Inherited IL-17RC deficiency in patients with chronic mucocutaneous candidiasis

Abstract

Chronic mucocutaneous candidiasis (CMC) is characterized by recurrent or persistent infections of the skin, nail, oral, and genital mucosae with Candida species, mainly C. albicans. Autosomal-recessive (AR) IL-17RA and ACT1 deficiencies and autosomal-dominant IL-17F deficiency, each reported in a single kindred, underlie CMC in otherwise healthy patients. We report three patients from unrelated kindreds, aged 8, 12, and 37 yr with isolated CMC, who display AR IL-17RC deficiency. The patients are homozygous for different nonsense alleles that prevent the expression of IL-17RC on the cell surface. The defect is complete, abolishing cellular responses to IL-17A and IL-17F homo- and heterodimers. However, in contrast to what is observed for the IL-17RA- and ACT1-deficient patients tested, the response to IL-17E (IL-25) is maintained in these IL-17RC-deficient patients. These experiments of nature indicate that human IL-17RC is essential for mucocutaneous immunity to C. albicans but is otherwise largely redundant.

Figure 1.

Three kindreds with AR IL-17RC deficiency. (A) Pedigrees and familial segregation of the identified IL-17RC nonsense mutations. The proband is indicated in black and by an arrow. I, II, and III indicate the generations. E? indicates individuals whose genetic status could not be evaluated. (B) Schematic diagram of the IL-17RC protein, showing the signal sequence (SS), extracellular (EC), transmembrane (TM), intracellular (IC), and SEFIR (expression similar to fibroblast growth factor IL-17R) domains and the exons and amino acids affected by the mutations. (C) Recurrent thrush on tongue of P1 (kindred A.II.5) and P3 (kindred C.II.1).

Figure 2.

Normal ex vivo development and in vitro differentiation of IL-17– and IL-22–producing T cells from P1. (A–C) Each symbol represents a value from a healthy control individual (black circles), a heterozygous (WT/Q138*) relative of P1 (black triangles), and P1 (black squares). Horizontal bars represent means. (A and B) Percentages are shown of CD3⁺/IL-17A⁺, CD3⁺/IL-17F⁺, CD3⁺/IL-22⁺, and CD3⁺/IFN-γ⁺ (A) and CD4⁺/IL-17A⁺, CD4⁺/IL-17F⁺, and CD4⁺/IL-22⁺ (B) cells, as determined by flow cytometry, among nonadherent PBMCs activated by incubation for 12 h with PMA and ionomycin. (C) Percentages are shown of IL-17A⁺, IL-22⁺, and IFN-γ⁺ T cell blasts after in vitro expansion in the presence of anti-CD3 antibody, IL-2, IL-1β, and IL-23 for 5 d, followed by 12 h of stimulation with PMA and ionomycin. The experiments were repeated at least three times.

Figure 3.

Normal secretion of IL-17A and IL-22 by whole blood cells from patients. (A–E) IL-17A (A–C) and IL-22 (D and E) secretion was determined by ELISA, in the absence of stimulation (open symbols) and after stimulation with PMA and ionomycin for 24 h (closed symbols). Horizontal bars represent medians. Each symbol represents a value from healthy control individuals (circles), heterozygous patients’ relatives (triangles), or patients (squares). The experiments were repeated at least three times.

Figure 4.

Expression of IL-17RC in fibroblasts from controls and patients. (A and B) Amounts of IL17RC cDNA generated from SV40-immortalized fibroblasts from two controls and two patients (P1 and P2), as determined by full-length RT-PCR (A) and TaqMan assays (B). (C) Amounts of IL17RC cDNA obtained from the SV40 fibroblasts of P1 and P2 either left untransfected (NT) or transfected with pUNO1, either empty (MCS) or encoding the WT, Q138*, R376*, or R378* IL-17RC. Results are also shown for the SV40 fibroblasts of two controls tested in parallel (C1 and C2). Means ± SD (error bars) of three independent experiments, as detected by quantitative PCR, are shown. β-ACTIN and GUS were used as endogenous controls. The experiments were repeated at least three times. (D) IL-17RC expression in P1’s and P2’s SV40 fibroblasts transfected with WT or mutant IL17RC alleles, as assessed by Western blotting. IL-17RC protein levels in SV40 fibroblasts from P1 and P2 transfected with the empty pUNO1mcs plasmid (mock) or the pUNO1 plasmid, encoding the WT or one of the three mutant (Q138*, R376*, or R378*) IL-17RC proteins, as determined by Western blotting with an anti–IL-17RC antibody (directed against amino acids 113–258). The anti-GAPDH antibody was used as a control for protein loading. These experiments were repeated at least three times.

Figure 5.

Expression of WT, mutant IL-17RC, and IL-17RA at the cell surface. (A) IL-17RC expression in HEK-293T cells transfected with V5 tag plasmid encoding IL-17RC WT or mutant alleles, as assessed by TIRF imaging. HEK-293T cells were transfect with V5 tag–IL17RC-pcDNA 3.1 encoding WT or mutant (Q138*, R376*, or R378*) IL-17RC. The “Epi” was assessed by epifluorescence illumination, and the “TIRF” was detected by TIRF microscopy. DAPI binds double-stranded DNA, and phalloidin binds F-actin. The “pseudocolor” scales were used to indicate the intensity staining in TIRF. For each setting condition, 20 cells have been analyzed from cumulating three independent experiments. Bars, 5 µm. (B) IL-17RA expression on SV40-immortalized fibroblasts from a control, P1 (Q138*/Q138*), P2 (R376*/R376*), and an IL-17RA–deficient patient (Q284*/Q284*), as assessed by flow cytometry. Isotype control, gray dotted lines; IL-17RA antibody, black lines. The experiments were repeated at least three times.

Figure 6.

Responses of P1’s and P2’s fibroblasts to IL-17 cytokines. (A and B) IL-6 (A) and GRO-α (B) production by SV40-immortalized fibroblasts from controls, P1 (IL-17RC Q138*/Q138*), P2 (IL-17RC Q376*/Q376*), and an IL-17RA–deficient patient (IL-17RA Q284*/Q284*), after 24 h of stimulation with IL-17A (10 and 100 ng/ml), IL-17F (10 and 100 ng/ml), and IL-17A/IL-17F (10 and 100 ng/ml), as determined by ELISA on supernatants. Means ± SD (error bars) of three independent experiments are shown. Statistical analyses were performed by the nonparametric statistical test (Mann–Whitney test), comparing NS (nonstimulated) and activated samples (*, P < 0.05; **, P < 0.01). The experiments were repeated at least three times.

Figure 7.

Production of IL-6 and GRO-α by the patients’ fibroblasts in responses to IL-17 cytokines, after transfection with the WT or mutant IL17RC alleles. (A and B) IL-6 and GRO-α production by SV40-immortalized fibroblasts from a control, P1, P2, and an IL-17RA–deficient patient transfected with an empty vector (mock) or an IL-17RC vector encoding the WT or each one of the mutant (Q138*, R376*, or R378*) proteins, after 24 h of stimulation with 100 ng/ml IL-17A, as determined by ELISA on supernatants. NS, nonstimulated; NT, not transfected. Means ± SD (error bars) of three independent experiments are shown. Statistical analyses were performed by the nonparametric statistical test (Mann–Whitney test; **, P < 0.01). The experiments were repeated at least three times.

Figure 8.

IL-17E/IL-25 response of the patients’ T cells. PBMCs from 14 controls, P2 and P3, 11 healthy heterozygous relatives, and an IL-17RA–deficient patient were cultured in thymic stromal lymphopoietin for 24 h, harvested, and restimulated with IL-2 and IL-17E/IL-25 for an additional 72 h. IL-5 concentrations in the culture supernatants were determined by ELISA. Errors bars represent SEM. Statistical analyses were performed by the nonparametric statistical test (Mann–Whitney test; **, P < 0.01). The experiments were repeated at least three times.

Figure 9.

Normal IL-6, IFN-γ, and IL-17A production by P2’s whole blood upon 48 h of stimulation with fungal compounds. (A–C) Whole blood from a local control (white bars), a travel control (gray bars), and P2 (black bars) were stimulated with fungal compounds (Curdlan, heat-killed C. albicans, S. cerevisiae, E. dermatitidis: a black yeast), as well as heat-killed S. aureus, VSV, BCG, or PMA/ionomycin for 48 h. IL-6 (A), IFN-γ (B), and IL-17A (C) were measured by ELISA. The experiments were repeated two times.

Figure 10.

Normal TNF production by P2’s and P3′s MDDCs upon 48 h of stimulation with fungal compounds. (A and B) MDDCs from four healthy control individuals (white bars), four heterozygous patients’ relatives (gray bars), and P2 or P3 (black bars) were stimulated with fungal compounds (zymosan, heat-killed S. cerevisiae [HKSC], C. albicans [HKCA and SC5314], E. dermatitidis, and Curdlan), as well as with VSV, BCG, or LPS for 48 h. TNF was measured by ELISA. Error bars represent SEM. The experiments were repeated two times.