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Antifungal Activity of Plasmacytoid Dendritic Cells against Cryptococcus neoformans In Vitro Requires Expression of Dectin-3 (CLEC4D) and Reactive Oxygen Species Camaron R. Hole,a,b Chrissy M. Leopold Wager,a,b Andrew S. Mendiola,a,b Karen L. Wozniak,a,b Althea Campuzano,a,b Xin Lin,c,d Floyd L. Wormley, Jr.a,b Department of Biology, The University of Texas at San Antonio, San Antonio, Texas, USAa; South Texas Center for Emerging Infectious Diseases, The University of Texas at San Antonio, San Antonio, Texas, USAb; Department of Molecular and Cellular Oncology, The University of Texas MD Anderson Cancer Center, Houston, Texas, USAc; Cancer Biology Program, The University of Texas Graduate School of Biomedical Sciences, Houston, Texas, USAd

Conventional dendritic cells (cDCs) are critical for protection against pulmonary infection with the opportunistic fungal pathogen Cryptococcus neoformans; however, the role of plasmacytoid dendritic cells (pDCs) is unknown. We show for the first time that murine pDCs have direct activity against C. neoformans via reactive oxygen species (ROS), a mechanism different from that employed to control Aspergillus fumigatus infections. The anticryptococcal activity of murine pDCs is independent of opsonization but appears to require the C-type lectin receptor Dectin-3, a receptor not previously evaluated during cryptococcal infections. Human pDCs can also inhibit cryptococcal growth by a mechanism similar to that of murine pDCs. Experimental pulmonary infection of mice with a C. neoformans strain that induces protective immunity demonstrated that recruitment of pDCs to the lungs is CXCR3 dependent. Taken together, our results show that pDCs inhibit C. neoformans growth in vitro via the production of ROS and that Dectin-3 is required for optimal growth-inhibitory activity.

C

ryptococcus neoformans, the predominant etiological agent of cryptococcosis, is the most common disseminated fungal pathogen in AIDS patients and remains the third most common cause of invasive fungal infection in organ transplant recipients (1, 2). Approximately 1 million cases of cryptococcal meningitis occur each year, resulting in ⬎620,000 deaths (3). C. neoformans is typically believed to cause cryptococcosis only in immunocompromised individuals, but increasing evidence shows that the disease occurs in immunocompetent hosts as well (4–6). At least one-third of patients with cryptococcal meningitis who receive appropriate therapy will undergo mycologic and/or clinical failure (7–10), illustrating the urgent need for more-effective drugs, immune therapies, and/or vaccines to combat cryptococcosis. Experimental murine studies using a C. neoformans strain engineered to express gamma interferon (IFN-␥), designated H99␥, have shown that pulmonary infection with H99␥ results in the induction of Th1-type cytokine responses in the lung, lymphocyte recruitment, reduced fungal burdens, and complete protection against an otherwise lethal challenge with C. neoformans (11–13). Cell-mediated immunity (CMI) by Th1-type CD4⫹ T cells is important for the induction of protective immunity against C. neoformans (12, 14). Dendritic cells (DCs) are necessary to drive Th1-type CD4⫹ T cell responses and the resulting protective immune responses against C. neoformans. Two main subsets of DCs are found in the lungs: conventional DCs (cDCs) and plasmacytoid DCs (pDCs). pDCs are a rare cell type, comprising 0.2% to 0.8% of the total population of peripheral blood mononuclear cells (PBMCs) in circulation (15). They are generally considered to be important for the protective immune response to viral infections, producing copious amounts of antiviral type I interferons. Upon stimulation, pDCs produce more type I interferons than any other cell type, as much as 1,000fold more, which is why they are also known as type I interferon-

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producing cells (IPC) (16, 17). While a fair amount is known about the roles that pDCs play during the immune response to viral infections, there is increasing evidence that they are also important in responding to bacterial infections, including Chlamydia pneumoniae (18), Legionella pneumophila (19), and Klebsiella pneumoniae (20) infections. However, little is known about the role of pDCs in response to fungal infections. Ramirez-Ortiz et al. have shown that pDCs are indispensable for protection against Aspergillus fumigatus infections in mice (21). pDCs mediate direct antifungal activity against A. fumigatus hyphae and conidia via the release of calprotectin, and depletion of pDCs in infected mice resulted in a significant decrease in survival from that of control mice with intact pDC numbers (21). Nonetheless, the role of pDCs during the protective immune response to cryptococcal infection has not been investigated. Our studies showed that pDCs have direct anticryptococcal activity via the production of reactive oxygen species (ROS), a mechanism different from that employed by pDCs to control pulmonary A. fumigatus infections. Opsonization was not required for the uptake of C. neoformans, and the inhibition of its growth, by pDCs. However, inhibition of C. neoformans growth by pDCs

Received 4 February 2016 Returned for modification 23 February 2016 Accepted 9 June 2016 Accepted manuscript posted online 20 June 2016 Citation Hole CR, Leopold Wager CM, Mendiola AS, Wozniak KL, Campuzano A, Lin X, Wormley FL, Jr. 2016. Antifungal activity of plasmacytoid dendritic cells against Cryptococcus neoformans in vitro requires expression of Dectin-3 (CLEC4D) and reactive oxygen species. Infect Immun 84:2493–2504. doi:10.1128/IAI.00103-16. Editor: L. Pirofski, Albert Einstein College of Medicine Address correspondence to Floyd L. Wormley, Jr., [email protected]. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

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appeared to require expression of the C-type lectin pattern recognition receptor (PRR) Dectin-3. pDC recruitment to the lungs appeared to be dependent on the chemokine receptor CXCR3. Taken together, our data demonstrate a novel mechanism by which pDCs mediate antifungal activity against C. neoformans. MATERIALS AND METHODS Mice. Female BALB/c (H-2d) mice (National Cancer Institute/Charles River Laboratories, Boston, MA, and The Jackson Laboratory, Bar Harbor, ME), inducible nitric oxide synthase (iNOS) knockout (KO) mice (B6;129P2-Nos2tm1Lau/J) and their appropriate control mice (B6129PF2/ J), ROS KO mice (B6.129S6-Cybbtm1Din/J), and C57BL/6J mice from The Jackson Laboratory were used throughout these studies. Mice were housed at The University of Texas at San Antonio Small Animal Laboratory Vivarium. Hind legs from Dectin-1 KO (129/SvEv-C57BL/6) mice were a kind gift from Chad Steele (University of Alabama at Birmingham). Dectin-2 KO mouse hind legs and Dectin-3 KO mice, both on the C57BL/6 background, were a kind gift from Marcel Wuethrich (University of Wisconsin—Madison). All animal experiments were approved by The University of Texas at San Antonio Institutional Animal Care and Use Committee (IACUC), and mice were handled according to IACUC guidelines. Strains and media. C. neoformans strains H99 (serotype A, mating type ␣) and H99␥ (serotype A, mating type ␣; a gamma interferon-producing strain derived from C. neoformans H99 [13]) and C. neoformans strain 52D (serotype D) were recovered from 15% glycerol stocks stored at ⫺80°C and were maintained on yeast extract-peptone-dextrose (YPD) medium (1% yeast extract, 2% peptone, 2% dextrose, and 2% Bacto agar). Yeast cells were grown for 15 to 17 h at 30°C with shaking in YPD broth (Becton Dickinson and Company, Sparks, MD), collected by centrifugation, and washed three times with sterile phosphate-buffered saline (PBS), and viable yeast cells were quantified using trypan blue dye exclusion in a hemacytometer. Pulmonary infections. Pulmonary C. neoformans infections were initiated by nasal inhalation as described previously (11, 12, 22). Mice were anesthetized with 2% isoflurane using a rodent anesthesia device (Eagle Eye Anesthesia, Jacksonville, FL) and were then given a yeast inoculum of 1 ⫻ 104 CFU of C. neoformans strain H99 or H99␥ in 50 ␮l of sterile PBS pipetted directly into the nostril. The inocula used were verified by quantitative culture on YPD agar. The mice were fed ad libitum and were monitored by inspection twice daily. Mice were euthanized at specific time points postinoculation by CO2 inhalation followed by cervical dislocation, and lung tissues were excised using aseptic technique. The left lungs were homogenized in 1 ml of sterile PBS, and 10-fold dilutions of each tissue were cultured on YPD agar supplemented with chloramphenicol. CFU were enumerated following incubation at 30°C for 48 h. Pulmonary leukocyte isolation. Lungs were excised at specific time points postinoculation and were enzymatically digested at 37°C for 30 min in 10 ml of digestion buffer (RPMI 1640 and 1 mg/ml of collagenase type IV) with intermittent stomacher homogenization. The digested tissues were then successively passed through sterile nylon strainers of various pore sizes (70 and 40 ␮m) (BD Biosciences, San Jose, CA). Erythrocytes were lysed by incubation in NH4Cl buffer (0.859% NH4Cl, 0.1% KHCO3, 0.0372% Na2 EDTA [pH 7.4]; Sigma-Aldrich) for 3 min on ice, followed by the addition of a 2-fold excess of PBS. The cells were then collected by centrifugation, resuspended in sterile PBS plus 2% heat-inactivated fetal bovine serum (flow cytometry buffer), and enumerated in a hemacytometer using trypan blue dye exclusion. CXCR3 blocking. For CXCR3-blocking experiments, mice received either 500 ␮g of an anti-CXCR3 antibody (clone CXCR3-17; BioXCell) or 500 ␮g of an isotype control antibody (Armenian hamster IgG; BioXCell) in a volume of 100 ␮l, injected intraperitoneally beginning 24 h before inoculation and continuing every other day throughout the study. Preparation of bone marrow-derived pDCs. Bone marrow-derived pDCs were prepared and cultured as described previously (23). Briefly,

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bone marrow was flushed from the femurs and tibiae of mice. Cells were washed, quantified by trypan blue dye exclusion in a hemacytometer, and plated at a concentration of 2 ⫻106/ml in complete medium (RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum, 2 mM L-glutamine, 100 U penicillin/ml, 100 ␮g of streptomycin/ml, and 50 mM 2-mercaptoethanol) supplemented with 200 ng/ml recombinant murine Flt3L (Peprotech, Rocky Hill, NJ). Cells were incubated at 37°C in a humidified environment supplemented with 5% CO2. The cells were harvested on day 7 following plating. The pDCs were then purified by positive selection using magnetically labeled antibodies against PDCA-1 according to the manufacturer’s protocol (Miltenyi Biotec, Auburn, CA). The purity of pDCs was evaluated using flow cytometry and was routinely observed to be ⬎90%. Isolation of human pDCs. Human pDCs were isolated from the whole blood of deidentified healthy donors, which was purchased from a commercial source (Innovative Research, Novi, MI). PBMCs were isolated from whole blood by Ficoll density gradient separation (BD Biosciences, San Diego, CA) using SepMate tubes (Stemcell Technologies Inc., Vancouver, BC, Canada). pDCs were double purified from PBMCs by positive selection using magnetically labeled antibodies against CD304 (BDCA-4/Neuropilin-1) according to the manufacturer’s protocol (Miltenyi Biotec, Auburn, CA). Following purification, cell culture assays were performed using complete medium supplemented with 10 mM HEPES buffer. The purity of pDCs was evaluated using flow cytometry and was routinely observed to be ⬎90%. Cytokine analysis. Cytokine levels in lung tissues were analyzed using an enzyme-linked immunosorbent assay (ELISA). Briefly, lung tissue was excised and homogenized in ice-cold sterile PBS (1 ml). An antiprotease buffer solution (1 ml) containing PBS, protease inhibitors (inhibiting cysteine, serine, and other metalloproteinases), and 0.05% Triton X-100 was added to the homogenate. Samples were then clarified by centrifugation (at 3,500 rpm) for 10 min. Pulmonary homogenates were assayed for cytokine production by use of ELISAs (R&D Systems, Minneapolis, MN) for CXCL9, CXCL10, and CXCL11 according to the manufacturer’s instructions or with the Bio-Plex Protein Array system (Bio-Rad Laboratories, Hercules, CA). Flow cytometry. Pulmonary leukocytes at 1 ⫻ 106 in 100 ␮l 1⫻ PBS plus 2% fetal bovine serum (flow cytometry buffer) were incubated with CD16/CD32 (Fc Block; BD Biosciences, San Jose, CA) in 96-well U-bottom plates for 5 min. The cells were then incubated with optimal concentrations of antibodies against B220 (RA3-6B2; BD Biosciences, San Jose, CA), CD11c (N418), CD45 (30-F11), or PDCA-1 (eBio129c; Affymetrix eBioscience Inc., San Diego, CA), conjugated to fluorochromes (phycoerythrin [PE], allophycocyanin [APC], Alexa Fluor 647, or PE-Cy7), in various combinations for 30 min at 4°C. Following incubation, samples were washed and were fixed in 2% ultrapure formaldehyde. The absolute number of leukocytes in each subset was then determined by multiplying the absolute number of CD45⫹ cells by the percentage of cells stained with fluorochrome-labeled antibodies for each cell population analyzed using BD FACSArray software on a BD FACSArray flow cytometer (BD Biosciences, San Jose, CA). pDCs were identified as CD45⫹ CD11cInt B220⫹ PDCA-1⫹ cells. Growth inhibition assay. pDCs were differentiated from the bone marrow of mice and were purified or isolated from human blood as described above. The purified pDCs (1 ⫻ 104/well) were cultured, in triplicate, within individual wells of a 96-well U-bottom tissue culture plate in RPMI complete medium alone or in RPMI complete medium containing C. neoformans strain H99 with or without an anti-cryptococcal GXM antibody (MAb F12D2 IgG1; a kind gift of Tom Kozel, University of Nevada, Reno) at effector-to-target ratios of 100:1, 10:1, and 1:1. H99 in RPMI complete medium without pDCs was used as a control. After 6 h, the contents of each well were centrifuged and the supernatants removed. The pDCs in the cell pellet were then lysed by washing three times with sterile water (100 ␮l) and incubating in water for 20 min. The remaining yeast

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cells were diluted in PBS and were plated onto YPD agar in order to quantify the live cryptococcal cells remaining. pDC-blocking treatment. Purified murine or human pDCs were pretreated with exogenous polysaccharides as described previously (24). Briefly, pDCs were treated with 0.5 mg/ml dextran, 0.5 mg/ml laminarin, 1 mg/ml mannan, or both laminarin and mannan (all from Sigma-Aldrich Co., St. Louis, MO) for 30 min at 37°C in a humidified environment supplemented with 5% CO2 prior to the performance of the growth inhibition assay as described above. Human pDCs were treated with 200 ng of a monoclonal antibody against Dectin-3 (25) or an isotype control antibody (IgG2a) for 30 min at 37°C in a humidified environment supplemented with 5% CO2 prior to the performance of the growth inhibition assay as described above. ROS inhibition and detection. In addition to deriving pDCs from ROS KO mice, we inhibited ROS production in pDCS as described previously (26). Purified murine or human pDCs were treated with a ROS inhibitor cocktail of 80 ␮g/ml purified bovine liver catalase, 8 ␮g/ml superoxide dismutase, and 100 mM D-mannitol (all from Sigma-Aldrich Co., St. Louis, MO), and the growth inhibition assay was performed as described above. Fluorescent labeling of C. neoformans. Cryptococci were labeled as described previously (27). Live organisms were washed with sterile 0.1 M sodium bicarbonate buffer, pH 8.0 (staining buffer), counted, and resuspended to 5 ⫻ 108/ml. C. neoformans yeast cells were incubated with 2 ␮g/ml Alexa Fluor 568 (Molecular Probes, Eugene, OR) at room temperature in the dark for 2 h. The organisms were then washed three times with sterile PBS, counted, and resuspended in sterile PBS to the concentration needed for each experiment. Confocal analysis. The in vitro growth inhibition assay was set up as described above with Alexa Fluor 568-stained C. neoformans. At various time points, the wells were incubated with trypan blue (0.02%) to quench the fluorescence of extracellular organisms and were fixed with 2% paraformaldehyde in PBS for 10 min at room temperature. After fixation, the cells were washed and were permeabilized with 0.1% saponin. While permeabilized, the cells were intracellularly stained with anti-LAMP-1 (mouse; Affymetrix eBioscience Inc., San Diego, CA). Images were acquired on a Zeiss LSM510 confocal laser scanning microscope using Zen 2009 (Zeiss, Oberkochen, Germany) acquisition software. Images were analyzed using Imaris 3D/4D software, version 7.2 (Bitplane, Zurich Switzerland). Statistical analysis. The unpaired Student t test (two-tailed) was used to analyze fungal burden, pulmonary cell populations, and cytokine/ chemokine data using GraphPad Prism, version 5.00 for Windows (GraphPad Prism Software, San Diego, CA, USA). For multiple comparisons, one-way analysis of variance (ANOVA) with Tukey’s multiplecomparison test was performed. Significant differences were defined as a P value of ⬍0.05.

RESULTS

pDCs inhibit cryptococcal growth in vitro. It is well established that cDCs exhibit anticryptococcal activity both in vitro and in vivo (26–29); however, the anticryptococcal activity of pDCs is unknown. To ascertain pDC anticryptococcal activity, purified bone marrow-derived pDCs (Fig. 1A) were cultured with wildtype (WT) C. neoformans strain H99 at different effector-to-target ratios for 6 h, and cryptococcal viability was determined. We observed that pDCs are able to inhibit the growth of C. neoformans in vitro in a dose-dependent manner (Fig. 1B). The levels of growth inhibition (given as means ⫾ standard errors of the means [SEM]) were 62.77% ⫾ 10.75% at an effector-to-target ratio of 100:1 (100 pDCs to 1 cryptococcal cell), 44.55% ⫾ 6.89% at 10:1 (10 pDCs to 1 cryptococcal cell), and 35.62% ⫾ 5.56% at 1:1 (1 pDC to 1 cryptococcal cell). There was no difference in viability between pDCs cultured with C. neoformans at a 1:1 ratio and pDCs cul-

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tured in medium alone (Fig. 1C). This is noteworthy in view of the fact that inhibition of the growth of A. fumigatus by pDCs has been shown to be mediated by calprotectin, which was released as a result of pDC death (21), and by the formation of extracellular traps by pDCs (24). Growth inhibition and phagocytosis are not dependent on opsonization. The uptake of C. neoformans by phagocytic cells is facilitated by opsonization of the yeast with complement proteins and/or antibodies (26). While human pDCs possess some Fc and complement receptors, the full array of murine pDC surface receptors is not well defined (30). In order to determine whether opsonization of C. neoformans is necessary for uptake or growth inhibition by pDCs, yeast cells were either left untreated or treated with an opsonizing antibody against the capsule component GXM and were cultured with pDCs for 6 h. There was no difference in the level of growth inhibition by pDCs cultured with H99 between the presence and the absence of an opsonizing antibody at an effector-to-target ratio of 100:1 (Fig. 1D) or at any other effectorto-target ratio tested (data not shown). Complement appears to not be a factor, since heat-inactivated serum was used to prepare the cell culture medium. To test whether pDCs are able to phagocytose nonopsonized C. neoformans, fluorescently labeled yeast cells were incubated with bone marrow-derived pDCs at an effector-to-target ratio of 1:1, and the uptake of the yeast was visualized using confocal microscopy. Trypan blue was used to quench the fluorescence of extracellular yeast cells that were not phagocytosed by the pDCs, allowing us to distinguish between extracellular and intracellular (phagocytosed) yeast. LAMP-1 staining was used to observe the location of yeast in relation to the lysosomes of pDCs. We showed that pDCs are able to phagocytose C. neoformans in as short a time as 30 min and that by 1 h, LAMP-1-positive compartments have fused with the C. neoformans-containing phagosome (Fig. 1E and F). This indicates that opsonization is not required for cryptococcal uptake by pDCs. We have shown previously that cDCs are able to phagocytose C. neoformans and that by 1 h postphagocytosis, the yeast acquires a crescent shape, which is indicative of fungal degradation and death (28). The pDCs contained several cryptococci by 6 h, but we observed no signs of yeast degradation or pDC death (Fig. 1E). ROS production is critical for the inhibition of C. neoformans growth by pDCs. To elucidate the mechanism used by pDCs to inhibit the growth of C. neoformans, known mediators of anticryptococcal activity were tested. Nitric oxide (NO) has been shown to be important in the control of cryptococcal infection in phagocytic cells (31–34). The enzyme responsible for the production of NO in phagocytic cells is inducible nitric oxide synthase (iNOS). To test if NO played a role in the inhibition of cryptococcal growth by pDCs, pDCs were differentiated from the bone marrow of iNOS KO mice (B6;129P2-Nos2tm1Lau/J) and their appropriate control mice (B6129PF2/J) and were cultured with H99 for 6 h. There was no defect in cryptococcal growth inhibition by pDCs derived from iNOS KO mice relative to inhibition by pDCs from control mice (Fig. 2A). These data suggest that NO is not critical for the inhibition of C. neoformans growth by pDCs. Additional known mediators of cDC anticryptococcal activity are reactive oxygen species (ROS) (26). To test if ROS are responsible for the cryptococcal growth inhibition observed, pDCs were grown from the bone marrow of mice that lack phagocyte superoxide production (B6.129S6-Cybbtm1Din/J) or their WT controls

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FIG 1 pDCs inhibit C. neoformans growth in vitro independently of opsonization. pDCs were differentiated from bone marrow cells, purified, and cultured with H99. Fungal burden was assessed 6 h later. (A) Purified pDCs. (B) The percentage of growth inhibition was calculated by dividing the CFU of H99 cultured with pDCs by the CFU of H99 alone and then multiplying by 100. (C) The viability of pDCs (effector-to-target ratio, 1:1) was assessed with the fixable viability dye

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FIG 2 The inhibition of C. neoformans growth by murine pDCs is dependent on ROS. pDCs were differentiated from bone marrow cells and were cultured with H99. Fungal burden was assessed 6 h later. (A) pDCs derived from WT (B6129PF2/J) and iNOS KO (B6;129P2-Nos2tm1Lau/J) mice were grown. (B) pDCs were differentiated from the bone marrow cells of WT (B6.129S6/J) and ROS KO (B6.129S6-Cybbtm1Din/J) mice. (C) WT pDCs were treated with a ROS inhibitor cocktail consisting of 80 ␮g/ml purified bovine liver catalase, 8 ␮g/ml superoxide dismutase, and 100 mM D-mannitol, and a growth inhibition assay was performed as described in Materials and Methods. Data shown are from two or three experiments with triplicate wells and are presented as means ⫾ SEM. *, P ⬍ 0.05.

(C57BL/6J) and were cultured with H99 for 6 h. pDCs from ROS KO mice exhibited a significant decrease in cryptococcal growth inhibition from that for WT pDCs (Fig. 2B). To confirm that the inhibition of C. neoformans growth by pDCs is dependent on ROS production, we performed the C. neoformans growth inhibition assay using pDCs derived from BALB/c mice in the absence or presence of ROS inhibitors (26). We observed a significantly lower level of cryptococcal growth inhibition by pDCs cultured in the presence of inhibitors of ROS production than by untreated pDCs (Fig. 2C). pDCs derived from ROS KO mice and pDCs cultured in the presence of ROS inhibitors exhibited similar defects in growth inhibition, suggesting that ROS production is required for cryptococcal growth inhibition by pDCs. Dectin-3 (CLEC4D) is necessary for cryptococcal growth inhibition and uptake by murine pDCs. Because opsonization was not required for the recognition and uptake of C. neoformans by pDCs, we hypothesized that a surface pattern recognition receptor may be involved in cryptococcal recognition and uptake by pDCs. The fungal cell surface is covered with carbohydrates, including ␤-glucans and mannan, which are recognized by C-type lectin receptors (CLRs) (35). To distinguish which carbohydrate moiety is recognized, we pretreated pDCs with laminarin (a ␤-glucan) alone, mannan alone, or both laminarin and mannan to block all receptors associated with these carbohydrates. pDCs treated with a nonspecific polysaccharide (dextran) or untreated pDCs were used as controls. There was no significant change in Cryptococcus growth inhibition by pDCs pretreated with dextran or laminarin from that by untreated pDCs (Fig. 3A). Pretreatment of pDCs with mannan alone or with mannan and laminarin together resulted in complete abrogation of their Cryptococcus growth inhibition activity (Fig. 3A). These data suggest that receptors recognizing mannan are responsible for the recognition of C. neoformans by pDCs. Murine pDCs have been shown to express Dectin-1, mannose receptor, Dectin-2, and other members of the Dectin-2 family (36–38). To determine which C-type lectin receptor (CLR) is necessary for the recognition and uptake of C. neoformans, and for

cryptococcal growth inhibition, by pDCs, we performed C. neoformans growth inhibition assays using pDCs derived from the bone marrow of mice deficient in Dectin-1, Dectin-2, Dectin-3, or mannose receptor. pDCs derived from Dectin-1 KO mice failed to exhibit any significant difference in cryptococcal growth inhibition from pDCs obtained from WT mice (Fig. 3B). Considering that Dectin-1 recognizes ␤-glucans, this result was expected, since the laminarin treatment had no effect on growth inhibition (Fig. 3A). We next tested for a potential role of CLRs known to recognize mannan. pDCs derived from mannose receptor KO or Dectin-2 KO mice also failed to exhibit any significant differences in cryptococcal growth inhibition from the pDCs of WT mice (Fig. 3C and D). However, pDCs derived from Dectin-3 KO mice demonstrated a significantly lower level of inhibition of C. neoformans growth than pDCs derived from WT mice (Fig. 3D). These data show a role for Dectin-3 in the inhibition of C. neoformans growth by pDCs. To test if the reduction in growth inhibition was due to defects in cryptococcal uptake, fluorescently labeled yeast cells were incubated with pDCs derived from the bone marrow of WT or Dectin-3 KO mice at an effector-to-target ratio of 1:1, and the uptake of the yeast was visualized using confocal microscopy. After 6 h of incubation, WT pDCs had phagocytosed several cryptococci, whereas the Dectin-3 KO pDCs exhibited a severe defect in cryptococcal uptake (Fig. 3E and F). To determine the requirement of Dectin-3 for protection against infection with C. neoformans, we inoculated WT and Dectin-3 KO mice with C. neoformans strain H99 via intranasal inhalation and monitored susceptibility to infection. We observed no significant difference in susceptibility to infection between Dectin-3 KO mice and WT mice (Fig. 4). These data suggest that while Dectin-3 is necessary for optimal inhibition of C. neoformans growth by pDCs, it may not be required for protection against infection with C. neoformans. Human pDCs inhibit the growth of C. neoformans. Our studies demonstrated that murine pDCs inhibit the growth of C. neoformans. Consequently, we sought to determine if human pDCs were also able to inhibit the growth of C. neoformans. Human

eFluor 780 via flow cytometry. (D) pDCs were incubated with C. neoformans yeast cells with or without 1 ␮g/ml opsonizing antibody (Ab). The data shown in panels B through D are from three experiments with triplicate wells and are presented as means ⫾ SEM. (E) pDCs were incubated with Alexa Fluor 568-labeled C. neoformans yeast cells without opsonizing antibody for the indicated times. Extracellular yeast cells were quenched by trypan blue, and pDCs were stained for LAMP-1. Confocal images are representative of two experiments performed with a 63⫻ lens objective with 1.5⫻ zoom. (F) Quantification of data from confocal images from two experiments and 10 fields per experiment.

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FIG 3 The inhibition of C. neoformans growth by murine pDCs is dependent on Dectin-3. pDCs were differentiated from bone marrow cells and were cultured with H99. Fungal burden was assessed 6 h later. (A) pDCs were pretreated with either 0.5 mg/ml dextran, 0.5 mg/ml laminarin, 1 mg/ml mannan, or both laminarin and mannan for 30 min at 37°C in a humidified environment supplemented with 5% CO2 prior to the performance of the growth inhibition assay as described in Materials and Methods. (B) pDCs were differentiated from the bone marrow cells of WT and Dectin-1 KO mice. (C) pDCs were differentiated from the bone marrow cells of WT and mannose receptor KO (MR KO) mice. (D) pDCs were differentiated from the bone marrow cells of WT, Dectin-2 KO, and Dectin-3 KO mice. The data shown are from two or three experiments with triplicate wells and are presented as means ⫾ SEM. (E) pDCs were incubated with Alexa Fluor 568-labeled C. neoformans yeast cells without opsonizing antibody for 6 h. Extracellular yeast cells were quenched by trypan blue, and pDCs were stained for LAMP-1. Confocal images are representative of two experiments performed with a 63⫻ lens objective with 1.5⫻ zoom. (F) Quantification of data from confocal images from two experiments with 10 fields per experiment. Asterisks indicate significant differences in growth inhibition or uptake. *, P ⬍ 0.05; ***, P ⬍ 0.0001.

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FIG 4 Survival of Dectin-3 KO mice compared to that of WT mice. C57BL/6J (WT) and Dectin-3 KO mice were inoculated intranasally with 1 ⫻ 104 CFU of C. neoformans strain H99. Mice were observed for survival analysis. Data are cumulative for two experiments using 5 mice per group per time point.

pDCs were isolated from the blood of healthy donors (Fig. 5A), cultured with C. neoformans strain H99, and subsequently measured for their capacity to inhibit the growth of C. neoformans. Our results showed that human pDCs were also able to inhibit C. neoformans growth at an effector-to-target ratio of 100:1 (100 pDCs to 1 cryptococcal cell); however, some patient-to-patient variability was observed (Fig. 5B). Effector-to-target ratios of 10:1 (10 human pDCs to 1 cryptococcal cell) and 1:1 (1 human pDC to 1 cryptococcal cell) showed no growth inhibition (data not shown). There was no difference in viability between pDCs cultured with C. neoformans at an effector-to-target ratio of 100:1 and pDCs cultured in medium alone (Fig. 5C). To test whether cryptococcal growth inhibition was due to ROS production, human pDCs were either left untreated or treated with a ROS inhibition cocktail. As was found for murine pDCs, inhibition of ROS production by human pDCs resulted in a significant decrease in cryptococcal growth inhibition (Fig. 5D). However, the defect in growth inhibition was not as significant as that exhibited by murine pDCs, suggesting that ROS may not be the only mechanism employed by human pDCs to inhibit C. neoformans. Human pDCs have been shown to express an array of PRRs different from that of murine pDCs. In order to elucidate which PRR on human pDCs may be responsible for cryptococcal recognition, we treated human pDCs with laminarin (a ␤-glucan) alone, mannan alone, or both laminarin and mannan in order to block all receptors associated with these carbohydrates. As with murine pDCs, mannan receptors appeared to be important for the recognition of C. neoformans, as evidenced by the fact that treatment with mannan completely abolished growth inhibition (Fig. 5E). Since growth inhibition by pDCs was dependent on mannan receptors, we sought to ascertain the requirement of Dectin-3 for cryptococcal growth inhibition by human pDCs. Human pDCs were isolated from the blood of healthy donors, cultured with C. neoformans strain H99 in the presence of a Dectin-3-blocking antibody or an isotype control antibody, and subsequently measured for their capacity to inhibit the growth of C. neoformans. We observed a significantly lower level of inhibition of C. neoformans growth by pDCs treated with the Dectin-3-blocking antibody than by isotype-treated pDCs (Fig. 5F). These data suggest that Dectin-3 is required for optimal inhibition of C. neoformans growth by human pDCs. To test if the reduction in growth inhibition was due to defects in cryptococcal uptake, fluorescently labeled yeast cells were incubated with human pDCs in the presence of a Dectin-3-blocking

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antibody or an isotype control antibody, and the uptake of the yeast was visualized using confocal microscopy. After 6 h of incubation, we observed no defect in cryptococcal uptake by human pDCs treated with the Dectin-3-blocking antibody relative to that by isotype control-treated human pDCs (data not shown). These data suggest that while Dectin-3 is required for optimal inhibition of C. neoformans growth by human pDCs, the mechanism differs from that employed by murine pDCs. CXCR3 is needed for pDC recruitment during pulmonary cryptococcal infection. The data presented above demonstrate that both murine and human pDCs have anticryptococcal activity. We next sought to determine if pDCs traffic to the lungs during a cryptococcal infection. BALB/c mice received an intranasal inoculation with 104 CFU of C. neoformans strain H99 or H99␥. We chose to use H99␥ because we have shown previously that cells from H99␥-infected mice have higher transcript levels of the chemokines CXCL9/MIG and CXCL10/IP10 than cells from mice infected with C. neoformans strain H99 (31, 39, 40). These chemokines have been shown to induce pDC recruitment (41, 42). Pulmonary leukocytes were then isolated on days 7 and 14 postinoculation and were analyzed by flow cytometry in order to determine the absolute number of pDCs recruited to the lungs during infection. We observed that experimental pulmonary infection with H99␥ elicits a significantly higher absolute number of pDCs recruited to the lungs at day 7 postinoculation than infection with WT (H99) cryptococci (Fig. 6A). The absolute number of pDCs in mice inoculated with H99␥ was observed to decrease by day 14 postinoculation (Fig. 6A), a decrease that appeared to correlate with a decline in pulmonary fungal burdens (Fig. 6B). However, in mice inoculated with H99, the absolute number of pDCs never rose above that observed in uninfected, naïve mice (Fig. 6A), suggesting that pDCs are not typically recruited to the lungs during pulmonary C. neoformans infection. To further test this, BALB/c mice were infected with H99 or the C. neoformans clinical isolate 52D. Experimental pulmonary infection with 52D is generally controlled in BALB/c mice (33, 43). We observed no significant difference in pDC recruitment to the lungs between mice infected with 52D and mice infected with H99 (data not shown). Studies to evaluate the necessity of pDCs for protection in H99␥-infected mice by depleting pDCs, either by use of depletion antibodies or by treatment of BDCA2-diphtheria toxin receptor transgenic mice with diphtheria toxin, were not technically feasible, due to incomplete depletion of pDCs in H99␥-infected mice or the deleterious sensitivity of the transgenic mice to long-term diphtheria toxin treatment (data not shown). To test whether the enhanced recruitment of pDCs in H99␥infected mice was due to CXCL9/MIG, CXCL10/IP10, or CXCL11/I-TAC, we next chose to measure the protein levels of these chemokines in the lungs of mice during WT or H99␥ infection. BALB/c mice received an intranasal inoculation with C. neoformans strain H99␥ or H99, and protein levels of CXCL9, CXCL10, and CXCL11 in pulmonary homogenates were determined on day 7 postinoculation. We observed that experimental pulmonary infection with H99␥ elicits significant increases in CXCL9, CXCL10, and CXCL11 protein levels over those with WT infection (Fig. 6C to E), increases that correlate with the increases in the number of pulmonary pDCs (Fig. 6A). Since infection with H99␥ elicits significant increases in the protein levels of CXCL9, CXCL10, and CXCL11 over those with WT infection (Fig. 6C to E), correlating with the increases

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FIG 5 Human pDCs inhibit the growth of C. neoformans. Human pDCs were isolated from the blood of healthy donors and were cultured with H99. Fungal burden was assessed 6 h later. (A) Isolation of human pDCs. (B) The percentage of growth inhibition was calculated by dividing the CFU of H99 cultured with human pDCs by the CFU of H99 alone and then multiplying by 100. Data from seven patients (with triplicate wells) are shown. (C) The viability of pDCs (effector-to-target ratio, 100:1) was assessed with the fixable viability dye eFluor 780 via flow cytometry. (D) Human pDCs were treated with a ROS inhibitor cocktail consisting of 80 ␮g/ml purified bovine liver catalase, 8 ␮g/ml superoxide dismutase, and 100 mM D-mannitol, and a growth inhibition assay was performed as described in Materials and Methods. (E) Human pDCs were pretreated with either 0.5 mg/ml dextran, 0.5 mg/ml laminarin, 1 mg/ml mannan, or both laminarin and mannan for 30 min at 37°C in a humidified environment supplemented with 5% CO2 prior to the performance of the growth inhibition assay as described in Materials and Methods. (F) Human pDCs were treated with 200 ng of an anti-Dectin-3 monoclonal antibody or an isotype control antibody (IgG2a) for 30 min at 37°C in a humidified environment supplemented with 5% CO2 prior to the performance of the growth inhibition assay as described in Materials and Methods. The data shown are from two to three donors (with triplicate wells) and are presented as means ⫾ SEM. Asterisks indicate significant differences in growth inhibition. *, P ⬍ 0.05; **, P ⬍ 0.001; ***, P ⬍ 0.0001.

in the number of pulmonary pDCs (Fig. 6A), we chose to evaluate the impact of blockage of the chemokine receptor CXCR3 on the infiltration of the lungs of H99␥-infected mice by pDCs. We elected to inhibit CXCR3 by use of anti-CXCR3 antibodies, because commercially available CXCR3 KO mice are on a C57BL/6 background, and C57BL/6 mice are naturally deficient in the chemokine CXCL11 (44). BALB/c mice were treated with isotype control or anti-CXCR3 antibodies at 24 h

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prior to infection with H99␥ and every 48 h thereafter. Treatment with anti-CXCR3 antibodies led to a significant reduction in the percentage of pDCs recruited to the lungs (Fig. 6F), and a significant increase in the pulmonary fungal burden (Fig. 6G), relative to those for isotype control-treated mice on day 7 postinoculation. These data suggest that CXCR3 expression is needed for pDC recruitment to the lungs of mice during the protective anti-C. neoformans immune response.

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FIG 6 CXCR3 is needed for pDC recruitment during pulmonary cryptococcal infection. BALB/c mice were inoculated via intranasal inhalation with 104 CFU of C. neoformans strain H99␥ or H99. Pulmonary leukocytes were then isolated from the right lobes of enzymatically digested lungs on days 7 and 14 postinoculation and were analyzed by flow cytometry. Fungal burden was quantified from lung homogenates. (A) The absolute numbers of plasmacytoid dendritic cells were determined. pDCs were characterized by surface markers (CD11cInt B220⫹ PDCA-1⫹). (B) Fungal burden results are expressed as mean log CFU per milliliter. (C to E) Pulmonary homogenates were prepared on day 7 postinoculation, and levels of CXCL9, -10, and -11 proteins were quantified by ELISA. (F and G) BALB/c mice were inoculated via intranasal inhalation with 104 CFU of C. neoformans strain H99␥. Prior to and during infection, mice were treated either with an isotype control antibody or with an anti-CXCR3 antibody. Lungs were excised at day 7 postinoculation, and percentages of pDCs (F) and pulmonary cryptococcal burdens (G) were quantified. The data shown are from three experiments with 4 to 5 mice per group and are presented as means ⫾ SEM. *, P ⬍ 0.05; **, P ⬍ 0.001; ***, P ⬍ 0.0001.

DISCUSSION

Plasmacytoid dendritic cells (pDCs) are known to be essential for protective immune responses to viruses. However, there is limited information regarding a role for pDCs in mediating the host de-

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fense against fungal pathogens. During Aspergillus infections, pDCs are required for protection (21), and mice resistant to Paracoccidioides brasiliensis exhibited higher numbers of pDCs in the lungs 96 h postinfection than mice that succumbed to the infec-

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tion (45). We have established that pDCs are able to inhibit cryptococcal growth, in contrast to conventional DCs, which are able to kill C. neoformans. This distinction has also been observed with Aspergillus fumigatus. Ramirez-Ortiz et al. showed in 2011 that pDCs are capable of inhibiting A. fumigatus hyphal growth, in contrast to neutrophils and monocytes, which are able to kill A. fumigatus (21). The inhibition of A. fumigatus growth by pDCs was shown to be mediated by calprotectin, which was released as a result of pDC death, induced by a gliotoxin produced by the fungus (21). Recently, it has been shown that following the stimulation of pDCs with A. fumigatus hyphae, pDCs are able to undergo ETosis and form extracellular traps (24). Unlike A. fumigatus, C. neoformans does not induce pDC death (Fig. 5C). We demonstrated that cryptococcal growth inhibition by pDCs is mediated by the generation of ROS. We have confirmed this both by using pDCs derived from the bone marrow of mice deficient in ROS production and by treating pDCs with ROS inhibitors. It has been shown that inhibition of ROS in cDCs leads to reduced anticryptococcal activity (26). Ablation of nitric oxide had no effect on growth inhibition by pDCs. Cryptococcus neoformans is surrounded by a thick polysaccharide capsule that inhibits phagocytosis of the yeast. Consequently, cryptococcal uptake by phagocytic cells normally requires opsonization by antibodies or complement (26). We observed that growth inhibition and uptake by pDCs were not dependent on opsonization. This finding has also been observed with pDC–A. fumigatus interactions, where hyphal recognition did not require opsonization (21). Pretreatment of pDCs with mannan completely abrogated the growth inhibition observed with mocktreated or dextran-treated pDCs, suggesting that pDCs recognize C. neoformans via receptors that recognize mannan. There are multiple receptors that recognize mannan, including Dectin-2, mannose receptor (CD206), dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin (DC-SIGN), and Dectin-3, also known as macrophage C-type lectin (MCL) and CLEC4D. Dectin-2 KO mice infected with C. neoformans exhibited higher levels of Th2-type cytokines, which are associated with a nonprotective immune response, than infected WT mice (46). Both mannose receptor and DC-SIGN have been shown to recognize heavily mannosylated cryptococcal mannoproteins (47), and mannose receptor KO mice succumb to C. neoformans infection significantly faster than WT mice (48). Dectin-3 recognizes mycobacterial cord factor (49) and has recently been shown to be a key component of antimycobacterial immunity (50). Dectin-3 can also form a heterodimer with Dectin-2 (25) and has been associated with protection against Candida albicans (25) and K. pneumoniae (51). The role of Dectin-3 in cryptococcal infections has not been investigated previously. We observed no significant reduction in the level of inhibition of C. neoformans growth with pDCs derived from mannose receptor KO or Dectin-2 KO mice; however, Dectin-3 KO pDCs exhibited a significant defect in growth inhibition. Dectin-3 KO pDCs also showed a defect in C. neoformans uptake. Conversely, treatment with mannan completely abrogated cryptococcal growth inhibition, while Dectin-3 KO pDCs still showed some inhibition, suggesting that Dectin-3 is not the only receptor that pDCs use to recognize C. neoformans. In line with this finding, there was no difference in survival between Dectin-3 KO mice and WT mice, indicating compensation by other receptors. Human studies do not always recapitulate rodent studies; how-

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ever, we found that human pDCs also inhibit cryptococcal growth. ROS production by human pDCs was also required for optimal growth inhibition; however, ROS did not appear to be the only mechanism used by human pDCs to inhibit C. neoformans. In addition, recognition of C. neoformans via CLRs recognizing mannan appeared to be required for cryptococcal growth inhibition by human pDCs. In line with the murine studies, blocking Dectin-3 on pDCs led to a significant defect in growth inhibition. Taking our findings together, we demonstrated that pDCs are able to inhibit cryptococcal growth in vitro. Optimal cryptococcal growth inhibition is dependent on ROS production but not on NO production. Dectin-3 is also required for growth inhibition. Human pDCs are able to inhibit cryptococcal growth in a manner and by a mechanism similar to those utilized by murine pDCs and also require Dectin-3 for optimal Cryptococcus growth-inhibitory activity but not for Cryptococcus uptake. Finally, pDC recruitment is dependent on the chemokine receptor CXCR3. These data suggest that pDCs could play a role in protection against C. neoformans. FUNDING INFORMATION This work, including the efforts of Floyd L. Wormley, was funded by HHS | National Institutes of Health (NIH) (AI071752). This work, including the efforts of Floyd L. Wormley, was funded by HHS | National Institutes of Health (NIH) (G12MD00759). This work, including the efforts of Floyd L. Wormley, was funded by DOD | Army Research Office (ARO).

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Infection and Immunity

September 2016 Volume 84 Number 9