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DAP12 Inhibits Pulmonary Immune Responses to Cryptococcus neoformans Lena J. Heung,a Tobias M. Hohla,b Infectious Diseases Service, Department of Medicine,a and Immunology Program, Sloan Kettering Institute,b Memorial Sloan Kettering Cancer Center, New York, New York, USA

Cryptococcus neoformans is an opportunistic fungal pathogen that is inhaled into the lungs and can lead to life-threatening meningoencephalitis in immunocompromised patients. Currently, the molecular mechanisms that regulate the mammalian immune response to respiratory cryptococcal challenge remain poorly defined. DAP12, a signaling adapter for multiple pattern recognition receptors in myeloid and natural killer (NK) cells, has been shown to play both activating and inhibitory roles during lung infections by different bacteria and fungi. In this study, we demonstrate that DAP12 plays an important inhibitory role in the immune response to C. neoformans. Infectious outcomes in DAP12ⴚ/ⴚ mice, including survival and lung fungal burden, are significantly improved compared to those in C57BL/6 wild-type (WT) mice. We find that eosinophils and macrophages are decreased while NK cells are increased in the lungs of infected DAP12ⴚ/ⴚ mice. In contrast to WT NK cells, DAP12ⴚ/ⴚ NK cells are able to repress C. neoformans growth in vitro. Additionally, DAP12ⴚ/ⴚ macrophages are more highly activated than WT macrophages, with increased production of tumor necrosis factor (TNF) and CCL5/RANTES and more efficient uptake and killing of C. neoformans. These findings suggest that DAP12 acts as a brake on the pulmonary immune response to C. neoformans by promoting pulmonary eosinophilia and by inhibiting the activation and antifungal activities of effector cells, including NK cells and macrophages.

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he environmental encapsulated yeast Cryptococcus neoformans is a common cause of invasive fungal infections in immunocompromised patients, including those with AIDS, solid organ transplants, and hematologic malignancies (1). When C. neoformans cells are inhaled into the lung, healthy individuals typically clear the infectious particles in an asymptomatic manner. However, in immunocompromised hosts, C. neoformans can proliferate in the lung, disseminate to the brain, and cause meningoencephalitis. Despite contemporary combination antifungal therapy, the survival rate for disseminated cryptococcosis approaches only 70% (2). Furthermore, the at-risk population for cryptococcosis is expanding due to the development and increased availability of novel immunosuppressive regimens for autoimmunity, transplantation, and cancer. An important approach to developing more efficacious therapies for cryptococcosis is to decipher the initial immune response in the lung that regulates fungal proliferation and tissue invasion. Classically, host defenses are triggered when fungal pathogen-associated molecular patterns (PAMPs) are detected by immune cell pattern recognition receptors (PRRs), such as C-type lectin receptors (CLRs) or Toll-like receptors (TLRs). Studies on the intracellular signaling molecules CARD9 (3) and MyD88 (4–6), which can mediate events downstream of CLRs and TLRs or interleukin-1 receptor superfamily members, respectively, suggest that these pathways play a role in mounting an effective immune response to C. neoformans. However, so far, only TLR9 and mannose receptor, which does not contain intracellular signaling motifs, have been identified as potential PRRs for C. neoformans in vivo (6–10). There are conflicting reports regarding the importance of TLR2 (4, 5, 11), and other PRRs for fungal cell wall components, including Dectin-1, Dectin-2, and TLR4, were found to have limited roles, if any, in disease outcomes after C. neoformans challenge (4, 5, 11–14). Furthermore, cryptococcosis has not been a prominent infectious complication in humans with Mendelian

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defects in CARD9 and MyD88 signaling (15, 16). Thus, work remains to identify key signaling pathways that regulate the immune response to C. neoformans. DAP12 is a transmembrane signaling adapter that contains a cytoplasmic immunoreceptor tyrosine-based activation motif (ITAM) and pairs to numerous receptors in myeloid and natural killer (NK) cells, including CLRs (e.g., MDL-1 and Ly49 receptors) and members of the immunoglobulin superfamily (e.g., TREM-1, -2, and -3) (17–20). DAP12 has been shown to have both activating and suppressive effects on myeloid and NK cell function (17–21). In the context of host-pathogen interactions, DAP12 signaling can have both beneficial and detrimental consequences. For example, adenovirus-mediated overexpression of DAP12 can enhance clearance of the pathogenic mold Aspergillus fumigatus in a neutropenic murine model of invasive pulmonary aspergillosis (22). The absence of DAP12 signaling increases the susceptibility of mice to influenza virus (23), murine cytomegalovirus (24), and the bacterium Salmonella enterica (25). In contrast, several studies have shown that the absence of DAP12 improves the immune response to pulmonary mycobacterial infection (26,

Received 4 April 2016 Accepted 5 April 2016 Accepted manuscript posted online 11 April 2016 Citation Heung LJ, Hohl TM. 2016. DAP12 inhibits pulmonary immune responses to Cryptococcus neoformans. Infect Immun 84:1879 –1886. doi:10.1128/IAI.00222-16. Editor: G. S. Deepe, Jr., University of Cincinnati Address correspondence to Tobias M. Hohl, [email protected]. Supplemental material for this article may be found at http://dx.doi.org/10.1128 /IAI.00222-16. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

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27) and to systemic infection with the bacterium Listeria monocytogenes (28). The goal of this study was to determine whether DAP12 signaling plays a functional role in the host response to C. neoformans. Here, we present data that indicate a detrimental role for DAP12 signaling during respiratory C. neoformans challenge. Infectious outcomes of survival and lung fungal burden are improved in DAP12⫺/⫺ mice, and, as specific mechanisms, we find that deletion of DAP12 reduces pulmonary eosinophilia and improves the anticryptococcal activity of NK cells and macrophages. Thus, our findings reveal a novel pathway by which C. neoformans may subvert mammalian signaling mechanisms to suppress an effective host immune response in the lung. MATERIALS AND METHODS Chemicals and reagents. Chemicals were from Sigma-Aldrich, cell culture reagents were from Life Technologies/Gibco, and microbiological culture media were from BD Biosciences unless otherwise noted. Antibodies for flow cytometry were purchased from BD Biosciences or eBioscience unless otherwise indicated. Mice. C57BL/6 and RAG1⫺/⫺ mice were purchased from Jackson Laboratory. DAP12⫺/⫺ mice (29) were provided by Merck Sharp & Dohme Corporation. All mouse strains were bred and housed in the Research Animal Resource Center of the Memorial Sloan Kettering Cancer Center (MSKCC) under specific-pathogen-free conditions. All animal experiments were conducted with sex- and age-matched mice and performed with approval from the MSKCC Institutional Animal Care and Use Committee. Infection with Cryptococcus neoformans. C. neoformans serotype A strain H99 was a generous gift from Joseph Heitman (Duke). A green fluorescent protein (GFP)-expressing C. neoformans H99 strain (30) was a generous gift from Robin May (University of Birmingham). All C. neoformans strains were maintained as frozen glycerol stocks at ⫺80°C and then grown on Sabouraud dextrose agar plates, followed by overnight culture in YPD medium (1% yeast extract, 2% peptone, 2% dextrose) on a shaker at 37°C. Cells were washed twice in sterile phosphate-buffered saline (PBS) and resuspended in PBS at a concentration of 103 cells per 50-␮l volume, unless otherwise noted. The concentration was confirmed by plating serial dilutions of the suspension on Sabouraud dextrose agar. Mice were anesthetized with inhaled isoflurane, and 50 ␮l of the fungal cell suspension was given intratracheally using a blunt-ended 20-gauge needle. Analysis of infected mice. To assess fungal burden, murine lungs and brains were mechanically homogenized in PBS using a PowerGen 125 homogenizer (Fisher). Lymph nodes were dissociated using ground glass slides. CFU in all tissues were counted after plating serial dilutions of the homogenates on Sabouraud dextrose agar. For flow cytometry analysis, single-cell lung suspensions were prepared as previously described (31) using digestion with DNase I (Roche) and collagenase type 4 (Worthington Biochemical) along with mechanical disruption using a gentleMACS dissociator (Miltenyi Biotec). Lung cells were counted and stained using the following antibodies: anti-CD45 (clone 30-F11), anti-Ly6G (clone 1A8), anti-CD11b (clone M1/70), antiCD11c (clone HL3), anti-major histocompatibility complex (MHC) class II (clone M5/114.15.2), anti-Ly6C (clone AL-21), anti-CD103 (clone 2E7), anti-SiglecF (clone E50-2440), anti-CD3ε (clone 17A2), anti-CD5 (clone 53-7.3), anti-CD8 (clone 53-6.7), anti-CD19 (clone eBoi1D3), anti-NK1.1 (clone PK136), anti-CD90.2 (clone 53-2.1), anti-CD127 (clone A7R34), and anti-TcR␤ (clone H57-597). Flow cytometry data were collected on a BD LSR II flow cytometer and analyzed with FlowJo (v9.7.6). Histopathology. The lungs of euthanized mice were perfused with 4% paraformaldehyde (PFA) in situ via a catheter inserted through an incision in the trachea. The entire lung was then harvested and fixed by immersion

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in 4% PFA. Lungs were then processed by the MSKCC Molecular Cytology Core Facility (MCCF) to generate 4-␮m sections of paraffin-embedded lungs stained with hematoxylin and eosin (H&E). Slides were scanned using a Zeiss Mirax Midi slide scanner with 20⫻/0.8-numerical-aperture (NA) objective in the MCCF. Slides were reviewed by a pathologist in the MSKCC Laboratory of Comparative Pathology. Morphometry analysis was carried out on scanned slide images using Pannoramic Viewer (v1.15.3; 3DHISTECH). Areas of lung inflammation were measured and expressed as the percentage of total lung area in each histologic section. NK cell assays. To isolate NK cells, spleens from DAP12⫺/⫺ or C57BL/6 mice were extruded through fine-mesh metal screens in RPMI with 10% fetal bovine serum (FBS) and 0.1 mg/ml DNase I. Red blood cells (RBCs) were lysed using RBC lysis buffer (Tonbo). The remaining splenocytes were enriched for NK cells by incubating with rat IgG antimouse CD4, CD8, CD19, and Ter119 followed by BioMag goat anti-rat IgG magnetic beads (Qiagen) and magnetic depletion to remove T and B cells and RBCs as previously described (32); all reagents were generously provided by Joseph Sun (MSKCC). The NK cell-enriched suspension was then stained with DAPI (4=,6=-diamidino-2-phenylindole) and antiCD45, anti-CD3, anti-CD5, anti-CD19, anti-NK1.1, anti-CD11c, and anti-TcR␤ antibodies. NK cells were sorted using a BD Aria cell sorter in the MSKCC Flow Cytometry Core Facility. Purified NK cells were then incubated with GFP-expressing C. neoformans strain H99 in 96-well tissue culture-treated flat-bottom plates for 24 h in RPMI with 10% FBS at 37°C with 5% CO2. To measure killing, cells were harvested from each experimental well and incubated for 30 min at room temperature in sterile distilled water to lyse all murine cells. Serial dilutions were then grown on Sabouraud dextrose agar at 37°C, and CFU were counted. Recombinant mouse interleukin-12 (IL-12) (R&D) was added to some experimental wells at 10 ng/ml. BMDM assays. To generate bone marrow-derived macrophages (BMDM), total bone marrow cells from DAP12⫺/⫺ or C57BL/6 mice were cultured in RPMI containing 30% L929-cell conditioned medium and 20% FBS at 37°C with 5% CO2 (the procedure was adapted from that described in reference 33). Medium was replaced on day 3 of culture. On day 5 of culture, adherent BMDM were harvested by gently washing cells off the plate with PBS. BMDM were then incubated with GFP-expressing C. neoformans strain H99 in 24-well flat-bottom culture plates for 24 h in RPMI with 10% FBS at 37°C with 5% CO2. The murine monoclonal antibody 18B7 (a generous gift from Arturo Casadevall at Johns Hopkins), which binds glucuronoxylomannan in the cryptococcal capsule and facilitates phagocytosis by macrophages (34), was added to all wells at a concentration of 10 ␮g/ml. To measure uptake of C. neoformans by BMDM, cells were harvested from each experimental well and stained with anti-CD45, anti-CD11b, and anti-F4/80 (clone BM8) antibodies. Samples were analyzed by flow cytometry for uptake of the fungal GFP signal by CD45⫹ CD11b⫹ F4/80⫹ macrophages. To measure killing of C. neoformans by BMDM, cells were harvested from each experimental well and incubated for 30 min at room temperature in sterile distilled water to lyse all murine cells. Serial dilutions were then grown on Sabouraud dextrose agar at 37°C, and CFU were counted. Cytokine measurements. Levels of cytokines were measured by enzyme-linked immunosorbent assay (ELISA) of lung homogenate supernatant or BMDM cell culture supernatant using Ready-Set-Go ELISA kits (eBioscience), except that DuoSet ELISA development systems kits (R&D) were used to measure CCL5/RANTES, IL-5, IL-1␣, granulocytemacrophage colony-stimulating factor (GM-CSF), CXCL1/KC, CXCL2/ MIP2, and CCL3/MIP1␣. Whole lungs were homogenized in PBS containing cOmplete protease inhibitor cocktail (Roche). Statistical analysis. All results are expressed as mean ⫾ standard error of the mean (SEM). A Mann-Whitney U test was used for statistical analysis of two-group comparisons. Survival data were analyzed with the Mantel-Cox test. All statistical analyses were performed with

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FIG 2 DAP12 does not regulate lung histopathology after C. neoformans chalFIG 1 DAP12 deletion improves survival and lung fungal burden after C.

neoformans challenge. DAP12⫺/⫺ mice (black circles) and WT mice (white circles) were challenged with C. neoformans strain H99 via the intratracheal route. (A) Kaplan-Meier survival plot of mice given 103 C. neoformans cells. Data were pooled from two independent experiments (n ⫽ 10 or 11 total mice per group). (B) Kaplan-Meier survival plot of mice given 104 C. neoformans cells. Data represent one experiment (n ⫽ 4 or 5 mice per group). (C) CFU in lung homogenates on days 7 and 14 p.i. from mice infected with 103 C. neoformans cells. Data were pooled from five independent experiments (n ⫽ 18 to 22 total mice per group). **, P ⬍ 0.01; ****, P ⬍ 0.0001.

GraphPad Prism software, v6.0f. A P value of ⬍0.05 was considered significant.

RESULTS

DAP12 deletion improves survival and reduces lung fungal burden after respiratory challenge with C. neoformans. To determine if DAP12 signaling is beneficial or detrimental following C. neoformans challenge, DAP12⫺/⫺ and C57BL/6 wild-type (WT) mice were infected with the highly virulent C. neoformans strain H99 via the intratracheal route and monitored for survival. After challenge with a low inoculum of 103 C. neoformans cells, DAP12⫺/⫺ mice survived for a median of 31 days, compared to 26 days for WT mice, resulting in a difference of 5 days (Fig. 1A). After challenge with a high inoculum of 104 C. neoformans cells, DAP12⫺/⫺ mice survived for a median of 24 days, and WT mice survived for 20 days, resulting in a difference of 4 days (Fig. 1B). Based on these results, we chose an inoculum of 103 C. neoformans cells for use in all subsequent in vivo experiments.

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lenge. (A) Representative images of whole-lung tissue sections from DAP12⫺/⫺ and WT mice on day 14 p.i. stained with H&E (scale bars ⫽ 2,000 ␮m). Insets show an area of inflammation in each lung at a magnification of ⫻40 (scale bars ⫽ 50 ␮m). C. neoformans cells are indicated by black arrowheads. (B) The percent area of lung inflammation was calculated from three sections per lung on day 14 p.i. using morphometry (n ⫽ 3 per group).

In accordance with the survival data, the lung fungal burden was significantly lower in DAP12⫺/⫺ mice than in WT controls on day 14 postinfection (p.i.) (Fig. 1C). There was a trend toward a lower fungal burden in the mediastinal lymph nodes in DAP12⫺/⫺ mice than in WT mice on days 7 and 14 p.i. (see Fig. S1A in the supplemental material). Brain dissemination was minimal at day 14 p.i. in this respiratory challenge model of cryptococcosis, but there was a trend toward lower fungal burden in DAP12⫺/⫺ brains than in those of WT mice (see Fig. S1B in the supplemental material). Collectively, these studies indicate that DAP12⫺/⫺ mice are more resistant to a lethal inoculum of C. neoformans and achieve fungal control within the lung for a longer period of time than WT mice. DAP12 does not regulate lung histopathology and cytokine responses. To assess whether DAP12 signaling contributes to histopathology in the lung following C. neoformans challenge, histologic examination of H&E-stained lung sections from DAP12⫺/⫺ and WT mice on day 14 p.i. was performed. There were no significant differences upon microscopic examination (Fig. 2A). Within all sections analyzed, there were moderate, multifocal, and randomly distributed perivascular and peribronchiolar inflammatory aggregates composed of a mixed population of

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FIG 3 Lung cytokine responses after C. neoformans challenge are not altered by DAP12 deletion. Cytokines were measured in the supernatants of whole lung homogenates from DAP12⫺/⫺ and WT mice on day 7 (A) and day 14 (B) p.i.

macrophages and multinucleated giant cells, eosinophils, and fewer numbers of neutrophils, lymphocytes, and plasma cells. Necrosis was not a prominent feature in any of the sections, but alveolar edema and fibrin accumulation were present in the more severely affected areas. Quantifying the area of inflammation by morphometry revealed a slight trend toward less inflammation in DAP12⫺/⫺ than in WT lungs (Fig. 2B), consistent with a lower fungal burden in DAP12⫺/⫺ mice at this time point. To determine if proinflammatory signals are altered in the lung after C. neoformans challenge, global cytokine profiling was carried out on supernatants from whole lung homogenates isolated from infected DAP12⫺/⫺ and WT mice. The results showed no significant differences between DAP12⫺/⫺ and WT mice on day 7 or 14 p.i. (Fig. 3A and B). In addition to the cytokines shown, IL-6, IL-1␣, GM-CSF, CXCL1/KC, CXCL2/MIP2, and CCL3/MIP1a were measured on day 7 p.i. and showed no significant differences between DAP12⫺/⫺ and WT mice (data not shown). Thus, the histopathology and analysis of whole-lung cytokine profiles were similar for DAP12⫺/⫺ and WT mice. DAP12 acts in innate immune cells to promote detrimental effects on murine survival during cryptococcosis. DAP12 is expressed predominantly in myeloid cells and in NK cells, but there is some expression in T and B cells (17). To determine whether DAP12 signaling in T and B cells contributes to detrimental effects after respiratory challenge with C. neoformans, we compared the survival of recombinase-activating gene 1 knockout (RAG1⫺/⫺) mice, which lack T and B cells, to the survival of C57BL/6 WT controls. We did not observe a difference in survival between the two mouse strains after they were challenged with 103 C. neoformans cells (see Fig. S2 in the supplemental material). Thus, loss of

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DAP12 signaling in T and B cells does not account for the improvement in infectious outcomes observed in DAP12⫺/⫺ mice. This result is consistent with the notion that DAP12 acts in myeloid or NK cells to repress the pulmonary immune response to C. neoformans. DAP12 deletion alters recruitment of leukocyte subsets to the lungs. Although there were no differences in lung histopathology or global cytokine production, this does not preclude differences in absolute numbers of immune cell subsets in the lungs. Given the similar survival of RAG1⫺/⫺ and WT mice, we focused our analysis on innate immune cells and quantified leukocyte subsets in the lungs of DAP12⫺/⫺ and WT mice challenged with C. neoformans (gating strategies are shown in Fig. S3 in the supplemental material). We determined that there were no significant differences in total number of CD45⫹ cells, neutrophils, Ly6Chi monocytes, CD11b⫹ dendritic cells (DCs), CD103⫹ DCs, and total innate lymphoid cells (ILCs) in the lungs of DAP12⫺/⫺ and WT mice on day 7 or 14 p.i. (Fig. 4). However, the lungs of DAP12⫺/⫺ mice contained fewer macrophages (CD45⫹ Ly6G⫺ MHCIIlo SSC-Ahi SiglecF⫹ CD11chi) than control lungs at both time points examined (Fig. 4). On day 14 p.i., eosinophils (CD45⫹ Ly6G⫺ MHCIIlo SSC-Ahi SiglecF⫹ CD11clo) were significantly lower in DAP12⫺/⫺ lungs, and, conversely, NK cells (Lin⫺ CD45⫹ TcR␤⫺ NK1.1⫹) were significantly higher in DAP12⫺/⫺ lungs, than in WT lungs (Fig. 4). Thus, loss of DAP12 signaling is associated with a decrease in eosinophil and macrophage recruitment, while NK cell recruitment is enhanced after C. neoformans challenge. DAP12 deletion imparts fungistatic activity to NK cells against C. neoformans. Since NK cells were more highly recruited to the lungs of DAP12⫺/⫺ mice than to those of WT mice during infection, we next examined NK cell antifungal activity on the basis of DAP12 expression. Splenic NK cells were sorted from DAP12⫺/⫺ and WT mice to a purity of 91 to 99% and cultured in vitro for 24 h with GFP-expressing C. neoformans cells at an NK/ fungal cell ratio of 100:1. C. neoformans cells incubated with DAP12⫺/⫺ NK cells had decreased growth compared to those incubated with WT NK cells (Fig. 5). This appears to be a fungistatic effect, since DAP12⫺/⫺ NK cells did not lower the number of CFU below that of C. neoformans cultured in the absence of NK cells (Fig. 5). Because IL-12 has been demonstrated to regulate the antifungal properties of NK cells (35), we next added 10 ng/ml recombinant mouse IL-12 to the cocultures, but we did not observe a significant change in NK cell anticryptococcal activity in our assay (data not shown). Overall, these data indicate that DAP12⫺/⫺ NK cells have fungistatic activity against C. neoformans in vitro, in contrast to their WT counterparts. DAP12 deletion enhances macrophage cytokine production and fungicidal activity against C. neoformans. Given our observation that lung macrophages are decreased in infected DAP12⫺/⫺ mice compared to WT mice, we tested whether DAP12⫺/⫺ macrophages were permissive for C. neoformans growth in vitro. Bone marrow-derived macrophages (BMDM) from DAP12⫺/⫺ and WT mice were cultured in vitro with GFP-expressing C. neoformans cells at two different BMDM/fungal cell ratios. Analysis of culture supernatants by ELISA demonstrated that DAP12⫺/⫺ BMDM produced significantly increased amounts of cytokines, including tumor necrosis factor (TNF) (Fig. 6A) and CCL5/RANTES (Fig. 6B), compared to those produced by WT BMDM. Gamma interferon (IFN-␥), IL-6, IL-10, IL-12, and IL-23 levels were also measured, but the majority of the samples had cytokine levels

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FIG 4 DAP12 regulates the recruitment of innate immune cells to the lungs after C. neoformans challenge. Innate immune cells were quantitated by flow

cytometry of lungs from DAP12⫺/⫺ and WT mice on days 7 and 14 p.i. Data on macrophages and eosinophils from day 7 p.i. represent one experiment (n ⫽ 5 per group). Remaining data were pooled from two independent experiments (n ⫽ 7 to 9 total mice per group). *, P ⬍ 0.05; **, P ⬍ 0.01.

below the limit of detection. Interestingly, DAP12⫺/⫺ BMDM exhibited a 60 to 90% increase in cryptococcal uptake (Fig. 6C) and an ⬃40% improvement in cryptococcal killing (Fig. 6D) compared to WT BMDM. Therefore, the absence of DAP12 signaling in macrophages increases their effector functions, as measured by cytokine production and by cryptococcal uptake and killing. DISCUSSION

Establishing the molecular routes by which C. neoformans triggers and subverts host immune defense mechanisms is essential to de-

FIG 5 DAP12 deletion enhances the fungistatic activity of NK cells. Purified splenic NK cells from DAP12⫺/⫺ (black circles) and WT (white circles) mice were incubated with C. neoformans for 24 h in vitro. For reference, the growth of C. neoformans in the absence of NK cells is shown (gray circles). Data were pooled from two independent experiments (n ⫽ 8 to 11 total replicates per group). ****, P ⬍ 0.0001. n/a, not applicable.

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fining immune modulatory strategies to improve the treatment of cryptococcosis. In this work, we have discovered that DAP12 signaling following C. neoformans challenge contributes to an immune response that is detrimental to the host. Although other studies have demonstrated that DAP12 can be either beneficial or detrimental depending on the microbial and tissue context (22– 28), this study represents the first evidence that DAP12 signaling can be unfavorable to the host during a fungal infection. The downstream effects of this DAP12-mediated pathway appear to be 3-fold: increased pulmonary eosinophilia and suppression of the antifungal activity of both NK cells and macrophages. Our results show that DAP12 deletion is associated with a decrease in pulmonary eosinophilia, which, in turn, correlates with prolonged survival. In humans, peripheral eosinophilia has been reported in disseminated cryptococcosis (36–38) and in cryptococcal pneumonia (39, 40), and eosinophils have been found in pleural effusions associated with pulmonary cryptococcosis (39) and in cryptococcal epidural abscesses (41). In mouse models of pulmonary cryptococcosis, pulmonary eosinophilia is observed as well (42–45). The degree of eosinophilia appears to correlate directly with susceptibility of different mouse strains to C. neoformans (42), and increasing eosinophils by IL-5 overexpression worsens infectious outcomes (46). Eosinophils may cause detrimental effects by promoting Th2 instead of protective Th1 responses (47). It has been reported that DAP12 is expressed in eosinophils (17, 19), so it is possible that deletion of DAP12 disrupts intrinsic mechanisms that would otherwise enable their recruitment and Th2 activation. Future studies using con-

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FIG 6 DAP12 deletion improves the response of bone marrow-derived macrophages to C. neoformans. BMDM generated from DAP12⫺/⫺ (black bars) and WT (white bars) mice were incubated for 24 h in vitro with GFP-expressing C. neoformans at two BMDM/fungal cell ratios. (A and B) Culture supernatants were analyzed for levels of cytokines TNF (WT mean ⫽ 19.4 pg/ml at 40:1 and 6.3 pg/ml at 80:1) (A) and CCL5/RANTES (WT mean ⫽ 188.9 pg/ml at 40:1 and 115.3 pg/ml at 80:1) (B). (C) Uptake of C. neoformans by BMDM was measured by quantitation of BMDM associated with the fungal GFP signal using flow cytometry. (D) Killing of C. neoformans was analyzed by measuring CFU. For reference, growth of C. neoformans in the absence of BMDM is shown (gray bars). Data for all experiments were pooled from three independent trials (n ⫽ 8 to 11 total replicates per group). All results were normalized to the mean for WT samples. ***, P ⬍ 0.001; ****, P ⬍ 0.0001. n/a, not applicable.

ditional DAP12 deletion in different myeloid cell subsets may help address these questions. In our study, we observed an increase in NK cells in DAP12⫺/⫺ lungs compared to WT lungs after C. neoformans challenge. In addition, the deletion of DAP12 enhances the ability of NK cells to inhibit the growth of C. neoformans in vitro. This result is in agreement with studies showing that DAP12 deletion improves the cytolytic activity of NK cells, as measured by 51Cr release assays (18). Taken together, these results support the idea that DAP12 signaling in NK cells inhibits anticryptococcal activity. In humans, it has been observed that NK cells from HIV-positive patients are unable to inhibit the growth of C. neoformans as effectively as those from healthy subjects in vitro, though this defect can be reversed by the addition of IL-12 (35). However, whether DAP12 regulates human NK cell responses to C. neoformans remains to be determined. In murine models, wild-type NK cells may have a role in the early stages of the immune response after systemic infection with C. neoformans (48–50), but ultimately the survival of NK celldepleted mice is no different from that of control mice following systemic infection (48). There are also no differences in CFU in target organs following intratracheal infection (48). Our results suggest that genetic deletion of DAP12 is likely to enhance NK cell

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fungistasis or, alternatively, eliminate their permissiveness for fungal growth. A possible explanation for the modest effect of DAP12 deletion on NK cell activity is functional overlap with the transmembrane signaling adapter DAP10. DAP10 has homology to DAP12 in the transmembrane region but contains a cytoplasmic YINM signaling motif. Interestingly, it has been shown that many receptors, including Ly49 receptors, can signal through either DAP12 or DAP10, although this signaling may be less efficient through one versus the other (17). Finally, the NK cell preparation for our in vitro studies had high purity (91 to 99% by postsort analysis), but we cannot completely exclude the presence of other cells that may have influenced NK cell function in our assay. We also found in our study that DAP12⫺/⫺ macrophages are more highly activated in response to C. neoformans. Hamerman and colleagues have shown previously that DAP12⫺/⫺ macrophages secrete more proinflammatory cytokines in response to TLR agonists, including bacterial antigens and CpG DNA (28). Our study extends the concept that DAP12 negatively regulates macrophage activation to an in vivo fungal challenge model and demonstrates that DAP12 deletion improves macrophage phagocytosis and fungicidal activity. Despite the observed increase in macrophage activation, it is interesting that the numbers of mac-

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rophages are decreased in infected DAP12⫺/⫺ lungs compared to WT lungs. Since DAP12⫺/⫺ macrophages are more effective in killing C. neoformans cells, it is possible that there is a feedback mechanism that suppresses further recruitment to the lungs or that lower macrophage numbers in DAP12⫺/⫺ mice may simply reflect the lower fungal burden seen at day 14 p.i. This is a subject for further studies, and the development of conditional targeting strategies for DAP12 would facilitate a more precise understanding of the role of macrophage-intrinsic DAP12 versus macrophage-extrinsic DAP12 signaling in host defense in vivo. In summary, we find that DAP12 acts as a brake on an effective immune response to C. neoformans through its influence on eosinophils, NK cells, and macrophages. Although DAP12⫺/⫺ mice have improved outcomes, ultimately they do succumb to lethal cryptococcal challenge in our model using the highly virulent C. neoformans H99 strain. Thus, targeting DAP12 would likely be one prong in a multifaceted approach to prevent mortality from C. neoformans. To expand the potential targets for immunotherapy, future investigations should include candidate receptors known to pair with DAP12 (e.g., TREM-2 [51]) for a possible role in innate immune regulation following C. neoformans challenge. ACKNOWLEDGMENTS

6.

7.

8.

9.

10.

11.

We thank Jessica Hamerman (Benaroya Research Institute), Joseph Heitman (Duke), Robin May (University of Birmingham), Arturo Casadevall (Johns Hopkins), Joseph Sun (MSKCC), and Eric Pamer (MSKCC) for reagents and helpful discussions, Julie White (MSKCC Laboratory of Comparative Pathology) for assistance with histopathology, the MSKCC Molecular Cytology Core Facility for histology slide preparation, the MSKCC Flow Cytometry Core Facility for assistance with cell sorting, and Ingrid Leiner and Anand Salem for technical assistance.

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FUNDING INFORMATION This work, including the efforts of Tobias Hohl, was funded by HHS | National Institutes of Health (NIH) (R01 AI093808, R21 AI105617, and P30 CA008748). This work, including the efforts of Tobias Hohl, was funded by Burroughs Wellcome Fund (BWF). This work, including the efforts of Lena J. Heung, was funded by Stony Wold-Herbert Fund, Inc. This work, including the efforts of Lena J. Heung, was funded by Dana Foundation (MSKCC Clinical Scholars Biomedical Research Training Program fellowship). The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

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DAP12 Inhibits Pulmonary Immune Responses to Cryptococcus neoformans.

Cryptococcus neoformans is an opportunistic fungal pathogen that is inhaled into the lungs and can lead to life-threatening meningoencephalitis in imm...
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