PMC:4419340 / 4337-24757 JSONTXT

{"project":"2_test","denotations":[{"id":"25918342-17701901-59633017","span":{"begin":750,"end":754},"obj":"17701901"},{"id":"25918342-23509278-59633018","span":{"begin":7698,"end":7702},"obj":"23509278"}],"text":"RESULTS\n\nClinical reports\nThe experiments described here were conducted in accordance with French regulations and were approved by the French Ministry of Research (IE-2010-547), the French Ethics committee (ID-RCB 2010-A00636-33), and ANSM (French National Agency for Medicines and Health Products Safety; B100712-40). Informed consent was obtained from all patients or their families, in the case of minors, in accordance with the World Medical Association, the Helsinki Declaration, and EU directives.\nThe index patient of kindred A (P1, kindred A.II.5, Fig. 1 A) is a 37-yr-old woman of Argentinean origin, living in Argentina, with no known parental consanguinity. The calculated homozygosity rate for P1 was 0.008 (not depicted; Purcell et al., 2007), providing further support for the notion that P1 was not from closely related parents. However, the mutation was located in a homozygous region of 7.1 Mb, which may be consistent with a cryptic consanguinity. Since the age of 4 yr, P1 has suffered from chronic intertrigo and pustules on her face, but with a normal scalp, nails, teeth, and sweating. She also developed recurrent oral (P1, kindred A.II.5, Fig. 1 C) and esophageal thrush. She had never suffered from any severe bacterial (staphylococcal, in particular), viral, or other fungal infection. P1 was treated with oral fluconazole. The response to treatment was good, with no drug resistance, but relapses occurred when treatment was stopped. Laboratory investigations showed normal immunophenotype (T, B, and NK). The T cell proliferations in response to phytohemagglutinin and antigens (tuberculin, candidin, and anatoxin) were normal. No endocrine, metabolic, or autoimmune abnormalities were reported. In particular, the patient did not display anti-DNA, antinuclear, or antithyroglobulin antibodies and had normal TSH levels. P1 had normal proportions of circulating IL-17A–, IL-17F–, IL-22–producing CD3+ and CD4+ T cells (Fig. 2, A–C) and displayed normal IL-17A production by freshly prepared leukocytes (Fig. 3 A). None of her sisters, parents, or sons had any particular relevant clinical history.\nFigure 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).\nFigure 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.\nFigure 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. The index patient of kindred B (P2, kindred B.II.1, Fig. 1 A) is a 12-yr-old boy born to Turkish parents, with no known parental consanguinity. The calculated homozygosity rate of P2 was 0.021 (not depicted), consistent with P2 originating from a family without close consanguinity. P2 has suffered from CMC since the age of 2 mo, with chronic intertrigo, aphthous stomatitis, oral thrush, and onychomycosis, but with no reported severe bacterial (staphylococcal in particular), viral, or other fungal infections. CMC was long lasting in this patient. It was treated with courses of oral and i.v. fluconazole and local nystatin, and a response to treatment was observed, but with relapse after treatment cessation. Routine laboratory investigations showed that lymphocyte counts were normal and serum IgG, IgA, IgM, and IgE levels were within the normal ranges. Thyroid function, as evaluated by determining TSH (2.4 IU/l) and T4 (1.19 ng/dl) levels, was normal. Normal levels of IL-17A and IL-22 were produced after ex vivo stimulation with PMA and ionomycin by freshly prepared leukocytes of P2 and other members of his family (Fig. 3, B and D). No endocrine, metabolic, or autoimmune abnormalities were reported. Neither the parents nor the siblings of this patient have ever suffered from severe infections.\nThe index patient of kindred C (P3, kindred C.II.1, Fig. 1 A) is an 8-yr-old boy from a third-degree consanguineous Turkish family. This information is consistent with the homozygosity rate of 0.039 obtained for this patient (not depicted). P3 displayed persistent oral candidiasis, with white plaques all over the buccal mucosa and dorsal surface of the tongue since early infancy (P3, kindred C.II.1, Fig. 1 C) and pustules on the skin and scalp. P3 had no history of any other significant infectious (bacterial, staphylococcal in particular, viral, or other fungal) disease or significant developmental defects, with normal hair, nails, teeth, and sweating. Leukocyte counts and absolute neutrophil and lymphocyte counts were normal. Serum IgG, IgA, and IgM levels were normal. No autoimmune antibodies, such as antinuclear, antiperoxisomal (anti-TPO and anti-TG), and antiparietal antibodies, were detected in the serum. Peripheral blood lymphocyte subsets were within the normal range. In vitro T cell proliferation tests showed that the percentages of CD3+CD25+ (66%; normal 43–97%) and CD3+CD69+ (68%; normal 45–100%) cells were within the normal ranges after stimulation with phytohemagglutinin. Normal levels of IL-17A and IL-22 secretion by freshly prepared leukocytes after ex vivo stimulation with PMA and ionomycin were observed for P3 and other members of his family (Fig. 3, C and E). No endocrine or metabolic abnormalities were found. Systemic antifungal therapy with oral fluconazole was initiated and moniliasis resolved. However, oral moniliasis recurred and persisted for some time after the cessation of treatment. Neither the parents nor the siblings of P3 had ever suffered from a severe infectious disease.\n\nHomozygous nonsense mutations of IL17RC\nWES revealed the presence of different homozygous nonsense mutations of IL17RC in these patients. We chose to investigate these mutations further as potential disease-causing mutations because of their likely impact on IL-17 immunity. In addition, IL17RC displayed a very short biological distance between the known CMCD-causing genes IL17RA and ACT1 (P-values of 0.0005 and 0.0007, respectively), making it a very likely novel candidate gene for CMCD (Itan et al., 2013). P1 was found to be homozygous for the c.412C\u003eT nonsense mutation in exon 3 of the IL17RC gene. This mutation replaces the glutamine codon at position 138 with a premature stop codon (Q138*; Fig. 1 B). The healthy parents, tested siblings, and children of P1 were all heterozygous (WT/Q138*) for the mutant allele, consistent with AR inheritance for this trait (Fig. 1 A). P2 was found to be homozygous for the c.1126C\u003eT nonsense mutation in exon 11 of the IL17RC gene. This mutation replaces the arginine codon in position 376 with a premature stop codon (R376*; Fig. 1 B). The parents and sister of P2 are healthy and heterozygous for the mutant allele, consistent with AR inheritance (Fig. 1 A). P3 was found to be homozygous for the c.1132C\u003eT nonsense mutation in exon 11 of the IL17RC gene, replacing the arginine codon in position 378 with a premature stop codon (R378*; Fig. 1 B). The parents and sister of P3 are healthy and heterozygous for the mutant allele, consistent with AR inheritance (Fig. 1 A). The mutant allele (Q138*) from P1 was not found in any of the various public databases (Human Gene Mutation Database, Ensembl, NHLBI GO Exome Sequencing Project [ESP], 1000 Genomes Project, and the Exome Aggregation Consortium [ExAC]) or in our in-house WES database (∼1,800 exomes), ruling out the possibility of an irrelevant polymorphism and suggesting that this mutation may define a rare AR CMCD-causing allele. The R376* mutant allele, found in P2, has been reported in the ExAC database, only at the heterozygous state, in 5 out of 59,269 individuals. The R378* mutant allele found in P3 has been reported previously (dbSNP accession no. rs143600903), but with a low frequency (0.057%); only one European from a population of 59,318 individuals has been found to be homozygous for this variant, consistent with the notion that this mutation may define a rare AR CMCD-causing allele. The three premature stop codons are located upstream from the segment encoding the transmembrane domain of IL-17RC (Fig. 1 B). No rare coding mutations were found in STAT1, IL17RA, IL17A, IL17F, ACT1, IL22, or IL22RA1 by WES in the three patients.\n\nImpaired production of IL17RC mRNA and protein\nWe used RT-PCR or TaqMan assays to determine the levels of full-length IL17RC mRNA, which we found to be lower in fibroblasts from the patients than in cells from healthy controls (Fig. 4, A and B). However, no band of the correct size corresponding to IL-17RC could be detected by Western blot in control fibroblasts without transfection. Therefore, the patients’ fibroblasts were then transfected with various expression vectors: an empty vector or vectors encoding the WT or any of the three mutant alleles (Q138*, R376*, or R378*). In this overexpression system, the IL17RC mRNAs generated by transcription from the WT allele or the three mutant alleles were detected equally well by qPCR (Fig. 4 C). A product of ∼85 kD, corresponding to isoform 1, was detected in cells transfected with the WT IL17RC allele, whereas no product of this molecular mass was detected in fibroblasts transfected with any of the three mutant IL17RC alleles (Fig. 4 D). No product with a lower molecular mass was detected in cells transfected with the Q138* IL-17RC–encoding construct, with an anti–IL-17RC antibody directed against the epitope covering amino acids 113–258. A product with a lower molecular mass (∼40 kD), corresponding to a C-terminally truncated protein, was detected in cells transfected with the constructs encoding R376* or R378* IL-17RC. A product of ∼80 kD was detected in cells transfected with the WT allele and a product of ∼35 kD was detected in cells transfected with the R376* or R378* allele. These products were probably the result of posttranscriptional modifications. In addition, HEK-293T cells transfected with various constructs encoding IL-17RC WT, Q138*, R376*, or R378* displayed a strong membrane expression, as detected by total internal reflection fluorescence (TIRF) imaging, only when transfected with the WT allele but none of the mutants alleles (Fig. 5 A). The absence of IL-17RC had no impact on the expression of IL-17RA, which was found to be expressed to similar levels on the fibroblasts of patients and controls (Fig. 5 B).\nFigure 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.\nFigure 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.\n\nImpaired responses to IL-17A and IL-17F, but normal responses to IL-17E/IL-25\nWe investigated whether the three IL17RC mutations identified had any functional consequences in terms of the response to IL-17 cytokines by testing the responses of the patients’ fibroblasts to various concentrations (10 and 100 ng/ml) of recombinant IL-17A and IL-17F homodimers and IL-17A/F heterodimers. Unlike fibroblasts from healthy controls, the patients’ fibroblasts did not respond to any of the three IL-17 dimers, whatever the concentration of cytokine used. These results were similar to those obtained for the IL-17RA–deficient patient carrying the homozygous Q284* mutation tested in parallel, in terms of IL-6 and GRO-α (growth-regulated oncogene-α) induction, as assessed by ELISA (Fig. 6, A and B). In contrast, the responses of the patients’ fibroblasts to IL-1β stimulation were within the same range as the controls. Transfection of the patients’ fibroblasts with a WT IL17RC construct, but not with an empty vector or with any of the three mutant constructs, restored the response to IL-17 cytokines in the patients’ fibroblasts (Fig. 7, A and B). In contrast, PBMCs from P2 and P3 stimulated with IL-17E/IL-25 in the presence of IL-2 produced IL-5 to levels in the control range, in contrast to what was observed for PBMCs from an IL-17RA–deficient patient. Thus, IL-17E/IL-25 signaling in humans is dependent on IL-17RA but not IL-17RC (Fig. 8). The three patients described here display a complete form of AR IL-17RC deficiency, with a lack of cellular responses to IL-17A and IL-17F homo- and heterodimers but normal responses to IL-17E/IL-25.\nFigure 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 \u003c 0.05; **, P \u003c 0.01). The experiments were repeated at least three times.\nFigure 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 \u003c 0.01). The experiments were repeated at least three times.\nFigure 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 \u003c 0.01). The experiments were repeated at least three times.\n\nNormal cellular responses to fungal compounds\nFinally, we investigated IL-6, IFN-γ, and IL-17A production by P2’s whole blood upon 48 h of stimulation with fungal compounds (Curdlan, heat-killed C. albicans, Saccharomyces cerevisiae, and Exophiala dermatitidis), as well as heat-killed Staphylococcus aureus, vesicular stomatitis virus (VSV), bacillus Calmette–Guérin (BCG), and PMA/ionomycin. The levels of all three cytokines produced were comparable with those obtained after whole blood stimulation of a local or a travel control (Fig. 9, A–C). Similarly, monocyte-derived DCs (MDDCs) from P2, P3, and their relatives, activated with various fungal compounds (including zymosan, heat-killed S. cerevisiae, C. albicans, E. dermatitidis, and Curdlan), as well as VSV, BCG, or LPS, produced levels of TNF comparable with the healthy controls tested in the same conditions (Fig. 10, A and B). Altogether, these results suggest that IL-17RC deficiency does not impair the whole blood or MDDC response to fungal compounds, at least for the cytokines measured, including IL-17A. Altogether, these data suggest that the patients’ defective IL-17RC–dependent responsive pathway is primarily responsible for CMC, as it does not affect the production of IL-17A and other cytokines by leukocytes and MDDCs in response to stimulation by C. albicans.\nFigure 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.\nFigure 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.\n\nDI"}