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

<div><p><i>Candida</i> 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 <i>Candida</i> infection. We phenocopied the human susceptibility in <i>Card9</i>^<i>-/-</i> mice, which develop uncontrolled brain candidiasis with diminished neutrophil accumulation. The induction of neutrophil-recruiting CXC chemokines is significantly impaired in infected <i>Card9</i>^<i>-/-</i> 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.</p></div

CARD9 deficiency results in a striking absence of neutrophils from the infected CSF that is not caused by peripheral neutropenia or decreased neutrophil survival.

<p><b>(A)</b> Representative cytopathology image of the patient’s infected CSF showing lymphocytes, eosinophils and mononuclear phagocytes, but no neutrophils. <b>(B)</b> Immunophenotype of the patient’s infected CSF, assessed by FACS. <b>(C)</b> 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 <i>Candida</i> meningitis post-Ommaya reservoir placement (grey bar) and those typically reported for <i>Candida</i> meningoencephalitis in patients without CARD9 deficiency (red bar) are also shown. <b>(D)</b> The percentages of neutrophils within leukocytes in peripheral blood of the CARD9-deficient patient over time. <b>(E)</b> 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⁺ PI^- apoptotic (left panels) and Annexin V⁺ PI⁺ dead (right panels) cells (n = 3–4; 3 independent experiments). All quantitative data represent mean ± SEM.</p

CARD9 mediates killing of <i>Candida</i> yeasts by human phagocytes but it is dispensable for the killing of invasive <i>Candida</i> hyphal forms that are found in infected tissue.

<p>Killing of opsonized and unopsonized <i>Candida</i> yeasts <b>(A)</b> and hyphal forms <b>(B)</b> by healthy donor and patient neutrophils (n = 5–7). Killing of opsonized and unopsonized <i>Candida</i> yeasts <b>(C)</b> and hyphal forms <b>(D)</b> by healthy donor and patient CD14⁺ monocytes (n = 5–8). <b>(E)</b> Internalization of opsonized (left panel) and unopsonized (right panel) <i>Candida</i> 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. *<i>P</i> < 0.05; **<i>P</i> = 0.01; ***<i>P</i> < 0.001. All quantitative data represent mean ± SEM.</p

CARD9 is required for neutrophil-targeted chemokine production during fungal infection of the CNS.

<p><b>(A</b>) Whole brain homogenates were analyzed for Cxcl1, Cxcl2 and Cxcl5 in WT (filled bars, n = 6) and <i>Card9</i>^<i>-/-</i> (empty bars, n = 6) mice by Luminex array. Chemokine concentration (ng/g) was expressed relative to the mean brain fungal burden (CFU/g). Mice analyzed at 24 hours were infected with 1.3x10⁵ CFU SC5314, and mice analyzed at 72 hours infected with 7x10⁴ CFU. Data is pooled from two independent experiments and analyzed by two-way ANOVA (with Bonferroni correction). (<b>B</b>) Relative expression of <i>Card9</i> mRNA by indicated cell populations FACS-sorted from WT brains at 24 hours post-infection (n = 3). (<b>C</b>) Neutrophils, Ly6C^hi monocytes, microglia and CD45^- cells were FACS-sorted from WT and <i>Card9</i>^<i>-/-</i> brains at 24 hours post-infection (inoculum: 1.3x10⁵) and RNA purified for qRT-PCR analysis of <i>Cxcl1</i>, <i>Cxcl2</i> and <i>Cxcl5</i> expression. See <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005293#ppat.1005293.s011" target="_blank">S11 Fig</a> for purity analysis. Data is pooled from nine (neutrophils, Ly6C^hi monocytes, microglia) or six sorts (CD45^- cells) and analyzed by two-way ANOVA (with Bonferroni correction). (<b>D</b>) Transgenic Cxcl2-GFP reporter animals (black histograms) were infected with 1.3x10⁵ CFU and brain leukocytes analyzed by FACS at 24 hours post-infection. Expression of GFP within the indicated cell populations was compared to non-transgenic WT animals (grey histograms). Histograms are representative of two independent experiments. (<b>E</b>) Quantification of the frequency of GFP⁺ cells within the indicated cell populations and (<b>F</b>) percentage of GFP⁺ neutrophils within the WT and <i>Card9</i>^<i>-/-</i> brain; nd: not detected. Data pooled from two independent experiments (n = 5) and analyzed by unpaired t-tests; data represent mean ± SEM. *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.005, ****<i>P</i><0.001.</p

CARD9 is critical for the generation of neutrophil-targeted chemokines in the infected CSF but it is dispensable for intrinsic chemotaxis of human neutrophils.

<p><b>(A)</b> Pooled data showing chemotaxis of healthy donor (n = 40) and patient (n = 2) neutrophils towards IL-8 (left panel) and fMLP (right panel) assessed by the EZ-TAXIScan system. <b>(B)</b> EZ-TAXIScan data for individual neutrophils moving towards either IL-8 (left panels) or fMLP (right panels); individual tracks are represented by different colours and were recorded over 1 hour. <b>(C)</b> Chemotaxis of healthy donor (n = 13–15) 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). <b>(D)</b> 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 <i>Candida</i> meningitis post-Ommaya reservoir placement (grey bar) and previously reported for neutrophilic meningitis in patients without CARD9 deficiency (red bar) are also shown. <b>(E)</b> 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. <b>(F)</b> The CSF of the CARD9^+/+<i>Candida</i>-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. **<i>P</i> < 0.01 by unpaired t-tests. All quantitative data represent mean ± SEM. fMLP, formyl-methionyl-leucyl-phenylalanine; LTB4, leukotriene B4; PMNs, neutrophils.</p

The autosomal recessive c.170G>A <i>CARD9</i> missense mutation results in impaired pro-inflammatory cytokine responses in PBMCs upon fungal stimulation.

<p><b>(A)</b> Grocott-Gomori methenamine-silver stain of the patient’s T12 vertebral body biopsy sample showing filamentous fungal elements consistent with <i>C</i>. <i>albicans</i> that grew in culture (magnification, ×400). <b>(B)</b> Sagittal magnetic resonance image of the cervical spine showing leptomeningeal enhancement (black arrows) and a C5-T1 syrinx (white arrow). <b>(C)</b> Sagittal magnetic resonance image of the brain showing an abscess (arrow). <b>(D)</b> Sequencing chromatograms of <i>CARD9</i> at position 170 for the patient, her parents and a healthy donor control. <b>(E)</b> Pedigree of the patient with <i>Candida</i> meningoencephalitis. The patient is indicated in black and by arrow and the genotype of <i>CARD9</i> alleles is indicated below each family member. <b>(F)</b> FACS histograms of CARD9 staining in neutrophils and CD14⁺ monocytes of healthy donors and the patient (n = 2). <b>(G)</b> 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). <b>(H-J)</b> 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 <i>C</i>. <i>albicans</i> 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. **<i>P</i> < 0.01; ***<i>P</i> < 0.001. <b>(K)</b> 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.</p

Neutrophil recruitment is critical for control of fungal brain infection and directly correlates with the extent of brain fungal burden in mice.

<p>WT animals were injected intravenously with 100 μg of 1A8 antibody (red bars, n = 10) or 2A3 isotype control antibody (grey bars, n = 8) at 24 hours prior to infection and at the time of infection. At 24 hours post-infection, brains from these mice were analyzed for (<b>A</b>) recruitment of myeloid cells by FACS and (<b>B</b>) brain fungal burden. Data pooled from two independent experiments and analyzed by two-way ANOVA (<b>A</b>) or Mann Whitney U-test (<b>B</b>). WT animals were systemically infected with a low (7x10⁴) or a high inoculum (1x10⁶) of <i>C</i>. <i>albicans</i> SC5314 and brains assessed for (<b>C</b>) fungal burden, (<b>D</b>) neutrophil recruitment by FACS and (<b>E</b>) production of neutrophil-targeted chemokines by Luminex array at 72 hours post-infection. Data shown is representative of 2 independent experiments and analyzed by unpaired t-tests (<b>C-E</b>). *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.005, ****<i>P</i><0.001. Data represent mean ± SEM.</p

CARD9 deficiency does not affect cell-intrinsic neutrophil chemotaxis, survival or expression of adhesion molecules.

<p><b>(A</b>) The ratio of CD45.1⁺ WT neutrophils to CD45.2⁺<i>Card9</i>^<i>-/-</i> neutrophils in mixed bone marrow chimeras was determined by FACS in the blood prior to infection and in the blood and brain at 24 (left panel) and 72 hours (right panel) post-infection (n = 7–8 per time point). (<b>B</b>) Expression of various adhesion molecules in whole brain homogenates of uninfected and infected WT (filled bars, n = 6) and <i>Card9</i>^<i>-/-</i> (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 1.3x10⁵ CFU SC5314, and mice analyzed at 72 hours infected with 7x10⁴ CFU. (<b>C</b>) Neutrophil survival in the brain of 24-hour infected (dose: 1.3x10⁵ CFU) WT (filled bars, n = 6) and <i>Card9</i>^<i>-/-</i> (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. *<i>P</i><0.05 by two-way ANOVA.</p