Inherited CARD9 deficiency in otherwise healthy children and adults with Candida species-induced meningoencephalitis, colitis, or both

Abstract

Background: Invasive infections of the central nervous system (CNS) or digestive tract caused by commensal fungi of the genus Candida are rare and life-threatening. The known risk factors include acquired and inherited immunodeficiencies, with patients often displaying a history of multiple infections. Cases of meningoencephalitis, colitis, or both caused by Candida species remain unexplained.

Objective: We studied 5 previously healthy children and adults with unexplained invasive disease of the CNS, digestive tract, or both caused by Candida species. The patients were aged 39, 7, 17, 37, and 26 years at the time of infection and were unrelated, but each was born to consanguineous parents of Turkish (2 patients), Iranian, Moroccan, or Pakistani origin. Meningoencephalitis was reported in 3 patients, meningoencephalitis associated with colitis was reported in a fourth patient, and the fifth patient had colitis only.

Methods: Inherited caspase recruitment domain family, member 9 (CARD9) deficiency was recently reported in otherwise healthy patients with other forms of severe disease caused by Candida, Trichophyton, Phialophora, and Exophiala species, including meningoencephalitis but not colitis caused by Candida and Exophiala species. Therefore we sequenced CARD9 in the 5 patients.

Results: All patients were found to be homozygous for rare and deleterious mutant CARD9 alleles: R70W and Q289* for the 3 patients with Candida albicans-induced meningoencephalitis, R35Q for the patient with meningoencephalitis and colitis caused by Candida glabrata, and Q295* for the patient with Candida albicans-induced colitis. Regardless of their levels of mutant CARD9 protein, the patients' monocyte-derived dendritic cells responded poorly to CARD9-dependent fungal agonists (curdlan, heat-killed C albicans, Saccharomyces cerevisiae, and Exophiala dermatitidis).

Conclusion: Invasive infections of the CNS or digestive tract caused by Candida species in previously healthy children and even adults might be caused by inherited CARD9 deficiency.

Keywords: Candida species; Inborn error of immunity; central nervous system; colitis; human; inherited CARD9 deficiency; invasive fungal diseases; primary immunodeficiency.

Figure 1

A. Pedigrees of five kindreds with invasive fungal infection and CARD9 mutations Each kindred is designated by a letter (A–E), each generation is designated by a Roman numeral (I–III) and each individual by an Arabic numeral (1–4). Patients with invasive fungal infections are indicated in black. The probands are indicated by arrows. The genotype of CARD9 is indicated below each individual. E? indicates that no DNA was available. B. Schematic diagram of the human CARD9 (isoform 1) gene and its mutations The human CARD9 isoform 1 gene is shown, with its known pathogenic mutations. Coding exons are numbered with roman numerals. Regions corresponding to the CARD domain and coiled-coil (CC) domain are indicated. Mutations associated with invasive fungal (Candida spp., Exophiala spp.) infections are indicated in red. Mutations associated with invasive fungal (Candida spp.) infections or deep dermatophytosis are underlined. Mutations previously reported in patients with deep dermatophytosis are indicated in blue or underlined, and mutations associated with subcutaneous phaeohyphomycosis are indicated in black.

Figure 2. Clinical, pathological and radiological features of patients

A. Brain abscess of P1, and B. Brain computed tomography scan of P3, and C.a–d Colonoscopy results for P3, and D Brain MRI of P4

Figure 3. Histology A–D

Histology results for the brain biopsy carried out on P1. Arrows indicate fungal agents. A–B. Epithelioid granuloma containing pseudohyphae and displaying Gomori-Grocott staining. C. Epithelioid granuloma containing pseudohyphae, stained with hematoxylin-eosin-safran and D. Periodic acid-Schiff stain. E–G. Histology results for the ileum mucosa biopsy of patient P2. E,F. Round yeasts, measuring up to 4 μ;m in diameter, identified by PAS staining. G. Anti-Candida immunohistochemistry.

Figure 4. Impact of CARD9 mutations on CARD9 protein levels and function

A–B. Impact of CARD9 mutations on CARD9 protein levels. A. Immunoblot analysis of CARD9 in whole-cell extracts of HEK-293T cells co-transfected with pcDNA3.1 V5 (C-terminally tagged), either empty or carrying the wild-type (WT) or mutant (R35Q, R70W, Q289* and Q295*) CARD9 alleles, together with a CFP plasmid, as a transfection control. Antibodies against CARD9, V5, CFP and GAPDH (as a loading control) were used. B. Flow cytometry analysis of CARD9 expression in monocyte-derived dendritic cells (MDDCs) from patient P1 and a control. C–D. Impact of CARD9 mutations on CARD9 protein function. C. TNF-α (top panel) and Interleukin (IL)-6 (bottom panel) production by whole blood cells after 24 hours of stimulation with zymosan, heat-killed S. cerevisiae, C. albicans, E. dermatitidis, S. aureus, VSV, BCG, LPS, and PMA plus ionomycin for P1, P2, P5, P2’s father and 7 controls. D. TNF-α production after 24 hours of stimulation of MDDCs with curdlan, zymosan, S. cerevisiae, C. albicans, E. dermatitidis, S. aureus, LPS and VSV for P1, P2 and P5, P2’s parents, and 6 healthy controls tested in parallel.

Figure 4. Impact of CARD9 mutations on CARD9 protein levels and function

A–B. Impact of CARD9 mutations on CARD9 protein levels. A. Immunoblot analysis of CARD9 in whole-cell extracts of HEK-293T cells co-transfected with pcDNA3.1 V5 (C-terminally tagged), either empty or carrying the wild-type (WT) or mutant (R35Q, R70W, Q289* and Q295*) CARD9 alleles, together with a CFP plasmid, as a transfection control. Antibodies against CARD9, V5, CFP and GAPDH (as a loading control) were used. B. Flow cytometry analysis of CARD9 expression in monocyte-derived dendritic cells (MDDCs) from patient P1 and a control. C–D. Impact of CARD9 mutations on CARD9 protein function. C. TNF-α (top panel) and Interleukin (IL)-6 (bottom panel) production by whole blood cells after 24 hours of stimulation with zymosan, heat-killed S. cerevisiae, C. albicans, E. dermatitidis, S. aureus, VSV, BCG, LPS, and PMA plus ionomycin for P1, P2, P5, P2’s father and 7 controls. D. TNF-α production after 24 hours of stimulation of MDDCs with curdlan, zymosan, S. cerevisiae, C. albicans, E. dermatitidis, S. aureus, LPS and VSV for P1, P2 and P5, P2’s parents, and 6 healthy controls tested in parallel.

Figure 4. Impact of CARD9 mutations on CARD9 protein levels and function

A–B. Impact of CARD9 mutations on CARD9 protein levels. A. Immunoblot analysis of CARD9 in whole-cell extracts of HEK-293T cells co-transfected with pcDNA3.1 V5 (C-terminally tagged), either empty or carrying the wild-type (WT) or mutant (R35Q, R70W, Q289* and Q295*) CARD9 alleles, together with a CFP plasmid, as a transfection control. Antibodies against CARD9, V5, CFP and GAPDH (as a loading control) were used. B. Flow cytometry analysis of CARD9 expression in monocyte-derived dendritic cells (MDDCs) from patient P1 and a control. C–D. Impact of CARD9 mutations on CARD9 protein function. C. TNF-α (top panel) and Interleukin (IL)-6 (bottom panel) production by whole blood cells after 24 hours of stimulation with zymosan, heat-killed S. cerevisiae, C. albicans, E. dermatitidis, S. aureus, VSV, BCG, LPS, and PMA plus ionomycin for P1, P2, P5, P2’s father and 7 controls. D. TNF-α production after 24 hours of stimulation of MDDCs with curdlan, zymosan, S. cerevisiae, C. albicans, E. dermatitidis, S. aureus, LPS and VSV for P1, P2 and P5, P2’s parents, and 6 healthy controls tested in parallel.

Figure 5. NF-κB transcriptional activity

NF-κB–luciferase assay in 293 HEK cells transfected with NF-κB–luciferase and pRL-SV40 vectors alone (A); with DECTIN1, SYK, and BCL10 constructs (B); with DECTIN-1, SYK, BCL10, and CARD9 WT constructs; and with DECTIN-1, SYK, BCL10, and CARD9 mutant constructs (R35Q, R70W, Q289* or Q295*). Cells were left unstimulated or were stimulated with 25 μg/ml curdlan, or 10⁷ particles/ml of S. cerevisae or C. albicans. Results are representative of two independent experiments performed and are expressed as means ± SEM of the ratio of Renilla luciferase and firefly control luciferase activities. Abbreviation: RLU, relative light units.

Figure 6. IL-17 production

A. Percentage of CD3⁺/IL-17⁺ cells of 10 controls, P2’s parents (CARD9 R70W/WT), P1, P2 and P5 measured by flow cytometry after 12 hours of stimulation with PMA/ionomycin. B. IL-17A production by the whole blood of 7 controls, P2′ father, P1, P2 and P5 measured after 24 hours of stimulation with PMA/ionomycin by ELISA.