PMC:4419340 / 30384-39922 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":"Molecular genetics.\nGenomic DNA was isolated from whole blood by a phenol/chloroform extraction method. IL17RC cDNA was amplified with specific primers. PCR products were analyzed by electrophoresis in 1% agarose gels, sequenced with the Big Dye Terminator cycle sequencing kit (Applied Biosystems), and analyzed on an ABI Prism 3700 apparatus (Applied Biosystems).\n\nMassively parallel sequencing.\n3 µg genomic DNA was extracted from the peripheral blood cells of the patients and sheared with an S2 Ultrasonicator (Covaris). An adaptor-ligated library was prepared with the Paired-End Sample Prep kit V1 (Illumina). Exome capture was performed with the SureSelect Human All Exon kit (Agilent Technologies). Single-end sequencing was performed on a Genome Analyzer IIx (Illumina), generating 72 base reads.\n\nCell purification and culture.\nHuman PBMCs were isolated from whole blood by Ficoll-Hypaque density gradient centrifugation (GE Healthcare). Primary human fibroblasts, obtained from skin biopsy specimens, were immortalized with SV40T antigen (SV40 fibroblasts) and cultured in DMEM (Gibco, Invitrogen) supplemented with 10% FBS (Gibco, Invitrogen). HEK-293T cells were cultured in DMEM supplemented with 10% FBS. All cells were grown at 37°C, under an atmosphere containing 5% CO2.\n\nRT-PCR for full-length IL-17RC and TaqMan probe detection.\nTotal RNA was extracted with the RNeasy Mini kit (QIAGEN) and reversed-transcribed to generate cDNA with the High-Capacity cDNA Reverse Transcription kit (Invitrogen). Platinum High-Fidelity Taq DNA Polymerase (Invitrogen) was then used to amplify the full-length IL17RC cDNA.\nThe following primers were used: F, 5′-CTGAAGAGGGATTCCAGCCC-3′ and R, 5′-GTAGAAAAACAGCGTCTGCC-3′. TaqMan probes for IL-17RC (Invitrogen) were used to detect mRNA, and the results were normalized against those for GUS (Human GUSB Endogenous Control VIC/MGB Probe; Primer Limited, Invitrogen).\n\nIL-17RC detection by Western blotting.\nTotal protein extracts were prepared from SV40 fibroblasts or HEK-293T cells 48 h after transfection. The proteins were separated by electrophoresis. The bands were electroblotted onto a membrane, which was then probed with an antibody against IL-17RC (Sigma-Aldrich) or GAPDH (Santa Cruz Biotechnology, Inc.).\n\nFibroblast activation.\nSV40-transformed fibroblasts were plated in 48-well plates at a density of 50,000 cells/well in 0.5 ml DMEM/10% FBS per well. The cells were incubated for 24 h and were then left unstimulated or were stimulated for 24 h with various amounts of IL-17 cytokines, recombinant human IL-17A, IL-17F, IL-17A/F, and IL-1β (from R\u0026D Systems). 24 h later, supernatants were collected and IL-6 (Sanquin or R\u0026D Systems) and GRO-α (R\u0026D Systems) levels were assessed with ELISA kits, according to the manufacturer’s instructions.\n\nPBMC culture.\nFrozen PBMCs were cultured in the presence of 100 ng/ml thymic stromal lymphopoietin (R\u0026D Systems) in X-VIVO 15 (Lonza) plus 5% human AB serum (Lonza), as previously described (Rickel et al., 2008). PBMCs were collected, washed, and resuspended at a density of 4 × 106 cells/well in 48-well plates, in a final volume of 0.5 ml/well, in the presence of 10 ng/ml recombinant human IL-2 (R\u0026D Systems) and 10 ng/ml recombinant human IL-17E/IL-25 (R\u0026D Systems). After 3 d, the amount of IL-5 secreted into the medium was determined with ELISA kits (R\u0026D Systems).\n\nPBMC activation.\nThe levels of IL-17A– and IL-22–producing T cells were evaluated by intracellular staining or by ELISA, as previously described (de Beaucoudrey et al., 2008). In brief, PBMCs were purified by centrifugation on a gradient (Ficoll-Paque PLUS; GE Healthcare) and resuspended in RPMI supplemented with 10% FBS (RPMI/10% FBS; Invitrogen). Adherent monocytes were removed from the PBMC preparation by incubation for 2 h at 37°C, under an atmosphere containing 5% CO2. For the ex vivo evaluation of IL-17– and IL-22–producing T cells by flow cytometry, we resuspended 5 × 106 nonadherent cells in 5 ml RPMI/10% FBS in 25-cm2 flasks and stimulated them by incubation with 40 ng/ml PMA (Sigma-Aldrich) and 10−5 M ionomycin (Sigma-Aldrich) in the presence of a secretion inhibitor (1 µl/ml GolgiPlug; BD) for 12 h. For the evaluation of the IL-17– and IL-22–producing T cell blasts after in vitro differentiation, the nonadherent PBMCs were dispensed into 24-well plates at a density of 2.5 × 106 cells/ml in RPMI/10% FBS and activated by incubation with 2 µg/ml of an antibody directed against CD3 (Orthoclone OKT3; Janssen-Cilag) either alone or together with 20 ng/ml IL-23 (R\u0026D Systems), 50 ng/ml IL-6 (R\u0026D Systems), 10 ng/ml IL-1β (R\u0026D Systems), or combinations of these three cytokines. After incubation for 3 d, the cells were restimulated in the same activation conditions, except that the anti-CD3 antibody was replaced with 40 IU/ml IL-2 (Proleukin i.v.; Chiron). We added 1 ml of the appropriate medium, resuspended the cells by gentle pipetting, and then split the cell suspension from each well into two. Flow cytometry was performed on one of the duplicated wells 2 d later, after stimulation by incubation for 12 h with 40 ng/ml PMA and 10−5 M ionomycin in the presence of 1 µl/ml GolgiPlug. Cells were washed in cold PBS, and surface labeling was achieved by incubating the cells with VioBlue-anti–human CD3 (Miltenyi Biotec) or FITC-anti–human CD4 (BD) antibody in 2% FBS in PBS for 20 min on ice. The cells were then washed twice with 2% FBS in cold PBS, fixed by incubation with 100 µl Cytofix for 30 min on ice, and washed twice with Cytoperm (Cytofix/Cytoperm Plus fixation/permeabilization kit; BD). The cells were then incubated for 1 h on ice with Alexa Fluor 488–conjugated anti–human IL-17A (eBioscience), IL-17F PE-conjugated anti–human IL-22 (R\u0026D Systems), or PE-conjugated anti–human IFN-γ (R\u0026D Systems) antibodies, washed twice with Cytoperm, and analyzed with a FACSCanto II system (BD).The contents of the other duplicated well were split into two, with one half left unstimulated and the other stimulated by incubation with 40 ng/ml PMA and 10−5 M ionomycin for another 2 d. Supernatants were collected after 48 h of incubation for ELISA.\n\nFlow cytometry.\nSV40 fibroblasts were plated in 24-well plates at a density of 50,000 cells/well, in 0.5 ml of 10% FBS in DMEM per well. The plates were incubated for 24 h. The cells were then subjected to flow cytometry. SV40-transformed fibroblasts or PBMCs were labeled with PE-mouse IgG1 (eBioscience), APC-mouse IgG1 (eBioscience), Alexa Fluor 647 mouse IgG1, κ isotype ctrl (FC) antibody (Sony Biotechnology, Inc.), VioGreen-mouse IgG2a (Miltenyi Biotec), VioBlue-mouse IgG2a (Miltenyi Biotec), FITC-mouse IgG1 (BD), FITC-mouse IgG2b (BD), and PE-mouse IgG1 (BD) antibodies or Alexa Fluor 647–mouse IgG1 (eBioscience) isotype antibodies as a control. PE-anti–human IL-17RA (clone J10MBS; eBioscience), APC-anti–human IL-17RA (clone 424LTS; eBioscience), Alexa Fluor 647 anti–human CD217 (IL-17RA) antibody (Sony Biotechnology, Inc.), VioGreen-anti–human CD14 (Miltenyi Biotec), VioBlue-anti–human CD3 (Miltenyi Biotec), FITC-anti–human CD4 (BD), FITC-anti–human CD8 (BD), FITC-anti–human CD14 (BD), FITC-anti–human CD19 (BD), and FITC-anti–human CD56 (BD) specific antibodies were used according to the manufacturers’ instructions, with the results analyzed by flow cytometry on a FACSCanto II system.\n\nThe TIRF assay.\nThe TIRF assay was performed on the transfected HEK-293T cells as described in “Cell purification and culture.” V5 tag–IL-17RC WT and mutant (Q138*, R376*, and R378*) vectors were constructed at the N terminus by the pcDNA3.1 Directional TOPO Expression kit (Life Technologies). After being fixed in 3.7% paraformaldehyde, the cells coated glass coverslips and were incubated with anti-V5 antibody (Life Technologies) and Phalloidin (Life Technologies) for 3 h at room temperature. After being washed, the glass coverslips were incubated with anti–mouse Alexa Fluor 488 (Life Technologies) for 1 h at room temperature and then stained with DAPI (Sigma-Aldrich). Fluorescence data were acquired with the Eclipse Ti-E TIRF imaging system (Nikon). Images were recorded with the QuanTEM 512 SC camera (Roper Scientific) and acquired with NIS-Elements AR software (version 3.1; Nikon). Image sets were processed with ImageJ version 1.43 software (National Institutes of Health).\n\nCell complementation.\nIL-17RC–deficient and IL-17RA–deficient SV40 fibroblasts were transfected with empty pUNO1-msc vectors, pUNO1–hIL-17RCa encoding WT human IL-17RC, or pUNO1–hIL-17RA encoding WT human IL-17RA (InvivoGen) with the Lipofectamine LTX transfection kit (Invitrogen), used according to the manufacturer’s instructions. The cells were incubated for 24 h and then stimulated with IL-17A, IL-17F, and IL-17A/F (R\u0026D Systems) at a concentration of 10 or 100 ng/ml. After 24 h, supernatants were harvested for the assessment of IL-6 and Gro-α levels by ELISA, and cells were collected for the evaluation of IL-17RC expression by FACS analysis.\n\nStatistical analysis.\nData were analyzed with the nonparametric statistical test (Mann–Whitney test) with Prism software (GraphPad Software), and P \u003c 0.05 or P \u003c 0.01 was considered statistically significant. Biological proximities between IL17RC and the known CMC-causing genes IL17RA and ACT1 were estimated using the human gene connectome method and its online server (Itan et al., 2014).\n\nHealthy controls.\nThe healthy controls were volunteer blood donors originating from the European Union, Turkey, or Argentina. 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