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


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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Fig. 1: Functional redundancy of CARD9-coupled CLRs for protective neutrophil recruitment to the fungus-infected brain.
Fig. 2: IL-1β and CXCL1 mediate protective neutrophil recruitment to the fungus-infected brain.
Fig. 3: Production of CXCL1 is dependent on IL-1β in the fungus-infected brain.
Fig. 4: Fungus-derived Candidalysin promotes neutrophil recruitment and control of fungal growth in the brain.
Fig. 5: Microglia produce IL-1β and CXCL1 in a Candidalysin-dependent manner.
Fig. 6: Candidalysin activates microglial IL-1β production via p38–c-Fos signaling and promotes microglial CXCL1 production through astrocyte interactions.
Fig. 7: CARD9 is required for microglial pro-IL-1β transcription, inflammasome activation and CXCL1 production in the fungus-infected brain.
Fig. 8: CARD9 is required specifically in microglia for neutrophil recruitment and control of fungal invasion into the CNS.

Data availability

The data that support the findings of this study are available from the corresponding authors upon request.


  1. 1.

    Lionakis, M. S. & Levitz, S. M. Host control of fungal infections: lessons from basic studies and human cohorts. Annu. Rev. Immunol. 36, 157–191 (2018).

    CAS  Article  Google Scholar 

  2. 2.

    Drummond, R. A. & Lionakis, M. S. Mechanistic insights into the role of C-type lectin receptor/CARD9 signaling in human antifungal immunity. Front. Cell Infect. Microbiol. 6, 39 (2016).

    Article  Google Scholar 

  3. 3.

    Glocker, E. O. et al. A homozygous CARD9 mutation in a family with susceptibility to fungal infections. N. Engl. J. Med. 361, 1727–1735 (2009).

    CAS  Article  Google Scholar 

  4. 4.

    Lanternier, F. et al. Deep dermatophytosis and inherited CARD9 deficiency. N. Engl. J. Med. 369, 1704–1714 (2013).

    CAS  Article  Google Scholar 

  5. 5.

    Drummond, R. A. et al. CARD9-dependent neutrophil recruitment protects against fungal invasion of the central nervous system. PLoS Pathog. 11, e1005293 (2015).

    Article  Google Scholar 

  6. 6.

    Li, X. et al. The β-glucan receptor Dectin-1 activates the integrin Mac-1 in neutrophils via Vav protein signaling to promote Candida albicans clearance. Cell Host Microbe 10, 603–615 (2011).

    CAS  Article  Google Scholar 

  7. 7.

    Drewniak, A. et al. Invasive fungal infection and impaired neutrophil killing in human CARD9 deficiency. Blood 121, 2385–2392 (2013).

    CAS  Article  Google Scholar 

  8. 8.

    Altmeier, S. et al. IL-1 coordinates the neutrophil response to C. albicans in the oral mucosa. PLOS Pathog. 12, e1005882 (2016).

    Article  Google Scholar 

  9. 9.

    Karki, R. et al. Concerted activation of the AIM2 and NLRP3 inflammasomes orchestrates host protection against Aspergillus infection. Cell Host Microbe 17, 357–368 (2015).

    CAS  Article  Google Scholar 

  10. 10.

    Biondo, C. et al. The interleukin-1β/CXCL1/2/neutrophil axis mediates host protection against group B streptococcal infection. Infect. Immun. 82, 4508–4517 (2014).

    CAS  Article  Google Scholar 

  11. 11.

    Nemeth, T., Futosi, K., Sitaru, C., Ruland, J. & Mocsai, A. Neutrophil-specific deletion of the CARD9 gene expression regulator suppresses autoantibody-induced inflammation in vivo. Nat. Commun. 7, 11004 (2016).

    CAS  Article  Google Scholar 

  12. 12.

    Wang, X. et al. Impaired specific antifungal immunity in CARD9-deficient patients with phaeohyphomycosis. J. Invest. Dermatol. 138, 607–617 (2018).

    CAS  Article  Google Scholar 

  13. 13.

    Lionakis, M. S. et al. Chemokine receptor Ccr1 drives neutrophil-mediated kidney immunopathology and mortality in invasive candidiasis. PLoS Pathog. 8, e1002865 (2012).

    CAS  Article  Google Scholar 

  14. 14.

    Lee, E. K. S. et al. Leukotriene B4-mediated neutrophil recruitment causes pulmonary capillaritis during lethal fungal sepsis. Cell Host Microbe 23, 121–133.e124 (2018).

    CAS  Article  Google Scholar 

  15. 15.

    Swamydas, M. et al. CXCR1-mediated neutrophil degranulation and fungal killing promote Candida clearance and host survival. Sci. Trans. Med. 8, 322ra310–322ra310 (2016).

    Article  Google Scholar 

  16. 16.

    Ngo, L. Y. et al. Inflammatory monocytes mediate early and organ-specific innate defense during systemic candidiasis. J. Infect. Dis. 209, 109–119 (2014).

    CAS  Article  Google Scholar 

  17. 17.

    Erwig, L. P. & Gow, N. A. R. Interactions of fungal pathogens with phagocytes. Nat. Rev. Microbiol. 14, 163–176 (2016).

    CAS  Article  Google Scholar 

  18. 18.

    Zheng, X., Wang, Y. & Wang, Y. Hgc1, a novel hypha-specific G1 cyclin-related protein regulates Candida albicans hyphal morphogenesis. EMBO J. 23, 1845–1856 (2004).

    CAS  Article  Google Scholar 

  19. 19.

    Moyes, D. L. et al. Candidalysin is a fungal peptide toxin critical for mucosal infection. Nature 532, 64–68 (2016).

    CAS  Article  Google Scholar 

  20. 20.

    Verma, A. H. et al. Oral epithelial cells orchestrate innate type 17 responses to Candida albicans through the virulence factor candidalysin. Sci. Immunol. 2, eaam8834 (2017).

    Article  Google Scholar 

  21. 21.

    Richardson, J. P. et al. Candidalysin drives epithelial signaling, neutrophil recruitment, and immunopathology at the vaginal mucosa. Infect. Immun. 86, e00645–17 (2017).

    Article  Google Scholar 

  22. 22.

    Naglik, J. R., Challacombe, S. J. & Hube, B. Candida albicans secreted aspartyl proteinases in virulence and pathogenesis. Microbiol. Mol. Biol. Rev. 67, 400–428 (2003).

    CAS  Article  Google Scholar 

  23. 23.

    Gabrielli, E. et al. In vivo induction of neutrophil chemotaxis by secretory aspartyl proteinases of Candida albicans. Virulence 7, 819–825 (2016).

    CAS  Article  Google Scholar 

  24. 24.

    Pericolini, E. et al. Secretory aspartyl proteinases cause vaginitis and can mediate vaginitis caused by Candida albicans in mice. mBio 6, e00724–15 (2015).

    CAS  Article  Google Scholar 

  25. 25.

    Henn, A. et al. The suitability of BV2 cells as alternative model system for primary microglia cultures or for animal experiments examining brain inflammation. Altex-Alternativen Zu Tierexperimenten 26, 83–94 (2009).

    Google Scholar 

  26. 26.

    Hennessy, E., Griffin, É. W. & Cunningham, C. Astrocytes are primed by chronic neurodegeneration to produce exaggerated chemokine and cell infiltration responses to acute stimulation with the cytokines IL-1β and TNF-α. J. Neurosci 35, 8411–8422 (2015).

    CAS  Article  Google Scholar 

  27. 27.

    Pineau, I., Sun, L., Bastien, D. & Lacroix, S. Astrocytes initiate inflammation in the injured mouse spinal cord by promoting the entry of neutrophils and inflammatory monocytes in an IL-1 receptor/MyD88-dependent fashion. Brain Behav. Immun. 24, 540–553 (2010).

    CAS  Article  Google Scholar 

  28. 28.

    Omari, K. M., John, G., Lango, R. & Raine, C. S. Role for CXCR2 and CXCL1 on glia in multiple sclerosis. Glia 53, 24–31 (2005).

    Article  Google Scholar 

  29. 29.

    Poeck, H. et al. Recognition of RNA virus by RIG-I results in activation of CARD9 and inflammasome signaling for interleukin 1β production. Nat. Immunol. 11, 63–69 (2009).

    Article  Google Scholar 

  30. 30.

    Pereira, M., Tourlomousis, P., Wright, J., P Monie, T. & Bryant, C. E. CARD9 negatively regulates NLRP3-induced IL-1β production on Salmonella infection of macrophages. Nat. Commun. 7, 12874–12874 (2016).

    CAS  Article  Google Scholar 

  31. 31.

    Kasper, L. et al. The fungal peptide toxin Candidalysin activates the NLRP3 inflammasome and causes cytolysis in mononuclear phagocytes. Nat. Commun. 9, 4260 (2018).

    Article  Google Scholar 

  32. 32.

    Parkhurst, C. N. et al. Microglia promote learning-dependent synapse formation through BDNF. Cell 155, 1596–1609 (2013).

    CAS  Article  Google Scholar 

  33. 33.

    Pappas, P. G., Lionakis, M. S., Arendrup, M. C., Ostrosky-Zeichner, L. & Kullberg, B. J. Invasive candidiasis. Nat. Rev. Dis. Primers 4, 18026 (2018).

    Article  Google Scholar 

  34. 34.

    Lionakis, M. S., Netea, M. G. & Holland, S. M. Mendelian genetics of human susceptibility to fungal infection. Cold Spring Harbor Perspect. Med. 4, a019638 (2014).

    Article  Google Scholar 

  35. 35.

    McCarthy, M. W., Kalasauskas, D., Petraitis, V., Petraitiene, R. & Walsh, T. J. Fungal infections of the central nervous system in children. J. Pediatr. Infect. Dis. Soc. 6, e123–e133 (2017).

  36. 36.

    Drummond, R. A. & Lionakis, M. S. Candidiasis of the central nervous system in neonates and children with primary immunodeficiencies. Curr. Fungal Infect. Rep. 12, 92–97 (2018).

    Article  Google Scholar 

  37. 37.

    Cetinkaya, P. G. et al. A young girl with severe cerebral fungal infection due to card 9 deficiency. Clin. Immunol. 191, 21–26 (2018).

    CAS  Article  Google Scholar 

  38. 38.

    Lanternier, F. et al. Inherited CARD9 deficiency in otherwise healthy children and adults with Candida species-induced meningoencephalitis, colitis, or both. J. Allergy Clin. Immunol. 135, 1558–1568 (2015).

    CAS  Article  Google Scholar 

  39. 39.

    Del Rio, L., Bennouna, S., Salinas, J. & Denkers, E. Y. CXCR2 deficiency confers impaired neutrophil recruitment and increased susceptibility during Toxoplasma gondii infection. J. Immunol. 167, 6503–6509 (2001).

    Article  Google Scholar 

  40. 40.

    Bonnett, C. R., Cornish, E. J., Harmsen, A. G. & Burritt, J. B. Early neutrophil recruitment and aggregation in the murine lung inhibit germination of Aspergillus fumigatus conidia. Infect. Immun. 74, 6528–6539 (2006).

    CAS  Article  Google Scholar 

  41. 41.

    Lévesque, S. A. et al. Myeloid cell transmigration across the CNS vasculature triggers IL-1β-driven neuroinflammation during autoimmune encephalomyelitis in mice. J. Exp. Med. 213, 929–949 (2016).

    Article  Google Scholar 

  42. 42.

    Hanamsagar, R., Aldrich, A. & Kielian, T. Critical role for the AIM2 inflammasome during acute CNS bacterial infection. J. Neurochem. 129, 704–711 (2014).

    CAS  Article  Google Scholar 

  43. 43.

    Prinz, M., Erny, D. & Hagemeyer, N. Ontogeny and homeostasis of CNS myeloid cells. Nat. Immunol. 18, 385–392 (2017).

    CAS  Article  Google Scholar 

  44. 44.

    Shinozaki, Y. et al. Transformation of astrocytes to a neuroprotective phenotype by microglia via P2Y1 receptor downregulation. Cell Rep. 19, 1151–1164 (2017).

    CAS  Article  Google Scholar 

  45. 45.

    Rothhammer, V. et al. Microglial control of astrocytes in response to microbial metabolites. Nature 557, 724–728 (2018).

    CAS  Article  Google Scholar 

  46. 46.

    Mao, L. et al. Pathogenic fungus Microsporum canis activates the NLRP3 inflammasome. Infect. Immun. 82, 882–892 (2014).

    Article  Google Scholar 

  47. 47.

    Goodridge, H. S. et al. Differential use of CARD9 by Dectin-1 in macrophages and dendritic cells. J. Immunol. 182, 1146–1154 (2009).

    CAS  Article  Google Scholar 

  48. 48.

    Weinblatt, M. E. et al. An oral spleen tyrosine kinase (Syk) inhibitor for rheumatoid arthritis. N. Engl. J. Med. 363, 1303–1312 (2010).

    CAS  Article  Google Scholar 

  49. 49.

    Flynn, R. et al. Targeting Syk-activated B cells in murine and human chronic graft-versus-host disease. Blood 125, 4085–4094 (2015).

    CAS  Article  Google Scholar 

  50. 50.

    Ruland, J., Duncan, G. S., Wakeham, A. & Mak, T. W. Differential requirement for Malt1 in T and B cell antigen receptor signaling. Immunity 19, 749–758 (2003).

    CAS  Article  Google Scholar 

  51. 51.

    Tay, T. L. et al. A new fate mapping system reveals context-dependent random or clonal expansion of microglia. Nat. Neurosci. 20, 793–803 (2017).

    CAS  Article  Google Scholar 

  52. 52.

    Goldmann, T. et al. A new type of microglia gene targeting shows TAK1 to be pivotal in CNS autoimmune inflammation. Nat. Neurosci. 16, 1618–1626 (2013).

    CAS  Article  Google Scholar 

  53. 53.

    Lionakis, M. S., Lim, J. K., Lee, C. C. R. & Murphy, P. M. Organ-specific innate immune responses in a mouse model of invasive candidiasis. J. Innate Immun. 3, 180–199 (2011).

    CAS  Article  Google Scholar 

  54. 54.

    Cougnoux, A. et al. Microglia activation in Niemann–Pick disease, type C1 is amendable to therapeutic intervention. Hum. Mol. Genet. 27, 2076–2089 (2018).

    CAS  Article  Google Scholar 

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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.

Author information




R.A.D. and M.S.L. designed the study. R.A.D., M.S., V.O., B.Z. and I.M.D. performed the experiments. R.A.D., M.S., V.O., B.Z., I.M.D., T.M.H. and M.S.L. analyzed the data. B.C.S., A.C.B., K.D.M.-B., S.A.L., Y.I., S.G.F., G.D.B., B.H., J.R.N. and T.M.H. provided key reagents/mouse lines and intellectual input into the experimental design regarding their use. R.A.D. and M.S.L. wrote the manuscript.

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Correspondence to Rebecca A. Drummond or Michail S. Lionakis.

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Integrated supplementary information

Supplementary Figure 1 Microglia express CARD9-coupled C-type lectin receptors.

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).

Supplementary Figure 5 Abundance and activation markers of Card9–/– microglia.

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.

Supplementary Figure 6 Microglia-specific deletion of Card9 in Card9fl/flCx3cr1CreER+/– mice.

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).

Supplementary Figure 7 CARD9-dependent glial antifungal immunity in the CNS.

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.

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Drummond, R.A., Swamydas, M., Oikonomou, V. et al. CARD9+ microglia promote antifungal immunity via IL-1β- and CXCL1-mediated neutrophil recruitment. Nat Immunol 20, 559–570 (2019).

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