containing: First Strand Buffer (Invitrogen-Life Technologies), 2 mM dNTPs (Pharmacia, Germany), 30 U RNAse inhibitor (Roche Applied Science, Indianapolis, IN, USA) and 400 U M- MLV reverse transcriptase (Invitrogen - Life Technologies, The Netherlands). The total reaction mix (50 ml) was incubated at 37uC for 60 min, heated to 95uC for 10 min and stored at 220uC until use. PCR primers (Eurogentec, Belgium) were designed using the Universal Probe Library of Roche (https://www.roche-applied- science.com) on the basis of the reported mRNA sequences. For the human cell cultures we used the following primers: IRAK1 (forward: gcccgaggagtacatcaaga; reverse: ctctgaccagccaaggtctc), IRAK2 (forward: cctcctctgaggcctgtgt; reverse: tgatctcaatttgccac- gaa), TNF receptor associated factor 6 (TRAF6; forward: tggcattacgagaagcagtg; reverse: tggacatttgtgacctgcat:), interleukin 6 (IL-6; forward: ctcagccctgagaaaggaga; reverse: tttcagccatctttg- gaagg), cyclooxygenase-2 (COX-2; forward: gaatggggtgatgagcagtt; reverse: gccactcaagtgttgcacat), elongation factor 1-alpha (EF1a; foward: atccacctttgggtcgcttt; reverse: ccgcaactgtctgtctcatatcac) and hypoxanthine phosphoribosyl transferase (HPRT; forward: tggcgtcgtcgtgattagtgatg; reverse: tgtaatccagcaggtcagca). For each PCR, a mastermix was prepared on ice, containing per sample: 1 ml cDNA, 2.5 ml of FastStart Reaction Mix SYBR Green I (Roche Applied Science, Indianapolis, IN, USA), 0.4 mM of both reverse and forward primers. The final volume was adjusted to 5 ml with H2O (PCR grade). The LightCyclerH 480 Real-Time PCR System (Roche-applied-science) was used with a 384-multi- well plate format. The cycling conditions were carried out as follows: initial denaturation at 95uC for 5 min, followed by 45 cycles of denaturation at 95uC for 15 s, annealing at 55–60uC for 5 s and extension at 72uC for 10 s. The fluorescent product was measured by a single acquisition mode at 72uC after each cycle. For distinguishing specific from non-specific products and primer dimers, a melting curve was obtained after amplification by holding the temperature at 65uC for 15 s followed by a gradual increase in temperature to 95uC at a rate of 2.5uC s21, with the signal acquisition mode set to continuous [30]. Quantification of data was performed using the computer program LinRegPCR in which linear regression on the Log(fluorescence) per cycle number data is applied to determine the amplification efficiency per sample [31]. The starting concentration of each specific product was divided by the starting concentration of reference genes (EF1a and HPRT) and this ratio was compared between patient/control groups. miRNA (miR-146a and the U6B small nuclear RNA gene, rnu6b) expression was analyzed using Taqman microRNA assays (Applied Biosystems, Foster City, CA). cDNA was generated using Taqman MicroRNA reverse transcription kit (Applied Biosystems, Foster City, CA) according to manufacturer’s instructions and the PCRs were run on a Roche Lightcycler 480 (Roche Applied Science, Basel, Switzerland), according to the instructions of the manufacturer. Data analysis was performed with the software provided by the manufacturer. Cell Cultures For astrocytes-enriched human cell cultures, fetal brain tissue (15–23 weeks of gestation) was obtained from spontaneous or medically induced abortions with appropriate maternal written consent for brain autopsy. Tissue was obtained in accordance with the Declaration of Helsinki and the AMC Research Code provided by the Medical Ethics Committee of the AMC. Resected tissue samples were collected in Dulbecco’s modified Eagle’s medium (DMEM)/HAM F10 (1:1) medium (Gibco, Life Tech- nologies), supplemented with 50 units/ml penicillin and 50 mg/ml miR-146a and Astrocyte Inflammatory Response streptomycin and 10% fetal calf serum (FCS). Cell isolation was performed as previously described [32,33]. Briefly, after removal of meninges and blood vessels, tissue was minced and dissociated by incubation at 37uC for 20 min in a Hank’s balanced salt solution containing 2.5 mg/ml trypsin (Sigma, St. Louis, MO, USA) and 0.1 mg/ml bovine pancreatic Dnase I (Boehringer Mannheim, Germany). Tissue was triturated and washed with DMEM/HAM F10 medium, supplemented with 50 units/ml penicillin and 50 mg/ml streptomycin and 10% FCS. Cell suspension (containing , 0.5 g wet weight tissue/10 ml culture medium) was passed through a 70 mm cell sieve (Becton Dickinson, USA) and plated into poly-L-lysine (PLL; 15 mg/ml, Sigma) pre-coated 25 cm2 flasks (Falcon, Lincoln Park, NJ) and maintained in a 5% CO2 incubator at 37uC. After 48 h the culture medium was replaced by fresh medium and cultures were subsequently refreshed twice a week. Cultures reached confluence after 2–3 weeks. Secondary astrocyte cultures were established by trypsinizing confluent cultures and sub-plating onto PLL-pre- coated 6 and 24-well plates (Costar; 0.56106 cell/well in a 6-well plate for western blot analysis or 0.16106 cell/well in a 24-well plate for RNA isolation and PCR) and simultaneously into PLL- precoated 12 mm coverslips (Sigma) in 24-well plates (Costar; 26104 cell/well; for immunocytochemistry). More than 98% of the cells in primary culture, as well as in the successive 12 passages were strongly immunoreactive for the astrocytic marker GFAP and S100b. In the present study astrocytes were used for immunocytochemical analyses at passage 3–4. The astrocytoma cell line U373 was obtained from the American Type Culture Collection (Rockville, MD, USA); cells were cultured in (DMEM)/HAM F10 (1:1) supplemented with 50 units/ml penicillin, 50 mg/ml streptomycin and 10% FCS. Treatment of Cell Cultures Human recombinant (r)IL-1ß (Peprotech, NJ, USA; 10 ng/ml) was applied and maintained for different time periods (from 10 min to 48 h) before harvesting the cells for RNA isolation or western blot analysis. Medium was collected to perform enzyme- linked immunosorbent assay (ELISA). In some experiments lipo- polysaccharide (LPS; 100 ng/ml; Sigma, St. Louis, USA), rIL-6 (10 ng/ml; Strathmann Biotec A.G., Hamburg, Germany), tumor necrosis factor a (TNFa; 1 ng/ml; Peprotech, NJ, USA) and High mobility group box 1 (HMGB1; 40 nM; HMGBiotech S.r.l., Milan, Italy) alone or together with IL-1 ß were applied and maintained in the medium for 24 before harvesting the cells for RNA isolation. In some experiments cells stimulated with LPS were treated with LPS-RS (a TLR4 antagonist from the photosynthetic bacterium Rhodobacter sphaeroides; Invivogen, Tou- louse, France; 10 mg/ml), applied 1 h before LPS. Human IL-1 receptor antagonist (IL-1Ra; 1 mg/ml; Peprotech, NJ, USA) was used to neutralize IL-1b activity (applied 1 h before IL-1b). As previously shown [33], the viability of human astrocytes in culture was not influenced by the treatments. Transfection of Cells Cells in 12-well plates were transfected either with anti- miR146a LNA (FAM – AacCcaTggAauTcaGuuCucA; Ribotask ApS, Odense, Denmark) or scrambled LNA (FAMTM dye-labeled Anti-miRTM Negative Control #1; Applied Biosystems, Carlsbad, CA, USA) in blocking experiments or with miR146a precursor (pre-mir146a; mir146 mimic, Applied Biosystems, Carlsbad, CA, USA) or scrambled pre-mir (FAMTM dye-labeled Pre-miRTM Negative Control #1; Applied Biosystems, Carlsbad, CA, USA) in overexpression experiments using Lipofectamine (Invitrogen, USA) according to manufacturer’s instructions. Briefly, 5 ml PLOS ONE | www.plosone.org 3 September 2012 | Volume 7 | Issue 9 | e44789