PMC:3279418 / 26129-33151 JSONTXT

{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/3279418","sourcedb":"PMC","sourceid":"3279418","source_url":"http://www.ncbi.nlm.nih.gov/pmc/3279418","text":"Materials and Methods\n\nEthics Statement\nAll mouse work was carried out in strict accordance with protocols approved by the Institutional Animal Care and Use Committees.\n\nMice\nSpecific-pathogen free colonies of C57BL/KaLawRij-Sharpincpdm/RijSunJ (JR#7599) and WT mice were obtained from The Jackson Laboratory (Bar Harbor, ME) and were maintained in a barrier facility. Normal littermate controls were either +/+ or +/Sharpincpdm. These control animals were phenotypically indistinguishable and are referred to as WT. Sex-matched WT and mutant mice were used at 6–10 weeks of age. For some experiments, cpdm mice were crossed with transgenic mice with a bacterial artificial chromosome (BAC) containing the Sharpin gene (FVB/NJ-Tg(RP24-173I23)1Sun/Sun, JR#8279). These mice were backcrossed onto the C57BL/KaLawRij-Sharpincpdm/RijSunJ background and N4 mice were used in the experiments reported here. All mouse work was carried out in strict accordance with the approved protocols by the Institutional Animal Care and Use Committee.\n\nConstructs and transfection\nThe complementary DNA (cDNA) of the mouse Sharpin gene was cloned and amplified from RAW264.7 RNA extracts. The primer sequences were forward, 5′-CC ATG GCG ATG TCG CCG CCC GCC GGC GGT; reverse, 5′- AAG CTT CTA GGT GGA AGC TGC AGC AAG A. The Sharpin cDNA was cloned into expression vector pFLAG-CMV-2. Murine fibroblasts and macrophages were were used to express recombinant SHARPIN protein. Cells (2×104) were seeded in 96-well treated plates the day before transfection. After overnight incubation, 200 ng pFLAG-SHARPIN plasmids were transfected with 0.5 ul Lipofectamine 2000. 24 hours later, cells were incubated with fresh culture medium. After another 24 hours, cells were lysed to confirm the FLAG-SHARPIN expression by immunoblots with anti-FLAG. Cells with no transfection and transfected with empty vector pFLAG-CMV-2 were used as negative control in all transfection experiments.\n\nGeneration of BMDC\nBMDC were developed as previously reported [54], [55]. Cells were collected after 10–12 days of culture. The cell yield was 2–3×107 cells/mouse with 80–95% BMDC.\n\nPhenotype of BMDC and splenic DC subsets\nThe phenotype of BMDC was determined before and after 24 hour culture in the presence of 100 ng/mL LPS. The cells were incubated in PBS with 0.1% NaN3, 1% BSA and 10% normal rabbit serum for 20 minutes on ice, washed and incubated for 30 minutes on ice with Alex Fluor-labeled anti-CD11c (MCD11C20, CALTAG) in combination with PE- anti-CD40 (3/23, BD Biosciences), PE-anti-CD80 (16-10A1, eBioscience), PE-anti-CD86 (P03.1, eBioscience), PE-anti-CD14 (Sa2.8, eBioscience) and biotinylated anti-TLR4 (BioLegend). The cells incubated with biotinylated anti-TLR4/MD-2 were washed twice and incubated for 30 minutes on ice with avidin-PE. To isolate splenic DC, spleens were collagenase digested and subject to Percoll gradient centrifugation. The bands at the 35–55% interface were collected to stain with PE-anti-CD11c (HL3, BioLegend), APC-anti-PDCA-1 (927, BioLegend) and FITC-anti-CD8α (53–6.7, BD BioScience). The cells were washed twice, fixed in 2% paraformaldehyde, and stored at 4°C until analysis. Flow cytometry was performed in an Excel (Coulter) instrument. Dead cells were omitted from the analysis by gating on forward and 90° light scatter, and 10,000 cells were analyzed by FlowJo software.\n\nBMDC-T cell in vitro interaction\nWT and cpdm BMDC (5×104) were stimulated with medium, 1 µg/mL LPS, 25 µg/mL and 5 µg/mL Pam3CYS. 24 hours later, cells were collected and washed with PBS. Allogeneic naïve CD4+ T cells were isolated from spleens of BALB/c mice by negative selection kit (Invitrogen) and were then added at 2.5×105 and co-cultured with activated BMDC. After 5 days, supernatant was collected and the secretion of IFNγ, IL4, IL2, and IL17A measured by ELISA. Negative controls are 1) stimulated BMDC without co-culture with allogeneic CD4+ T cells; 2) allogeneic CD4+ T cells without co-culture with stimulated BMDC. Both negative controls show no production of aforementioned cytokines.\n\nRNA expression by BMDC\nBMDC were cultured at 106 cells/mL in 10 mL of RPMI-1640 complete medium in the presence or absence of 100 ng/mL LPS or 25 µg/mL poly I:C. After 1 and 2 hours, RNA was isolated with TRI-reagent (Sigma) according to the manufacturer's instructions. The expression of Il6, Il12p40, Gmcsf, and Sharpin mRNA was determined by qRT-PCR. Primers and probes were purchased from Applied Biosystems. Reverse transcription was performed at 42°C for 60 minutes with the final denaturation step at 90°C for 5 minutes in 30 µl containing 0.5 µg of total RNA. dNTPs, oligo(dT)15 primer, recombinant RNasin Ribonuclease Inhibitor, and M-MLV Reverse Transcriptase (all from Promega, Madison, WI) were used according to the manufacturer's instruction. Reverse transcription was done in a PTC-200 Peltier Thermal Cycler (MJ Research, Watertown, MA). qRT-PCR was performed in ABI Prism 7700 Sequence Detection System with TaqMan® Gene Expression Assays (Applied Biosystems, Foster City, CA) for mouse Actb, IL6, IL12p40, Ifnβ, Gmcsf, and Sharpin according to the manufacturer's protocol. The endogenous standard for normalization of the target gene was β-actin. Relative gene expression was calculated using the 2−ΔΔCt method [56].\n\nIn vitro cytokine secretion by BMDC\nThe BMDC were cultured in triplicate wells of 24-well or 96-well plates at 106 or 105 cells/mL, respectively, in RPMI-1640 complete medium. The cells were washed and stimulated with 100 ng/mL LPS or 25 µg/mL poly I:C. After 24 hours, supernatants were harvested for ELISA analysis. The presence of nitrite in the supernatants was determined using the Griess reagent.\n\nImmunofluorescence\nFibroblast and macrophage cells were transfected with pFLAG-SHARPIN. 48 hours later, cells were washed briefly with PBS, then fixed in cold methanol at −20°C for 10 minutes and cold acetone at -20°C for 1 minute. Incubate cells with PBS+1% BSA for 15 minutes to block non-specific binding. Fixed cells were then incubated with anti-FLAG (1∶100) as the primary antibody at room temperature for 1 hour, followed by three 5-minute washes. Cells were further incubated with goat anti-rabbit IgG-FITC (1∶100) as the secondary antibody at room temperature for 30 minutes, followed by three 5-minute washes. Cells were then examined using inverse fluorescence microscope.\n\nImmunoblots\nAfter adding stimulating ligands, 100 ng/mL LPS or 25 µg/mL poly I:C, BMDC were collected and lysed at 0-, 15-, 30-, and 60-minute time points. Immunoblots were performed with antibodies (Cell Signaling Technology) against p-IKK1/2(#2697), p-IκBα (#9246), IκBα (#4814), p-TBK1(#5483), p-p38 (#9216), p38 (#9212), p-ERK1/2 (#4376), and ERK1/2 (#4695). Beta-actin (sc-47778, Santa Cruz Biotechnology) was used as loading control.\n\nStatistical analysis\nData are expressed as mean ± SD. The statistical significance of differences of means between experimental groups was determined by Students' t-test. 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