As self RNA can under certain conditions activate TLR7 and trigger autoimmunity, our results identify furin-like PCs as a possible target to attenuate TLR7-dependent autoimmunity and other immune pathologies. differentiation of the CD14+ subpopulation of PBMCs as described previously (Mohty et al., 2003). the C-terminus with HA was cloned into the lentiviral vector pHR-SIN-IRES-Em (Demaison et al., 2002). Double mutations were inserted using the quick QuikChange II XL Site-Directed Mutagenesis Kit (Stratagene) according to the manufacturers conditions. MISSION shRNA lentiviral vectors were from Sigma-Aldrich. Lentiviral vectors encoding FLAG-tagged Unc93B1 or MyD88 were from GeneCopoeia. Cells transduces with lentiviruses were selected by FACS Mouse monoclonal to FABP4 sorting or puromycin selection. Cell Lysis and Quantitative Immunoblot Analysis 106 cells were lysed in 50 l 1% (v/v) Triton X-100 based lysis buffer. Where indicated, samples were digested with PNGAse F (NEB). Proteins were separated by SDS-PAGE and standard Western Blot analysis was performed. To calculate the percentage of truncated TLR7 per lane, the intensity of the band of processed TLR7 was divided by the intensity of the band of total TLR7 (shorter + longer fragment). Large-scale Immunoprecipitation and Tandem Mass Spectrometry Lysate of PMA-differentiated THP-TLR7 cells was pre-cleared with mouse IgG-Agarose (Sigma), and immunoprecipitated using anti-HA-Agarose Clone HA-7 (Sigma). Eluted polypeptides were visualized by silver staining on SDS-PAGE, and then tandem mass spectrometry was performed on bands of H3B-6527 interest. IL-8, IFN- and mIL12-p40 Assay Cells were stimulated for 24 hrs with indicated TLR agonists. Conditioned medium was collected and secretion of hIL-8, hIFN- or mIL-12p40 was analyzed by enzyme-linked immunosorbent assay (ELISA). Phagosome Isolation Latex beads were fed to PMA differentiated THP-1 cells, and cells were then disrupted by dounce homogenization. Latex-bead-containing phagosomes were isolated on a 60-10% sucrose step gradient after ultracentrifugation. Phagosomes were lysed in lysis buffer, and proteins were separated by SDS-PAGE and visualized by immunoblot. Transient Transfection and Antibiotic Selection LoVo cells (70-90 % confluence) were transiently transfected by mixing either 1 or 2 2 g each of cDNAs of ppPC5 (Nie et al., 2003), or ppPC7 (Zhong et al., 1999) with 6 l FuGENE-6 as described by the manufacturer. After 12 hrs successfully transfected cells were selected by adding G418. Confocal microscopy PMA differentiated THP-1 cells were fixed, permeabilized with 0.5 % Triton-X 100, blocked, and then stained with primary followed by secondary antibodies. Images were taken using a confocal microscope and Lis coefficient was calculated to measure the extend of co-localization. Semi-quantitative RT-PCR Semi-quantitative RT-PCR for amplification of ppPC5 and ppPC7, and for GAPDH as housekeeping gene was performed as described in the Supplemental Information. Statistics All values are represented as H3B-6527 the mean S.D. An unpaired two-way Student’s t test was performed to determine difference between the control and treated group. Significance was accepted at p 0.05 versus control. *, p 0.05; **, p 0.01; ***, p 0.001; ****, p 0.0001. ? Highlights C Human TLR7 is cleaved and accumulates in endosomes independently of low pHC Calcium-dependent furin-like proprotein convertases (PCs) process TLR7C Inhibition or knockdown of furin-like PCs reduces responsiveness to TLR7 agonistsC Mutating a furin-like PC recognition site in TLR7 reduces receptor processing Supplementary Material Supplemental InformationClick here to view.(1.5M, pdf) Acknowledgements The authors thank Carmela De Santo for generous practical and advisory input, Sarah Booth, Giorgio Napolitani for advisory input, and Moira Johnson for critical reading and editing of the manuscript. We thank Graeme Ball (Micron H3B-6527 Oxford, Advanced Bioimaging Unit) for helping with the Fiji analysis, and Laurence Chaperot as well as Jo?l Plumas (Universit Joseph Fourier, Grenoble, France) for experimental help. This work was supported by the Medical Research Council, UK, CRUK (Programme Grant # C399/A2291 to VC and LRI core support to CRS), DC-THERA, European Commission Sixth Framework Programme (Project Number 512074), the Harry Mahon Cancer Research Trust, UK, CIHR grant # MOP 44363, and a Canada Chair # 216684 (to NGS). B.M.K. is supported by the Biomedical Research Centre (NIHR), Oxford, UK..
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