CARD9-Dependent Neutrophil Recruitment Protects against Fungal Invasion of the Central Nervous System

Candida is the most common human fungal pathogen and causes systemic infections that require neutrophils for effective host defense. Humans deficient in the C-type lectin pathway adaptor protein CARD9 develop spontaneous fungal disease that targets the central nervous system (CNS). However, how CARD9 promotes protective antifungal immunity in the CNS remains unclear. Here, we show that a patient with CARD9 deficiency had impaired neutrophil accumulation and induction of neutrophil-recruiting CXC chemokines in the cerebrospinal fluid despite uncontrolled CNS Candida infection. We phenocopied the human susceptibility in Card9 -/- mice, which develop uncontrolled brain candidiasis with diminished neutrophil accumulation. The induction of neutrophil-recruiting CXC chemokines is significantly impaired in infected Card9 -/- brains, from both myeloid and resident glial cellular sources, whereas cell-intrinsic neutrophil chemotaxis is Card9-independent. Taken together, our data highlight the critical role of CARD9-dependent neutrophil trafficking into the CNS and provide novel insight into the CNS fungal susceptibility of CARD9-deficient humans.


Introduction
Human systemic fungal infections are typically opportunistic and primarily affect patients with acquired immunodeficiency (i.e. commonly due to HIV infection or modern medical interventions such as cancer chemotherapy and transplantation) or inborn errors of immunity [1,2]. Systemic candidiasis, in particular, is the most common deep-seated fungal infection in the developed world and is now the most common cause of nosocomial bloodstream infection in the US [3]. Despite the availability of potent antifungal drugs, clinical outcomes of infected patients are often poor leading to significant morbidity and unacceptably high mortality rates [1]. Adjunctive immune-based therapies are therefore becoming increasingly desirable, yet their development and success depends on improving our understanding of the cellular and molecular basis of human antifungal immunity.
Fungi are recognized by the host immune system via innate pattern recognition receptors (PRRs) that are predominantly expressed by myeloid cells, including neutrophils, monocytes/ macrophages and dendritic cells. C-type lectin receptors (CLRs) are a large class of PRRs of which several members including Dectin-1, Dectin-2 and Mincle have been shown to play crucial roles in antifungal immunity [4]. These receptors bind components of the fungal cell wall and this innate recognition event leads to the initiation of intracellular signaling cascades that stimulate cellular responses such as phagocytosis, induction of the respiratory burst, and production of pro-inflammatory chemokines/cytokines [4]. Although different CLRs exhibit independent functions, they utilize a common signaling pathway involving the kinase Syk and the signaling adaptor, CARD9 [5].
CARD9 is one of the most crucial mammalian antifungal immune molecules identified to date. Genetic deletion of Card9 in mice results in defective pro-inflammatory cytokine production by myeloid cells and leads to a substantial increase in mortality following C. albicans challenge compared to WT mice [6]. CARD9 is also indispensable for antifungal defense in peripheral blood revealed normal monocyte, neutrophil, eosinophil, CD4 + T, CD8 + T, CD19 + B, and natural killer lymphocyte numbers. Phagocyte oxidative burst assessed by the dihydrorhodamine (DHR) assay was normal. T-lymphocyte proliferations were normal in response to PHA and PWM but decreased or absent in response to Candida and tetanus antigens. The levels of the patient's IgA, IgE, IgG and IgM were normal. Neither her parents nor her siblings had a history of infections.
Since autosomal recessive CARD9 deficiency has been shown to predispose to Candida meningoencephalitis [7][8][9]12], we sequenced CARD9 exons (S1 Table) and found a homozygous CARD9 missense mutation, c.170G>A in exon 3 causing a substitution of histidine for arginine at position 57 (R57H), a highly conserved residue within the CARD domain of the CARD9 protein (Fig 1D and S1 Fig). The patient's parents and brother are healthy and heterozygous for the mutation (WT/c.170G>A) whereas the patient's healthy sister has WT CARD9 alleles (Fig 1D and 1E), consistent with autosomal recessive CARD9 deficiency with complete clinical penetrance. The c.170G>A mutation was not found in the 1000 Genomes or ExAC databases covering 117,000 chromosomes in the specific CARD9 exon, thus this mutation is unlikely to be an irrelevant polymorphism. In addition, it was predicted to be deleterious by SIFT (deleterious), CADD [PHRED score 17.7] [20] and PolyPhen 2 [highest possible score of 1] [21]. Collectively, these data strongly suggest that the patient is homozygous for a rare and deleterious mutant CARD9 allele.
The c.170G>A CARD9 Mutation Results in Generation of Full-Length CARD9 Protein with Impaired Function Various CARD9 mutations that lead to production of full-length, truncated or absent CARD9 proteins have been reported to date [7,8,12,13]. Hence, we next investigated the impact of the c.170G>A mutation on CARD9 protein levels by FACS and Western blot analyses on the patient neutrophils and CD14 + monocytes and compared them to that of healthy donor myeloid cells. FACS revealed similar expression of CARD9 in WT and patient neutrophils and CD14 + monocytes (Fig 1F), whereas, as expected, no significant CARD9 expression was observed on T lymphocytes (S2 Fig). Similarly, immunoblot analyses for CARD9 on whole-cell extracts of the patient neutrophils and CD14 + monocytes demonstrated that CARD9 protein expression levels and molecular weight were similar with that of WT cells (Fig 1G). These data are in agreement with previous reports in which nonsense mutations prevented the generation of full-length CARD9 protein, whereas missense mutations did not predictably do so [7,8,12,13].
We next sought to examine the functional consequences of the c.170G>A mutation by measuring the production of pro-inflammatory cytokines and chemokines from peripheral blood mononuclear cells (PBMCs) stimulated with either heat-killed C. albicans or LPS. Consistent Sequencing chromatograms of CARD9 at position 170 for the patient, her parents and a healthy donor control. (E) Pedigree of the patient with Candida meningoencephalitis. The patient is indicated in black and by arrow and the genotype of CARD9 alleles is indicated below each family member. (F) FACS histograms of CARD9 staining in neutrophils and CD14 + monocytes of healthy donors and the patient (n = 2). (G) Representative Western blots of CARD9 protein expression in neutrophils and CD14 + monocytes of healthy donors and the patient. GAPDH is shown as loading control (n = 3). (H-J) IL-1β, IL-6 and TNFα production by healthy donor (n = 10) and patient (n = 4) PBMCs after 48 hours of stimulation with heat-killed C. albicans or LPS or after 48 hours without stimulation (n = 4-8 independent experiments). Data is analyzed by Mann Whitney U-test or unpaired t-test, where appropriate. **P < 0.01; ***P < 0.001. (K) Percentage of CD4 + IL-17A + T cells (within CD4 + T-cells) of the patient and 9 healthy donors following stimulation with PMA/ionomycin, assessed by FACS. All quantitative data represent mean ± SEM.
with previously reported CARD9 mutations [8,12,13], the c.170G>A mutation resulted in dramatically decreased IL-1β, IL-6 and TNF-α production after 48 hours of stimulation with C. albicans compared with 10 healthy donors, while cytokine production following LPS stimulation was comparable in the patient and healthy donor PBMCs (Fig 1H-1J). Furthermore, the production of GM-CSF, IFN-γ, IL-1α, IL-2, IL-10, CCL3, CCL4, CCL5, CCL7 and CXCL8 was also found to be CARD9-dependent in human PBMCs following fungal stimulation, whereas production of IL-4, IL-7, IL-15, IL-16, CCL2, CXCL1, CXCL2 and CXCL5 was CARD9-independent (S3 Fig). Since decreased proportions of IL-17 + CD4 + T cells have been reported in some, but not all, CARD9-deficient patients [7,8,10,12,13], we evaluated the production of IL-17A by the patient's CD4 + T cells following PMA/ionomycin stimulation ex vivo using FACS, and found no impairment relative to 9 healthy donors ( Fig 1K). Taken together, these data show that the c.170G>A mutation results in the generation of full-length CARD9 protein which exhibits impaired capacity for production of pro-inflammatory mediators upon fungal stimulation.
Human Phagocytes Require CARD9 for Killing Fungal Yeasts but Not Hyphae, which Are Present in Infected Tissue Neutrophils and monocytes/macrophages are the critical cellular mediators of host defense against systemic C. albicans infection [19,22,23]. Therefore, we next investigated the effects of the c.170G>A mutation on the ability of neutrophils and monocytes to kill the two major morphological forms of C. albicans, yeasts and hyphae. Hyphae are the invasive filamentous fungal forms present in human infected CNS tissue (Fig 1A). It has been previously shown that CARD9 deficiency impairs the killing capacity of neutrophils against unopsonized, but not opsonized, C. albicans yeasts [7,15]. Indeed, we found decreased killing by the patient's neutrophils of unopsonized C. albicans yeasts whereas neutrophil killing was intact against opsonized yeasts (Fig 2A). However, CARD9-deficient neutrophils did not exhibit impaired killing capacity against opsonized or unopsonized C. albicans hyphae (Fig 2B), which are the predominant form found in infected CNS tissue of mice and humans ( Fig 1A) [24,25]. We then examined the impact of CARD9 deficiency on the ability of CD14 + monocytes to kill C. albicans yeasts and hyphae. CD14 + monocytes demonstrated inferior killing capacity of C. albicans relative to neutrophils (Fig 2A-2D). As shown in Fig 2C and 2D, CD14 + monocytes from the patient exhibited a modest decrease in killing of C. albicans yeasts, but hyphal killing was intact. The decreases observed in killing of yeasts by the patient's neutrophils and CD14 + monocytes were not due to impaired fungal internalization ( Fig 2E). Collectively, these data show that although CARD9-deficient neutrophils have a decreased ability to kill unopsonized C. albicans yeasts, CARD9 is dispensable for killing of the invasive hyphal forms found in infected CNS tissue. Therefore, other factors in addition to CARD9-mediated C. albicans yeast killing by phagocytes are likely to also contribute to the susceptibility of CARD9-deficient patients to Candida infection of the CNS.

CARD9 Deficiency Results in a Striking Absence of Neutrophils in the Infected CNS
Notably, we observed that the patient had a striking absence of neutrophils in the infected CSF, despite uncontrolled fungal disease. Fig 3A shows a representative cytopathology image of the infected CSF, in which lymphocytes, eosinophils and mononuclear phagocytes could be found, but neutrophils were absent. Indeed, FACS analysis of the infected CSF revealed that the predominant accumulating leukocytes were T-lymphocytes and eosinophils (Fig 3B), which are not critical mediators of host defense against systemic C. albicans infection [19]. This finding is in agreement with other reports of high numbers and predominance of lymphocytes and eosinophils in the CSF of Candida-infected CARD9-deficient patients [7,9]. Monocytes, dendritic cells and B-cells were also present (5-10% each), whereas neutrophils comprised <1% of total leukocytes ( Fig 3B). Over time, the lack of neutrophils was consistent with these cells never exceeding 7% of total leukocytes in the infected CSF (range: 0-7%) despite continued inflammation (range of nucleated cells in CSF: 108-422/μl; range of % of eosinophils within leukocytes in CSF: 3-40%; range of lymphocytes within leukocytes in CSF: 25-72%; range of protein levels in CSF: 161-845 mg/dL) and extremely high fungal load indicated by persistently elevated CSF BDG (Fig 3C). Persistent Candida tissue hyphal invasion and absence of neutrophil infiltration were confirmed in the patient's CNS tissue by brain biopsy (S4 Fig). The decreased neutrophil accumulation noted in our patient is grossly suboptimal compared to the robust neutrophil accumulation seen in patients without CARD9 deficiency when they develop C. albicans meningitis, typically post-neurosurgical procedures (Fig 3C; red bar) [26][27][28][29]. CARD9 mediates killing of Candida yeasts by human phagocytes but it is dispensable for the killing of invasive Candida hyphal forms that are found in infected tissue. Killing of opsonized and unopsonized Candida yeasts (A) and hyphal forms (B) by healthy donor and patient neutrophils (n = 5-7). Killing of opsonized and unopsonized Candida yeasts (C) and hyphal forms (D) by healthy donor and patient CD14 + monocytes (n = 5-8). (E) Internalization of opsonized (left panel) and unopsonized (right panel) Candida yeasts by healthy donor and patient neutrophils and CD14 + monocytes (n = 3 independent experiments). Data is analyzed by Mann Whitney U-test or unpaired t-test, where appropriate. *P < 0.05; **P = 0.01; ***P < 0.001. All quantitative data represent mean ± SEM. Consistent with that, a patient at NIH who developed C. albicans meningitis post-Ommaya reservoir placement exhibited pleiocytosis (133-1091 nucleated cells/μl) and significant neutrophil mobilization in the infected CSF (56-73% of total leukocytes) ( Fig 3C; grey bar).
We next sought to investigate the etiology of suboptimal neutrophil accumulation in the infected CSF in the setting of CARD9 deficiency. The patient did not manifest peripheral neutropenia (range of absolute neutrophil count in the blood: 1,960-5,340/mm 3 ), ruling out this possibility ( Fig 3D). We next examined whether CARD9 is a survival factor for human neutrophils. For that, we harvested neutrophils from the peripheral blood of the patient and healthy donors and determined differential cell apoptosis and death at 3 and 6 hours after serum starvation, TNF-α stimulation or live C. albicans challenge ex vivo. Neutrophils were also directly exposed to the patient's infected CSF to evaluate for the presence of potential soluble pro-apoptotic factors within the infected tissue. We found that CARD9-deficient neutrophils did not exhibit enhanced apoptosis or death under any of the tested conditions relative to WT neutrophils ( Fig 3E). Therefore, these data demonstrate a striking absence of neutrophil accumulation in the Candida-infected CSF in human CARD9 deficiency, which is not caused by peripheral neutropenia or defective neutrophil survival.

CARD9 Is Critical for the Production of Neutrophil-Targeted Chemoattractants in the Infected CSF
We reasoned that the decreased neutrophil recruitment in the CNS of the patient could be explained by the net effect of (1) impaired intrinsic chemotactic ability of CARD9-deficient human neutrophils and/or (2) defective production of neutrophil-targeted chemoattractants in the infected tissue. Thus, we tested whether CARD9-deficient neutrophils have cell-intrinsic chemotactic defects and/or whether the neutrophil-targeted chemoattractants are not produced in order to generate the chemotactic gradient that is necessary for cell trafficking into the infected CNS. We first assessed neutrophil chemotaxis using both the EZ-TAXIScan system and a modified Boyden chamber, the 96-well Neuroprobe chamber. The EZ-TAXIScan instrument allows for simultaneous measurement of cell velocity and directionality by observing the spatial and temporal movement of individual cells toward a chemoattractant. Using this method, we found no defect in chemotaxis of CARD9-deficient neutrophils toward IL-8 or formyl-methionyl-leucyl-phenylalanine (fMLP) (Fig 4A and 4B), two prototypic chemotactic factors for human neutrophils. We then used the 96-well Neuroprobe chamber and examined neutrophil chemotaxis toward IL-8, fMLP, the leukotriene B4 (LTB4), and the complement factor C5a. Similar to our EZ-TAXIScan data, no impairment was found in the chemotaxis of CARD9-deficient neutrophils relative to WT neutrophils using the 96-well Neuroprobe chamber when correcting for multiple comparisons (Fig 4C).
Since the patient's neutrophils did not exhibit a primary chemotaxis defect, we next measured levels of the neutrophil-targeted chemokines CXCL1, CXCL2, CXCL5 and IL-8 in the infected CSF by Luminex array at different prospective time-points during uncontrolled fungal infection. Strikingly, we found absent to very low levels of these chemokines (Fig 4D). IL-8 was Percentages of neutrophils within leukocytes in the CARD9-deficient patient's infected CSF over time. The frequency range of neutrophils in the CSF found in our CARD9 +/+ patient with Candida meningitis post-Ommaya reservoir placement (grey bar) and those typically reported for Candida meningoencephalitis in patients without CARD9 deficiency (red bar) are also shown. (D) The percentages of neutrophils within leukocytes in peripheral blood of the CARD9-deficient patient over time. (E) Healthy donor and patient neutrophils were cultured for 3 hours (upper panels) or 6 hours (lower panels) under the indicated conditions and FACS used to assess the percentage of Annexin V + PIapoptotic (left panels) and Annexin V + PI + dead (right panels) cells (n = 3-4; 3 independent experiments). All quantitative data represent mean ± SEM. doi:10.1371/journal.ppat.1005293.g003 detected at higher levels (mean: 267.6 pg/mL) than all other chemokines (mean: 13.3 pg/mL for CXCL1, 10.6 pg/mL for CXCL2, 16.3 pg/mL for CXCL5), but these IL-8 levels were comparable to previously reported IL-8 levels in uninfected CSF from healthy donors [30]. Interestingly, the measured levels of IL-8 are significantly lower than those detected in patients with neutrophilic meningitis in the absence of CARD9 deficiency ( Fig 4D; red bar) [30]. Consistent with the absence of induction of neutrophil-targeted chemokines, CSF from the infected patient did not exhibit any greater chemotactic activity toward WT and patient neutrophils than that observed in buffer, CSF from a healthy subject or CSF from two patients with lymphocytic (non-neutrophilic) meningitis ( Fig 4E).
Instead, our CARD9 +/+ patient who developed C. albicans meningitis post-Ommaya reservoir placement associated with neutrophil mobilization in the CSF exhibited significant induction of CXC neutrophil-recruiting chemokines in the infected CSF ( Fig 4D; grey bar). Specifically, the CSF levels of IL-8, CXCL1, CXCL2 and CXCL5 were 17.1-fold (4576.8 pg/ mL), 294.4-fold (3120.6 pg/mL), 16-fold (261.2 pg/mL) and 93.4-fold (1523.1 pg/mL) greater, respectively, relative to those detected in the CSF of our CARD9-deficient patient. In line with the induction of neutrophil-targeted CXC chemokines, Candida-infected CSF from the CARD9 +/+ patient exhibited chemotactic activity toward WT neutrophils ex vivo ( Fig 4F). Altogether, these data suggest that the absence of neutrophil accumulation in the infected CNS despite uncontrolled fungal disease correlates with a defect at the level of chemoattractant production in the infected tissue, whereas CARD9 is dispensable for cell-intrinsic neutrophil chemotaxis.

Neutrophils Are Critical for Control of Fungal CNS Infection in Wild-Type (WT) Mice
To further examine the mechanistic role of CARD9 in antifungal defense of the CNS, we utilized a well-established mouse model of systemic C. albicans infection. This model results in fungal dissemination to the brain associated with an early influx of neutrophils, whose accumulation decreases later on as the infection is controlled [24]. To directly examine whether the recruitment of neutrophils to the CNS is critical for control of fungal proliferation in this tissue, we depleted neutrophils in WT mice using the 1A8 clone of the anti-Ly6G antibody [31]. We found that administration of 1A8 successfully depleted >90% of neutrophils in the brain of infected mice compared to infected mice treated with an isotype control antibody, and had no effect on the recruitment of other myeloid cells such as Ly6C hi monocytes ( Fig 5A). Analysis of brain fungal burdens in these animals revealed that neutrophil depletion by 1A8 led to a significant increase in brain fungal load compared to control animals ( Fig 5B). Therefore, these data demonstrate that neutrophil recruitment to the CNS post-infection is critical for control of fungal brain infection.  [13][14][15] and patient (n = 2) neutrophils towards fMLP, C5a, IL-8 and LTB4 at the indicated concentrations, assessed by the 96-well Neuroprobe chamber. Data is analyzed by two-way ANOVA (with Bonferroni correction). (D) Levels of neutrophil-targeted chemokines in the CARD9-deficient patient's infected CSF (n = 3). The range of IL-8 in the CSF found in our CARD9 +/+ patient with Candida meningitis post-Ommaya reservoir placement (grey bar) and previously reported for neutrophilic meningitis in patients without CARD9 deficiency (red bar) are also shown. (E) The patient's infected CSF is not chemotactic toward healthy donor and patient neutrophils, as assessed by the 96-well Neuroprobe chamber (n = 2). Resting indicates neutrophil migration toward buffer; nd: not detected. (F) The CSF of the CARD9 +/+ Candida-infected patient post-Ommaya reservoir, but not the CSF of the CARD9-deficient patient, is chemotactic toward healthy donor neutrophils, as assessed by the 96-well Neuroprobe chamber (n = 2-4). Resting indicates neutrophil migration toward buffer. **P < 0.01 by unpaired t-tests. All quantitative data represent mean ± SEM. fMLP, formyl-methionyl-leucyl-phenylalanine; LTB4, leukotriene B4; PMNs, neutrophils. doi:10.1371/journal.ppat.1005293.g004

Fungal Infection of WT Mouse CNS Results in Chemokine Production and Neutrophil Recruitment That Correlate with the Extent of Brain Fungal Burden
We further analyzed the fungal-load dependence of the neutrophil response in the infected WT mouse CNS by infecting WT mice with either a low (7x10 4 ) or a high (1x10 6 ) inoculum of C. albicans. WT mice infected with the high inoculum developed significantly greater brain fungal burden compared to those infected with the low inoculum ( Fig 5C). Using FACS, we assessed the number of recruited neutrophils in the brains of animals with high or low tissue burden. We found that animals infected with the high inoculum, which developed high brain fungal proliferation, had nearly 10-fold greater neutrophil numbers in the brain than observed in the mice which received the low inoculum and had lower tissue fungal burden ( Fig 5D). Accordingly, we found that the levels of the major mouse neutrophil-targeted chemokines Cxcl1 (KC) and Cxcl2 (MIP-2) were significantly increased in the brains of mice with the higher tissue fungal burden ( Fig 5E). In contrast, LPS-induced chemokine (LIX), the mouse homologue of CXCL5 which can also recruit neutrophils, was similar in all brains analyzed and had no correlation to the extent of brain fungal burden (Fig 5E), suggesting that Cxcl5 induction is not fungal-load dependent in the infected mouse brain. Taken together, these data show that neutrophil-targeted chemokine production and neutrophil mobilization to the infected CNS directly correlates with the extent of tissue fungal burden.

Card9 Is Required for CNS Neutrophil Accumulation and Control of Fungal Brain Infection
We next sought to understand the functional role of Card9 in the mouse brain during C. albicans infection. For this, we used Card9 -/mice that are significantly more susceptible to systemic candidiasis (Fig 6A), as has been previously shown [6]. Importantly, we phenocopied for the first time the human CNS susceptibility to Candida infection of CARD9-deficient patients in Card9 -/mice which developed uncontrolled brain infection, evidenced by significantly higher brain fungal burdens as early as 24 hours post-infection (Fig 6B). At 72 hours postinfection, Card9 -/mice exhibited dramatically greater brain fungal burdens (over 1000-fold) compared to WT animals. In agreement, histopathological analysis of infected brains revealed extensive tissue invasion in the Card9 -/brain by hyphae, which were the predominant fungal morphology observed in the infected brain ( Fig 6C).
Since our CARD9-deficient patient had limited numbers of neutrophils in the infected CSF, we next investigated whether the same defect was present in the infected Card9 -/mouse brain. Indeed, we found a profound decrease in neutrophil accumulation in the Card9 -/brain at 24 hours post-infection compared with WT animals (Fig 6D and S5A Fig). Notably, while WT mice recruited high numbers of neutrophils to the brain early after infection, there was no significant increase in neutrophil numbers or frequency between uninfected and infected Card9 -/brains at any time point after infection, despite dramatic tissue fungal proliferation ( Fig 6D  and S5A Fig). Indeed, histopathology revealed that neutrophils could be seen to crowd around hyphae in WT brains at 24 hours post-infection, whereas very few of these cells were observed in Card9 -/brains ( Fig 6E). Card9 -/brains had similar neutrophil numbers with WT brains at 72 hours post-infection; strikingly, these numbers were similar to those observed in the uninfected brain (Fig 6D). This reflects a markedly defective response in Card9 -/mice since these animals have over 1000-fold increase in brain fungal burden compared to WT ( Fig 6B); hence, based on the inoculum-dependence of neutrophil accumulation in the mouse brain (Fig 5), these animals should have mobilized significantly more neutrophils into this tissue. In our patient and in other reported patients' CSF [7,9], in addition to a lack of neutrophils, we and others also observed high numbers of lymphocytes and eosinophils. To investigate if this was also true in Card9 -/mice, we calculated the relative frequency of multiple lymphoid and myeloid cell populations in the brain following infection by FACS. Besides a significant reduction in neutrophils and a corresponding increase in microglia percentages in Card9 -/brains, we did not find any other significant differences in the frequency of other leukocyte populations between WT and Card9 -/brains at the tested time points (S5B Fig). Collectively, these data show that, consonant to human findings, Card9 is required for the control of C. albicans infection of the CNS and for promoting neutrophil accumulation in the infected brain.

Card9 Mediates Tissue-Specific and Fungal-Specific Neutrophil Accumulation into the Infected CNS
We next investigated whether neutrophil accumulation into the C. albicans-infected kidney is Card9-dependent. We found that, in line with previously reported findings [6], Card9 -/mice had significantly increased kidney fungal burdens at both 24 and 72 hours post-infection (S6A Fig). However, unlike the brain, Card9 -/mice were able to mobilize significant numbers of neutrophils into the kidney at 72 hours post-infection, approximately 3-fold greater than in WT infected kidneys at the same time-point post-infection, and nearly 100-fold greater than in uninfected control kidneys (S6B Fig). Although the extent of neutrophil accumulation may still be to a degree suboptimal relative to the higher kidney fungal load seen in Card9 -/kidneys [22,24,32], the data collectively indicate that Card9 plays organ-specific roles in neutrophil accumulation after systemic candidiasis, in agreement with a recent report showing neutrophil mobilization into the C. tropicalis infected Card9 -/kidney [33].
We then assessed whether the defect in neutrophil accumulation into the infected brain was specific to fungi or could be recapitulated with non-fungal pathogens. For this, we infected WT and Card9 -/animals intravenously with Staphylococcus aureus, an important human bacterial pathogen. We found that Card9 -/mice had similar bacterial burdens and accumulation of neutrophils in the brain compared to WT mice at 48 hours post-infection (S7 Fig). These data are in agreement with the lack of development of bacterial CNS infections in patients with CARD9 deficiency and show that the neutrophil accumulation defect we observed during C. albicans infection is specific for fungal pathogens.

Card9 Is Not Required for Neutrophil Production and Egress from Bone Marrow
We next began to investigate possible mechanisms of the Card9-dependent accumulation of neutrophils in the brains of C. albicans infected mice. We first assessed whether Card9 is critical for the production of neutrophil progenitors in the bone marrow and egress of these cells into the peripheral blood after infection. We found that C. albicans infection led to a significant increase in neutrophil progenitors in the bone marrow (S8 Fig) and a significant induction of neutrophilia in the blood of WT mice (Fig 6F). No differences were observed in neutrophil numbers in the bone marrow (S8 Fig) and peripheral blood (Fig 6F) between WT and Card9 -/animals, either at steady state or after infection. Since neutrophil production and mobilization of large numbers of neutrophils from the bone marrow into the blood of Card9 -/mice following C. albicans infection did not translate into accumulation of these cells into the infected CNS (Fig 6D-6F), these data collectively suggest that Card9 is critical for the trafficking of neutrophils from the blood into the infected CNS.

Card9 -/-Neutrophils Exhibit Normal Chemotaxis, Adhesion and Survival In Vivo
We showed earlier that CARD9-deficient human neutrophils had normal chemotaxis toward four major neutrophil-targeted chemoattractants ex vivo (Fig 4A-4C). To analyze the recruitment of WT and Card9 -/neutrophils into the fungal-infected CNS in vivo, we generated mixed bone marrow chimeras in order to directly assess whether Card9 -/neutrophils have a cell-intrinsic chemotactic defect. WT animals were lethally irradiated and subsequently injected with a 1:1 ratio of CD45.1 + WT and CD45.2 + Card9 -/bone marrow cells. Following the adoptive transfer, animals were left to reconstitute for 8 weeks prior to C. albicans infection (S9 Fig). The relative recruitment of WT and Card9 -/neutrophils to the brain of infected chimeras was then assessed by FACS, with congenic CD45 isoform expression used to distinguish between WT and Card9 -/neutrophils. Using this approach, we found no defect in the ability of Card9 -/neutrophils to traffic into the infected brain, since the relative ratio of WT:Card9 -/neutrophils remained unchanged before and after infection ( Fig 7A). These data indicate that neutrophil-intrinsic Card9 is dispensable for neutrophil recruitment during fungal infection of the CNS.
The mixed chimera experiment results ( Fig 7A) implied that Card9 -/neutrophils are able to upregulate key adhesion molecules that allow their transmigration from the blood into the infected brain. To further test this, we next analyzed whether expression of various adhesion molecules that are required for neutrophil adhesion and rolling on the endothelial surface for transmigration into inflamed tissues was disrupted in the absence of Card9. Using qRT-PCR on whole brain homogenates, we found that all adhesion molecules analyzed were induced in the brain of WT mice following infection (Fig 7B). While we observed a delayed induction in the expression of α-integrin X and α-integrin L in infected Card9 -/brains, these differences were not evident at 72 hours post-infection. Since β-integrin 2 induction was greater in Card9 -/brains at 72 hours post-infection, we assessed whether its induction in the Candidainfected brain is fungal load-dependent by infecting WT mice with high and low brain fungal burdens. Indeed, induction of β-integrin 2, ICAM-1 and L-selectin was significantly greater in animals with higher brain fungal burden, whereas induction of α-integrin X, α-integrin L and P-selectin was not fungal load-dependent (S10 Fig). These data suggest that the higher levels of β-integrin 2 and the trend toward higher induction of ICAM-1 observed in Card9 -/brains ( Fig 7B) may reflect greater tissue fungal proliferation in these mice. Collectively, we did not observe consistent or profound defects in the expression of neutrophil-targeted adhesion molecules in the infected Card9 -/brain (Fig 7B).
The mixed chimera experiment results (Fig 7A) also suggested that Card9 -/neutrophils do not have a survival defect in vivo. To directly rule out the possibility that reduced neutrophil numbers in the Card9 -/brain is caused by enhanced neutrophil apoptosis and/or death following their recruitment into the CNS, we analyzed the extent of apoptosis and death in neutrophils recruited to the brain at 24 hours post-infection using Annexin-V and 7-AAD staining. We found that the frequency of apoptotic and dead neutrophils was similar in WT and Card9 -/brains (Fig 7C), in line with our patient data that showed that CARD9 is not a survival factor in human neutrophils (Fig 3E).
Taken together, these data show that Card9 deficiency does not adversely affect cell-intrinsic neutrophil trafficking to the brain, expression of neutrophil-targeted adhesion molecules, or neutrophil survival during C. albicans infection of the CNS in mice. Expression of various adhesion molecules in whole brain homogenates of uninfected and infected WT (filled bars, n = 6) and Card9 -/-(empty bars, n = 6) mice, as determined by qRT-PCR. Gene expression was calculated relative to Gapdh. Mice analyzed at 24 hours were infected with 1.3x10 5 CFU SC5314, and mice analyzed at 72 hours infected with 7x10 4 CFU. (C) Neutrophil survival in the brain of 24-hour infected (dose: 1.3x10 5 CFU) WT (filled bars, n = 6) and Card9 -/-(empty bars, n = 6) mice was determined by Annexin-V and 7-AAD staining and analysis by FACS. Apoptotic neutrophils were defined as Annexin-V + 7-AAD -, while dead neutrophils were defined as Annexin-V + 7-AAD + . All data is pooled from two independent experiments; data represent mean ± SEM. *P<0.05 by two-way ANOVA. doi:10.1371/journal.ppat.1005293.g007

CARD9 Is Required for Neutrophil-Targeted Chemokine Production in the Infected CNS
Since Card9 -/neutrophils did not exhibit a cell-intrinsic chemotactic defect, and as we found our patient's infected CSF to contain low concentrations of neutrophil-targeted CXC chemokines, we next investigated if this defect could also be modeled in the brains of Card9 -/mice. Thus, we analyzed whole brain homogenates for expression of Cxcl1, Cxcl2 and Cxcl5 by Luminex array in uninfected and C. albicans-infected mice. We found that both Cxcl1 and Cxcl2 were significantly induced following infection in both WT and Card9 -/mice whereas Cxcl5 was poorly induced (S11 Fig). At 24 hours post-infection, WT and Card9 -/brains had similar levels of these chemokines despite a 10-fold higher fungal load in Card9 -/brain tissue (S11 Fig). At 72 hours post-infection, chemokine levels decreased in WT mice in parallel with falling tissue fungal burden (Fig 6B), whereas chemokine levels in Card9 -/brains remained stable relative to 24 hours post-infection (S11 Fig) despite a further dramatic increase in brain fungal burden (Fig 6B). When chemokine production was expressed as a concentration relative to fungal load, Cxcl1 and Cxcl2 levels were dramatically decreased in Card9 -/brains ( Fig 8A).
We then examined CXC chemokine induction in the kidney and found that Cxcl1 and Cxcl2 were significantly increased in the infected Card9 -/kidneys compared to WT (S6C Fig). Although induction of Cxcl1 and Cxcl2 was to a degree impaired relative to the kidney fungal load in Card9 -/mice (S6A and S6C Fig), renal CXC chemokine induction was sufficient for mediating neutrophil recruitment into the Candida-infected Card9 -/kidney (S6B Fig). Since the IL-17/IL-22 pathway has also been shown to promote neutrophil recruitment into infected tissues [34], and some CARD9-deficient patients have reduced Th17 cells in the peripheral blood [7,9,13], we determined the levels of IL-17 and IL-22 in the brains of WT and Card9 -/mice following infection. However, we found no consistent induction of these cytokines compared to uninfected control brain, at either the mRNA or protein levels. Taken together, Card9 -/mice display a marked insufficiency in the induction of Cxcl1 and Cxcl2 in the brain during C. albicans infection relative to the extent of tissue fungal burden.

Neutrophils and CD45 -Cells Produce Neutrophil-Targeted Chemoattractants in a Card9-Dependent Manner
Since we identified that CXC chemokines were reduced in the CNS of both mice and humans with CARD9 deficiency, we next sought to understand the cellular source of these chemokines in a bid to further define the antifungal immune response within the brain. We used FACSsorting (S12 Fig) to purify the major recruited myeloid cell populations from infected brains (neutrophils and Ly6C hi monocytes), and then used qRT-PCR to quantify relative expression of Cxcl1, Cxcl2 and Cxcl5. We also FACS-sorted and analyzed resident cell populations from infected brains, as microglia and astrocytes have been shown to be major producers of cytokines and chemokines during CNS inflammation [35][36][37]. Firstly, we found that all cell populations expressed Card9, with the highest levels detected in resident microglia (Fig 8B). Interestingly we also found low, albeit detectable, expression of Card9 in the CD45population (Fig 8B), which is predominantly made up of resident glial cells as determined by the presence of transcripts for glial acidic fibrillary protein (Gfap; astrocyte marker) and myelin oligodendrocyte glycoprotein (Mog; oligodendrocyte marker), while endothelial cells were absent as determined by CD31/CD102 staining (S12C and S12D Fig). We found that neutrophils were the major cellular source of Cxcl2 in the infected brain in WT animals, while CD45cells predominantly produced Cxcl1 and Cxcl5 (Fig 8C). In infected Card9 -/brains, we found a significant decrease in levels of Cxcl1 and Cxcl2 from purified Card9 -/neutrophils, and further profound decreases in Cxcl1 and Cxcl5 in the Card9 -/-CD45population (Fig 8C). To visualize the cellular sources of Cxcl2 in the Candida-infected brain in vivo, we made use of a recently described transgenic Cxcl2-GFP reporter mouse [38]. In line with our qRT-PCR data, we found that neutrophils in the infected WT brain were the predominant producers of Cxcl2 following infection, whereas Cxcl2 was undetectable in other tested cell populations ( Fig  8D and 8E). When we infected Card9 -/mice on this genetic background (Card9 -/-Cxcl2-GFP), we found decreased proportion of Cxcl2-expressing Card9 -/neutrophils in the infected brain ( Fig 8F). Thus, our data indicate that resident CD45cells and recruited neutrophils are the major producers of neutrophil-targeted CXC chemokines in the C. albicans-infected mouse brain. Taken together, because CNS tissue does not contain large numbers of resident neutrophils at steady state, our data are consistent with a model by which an early wave of Card9-dependent chemotactic signals by resident cells is followed by a subsequent positive feedback loop of neutrophil-driven cell chemoattraction that is defective in the Card9 -/brain.

Discussion
In the present study, we show that the antifungal adaptor molecule CARD9 is critical for neutrophil recruitment from the blood into the CNS during fungal infection in mice and humans. CARD9 appears to act by promoting production of neutrophil-targeted chemoattractants in the infected CNS tissue, while cell-intrinsic neutrophil chemotaxis is CARD9-independent. Our conclusions are based on detailed analysis of differences in clinical, microbiological, pathological, immunological and molecular parameters between Card9 +/+ and Card9 -/mice, and are corroborated by analogous functional defects identified in a CARD9-deficient patient with Candida meningoencephalitis. Our study is the first to uncover the crucial role of CARD9 on neutrophil accumulation in the CNS during systemic fungal infection in mice and humans, and provides novel insight into the mechanisms of spontaneous susceptibility to CNS fungal infection seen in autosomal recessive CARD9 deficiency.
Here, we describe a novel CARD9 missense mutation, c.170G>A (p.R57H), which further adds to the list of CARD9 mutations associated with the development of systemic fungal infections [7][8][9][10][11][12][13][14]18]. Multiple missense and nonsense CARD9 mutations have now been described that reside in both the coiled-coil and CARD domains [7][8][9][10][11][12][13][14]. There has been thus far no correlation between specific CARD9 mutations and development of systemic fungal infections, and therefore more research will be required to define the potential differential functional roles of the coiled-coil and CARD domains of CARD9 on promoting mucosal versus systemic immunity against C. albicans and other fungi.
Despite several reports of CARD9-deficient patients with CNS infections, the mechanisms of susceptibility in these patients are not well understood, which prompted us to investigate phagocyte function, since these cells are required components of systemic antifungal immune defense against Candida species [24]. We found that monocytes from the CARD9-deficient patient had a profound defect in pro-inflammatory cytokine production specifically in response to fungal components, similar to other reported findings [7][8][9][10][11][12][13][14]. In addition, we show for the first time a modest, yet significant, defect in killing of C. albicans yeasts by monocytes, the mechanisms of which merit future investigation. Similarly, in agreement with a previous report [7], we found a defect in killing of unopsonized yeasts by CARD9-deficient human neutrophils, although opsonization could restore this defect. We found no impact of CARD9 deficiency on the killing of hyphal C. albicans forms by either neutrophils or monocytes. These results confirm recently reported findings using CARD9-deficient neutrophils from a patient nd: not detected. Data pooled from two independent experiments (n = 5) and analyzed by unpaired t-tests; data represent mean ± SEM. *P<0.05, **P<0.01, ***P<0.005, ****P<0.001. doi:10.1371/journal.ppat.1005293.g008 infected with Phialophora, in which only killing of unopsonized conidia was defective, while hyphal killing was intact [14]. Collectively, the impaired killing of CARD9-deficient neutrophils against unopsonized Candida yeasts has led to the hypothesis that CARD9 deficiency may predispose humans to CNS infection since opsonization is suboptimal in this tissue. However, because the predominant Candida morphology in the infected brain in mice and humans is hyphae (Fig 1A, Fig 6C and S4 Fig) [9,24], and because phagocyte hyphal killing is intact in these patients, this current explanation appears insufficient to fully account for why these patients develop spontaneous CNS fungal disease.
Strikingly, we found that CARD9 deficiency resulted in a profound absence of neutrophils in the infected CNS (Fig 3, Fig 6 and S4 Fig); instead, eosinophils and lymphocytes were the predominant leukocytes in the infected CSF, in agreement with recently published findings in other CARD9-deficient patients with Candida CNS infection [7,9]. This profound lack of neutrophil accumulation in the infected CNS is in stark contrast to the robust accumulation of neutrophils in the Candida-infected CNS of mice and humans without CARD9 deficiency [24,29,39]. In our CARD9-deficient patient, this CNS-specific neutropenia could not be explained by a decrease in circulating neutrophils or neutrophil survival, nor a cell-intrinsic chemotaxis defect. Instead, we found that neutrophil-targeted CXC chemokines were not induced in the infected CSF. In patients with neutrophilic meningitis, levels of IL-8 in the CSF have been reported to reach up to 45,000 pg/mL (median, 13,900 pg/mL) [30], and the infected CSF has been shown to be chemotactic toward neutrophils ex vivo [40]. Instead, our patient's CSF lacked chemotactic activity toward neutrophils ex vivo and had approximately 300 pg/mL of IL-8, which is within the range of concentrations seen in the CSF of uninfected control individuals [30], and less than 20 pg/mL of CXCL1, CXCL2 and CXCL5. This is in contrast to the induction of CXC chemokines in the Candida-infected CSF of another patient without CARD9 deficiency who developed meningitis post-Ommaya reservoir placement at NIH, whose CSF was chemotactic toward neutrophils ex vivo. Therefore, our data indicate that CARD9 is required for proper production of these chemoattractants in response to CNS fungal infection. However, more work assessing CSF leukocyte immunophenotype and measuring these chemoattractants in the CSF of additional patients with CNS fungal infections, with and without CARD9-deficiency, is needed.
To directly examine the role of CARD9 in CNS antifungal defense, we moved to a mouse model of systemic candidiasis using Card9 -/mice [41]. We show for the first time that these animals develop significant brain fungal infection with a profound absence of neutrophil accumulation in the infected CNS. Furthermore, we also found that Card9 -/neutrophils could mobilize from the bone marrow into the blood, thus demonstrating a Card9-dependent defect of cell trafficking from the blood into the CNS, which is tissue-and fungus-specific. In line with our human data, we showed that Card9 -/neutrophils did not have a survival defect nor were they intrinsically defective in trafficking from the blood into the Candida-infected brain, since they were able to mobilize into WT brains in mixed bone marrow chimera experiments.
Since our human data suggested that neutrophil-targeted chemokine production depended on CARD9, we investigated the production and source of these molecules in the infected CNS using our mouse model. We found that production of neutrophil-targeted chemokines was grossly suboptimal in the Card9 -/brain relative to the tissue fungal burden. These data suggest that the lack of neutrophils in the Card9-deficient CNS is caused, at least in part, by impaired chemoattractant production. Recent work has demonstrated dependency on both Card9 and IL-1R/Myd88 signaling for neutrophil-targeted chemokine production and neutrophil accumulation in the Aspergillus-infected mouse lung [38,42]. Specifically, Card9 -/mice infected with Aspergillus recruited normal numbers of neutrophils to the lung in the early phase of the infection driven via IL-1R/Myd88-dependent CXC chemokine production, whereas at the later stages of the infection a 40% decrease in neutrophil accumulation was observed, which was due to decreased CXC chemokine production by Card9 -/hematopoietic cells [38,42]. In stark contrast to this partial dependency on Card9 for neutrophil recruitment during pulmonary aspergillosis, we show here that Card9 deficiency almost abolishes neutrophil recruitment to the CNS throughout the entire course of C. albicans infection. Taken together, these data further underscore the tissue-specific nature of antifungal innate immune responses [24] and provide a potential explanation why CARD9-deficient patients, including our patient reported here, have not been reported to have increased susceptibility to pulmonary infections with ubiquitous inhaled molds.
We next investigated the cellular source of neutrophil-targeted chemokines in the CNS, in a bid to further understand how neutrophils are recruited to this site during fungal infection. We found that neutrophils were the predominant source of Cxcl2 and to a lesser extent of Cxcl1, in agreement with previous reports that neutrophils can create positive-feedback loops for their continued recruitment into fungal-infected tissue [32]. We next investigated if Card9 was required for this phenomenon, and found that Card9 -/neutrophils produced less Cxcl1 and Cxcl2. Interestingly, we also found significant decreases in Cxcl1 and Cxcl5 transcript levels in the CD45resident cell population purified from Card9 -/brains, which we found were positive for transcripts encoding glial markers Gfap and Mog. Therefore, our data point to a two-step chemokine-driven neutrophil recruitment process in the WT CNS, initially promoted by resident stromal cells and followed by cell-intrinsic neutrophil chemokine production. In Card9 -/brains, an initial 'hit' of defective chemokine production by the resident CD45compartment may lead to poor neutrophil recruitment, which appears to be further exacerbated by the absence of neutrophil-mediated Cxcl1 and Cxcl2 production in the Card9 -/brain.
Future studies should also aim to examine other neutrophil-targeted chemoattractants that may also contribute to Card9-dependent neutrophil recruitment into the infected CNS, as well as their myeloid and non-myeloid cellular sources and their Card9-dependent organ-specific localization in tissue. In particular, our work has indicated that the resident cells in the brain are integral for neutrophil-targeted chemokine production, and their identification and Card9-dependent functions within antifungal immunity is an area of ongoing investigation in our laboratory. Additionally, future work should also focus on whether CARD9 is equally critical for neutrophil recruitment to the CNS in CARD9-deficient patients and mice infected with other fungal pathogens, such as the recently reported Exophiala [10].
In summary, we have identified a novel critical role for CARD9 in tissue-and fungus-specific neutrophil recruitment to the CNS during C. albicans infection in mice and humans. CARD9 is important for the production of chemoattractants in the CNS by neutrophils and resident cells, whereas other neutrophil-intrinsic functions such as phagocytosis, oxidative burst, killing, chemotaxis and survival, appear largely CARD9-independent. Our work highlights the importance of the CLR/CARD9 immune pathway in the neutrophil-dependent control of C. albicans infection of the CNS, and forms the foundation for devising immune-based therapies for bypassing CARD9 in the production of neutrophil-targeted chemokines such as via the potential direct intrathecal delivery of these molecules in infected CARD9-deficient patients.

Ethics
The patient, her relatives and the healthy donors were enrolled in protocols approved by the National Institute of Allergy and Infectious Diseases and National Cancer Institute Institutional Board Review (IRB) committees, and provided written informed consent for participation in the study. This study was conducted in accordance with the Helsinki Declaration. Animal studies were performed in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health, under the auspices of protocol LCID14E approved by the Animal Care and Use Committee of the National Institute of Allergy and Infectious Diseases. Animals were euthanized by cervical dislocation following administration of ketamine/xylazine cocktail.
Isolation of DNA and CARD9 Sequencing DNA was harvested from Ammonium-Chloride-Potassium (ACK)-lysed whole blood from the CARD9-deficient patient, her parents and two siblings using the Gentra Puregene Blood DNA isolation Kit (Qiagen) per the manufacturer's instructions. Genomic amplification was performed in 15 μL reactions using Platinum PCR SuperMix High Fidelity (Life Technologies), 0.625 mM of each primer and 10-200 ng of DNA. Cycling conditions were 95°C for 3 minutes followed by 35 cycles of 95°C for 20 seconds, and 68°C for 2:45. PCR products were purified using ExoSAP-IT (USB products by Affymetrix), sequenced using Big Dye Terminators v3.1 (Life Technologies), cleaned up using Performa DTR Ultra spin plates and run on an ABI 3730XL. Amplification and sequencing primers are shown in S1 Table. Isolation of Human PBMC, CD14 + Monocytes and Neutrophils from Peripheral Whole Blood PBMC were harvested from whole blood by gradient centrifugation using Lymphocyte Separation Media (Lonza), according to the manufacturer's instructions. PBMC were washed in PBS and quantified prior to use in stimulation assays and flow cytometry (see below) or downstream sorting of CD14 + monocytes. CD14 + monocytes were magnetically sorted using negative selection via the Monocyte Isolation Kit II (Miltenyi) according to the manufacturer's instructions. Purity and viability of sorted monocytes was >84% and >92%, respectively. Neutrophils were isolated using 3% Dextran in 0.85% sodium chloride and red blood cells lysed using sequential exposure to 0.2% and 1.6% NaCl solutions. Purity and viability of isolated neutrophils exceeded 95%. CD14 + monocytes and neutrophils were quantified prior to use in FACS or Western blot analyses or downstream functional assays, including internalization assays, killing assays, survival studies and chemotaxis studies (see below).

PBMC Stimulation and Cytokine Determination by Luminex Array
To determine whether CARD9 deficiency affects the ability of PBMCs to produce pro-inflammatory cytokines and chemokines, we used a Luminex-based assay. In brief, 5 x 10 5 PBMCs from healthy donors or the CARD9-deficient patient were incubated in duplicate in a roundbottom 96-well plate (Corning Inc.) at 37°C in a 5% CO 2 incubator in RPMI 1640 containing 10% fetal bovine serum (FBS, Gibco), 100 U/mL of penicillin, and 100 μg/mL of streptomycin (unstimulated), or RPMI 1640 with 10% FBS/antibiotics containing LPS (100 ng/mL), or heatkilled C. albicans SC5314 yeasts (1 x 10 6 /mL). After 48 hours of stimulation, PBMCs were pelleted and the supernatant was collected and stored at -80°C until analysis. Luminex analysis was done via a multiplex bead array assay with antibodies and cytokine standards to generate known concentration curves (R&D Systems, Peprotech). Individual Luminex bead sets (Luminex) were coupled to cytokine-specific capture antibodies according to manufacturer's protocols and biotinylated polyclonal antibodies were used at twice the recommended concentrations for a classical ELISA according to the manufacturer's instructions. The assay was run with 1200 beads per set of cytokines in a volume of 50 μL. The plates were read on a Luminex MAGPIX platform where more than 50 beads were collected per bead set. The median fluorescence intensity of the beads was then measured for each individual bead, which was analyzed with the Millipex software using a 5P regression algorithm.

Candida Internalization by Human Neutrophils and CD14 + Monocytes
To determine whether CARD9 deficiency affects the capacity of peripheral blood neutrophils and/or CD14 + monocytes to internalize C. albicans, 5 x 10 4 cells from healthy donors or the CARD9-deficient patient were incubated in a polystyrene round-bottom FACS tube (BD Falcon) in a 37°C shaking water bath for 60 minutes in RPMI 1640 containing 100 U/mL of penicillin and 100 μg/mL of streptomycin with dTomato-expressing Candida that had been suspended in RPMI 1640 with antibiotics without (unopsonized) or with 5% human serum (opsonized) at a cell:yeast ratio of 1:25 or 1:1, respectively. Samples were then stained with a FITC-conjugated anti-Candida antibody (cat #21164; Abcam) and APC-conjugated anti-CD14 (for monocytes) or anti-CD15 (for neutrophils) antibodies and subsequently washed. FACS was performed on a 5-laser LSRFortessa. The percentage of Candida internalization by myeloid cells was then determined by initially gating on APC + monocytes or neutrophils and then by defining the percentage of APC + cells that had internalized Candida (APC + PE + FITC -) versus those that had Candida bound on the cell surface (APC + PE + FITC + ).

Candida Killing by Human Neutrophils and CD14 + Monocytes
To determine whether CARD9 deficiency affects the capacity of peripheral blood neutrophils and/or CD14 + monocytes to kill Candida, we used the alamarBlue-based fluorescence assay as previously described [22]. Briefly, neutrophils and monocytes were harvested from peripheral blood as described above, and 5 x 10 4 cells from healthy donors or the CARD9-deficient patient were incubated in flat-bottom 96-well plates (Genesee Scientific) for 2.5 hours with opsonized or unopsonized Candida yeasts or 3-hour pre-grown pseudohyphae at a cell:Candida ratio ranging from 1:4 to 4:1 (see figure legends). The wells were then treated with 0.02% Triton X-100 in water to lyse the neutrophils/monocytes, washed twice with PBS and incubated with 1x alamarBlue (Invitrogen) in PBS for 18 hours at 37°C. Fluorescence was measured using a POLARstar OPTIMA plate reader (BMG Labtech). Killing was calculated by comparing the fluorescence of Candida incubated with neutrophils or monocytes with that of Candida incubated without myeloid cells.

Survival Studies of Human Neutrophils
Neutrophils from healthy donors and the CARD9-deficient patient were harvested as described above. To determine whether CARD9 deficiency impairs survival of neutrophils, cells were cultured for 3 or 6 hours at 37°C in a 5% CO 2 incubator in RPMI 1640 without FBS (serum starvation) or in RPMI 1640 with 10% FBS with tumor necrosis factor-α (50 ng/mL) or live Candida albicans SC5314 (1 x 10 5 cells/mL) or in infected CSF from the CARD9-deficient patient to assess the extent of cell apoptosis and death. Cells were stained with Annexin V and propidium iodide (BD Biosciences) per the manufacturer's instructions, and flow cytometry was performed on a 5-laser LSRFortessa.

Chemotaxis Assays of Neutrophils
Chemotaxis assays were performed on freshly isolated neutrophils using the EZ-TAXIScan (ECI, Inc., Japan) or a modified Boyden chamber (NeuroProbe). In brief, 5 x 10 3 neutrophils were placed in each well of the EZ-TAXIScan and 1.0 μL of either buffer or fMLF (5x10 -8 M) or IL-8 (5x10 -8 M) was added to the opposing well. Images of cellular migration were captured every 30 sec for 60 min at 37°C and individual cells (10 picked at random) were tracked digitally using ImageJ software. The paths of the migrating cells were plotted with the position of each cell at t = 0 anchored at the origin. Using the coordinates obtained at each time point, the migratory path of each cell was resolved into a random migrational vector (orthogonal to the direction of the chemoattractant, along the x-axis) and a directed migrational vector (parallel to the direction of the chemoattractant, along the y-axis). In the absence of chemoattractant, the directed migrational vector should be equivalent to the random migrational vector. For studies analyzing chemotaxis to several chemoattractants simultaneously, chemoattractants (fMLF, LTB 4 , C5a, and IL-8) were prepared at the indicated concentrations in PBS with 0.1% human serum albumin and loaded in triplicate to the bottom well of a 96-well ChemoTx disposable chemotaxis plate (Neuro Probe, Inc.). Neutrophils were labeled with calcein-AM (10 μg/mL) and 7.5 x10 4 added on top of the wells, separated from the chemoattractants by a 5 μm polycarbonate filter. Plates were incubated for 45 min at 37°C, and the fluid in upper chamber removed. Cells attached to the underside of the filter were detached by washing in 2 mM EDTA. After removing the filter, fluorescence of each well was determined in a multi-well fluorescent plate reader (SpectraMAX Gemini EM). The number of migrating cells was determined using a standard curve made with known quantities of labeled cells. To test whether CSF from our CARD9-deficient Candida-infected patient or CSF from a healthy uninfected donor or CSF from our CARD9 +/+ Candida-infected patient post-Ommaya reservoir placement or CSF from two patients with non-neutrophilic (lymphocytic) meningitis was chemotactic itself toward WT and/or CARD9-deficient neutrophils, the NeuroProbe protocol was used, using a 50% dilution of CSF as the chemoattractant.

Expression of CARD9 on Neutrophils and CD14 + Monocytes Using FACS Analysis
Neutrophils and PBMCs were harvested from healthy donors and the CARD9-deficient patient as described above. Cells were first stained with Live/Dead fluorescent dye (Invitrogen) for 10 minutes on ice, followed by anti-CD16/32 for 10 min to block Fc receptors, and then by APCconjugated anti-human CD14 (clone M5E2; BD Biosciences). Cells were then fixed with 2% paraformaldehyde (Affymetrix) at room temperature for 10 minutes and subsequently permeabilized with 1X FACS permeabilizing solution 2 (BD Biosciences) according to the manufacturer's instructions. The cells were then incubated for 30 minutes with rabbit monoclonal anti-CARD9 (clone EPR6489; Origene) or monoclonal rabbit IgG isotype control (clone EPR25A; Abcam), washed and then stained with PE-conjugated secondary anti-rabbit antibody (eBioscience) for 30 minutes. Samples were acquired using a BD LSRFortessa.

Expression of CARD9 on Neutrophils and CD14 + Monocytes Using Western Blot Analysis
Neutrophils and CD14 + monocytes were harvested from healthy donors and the CARD9-deficient patient as described above. Cells were washed once with ice-cold PBS and lysed in buffer containing 50 mM HEPES, 50 mM NaCl, 10% glycerol, 0.5% Nonidet P-40, 2 mM EDTA, and a Protease and Phosphatase Inhibitor Cocktail (cat# 78440; Thermo Scientific). Cell lysates were centrifuged at 13,200 g for 10 minutes at 4°C, and equal protein amounts from the supernatant were resuspended in SDS loading buffer with 5% β-mercaptoethanol for Western blot analysis. Proteins were analyzed by SDS-PAGE and transferred to Immobilon P, polyvinylidene difluoride membrane (Millipore, Billerica). Membranes were blocked in TBS-T containing 5% nonfat dry milk, and then incubated with polyclonal rabbit anti-human CARD9 (cat# 12892-1-AP; ProteinTech Group, Chicago, IL) and rabbit monoclonal antibody against GAPDH (cat# 2118; Cell Signaling Technology) in TBS-T containing 5% BSA for 2 hours at room temperature under continuous agitation. Membranes were then washed 3 times with TBS-T, and incubated with horseradish peroxidase-conjugated anti-rabbit secondary antibody (Southern Biotech) in TBS-T containing 5% nonfat dry milk for 1 hour at room temperature. Membranes were subsequently washed, and detection of immunoreactive bands was performed using the SuperSignal West Fempto Maximum Sensitivity Substrate chemiluminescence (ECL) detection kit according to the manufacturer's instructions (Pierce Biotechnology).

Determination of IL-17A + CD4 + T Cells
PBMCs from healthy donors and the CARD9-deficient patient were isolated as described above and cultured in RPMI 1640 with 10% FBS, 2mM L-glutamine, 100 U/mL of penicillin, and 100 μg/mL of streptomycin (Gibco) at 37°C in a humidified 5% CO 2 incubator. Intracellular staining for IL-17A was performed on PBMCs stimulated for 6 hours with PMA (20 ng/ml; Sigma) and ionomycin (1 μM; Life Technologies) in the presence of Brefeldin A (10 μg/ml; Sigma). FITC-labeled anti-CD4 (clone RPA-T4; eBioscience) was added for the final 30 minutes of culture before the fix and permeabilization step. Cells were then washed with PBS and then fixed and made permeable with Foxp3 staining set (eBioscience) according to the manufacturer's instructions. Cells were then incubated with PE-labeled anti-IL-17A (clone eBio64-CAP17; eBioscience) for 1 hour at 4°C, and acquired on a FACSCanto (BD Biosciences). [8][9][10][11][12] week old female C57BL/6 (Taconic) and Card9 -/- [41] mice were maintained in individually ventilated cages under specific pathogen-free conditions at the National Institutes of Health (Bethesda, MD, USA). Cxcl2-GFP reporter animals [38] were bred and housed in the Memorial Sloan Kettering Cancer Center Comparative Medicine Shared Resources under specific pathogen-free conditions. Candida albicans strain SC5314 was used for all infections. Yeast was serially passaged 3 times in YPD (yeast extract, bacto-peptone and dextrose) broth, grown at 30°C with shaking for 18-24 hours at each passage. Yeast cells were washed in PBS, counted, and injected intravenously via the lateral tail vein. Each animal received 0.7-1.3 x 10 5 yeast cells (details for doses used in individual experiments are presented in the figure legends). At 24 or 72 hours post-infection, animals were euthanized and following analyses performed: brain fungal burdens; FACS analysis on blood, brain and bone marrow cells; quantification of chemokines by Luminex array; histopathology; cell sorting; RNA isolation from brain tissue followed by qRT-PCR analysis.

Depletion of Neutrophils in Infected Mice by 1A8 Administration
WT mice were given 100 μg of 1A8 or 2A3 (isotype) antibody (Bio X Cell) intravenously at 24 hours prior to infection, and again at the time of infection. Antibody-treated mice were infected with 1 x 10 5 CFU of C. albicans SC5314, and analyzed at 24 hours post-infection for brain fungal burden and FACS analysis (see below).

Generation of Mixed Bone Marrow Chimeras
6-8 week old recipient C57BL/6.SJL (CD45.1 + ) mice were irradiated with 900 rad and left to rest for 4 hours. Bone marrow from gender-matched C57BL/6.SJL and Card9 -/-(CD45.2 + ) donor mice was isolated from the femurs, washed and counted using Trypan blue exclusion, and resuspended as a 1:1 ratio for transfer. Flow cytometry was used to confirm the ratio of CD45.1 + to CD45.2 + cells prior to use. 5 x 10 6 total bone marrow cells were transferred to each irradiated recipient mouse intravenously via the lateral tail vein approximately 4 hours postirradiation. Recipient animals were given trimethoprim/sulfamethoxazole in the drinking water for the first 4 weeks of reconstitution, and then switched to antibiotic-free water. Mice were left to reconstitute for a total of 8 weeks post-transfer, and chimera status was assessed by flow cytometry on a sample of peripheral blood. Chimeras were then infected with 1.3 x 10 5 CFU of SC5314, and euthanized for analysis at 24 or 72 hours post-infection. Brains and 300 μL peripheral blood were stained for flow cytometry analysis as above. WT and Card9 -/neutrophils (CD45 + CD11b + Ly6G hi ) were separated based on expression of congenic CD45.1 and CD45.2 markers and the ratio between them calculated.

Determination of Fungal Burdens from Mouse Brains
At 24 or 72 hours post-infection, animals were euthanized and brains and kidneys were weighed, homogenized in PBS, and serially diluted before plating onto YPD agar supplemented with Penicillin/Streptomycin (Invitrogen). Colonies were counted after incubation at 37°C for 24-48 hours.

Flow Cytometry of Mouse Bone Marrow, Blood and Brain
Leukocytes were isolated from brain, kidney, blood and bone marrow using previously described methods [24,43], resuspended in PBS and stained with fluorochrome-conjugated antibodies as described for the human samples. Anti-mouse antibodies used in this study were:

FACS Analysis of Neutrophils for Annexin V and 7-AAD Expression
Brain-isolated leukocytes were Fc-blocked and stained with fluorochrome-conjugated antibodies as described above, washed twice with PBS, and resuspended in Annexin V binding buffer (BD Biosciences). Samples were incubated at room temperature for 15 minutes with 5 μl each of FITC-conjugated Annexin V and 7-AAD per the manufacturer's instructions (BD Biosciences). FACS was performed on a 5-laser LSRFortessa.

Chemokine/Cytokine Quantification in Mouse Brains by Luminex Array
Brains and kidneys were weighed and then homogenized in PBS/0.05% Tween20 and a protease inhibitor cocktail (Roche). Cell debris was removed by two sequential centrifugation steps (3600 rpm for 5 minutes, 13200 rpm for 10 minutes), and the resulting supernatant filtered through 0.2 μm filters, snap-frozen on dry ice and stored at -80°C until analysis. Samples were analyzed by Luminex assay, as described above, and cytokine/chemokine concentrations were determined per gram of tissue.

Histopathology
Brains were removed from infected mice at the indicated time points and fixed in 10% formalin for 24 hours before embedding in paraffin wax. Tissue sections were stained with periodic acid-Schiff (PAS) and hematoxylin and eosin (H&E).

Analysis of Cxcl2 Expression by Brain Myeloid Cells Using Cxcl2-GFP Reporter Mice
Cxcl2-GFP reporter and non-transgenic WT littermates were infected as described above with 1.3 x 10 5 CFU SC5314 intravenously and analyzed at 24 hours post-infection. Brains from infected mice were processed as previously described [24], and stained for FACS analysis as described above. Cxcl2-GFP expression was determined in the major myeloid populations (neutrophils, Ly6C hi monocytes and microglia) by comparison with WT (GFP -) cells gated in the same way.

Staphylococcus aureus Infection Model
Community-acquired methicillin resistant Staphylococcus aureus USA300 (CA-MRSA, LAC strain from F. DeLeo, Rocky Mountain Laboratories, NIAID) was grown in brain heart infusion broth (BHI, Difco Laboratories, Detroit, Mich.) at 37°C with shaking at 230 rpm for 18 hours. The culture was then centrifuged at 3000 rpm for 10 minutes at room temperature and the pellet washed twice with PBS. WT and Card9 -/mice were infected intravenously with 2 x 10 7 CFU via the lateral tail vein, and animals euthanized at 48 hours post-infection and analyzed as described above.

Statistics
Statistical analyses were performed using GraphPad Prism 6.0 software. Details of individual tests are included in the figure legends. In general, data was tested for normal distribution by Kolmogorov-Smirnov normality test and analyzed accordingly by unpaired two-tailed t-test or Mann Whitney U-test. In cases where multiple data sets were analyzed, two-way ANOVA was used with Bonferroni correction. In all cases, P values <0.05 were considered significant.