CARD9: at the intersection of mucosal and systemic antifungal immunity

In this issue of Blood, Drewniak et al¹  demonstrate that neutrophil killing of Candida depends on caspase recruitment domain–containing protein 9 (CARD9) function but not on the production of reactive oxidants. Furthermore, although CARD9 is required for Candida killing, it is dispensable for killing of Staphylococcus aureus and Aspergillus.

Candida inhabits the mucosal surfaces in ∼50% of healthy individuals, typically without sign or symptom. However, when weakening or perturbation of host defenses occurs, it can cause a range of ailments, from localized mucosal disease to fatal systemic infections. Until now, the host factors involved in mucosal and systemic disease were not overlapping. Adaptive immunity, predominantly in the form of interleukin-17 (IL-17)–producing lymphocytes, controls Candida at the mucosal level by promoting the production of neutrophil-recruiting chemotactic factors and the generation of epithelial anti-Candida antimicrobial peptides.²  In stark contrast, innate immunity controls systemic candidiasis through oxidative and nonoxidative phagocyte pathways. Consistent with this dichotomy, patients with AIDS develop mucosal but not systemic candidiasis, whereas patients with neutropenia develop systemic but not mucosal candidiasis.

Chronic mucocutaneous candidiasis (CMC), but not systemic candidiasis, is associated with mutations in STAT1, STAT3, AIRE, DECTIN-1, IL-17RA, and IL-17F, as well as in patients with autoantibodies to IL-17, all situations in which IL-17 immunity is compromised.²  In contrast, patients with chronic granulomatous disease and complete myeloperoxidase deficiency, who have defects in phagocyte oxidative cytotoxicity, infrequently develop systemic candidiasis but do not develop CMC.² 

In contrast to the immunodeficiencies that segregate cleanly between CMC and systemic candidiasis, CARD9 deficiency is unique in having both heightened susceptibility to CMC and systemic candidiasis.³  CARD9 is the critical adaptor protein that operates downstream of several immunoreceptor tyrosine-based activation motif (ITAM)–associated C-type lectin receptors (CLRs) including dectin-1, dectin-2, and mincle. Following receptor engagement and syk kinase phosphorylation, CARD9 forms a complex with BCL10 and MALT1, which transduces non–Toll-like receptor (TLR) signaling to the canonical nuclear factor–κB (NF-κB) pathway.⁴  Therefore, the syk-CARD9 pathway is at the convergence of innate and adaptive antifungal immune responses downstream of multiple receptors. This central positioning of CARD9 in multiple signal pathways explains why lack of CARD9 results in severe susceptibility to both mucosal and systemic candidiasis.

Card9 is indispensable for development of T-helper 17 (Th17) responses to Candida in mice, mostly through dectin-2, and, to a lesser extent, dectin-1 signaling.⁵  Similarly, patients with autosomal-recessive homozygous CARD9 Q295X mutation had decreased circulating IL-17⁺ T lymphocytes.³  Although IL-17 production by lymphocytes was impaired in this case of CARD9 deficiency, stimulation of CARD9-deficient monocytes with Candida elicited selectively diminished IL-1β and IL-6, which are pivotal cytokines for priming downstream Th17 differentiation.¹  Surprisingly, CARD9-deficient neutrophils display a killing defect that is unmasked by Candida but inapparent against Aspergillus or bacteria. Consonant with these in vitro data, patients with CARD9 deficiency but not those with DECTIN-1 mutations develop systemic candidiasis.^2,3  Likewise, Card9^−/− mice appear more susceptible to systemic candidiasis than dectin-1^−/− or dectin-2^−/− mice.^5-7  Therefore, the CLR(s) upstream of CARD9, which mediate(s) neutrophil anti-Candida activity, either alone or in synergy with dectin-1 and/or dectin-2, remain(s) elusive. Mutations in syk, BCL10, and MALT1 have not yet been identified, but might be expected to be phenocopies of CARD9 deficiency.

A curious feature of candidiasis in human CARD9 deficiency is central nervous system involvement, in contrast to the hepatosplenic candidiasis seen in chemotherapy-associated neutropenia, especially for hematologic malignancies. This tissue specificity is not due to brain-restricted CARD9 expression in humans⁴  but may reflect low levels of opsonins in the central nervous system, as impaired Candida killing was seen against unopsonized but not opsonized yeasts.¹ 

Human CARD9 mutations also predispose to cutaneous and invasive infections with dermatophytes,^3,8  suggesting that antifungal control of these fungi is similar and also dependent on CARD9. In contrast, no infections by endemic dimorphic fungi have yet been reported in human CARD9 deficiency, implying that syk-CARD9 signaling is independent of the IL-12/interferon-γ (IFN-γ)/signal transducer and activator of transcription 1 (STAT1) axis in macrophages, which is critical for control of these intracellular fungi.²  Indeed, differential use of the CLR-syk-CARD9 axis by different myeloid cells has previously been reported.⁹  Similarly, CARD9-deficient patients have not reported infections with ubiquitous inhaled molds and their neutrophils handle Aspergillus normally in vitro. Reemphasizing the important differences between mice and humans, the syk/Card9 pathway is indispensable for murine host defense against Aspergillus.¹⁰  This human disease underscores the distinct ways in which human phagocytes control different pathogenic yeasts and molds, as well as its divergence from some of the mechanisms that are so critical in mice.

The explosion of knowledge of human genetic defects that confer susceptibility to mucosal and systemic fungal infection over the past decade continues to rumble. However, it is not time for complacency. We have not yet explained how or why different fungi are handled differently in different anatomical compartments at different times (eg, menses). We are also somewhat in the dark about why such a select few fungal species cause human disease at all. The intersection through which both mucosal and systemic antifungal protection pass is CARD9, showing us where antifungal immune signaling pathways are integrated. By following these critical signals in both directions, from the initial encounter with the fungus, to the final lysis of the fungal cell wall will eventually allow us to devise novel strategies for risk assessment, diagnosis, prognostication, and immunotherapy of fungal infections in humans.