hil 17ra  (Sino Biological)

Journal: The Journal of Experimental Medicine

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

doi: 10.1084/jem.20141065

Figure Lengend Snippet: 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.

Article Snippet: IL-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.

Techniques: Expressing, Generated, Reverse Transcription Polymerase Chain Reaction, Transfection, Real-time Polymerase Chain Reaction, Mutagenesis, Western Blot, Plasmid Preparation

Journal: The Journal of Experimental Medicine

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

doi: 10.1084/jem.20141065

Figure Lengend Snippet: 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.

Article Snippet: IL-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.

Techniques: Expressing, Mutagenesis, Transfection, Plasmid Preparation, Imaging, Microscopy, Staining, Flow Cytometry

Journal: The Journal of Experimental Medicine

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

doi: 10.1084/jem.20141065

Figure Lengend Snippet: 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.

Article Snippet: IL-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.

Techniques: Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

Journal: The Journal of Experimental Medicine

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

doi: 10.1084/jem.20141065

Figure Lengend Snippet: 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.

Article Snippet: IL-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.

Techniques: Transfection, Mutagenesis, Plasmid Preparation, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

Journal: The Journal of Experimental Medicine

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

doi: 10.1084/jem.20141065

Figure Lengend Snippet: 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.

Article Snippet: IL-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.

Techniques: Cell Culture, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

Journal: Clinical & Translational Immunology

Article Title: IL‐17 drives salivary gland dysfunction via inhibiting TRPC1‐mediated calcium movement in Sjögren’s syndrome

doi: 10.1002/cti2.1277

Figure Lengend Snippet: Increased salivary IL‐17 levels negatively correlate with saliva flow rates (SFR) in primary Sjögren’s syndrome (pSS) patients. (a) IL‐17 concentrations in saliva from pSS patients and non‐SS subjects were measured ( n = 25 for non‐SS, n = 33 for pSS). The plot shows correlation analysis of salivary IL‐17 concentrations and SFRs. (b) Salivary IL‐17 concentrations in pSS patients with SFR values lower and higher than 0.1 mL min −1 are analysed ( n = 12 for SFR ≤0.1 mL min −1 , n = 21 for SFR > 0.1 mL min −1 ). (c) Representative confocal images showing IL‐17‐producing cells in labial gland biopsies from pSS patients. (d) Confocal images showing CD4 + IL‐17 + Th17 cells in labial gland biopsies from pSS patients. (e) Numbers of total IL‐17 + cells and CD4 + IL‐17 + Th17 cells in labial gland biopsies from pSS patients were analysed ( n = 9 for SFR ≤ 0.1 mL min −1 , n = 16 for SFR > 0.1 mL min −1 ). (f) Confocal images showing the expression of IL‐17 receptor A (IL‐17RA) and IL‐17 receptor C (IL‐17RC) in labial glands of pSS patients. Data are shown as mean ± SD; * P ‐value < 0.05; ** P ‐value < 0.01.

Article Snippet: The following antibodies were used: anti‐hCD4 (clone A161A1, Biolegend, San Diego, CA, USA), anti‐hIL‐17 (clone BL168, Biolegend), anti‐hIL‐17RA (clone J10MBS, Thermo Fisher Scientific), anti‐hIL‐17RC (clone 309822, R&D Systems, Minneapolis, MN, USA), anti‐mCD4 (clone GK1.5, Biolegend), anti‐mIL‐17RA (clone PAJ‐17R, Thermo Fisher Scientific), anti‐mIL‐17RC (FAB2270A, R&D Systems), anti‐AQP5 (clone H‐200; Santa Cruz Biotechnology, Dallas TX, USA) and anti‐TRPC1 (clone E‐6, Santa Cruz Biotechnology).

Techniques: Expressing

Journal: Clinical & Translational Immunology

Article Title: IL‐17 drives salivary gland dysfunction via inhibiting TRPC1‐mediated calcium movement in Sjögren’s syndrome

doi: 10.1002/cti2.1277

Figure Lengend Snippet: IL‐17 signalling is critical for salivary gland dysfunction during experimental Sjögren’s syndrome (ESS) development. (a) IL‐17‐secreting cells in salivary glands (SGs) from naïve and ESS mice were analysed by flow cytometry. Kinetic changes of saliva flow rates (red line) and numbers of IL‐17‐secreting cells (blue line) and Th17 cells (histogram) in SGs were analysed during ESS development ( n = 6 for each group, asterisks indicate comparisons with naïve mice). (b) Flow cytometric profiles showing IL‐17RA + IL‐17RC + cells within CD45 + leukocytes and AQP5 + SG epithelial cells in SG tissues are presented. The frequencies of IL‐17RA + IL‐17RC + cells in SG from naïve and ESS mice were analysed ( n = 5 for each group). (c) Representative confocal images showing IL‐17RA and IL‐17RC expression in AQP5 + SG epithelial cells. (d) Saliva flow rates of wild‐type (WT), IL‐17‐deficient and IL‐17RC‐deficient mice with or without ESS induction were measured ( n = 5 for each group). (e) The schematic diagram showing the generation of chimeric mice with IL‐17RC deficiency in non‐hematopoietic and hematopoietic cells is presented. Flow cytometric analysis shows more than 95% of donor‐derived leukocytes in recipient chimeric mice after reconstitution. (f) Saliva flow rates were measured in the chimeric mice after ESS induction ( n = 8 for each group). Data are shown as mean ± SD; ns, not significant; * P ‐value < 0.05; ** P ‐value < 0.01; *** P ‐value < 0.001.

Article Snippet: The following antibodies were used: anti‐hCD4 (clone A161A1, Biolegend, San Diego, CA, USA), anti‐hIL‐17 (clone BL168, Biolegend), anti‐hIL‐17RA (clone J10MBS, Thermo Fisher Scientific), anti‐hIL‐17RC (clone 309822, R&D Systems, Minneapolis, MN, USA), anti‐mCD4 (clone GK1.5, Biolegend), anti‐mIL‐17RA (clone PAJ‐17R, Thermo Fisher Scientific), anti‐mIL‐17RC (FAB2270A, R&D Systems), anti‐AQP5 (clone H‐200; Santa Cruz Biotechnology, Dallas TX, USA) and anti‐TRPC1 (clone E‐6, Santa Cruz Biotechnology).

Techniques: Flow Cytometry, Expressing, Derivative Assay