Joachim Morschhauser for providing C. albicans and C. dublin-

iensis clinical isolates and C. albicans strain SCADH1G4A, as well as

plasmids pADH1E1 and pNIM1. We also thank Toni Kaulfuß and

Christina Taubert for technical assistance, and the Department of In-

fection Biology (Hans Knoll Institute, Jena, Germany) for access to

their microscope.

DisclosuresThe authors have no financial conflicts of interest.

References1. Sullivan, D. J., G. P. Moran, E. Pinjon, A. Al-Mosaid, C. Stokes, C. Vaughan,

and D. C. Coleman. 2004. Comparison of the epidemiology, drug resistancemechanisms, and virulence of Candida dubliniensis and Candida albicans.FEMS Yeast Res. 4: 369–376.

2. Sullivan, D. J., T. J. Westerneng, K. A. Haynes, D. E. Bennett, andD. C. Coleman. 1995. Candida dubliniensis sp. nov.: phenotypic and molecularcharacterization of a novel species associated with oral candidosis in HIV-infected individuals. Microbiology 141: 1507–1521.

3. Staib, P., and J. Morschhauser. 2007. Chlamydospore formation in Candidaalbicans and Candida dubliniensis—an enigmatic developmental programme.Mycoses 50: 1–12.

4. Koga-Ito, C. Y., E. Y. Komiyama, C. A. Martins, T. C. Vasconcellos, A. O. Jorge,Y. R. Carvalho, R. F. do Prado, and I. Balducci. 2011. Experimental systemicvirulence of oral Candida dubliniensis isolates in comparison with Candidaalbicans, Candida tropicalis and Candida krusei. Mycoses 54: e278–e285.

5. Jayatilake, J. A. M. S., Y. H. Samaranayake, and L. P. Samaranayake. 2008. Acomparative study of candidal invasion in rabbit tongue mucosal explants andreconstituted human oral epithelium. Mycopathologia 165: 373–380.

6. Stokes, C., G. P. Moran, M. J. Spiering, G. T. Cole, D. C. Coleman, andD. J. Sullivan. 2007. Lower filamentation rates of Candida dubliniensis con-tribute to its lower virulence in comparison with Candida albicans. FungalGenet. Biol. 44: 920–931.

7. Vilela, M. M. S., K. Kamei, A. Sano, R. Tanaka, J. Uno, I. Takahashi, J. Ito,K. Yarita, and M. Miyaji. 2002. Pathogenicity and virulence of Candida dubliniensis:comparison with C. albicans. Med. Mycol. 40: 249–257.

8. Gilfillan, G. D., D. J. Sullivan, K. Haynes, T. Parkinson, D. C. Coleman, andN. A. Gow. 1998. Candida dubliniensis: phylogeny and putative virulence fac-tors. Microbiology 144: 829–838.

9. Moran, G., C. Stokes, S. Thewes, B. Hube, D. C. Coleman, and D. Sullivan.2004. Comparative genomics using Candida albicans DNA microarrays revealsabsence and divergence of virulence-associated genes in Candida dubliniensis.Microbiology 150: 3363–3382.

10. Jackson, A. P., J. A. Gamble, T. Yeomans, G. P. Moran, D. Saunders, D. Harris,M. Aslett, J. F. Barrell, G. Butler, F. Citiulo, et al. 2009. Comparative genomicsof the fungal pathogens Candida dubliniensis and Candida albicans. GenomeRes. 19: 2231–2244.

11. O’Connor, L., N. Caplice, D. C. Coleman, D. J. Sullivan, and G. P. Moran. 2010.Differential filamentation of Candida albicans and Candida dubliniensis Is gov-erned by nutrient regulation of UME6 expression. Eukaryot. Cell 9: 1383–1397.

12. Nordenfelt, P., and H. Tapper. 2011. Phagosome dynamics during phagocytosisby neutrophils. J. Leukoc. Biol. 90: 271–284.

13. Robinson, J. M. 2009. Phagocytic leukocytes and reactive oxygen species.Histochem. Cell Biol. 131: 465–469.

14. Urban, C. F., U. Reichard, V. Brinkmann, and A. Zychlinsky. 2006. Neutrophilextracellular traps capture and kill Candida albicans yeast and hyphal forms.Cell. Microbiol. 8: 668–676.

15. Netea, M. G., G. D. Brown, B. J. Kullberg, and N. A. R. Gow. 2008. An inte-grated model of the recognition of Candida albicans by the innate immunesystem. Nat. Rev. Microbiol. 6: 67–78.

16. Silva, M. T. 2010. When two is better than one: macrophages and neutrophilswork in concert in innate immunity as complementary and cooperative partnersof a myeloid phagocyte system. J. Leukoc. Biol. 87: 93–106.

17. Peltroche-Llacsahuanga, H., N. Schnitzler, S. Schmidt, K. Tintelnot,R. Lutticken, and G. Haase. 2000. Phagocytosis, oxidative burst, and killingof Candida dubliniensis and Candida albicans by human neutrophils. FEMSMicrobiol. Lett. 191: 151–155.

18. Geiger, J., D. Wessels, S. R. Lockhart, and D. R. Soll. 2004. Release of a potentpolymorphonuclear leukocyte chemoattractant is regulated by white-opaqueswitching in Candida albicans. Infect. Immun. 72: 667–677.

2510 NEUTROPHIL RESPONSES TO C. ALBICANS AND C. DUBLINIENSIS

by guest on February 7, 2016http://w

ww

.jimm

unol.org/D

ownloaded from

19. Jayatilake, J. A. M. S., Y. H. Samaranayake, L. K. Cheung, and L. P. Samaranayake.2006. Quantitative evaluation of tissue invasion by wild type, hyphal and SAPmutants of Candida albicans, and non-albicans Candida species in reconstitutedhuman oral epithelium. J. Oral Pathol. Med. 35: 484–491.

20. Jayatilake, J. A. M. S., L. P. Samaranayake, Q. Lu, and L. J. Jin. 2007. IL-1alpha,IL-1ra and IL-8 are differentially induced by Candida in experimental oralcandidiasis. Oral Dis. 13: 426–433.

21. Moyes, D. L., C. Murciano, M. Runglall, A. Kohli, A. Islam, and J. R. Naglik.2012. Activation of MAPK/c-Fos induced responses in oral epithelial cells isspecific to Candida albicans and Candida dubliniensis hyphae. Med. Microbiol.Immunol. (Berl.) 201: 93–101.

22. Gillum, A. M., E. Y. Tsay, and D. R. Kirsch. 1984. Isolation of the Candidaalbicans gene for orotidine-59-phosphate decarboxylase by complementation ofS. cerevisiae ura3 and E. coli pyrF mutations. Mol. Gen. Genet. 198: 179–182.

23. Slutsky, B., M. Staebell, J. Anderson, L. Risen, M. Pfaller, and D. R. Soll. 1987.“White-opaque transition”: a second high-frequency switching system in Can-dida albicans. J. Bacteriol. 169: 189–197.

24. Staib, P., and J. Morschhauser. 1999. Chlamydospore formation on Staib agar asa species-specific characteristic of Candida dubliniensis. Mycoses 42: 521–524.

25. Morschhauser, J., M. Ruhnke, S. Michel, and J. Hacker. 1999. Identification ofCARE-2-negative Candida albicans isolates as Candida dubliniensis. Mycoses42: 29–32.

26. Dabas, N., and J. Morschhauser. 2007. Control of ammonium permease ex-pression and filamentous growth by the GATA transcription factors GLN3 andGAT1 in Candida albicans. Eukaryot. Cell 6: 875–888.

27. Reuss, O., and J. Morschhauser. 2006. A family of oligopeptide transporters isrequired for growth of Candida albicans on proteins.Mol. Microbiol. 60: 795–812.

28. Park, Y.-N., and J. Morschhauser. 2005. Tetracycline-inducible gene expressionand gene deletion in Candida albicans. Eukaryot. Cell 4: 1328–1342.

29. Reuss, O., A. Vik, R. Kolter, and J. Morschhauser. 2004. The SAT1 flipper, anoptimized tool for gene disruption in Candida albicans. Gene 341: 119–127.

30. Losse, J., E. Svobodova, A. Heyken, B. Hube, P. F. Zipfel, and M. Jozsi. 2011.Role of pH-regulated antigen 1 of Candida albicans in the fungal recognitionand antifungal response of human neutrophils. Mol. Immunol. 48: 2135–2143.

31. Sudbery, P. E. 2011. Growth of Candida albicans hyphae. Nat. Rev. Microbiol. 9:737–748.

32. Gaffen, S. L., N. Hernandez-Santos, and A. C. Peterson. 2011. IL-17 signaling inhost defense against Candida albicans. Immunol. Res. 50: 181–187.

33. Edens, H. A., C. A. Parkos, T. W. Liang, A. J. Jesaitis, J. E. Cutler, andH. M. Miettinen. 1999. Non-serum-dependent chemotactic factors produced byCandida albicans stimulate chemotaxis by binding to the formyl peptide re-ceptor on neutrophils and to an unknown receptor on macrophages. Infect.Immun. 67: 1063–1071.

34. Sato, T., K. Iwabuchi, I. Nagaoka, Y. Adachi, N. Ohno, H. Tamura, K. Seyama,Y. Fukuchi, H. Nakayama, F. Yoshizaki, et al. 2006. Induction of human neu-trophil chemotaxis by Candida albicans-derived beta-1,6-long glycoside side-chain-branched beta-glucan. J. Leukoc. Biol. 80: 204–211.

35. Wozniok, I., A. Hornbach, C. Schmitt, M. Frosch, H. Einsele, B. Hube, J. Loffler,and O. Kurzai. 2008. Induction of ERK-kinase signalling triggers morphotype-specific killing of Candida albicans filaments by human neutrophils. Cell.Microbiol. 10: 807–820.

36. Mayadas, T. N., and X. Cullere. 2005. Neutrophil beta2 integrins: moderators oflife or death decisions. Trends Immunol. 26: 388–395.

37. Wellington, M., J. M. Bliss, and C. G. Haidaris. 2003. Enhanced phagocytosis ofCandida species mediated by opsonization with a recombinant human antibodysingle-chain variable fragment. Infect. Immun. 71: 7228–7231.

38. Kennedy, A. D., J. A. Willment, D. W. Dorward, D. L. Williams, G. D. Brown,and F. R. DeLeo. 2007. Dectin-1 promotes fungicidal activity of human neu-trophils. Eur. J. Immunol. 37: 467–478.

39. Taylor, P. R., S. V. Tsoni, J. A. Willment, K. M. Dennehy, M. Rosas, H. Findon,K. Haynes, C. Steele, M. Botto, S. Gordon, and G. D. Brown. 2007. Dectin-1 isrequired for beta-glucan recognition and control of fungal infection. Nat.Immunol. 8: 31–38.

40. Drummond, R. A., and G. D. Brown. 2011. The role of Dectin-1 in the hostdefence against fungal infections. Curr. Opin. Microbiol. 14: 392–399.

41. van Bruggen, R., A. Drewniak, M. Jansen, M. van Houdt, D. Roos, H. Chapel,A. J. Verhoeven, and T. W. Kuijpers. 2009. Complement receptor 3, not Dectin-1, is the major receptor on human neutrophils for beta-glucan-bearing particles.Mol. Immunol. 47: 575–581.

42. Brinkmann, V., U. Reichard, C. Goosmann, B. Fauler, Y. Uhlemann, D. S. Weiss,Y. Weinrauch, and A. Zychlinsky. 2004. Neutrophil extracellular traps killbacteria. Science 303: 1532–1535.

43. Fuchs, T. A., U. Abed, C. Goosmann, R. Hurwitz, I. Schulze, V. Wahn,Y. Weinrauch, V. Brinkmann, and A. Zychlinsky. 2007. Novel cell death programleads to neutrophil extracellular traps. J. Cell Biol. 176: 231–241.

44. Papayannopoulos, V., K. D. Metzler, A. Hakkim, and A. Zychlinsky. 2010.Neutrophil elastase and myeloperoxidase regulate the formation of neutrophilextracellular traps. J. Cell Biol. 191: 677–691.

45. Bruns, S., O. Kniemeyer, M. Hasenberg, V. Aimanianda, S. Nietzsche,A. Thywissen, A. Jeron, J.-P. Latge, A. A. Brakhage, and M. Gunzer. 2010.Production of extracellular traps against Aspergillus fumigatus in vitro and in

infected lung tissue is dependent on invading neutrophils and influenced byhydrophobin RodA. PLoS Pathog. 6: e1000873.

46. Metzler, K. D., T. A. Fuchs, W. M. Nauseef, D. Reumaux, J. Roesler, I. Schulze,V. Wahn, V. Papayannopoulos, and A. Zychlinsky. 2011. Myeloperoxidase isrequired for neutrophil extracellular trap formation: implications for innateimmunity. Blood 117: 953–959.

47. Menegazzi, R., E. Decleva, and P. Dri. 2012. Killing by neutrophil extracellulartraps: fact or folklore? Blood 119: 1214–1216.

48. Remijsen, Q., T. Vanden Berghe, E. Wirawan, B. Asselbergh, E. Parthoens,R. De Rycke, S. Noppen, M. Delforge, J. Willems, and P. Vandenabeele. 2011.Neutrophil extracellular trap cell death requires both autophagy and superoxidegeneration. Cell Res. 21: 290–304.

49. Yost, C. C., M. J. Cody, E. S. Harris, N. L. Thornton, A. M. McInturff,M. L. Martinez, N. B. Chandler, C. K. Rodesch, K. H. Albertine, C. A. Petti,et al. 2009. Impaired neutrophil extracellular trap (NET) formation: a novelinnate immune deficiency of human neonates. Blood 113: 6419–6427.

50. Brand, A. 2012. Hyphal growth in human fungal pathogens and its role in vir-ulence. Int. J. Microbiol. 2012: 517529.

51. Kumamoto, C. A., and M. D. Vinces. 2005. Contributions of hyphae and hypha-co-regulated genes to Candida albicans virulence. Cell. Microbiol. 7: 1546–1554.

52. Romani, L. 2004. Immunity to fungal infections. Nat. Rev. Immunol. 4: 1–23.53. Cheng, S.-C., F. L. van de Veerdonk, M. Lenardon, M. Stoffels, T. Plantinga,

S. Smeekens, L. Rizzetto, L. Mukaremera, K. Preechasuth, D. Cavalieri, et al.2011. The dectin-1/inflammasome pathway is responsible for the induction ofprotective T-helper 17 responses that discriminate between yeasts and hyphae ofCandida albicans. J. Leukoc. Biol. 90: 357–366.

54. Sato, K., X. L. Yang, T. Yudate, J. S. Chung, J. Wu, K. Luby-Phelps,R. P. Kimberly, D. Underhill, P. D. Cruz, Jr., and K. Ariizumi. 2006. Dectin-2 isa pattern recognition receptor for fungi that couples with the Fc receptor gammachain to induce innate immune responses. J. Biol. Chem. 281: 38854–38866.

55. Huang, W., L. Na, P. L. Fidel, and P. Schwarzenberger. 2004. Requirement ofinterleukin-17A for systemic anti-Candida albicans host defense in mice. J.Infect. Dis. 190: 624–631.

56. Eyerich, K., S. Foerster, S. Rombold, H.-P. Seidl, H. Behrendt, H. Hofmann,J. Ring, and C. Traidl-Hoffmann. 2008. Patients with chronic mucocutaneouscandidiasis exhibit reduced production of Th17-associated cytokines IL-17 andIL-22. J. Invest. Dermatol. 128: 2640–2645.

57. Conti, H. R., F. Shen, N. Nayyar, E. Stocum, J. N. Sun, M. J. Lindemann,A. W. Ho, J. H. Hai, J. J. Yu, J. W. Jung, et al. 2009. Th17 cells and IL-17 re-ceptor signaling are essential for mucosal host defense against oral candidiasis.J. Exp. Med. 206: 299–311.

58. Zelante, T., A. De Luca, C. D’Angelo, S. Moretti, and L. Romani. 2009. IL-17/Th17 in anti-fungal immunity: what’s new? Eur. J. Immunol. 39: 645–648.

59. van de Veerdonk, F. L., R. J. Marijnissen, B. J. Kullberg, H. J. P. M. Koenen, S.-C. Cheng, I. Joosten, W. B. van den Berg, D. L. Williams, J. W. M. van der Meer,L. A. B. Joosten, and M. G. Netea. 2009. The macrophage mannose receptorinduces IL-17 in response to Candida albicans. Cell Host Microbe 5: 329–340.

60. van der Graaf, C. A. A., M. G. Netea, I. Verschueren, J. W. M. van der Meer, andB. J. Kullberg. 2005. Differential cytokine production and Toll-like receptorsignaling pathways by Candida albicans blastoconidia and hyphae. Infect.Immun. 73: 7458–7464.

61. Gross, O., H. Poeck, M. Bscheider, C. Dostert, N. Hannesschlager, S. Endres,G. Hartmann, A. Tardivel, E. Schweighoffer, V. Tybulewicz, et al. 2009. Sykkinase signalling couples to the Nlrp3 inflammasome for anti-fungal host de-fence. Nature 459: 433–436.

62. Laan, M., Z. H. Cui, H. Hoshino, J. Lotvall, M. Sjostrand, D. C. Gruenert,B. E. Skoogh, and A. Linden. 1999. Neutrophil recruitment by human IL-17 viaC-X-C chemokine release in the airways. J. Immunol. 162: 2347–2352.

63. Saville, S. P., A. L. Lazzell, C. Monteagudo, and J. L. Lopez-Ribot. 2003.Engineered control of cell morphology in vivo reveals distinct roles for yeast andfilamentous forms of Candida albicans during infection. Eukaryot. Cell 2: 1053–1060.

64. Naglik, J. R., C. A. Rodgers, P. J. Shirlaw, J. L. Dobbie, L. L. Fernandes-Naglik,D. Greenspan, N. Agabian, and S. J. Challacombe. 2003. Differential expressionof Candida albicans secreted aspartyl proteinase and phospholipase B genes inhumans correlates with active oral and vaginal infections. J. Infect. Dis. 188:469–479.

65. Wachtler, B., D. Wilson, K. Haedicke, F. Dalle, and B. Hube. 2011. From at-tachment to damage: defined genes of Candida albicans mediate adhesion, in-vasion and damage during interaction with oral epithelial cells. PLoS ONE 6:e17046.

66. Spiering, M. J., G. P. Moran, M. Chauvel, D. M. Maccallum, J. Higgins,K. Hokamp, T. Yeomans, C. d’Enfert, D. C. Coleman, and D. J. Sullivan. 2010.Comparative transcript profiling of Candida albicans and Candida dubliniensisidentifies SFL2, a C. albicans gene required for virulence in a reconstitutedepithelial infection model. Eukaryot. Cell 9: 251–265.

67. Moran, G. P., D. M. MacCallum, M. J. Spiering, D. C. Coleman, andD. J. Sullivan. 2007. Differential regulation of the transcriptional repressorNRG1 accounts for altered host-cell interactions in Candida albicans andCandida dubliniensis. Mol. Microbiol. 66: 915–929.

The Journal of Immunology 2511

by guest on February 7, 2016http://w

ww

.jimm

unol.org/D

ownloaded from