The C-type lectin receptor–Syk (spleen tyrosine kinase) adaptor CARD9 facilitates protective antifungal immunity within the central nervous system (CNS), as human deficiency in CARD9 causes susceptibility to fungus-specific, CNS-targeted infection. CARD9 promotes the recruitment of neutrophils to the fungus-infected CNS, which mediates fungal clearance. In the present study we investigated host and pathogen factors that promote protective neutrophil recruitment during invasion of the CNS by Candida albicans. The cytokine IL-1β served an essential function in CNS antifungal immunity by driving production of the chemokine CXCL1, which recruited neutrophils expressing the chemokine receptor CXCR2. Neutrophil-recruiting production of IL-1β and CXCL1 was induced in microglia by the fungus-secreted toxin Candidalysin, in a manner dependent on the kinase p38 and the transcription factor c-Fos. Notably, microglia relied on CARD9 for production of IL-1β, via both transcriptional regulation of Il1b and inflammasome activation, and of CXCL1 in the fungus-infected CNS. Microglia-specific Card9 deletion impaired the production of IL-1β and CXCL1 and neutrophil recruitment, and increased fungal proliferation in the CNS. Thus, an intricate network of host–pathogen interactions promotes antifungal immunity in the CNS; this is impaired in human deficiency in CARD9, which leads to fungal disease of the CNS.
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This work was supported by the Intramural Research Program of the National Institute of Allergy and Infectious Disease, National Institutes of Health, as well as NIH grants awarded to T.M.H. (no. R01 093808), S.G.F. (no. R01AI124566) and S.A.L. (no. R01CA161373). Additional funding was provided by the Burroughs Wellcome Fund (awarded to T.M.H.), the Wellcome Trust (nos. 102705 and 097377; awarded to G.D.B.), the MRC Centre for Medical Mycology and the University of Aberdeen (no. MR/N006364/1; awarded to G.D.B.). The authors additionally thank C. Huaman for care and screening of the Malt1–/– mice, which were a kind gift to B.C.S. from T. Mak and the University Health Network (Canada), and D. McGavern and F. Crews for providing the murine glial cell lines.
Integrated supplementary information
Microglia were MACS-sorted from uninfected wild-type brains and analyzed for expression of the indicated C-type lectin receptors (and Card9 as control) by qRT-PCR, relative to Gapdh. Each point represents pooled microglia from an individual mouse (n = 4 animals). Data presented as mean +/− SEM.
Supplementary Figure 2 Gating strategy used to define myeloid cells in the C. albicans-infected brain.
Gates and their associated frequency are outlined in blue. Titles in blue above each plot denote the cell population being defined in that plot.
Supplementary Figure 3 Optimal neutrophil phagocytosis of Candida requires Dectin-1 and Dectin-2 in the C. albicans-infected brain.
Dectin-1 (n = 4 animals) and Dectin-2-deficient (n = 5 animals) mice and their wild-type controls (n = 13 animals) were infected with 5 × 106 green fluorescent protein (GFP)-expressing C. albicans and brain leukocytes analyzed 2 h later by flow cytometry, using the gating strategy shown in Supplementary Fig. 2. The percentage of GFP+ neutrophils was used as a measure of in vivo phagocytosis. Each point represents an individual mouse. Data is pooled from 2-3 experiments and shown as mean +/− SEM. Histograms are gated on live CD45+ Ly6G+ CD11b+ neutrophils. *P = 0.0314 (WT vs Clec7a–/–), *P=0.0192 (WT vs Clec4n–/–) by unpaired two-tailed t-test.
Supplementary Figure 4 Chemoattractant receptors LTB4R1 and FPR1 are not required for protective neutrophil recruitment to the fungus-infected brain.
Animals deficient in chemoattractant receptors were infected and analyzed as described for Fig. 1 for (a) neutrophil recruitment to the brain at 24 h (WT n = 9 animals, Ltb4r1–/– n = 9 animals; WT n = 6 animals, Fpr1–/– n = 8 animals), and (b) fungal burdens at 72 h post-infection (24 h: WT n = 9 animals, Ltb4r1–/– n = 6 animals; WT n = 6 animals, Fpr1–/– n = 8 animals; 72 h: WT n = 8 animals, Ltb4r1–/– n = 5 animals; WT n = 6 animals, Fpr1–/– n = 6 animals). ‘Wild type’ refers to appropriate matched control animals for each knock-out line for gender, age and genetic background. Individual points represent different mice. Data is pooled from 2-3 independent experiments and shown as mean +/− SEM, and analyzed by unpaired two-tailed t-test (panel a) or two-tailed Mann Whitney U-test (panel b).
The number of microglia from Card9+/+ and Card9–/– mice were determined at the steady-state and during infection at the indicated time points (left; 0 h: WT n = 7 animals, Card9–/– n = 5 animals; 24 h : WT n = 10 animals, Card9–/– n = 11 animals; 72 h: WT n = 13 animals, Card9–/– n = 12 animals). Expression levels of lineage and activation markers were determined in 8-10-week-old uninfected mice by flow cytometry (right; WT n = 7 animals, Card9–/– n = 7 animals). Data shown is pooled from 2-3 independent experiments and presented as mean +/− SEM. Each point represents an individual animal.
Microglia and Ly6Chi monocytes were FACS-sorted from 24 h infected brains from Card9fl/flCx3cr1CreER–/– and Card9fl/flCx3cr1CreER+/– littermates that had been tamoxifen-pulsed 4 weeks earlier. Cells were pooled from 4-5 brains per group, and Card9 expression quantified by qRT-PCR relative to Gapdh. Data is pooled from 2 independent sorting experiments. Bar charts show the mean of the data, points represent the result from each independent sorting experiment (n = 2 independent experiments).
Schematic depiction of the Candidalysin–IL-1β–CXCL1 protective immune axis acting via CARD9-expressing microglia to mediate protective neutrophil accumulation to the brain following invasive C. albicans infection of the CNS.