PMC:4419340 / 18648-22366 JSONTXT

{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/4419340","sourcedb":"PMC","sourceid":"4419340","source_url":"https://www.ncbi.nlm.nih.gov/pmc/4419340","text":"Impaired 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\nN","divisions":[{"label":"Title","span":{"begin":0,"end":77}},{"label":"Figure caption","span":{"begin":1648,"end":2377}},{"label":"Figure caption","span":{"begin":2376,"end":3158}},{"label":"Figure caption","span":{"begin":3157,"end":3717}}],"tracks":[]}