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{"project":"UBERON-AE","denotations":[{"id":"T4645","span":{"begin":10958,"end":10963},"obj":"http://purl.obolibrary.org/obo/UBERON_0001977"},{"id":"T2903","span":{"begin":7052,"end":7057},"obj":"http://purl.obolibrary.org/obo/UBERON_0001977"},{"id":"T2902","span":{"begin":6604,"end":6609},"obj":"http://purl.obolibrary.org/obo/UBERON_0001977"},{"id":"T3465","span":{"begin":7788,"end":7799},"obj":"http://purl.obolibrary.org/obo/UBERON_0012131"},{"id":"T2809","span":{"begin":6258,"end":6263},"obj":"http://purl.obolibrary.org/obo/UBERON_0000178"},{"id":"T2808","span":{"begin":6199,"end":6204},"obj":"http://purl.obolibrary.org/obo/UBERON_0000178"},{"id":"T2807","span":{"begin":6175,"end":6180},"obj":"http://purl.obolibrary.org/obo/UBERON_0000178"},{"id":"T924","span":{"begin":5555,"end":5560},"obj":"http://purl.obolibrary.org/obo/UBERON_0000178"}],"text":"Regulation of Mycobacterium tuberculosis-Dependent HIV-1 Transcription Reveals a New Role for NFAT5 in the Toll-Like Receptor Pathway\nNFAT5 Modulation of MTb-Induced HIV-1 Replication\n\nAbstract\nTuberculosis (TB) disease in HIV co-infected patients contributes to increased mortality by activating innate and adaptive immune signaling cascades that stimulate HIV-1 replication, leading to an increase in viral load. Here, we demonstrate that silencing of the expression of the transcription factor nuclear factor of activated T cells 5 (NFAT5) by RNA interference (RNAi) inhibits Mycobacterium tuberculosis (MTb)-stimulated HIV-1 replication in co-infected macrophages. We show that NFAT5 gene and protein expression are strongly induced by MTb, which is a Toll-like receptor (TLR) ligand, and that an intact NFAT5 binding site in the viral promoter of R5-tropic HIV-1 subtype B and subtype C molecular clones is required for efficent induction of HIV-1 replication by MTb. Furthermore, silencing by RNAi of key components of the TLR pathway in human monocytes, including the downstream signaling molecules MyD88, IRAK1, and TRAF6, significantly inhibits MTb-induced NFAT5 gene expression. Thus, the innate immune response to MTb infection induces NFAT5 gene and protein expression, and NFAT5 plays a crucial role in MTb regulation of HIV-1 replication via a direct interaction with the viral promoter. These findings also demonstrate a general role for NFAT5 in TLR- and MTb-mediated control of gene expression.\n\nAuthor Summary\nThe major cause of AIDS deaths globally has been tuberculosis (TB), which is caused by the bacterium Mycobacterium tuberculosis (MTb). Co-infection with MTb exacerbates human immunodeficiency virus type1 (HIV-1) replication and disease progression via both innate and adaptive host immune responses to MTb infection. In this report, we present evidence that the transcription factor NFAT5 plays a crucial role in MTb-induced HIV-1 replication in human peripheral blood cells and monocytes. We also show that MTb infection itself stimulates NFAT5 gene expression in human monocytes and that its expression involves the TLR signalling pathway and requires the downstream adaptor proteins MyD88, IRAK1, and TRAF6. This identification of a novel role for NFAT5 in TB/HIV-1 co-infection reveals that NFAT5 is a major mediator of TLR-dependent gene expression and thus provides a potential new therapeutic target for treatment of HIV-1 and possibly other diseases.\n\nIntroduction\nMycobacterium tuberculosis (MTb), the causative agent of tuberculosis (TB), is the most common co-infection and cause of death in patients infected with human immunodeficiency virus type 1 (HIV-1) [1], [2]. Direct engagement of pathogen recognition receptors (PRRs) by MTb on mononuclear phagocytes activates signaling cascades that directly induce transcription from the proviral LTR (reviewed in [3]). Furthermore, inflammatory cytokines and chemokines produced by the human host in response to MTb infection activate signal transduction pathways in CD4 T cells and monocytic cells that also result in transcriptional activation of the HIV-1 LTR [4]–[6]. Activation of HIV-1 replication via these MTb-induced pathways ultimately leads to higher viral loads and, in turn, expedited CD4 T cell loss and progression to AIDS ([7], reviewed in [8]–[10]). Furthermore, the progressive immune compromise associated with HIV-1 infection itself is a major cause of latent MTb reactivation, as well as increased susceptibility to primary TB infection ([11]–[15], reviewed in [8]).\nThe primary PRR on monocytic cells triggered by MTb infection is toll-like receptor (TLR) 2 [16]–[20]. Engagement of TLR2 results in engagement of the adaptor protein MyD88 and the subsequent recruitment of several kinases, including IRAK1 and IRAK4, and the ubiquitin ligase TRAF6 ([21]–[23], reviewed in [10], [24]). TRAF6 activates IκB kinase (IKK) and mitogen-activated protein (MAP) kinases that, in turn, ultimately induce activation of specific transcription factor families, including the NF-κB and AP-1 families, which have been shown to associate with the HIV-1 LTR and to drive its transcription ([22], [25]–[27], reviewed in [10]).\nNotably, HIV-1 comprises several subtypes, and the LTR of each subtype is unique with respect to the number and organization of activator binding sites. For example, HIV-1 subtype B, the most highly characterized viral subtype and the primary cause of infection in the Americas, Europe, Japan, and Australia, has two tandem NF-κB motifs in its LTR. By contrast, HIV-1 subtypes C and E, which have spread disproportionately in TB-burdened sub-Saharan Africa and southeast Asia, have three and one NF-κB binding sites, respectively [1], [28]–[30].\nWe previously showed that the most primordial member of the nuclear factor of activated T cells (NFAT) family, NFAT5 (also known as TonEBP), binds to a site within the HIV-1 LTR that is highly conserved across all HIV-1 subtypes, and is also conserved in HIV-2 and SIV LTRs. This NFAT5 site overlaps the core NF-κB binding motifs in the LTR and is required for constitutive replication of representative HIV-1 subtype B, C, and E isolates in human primary monocyte-derived macrophages (MDM) [31]. Given that NFAT5 has previously been shown to be transcriptionally activated by the MAP kinase p38, which is downstream of MyD88 signaling, [32], we speculated that NFAT5 may also be involved in MTb-induced activation of HIV-1 replication via a TLR-mediated pathway in monocytes and peripheral blood mononuclear cells (PBMC).\nHere, we show that NFAT5 and its cognate binding site are of crucial importance for efficient MTb-induced stimulation of HIV-1 replication in human MDM and PBMC. Moreover, we demonstrate that MTb infection increases NFAT5 gene expression in human monocytes in a MyD88-dependent manner. Thus, these results expand the known stimuli of NFAT5 expression to the PRR-mediated innate immune response, and demonstrate that NFAT5 is a critical modulator of MTb-induced enhancement of HIV-1 replication.\n\nMaterials 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 significant.\n\nResults\n\nMTb increases HIV-1 LTR activity of HIV-1 subtypes B, C, and E\nTo compare the functional impact of MTb stimulation on subtype-specific HIV-1 LTR activity, we first constructed reporter plasmids containing viral subtype B, C, and E LTRs (−208 to + 64 nt relative to the transcription start site) linked to the firefly luciferase reporter gene. After transfection of the monocytic THP-1 cell line with these plasmids, cells were stimulated with an irradiated whole cell lysate of MTb (H37Rv). We note that MTb lysate induces inflammatory responses in monocytes that resemble those induced in response to live MTb (see for example, [35]–[37]). Upon stimulation, the B, C, and E LTR-driven reporters demonstrated a significant enhancement in luciferase activity (Figure 1A) and the magnitude of this effect was subtype-specific. Subtype C LTRs displayed the strongest activity, while the LTRs from subtype E isolates consistently showed the weakest activity (Figure 1A), consistent with previous studies demonstrating subtype-specific LTR activity that used TNF as a stimulus [38], [39].\n10.1371/journal.ppat.1002620.g001 Figure 1 NFAT5 interaction with the LTR is important for MTb-induced HIV-1 transcription.\n(A) MTb stimulation increases activity of LTRs derived from HIV-1 subtypes B, C, and E. HIV-1 LTRs (−208 to +64 nt relative to the transcription start site) from representative subtype B, C, and E viral isolates were cloned into plasmid pGL3. THP-1 cells (0.8×106/ml) were transfected with each reporter plasmid (0.3 µg/ml) plus the Renilla luciferase control plasmid pRL-TK (0.03 µg/ml) and incubated at 37°C for 16 hours. Cells were then either left untreated or treated with 10 µg/ml MTb lysate for 8 hours before termination of the cultures. In the histogram, open bars represent individual LTR activities in untreated cells. Light grey bars represent mean values of LTR activities from each subtype in untreated cells. Black bars represent individual LTR activities in MTb lysate-treated cells, and dark grey bars represent mean values of LTR activities from each subtype in cells treated with MTb lysate. LTR transcriptional activity for all of the representative LTRs tested was significantly increased in cultures treated with MTb lysate in comparison to untreated cultures. Results are from three independent experiments performed in duplicate (*, p\u003c0.05; **, p\u003c0.01 as compared to unstimulated cultures). (B) Specific disruption of the NFAT5 binding site significantly reduces LTR-reporter gene activity in monocytic cells in response to MTb lysate treatment. THP-1 cells were transfected with luciferase expression vectors encoding nucleotides 208 to +64 of the wild-type HIV-1Lai LTR and an HIV-1Lai LTR containing the NFAT5 binding site mutations (N5-Mut). After 16 hours, the cells were left untreated or exposed to 10 µg/ml MTb lysate for 8 hours at 37°C. Disruption of NFAT5 binding to the enhancer region significantly suppressed LTR-driven reporter gene expression in comparison to the wild-type LTR when cells were treated with MTb lysate (p\u003c0.01). LTR activity was also suppressed in the untreated cells but to a lesser extent (p\u003c0.05). Results are from three independent experiments performed in duplicate and adjusted to Renilla luciferase control expression (*, p\u003c0.05; **, p\u003c0.01). Nucleotide sequences representing the wild-type and NFAT5 binding site-mutated HIV-1Lai LTRs are shown at the bottom of the figure. (C) MTb lysate increases NFAT5 protein levels in monocytic cells. THP-1 cells were left untreated (control) or exposed to 10 µg/ml MTb lysate for 8 or 24 hours at 37°C. Whole cell extracts were collected and analyzed by western blot with anti-NFAT5"}

GO-BP

GO-MF

GO-CC

sentences

ICD10

{"project":"ICD10","denotations":[{"id":"T1035","span":{"begin":2515,"end":2527},"obj":"http://purl.bioontology.org/ontology/ICD10/A15-A19.9"},{"id":"T116","span":{"begin":593,"end":605},"obj":"http://purl.bioontology.org/ontology/ICD10/A15-A19.9"},{"id":"T115","span":{"begin":28,"end":40},"obj":"http://purl.bioontology.org/ontology/ICD10/A15-A19.9"},{"id":"T1037","span":{"begin":2660,"end":2676},"obj":"http://purl.bioontology.org/ontology/ICD10/D84.9"},{"id":"T1036","span":{"begin":2558,"end":2570},"obj":"http://purl.bioontology.org/ontology/ICD10/A15-A19.9"}],"text":"Regulation of Mycobacterium tuberculosis-Dependent HIV-1 Transcription Reveals a New Role for NFAT5 in the Toll-Like Receptor Pathway\nNFAT5 Modulation of MTb-Induced HIV-1 Replication\n\nAbstract\nTuberculosis (TB) disease in HIV co-infected patients contributes to increased mortality by activating innate and adaptive immune signaling cascades that stimulate HIV-1 replication, leading to an increase in viral load. Here, we demonstrate that silencing of the expression of the transcription factor nuclear factor of activated T cells 5 (NFAT5) by RNA interference (RNAi) inhibits Mycobacterium tuberculosis (MTb)-stimulated HIV-1 replication in co-infected macrophages. We show that NFAT5 gene and protein expression are strongly induced by MTb, which is a Toll-like receptor (TLR) ligand, and that an intact NFAT5 binding site in the viral promoter of R5-tropic HIV-1 subtype B and subtype C molecular clones is required for efficent induction of HIV-1 replication by MTb. Furthermore, silencing by RNAi of key components of the TLR pathway in human monocytes, including the downstream signaling molecules MyD88, IRAK1, and TRAF6, significantly inhibits MTb-induced NFAT5 gene expression. Thus, the innate immune response to MTb infection induces NFAT5 gene and protein expression, and NFAT5 plays a crucial role in MTb regulation of HIV-1 replication via a direct interaction with the viral promoter. These findings also demonstrate a general role for NFAT5 in TLR- and MTb-mediated control of gene expression.\n\nAuthor Summary\nThe major cause of AIDS deaths globally has been tuberculosis (TB), which is caused by the bacterium Mycobacterium tuberculosis (MTb). Co-infection with MTb exacerbates human immunodeficiency virus type1 (HIV-1) replication and disease progression via both innate and adaptive host immune responses to MTb infection. In this report, we present evidence that the transcription factor NFAT5 plays a crucial role in MTb-induced HIV-1 replication in human peripheral blood cells and monocytes. We also show that MTb infection itself stimulates NFAT5 gene expression in human monocytes and that its expression involves the TLR signalling pathway and requires the downstream adaptor proteins MyD88, IRAK1, and TRAF6. This identification of a novel role for NFAT5 in TB/HIV-1 co-infection reveals that NFAT5 is a major mediator of TLR-dependent gene expression and thus provides a potential new therapeutic target for treatment of HIV-1 and possibly other diseases.\n\nIntroduction\nMycobacterium tuberculosis (MTb), the causative agent of tuberculosis (TB), is the most common co-infection and cause of death in patients infected with human immunodeficiency virus type 1 (HIV-1) [1], [2]. Direct engagement of pathogen recognition receptors (PRRs) by MTb on mononuclear phagocytes activates signaling cascades that directly induce transcription from the proviral LTR (reviewed in [3]). Furthermore, inflammatory cytokines and chemokines produced by the human host in response to MTb infection activate signal transduction pathways in CD4 T cells and monocytic cells that also result in transcriptional activation of the HIV-1 LTR [4]–[6]. Activation of HIV-1 replication via these MTb-induced pathways ultimately leads to higher viral loads and, in turn, expedited CD4 T cell loss and progression to AIDS ([7], reviewed in [8]–[10]). Furthermore, the progressive immune compromise associated with HIV-1 infection itself is a major cause of latent MTb reactivation, as well as increased susceptibility to primary TB infection ([11]–[15], reviewed in [8]).\nThe primary PRR on monocytic cells triggered by MTb infection is toll-like receptor (TLR) 2 [16]–[20]. Engagement of TLR2 results in engagement of the adaptor protein MyD88 and the subsequent recruitment of several kinases, including IRAK1 and IRAK4, and the ubiquitin ligase TRAF6 ([21]–[23], reviewed in [10], [24]). TRAF6 activates IκB kinase (IKK) and mitogen-activated protein (MAP) kinases that, in turn, ultimately induce activation of specific transcription factor families, including the NF-κB and AP-1 families, which have been shown to associate with the HIV-1 LTR and to drive its transcription ([22], [25]–[27], reviewed in [10]).\nNotably, HIV-1 comprises several subtypes, and the LTR of each subtype is unique with respect to the number and organization of activator binding sites. For example, HIV-1 subtype B, the most highly characterized viral subtype and the primary cause of infection in the Americas, Europe, Japan, and Australia, has two tandem NF-κB motifs in its LTR. By contrast, HIV-1 subtypes C and E, which have spread disproportionately in TB-burdened sub-Saharan Africa and southeast Asia, have three and one NF-κB binding sites, respectively [1], [28]–[30].\nWe previously showed that the most primordial member of the nuclear factor of activated T cells (NFAT) family, NFAT5 (also known as TonEBP), binds to a site within the HIV-1 LTR that is highly conserved across all HIV-1 subtypes, and is also conserved in HIV-2 and SIV LTRs. This NFAT5 site overlaps the core NF-κB binding motifs in the LTR and is required for constitutive replication of representative HIV-1 subtype B, C, and E isolates in human primary monocyte-derived macrophages (MDM) [31]. Given that NFAT5 has previously been shown to be transcriptionally activated by the MAP kinase p38, which is downstream of MyD88 signaling, [32], we speculated that NFAT5 may also be involved in MTb-induced activation of HIV-1 replication via a TLR-mediated pathway in monocytes and peripheral blood mononuclear cells (PBMC).\nHere, we show that NFAT5 and its cognate binding site are of crucial importance for efficient MTb-induced stimulation of HIV-1 replication in human MDM and PBMC. Moreover, we demonstrate that MTb infection increases NFAT5 gene expression in human monocytes in a MyD88-dependent manner. Thus, these results expand the known stimuli of NFAT5 expression to the PRR-mediated innate immune response, and demonstrate that NFAT5 is a critical modulator of MTb-induced enhancement of HIV-1 replication.\n\nMaterials 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 significant.\n\nResults\n\nMTb increases HIV-1 LTR activity of HIV-1 subtypes B, C, and E\nTo compare the functional impact of MTb stimulation on subtype-specific HIV-1 LTR activity, we first constructed reporter plasmids containing viral subtype B, C, and E LTRs (−208 to + 64 nt relative to the transcription start site) linked to the firefly luciferase reporter gene. After transfection of the monocytic THP-1 cell line with these plasmids, cells were stimulated with an irradiated whole cell lysate of MTb (H37Rv). We note that MTb lysate induces inflammatory responses in monocytes that resemble those induced in response to live MTb (see for example, [35]–[37]). Upon stimulation, the B, C, and E LTR-driven reporters demonstrated a significant enhancement in luciferase activity (Figure 1A) and the magnitude of this effect was subtype-specific. Subtype C LTRs displayed the strongest activity, while the LTRs from subtype E isolates consistently showed the weakest activity (Figure 1A), consistent with previous studies demonstrating subtype-specific LTR activity that used TNF as a stimulus [38], [39].\n10.1371/journal.ppat.1002620.g001 Figure 1 NFAT5 interaction with the LTR is important for MTb-induced HIV-1 transcription.\n(A) MTb stimulation increases activity of LTRs derived from HIV-1 subtypes B, C, and E. HIV-1 LTRs (−208 to +64 nt relative to the transcription start site) from representative subtype B, C, and E viral isolates were cloned into plasmid pGL3. THP-1 cells (0.8×106/ml) were transfected with each reporter plasmid (0.3 µg/ml) plus the Renilla luciferase control plasmid pRL-TK (0.03 µg/ml) and incubated at 37°C for 16 hours. Cells were then either left untreated or treated with 10 µg/ml MTb lysate for 8 hours before termination of the cultures. In the histogram, open bars represent individual LTR activities in untreated cells. Light grey bars represent mean values of LTR activities from each subtype in untreated cells. Black bars represent individual LTR activities in MTb lysate-treated cells, and dark grey bars represent mean values of LTR activities from each subtype in cells treated with MTb lysate. LTR transcriptional activity for all of the representative LTRs tested was significantly increased in cultures treated with MTb lysate in comparison to untreated cultures. Results are from three independent experiments performed in duplicate (*, p\u003c0.05; **, p\u003c0.01 as compared to unstimulated cultures). (B) Specific disruption of the NFAT5 binding site significantly reduces LTR-reporter gene activity in monocytic cells in response to MTb lysate treatment. THP-1 cells were transfected with luciferase expression vectors encoding nucleotides 208 to +64 of the wild-type HIV-1Lai LTR and an HIV-1Lai LTR containing the NFAT5 binding site mutations (N5-Mut). After 16 hours, the cells were left untreated or exposed to 10 µg/ml MTb lysate for 8 hours at 37°C. Disruption of NFAT5 binding to the enhancer region significantly suppressed LTR-driven reporter gene expression in comparison to the wild-type LTR when cells were treated with MTb lysate (p\u003c0.01). LTR activity was also suppressed in the untreated cells but to a lesser extent (p\u003c0.05). Results are from three independent experiments performed in duplicate and adjusted to Renilla luciferase control expression (*, p\u003c0.05; **, p\u003c0.01). Nucleotide sequences representing the wild-type and NFAT5 binding site-mutated HIV-1Lai LTRs are shown at the bottom of the figure. (C) MTb lysate increases NFAT5 protein levels in monocytic cells. THP-1 cells were left untreated (control) or exposed to 10 µg/ml MTb lysate for 8 or 24 hours at 37°C. Whole cell extracts were collected and analyzed by western blot with anti-NFAT5"}

simple1

BioNLP16_DUT

BioNLP16_Messiy

DLUT931

bionlp-st-ge-2016-test-ihmc

bionlp-st-ge-2016-spacy-parsed

bionlp-st-ge-2016-test-tees

testone

test3

PMC:3320587 / 0-19038 JSONTXT

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PMC:3320587 / 0-19038 JSON TXT