CARD9+ microglia promote antifungal immunity via IL-1β- and CXCL1-mediated neutrophil recruitment

Abstract

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.

Fig. 1:. Functional redundance of CARD9-coupled C-type lectin receptors for protective neutrophil recruitment to the fungal-infected brain.

a, Card9^−/− mice (n=4 animals) and their wild-type controls (n=4 animals) were intravenously infected with C. albicans SC5314 and analyzed for neutrophil counts by flow cytometry at 24 h post-infection (left; dose: 1.3 × 10⁵ CFU) and fungal growth within the brain at 72 h post-infection (right; dose: 7 × 10⁴ CFU). b, Animals of the indicated genotype (WT n = 9 animals, Clec7a^−/− n = 6 animals; WT n = 12 animals, Clec4n^−/− n = 10 animals; WT n = 6 animals, Clec4d^−/− n = 6 animals; WT n = 6 animals, Clec4e^−/− n = 8 animals; WT n = 10 animals, Clec7a^−/−Fcer1g^−/− n = 9 animals) were intravenously infected with C. albicans SC5314 (2 × 10⁵ CFU for Clec4d^−/− and Clec7a^−/−Fcer1g^−/− and their controls; 1.3 × 10⁵ all others) and analyzed for neutrophil counts by flow cytometry at 24 h post-infection and c, fungal burdens in the brain at 24 and 72 h post-infection (WT n = 9 animals, Clec7a^−/− n = 6 animals; WT n = 10 animals, Clec4n^−/− n = 10 animals; WT n = 10 animals, Clec4d^−/− n = 10 animals; WT n = 6 animals, Clec4e^−/− n = 4 animals; WT n = 10 animals, Clec7a^−/−Fcer1g^−/− n = 9 animals). d, Malt1^−/− mice and their littermate controls were infected as above and analyzed for fungal burdens in the brain (right; WT n = 7 animals, Malt1^−/− n = 5 animals) and neutrophil recruitment to the brain at 24 h post-infection (left; WT n = 5 animals, Malt1^−/− n = 5 animals). In all cases, ‘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 independent experiments and is shown as mean +/− SEM, and analyzed by unpaired two-tailed t-test (panel a [left], b) or two-tailed Mann Whitney U-test (panel a [right], c, d). *P<0.05, **P<0.01, ***P<0.005, ****P<0.001.

Fig. 2:. IL-1β and CXCL1 mediate protective neutrophil recruitment to the fungal-infected brain.

a,b, Animals deficient in elements of the IL-1R signaling pathway or c,d, chemokine receptors and their ligands, were infected and analyzed for neutrophil recruitment at 24 h post-infection (a,c) and control of fungal brain infection (b,d) as in Fig. 1. ‘Wild type’ refers to appropriate matched control animals for each knock-out line for gender, age and genetic background. Individual points represent different mice; (a) WT n = 8 animals, Il1r^−/− n = 8 animals; WT n = 7 animals, Myd88^−/− n = 6 animals; WT n = 12 animals, Il1a^−/− n = 12 animals; WT n = 6 animals, Il1b^−/− n = 6 animals; WT n = 8 animals, Il1a^−/−Il1b^−/− n = 6 animals. (b) WT n = 8 animals, Il1r^−/− n = 7 animals; WT n = 7 animals, Myd88^−/− n = 6 animals; WT n = 3-8 animals, Il1a^−/− n = 4-8 animals; WT n = 6 animals, Il1b^−/− n = 6 animals; WT n = 6 animals, Il1a^−/−Il1b^−/− n = 6 animals. (c) WT n = 6 animals, Ccr1^−/− n = 6 animals; WT n = 11 animals, Cxcr1^−/− n = 11 animals; WT n = 14 animals, Cxcr2^−/− n = 16 animals; WT n = 8 animals, Cxcl1^−/− n = 7 animals. (d) WT n = 6-7 animals, Ccr1^−/− n = 6-7 animals; WT n = 7-10 animals, Cxcr1^−/− n = 7-10 animals; WT n = 8-10 animals, Cxcr2^−/− n = 7-10 animals; WT n = 6-8 animals, Cxcl1^−/− n = 5-7 animals. Data is pooled from 2-3 independent experiments and shown as mean +/− SEM, analyzed by unpaired two-tailed t-test (panel a, c) or two-tailed Mann Whitney U-test (panel b, d). *P<0.05, **P<0.01, ***P<0.005, ****P<0.001.

Fig. 3:. Production of CXCL1 is dependent on IL-1β in the fungal-infected brain.

a, Wild type (n = 6/7 animals), Cxcl1^−/− (n = 5 animals) and Il1b^−/− (n = 6 animals) animals were infected as in Fig. 1 and brains isolated at 24 h post-infection and analyzed for CXCL1 or IL-1β production by ELISA. Data is pooled from 2 independent experiments and analyzed by unpaired two-tailed t-test. b, The relative proportions of myeloid cell populations (gated within live CD45⁺ singlets) in the uninfected ( n = 6 animals) and 24 h infected WT (n = 6 animals) brain (left), and the relative proportion of myeloid cell populations producing CXCL1 (n = 3 animals) or pro-IL-1β (n = 9 animals) in the 24 h infected brain (right). For the latter, total CD45⁺CXCL1(or IL-1β)⁺ cells were first gated and then cell types defined within this initial gate using lineage markers (see below), using samples from the unstimulated condition. Data is shown as the mean +/− SEM. c, Wild type (n = 3 animals) and Il1b^−/− mice (n = 4 animals) were infected with 2 × 10⁵ C. albicans and brain cells analyzed for CXCL1 production by intracellular flow cytometry 24 h later. Brain cells were restimulated ex vivo with 62.5 μg/mL depleted zymosan or 1 μg/mL LPS for 4 h in the presence of 5 μg/mL Brefeldin A. Representative plots from the LPS-stimulated condition are gated on microglia (top; CD45^int Ly6G⁻ CD11b⁺), Ly6C^hi monocytes (middle; CD45^hi Ly6C^hi Ly6G⁻ CD11b⁺) and neutrophils (bottom; CD45^hi Ly6C^int Ly6G^hi CD11b⁺), showing corresponding Cxcl1^−/− cells as gating controls. In all panels, ‘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 shown as mean +/− SEM, and analyzed by unpaired two-tailed t-test. *P<0.05, **P<0.01.

Fig. 4:. Fungal-derived Candidalysin promotes neutrophil recruitment and control of fungal growth in the brain.

a,b,c,e, Animals were infected with 2 × 10⁵ CFU of the indicated C. albicans strains (parental strains, closed symbols; deficient mutants, open symbols) and analyzed as in Fig. 1 for fungal burdens (hgc1Δ/Δ n = 8 animals, hgc1Δ/Δ + HGC1 n = 7 animals; BWP17 n = 10 animals, ece1Δ/Δ n = 11 animals, ece1Δ/Δ + ECE1 n = 10 animals, ece1Δ/Δ + ECE1_Δ184-279 n = 11 animals; CAI4 + CIp10 n = 6 animals, sap4/5/6Δ/Δ + CIp10 n = 6 animals) and neutrophil recruitment (hgc1Δ/Δ n = 10 animals, hgc1Δ/Δ + HGC1 n = 10 animals; BWP17 n = 7 animals, ece1Δ/Δ n = 7 animals, ece1Δ/Δ + ECE1 n = 7 animals, ece1Δ/Δ + ECE1_Δ184-279 n = 11 animals; CAI4 + CIp10 n = 6 animals, sap4/5/6Δ/Δ + CIp10 n = 7 animals). Histology shown in (c) is from 24 h post-infection, stained with PAS. Scale bar is 50 μm. d, Whole brain homogenates from animals infected with indicated strains were isolated at 24 h post-infection and analyzed for IL-1β and CXCL1 using ELISA (BWP17 n = 8-10 animals, ece1Δ/Δ n = 8-10 animals, ece1Δ/Δ + ECE1 n = 6-11 animals, ece1Δ/Δ + ECE1_Δ184-279 n = 7-11 animals). Individual points represent different mice. Data is pooled from 2-4 independent experiments and shown as mean +/− SEM, analyzed by unpaired two-tailed t-test, or two-tailed Mann Whitney U-test (panel a, left). *P<0.05, **P<0.01, ***P<0.005; ns = not significant.

Fig. 5:. Microglia produce IL-1β and CXCL1 in a Candidalysin-dependent manner.

Animals were infected with wild-type C. albicans (BWP17; closed bars) or a Candidalysin-null strain (ece1Δ/Δ; open bars), and brain cells isolated 24 h later. Brain leukocytes were restimulated as in Fig. 3, and intracellular staining for a, IL-1β (unstimulated, n = 8 animals; zymosan, n = 4 animals; LPS n = 6 animals) and b, CXCL1 (unstimulated, n = 12 animals; zymosan, n = 6 animals; LPS n = 9 animals) was analyzed by flow cytometry. Box-and-whisker plots show the minimum/maximum values (whiskers), the 25^th/75^th percentiles and the median. Data is pooled from 2-4 independent experiments and analyzed by unpaired two-tailed t-tests. *P<0.05, **P<0.01. Representative staining is shown for LPS-stimulated microglia (gated as in Fig. 3) from wild-type mice infected with indicated strains, or BWP17-infected cytokine-deficient mutants as control.

Fig. 6:. Candidalysin activates microglial IL-1β production via p38-cFos signaling and promotes microglial CXCL1 production through astrocyte interactions.

BV-2 microglia were seeded into 24-well plates at 5 × 10⁵ per well and left to adhere for 2 h in the presence of 50 n g/mL LPS (for priming) before the addition of purified Candidalysin at the indicated concentrations. Cell culture supernatants were analyzed for a, IL-1β production or b, LDH release after 24 h of stimulation. Data is shown with the mean +/− SEM, individual points represent individual culture wells (n = 4). c-d, In some experiments, BV-2 cells were co-cultured with 3 × 10⁵ C8-D1A astrocytes, and CXCL1 production analyzed in the supernatant by ELISA (n = 10 individual culture wells) or by intracellular flow cytometry (n = 3-5 individual culture wells; data shown with the mean). In d, microglia and astrocytes were distinguished by CD45 staining, and CXCL1 production assessed within CD45⁺ (microglia) and CD45⁻ (astrocyte) gates. Histogram is gated on CD45⁺ microglia. e, To measure cFos and pMKP½ activation, BV-2 cells were stimulated with the indicated Candidalysin concentrations for 30 or 120 min and BV-2 cells then lysed and analyzed for cFos and pMKP½ by immunoblot, normalizing to β-actin. Immunoblots shown are representative of 2 independent experiments. f, BV-2 cells were cultured in the presence of the indicated cFos and p38 inhibitors for 2 h prior to stimulating with 20 μM Candidalysin, and IL-1β measured in the supernatant by ELISA after 24 h (data shown as mean +/− SEM; n = 6 individual culture wells). All data is pooled from 2 independent experiments and analyzed by one-way ANOVA. *P<0.05, **P<0.01, ***P<0.005, ****P<0.0001.

Fig. 7:. CARD9 is required for microglial pro-IL-1β transcription, inflammasome activation, and CXCL1 production in the fungal-infected brain.

a,b, Card9^+/+ ( n = 13 animals) and Card9^−/− (n = 13 animals) animals were infected with 2 × 10⁵ CFU wild-type C. albicans (BWP17), and brain cells isolated 24 h later. Brain leukocytes were restimulated as in Fig. 4, and intracellular staining for pro-IL-1β and CXCL1 analyzed by flow cytometry in total CD45⁺ cells (LPS-stimulated condition shown) (a) or microglia alone, normalized to Card9^+/+ results (b). Panels a,b show pooled data from 4 independent experiments, analyzed with two-tailed unpaired t-test. Data shown as mean +/− SEM (a) or with minimum/maximum values (whiskers), the 25^th/75^th percentiles and the median (b). c, Microglia were FACS-sorted from pooled Card9^+/+ (n = 4 animals) and Card9^−/− animals (n = 4 animals) at 24 h post-infection and analyzed by unpaired two-tailed t-test for Il1b expression by qRT-PCR, or d,e, the indicated proteins by immunoblot (Caspase and IL-1β blots; WT n = 6 animals, Card9^−/− n = 7 animals; cFos blot; WT n = 10 animals, Card9^−/− n = 10 animals; NLRP3 blot; WT n = 8 animals, Card9^−/− n = 8 animals;). Graphs in (d,e) represent the band pixel density normalized to the wild type control, and are shown with mean +/− SEM and analyzed by unpaired two-tailed student t-tests. Example blots are representative of 3 independent FACS sorts/experiments; pooled data is shown in the graphs above. f, Nlrp3^−/− animals and their wild-type controls were infected with 1.3 × 10⁵ CFU C. albicans and analyzed by unpaired two-tailed t-tests for neutrophil recruitment to the brain 24 h later (left; WT n = 9 animals, Nlrp3^−/− n = 8 animals) and by two-tailed Mann-Whitney U-test for fungal brain burdens at 72 h post-infection (right; WT n = 14 animals, Nlrp3^−/− n = 14 animals), as described in Fig. 1. *P<0.05, **P<0.01, ***P<0.005.

Fig. 8:. CARD9 is required specifically in microglia for neutrophil recruitment and control of fungal invasion in the CNS.

a, Card9^fl/flCx3cr1^CreER−/− and Card9^fl/flCx3cr1^CreER+/- littermates (n=8-13) were tamoxifen-pulsed at 4-5 weeks of age, left to rest for 4-6 weeks and then infected with 1.3 × 10⁵ CFU C. albicans (SC5314) intravenously and analyzed for brain and kidney fungal burdens (Card9^fl/flCx3cr1^CreER−/− n = 8-10 animals; Card9^fl/flCx3cr1^CreER+/− n = 8 animals), b, neutrophil recruitment to the brain at 24 h post-infection (Card9^fl/flCx3cr1^CreER−/− n = 13 animals; Card9^fl/flCx3cr1^CreER+/− n = 9 animals), and c, intracellular staining for pro-IL-1β and CXCL1, as described in Fig. 3 (Card9^fl/flCx3cr1^CreER−/− n = 8 animals; Card9^fl/flCx3cr1^CreER+/− n = 8 animals). Data is pooled from 2-4 independent experiments and is shown as mean +/− SEM, analyzed by two-tailed Mann-Whitney U-tests (panel a) or two-tailed unpaired t-tests (panel b, c). *P<0.05, **P<0.01.