hsa-let-7b-5p facilitates Mycobacterium tuberculosis survival in THP-1 human macrophages by Fas downregulation

hsa-let-7b-5p facilitates Mycobacterium tuberculosis survival in THP-1 human macrophages by Fas... Abstract Tuberculosis continues to be one of the deadliest infectious diseases worldwide. MicroRNAs (miRNAs) are small non-coding entities that play critical role as post-transcriptional regulators and are transcriptionally deregulated upon mycobacterial infection. In this study, we found significant upregulation of hsa-let-7b-5p in Mycobacterium tuberculosis (MTB) infected THP-1 human macrophages. Concomitantly, we detected the reduced level of Fas protein, one of the targets of hsa-let-7b-5p, in MTB-infected THP-1 macrophages. Using luciferase assay, a direct interaction between hsa-let-7b-5p and the Fas 3΄-untranslated region (3΄-UTR) was established. Inhibition of hsa-let-7b-5p augmented the apoptosis of THP-1 cells enabling enhanced clearance of MTB. Our findings suggest that hsa-let-7b-5p helps intracellular survival of MTB in THP-1 cells by downregulating Fas protein level. This highlights hsa-let-7b-5p as a potential therapeutic target for tuberculosis treatment. M. tuberculosis, hsa-let-7b-5p, Fas, Apoptosis, THP-1 cells INTRODUCTION Tuberculosis (TB) is caused by Mycobacterium tuberculosis (MTB) which has co-evolved with host for several thousand years. This has led the bug better adapted for human host wherein it can survive for longer period of time asymptomatically in dormant stage. Infection of mammalian host usually occurs by the aerosol route and the lung is primarily the first affected organ. The resident macrophages of lung interact with bacilli and internalize them in the phagosome through pathogen-specific receptors. The MTB tends to survive and replicate inside macrophages instead of being eliminated bypassing the host innate defense system otherwise meant to clear pathogens. MTB interferes with host trafficking system by modulating events in endosomal/phagosomal maturation and creates a protected niche in the mycobacterial phagosome (Houben, Nguyen and Pieters 2006). MicroRNAs (miRNAs) are widely conserved small (18–22 nucleotides) non-coding RNAs that function as post-transcriptional regulators. They interact with their target coding mRNAs and inhibit translation by either degradation of the mRNAs or blocking translation without degrading the targets (Du and Zamore 2005). Conservation of individual miRNAs across different species highlights their functional relevance. Notably, miRNAs with altered expressions are involved in a broad spectrum of human diseases (Kanwar, Mahidhara and Rupinder 2009; Pauley, Seunghee and Chan 2009). A host of evidences have established that miRNAs are regulatory players in cell-mediated immune processes, thereby, qualifying these regulators as biomarkers candidates in immune disorders (Rodriguez et al.2007; Mittelbrunn et al.2011). Recently, miRNAs have been shown to play important roles in successful microbial infections (Yi et al.2012; Staedel and Darfeuille 2013; Abdalla et al.2016). Their significant roles in the control of gene expression have been described in different biological processes including development of the immune system and orchestration of anti-pathogenic responses (Taganov, Boldin and Baltimore 2007; Nathans et al.2009). However, a lot is still to be known about the role of miRNAs in intracellular survival of pathogens, immune regulation and pathogenesis of MTB in host (Ottenhoff 2012). This study was aimed to identify and characterize miRNAs facilitating the intracellular survival of MTB in macrophages. Using TaqMan Low Density Array (TLDA) of human miRNAs (Invitrogen, USA), we analyzed the expression of miRNAs in MTB-infected THP-1 macrophages. Of several miRNAs that appeared significantly upregulated in array analysis followed by TaqMan real time qRT-PCR validation, hsa-let-7b-5p was examined in details. Its targets were predicted in silico and the binding of hsa-let-7b-5p to Fas, one of the predicted targets, was validated using luciferase reporter assay and immunoblotting. Based on our data, we suggest that hsa-let-7b-5p helps in intracellular survival of MTB by downregulation of Fas receptor. MATERIALS AND METHODS Bacterial strains and culture MTB H37Rv and M. smegmatis mc2155 strains were obtained from departmental stock (Division of Microbiology, CSIR-CDRI, Lucknow, India) and cultured in Middlebrook 7H9 liquid medium supplemented with oleic acid albumin dextrose catalase (OADC) enrichment (Difco, USA). For growth on solid surface, cultures were spread on Middlebrook 7H11 plates supplemented with 10% OADC, 0.5% glycerol and 0.05% Tween-80 and incubated at 37°C. Escherichia coli strains were cultured in Luria Bertani (LB) broth at 37°C and spread on LB agar plates. Cell culture and infection Human THP-1 cells were maintained at 37°C and 5% CO2 in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS). To induce cell adherence and differentiation, 1 × 106 THP-1 cells were seeded per well in a 6-wells plate and treated with 30 ng/ml PMA for 48 h in culture medium. Mid log phase (0.7 OD600) bacterial cultures, carefully dislodged to single cell suspension, were used for infection. The mycobacteria and THP-1 cells were seeded at 10:1 multiplicity of infection, incubated for 4 h, washed three times, 5 min each, and incubated in complete RPMI-1640 media with amikacin (200 μg/ml) for 1 h to kill extracellular mycobacteria. After the last wash cells were harvested for 0 h time point, fresh medium was added and cells were incubated for 24–48 h at 37°C with 5% CO2. Viability of THP-1 cells was assessed by trypan blue dye exclusion assay. miRNAs profiling of MTB-infected THP-1 macrophages using TLDA and validation by TaqMan qRT-PCR Total RNA were isolated from uninfected and 24 h post-MTB-infected THP-1 cells using MirVana miRNA isolation kit (Cat #AM1560, Ambion, USA). RNA was assessed by NanoDrop spectrophotometer and Agilent RNA 6000 Nano Kit on Agilent 2100 Bioanalyser (Thermo Scientific, USA). One microgram total RNA per sample was reverse transcribed using Megaplex Human Pool A and Pool B stem loop RT primers (version 3.0) and TaqMan MicroRNA RT kit (Invitrogen, USA) according to the manufacturer's protocol. Expression analysis was performed on QuantStudio 12K Flex Real-Time PCR System (Applied Biosystems) using cDNAs on TLDA A and B cards (version 3.0) with validated TaqMan® miRNA probes. The TLDA cards measure miRNA expression based on TaqMan-enabled real-time analysis, and fold change in miRNAs expression of treated versus untreated samples were calculated (ΔΔCt method) using Expression Suite Software (version 1.0.3, Applied Biosystems). Data were normalized to expression of U6 small nuclear RNA. Only targets having threshold cycle (Ct) ≤ 35 were included in this study. Details of this experiment may be obtained from data submitted to NCBI GEO repository (accession number GSE94007). For TaqMan qRT-PCR, 10 ng total RNA per sample was reverse transcribed using TaqMan gene-specific stem loop primers provided with individual miRNA assay kit (Invitrogen, USA). Reactions were set up as per kit's protocol and run in StepOnePlus real-time PCR system (Thermo Fisher Scientific, USA). U6 snRNA was used as an internal control, and fold change in gene expression between treated and untreated samples were calculated using the comparative ΔΔCt method. Quantitative real-time qRT-PCR Real-time qRT-PCR was performed using total RNAs from THP-1 cells treated with MTB, hsa-let-7b mimics and hsa-let-7b inhibitors. Total RNA was isolated using RNeasy Mini Kit (Qiagen, India) and DNase treated using RNase free Turbo DNase (Ambion, USA). For each sample, 1 μg of DNA-free RNA was reverse transcribed using Thermo Scientific RevertAid cDNA Synthesis Kit (Cat # K1621) by oligo-dT18 primer as per kit's protocol. The qRT-PCR was performed with Fas gene-specific primers (Forward: 5΄-TCCTCCAGGTGAAAGGAAAGCT-3΄ and Reverse: 5΄-TCTGCACTTGGTATTCTGGGTC-3΄) using Power SYBR® Green PCR Master Mix (Thermo Fisher Scientific, Cat # 4367659) on StepOnePlus PCR system. A negative control was parallely run using RNA sample not reverse transcribed. Expression level was normalized by GAPDH (Forward: 5΄-ACCACAGTCCATGCCATCACTG-3΄ and Reverse: 5΄-TGCTTCACCACCTTCTTGATGT-3΄), and fold change in gene expression between treated and untreated samples were calculated using the comparative ΔΔCt method. Experiments were repeated twice using independent cultures, and each reaction was set up in triplicates (Fig. 1C). Figure 1. View largeDownload slide (A) Validations of differentially regulated miRNAs by TaqMan Real-time RT-PCR. Relative expression was analyzed using RNA samples from MTB infected and uninfected THP-1 cells. The expressions were normalized to U6 snRNA. Data were obtained from two biological replicates and each reaction was set up in triplicates. Values plotted are mean ± SD. (B) Western blot analysis of FAS protein in THP-1 cell transfected with M. smegmatis (MS) and M. tuberculosis (MTB), hsa-let-7b-5p mimics, hsa-let-7b-5p inhibitor and non-specific miRNA Cel-miR-39-3p. Experiments were repeated with two biological replicates. Densitometry values were normalized to the levels of β-Actin. Data are mean ± SD (**P < 0.01). (C) Downregulation of Fas expression in THP-1 cells upon MTB infection and after transfection with let-7b-5p mimic. Transfection with let-7b-5p inhibitor ameliorates the Fas transcripts level in THP-1 cells. Data represent the mean ± SD from two independent experiments, each reaction was set up in triplicates (**P < 0.01, ***P < 0.001). Figure 1. View largeDownload slide (A) Validations of differentially regulated miRNAs by TaqMan Real-time RT-PCR. Relative expression was analyzed using RNA samples from MTB infected and uninfected THP-1 cells. The expressions were normalized to U6 snRNA. Data were obtained from two biological replicates and each reaction was set up in triplicates. Values plotted are mean ± SD. (B) Western blot analysis of FAS protein in THP-1 cell transfected with M. smegmatis (MS) and M. tuberculosis (MTB), hsa-let-7b-5p mimics, hsa-let-7b-5p inhibitor and non-specific miRNA Cel-miR-39-3p. Experiments were repeated with two biological replicates. Densitometry values were normalized to the levels of β-Actin. Data are mean ± SD (**P < 0.01). (C) Downregulation of Fas expression in THP-1 cells upon MTB infection and after transfection with let-7b-5p mimic. Transfection with let-7b-5p inhibitor ameliorates the Fas transcripts level in THP-1 cells. Data represent the mean ± SD from two independent experiments, each reaction was set up in triplicates (**P < 0.01, ***P < 0.001). Western blot analysis THP-1 cells were harvested 24 h post-infection, lysed and total proteins were extracted as described earlier (Bettencourt et al.2013). Equal amount of proteins were separated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred to polyvinylidene difluoride membrane by semi dry method. Membranes were blocked and incubated overnight with primary antibodies: anti-FAS (Cat # sc-7886), anti-β-Actin (Cat # GX-5111M), (Santa Cruze Biotechnology INC. USA). Next day, membranes were washed and incubated with horseradish peroxidase-conjugated secondary antibodies. The blots were developed using the chemiluminescent substrate (Pierce), and the signals were captured on the Chemidoc system (Image Quant LAS 500, GE healthcare). Band intensities were quantified densitometrically using the ImageJ software. Densitometry values were normalized to the levels of β-Actin. Transient transfection of miRNA mimics and inhibitors and growth analysis miRNA mimics and inhibitors were procured from Exiqon (Part no. 470000-001, 479000-001). Fas siRNA (Cat # E-003776-00-0005) and non-specific siRNA (Cat # D-001910-01) were procured from Dhermacon, USA. Total 50 pmol mimics and 150 pmol inhibitors per 3 × 106 THP-1 cells were used for transfection of selected miRNA using Lipofectamine 3000® (Invitrogen, USA) as per manufacturer´s instructions. THP-1 cells were transfected with pmirGLO vector (Promega, USA), mimics, inhibitors, non-specific control miRNA Cel-39-3p, Fas siRNA and non-specific siRNA as and when needed. MTB infection was given to pre-transfected THP-1 cells when required. Cells were lysed after 24 h of infection for RNA and protein analysis. For CFU enumeration, MTB-infected THP-1 cells were lysed, bacilli pelleted, diluted with 7H9 medium and then seeded on 7H11 agar plates for bacterial colony counting after 3 weeks. Flow cytometry and Annexin V/propidium iodide staining THP-1 cells were seeded in 6-wells plate (1 × 106 cells/well) and transfected with hsa-let-7b-5p inhibitor and MTB. H2O2 treatment (500 μM) was given to THP-1 cells to induce apoptosis as described earlier (Xiang et al.2016). After 24 h of treatment, adherent cells were washed with ice-cold phosphate buffer saline and trypsinized (0.05% trypsin) to detach cells. Annexin V/propidium iodide staining was performed as per kit's protocol (Cat # 556419, BD Pharmingen, USA). Cloning of Fas 3΄-UTR and luciferase assay Human Fas gene sequence was retrieved from database (NCBI) and 1348 bp 3΄-UTR was deduced. Fas 3΄-UTR was amplified using 5΄-GTTGTTTAAACTTGGTCTAGAGTGA-3΄ (Fas 3΄UTR-F forward primer) and 5΄-ACTCGAGTAATTACGTACTTTTACC-3΄ (Fas 3΄UTR-R reverse primer) and amplicon was cloned at Pme1-Xba1 restriction sites downstream to firefly luciferase reporter gene into pmirGLO Dual-Luciferase miRNA Target Expression Vector (Promega, USA) to create pmirGLO:Fas3΄UTR (Fig. 2A). pmirGLO:Fas3΄UTRΔSS was created by deleting the seed sequence region of Fas 3΄UTR by Nde1-EcoR1 restriction digestion followed by blunting and ligation. Luciferase assay was performed using Promega's Dual-Glo Luciferase Assay System (Promega, USA) as per kit's protocol. Normalized firefly luciferase activity (firefly luciferase activity/Renilla luciferase activity) for each construct was compared to that of the pmirGLO vector control without insert. For each transfection, luciferase activity was averaged from three replicates. Figure 2. View largeDownload slide (A) Cloning of 1.34-kb Fas3΄UTR at Pme1-Xba1 sites in the pmirGLO vector. Seed sequences are highlighted in green. Predicted pairing of hsa-let-7b-5p seed sequences with its target is highlighted. (B) Confirmation of cloned 3΄UTR with seed sequences by sequencing. (C) Firefly luciferase assay after transfection of THP-1 cells with pmirGLO vector, pmirGLO:Fas3΄UTR, pmirGLO:Fas3΄UTRΔSS (seed sequences deleted in 3΄UTR), hsa-let-7b-5p mimics and inhibitor. Total luciferase activity obtained from THP-1 cells carrying pmirGLO basic vector is considered 100%. Normalized firefly luciferase activity (firefly luciferase activity/Renilla luciferase activity) for each construct was compared to that of the pmirGLO vector control. For each transfection, luciferase activity was averaged from three replicates (*P < 0.05). Figure 2. View largeDownload slide (A) Cloning of 1.34-kb Fas3΄UTR at Pme1-Xba1 sites in the pmirGLO vector. Seed sequences are highlighted in green. Predicted pairing of hsa-let-7b-5p seed sequences with its target is highlighted. (B) Confirmation of cloned 3΄UTR with seed sequences by sequencing. (C) Firefly luciferase assay after transfection of THP-1 cells with pmirGLO vector, pmirGLO:Fas3΄UTR, pmirGLO:Fas3΄UTRΔSS (seed sequences deleted in 3΄UTR), hsa-let-7b-5p mimics and inhibitor. Total luciferase activity obtained from THP-1 cells carrying pmirGLO basic vector is considered 100%. Normalized firefly luciferase activity (firefly luciferase activity/Renilla luciferase activity) for each construct was compared to that of the pmirGLO vector control. For each transfection, luciferase activity was averaged from three replicates (*P < 0.05). Data analysis Data are presented as mean ± standard deviation (SD). Statistical differences and significance were analyzed using one-way ANOVA followed by Newman-Kauls multiple comparison test and Student's t-test, respectively. RESULTS miRNA profiling of THP-1 cells infected with Mycobacterium tuberculosis H37Rv miRNAs profiling of MTB-infected THP-1 cells revealed deregulation of several miRNAs in comparison to uninfected control (GEO accession number GSE94007). Six miRNAs appeared significantly upregulated (≥1.5-fold, P < 0.01): hsa-miR-643, hsa-let-7b-5p, hsa-miR-148b, hsa-miR-639, hsa-miR-671-3p and hsa-miR-193a-3p, while more numbers of miRNAs were found to be downregulated (Table 1). This was duly validated by TaqMan real-time qRT-PCR, and similar trends were observed in their expressions (Fig. 1A). Among the upregulated miRNAs, we focused on hsa-let-7b-5p as this was recently reported to be significantly upregulated in pulmonary TB patients (Xin et al.2016). Table 1. TLDA miRNA profile of THP-1 macrophages after 24 h of MTB infection. Only select miRNAs are included in this table. Details are available at GEO database (accession number GSE94007). miRNAs  Fold changes  Up-regulated miRNAs  hsa-miR-643  4.255  hsa-let-7b-5p  4.134  hsa-miR-148b  2.536  hsa-miR-639  1.982  hsa-miR-671-3p  1.698  hsa-miR-193a-3p  1.599  Down-regulated miRNAs  hsa-miR-720  40.00  hsa-miR-196b  35.71  hsa-miR-151-3p  34.48  hsa-miR-146a  4.56  hsa-miR-155  4.42  hsa-miR-223  4.32  hsa-miR-20b  3.83  hsa-miR-494  3.50  hsa-miR-362  2.74  hsa-miR-454  2.57  hsa-miR-596  2.38  hsa-miR-503  2.29  hsa-miR-27a  2.26  hsa-miR-15a  2.07  miRNAs  Fold changes  Up-regulated miRNAs  hsa-miR-643  4.255  hsa-let-7b-5p  4.134  hsa-miR-148b  2.536  hsa-miR-639  1.982  hsa-miR-671-3p  1.698  hsa-miR-193a-3p  1.599  Down-regulated miRNAs  hsa-miR-720  40.00  hsa-miR-196b  35.71  hsa-miR-151-3p  34.48  hsa-miR-146a  4.56  hsa-miR-155  4.42  hsa-miR-223  4.32  hsa-miR-20b  3.83  hsa-miR-494  3.50  hsa-miR-362  2.74  hsa-miR-454  2.57  hsa-miR-596  2.38  hsa-miR-503  2.29  hsa-miR-27a  2.26  hsa-miR-15a  2.07  View Large hsa-let-7b-5p targets Fas and downregulates its expression upon MTB infection Fas was predicted hsa-let-7b-5p target by DIANA micro-T CDS v5, miRTarBase v7 and Miranda database target prediction tools (Ritchie and Rasko 2014). Since miRNAs regulate the target genes at post-transcriptional level, we speculated that the enhanced expression of hsa-let-7b-5p would lower the Fas protein level in MTB-infected THP-1 cells. Expectedly, in a western blot analysis using total proteins from MTB-infected THP-1 cells, the Fas protein level was found significantly down in comparison to uninfected THP-1 cells (Fig. 1B). Similar decline in Fas level was detected in THP-1 cells transfected with hsa-let-7b-5p mimics, while no changes were noticed when THP-1 cells were transfected with Cel-mir-39-3p, a non-specific miRNA, and hsa-let-7b-5p inhibitor (Fig. 1B). These results suggest that the MTB infection of THP-1 cells leads to enhanced expression of hsa-let-7b-5p, which in turn brings down the level of its target Fas protein. When hsa-let-7b-5p was antagonized with its inhibitor, the Fas level remains unaltered even after MTB infection, as it was noticed after co-transfection of THP-1 cells with MTB and hsa-let-7b-5p inhibitor (Fig. 1B). Interestingly, no decline in Fas level was noticed in non-pathogenic M. smegmatis infected THP-1 cells. The binding site of hsa-let-7b-5p in the Fas 3΄-UTR was detected by TargetScanHuman v 7.1 (www.targetscan.org). To examine the hsa-let-7b-5p and Fas 3΄-UTR interaction, 1.34-kb Fas 3΄-UTR containing complete binding region was cloned to the downstream of a firefly luciferase gene in a pmirGLO miRNA target expression vector (Fig. 2A). When THP-1 cells, transformed with the recombinant pmirGLO:Fas3΄UTR vector, were challenged with hsa-let-7b-5p mimics the luciferase activity was significantly reduced with respect to basal level pmirGLO:Fas3΄UTR expression (Fig. 2C), indicating the binding of miRNAs to the cloned miRNA target sequence. Renilla luciferase gene in the vector backbone is used as a control reporter for normalization of gene expression. To ensure that the regulation is indeed mediated through the predicted binding site, another vector construct was created in which the seed sequences were deleted (pmirGLO:Fas3΄UTRΔSS). It was reasoned that devoid of seed sequences Fas3΄UTR will desist hsa-let-7b-5p mimics. Expectedly, co-transfection of THP-1 cells with pmirGLO:Fas3΄UTRΔSS and hsa-let-7b-5p mimics did not alter the luciferase activity; rather, restored the basal level luciferase activity rendered by pmirGLO:Fas3΄UTR (Fig. 2C). These results clearly established that the hsa-let-7b-5p curtails the expression of Fas protein through interaction with its 3΄UTR in MTB-infected THP-1 cells. In order to assess whether hsa-let-7b-5p downregulates Fas by degradation of transcript or by translational inhibition, the Fas transcript level was examined by qRT-PCR in THP-1 cells, infected with MTB, let-7b-5p mimics and its inhibitor. An apparent decline in Fas transcripts was noticed in samples treated with MTB and let-7b-5p mimics with respect to untreated control, while treatment with let-7b-5p inhibitor enhanced the level of Fas transcripts significantly (Fig. 1C), suggesting that the let-7b-5p regulates Fas protein level by degrading the Fas transcripts instead of translational inhibition. hsa-let-7b-5p ameliorates the intracellular survival of MTB in THP-1 cells Macrophages tend to undergo apoptosis after mycobcterial infection as the induction of apoptosis induces direct killing of intracellular mycobacteria. Fas has been shown to play a central role in the regulation of apoptosis in mycobacteria (Oddo et al.1998). Since Fas level was brought down by enhanced expression of hsa-let-7b-5p in MTB-infected THP-1 cells, we sought to examine the apoptotic populations of THP-1 cells after inhibiting the hsa-let-7b-5p miRNA. Treatment of THP-1 cells with hsa-let-7b-5p inhibitor enhanced the apoptotic cells population markedly (20%) in comparison to uninfected control (4.9%) and MTB-infected THP-1 cells (9.0%) (Fig. 3A). When THP-1 cells treated with hsa-let-7b-5p inhibitor were infected with MTB the apoptotic population of THP-1 cells were reduced to 7.1% (Fig. 3A). This is likely as the ensuing upregulation of hsa-let-7b-5p after MTB infection of THP-1 cells would have antagonized the hsa-let-7b-5p inhibitor to a larger extent, and the prevailing level of hsa-let-7b-5p miRNA had brought the Fas to its basal level. This was duly demonstrated in the western blot analysis (Fig. 1B). Further, we examined the survival of MTB in THP-1 cells treated with hsa-let-7b-5p inhibitor in comparison to untreated THP-1 cells. Twenty-four hour post infection THP-1 cells were lysed, MTB bacilli were recovered and allowed to grow on 7H11 agar plates. A significant decline in bacterial load was obtained in bacilli recovered from MTB-infected THP-1 cells treated with hsa-let-7b-5p inhibitor vis-à-vis untreated ones (Fig. 3B). Similar results were obtained when THP-1 cells treated with Fas siRNA were infected with MTB (Fig. 3B). These results established that the hsa-let-7b-5p inhibitor reduces the MTB survival in THP-1 macrophage cells. Figure 3. View largeDownload slide (A) Apoptosis of THP-1 cells, stained with annexin V-FITC/PI, analyzed by FACS. Lower left quadrant, viable cells (annexin V-FITC and PI negative); lower right quadrant, early apoptotic cells (annexin V-FITC positive and PI negative); upper right quadrant, cells that have already died (annexin V-FITC and PI positive). Similar trends were observed in two separate experiments. (B) Survival of MTB in THP-1 macrophages treated with hsa-let-7b-5p inhibitor vs untreated. The THP-1 cells were first transfected with hsa-let-7b inhibitor, Fas siRNA and non-specific siRNA, then infected with MTB for 24 h and intracellular MTB survival was evaluated by CFU analysis. Data were obtained from two separate culture and each experiment was set up in triplicates. Significance was calculated by Student's t-test (**P < 0.01). Figure 3. View largeDownload slide (A) Apoptosis of THP-1 cells, stained with annexin V-FITC/PI, analyzed by FACS. Lower left quadrant, viable cells (annexin V-FITC and PI negative); lower right quadrant, early apoptotic cells (annexin V-FITC positive and PI negative); upper right quadrant, cells that have already died (annexin V-FITC and PI positive). Similar trends were observed in two separate experiments. (B) Survival of MTB in THP-1 macrophages treated with hsa-let-7b-5p inhibitor vs untreated. The THP-1 cells were first transfected with hsa-let-7b inhibitor, Fas siRNA and non-specific siRNA, then infected with MTB for 24 h and intracellular MTB survival was evaluated by CFU analysis. Data were obtained from two separate culture and each experiment was set up in triplicates. Significance was calculated by Student's t-test (**P < 0.01). DISCUSSION In this study, we performed miRNAs profiling in MTB-infected THP-1 cells using Invitrogen's TLDA followed by TaqMan real time qRT-PCR validation. Based on literature and an earlier report, we picked up one of the upregulated miRNAs, hsa-let-7b-5p, as this was found to be significantly upregulated in circulating miRNAs populations of TB patients (Xin et al.2016). hsa-let-7b-5p belongs to let-7 family of miRNAs which is widely conserved in mammals. Fas was identified as hsa-let-7b-5p target by more than one target predicting tools on the basis of eight nucleotides seed sequences in its 3΄UTR (Fig. 2A). It was likely as both Fas (Oddo et al.1998) and hsa-let-7b (Ham et al.2015) were reported to have roles in apoptosis. Fas-mediated apoptosis of macrophages was already reported in case of virulent MTB infection (Oddo et al.1998). Guided by these reports, we investigated the role of hsa-let-7b-5p in the regulation of Fas-mediated apoptosis in THP-1 cells. Western blot analysis and dual luciferase assay carrying pmirGLO:Fas3΄UTR vector clearly established that Fas is the direct target of hsa-let-7b-5p miRNA and its level is negatively regulated by hsa-let-7b-5p. When macrophage cells are infected with MTB and M. smegmatis, the later gets cleared within 24 h as it induces strong apoptosis (Bohsali et al.2010), while MTB tends to survive longer as it evades apoptosis (Keane, Remold and Kornfeld 2000). Fas-induced apoptosis of infected human macrophages has been shown to reduce the survival of intracellular MTB (Oddo et al.1998). Considering these facts, it is inferred that after MTB infection THP-1 cells induce hsa-let-7b-5p which lowers the Fas level in infected macrophages facilitating the intracellular survival of MTB. Since MS infection fails to alter the Fas basal level, it gets eliminated within 24 h of infection. True to this assumption, our western data showed unaltered Fas level in MS-infected THP-1 cells, while lower level of Fas protein was detected in MTB-infected THP-1 cells (Fig. 1B). The capacity of infected macrophages to undergo apoptosis is an efficient mechanism for innate immune response against mycobacteria (Briken and Jessica 2008). Apoptosis is a mode of clearance of mycobacteria from infected macrophages by direct killing (Molloy, Laochumroonvorapong and Kaplan 1994; Keane et al.1997). Mycobacteria contained in apoptotic bodies were taken up via phagocytosis by uninfected bystander and neighboring macrophages, thereby killing the bacteria more efficiently (Fratazzi et al.1997). Alveolar macrophages infected with virulent mycobacterial strains induce little apoptosis above threshold level (Balcewicz-Sablinska et al.1998; Keane, Remold and Kornfeld 2000). This helps virulent mycobacteria to survive for longer duration inside macrophages. The treatment of THP-1 cells with hsa-let-7b-5p inhibitor neutralized the intrinsic hsa-let-7b-5p population inflicting enhanced apoptosis of infected macrophages, but when THP-1 cells were co-transfected with hsa-let-7b-5p inhibitor and MTB, the ensuing overexpression of hsa-let-7b-5p antagonized the inhibitor effect and the basal level apoptosis of MTB-infected THP-1 cells was restored (Fig. 3A). This led to substantially reduced bacterial burden in THP-1 cells co-transfected with MTB and hsa-let-7b-5p inhibitor. The apoptosis of infected macrophages is associated with a reduction in viability of intracellular MTB is clearly evident in our apoptosis and CFU analysis (Fig. 3). Although the role of hsa-let-7b in suppression of apoptosis has been reported earlier through different targets like caspase-3 (Ham et al.2015), Ras and NF (Johnson et al.2005; Meng et al.2007), first time, in this study, we have shown that Fas is a direct target of hsa-let-7b and hsa-let-7b downregulates Fas level in MTB-infected THP-1 macrophages. In summary, we report the enhanced expression of hsa-let-7b-5p miRNA in THP-1 cells after infection with MTB. The upregulated hsa-let-7b-5p miRNA targets Fas and through interaction with its 3΄UTR downregulates its level in infected macrophages, which impedes the apoptosis of infected macrophages ameliorating the survival of intracellular MTB. Thus, we have been able to impart significance to enhanced expression of hsa-let-7b-5p in MTB-infected THP-1 macrophages. Finally, the reduced bacterial burden in THP-1 cells co-transfected with MTB and hsa-let-7b-5p inhibitor suggests that hsa-let-7b-5p could be a potential therapeutic target for TB. Acknowledgements AT and VS acknowledge their research fellowships from UGC and ICMR, respectively. Technical support from CDRI SAIF division in FACS experiments is duly acknowledged. This is manuscript No. 9634 of CDRI. Conflict of interest. None declared. REFERENCES Abdalla AE, Duan X, Deng W et al.   MicroRNAs play big roles in modulating macrophages response toward mycobacteria infection. Infect Genet Evol  2016; 45: 378– 82. Google Scholar CrossRef Search ADS PubMed  Balcewicz-Sablinska MK, Keane J, Kornfeld H et al.   Pathogenic Mycobacterium tuberculosis evades apoptosis of host macrophages by release of TNF-R2, resulting in inactivation of TNF-α. J Immun  1998; 161: 2636– 41. 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Tuberculosis  2016; 99: 63– 9. Google Scholar CrossRef Search ADS PubMed  Yi Z, Fu Y, Ji R et al.   Altered microRNA signatures in sputum of patients with active pulmonary tuberculosis. PLoS One  2012; 7: e43184. Google Scholar CrossRef Search ADS PubMed  © FEMS 2018. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png FEMS Microbiology Letters Oxford University Press

hsa-let-7b-5p facilitates Mycobacterium tuberculosis survival in THP-1 human macrophages by Fas downregulation

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Abstract

Abstract Tuberculosis continues to be one of the deadliest infectious diseases worldwide. MicroRNAs (miRNAs) are small non-coding entities that play critical role as post-transcriptional regulators and are transcriptionally deregulated upon mycobacterial infection. In this study, we found significant upregulation of hsa-let-7b-5p in Mycobacterium tuberculosis (MTB) infected THP-1 human macrophages. Concomitantly, we detected the reduced level of Fas protein, one of the targets of hsa-let-7b-5p, in MTB-infected THP-1 macrophages. Using luciferase assay, a direct interaction between hsa-let-7b-5p and the Fas 3΄-untranslated region (3΄-UTR) was established. Inhibition of hsa-let-7b-5p augmented the apoptosis of THP-1 cells enabling enhanced clearance of MTB. Our findings suggest that hsa-let-7b-5p helps intracellular survival of MTB in THP-1 cells by downregulating Fas protein level. This highlights hsa-let-7b-5p as a potential therapeutic target for tuberculosis treatment. M. tuberculosis, hsa-let-7b-5p, Fas, Apoptosis, THP-1 cells INTRODUCTION Tuberculosis (TB) is caused by Mycobacterium tuberculosis (MTB) which has co-evolved with host for several thousand years. This has led the bug better adapted for human host wherein it can survive for longer period of time asymptomatically in dormant stage. Infection of mammalian host usually occurs by the aerosol route and the lung is primarily the first affected organ. The resident macrophages of lung interact with bacilli and internalize them in the phagosome through pathogen-specific receptors. The MTB tends to survive and replicate inside macrophages instead of being eliminated bypassing the host innate defense system otherwise meant to clear pathogens. MTB interferes with host trafficking system by modulating events in endosomal/phagosomal maturation and creates a protected niche in the mycobacterial phagosome (Houben, Nguyen and Pieters 2006). MicroRNAs (miRNAs) are widely conserved small (18–22 nucleotides) non-coding RNAs that function as post-transcriptional regulators. They interact with their target coding mRNAs and inhibit translation by either degradation of the mRNAs or blocking translation without degrading the targets (Du and Zamore 2005). Conservation of individual miRNAs across different species highlights their functional relevance. Notably, miRNAs with altered expressions are involved in a broad spectrum of human diseases (Kanwar, Mahidhara and Rupinder 2009; Pauley, Seunghee and Chan 2009). A host of evidences have established that miRNAs are regulatory players in cell-mediated immune processes, thereby, qualifying these regulators as biomarkers candidates in immune disorders (Rodriguez et al.2007; Mittelbrunn et al.2011). Recently, miRNAs have been shown to play important roles in successful microbial infections (Yi et al.2012; Staedel and Darfeuille 2013; Abdalla et al.2016). Their significant roles in the control of gene expression have been described in different biological processes including development of the immune system and orchestration of anti-pathogenic responses (Taganov, Boldin and Baltimore 2007; Nathans et al.2009). However, a lot is still to be known about the role of miRNAs in intracellular survival of pathogens, immune regulation and pathogenesis of MTB in host (Ottenhoff 2012). This study was aimed to identify and characterize miRNAs facilitating the intracellular survival of MTB in macrophages. Using TaqMan Low Density Array (TLDA) of human miRNAs (Invitrogen, USA), we analyzed the expression of miRNAs in MTB-infected THP-1 macrophages. Of several miRNAs that appeared significantly upregulated in array analysis followed by TaqMan real time qRT-PCR validation, hsa-let-7b-5p was examined in details. Its targets were predicted in silico and the binding of hsa-let-7b-5p to Fas, one of the predicted targets, was validated using luciferase reporter assay and immunoblotting. Based on our data, we suggest that hsa-let-7b-5p helps in intracellular survival of MTB by downregulation of Fas receptor. MATERIALS AND METHODS Bacterial strains and culture MTB H37Rv and M. smegmatis mc2155 strains were obtained from departmental stock (Division of Microbiology, CSIR-CDRI, Lucknow, India) and cultured in Middlebrook 7H9 liquid medium supplemented with oleic acid albumin dextrose catalase (OADC) enrichment (Difco, USA). For growth on solid surface, cultures were spread on Middlebrook 7H11 plates supplemented with 10% OADC, 0.5% glycerol and 0.05% Tween-80 and incubated at 37°C. Escherichia coli strains were cultured in Luria Bertani (LB) broth at 37°C and spread on LB agar plates. Cell culture and infection Human THP-1 cells were maintained at 37°C and 5% CO2 in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS). To induce cell adherence and differentiation, 1 × 106 THP-1 cells were seeded per well in a 6-wells plate and treated with 30 ng/ml PMA for 48 h in culture medium. Mid log phase (0.7 OD600) bacterial cultures, carefully dislodged to single cell suspension, were used for infection. The mycobacteria and THP-1 cells were seeded at 10:1 multiplicity of infection, incubated for 4 h, washed three times, 5 min each, and incubated in complete RPMI-1640 media with amikacin (200 μg/ml) for 1 h to kill extracellular mycobacteria. After the last wash cells were harvested for 0 h time point, fresh medium was added and cells were incubated for 24–48 h at 37°C with 5% CO2. Viability of THP-1 cells was assessed by trypan blue dye exclusion assay. miRNAs profiling of MTB-infected THP-1 macrophages using TLDA and validation by TaqMan qRT-PCR Total RNA were isolated from uninfected and 24 h post-MTB-infected THP-1 cells using MirVana miRNA isolation kit (Cat #AM1560, Ambion, USA). RNA was assessed by NanoDrop spectrophotometer and Agilent RNA 6000 Nano Kit on Agilent 2100 Bioanalyser (Thermo Scientific, USA). One microgram total RNA per sample was reverse transcribed using Megaplex Human Pool A and Pool B stem loop RT primers (version 3.0) and TaqMan MicroRNA RT kit (Invitrogen, USA) according to the manufacturer's protocol. Expression analysis was performed on QuantStudio 12K Flex Real-Time PCR System (Applied Biosystems) using cDNAs on TLDA A and B cards (version 3.0) with validated TaqMan® miRNA probes. The TLDA cards measure miRNA expression based on TaqMan-enabled real-time analysis, and fold change in miRNAs expression of treated versus untreated samples were calculated (ΔΔCt method) using Expression Suite Software (version 1.0.3, Applied Biosystems). Data were normalized to expression of U6 small nuclear RNA. Only targets having threshold cycle (Ct) ≤ 35 were included in this study. Details of this experiment may be obtained from data submitted to NCBI GEO repository (accession number GSE94007). For TaqMan qRT-PCR, 10 ng total RNA per sample was reverse transcribed using TaqMan gene-specific stem loop primers provided with individual miRNA assay kit (Invitrogen, USA). Reactions were set up as per kit's protocol and run in StepOnePlus real-time PCR system (Thermo Fisher Scientific, USA). U6 snRNA was used as an internal control, and fold change in gene expression between treated and untreated samples were calculated using the comparative ΔΔCt method. Quantitative real-time qRT-PCR Real-time qRT-PCR was performed using total RNAs from THP-1 cells treated with MTB, hsa-let-7b mimics and hsa-let-7b inhibitors. Total RNA was isolated using RNeasy Mini Kit (Qiagen, India) and DNase treated using RNase free Turbo DNase (Ambion, USA). For each sample, 1 μg of DNA-free RNA was reverse transcribed using Thermo Scientific RevertAid cDNA Synthesis Kit (Cat # K1621) by oligo-dT18 primer as per kit's protocol. The qRT-PCR was performed with Fas gene-specific primers (Forward: 5΄-TCCTCCAGGTGAAAGGAAAGCT-3΄ and Reverse: 5΄-TCTGCACTTGGTATTCTGGGTC-3΄) using Power SYBR® Green PCR Master Mix (Thermo Fisher Scientific, Cat # 4367659) on StepOnePlus PCR system. A negative control was parallely run using RNA sample not reverse transcribed. Expression level was normalized by GAPDH (Forward: 5΄-ACCACAGTCCATGCCATCACTG-3΄ and Reverse: 5΄-TGCTTCACCACCTTCTTGATGT-3΄), and fold change in gene expression between treated and untreated samples were calculated using the comparative ΔΔCt method. Experiments were repeated twice using independent cultures, and each reaction was set up in triplicates (Fig. 1C). Figure 1. View largeDownload slide (A) Validations of differentially regulated miRNAs by TaqMan Real-time RT-PCR. Relative expression was analyzed using RNA samples from MTB infected and uninfected THP-1 cells. The expressions were normalized to U6 snRNA. Data were obtained from two biological replicates and each reaction was set up in triplicates. Values plotted are mean ± SD. (B) Western blot analysis of FAS protein in THP-1 cell transfected with M. smegmatis (MS) and M. tuberculosis (MTB), hsa-let-7b-5p mimics, hsa-let-7b-5p inhibitor and non-specific miRNA Cel-miR-39-3p. Experiments were repeated with two biological replicates. Densitometry values were normalized to the levels of β-Actin. Data are mean ± SD (**P < 0.01). (C) Downregulation of Fas expression in THP-1 cells upon MTB infection and after transfection with let-7b-5p mimic. Transfection with let-7b-5p inhibitor ameliorates the Fas transcripts level in THP-1 cells. Data represent the mean ± SD from two independent experiments, each reaction was set up in triplicates (**P < 0.01, ***P < 0.001). Figure 1. View largeDownload slide (A) Validations of differentially regulated miRNAs by TaqMan Real-time RT-PCR. Relative expression was analyzed using RNA samples from MTB infected and uninfected THP-1 cells. The expressions were normalized to U6 snRNA. Data were obtained from two biological replicates and each reaction was set up in triplicates. Values plotted are mean ± SD. (B) Western blot analysis of FAS protein in THP-1 cell transfected with M. smegmatis (MS) and M. tuberculosis (MTB), hsa-let-7b-5p mimics, hsa-let-7b-5p inhibitor and non-specific miRNA Cel-miR-39-3p. Experiments were repeated with two biological replicates. Densitometry values were normalized to the levels of β-Actin. Data are mean ± SD (**P < 0.01). (C) Downregulation of Fas expression in THP-1 cells upon MTB infection and after transfection with let-7b-5p mimic. Transfection with let-7b-5p inhibitor ameliorates the Fas transcripts level in THP-1 cells. Data represent the mean ± SD from two independent experiments, each reaction was set up in triplicates (**P < 0.01, ***P < 0.001). Western blot analysis THP-1 cells were harvested 24 h post-infection, lysed and total proteins were extracted as described earlier (Bettencourt et al.2013). Equal amount of proteins were separated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred to polyvinylidene difluoride membrane by semi dry method. Membranes were blocked and incubated overnight with primary antibodies: anti-FAS (Cat # sc-7886), anti-β-Actin (Cat # GX-5111M), (Santa Cruze Biotechnology INC. USA). Next day, membranes were washed and incubated with horseradish peroxidase-conjugated secondary antibodies. The blots were developed using the chemiluminescent substrate (Pierce), and the signals were captured on the Chemidoc system (Image Quant LAS 500, GE healthcare). Band intensities were quantified densitometrically using the ImageJ software. Densitometry values were normalized to the levels of β-Actin. Transient transfection of miRNA mimics and inhibitors and growth analysis miRNA mimics and inhibitors were procured from Exiqon (Part no. 470000-001, 479000-001). Fas siRNA (Cat # E-003776-00-0005) and non-specific siRNA (Cat # D-001910-01) were procured from Dhermacon, USA. Total 50 pmol mimics and 150 pmol inhibitors per 3 × 106 THP-1 cells were used for transfection of selected miRNA using Lipofectamine 3000® (Invitrogen, USA) as per manufacturer´s instructions. THP-1 cells were transfected with pmirGLO vector (Promega, USA), mimics, inhibitors, non-specific control miRNA Cel-39-3p, Fas siRNA and non-specific siRNA as and when needed. MTB infection was given to pre-transfected THP-1 cells when required. Cells were lysed after 24 h of infection for RNA and protein analysis. For CFU enumeration, MTB-infected THP-1 cells were lysed, bacilli pelleted, diluted with 7H9 medium and then seeded on 7H11 agar plates for bacterial colony counting after 3 weeks. Flow cytometry and Annexin V/propidium iodide staining THP-1 cells were seeded in 6-wells plate (1 × 106 cells/well) and transfected with hsa-let-7b-5p inhibitor and MTB. H2O2 treatment (500 μM) was given to THP-1 cells to induce apoptosis as described earlier (Xiang et al.2016). After 24 h of treatment, adherent cells were washed with ice-cold phosphate buffer saline and trypsinized (0.05% trypsin) to detach cells. Annexin V/propidium iodide staining was performed as per kit's protocol (Cat # 556419, BD Pharmingen, USA). Cloning of Fas 3΄-UTR and luciferase assay Human Fas gene sequence was retrieved from database (NCBI) and 1348 bp 3΄-UTR was deduced. Fas 3΄-UTR was amplified using 5΄-GTTGTTTAAACTTGGTCTAGAGTGA-3΄ (Fas 3΄UTR-F forward primer) and 5΄-ACTCGAGTAATTACGTACTTTTACC-3΄ (Fas 3΄UTR-R reverse primer) and amplicon was cloned at Pme1-Xba1 restriction sites downstream to firefly luciferase reporter gene into pmirGLO Dual-Luciferase miRNA Target Expression Vector (Promega, USA) to create pmirGLO:Fas3΄UTR (Fig. 2A). pmirGLO:Fas3΄UTRΔSS was created by deleting the seed sequence region of Fas 3΄UTR by Nde1-EcoR1 restriction digestion followed by blunting and ligation. Luciferase assay was performed using Promega's Dual-Glo Luciferase Assay System (Promega, USA) as per kit's protocol. Normalized firefly luciferase activity (firefly luciferase activity/Renilla luciferase activity) for each construct was compared to that of the pmirGLO vector control without insert. For each transfection, luciferase activity was averaged from three replicates. Figure 2. View largeDownload slide (A) Cloning of 1.34-kb Fas3΄UTR at Pme1-Xba1 sites in the pmirGLO vector. Seed sequences are highlighted in green. Predicted pairing of hsa-let-7b-5p seed sequences with its target is highlighted. (B) Confirmation of cloned 3΄UTR with seed sequences by sequencing. (C) Firefly luciferase assay after transfection of THP-1 cells with pmirGLO vector, pmirGLO:Fas3΄UTR, pmirGLO:Fas3΄UTRΔSS (seed sequences deleted in 3΄UTR), hsa-let-7b-5p mimics and inhibitor. Total luciferase activity obtained from THP-1 cells carrying pmirGLO basic vector is considered 100%. Normalized firefly luciferase activity (firefly luciferase activity/Renilla luciferase activity) for each construct was compared to that of the pmirGLO vector control. For each transfection, luciferase activity was averaged from three replicates (*P < 0.05). Figure 2. View largeDownload slide (A) Cloning of 1.34-kb Fas3΄UTR at Pme1-Xba1 sites in the pmirGLO vector. Seed sequences are highlighted in green. Predicted pairing of hsa-let-7b-5p seed sequences with its target is highlighted. (B) Confirmation of cloned 3΄UTR with seed sequences by sequencing. (C) Firefly luciferase assay after transfection of THP-1 cells with pmirGLO vector, pmirGLO:Fas3΄UTR, pmirGLO:Fas3΄UTRΔSS (seed sequences deleted in 3΄UTR), hsa-let-7b-5p mimics and inhibitor. Total luciferase activity obtained from THP-1 cells carrying pmirGLO basic vector is considered 100%. Normalized firefly luciferase activity (firefly luciferase activity/Renilla luciferase activity) for each construct was compared to that of the pmirGLO vector control. For each transfection, luciferase activity was averaged from three replicates (*P < 0.05). Data analysis Data are presented as mean ± standard deviation (SD). Statistical differences and significance were analyzed using one-way ANOVA followed by Newman-Kauls multiple comparison test and Student's t-test, respectively. RESULTS miRNA profiling of THP-1 cells infected with Mycobacterium tuberculosis H37Rv miRNAs profiling of MTB-infected THP-1 cells revealed deregulation of several miRNAs in comparison to uninfected control (GEO accession number GSE94007). Six miRNAs appeared significantly upregulated (≥1.5-fold, P < 0.01): hsa-miR-643, hsa-let-7b-5p, hsa-miR-148b, hsa-miR-639, hsa-miR-671-3p and hsa-miR-193a-3p, while more numbers of miRNAs were found to be downregulated (Table 1). This was duly validated by TaqMan real-time qRT-PCR, and similar trends were observed in their expressions (Fig. 1A). Among the upregulated miRNAs, we focused on hsa-let-7b-5p as this was recently reported to be significantly upregulated in pulmonary TB patients (Xin et al.2016). Table 1. TLDA miRNA profile of THP-1 macrophages after 24 h of MTB infection. Only select miRNAs are included in this table. Details are available at GEO database (accession number GSE94007). miRNAs  Fold changes  Up-regulated miRNAs  hsa-miR-643  4.255  hsa-let-7b-5p  4.134  hsa-miR-148b  2.536  hsa-miR-639  1.982  hsa-miR-671-3p  1.698  hsa-miR-193a-3p  1.599  Down-regulated miRNAs  hsa-miR-720  40.00  hsa-miR-196b  35.71  hsa-miR-151-3p  34.48  hsa-miR-146a  4.56  hsa-miR-155  4.42  hsa-miR-223  4.32  hsa-miR-20b  3.83  hsa-miR-494  3.50  hsa-miR-362  2.74  hsa-miR-454  2.57  hsa-miR-596  2.38  hsa-miR-503  2.29  hsa-miR-27a  2.26  hsa-miR-15a  2.07  miRNAs  Fold changes  Up-regulated miRNAs  hsa-miR-643  4.255  hsa-let-7b-5p  4.134  hsa-miR-148b  2.536  hsa-miR-639  1.982  hsa-miR-671-3p  1.698  hsa-miR-193a-3p  1.599  Down-regulated miRNAs  hsa-miR-720  40.00  hsa-miR-196b  35.71  hsa-miR-151-3p  34.48  hsa-miR-146a  4.56  hsa-miR-155  4.42  hsa-miR-223  4.32  hsa-miR-20b  3.83  hsa-miR-494  3.50  hsa-miR-362  2.74  hsa-miR-454  2.57  hsa-miR-596  2.38  hsa-miR-503  2.29  hsa-miR-27a  2.26  hsa-miR-15a  2.07  View Large hsa-let-7b-5p targets Fas and downregulates its expression upon MTB infection Fas was predicted hsa-let-7b-5p target by DIANA micro-T CDS v5, miRTarBase v7 and Miranda database target prediction tools (Ritchie and Rasko 2014). Since miRNAs regulate the target genes at post-transcriptional level, we speculated that the enhanced expression of hsa-let-7b-5p would lower the Fas protein level in MTB-infected THP-1 cells. Expectedly, in a western blot analysis using total proteins from MTB-infected THP-1 cells, the Fas protein level was found significantly down in comparison to uninfected THP-1 cells (Fig. 1B). Similar decline in Fas level was detected in THP-1 cells transfected with hsa-let-7b-5p mimics, while no changes were noticed when THP-1 cells were transfected with Cel-mir-39-3p, a non-specific miRNA, and hsa-let-7b-5p inhibitor (Fig. 1B). These results suggest that the MTB infection of THP-1 cells leads to enhanced expression of hsa-let-7b-5p, which in turn brings down the level of its target Fas protein. When hsa-let-7b-5p was antagonized with its inhibitor, the Fas level remains unaltered even after MTB infection, as it was noticed after co-transfection of THP-1 cells with MTB and hsa-let-7b-5p inhibitor (Fig. 1B). Interestingly, no decline in Fas level was noticed in non-pathogenic M. smegmatis infected THP-1 cells. The binding site of hsa-let-7b-5p in the Fas 3΄-UTR was detected by TargetScanHuman v 7.1 (www.targetscan.org). To examine the hsa-let-7b-5p and Fas 3΄-UTR interaction, 1.34-kb Fas 3΄-UTR containing complete binding region was cloned to the downstream of a firefly luciferase gene in a pmirGLO miRNA target expression vector (Fig. 2A). When THP-1 cells, transformed with the recombinant pmirGLO:Fas3΄UTR vector, were challenged with hsa-let-7b-5p mimics the luciferase activity was significantly reduced with respect to basal level pmirGLO:Fas3΄UTR expression (Fig. 2C), indicating the binding of miRNAs to the cloned miRNA target sequence. Renilla luciferase gene in the vector backbone is used as a control reporter for normalization of gene expression. To ensure that the regulation is indeed mediated through the predicted binding site, another vector construct was created in which the seed sequences were deleted (pmirGLO:Fas3΄UTRΔSS). It was reasoned that devoid of seed sequences Fas3΄UTR will desist hsa-let-7b-5p mimics. Expectedly, co-transfection of THP-1 cells with pmirGLO:Fas3΄UTRΔSS and hsa-let-7b-5p mimics did not alter the luciferase activity; rather, restored the basal level luciferase activity rendered by pmirGLO:Fas3΄UTR (Fig. 2C). These results clearly established that the hsa-let-7b-5p curtails the expression of Fas protein through interaction with its 3΄UTR in MTB-infected THP-1 cells. In order to assess whether hsa-let-7b-5p downregulates Fas by degradation of transcript or by translational inhibition, the Fas transcript level was examined by qRT-PCR in THP-1 cells, infected with MTB, let-7b-5p mimics and its inhibitor. An apparent decline in Fas transcripts was noticed in samples treated with MTB and let-7b-5p mimics with respect to untreated control, while treatment with let-7b-5p inhibitor enhanced the level of Fas transcripts significantly (Fig. 1C), suggesting that the let-7b-5p regulates Fas protein level by degrading the Fas transcripts instead of translational inhibition. hsa-let-7b-5p ameliorates the intracellular survival of MTB in THP-1 cells Macrophages tend to undergo apoptosis after mycobcterial infection as the induction of apoptosis induces direct killing of intracellular mycobacteria. Fas has been shown to play a central role in the regulation of apoptosis in mycobacteria (Oddo et al.1998). Since Fas level was brought down by enhanced expression of hsa-let-7b-5p in MTB-infected THP-1 cells, we sought to examine the apoptotic populations of THP-1 cells after inhibiting the hsa-let-7b-5p miRNA. Treatment of THP-1 cells with hsa-let-7b-5p inhibitor enhanced the apoptotic cells population markedly (20%) in comparison to uninfected control (4.9%) and MTB-infected THP-1 cells (9.0%) (Fig. 3A). When THP-1 cells treated with hsa-let-7b-5p inhibitor were infected with MTB the apoptotic population of THP-1 cells were reduced to 7.1% (Fig. 3A). This is likely as the ensuing upregulation of hsa-let-7b-5p after MTB infection of THP-1 cells would have antagonized the hsa-let-7b-5p inhibitor to a larger extent, and the prevailing level of hsa-let-7b-5p miRNA had brought the Fas to its basal level. This was duly demonstrated in the western blot analysis (Fig. 1B). Further, we examined the survival of MTB in THP-1 cells treated with hsa-let-7b-5p inhibitor in comparison to untreated THP-1 cells. Twenty-four hour post infection THP-1 cells were lysed, MTB bacilli were recovered and allowed to grow on 7H11 agar plates. A significant decline in bacterial load was obtained in bacilli recovered from MTB-infected THP-1 cells treated with hsa-let-7b-5p inhibitor vis-à-vis untreated ones (Fig. 3B). Similar results were obtained when THP-1 cells treated with Fas siRNA were infected with MTB (Fig. 3B). These results established that the hsa-let-7b-5p inhibitor reduces the MTB survival in THP-1 macrophage cells. Figure 3. View largeDownload slide (A) Apoptosis of THP-1 cells, stained with annexin V-FITC/PI, analyzed by FACS. Lower left quadrant, viable cells (annexin V-FITC and PI negative); lower right quadrant, early apoptotic cells (annexin V-FITC positive and PI negative); upper right quadrant, cells that have already died (annexin V-FITC and PI positive). Similar trends were observed in two separate experiments. (B) Survival of MTB in THP-1 macrophages treated with hsa-let-7b-5p inhibitor vs untreated. The THP-1 cells were first transfected with hsa-let-7b inhibitor, Fas siRNA and non-specific siRNA, then infected with MTB for 24 h and intracellular MTB survival was evaluated by CFU analysis. Data were obtained from two separate culture and each experiment was set up in triplicates. Significance was calculated by Student's t-test (**P < 0.01). Figure 3. View largeDownload slide (A) Apoptosis of THP-1 cells, stained with annexin V-FITC/PI, analyzed by FACS. Lower left quadrant, viable cells (annexin V-FITC and PI negative); lower right quadrant, early apoptotic cells (annexin V-FITC positive and PI negative); upper right quadrant, cells that have already died (annexin V-FITC and PI positive). Similar trends were observed in two separate experiments. (B) Survival of MTB in THP-1 macrophages treated with hsa-let-7b-5p inhibitor vs untreated. The THP-1 cells were first transfected with hsa-let-7b inhibitor, Fas siRNA and non-specific siRNA, then infected with MTB for 24 h and intracellular MTB survival was evaluated by CFU analysis. Data were obtained from two separate culture and each experiment was set up in triplicates. Significance was calculated by Student's t-test (**P < 0.01). DISCUSSION In this study, we performed miRNAs profiling in MTB-infected THP-1 cells using Invitrogen's TLDA followed by TaqMan real time qRT-PCR validation. Based on literature and an earlier report, we picked up one of the upregulated miRNAs, hsa-let-7b-5p, as this was found to be significantly upregulated in circulating miRNAs populations of TB patients (Xin et al.2016). hsa-let-7b-5p belongs to let-7 family of miRNAs which is widely conserved in mammals. Fas was identified as hsa-let-7b-5p target by more than one target predicting tools on the basis of eight nucleotides seed sequences in its 3΄UTR (Fig. 2A). It was likely as both Fas (Oddo et al.1998) and hsa-let-7b (Ham et al.2015) were reported to have roles in apoptosis. Fas-mediated apoptosis of macrophages was already reported in case of virulent MTB infection (Oddo et al.1998). Guided by these reports, we investigated the role of hsa-let-7b-5p in the regulation of Fas-mediated apoptosis in THP-1 cells. Western blot analysis and dual luciferase assay carrying pmirGLO:Fas3΄UTR vector clearly established that Fas is the direct target of hsa-let-7b-5p miRNA and its level is negatively regulated by hsa-let-7b-5p. When macrophage cells are infected with MTB and M. smegmatis, the later gets cleared within 24 h as it induces strong apoptosis (Bohsali et al.2010), while MTB tends to survive longer as it evades apoptosis (Keane, Remold and Kornfeld 2000). Fas-induced apoptosis of infected human macrophages has been shown to reduce the survival of intracellular MTB (Oddo et al.1998). Considering these facts, it is inferred that after MTB infection THP-1 cells induce hsa-let-7b-5p which lowers the Fas level in infected macrophages facilitating the intracellular survival of MTB. Since MS infection fails to alter the Fas basal level, it gets eliminated within 24 h of infection. True to this assumption, our western data showed unaltered Fas level in MS-infected THP-1 cells, while lower level of Fas protein was detected in MTB-infected THP-1 cells (Fig. 1B). The capacity of infected macrophages to undergo apoptosis is an efficient mechanism for innate immune response against mycobacteria (Briken and Jessica 2008). Apoptosis is a mode of clearance of mycobacteria from infected macrophages by direct killing (Molloy, Laochumroonvorapong and Kaplan 1994; Keane et al.1997). Mycobacteria contained in apoptotic bodies were taken up via phagocytosis by uninfected bystander and neighboring macrophages, thereby killing the bacteria more efficiently (Fratazzi et al.1997). Alveolar macrophages infected with virulent mycobacterial strains induce little apoptosis above threshold level (Balcewicz-Sablinska et al.1998; Keane, Remold and Kornfeld 2000). This helps virulent mycobacteria to survive for longer duration inside macrophages. The treatment of THP-1 cells with hsa-let-7b-5p inhibitor neutralized the intrinsic hsa-let-7b-5p population inflicting enhanced apoptosis of infected macrophages, but when THP-1 cells were co-transfected with hsa-let-7b-5p inhibitor and MTB, the ensuing overexpression of hsa-let-7b-5p antagonized the inhibitor effect and the basal level apoptosis of MTB-infected THP-1 cells was restored (Fig. 3A). This led to substantially reduced bacterial burden in THP-1 cells co-transfected with MTB and hsa-let-7b-5p inhibitor. The apoptosis of infected macrophages is associated with a reduction in viability of intracellular MTB is clearly evident in our apoptosis and CFU analysis (Fig. 3). Although the role of hsa-let-7b in suppression of apoptosis has been reported earlier through different targets like caspase-3 (Ham et al.2015), Ras and NF (Johnson et al.2005; Meng et al.2007), first time, in this study, we have shown that Fas is a direct target of hsa-let-7b and hsa-let-7b downregulates Fas level in MTB-infected THP-1 macrophages. In summary, we report the enhanced expression of hsa-let-7b-5p miRNA in THP-1 cells after infection with MTB. The upregulated hsa-let-7b-5p miRNA targets Fas and through interaction with its 3΄UTR downregulates its level in infected macrophages, which impedes the apoptosis of infected macrophages ameliorating the survival of intracellular MTB. Thus, we have been able to impart significance to enhanced expression of hsa-let-7b-5p in MTB-infected THP-1 macrophages. 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FEMS Microbiology LettersOxford University Press

Published: Apr 1, 2018

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