STAT1 Signaling within Macrophages Is Required for Antifungal Activity against Cryptococcus neoformans Chrissy M. Leopold Wager,a,b Camaron R. Hole,a,b Karen L. Wozniak,a,b Michal A. Olszewski,c,d Mathias Mueller,e Floyd L. Wormley, Jr.a,b Department of Biology, The University of Texas at San Antonio, San Antonio, Texas, USAa; The South Texas Center for Emerging Infectious Diseases, The University of Texas at San Antonio, San Antonio, Texas, USAb; VA Ann Arbor Health System, University of Michigan Health System, Ann Arbor, Michigan, USAc; Division of Pulmonary & Critical Care Medicine, Department of Internal Medicine, University of Michigan Health System, Ann Arbor, Michigan, USAd; Institute of Animal Breeding and Genetics, University of Veterinary Medicine Vienna, Vienna, Austriae

Cryptococcus neoformans, the predominant etiological agent of cryptococcosis, is an opportunistic fungal pathogen that primarily affects AIDS patients and patients undergoing immunosuppressive therapy. In immunocompromised individuals, C. neoformans can lead to life-threatening meningoencephalitis. Studies using a virulent strain of C. neoformans engineered to produce gamma interferon (IFN-␥), denoted H99␥, demonstrated that protection against pulmonary C. neoformans infection is associated with the generation of a T helper 1 (Th1)-type immune response and signal transducer and activator of transcription 1 (STAT1)-mediated classical (M1) macrophage activation. However, the critical mechanism by which M1 macrophages mediate their anti-C. neoformans activity remains unknown. The current studies demonstrate that infection with C. neoformans strain H99␥ in mice with macrophage-specific STAT1 ablation resulted in severely increased inflammation of the pulmonary tissue, a dysregulated Th1/Th2-type immune response, increased fungal burden, deficient M1 macrophage activation, and loss of protection. STAT1-deficient macrophages produced significantly less nitric oxide (NO) than STAT1-sufficient macrophages, correlating with an inability to control intracellular cryptococcal proliferation, even in the presence of reactive oxygen species (ROS). Furthermore, macrophages from inducible nitric oxide synthase knockout mice, which had intact ROS production, were deficient in anticryptococcal activity. These data indicate that STAT1 activation within macrophages is required for M1 macrophage activation and anti-C. neoformans activity via the production of NO.

C

ryptococcus neoformans is an opportunistic fungal pathogen which frequently causes life-threatening infection in individuals with impaired T cell function (i.e., persons with AIDS or lymphoid malignancies and recipients of immunosuppressive therapies) (1–13). Cryptococcosis is considered an AIDS-defining illness and the most common mycological cause of morbidity and mortality among AIDS patients (14). Global estimates show that one million cases of cryptococcal meningitis occur in AIDS patients each year, resulting in over 620,000 deaths (14). C. neoformans also remains the third most common invasive fungal infection among organ transplant recipients, accounting for 8% of fungal diseases in solid-organ transplant recipients from March 2001 through March 2006 (13, 15, 16). The development of drug resistance or the inability of the host’s immune system to participate in eradicating the pathogen necessitates the development of new therapeutic strategies that consider the immune status of the host population commonly impacted by cryptococcal disease (17–19). C. neoformans is ubiquitous throughout the environment, and exposure via the inhalation of desiccated basidiospores or yeast into the lung alveoli often occurs in early childhood (20–22). The overwhelming majority of exposures result in asymptomatic disease and possible latency in immunocompetent individuals. However, in immunocompromised individuals, C. neoformans can spread from the lungs to the central nervous system (CNS), resulting in life-threatening meningoencephalitis (9). The host is dependent on resident pulmonary phagocytes to contain the pathogen and prevent dissemination from the lungs. Protection against C. neoformans infections is associated with the induction of Th1type cytokine responses (interleukin-2 [IL-2], IL-12, gamma in-

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terferon [IFN-␥], tumor necrosis factor alpha [TNF-␣]), increased lymphocyte infiltration, and classical macrophage (M1) activation (23–29). In contrast, Th2-type cytokine responses against cryptococcosis are nonprotective and are associated with alternative macrophage (M2) activation (30–32). M1 macrophages are induced following IFN-␥/signal transducer and activator of transcription 1 (STAT1) pathway activation and can efficiently kill phagocytized microorganisms through the production of reactive oxygen and nitrogen species (24, 30, 33–37). M2 macrophages, however, are induced by IL-4 and/or IL-13 and are associated with wound healing and aid in promoting a Th2-type immune response (33, 36). These M2 macrophages are proficient at phagocytizing C. neoformans; however, they are not efficiently microbicidal against the yeast (24, 30, 34–39). The enzyme inducible nitric oxide synthase (iNOS), which is upregulated in M1 macrophages, and arginase 1 (Arg1), which is upregulated in M2

Received 20 July 2015 Returned for modification 20 July 2015 Accepted 3 September 2015 Accepted manuscript posted online 8 September 2015 Citation Leopold Wager CM, Hole CR, Wozniak KL, Olszewski MA, Mueller M, Wormley FL, Jr. 2015. STAT1 signaling within macrophages is required for antifungal activity against Cryptococcus neoformans. Infect Immun 83:4513–4527. doi:10.1128/IAI.00935-15. Editor: G. S. Deepe, Jr. Address correspondence to Floyd L. Wormley, Jr., [email protected]. Supplemental material for this article may be found at http://dx.doi.org/10.1128 /IAI.00935-15. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

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macrophages, compete for the substrate L-arginine to produce nitric oxide and L-citrulline or L-ornithine and urea, respectively (40). Thus, the local population of macrophages tends to polarize in one way or the other depending on the cytokine microenvironment. Infection with various C. neoformans strains, such as strain H99 (serotype A) and strain 1841 (serotype D), results in the induction of Th2-type responses characterized by increased IL-4 and IL-13 cytokine production, M2 macrophage polarization, increased fungal burden, dissemination, and exacerbation of disease (25, 26, 34). In addition, inoculation of C57BL/6 mice with strain 52D (serotype D) results in the induction of Th2-type responses, M2 macrophage activation, and progression of disease (30, 31, 41). Conversely, experimental pulmonary infection in mice with highly virulent C. neoformans strain H99 engineered to express IFN-␥, designated H99␥, results in Th1-type and IL-17A cytokine responses, M1 macrophage activation, and resolution of the acute infection (25, 42, 43). Additionally, mice protectively immunized with C. neoformans strain H99␥ and subsequently challenged with wild-type (WT) C. neoformans strain H99 developed an M1 macrophage phenotype which was associated with enhanced STAT1 activation and ensuing protection against the yeast (23). Studies utilizing STAT1-deficient mice inoculated with C. neoformans H99␥ or WT clinical isolate strain 52D revealed that STAT1 signaling is required for the control of fungal burden, prevention of dissemination, and survival (37). Clarification of the role played by STAT1 and its downstream effectors in facilitating protection against C. neoformans as well as other intracellular organisms has the potential to identify novel strategies for immune therapies that target the host, rather than the organism, to fight diseases like cryptococcosis. The objective of the studies presented here was to elucidate the role of STAT1 in specifically mediating M1 macrophage activation and anticryptococcal activity utilizing mice with macrophagespecific STAT1 ablation, namely, LysMCreSTAT1flfl mice. These studies demonstrated that the induction of STAT1 signaling within macrophages alone was required for M1 macrophage activation and the antifungal activity of macrophages against C. neoformans. Mice deficient in STAT1 signaling within macrophages were unable to control C. neoformans growth within the lungs and subsequent dissemination to the CNS. They also exhibited increased pulmonary fungal burden and proinflammatory responses during pulmonary infection with C. neoformans strain H99␥ that normally induces protective immunity in WT mice. Additionally, we show that NO production by M1-polarized macrophages is essential for the anti-C. neoformans activity of macrophages, providing evidence of an effector cell population and a mechanism by which the host can mount a protective immune response to C. neoformans. MATERIALS AND METHODS Mice. Male and female LysMCreSTAT1flfl [B6.129P2-Lyz2tm1(cre)Ifo /J-StattmBiat] and control STAT1flfl (B6.129P2-STAT1tmBiat) mice, a kind gift from M. Mueller (Institute of Animal Breeding and Genetics, University of Veterinary Medicine Vienna, Vienna, Austria), female iNOS knockout (KO) [CByJ.129P2(B6)-Nos2tm1Lau/J] mice, and control BALB/ cByJ mice (The Jackson Laboratory, Bar Harbor, ME) were used throughout these studies. Mice were housed at The University of Texas at San Antonio Small Animal Laboratory Vivarium and handled according to guidelines approved by the Institutional Animal Care and Use Committee.

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Strains and media. C. neoformans strain H99 and C. neoformans strain H99␥ (derived from H99 serotype A, mating type ␣) (43) were recovered from a 15% glycerol stock stored at ⫺80°C prior to use in the experiments described in this study. The strains were maintained on yeast extractpeptone-dextrose (YPD) medium agar plates (Becton Dickinson, Sparks, MD). Yeast cells were grown for 16 to 18 h at 30°C with shaking in liquid YPD broth, collected by centrifugation, and washed three times with sterile phosphate-buffered saline (PBS), and viable yeasts were quantified using trypan blue dye exclusion on a hemacytometer. Pulmonary cryptococcal infections. Mice were anesthetized with 2% isoflurane using a rodent anesthesia device (Eagle Eye Anesthesia, Jacksonville, FL) and then given an intranasal inoculation with 1 ⫻ 104 CFU of C. neoformans strain H99 or strain H99␥ in 50 ␮l of sterile PBS. The inocula used for nasal inhalation were verified by quantitative culture on YPD agar. Mice were euthanized on predetermined days by CO2 inhalation followed by cervical dislocation, and lung tissues were excised using an aseptic technique. For survival analyses, mice were inoculated as stated above. Fungal burden. For fungal burden analysis, the left lobe of the lung was removed and homogenized in 1 ml sterile PBS. A 50-␮l aliquot was removed, serially diluted in sterile PBS, and plated on YPD agar supplemented with chloramphenicol. Plates were incubated for 48 h at 30°C and fungal colonies recorded. Cytokine analysis. Cytokine production in lung tissues was analyzed using the Bio-Plex protein array system (Luminex-based technology; BioRad Laboratories, Hercules, CA). Briefly, the left lobe of the lung was excised and homogenized in ice-cold sterile PBS (1 ml). An aliquot (50 ␮l) was taken to quantify the pulmonary fungal burden, and an antiprotease buffer solution containing PBS, protease inhibitors (inhibiting cysteine, serine, and other metalloproteinases), and 0.05% Triton X-100 was added to the homogenate and then clarified by centrifugation (800 ⫻ g) for 10 min. Supernatants from pulmonary homogenates were assayed for the presence of IL-1␣, IL-1␤, IL-2, IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, IL12(p40), IL-12(p70), IL-13, IL-17A, CCL5/RANTES, Eotaxin, CXCL1/ KC, CCL3/MIP-1␣, CCL4/MIP-1␤, CCL2/monocyte chemoattractant protein 1 (MCP-1), granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage CSF (GM-CSF), TNF-␣, and IFN-␥. Pulmonary leukocyte isolation. Lungs were excised on days 7 and 10 postinoculation and digested enzymatically at 37°C for 30 min in 10 ml digestion buffer (RPMI 1640 and 1 mg/ml collagenase type IV; SigmaAldrich, St. Louis, MO) with intermittent (every 10 min) stomacher homogenizations. The digested tissues then were successively filtered through sterile 70- and 40-␮m nylon filters (BD Biosciences, San Diego, CA) to enrich for leukocytes, and then cells were washed with sterile Hanks’ balanced salt solution (HBSS). Erythrocytes were lysed by incubation in NH4Cl buffer (0.859% NH4Cl, 0.1% KHCO3, 0.0372% Na2EDTA [pH 7.4]; Sigma-Aldrich) for 3 min on ice, followed by a 2-fold excess of PBS. The resulting leukocyte population then was enriched for macrophages by positive selection by labeling cells with anti-F4/80-biotin antibody (eBioscience, Inc., San Diego, CA) and subsequent binding of antibiotin magnetic beads (Miltenyi Biotec, Auburn, CA) according to the manufacturer’s recommendations. Real-time PCR analysis. Total RNA was isolated from purified F4/ 80⫹ cells using TRIzol reagent (Invitrogen, Carlsbad, CA) and then DNase (Qiagen, Germantown, MD) treated to remove possible traces of contaminating DNA according to the manufacturer’s instructions. Total RNA subsequently was recovered using the Qiagen RNeasy kit. cDNA was synthesized from 1 ␮g total RNA using the oligo(dT) primer and reagents supplied in the SuperScript III RT kit (Invitrogen) according to the manufacturer’s instructions. The cDNA was used as a template for real-time PCR analysis using the TaqMan gene expression assay (Applied Biosystems, Foster City, CA) according to the manufacturer’s instructions. All real-time PCRs were performed using the 7300 real-time PCR system (Applied Biosystems). For each real-time PCR, a master mix was prepared on ice with TaqMan gene expression assays specific for iNOS, IFN-␥,

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TNF-␣, CXCL9, CXCL10, IRF-1, SOCS-1, IL-17A, Ym1, FIZZ1, Arg1, IL-4, IL-13, and CD206 (Applied Biosystems). TaqMan rodent glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Applied Biosystems) was used as an internal control. The thermal cycling parameters contained an initial denaturing cycle of 95°C for 10 min followed by 40 cycles of 95°C for 15 s and 60°C for 60 s. Results of the real-time PCR data were derived using the comparative threshold cycle (CT) method to detect relative gene expression as described previously (25). Histology. Mice were sacrificed according to approved protocols, and the lungs were immediately perfused with sterile PBS by transcardial perfusion through the right ventricle. The pericardium and trachea were exposed by dissection, and an incision was made in the trachea for the insertion of a sterile flexible cannula attached to a 3-ml syringe. The lungs were slowly inflated with 0.5 to 0.7 ml 10% ultrapure formaldehyde (Polysciences, Inc., Warrington, PA), excised, and immediately fixed in 10% ultrapure formaldehyde for 24 to 48 h. The lungs then were transferred to 70% ethanol and subsequently mounted into cassettes and paraffin embedded by personnel at The University of Texas Health Science Center at San Antonio Histology and Immunohistochemistry Laboratory. After paraffin embedding, 5-mm sections were cut and stained using hematoxylin-eosin (H&E) with mucicarmine at McClinchey Histology Labs, Stockbridge, MI. Sections were analyzed with light microscopy using a Nikon microscope, and microphotographs were taken using Digital Microphotography system DFX1200 with ACT-1 software (Nikon Co., Tokyo, Japan) at the Ann Arbor VA Health System. Macrophage anticryptococcal assay, ROS detection assay, and NO production assay. Cell viability and quantity of F4/80⫹-enriched macrophage populations were assessed using trypan blue exclusion in a hemacytometer. Macrophages were cultured at a density of 5 ⫻ 105 cells per well in a 96-well tissue culture plate in RPMI 1640 (without phenol red) (Life Technologies, Grand Island, NY) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 2 mM L-glutamine, and 100 ␮g/ml penicillin-streptomycin (R10). Macrophages were incubated in R10 at 37°C with 5% CO2. The initial fungal burden was determined by the lysis of the macrophages using sterile deionized water followed by serial dilution and plating on YPD agar supplemented with chloramphenicol (Mediatech, Manassas, VA) for 48 h at 30°C. After 24 h of incubation, cell supernatants were collected and an aliquot (50 ␮l) was used to determine NO production with Griess reagent (Sigma-Aldrich) according to the manufacturer’s instructions. Light absorbance values were measured at 540 nm using a BioTek Elx808 absorbance microplate reader with Gen5 v1.04.5 software. Alternatively, F4/80⫹ macrophages were isolated and adjusted to 5 ⫻ 105 cells per well as described above. Cells were mixed with CellROX deep red reagent (Life Technologies) according to the manufacturer’s instructions, and ROS levels were measured using BD FACSArray software on a BD FACSArray flow cytometer (BD Biosciences). NO inhibition. Macrophages were isolated and cultured at a density of 5 ⫻ 105 cells per well in a 96-well tissue culture plate as described above. Cells were incubated in R10 with or without N␻-nitro-L-arginine methyl ester hydrochloride (L-NAME; Sigma-Aldrich) (1 mM) to inhibit NO production. After 24 h of incubation, cell supernatants were collected and nitrite production was measured as described above. Additionally, cells were lysed and intramacrophage cryptococci were enumerated as described above. Flow cytometry. Standard methodology was employed for the direct immunofluorescence of pulmonary leukocytes. Briefly, in 96-well U-bottom plates, 100 ␮l containing 1 ⫻ 106 cells in PBS plus 2% FBS (fluorescence-activated cell sorter [FACS] buffer) were incubated with 100 ␮l of Fc block (BD Biosciences) diluted in FACS buffer for 5 min to block nonspecific binding of antibodies to cellular Fc receptors. Subsequently, an optimal concentration of fluorochrome-conjugated antibodies (between 0.06 and 0.5 ␮g per 1 ⫻ 106 cells) was added in various combinations to allow for dual or triple staining experiments, and cells were incubated for 30 min at 4°C. Following incubation, the cells were washed three times with FACS buffer and were fixed in 200 ␮l of 2% ultrapure formal-

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dehyde (Polysciences, Inc., Warrington, PA) diluted in FACS buffer (fixation buffer). Cells incubated with either FACS buffer alone or single fluorochrome-conjugated antibodies were used to determine positive staining and spillover/compensation calculations, and the flow cytometer determined background fluorescence. The samples were analyzed using BD FACSArray software on a BD FACSArray flow cytometer (BD Biosciences). Dead cells were excluded on the basis of forward angle and 90° light scatter. For data analyses, 30,000 events (cells) were evaluated from a predominantly leukocyte population identified by back-gating from CD45⫹-stained cells. The absolute number of total leukocytes was quantified by multiplying the total number of cells observed with a hemacytometer by the percentage of CD45⫹ cells determined by flow cytometry. The absolute number of leukocytes (CD45⫹ cells), CD4⫹/CD3⫹ T cells, CD8⫹/CD3⫹ T cells, CD19⫹/B220⫹ B cells, polymorphonuclear leukocytes (PMNs) (1A8⫹/CD45⫹), macrophages (F4/80⫹/CD45⫹), dendritic cells (DCs) (CD11bint/CD11c⫹/CD45⫹), NK cells (Nkp46⫹/DX5⫹), NKT cells (Nkp46⫹/DX5⫹/CD4⫹), and eosinophils (SiglecF⫹/CD11bint) was determined by multiplying the percentage of each gated population by the total number of CD45⫹ cells. Dendritic cell killing assay. Spleens were aseptically removed from 3 to 4 STAT1flfl mice and LysMCreSTAT1flfl mice and passed through a 70-␮m filter to enrich for leukocytes. Erythrocytes were lysed as described above. Leukocytes were depleted of T cells with CD3 microbeads according to the manufacturer’s instructions (Miltenyi) and then depleted of macrophages as described above. The remaining cells were enriched for dendritic cells using CD11c microbeads according to the manufacturer’s instructions (Miltenyi). The purified DCs (1 ⫻ 104/well) were cocultured, in triplicate, within individual wells of a 96-well U-bottom tissue culture plate in RPMI complete medium containing C. neoformans strain H99 with anticryptococcal GXM antibody (monoclonal antibody F12D2 IgG1; kind gift of Tom Kozel, University of Nevada, Reno) at effector-to-target ratios of 20:1 and with H99 in RPMI complete medium without DCs as a control. All conditions were performed with and without IFN-␥ at 100 ng/ml (R&D Systems, Minneapolis, MN). After 24 h, the content of each well was centrifuged and the supernatants were removed. The DCs in the cell pellet then were lysed with sterile water (100 ␮l) and incubated for 15 min. The remaining yeast was diluted in PBS and plated onto YPD agar to quantify the live cryptococcal cells remaining. Statistical analysis. The Mann-Whitney test was used to analyze fungal burden to detect statistically significant differences using GraphPad Prism version 5.00 for Windows (GraphPad Software, San Diego, CA). The Kruskal-Wallis with Dunn’s multiple-comparison test was used to analyze fungal burden in C. neoformans H99 compared to that in C. neoformans H99␥ in the STAT1flfl and LysMCreSTAT1flfl mice to detect statistically significant differences (GraphPad Software). The unpaired Student t test was used to analyze cytokine/chemokine levels, pulmonary cell populations, ROS, and NO assays (two-tailed) in C. neoformans H99␥-inoculated mice to detect statistically significant differences (GraphPad Software). One-way analysis of variance (ANOVA) with Tukey’s posttest was used for real-time PCR analysis and cytokine/ chemokine levels comparing C. neoformans H99- and C. neoformans H99␥-inoculated mice and the DC killing assay to detect significant differences (GraphPad software). Survival data were analyzed using the logrank test (GraphPad software). Significant differences were defined as P ⬍ 0.05 (*), P ⬍ 0.01 (**), or P ⬍ 0.001 (***).

RESULTS

STAT1 signaling in macrophages is required for protection against C. neoformans H99␥. Previous studies showed that the induction of STAT1 signaling is associated with classical macrophage activation and protective anticryptococcal immune responses (23, 37). To demonstrate that the loss of protective anticryptococcal immunity in STAT1-deficient mice (37) was due specifically to the absence of STAT1 signaling within macrophages, we sought to determine if the myeloid-specific ablation of

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FIG 1 Mice with macrophage-specific STAT1 ablation are not protected against infection with C. neoformans H99␥. STAT1flfl and LysMCreSTAT1flfl mice were given a pulmonary infection with 104 C. neoformans H99␥ yeast cells. Mice were observed for up to 60 days for survival analysis (A), or fungal burden was analyzed at days 7 and 10 postinfection (B). (C) Fungal burden also was enumerated as mice succumbed to infection or at the conclusion of the study. Experiments are cumulative of 2 experiments using 4 to 5 mice per group per time point (*, P ⬍ 0.05; **, P ⬍ 0.01; ***, P ⬍ 0.001).

STAT1 signaling would render mice susceptible to pulmonary infection with C. neoformans H99␥. To this end, LysMCreSTAT1flfl mice, which have STAT1 deleted in macrophages and neutrophils (44, 45), and control STAT1flfl mice were given an experimental pulmonary infection with C. neoformans strain H99␥. We observed 90% mortality of LysMCreSTAT1flfl mice with a median survival time of 18 days post-H99␥ infection (P ⬍ 0.001) (Fig. 1A). In contrast, we observed 100% survival of STAT1flfl mice. Pulmonary fungal burden was quantified on days 7 and 10 postchallenge, i.e., at time points prior to the onset of mortality to ensure accurate statistical analysis of the fungal burden. LysMCreSTAT1flfl mice had significantly higher pulmonary fungal burden on both days 7 and 10 postinoculation than STAT1flfl mice (P ⬍ 0.001) (Fig. 1B). Additionally, significantly more CFU were observed in the brain of LysMCreSTAT1flfl mice on day 10 postinoculation than in STAT1flfl mice (P ⬍ 0.01) (Fig. 1B). Upon conclusion of the survival analysis, LysMCreSTAT1flfl mice had significantly more yeast cells in the lungs than STAT1flfl mice, and only 2 of 10 STAT1flfl mice had detectable CFU in brain, whereas yeast cells were present in all LysMCreSTAT1flfl mice that were analyzed (P ⬍ 0.05 and P ⬍ 0.001, respectively) (Fig. 1C). We are confident that the loss of protection in LysMCreSTAT1flfl mice is due to STAT1-ablated signaling within macrophages, as no impact on pulmonary fungal burden is observed in H99␥-inoculated mice depleted of neutrophils (46). It has been shown that a portion of the DC population may be affected in the LysMCre system (45). However, we detected no defect in killing of C. neoformans strain H99 by DCs obtained from LysMCreSTAT1flfl mice compared to that from STAT1flfl mice (see Fig. S1 in the supplemental material). Consequently, these studies show that macrophage-specific STAT1 signaling is critical for protection against C. neoformans. Furthermore, these data suggest that STAT1 signaling in macrophages plays a role in the prevention of cryptococcal trafficking to the brain. STAT1 deletion in macrophages results in the development of severe infection and pulmonary pathology in mice infected with C. neoformans H99␥. Having determined that STAT1 deletion in macrophages was detrimental to mouse survival and control of C. neoformans growth in the lungs, we sought to evaluate the effect of macrophage-specific STAT1 ablation on the develop-

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ment of pathology in infected lungs. Histological analysis at 10 days postinoculation with C. neoformans H99␥ revealed minimal fungal presence and a very modest and contained inflammatory response in the lungs of STAT1flfl mice, consistent with the development of protective responses and rapid clearance of C. neoformans with minimal pulmonary pathology (Fig. 2A). This was in contrast to the widespread inflammatory response observed in the lungs of infected LysMCreSTAT1flfl mice (Fig. 2B). The alveolar architecture in the lungs of STAT1flfl mice contained inflammatory foci with a few inflammatory cells, predominantly macrophages, but otherwise was well preserved (Fig. 2C). In comparison, the inflammatory pathology in the lung of LysMCreSTAT1flfl mice was much more profound. Over 50% of the lung tissue in the visual field was consolidated with complete occlusion of the air space in the infected areas (Fig. 2B and D). Closer examination revealed that cryptococcal organisms were abundant regardless of the robust inflammatory infiltrates in these mice. High-power examination revealed that the leukocyte infiltrate characteristics were different between the two groups of mice. We observed mixed cellular infiltrates and an abundance of intra- and extracellular yeast in the lungs of LysMCreSTAT1flfl mice (Fig. 2F). Numerous eosinophils (a hallmark of a nonprotective Th2 response) were found in small groups within the cellular infiltrate. Macrophages within infected LysMCreSTAT1flfl mice were large and densely packed and sometimes were multinucleated, suggesting fused cell clusters, and were frequently laden with cryptococci with large, fused capsules consistent with active proliferation of microbes (Fig. 2F). The few cryptococcal organisms observed in the lung tissue of STAT1flfl mice were internalized and partially degraded by macrophages (Fig. 2E). Collectively, these pathological findings demonstrate that mice with STAT1-deficient macrophages were unable to control cryptococcal infection in the lungs regardless of the development of a robust inflammatory response and had profound inflammatory pathology. Loss of STAT1 signaling in macrophages does not affect the induction of a cell-mediated immune response but does contribute to an increased proinflammatory response following inoculation with C. neoformans H99␥. To determine if STAT1 signaling specifically in macrophages affects the cytokine microenvironment in pulmonary tissues, we analyzed lung homogenates from STAT1flfl and LysMCreSTAT1flfl mice at days 7 and

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FIG 2 STAT1 deletion in macrophages resulted in the development of severe infection and pulmonary pathology in mice inoculated with C. neoformans H99␥. STAT1flfl and LysMCreSTAT1flfl mice were challenged by inhalation of 104 CFU of C. neoformans H99␥, and the lungs were processed for histopathological analysis on day 10 postinoculation. Note that STAT1flfl mice show significantly less pronounced inflammatory responses over the entire lung field (A) than the LysMCreSTAT1flfl mice (B), in which over 50% of the lung field is consolidated. High-power images using 20⫻ objectives (C and D) and 40⫻ objectives (E and F) demonstrate that the alveolar architecture is largely preserved in the STAT1flfl mice (C), and very few cryptococci are present (black arrows) and are mostly internalized by macrophages (M⌽) showing various stages of degradation. (D and F) The alveoli of LysMCreSTAT1flfl mice are filled with cryptococcal organisms and are densely permeated by mixed-leukocyte infiltrate. The majority of cryptococcal cells are large, surrounded by thick capsules, and show evidence of growth within the infiltrates and/or macrophages. Note that eosinophils (Eo) are abundantly present in the lungs of LysMCreSTAT1flfl mice (F) but not STAT1flfl mice (E). Histological slides were stained with H&E and mucicarmine, and images were taken with an original magnification of 4⫻ (A and B), 20⫻ (C and D), and 40⫻ (E and F) objective power. Images are representative images of 2 independent experiments using 3 mice per group.

10 postinoculation with C. neoformans H99␥. Similar to STAT1 complete KO mice (37), the LysMCreSTAT1flfl mice showed no defects in Th1- or Th2-type cytokine production (Fig. 3). The LysMCreSTAT1flfl mice had significantly increased IFN-␥ production at day 10 and IL-12p70 production at days 7 and 10 postinoculation compared to production by STAT1flfl mice (Fig. 3A) (P ⬍ 0.05). The levels of Th2-type cytokine IL-4 were significantly

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increased in LysMCreSTAT1flfl mice at days 7 (P ⬍ 0.001) and 10 (P ⬍ 0.05) postinoculation (Fig. 3D). IL-5 and IL-10 levels were significantly higher in LysMCreSTAT1flfl mice than in STAT1flfl mice at day 10 postinoculation (Fig. 3E and F) (P ⬍ 0.05), although this may not be biologically relevant, as the levels were relatively low. LysMCreSTAT1flfl mice exhibited significantly increased levels of proinflammatory cytokines IL-1␤ (P ⬍ 0.01 at day 7; P ⬍ 0.05

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FIG 3 Macrophage-restricted STAT1 deletion results in a mixed Th1/Th2 cytokine profile in response to C. neoformans H99␥ infection. STAT1flfl and LysMCreSTAT1flfl mice were given a pulmonary infection with 104 C. neoformans H99␥ yeast cells. At days 7 and 10 postinoculation, lungs were excised and the right lobe was homogenized and analyzed for Th1- and Th2-type cytokines. Data shown are the means ⫾ standard errors of the means (SEM) from 2 independent experiments performed using 4 mice per group per time point (*, P ⬍ 0.05; **, P ⬍ 0.01; ***, P ⬍ 0.001).

at day 10), IL-6 (P ⬍ 0.05 at day 7; P ⬍ 0.01 at day 10), IL-17A (P ⬍ 0.05 at day 7; P ⬍ 0.01 at day 10), G-CSF (P ⬍ 0.05 at days 7 and 10), and GM-CSF (P ⬍ 0.001 at day 7; P ⬍ 0.01 at day 10) in lung homogenates compared to levels detected in STAT1flfl mice (Fig. 4B to D, I, and J). LysMCreSTAT1flfl mice also had significantly increased levels of the chemokines KC/CXCL1 and RANTES/CCL5 at day 10 (P ⬍ 0.01 and P ⬍ 0.05, respectively) and MIP-1␣/CCL3 (P ⬍ 0.001 at day 10), MIP-1␤/CCL4 (P ⬍ 0.01 at days 7 and 10), and MCP-1/CCL2 (P ⬍ 0.001 at days 7 and 10) at days 7 and 10 postinoculation compared to those of STAT1flfl mice (Fig. 4F to H, K, and L). We then determined the impact of elevated proinflammatory cytokine and chemokine production in the lungs of LysMCreSTAT1flfl mice on the recruitment of leukocytes to the lung during C. neoformans H99␥ infection using flow cytometry analysis at days 7 and 10 postinfection. Significantly more leukocytes were observed in the lungs of LysMCreSTAT1flfl mice than in STAT1flfl mice (Fig. 5A) (P ⬍ 0.05) by day 10 postinoculation, correlating with the robust proinflammatory infiltrate observed in the histology (Fig. 2). There was no significant difference in total macrophages, CD4⫹ or CD8⫹ T cells, B cells, or NK cells recruited to the lungs of LysMCreSTAT1flfl and STAT1flfl mice (Fig. 5B, D to F, and I). However, we observed significantly more dendritic cells (DCs) (P ⬍ 0.01 at day 7 and P ⬍ 0.05 at day 10) (Fig. 5C), neutrophils (P ⬍ 0.01 at day 7 and P ⬍ 0.05 at day 10) (Fig. 5G), and eosinophils (P ⬍ 0.05) (Fig. 5H) at days 7 and 10 postinoculation in the lung tissues of LysMCreSTAT1flfl mice compared to levels for control STAT1flfl mice. These data indicate that a lack of STAT1 signaling in macrophages is associated with a more pronounced cellular recruitment of DCs, neutrophils, and eosinophils in mice following infection with C. neoformans H99␥. We questioned whether the robust increase in inflammatory cytokines, chemokines, and subsequent pulmonary infiltrates in

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the LysMCreSTAT1flfl mice was a consequence of the IFN-␥ produced by the genetically modified strain of C. neoformans. Therefore, LysMCreSTAT1flfl mice and STAT1flfl mice were given a pulmonary inoculation with C. neoformans strain H99 and C. neoformans strain H99␥, and cytokines and chemokines were measured in the lung tissue at 10 days postinoculation. We observed no difference in proinflammatory cytokines IL-1␣, IL-␤, IL-6, IL17A, TNF-␣, KC/CXCL1, RANTES/CCL5, MCP-1/CCL2, G-CSF, GM-CSF, MIP-1␣/CCL3, or MIP-1␤/CCL4 between STAT1flfl mice and LysMCreSTAT1flfl mice inoculated with WT C. neoformans H99 (Fig. 6). Also, no difference in proinflammatory cytokine expression was observed between STAT1flfl mice and LysMCreSTAT1flfl mice inoculated with H99 compared to STAT1flfl mice inoculated with C. neoformans H99␥ (Fig. 6). In contrast, LysMCreSTAT1flfl mice inoculated with C. neoformans H99␥ had a significant increase in all proinflammatory cytokines and chemokines measured compared to STAT1flfl mice inoculated with C. neoformans H99␥ and both strains of mice inoculated with C. neoformans H99 (Fig. 6). A similar trend was observed with Th1-associated cytokines IL-12(p70), IL-2, and IFN-␥ and immunoregulatory IL-10 (see Fig. S2 in the supplemental material). Interestingly, LysMCreSTAT1flfl mice produced the same amount of elevated IL-4 and low levels of IL-12(p70) when inoculated with WT C. neoformans H99 compared to levels for STAT1flfl mice, indicating that STAT1 signaling in macrophages does not affect the generation of a typical cryptococcus-induced Th2 type response (see Fig. S2). As the robust inflammatory response was observed only in the LysMCreSTAT1flfl mice inoculated with C. neoformans H99␥, we sought to determine if the increase in mortality of the mice was due to the increased inflammation. LysMCreSTAT1flfl mice and STAT1flfl mice were given a pulmonary inoculation with C. neoformans strain H99 and C. neoformans strain H99␥, and animals

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FIG 4 Loss of STAT1 signaling in macrophages results in an augmented proinflammatory cytokine profile in response to C. neoformans H99␥ infection. STAT1flfl and LysMCreSTAT1flfl mice were given a pulmonary infection with 104 C. neoformans H99␥ yeast cells. At days 7 and 10 postinoculation, lungs were excised and the right lobe was homogenized and analyzed for proinflammatory cytokines. Data shown are the means ⫾ SEM from 2 independent experiments performed using 4 mice per group per time point (*, P ⬍ 0.05; **, P ⬍ 0.01; ***, P ⬍ 0.001).

were monitored for survival or fungal burden assessed at 10 days postinoculation. We observed a 100% mortality rate in LysMCreSTAT1flfl mice inoculated with WT C. neoformans H99 and a 90% mortality rate in these mice following inoculation with C. neoformans strain H99␥ with a median survival rate of 20 and 19 days, respectively (Fig. 7A). STAT1flfl mice inoculated with WT C. neoformans strain H99 suffered a 90% mortality rate with median survival of 22 days; however, the STAT1flfl mice inoculated with C. neoformans strain H99␥ had a 100% survival rate (Fig. 7A) (P ⬍ 0.001), consistent with previous findings (Fig. 1A). At day 10 postinoculation, we observed no significant difference in fungal burden between the LysMCreSTAT1flfl and STAT1flfl mice inoculated with WT strain H99 (Fig. 7B). However, we did observe significantly increased pulmonary fungal burden in the STAT1flfl mice infected with WT C. neoformans H99 compared to that in mice infected with strain H99␥ (Fig. 7B)

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(P ⬍ 0.5). In addition, we also observed a significant increase in fungal burden and mortality in LysMCreSTAT1flfl mice inoculated with strain H99␥ compared to those of STAT1flfl mice (Fig. 7A [P ⬍ 0.001] and B [P ⬍ 0.01]), correlating with previous findings (Fig. 1B). Thus, the lack of any difference in mortality between LysMCreSTAT1flfl and STAT1flfl mice inoculated with WT strain H99 and LysMCreSTAT1flfl mice infected with C. neoformans strain H99␥ could not be predominantly attributed to increased proinflammatory cytokine production in LysMCreSTAT1flfl mice infected with C. neoformans strain H99␥. Taken together, the most likely cause of death in all groups of C. neoformans-infected mice appears to be due to the increase in fungal burden. Loss of macrophage-specific STAT1 signaling results in increased M2 macrophage phenotype and deficient anticryptococcal activity. Alternatively activated macrophages are ill equipped to combat infection with C. neoformans, as they are in-

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FIG 5 Macrophage-specific STAT1 ablated mice exhibit an increase in leukocyte infiltration in response to C. neoformans H99␥ infection. STAT1flfl and LysMCreSTAT1flfl mice were given a pulmonary infection with 104 C. neoformans H99␥ yeast cells. At days 7 and 10 postinoculation, lungs were excised and analyzed for pulmonary infiltrates. Leukocytes were labeled with anti-CD45 antibodies (A) or dually labeled with anti-CD45 and antibodies for specific cell types (B to I) and analyzed by flow cytometry. Data shown are the means ⫾ SEM of absolute cell numbers from 3 independent experiments performed using 4 mice per group per time point (*, P ⬍ 0.05; **, P ⬍ 0.01).

efficient killers of the yeast, leading to increased pulmonary fungal burden (24, 30, 34–39). Previous studies indicate that alternative macrophage activation in response to pulmonary C. neoformans strain H99␥ infection occurs in mice with complete ablation of STAT1 (37). Nonetheless, we were not certain that STAT1 signaling within macrophages was directly required for the anticryptococcal activity of M1 macrophages. Consequently, leukocytes were isolated from enzymatic lung digests of STAT1flfl and LysMCreSTAT1flfl mice that were given an intranasal inoculation with C. neoformans strain H99␥ on days 7 and 10 postinoculation. We enriched for macrophages and performed real-time PCR to phenotype the expression of certain transcripts associated with macrophage activation. We observed no differences in the gene expression of the majority of markers typically associated with classical macrophage activation, including iNOS (Fig. 8A), IFN-␥, TNF-␣, and CXCL10 (see Fig. S3 in the supplemental material); however, significantly decreased levels of IL-17A transcripts were observed on day 10 postinoculation in LysMCreSTAT1flfl compared to the levels in STAT1flfl mice (see Fig. S3) (P ⬍ 0.01). We also observed a decrease in transcripts for M1 macrophage-associated chemokine CXCL9, although the difference was not significant.

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No differences in the majority of transcripts typically associated with alternative macrophage activation, including CD206, FIZZ1, YM1, IL-4, and IL-13, were detected, with the exception of Arg1 (Fig. 8; also see Fig. S3 in the supplemental material). Transcripts for Arg1 were significantly increased on day 7 postinoculation in LysMCreSTAT1flfl compared to the levels in STAT1flfl mice (Fig. 8A) (P ⬍ 0.05). Overall, these data suggest a role for macrophage-specific STAT1 signaling in the induction of IL-17A and repression of Arg1 gene expression during pulmonary C. neoformans infection. Both NO and reactive oxygen species (ROS) are by-products of M1 macrophages and are thought to be important for the killing of C. neoformans by host cells (23, 47–53). Previous studies in our laboratory have shown that mice infected with C. neoformans H99␥ have increased NO production and enhanced anticryptococcal activity by macrophages compared to macrophages from mice inoculated with WT C. neoformans H99 (23, 37). Furthermore, mice with a general STAT1 deficiency do not induce a protective response to C. neoformans H99␥ correlating with decreased NO production; however, ROS production by macrophages remains intact (37). This suggests that NO produced by M1 macrophages is critical for antifungal activity while ROS may be dispens-

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FIG 6 Mice with STAT1-deficient macrophages do not have an increased proinflammatory response to C. neoformans H99 infection. STAT1flfl and LysMCreSTAT1flfl mice were given a pulmonary infection with 104 C. neoformans H99 or H99␥ yeast cells. At day 10 postinoculation, lungs were excised and the right lobe was homogenized and analyzed for proinflammatory cytokines. Data shown are the means ⫾ SEM from 2 independent experiments performed using 5 mice per group per experiment (*, P ⬍ 0.05; **, P ⬍ 0.01; ***, P ⬍ 0.001).

able. To verify the role STAT1 signaling within macrophages plays concerning the production of NO and ROS and, thus, antifungal activity, lung tissues were excised from LysMCreSTAT1flfl mice and STAT1flfl mice at days 7 and 10 postinoculation with C. neoformans H99␥. Pulmonary macrophages were isolated and analyzed for ROS production or were cultured ex vivo for 24 h, and nitrite levels were measured in the supernatants. There was no difference in ROS production by the macrophages from the two groups of mice (Fig. 8B); however, macrophages from LysMCreSTAT1flfl mice produced significantly less NO than STAT1flfl mice (Fig. 8C) (P ⬍ 0.01 at day 7 and P ⬍ 0.001 at day 10). Macrophages cultured ex vivo for 24 h were lysed to enumerate cryptococcal cells confined within the phagocytes. Macrophages from LysMCreSTAT1flfl mice contained significantly more yeast cells than macrophages isolated from STAT1flfl mice at both days 7 and 10 postinoculation (Fig. 8D) (P ⬍ 0.001). This increase in intramacrophage cryptococci correlates with decreased NO, suggesting that NO, and not ROS, is important for the anticryptococcal activity of macrophages.

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NO is required for macrophage killing of C. neoformans. Since correlation does not always equal causation, we sought to determine the requirement of NO for macrophage killing of C. neoformans H99␥. For this, STAT1flfl mice were given an intranasal inoculation with C. neoformans H99␥ and pulmonary macrophages were isolated at day 7 postinoculation, the time point when the largest amount of NO was detected in these mice (Fig. 8C). The macrophages were cultured ex vivo for 24 h in medium alone or in the presence of the NO inhibitor L-NAME. Culture with L-NAME significantly decreased the amount of NO produced by these STAT1-sufficient macrophages (Fig. 9A) (P ⬍ 0.001). At the time of isolation and at 24 h postculture, macrophages were lysed to release and enumerate the intracellular cryptococci. Macrophages cultured with L-NAME and, thus, with decreased NO production had significantly increased intracellular cryptococci (Fig. 9B) (P ⬍ 0.01), demonstrating the importance of NO for the control of intramacrophage yeast proliferation. To further confirm these observations, mice deficient in iNOS, an enzyme necessary for NO production, and WT BALB/c mice

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FIG 7 Susceptibility of LysMCreSTAT1flfl mice to C. neoformans H99␥ is not due to overexuberant inflammatory responses alone. STAT1flfl and LysMCreSTAT1flfl mice were given a pulmonary infection with 104 C. neoformans H99 or H99␥ yeast cells. Mice were observed for up to 60 days for survival analysis (A), or pulmonary fungal burden was analyzed at day 10 postinfection (B). Experiments are cumulative of 2 experiments using 4 to 5 mice per group per experiment (*, P ⬍ 0.05; **, P ⬍ 0.01; ***, P ⬍ 0.001).

were inoculated with C. neoformans H99␥. At day 7 postinoculation, macrophages were isolated from the pulmonary tissue and analyzed for ROS production to ensure that the deletion of the iNOS gene did not disrupt the making of ROS. There was no difference in ROS production between the WT and iNOS KO mice (Fig. 10A). In addition, macrophages were cultured ex vivo for 24 h, and nitrite levels were measured in the supernatants. As expected, there was significantly less NO produced by iNOS KO mice than WT mice (Fig. 10B) (P ⬍ 0.001). Alternatively, macrophages were lysed at the time of isolation and intracellular cryptococci enumerated. There was no difference in intracellular cryptococci at the time of isolation (0 h) (Fig. 10C). However, macrophages deficient in iNOS contained significantly more in-

tracellular yeast than WT macrophages at 24 h postculture (Fig. 10C) (P ⬍ 0.001), indicating that these cells are unable to control the intracellular proliferation of Cryptococcus. DISCUSSION

STAT1 is an important signaling molecule that plays a major role in mediating proinflammatory responses following ligation of the Th1-type cytokine IFN-␥, which is important for modulating protective immune responses to multiple pathogens, to its receptor (54, 55). Resistance to infection with various intracellular pathogens, such as Listeria monocytogenes and Toxoplasma gondii, relies on signaling through the IFN-␥-JAK1/2-STAT1 signaling pathway (54, 56–58). We have recently shown that STAT1 signaling is

FIG 8 STAT1 is required for M1 macrophage activation and anticryptococcal NO production. LysMCreSTAT1flfl and STAT1flfl mice were inoculated with 104

CFU C. neoformans strain H99␥. Pulmonary F4/80⫹ macrophages were isolated from the lungs at days 7 and 10 postinoculation. (A) Total RNA was extracted and transcripts analyzed for iNOS and arginase 1. The indicated mRNA levels were normalized to GAPDH. (B) Alternatively, macrophages were analyzed for ROS production. Macrophages also were cultured ex vivo for 24 h and nitrite levels measured in the supernatant (C), and macrophages were lysed and intracellular cryptococci enumerated (D). Results are expressed as means ⫾ SEM and are cumulative of three experiments using 3 to 5 mice per group per time point (*, P ⬍ 0.05; **, P ⬍ 0.01; ***, P ⬍ 0.001).

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FIG 9 Inhibition of macrophage NO production results in loss of anticryptococcal activity. STAT1flfl mice were inoculated with 104 CFU C. neoformans strain H99␥. Pulmonary F4/80⫹ macrophages were isolated from the lungs at day 7 postinoculation. (A) Macrophages were cultured in the presence or absence of the iNOS inhibitor L-NAME for 24 h, and nitrite levels in the supernatant were measured. (B) After culture, macrophages were lysed and intracellular cryptococci enumerated. Results are expressed as means ⫾ SEM and are cumulative of three experiments using 3 mice per group per time point (*, P ⬍ 0.05; **, P ⬍ 0.01; ***, P ⬍ 0.001).

essential for protection against both a WT clinical isolate of C. neoformans (strain 52D) and a virulent strain genetically engineered to produce IFN-␥ (strain H99␥) (37). However, the necessity for STAT1 signaling specifically in macrophages for anticryptococcal activity and, thus, protection against C. neoformans has not been investigated. Here, we present evidence that macrophage-specific STAT1 signaling is required for the generation of a protective immune response to C. neoformans H99␥. Our data highlight that STAT1 signaling in macrophages during C. neoformans infection is critical for the induction of M1 macrophage activation and the production of antifungal NO. The present studies were designed to evaluate the putative role of STAT1 signaling in macrophages for mediating protection against pulmonary infection with C. neoformans H99␥. Our results demonstrate that mice with macrophage-restricted STAT1 ablation were unable to clear the infection from their lungs and had increased trafficking of the yeast to the brain compared to STAT1flfl mice. The increase in trafficking of the yeast to the brain in LysMCreSTAT1flfl mice suggests a role for STAT1 signaling in the prevention of dissemination. One theory posits that macrophages can act as a Trojan horse, carrying cryptococcal cells across the blood-brain barrier (reviewed in reference 59). Perhaps the inability of STAT1-deficient macrophages to efficiently kill the yeast cells allows for their translocation to the CNS. Even with STAT1 signaling intact in all other cell types, the LysMCreSTAT1flfl mice were rendered susceptible to C. neoformans H99␥ infection due to the lack of STAT1 in the macro-

phages, demonstrating the critical role of this signaling pathway in these phagocytes for protection. The data further show that loss of STAT1 signaling in macrophages does not result in deficient leukocyte accumulation in the lungs but actually shows a trend toward increased leukocyte recruitment and some, but not all, proinflammatory cytokines and chemokines compared to STAT1flfl mice following C. neoformans H99␥ inoculation. The LysMCreSTAT1flfl mice had increased levels of chemokines KC/CXCL1 and MCP-1/CCL2, which correlated with increased trafficking of neutrophils and DCs, respectively. Previous studies in our laboratory demonstrated that the depletion of neutrophils does not affect pulmonary fungal burden, and these phagocytes are not required for clearance of C. neoformans strain H99␥ (46). Our results correlate with another study which showed that neutropenic mice are no more susceptible to infection and that the lack of neutrophils actually may be a benefit to the host during C. neoformans infection (60). Major cryptococcal capsular polysaccharide glucuronoxylomannan (GXM) induces cytokine production, including IL-1␤, by neutrophils (61). The increased influx of neutrophils correlates with the robust production of IL-1␤ in the lungs of LysMCreSTAT1flfl mice in response to C. neoformans H99␥, which indicates the induction of the Nlrp3 inflammasome in various cell types. IL-1␤ and IL-1R␣ were upregulated in a genetically resistant mouse model which developed protective Th1-type responses to moderately virulent clinical isolate C. neoformans strain 52D; thus, it is associated with resistance (62). However, this is in opposition to our observations in which

FIG 10 NO is critical for the anticryptococcal activity of macrophages. iNOS-deficient and WT BALB/c mice were given a pulmonary infection with 104 C.

neoformans H99␥ yeast cells. Pulmonary F4/80⫹ macrophages were isolated from the lungs of WT and iNOS KO mice at day 7, and macrophages were examined for ROS production (A) or cultured ex vivo for 24 h, and supernatants were analyzed for the presence of nitrite (B). (C) Alternatively, macrophages were lysed at time zero and 24 h after ex vivo culture to release and enumerate intracellular cryptococci. Data are expressed as the means ⫾ SEM and are cumulative of three experiments using 3 to 4 mice per group per time point (***, P ⬍ 0.001).

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increased IL-1␤ is unable to induce a strong Th1-type immune response and subsequent protection in macrophage-specific STAT1-ablated mice. Additionally, we observed increased accumulation of DCs and eosinophils following macrophage-specific STAT1 deletion, findings previously linked to protective and nonprotective responses to C. neoformans, respectively (37, 63–67). The increased presence of eosinophils in the LysMCreSTAT1flfl mice is not surprising, as these mice are not undergoing a protective response to the infection with C. neoformans H99␥. Conversely, the increase in DCs, which are capable of killing C. neoformans (63–65), is surprising and indicates that DCs alone are unable to provide protection. It has been established that the LysMCre system can result in 16% deficiency of the gene of interest in splenic DCs (45). Here, we show that DCs from LysMCreSTAT1flfl mice are able to kill C. neoformans strain H99 as efficiently as DCs from the STAT1flfl mice ex vivo; therefore, fungal burden expansion is not due to an inability of the DCs to kill the yeast. Collectively, with the impaired clearance in LysMCreSTAT1flfl mice, the cellular accumulation data suggest that STAT1 signaling in macrophages plays a balancing role during cryptococcal infection by both supporting clearance of infection and counterregulating excessive inflammatory pathology associated with increased accumulation of leukocytes in the infected lungs. This conclusion is being made cautiously, because the IFN-␥ produced by the genetically modified strain of C. neoformans could be contributing to the massive proinflammatory response observed in the LysMCreSTAT1flfl mice. Our previous studies show no significant difference in IFN-␥ protein levels in the lungs of IFN-␥ KO mice compared to those of WT mice infected with C. neoformans H99␥ (37). Interestingly, only LysMCreSTAT1flfl mice inoculated with the C. neoformans strain H99␥ displayed the overexuberant proinflammatory response, and the levels of IFN-␥ in the lungs increased by only 20 pg/ml, which is low and likely not biologically relevant. However, the microenvironment immediately surrounding the IFN-␥-producing C. neoformans cell is likely to have a higher level of IFN-␥ protein, which could be binding to its receptor on the host immune cells, driving the production of Th1-associated and proinflammatory cytokines and contributing to pathology. Altogether, these data indicate that the inflammation observed during infection with the IFN-␥-producing strain likely is induced by the small boost the mutant strain provides. However, this IFN-␥ production and subsequent inflammation does not have a significant impact on the median survival or fungal burden compared to that of infection with the WT C. neoformans strain H99. This indicates that although the substantial inflammatory response in C. neoformans H99␥-infected LysMCreSTAT1flfl mice likely is contributing to pathology, the inability of the STAT1-deficient macrophages to kill the yeast cells plays the definitive role in the reduction in survival. Macrophage activation phenotype is a crucial determinant of the outcome of cryptococcal infections (23–25, 34, 35, 37, 42, 68). The present studies show that STAT1-deficient macrophages have a shift in the iNOS-to-Arg1 ratio in favor of Arg1, an indicator of M2 macrophage activation, as these two enzymes compete for the substrate L-arginine. M1-activated macrophages are classified by their production of NO and ROS in response to a myriad of pathogens. Many fungal pathogens, including C. neoformans, Histoplasma capsulatum, Blastomyces dermatitidis, and Coccidioides immitis, are sensitive to NO produced by M1 macrophages and have

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developed various mechanisms to suppress or prevent NO production (reviewed in reference 36). C. neoformans, for example, has been shown to downregulate macrophage iNOS gene expression, suppressing NO (69). In the absence of STAT1 signaling in macrophages, these phagocytes are unable to produce NO in response to C. neoformans infection, correlating with previous findings that the activation of STAT1 signaling within macrophages is required for NO production (70). It was interesting that although there was no difference in gene expression for iNOS, the activity of the enzyme in STAT1-sufficient macrophages was significantly increased, as evidenced by NO production. Additionally, deficiency in NO production, due to either STAT1 or iNOS deletion, resulted in increased intramacrophage fungal burden even in the presence of intact ROS production. These findings draw a parallel with previous studies indicating an important role for macrophage NO in killing and control of cryptococcal infections (23, 25, 37, 41, 59, 71). Here, we definitively show that NO production by STAT1-mediated classically activated macrophages is essential for protection against C. neoformans. Therefore, the development of therapeutics that induce M1 macrophage activation via an IFN-␥/ STAT1 mechanism may provide protection against a myriad of microbial pathogens that are susceptible to killing by M1 macrophages. There is a concern that treatments relying on production of NO by human macrophages may not be feasible due to the epigenetic gene silencing by CpG methylation, histone modifications, and chromatin compaction of the human iNOS gene (72). However, this contrasts with other studies which demonstrate the induction of iNOS and functional NO production in granulomatous tissues in both humans and cynomolgus macaques (73, 74). Cryptococcal granulomas in both rats and humans have been shown to house iNOS⫹ macrophages, indicating a role for NO in anticryptococcal properties of these granulomas (73, 75). Thus, immunotherapeutics targeting the stimulation of STAT1 in macrophages to promote NO production could prove fruitful against not only C. neoformans but also other pathogens where NO is an effective antimicrobial. Altogether, these data demonstrate that STAT1 signaling in macrophages plays a role in the regulation of the Th1/Th2 response, control of inflammation, and overall protection against C. neoformans H99␥, whether directly (by cytokine/chemokine production) or indirectly (by killing the yeast). Furthermore, NO production by STAT1-mediated M1 macrophages is critical for control of the intracellular growth of C. neoformans. Even in the presence of other immune cells with demonstrated anticryptococcal activity, including DCs, neutrophils, and NK cells (61, 63, 64, 76, 77), the loss of macrophages capable of polarizing to an M1 phenotype is crippling to the host’s ability to mount protective responses. We definitively show that STAT1-mediated M1 macrophage activation is crucial to anticryptococcal responses, providing an effector cell population and mechanism by which the host executes protective responses against C. neoformans. ACKNOWLEDGMENTS F.L.W. was supported by research grants 2RO1AI071752 and R21AI100893 from the National Institute of Allergy and Infectious Diseases (NIAID) of the National Institutes of Health (NIH) and by the Army Research Office of the Department of Defense, no. W911NF-11-1-0136 (F.L.W.). M.A.O. is supported by the Biomedical Research and Development Service, Department of Veterans Affairs, merit grant

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M1 Macrophages Protect against C. neoformans

5I01BX000656-06. M.M. is supported by SFB F28 from the Austrian Science Fund FWF. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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M1 Macrophages Protect against C. neoformans

Olszewski MA. 2013. Macrophage M1/M2 polarization dynamically adapts to changes in cytokine microenvironments in Cryptococcus neoformans infection. mBio 4:e00264-13. http://dx.doi.org/10.1128/mBio .00264-13. 72. Gross TJ, Kremens K, Powers LS, Brink B, Knutson T, Domann FE, Philibert RA, Milhem MM, Monick MM. 2014. Epigenetic silencing of the human NOS2 gene: rethinking the role of nitric oxide in human macrophage inflammatory responses. J Immunol 192:2326 –2338. http://dx .doi.org/10.4049/jimmunol.1301758. 73. Facchetti F, Vermi W, Fiorentini S, Chilosi M, Caruso A, Duse M, Notarangelo LD, Badolato R. 1999. Expression of inducible nitric oxide synthase in human granulomas and histiocytic reactions. Am J Pathol 154:145–152. http://dx.doi.org/10.1016/S0002-9440(10)65261-3. 74. Mattila JT, Ojo OO, Kepka-Lenhart D, Marino S, Kim JH, Eum SY, Via LE, Barry CE, III, Klein E, Kirschner DE, Morris SM, Jr, Lin PL, Flynn

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JL. 2013. Microenvironments in tuberculous granulomas are delineated by distinct populations of macrophage subsets and expression of nitric oxide synthase and arginase isoforms. J Immunol 191:773–784. http://dx .doi.org/10.4049/jimmunol.1300113. 75. Goldman D, Cho Y, Zhao M, Casadevall A, Lee SC. 1996. Expression of inducible nitric oxide synthase in rat pulmonary Cryptococcus neoformans granulomas. Am J Pathol 148:1275–1282. 76. Oykhman P, Timm-McCann M, Xiang RF, Islam A, Li SS, Stack D, Huston SM, Ma LL, Mody CH. 2013. Requirement and redundancy of the Src family kinases Fyn and Lyn in perforin-dependent killing of Cryptococcus neoformans by NK cells. Infect Immun 81:3912–3922. http://dx .doi.org/10.1128/IAI.00533-13. 77. Lipscomb MF, Alvarellos T, Toews GB, Tompkins R, Evans Z, Koo G, Kumar V. 1987. Role of natural killer cells in resistance to Cryptococcus neoformans infections in mice. Am J Pathol 128:354 –361.

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STAT1 signaling within macrophages is required for antifungal activity against Cryptococcus neoformans.

Cryptococcus neoformans, the predominant etiological agent of cryptococcosis, is an opportunistic fungal pathogen that primarily affects AIDS patients...
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