PMC:3320587 / 6083-15332 JSONTXT

{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/3320587","sourcedb":"PMC","sourceid":"3320587","source_url":"http://www.ncbi.nlm.nih.gov/pmc/3320587","text":"Materials and Methods\n\nEthics statement\nIn our studies we used unidentified human discarded blood cells (peripheral blood mononuclear cells, PBMC), which we obtained from the Blood Bank of Children's Hospital in Boston.\n\nCell culture\nPBMC from normal unidentified donors were isolated by Ficoll-Hypaque (Pharmacia Corporation, Peapack, NJ) density gradient centrifugation and were cultured in RPMI 1640 medium with 2 mM L-glutamine (BioWhittaker, Inc., Walkersville, MD) supplemented with 10% heat-inactivated fetal calf serum (FCS) (Gemini Bio-Products, www.gembio.com). Human monocytes were isolated from PBMC preparations by positive selection with CD14 microbeads from Miltenyi Biotec (www.miltenyibiotec.com) as described by the manufacturer, and were cultured at 1×106 cells per well in 6-well plates in Macrophage-SFM medium (Gibco, www.invitrogen.com) supplemented with 15 ng/ml recombinant human MCSF (R\u0026D, www.rndsystems.com) and 5% heat-inactivated human AB serum (Nabi, Boca Raton, FL). The cell cultures were incubated at 37°C and 5% CO2 for 5 days, after which supernatant was replaced with fresh medium lacking MCSF before manipulation. More than 95% of the adherent cells obtained with this technique were CD14+ macrophages as verified by flow cytometry. THP-1 cells were obtained from ATCC (www.atcc.org) and cultured in RPMI 1640 medium supplemented with 10% FCS (BioWhittaker, www.lonzabio.com). 293T cells were obtained from ATCC (www.atcc.org) and were maintained in Dulbecco's Modified Eagle's medium (DMEM) (Gibco, www.invitrogen.com) supplemented with 10% FCS.\n\nViruses\nHIV-1Bal, HIV-1Lai, HIV-193TH64, HIV-192TH51, HIV-192TH53, HIV-198CH01, and HIV-198IN22 were obtained from The Centralized Facility for AIDS Reagents, National Institute for Biological Standard and Control (NIBSC), United Kingdom. HIV-1KR25 was isolated in our laboratory as described before [33].\n\nLTR plasmid construction and reporter assay\nLTR reporter plasmids were constructed by inserting nucleotides −208 to +64 relative to the transcriptional initiation site of HIV-1Lai, HIV-1Bal (B subtype), HIV-198IN17, HIV-198IN22, HIV-198CH01, HIV-1CM9 (C subtype), HIV-193TH64, HIV-192TH53, HIV-192TH51, and HIV-1KR25 (E subtype) into the reporter vector pGL3 (Promega BioSciences, www.promega.com) using Xho I and Hind III restriction enzyme sites. Sequences were aligned and analyzed with CLUSTAL W (www.ebi.ac.uk/clustalw/). The HIV-1Lai NFAT5 binding site-mutant (N5-Mut) reporter plasmid was created by standard PCR-based mutagenesis methods [34]. THP-1 cells (0.8×106/ml) were transfected with 0.3 µg/ml LTR wild-type (WT) or mutated reporter plasmids in combination with 0.03 µg/ml Renilla luciferase (pRL-TK) control vector using Effectene transfection reagent (Qiagen; www.qiagen.com). Cells were incubated at 37°C for 16 hours after which they were stimulated with 10 µg/ml MTb CDC1551 lysate or left unstimulated for 8 hours. Reporter gene expression was quantitated by dual-luciferase reporter assay according to the manufacturer's protocol (Promega; www.promega.com).\n\nQuantitative DNase I footprinting\nRecombinant NFAT5 (amino acids 175–471) with an N-terminal 6× His tag was expressed in E. coli BL21(DE3) cells (Stratagene; www.stratagene.com) and purified under native conditions using Ni-NTA agarose (Qiagen). Recombinant p50 and p65 were purchased (Active Motif, www.activemotif.com). Quantitative DNase I footprinting was performed as previously described [31].\n\nHIV-1 infectious molecular clones\nThe plasmid encoding the full-length infectious molecular clone of HIV-1Lai was obtained from the NIH AIDS Reagent and Reference Program. The HIV-1Lai/Bal-Env infectious molecular clone was constructed by replacing the envelope (env) gp160 amino acids 103–717 of the HIV-1Lai (B subtype that utilizes CXCR4) molecular clone with the corresponding region of HIV-1Bal (B subtype that utilizes CCR5). The HIV-1Lai/Bal-Env chimeric virus uses CCR5 as a secondary receptor. The infectious molecular clone of HIV-198IN22 was constructed using DNA extracted from PBMC that were infected with a primary isolate of HIV-198IN22. HIV-1Lai/Bal-Env and HIV-198IN22 mutant viruses were constructed by introducing point mutations using standard PCR-based mutagenesis methods.\n\nsiRNA transfection of MDM\nAn siRNA was constructed (Ambion Inc., www.ambion.com) to target a sequence unique to the NFAT5 transcript: 5′-CAACATGCCTGGAATTCAA-3′ (nt 335 to 353) [31]. As described, a control for non-specific siRNA effects, we used an siRNA targeting the green fluorescent protein (GFP), 5′- GGCTACGTCCAGGAGCGCACC-3′. MDM were transfected in 6-well plates using 1 µM of the indicated siRNA in siPORT NeoFX transfection reagent (Ambion Inc., www.ambion.com), prepared as recommended by the manufacturer, in a final volume of 750 µl in Macrophage-SFM medium plus 5% heat-inactivated human AB serum. The cultures were left at 37°C overnight after which cells were washed and incubated in fresh medium. MDM were transfected two times for efficient knock down of NFAT5 expression before infection experiments were performed [31].\n\nStable THP-1 cells expressing shRNA\nThe lentiviral plasmid pLKO.1 expressing shRNA targeting human MyD88 was purchased from Open Biosystems (www.openbiosystems.com) and was validated in our laboratory. shRNA targeting human IRAK1 (forward primer 5′-CCGGAGCAGCTGTCCAGGTTTCGTCTCATAAAACCTGGACAGCTGCTCCTTTTTG-3′, reverse primer 5′-AATTCAAAAAGGAGCAGCTGTCCAGGTTTTATGAGACGAAACCTGGACAGCTGCT-3′ mRNA (IRAK1 mRNA target sequence is underlined) and human TRAF6 (forward primer 5′-CCGGAGAAACCTGTTGTGATTCGTCTCATAAATCACAACAGGTTTCTCCTTTTTG-3′, reverse primer 5′-AATTCAAAAAGGAGAAACCTGTTGTGATTTATGAGACGAATCACAACAGGTTTCT-3′ (TRAF6 mRNA target sequence is underlined) were designed in our laboratory and were cloned into the pLKO.1 plasmid. Lentiviruses encoding shRNA sequences were generated by transfecting the packaging cell line HEK-293T with the shRNA-encoding pLKO.1 plasmids in combination with the packaging plasmid psPAX2 and the envelope plasmid pMD2.G using Effectene transfection reagent (Qiagen, www.qiagen.com). Supernatants were collected 48 hours post-transfection, clarified by centrifugation, and stored at −80°C. THP-1 cells were transduced with the lentiviral particles by culturing the cells with supernatants from the virus-producing cells in the presence of 8 µg/ml polybrene (Millipore, www.millipore.com) and spinoculation for two hours at 2000 RPM. Successfully transduced cells were selected and expanded by treatment with 0.8 µg/ml puromycin.\n\nMTb culture\nThe MTb clinical strain CDC1551 was prepared by adding 100 µl of frozen bacteria stock into 10 ml of Middlebrook 7H9 medium (Difco BD, www.bd.com) supplemented with albumin dextrose complex (ADC) and 0.05% Tween 80 (Sigma-Aldrich, www.sigmaaldrich.com). The cultures were grown to an OD650 of 0.4 at 37°C to ensure that they were in the logarithmic growth phase. Bacteria were then plated, washed with PBS, resuspended in PBS, and passed through a 5 µm filter to ensure that the bacteria were in a single cell suspension. Bacterial cell numbers were determined by measurement of OD650 before further dilution with RPMI 1640 medium for cell infection studies at 10∶1 PBMC∶ bacilli or 1∶1 MDM∶ bacilli and THP-1∶bacilli. Colony-forming unit (CFU) analysis was performed and on days 4 and 7 the average CFU counts were 6×103 and 5×104, respectively, confirming that mycobacteria levels increased over the course of infection of primary MDM.\n\nWestern blot\nWhole cell extracts were collected with lysis buffer containing 150 mM NaCl, 50 mM Tris–HCl, pH 7.5, 1% Triton, 10% glycerol, and 1 tablet of Complete EDTA-free Protease Inhibitor Cocktail (Roche) per 25 ml of buffer. Extracts were boiled for 5 min in 1× Laemmli sample buffer with 5% v/v 2-mercaptoethanol and proteins were separated by SDS-PAGE. The gel was transferred to a nitrocellulose Trans-Blot Transfer Membrane (BioRad). The blot was then blocked for 1 h at 37°C in a solution of 4% BSA (Sigma) and 0.1% Tween-20 (BioRad) in a buffer containing 50 mM Tris and 150 mM NaCl at pH 7.6 (BSA/TBST). Primary incubation was carried out with a1∶200 dilution of rabbit anti-NFAT5 antibody (H-300) (Santa Cruz Biotechnology) and a 1∶500 dilution of goat anti-Lamin-B1 antibody (sc-6217; Santa Cruz Biotechnology) in BSA/TBST for 2 h at room temperature. The blot was washed 3×5 min in TBST and incubated in 1∶6000 donkey anti-goat-HRP (Santa Cruz Biotechnology) or goat anti-rabbit-HRP (BioRad) as appropriate for 1 h. The blot was again washed 3×5 min in TBST and developed with SuperSignal West Pico Chemiluminescent Reagent (Pierce).\n\nQuantitative PCR\nThe mRNA expression levels were determined by SYBR Green-based real-time PCR (Applied Biosystems, www.appliedbiosystems.com). The reaction conditions were 95°C for 10 min followed by 40 cycles of 95°C for 15 sec and 60°C for 1 min. The results were normalized using β-actin mRNA as an internal control and expressed as relative values.\n\nStatistical analysis\nWhere applicable, results are expressed as mean ± SEM. Comparison between two groups was performed using the paired Student t-Test with the aid of Microsoft Excel software. p≤0.05 was considered 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