may allow mycobacteria to survive in the macrophage. Although the exact cause of ER stress in tuberculosis is still unclear, a recent paper showed that ER stress chaperones, including CHOP, were found in Mtb-induced granulomas [24]. Torres et al. [42] previously reported macrophages processed heat-killed Mtb more rapidly and efficiently than live Mtb. This finding may also explain why administration of heat-killed Mtb suppressed the ER stress response in macrophages. Our data suggest that living Mtb can induce ER stress-mediated apoptosis, but macrophages stimulated with heat-killed Mtb may overcome the UPR. NO and ROS production is important to control of Mtb [22] and ER stress pathway are induced by NO or ROS [43,44]. We have shown here live Mtb infection induces NO and ROS production in Raw264.7 cells. Interestingly, CHOP induction was decreased by NAC, an ROS scavenger, whereas treatment with NO scavenger had no effect. It can be postulated that produced NO is not enough to cause ER stress mediated apoptosis of macrophages during Mtb infection. Although the mechanisms of ROS activate ER stress pathway remains to be more investigated, these findings suggest that ROS-induced ER stress-mediated apoptosis is involved in the pathogenesis of tuberculosis. We have demonstrated for the first time that live Mtb infection induces ROS productions and activates ER stress-mediated apoptosis. Specifically, regulation of the eIF2a/CHOP pathway plays an important role in intracellular survival of mycobacteria. Taken together, our observations reveal that ER stress pathway is one of the important components of host defense mechanisms against Mtb infection. Therefore, we suggest that the ER stress signaling pathway may be involved in Mtb-induced apoptosis to control intracellular growth of Mtb. Materials and Methods Mtb culture Mycobacterium tuberculosis strain H37Rv (ATCC 27294) was grown in Middlebrook 7H9 liquid medium supplemented with 10% OADC (oleic acid, albumin, dextrose, catalase), 5% glycerol, and 0.05% Tween-80 and resuspended in phosphate-buffered saline (PBS) at a concentration of 16108 CFU/mL. Aliquots were frozen at 270uC until used. Heat-killed Mtb H37Rv was prepared by heating live H37Rv in PBS at 80uC for 30 min. Cell culture and Mtb infection The murine macrophage cell line Raw264.7 cells and human lung adenocarcinoma epithelial cell line A549 (ATCC No. 185- CCL) were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FBS, penicillin (100 IU/mL), and streptomycin (100 mg/mL). The cells (16105) were cultured in 12-well polypropylene tissue culture plates overnight at 37uC, 5% CO2 to allow cell adherence before infection. Bone marrow derived macrophages (BMDMs) were isolated from femurs and tibias of C57BL/6 mice (6–8 weeks old) and then differentiated by growth for 3–5 days in medium containing M-CSF (25 mg/mL; R&D). The cells were infected for 3 h with Mtb H37Rv (ATCC 27294) at a MOI of 1:1,10:1. Then, cells were washed to remove noninfected bacteria and cultured with fresh complete medium without antibiotics and intracellular bacterial counts were determined at various times postinfection on Middlebrook 7H10 agar. Ethics Statement All animal procedures were reviewed and approved by the Institutional Animal Care and Use Committee of Chungnam National University (Permit Number: 2010-2-32). All animal ER Stress-Mediated Apoptosis in Mtb Infections experiments were performed in accordance with Korean Food and Drug Administration (KFDA) guidelines. Reagents Salubrinal, a selective inhibitor of eIF-2a, and tunicamycin (Tm, Calbiochem) were prepared as a concentrated stock solution (10 mg/mL) in dimethyl sulfoxide. N-nitro-L-arginine methyl ester (L-NAME, Sigma) and N-acetyl cysteine (NAC, Sigma) were dissolved in DMEM and diluted to the desired concentration directly in the culture medium. Raw264.7 cells were pretreated with indicated concentrations of salubrinal or NAC for 30 min before Mtb infection. DMSO alone was used as the negative control. RT-PCR analysis Total RNA was prepared from the cultured Raw264.7 cells, reverse transcribed, and cDNA was used to amplify CHOP, BiP, and XBP-1, with b-actin as an internal control. All amplification reactions were performed as previously described [7]. Immunoblotting analysis Immunoblotting was performed as previously described [7]. The primary antibodies were anti-CHOP (Cell Signaling, MA), anti-GRP78/BiP (Cell Signaling), anti-phospho(Ser-51)-eIF2a (Assay Designs), anti-caspase-12 (Cell Signaling), anti-caspase-9 (Cell Signaling), anti-caspase-3 (Cell Signaling), and anti-b-actin (Santa Cruz Biotechnology). The secondary antibodies used in the study are goat anti-rabbit-IgG-HRP (Cell signaling), rabbit anti- mouse-IgG-HRP (Calbiochem). The blots were quantitated with a Gel Doc 2000 gel-documentation system (Bio-Rad). Actin is shown as a control for protein loading. As a positive control, tunicamycin (2.5 mg/mL) treated for 6 h Detection of nitric oxide (NO) NO production was evaluated by nitrite accumulation in the supernatant using the Griess reaction. A portion (100 mL) of each culture supernatants was added to 100 mL of Griess reagent, and the absorbance at 540 nm was measured with a microplate reader. Reactive oxygen species (ROS) The production of ROS was detected at 24 h post infection with Mtb (MOI = 1) by 20 mM dihydroethidium (DHE) staining for 30 min. Briefly, Raw264.7 cells were infected for 3 h with Mtb (MOI = 1). Cells were washed 3 times with Hanks’ balanced salt solution, fixed with 4% paraformaldehyde and analyzed by FACSCanto II system (BD Biosciences, San Jose, CA, USA). Apoptosis analysis Apoptotic cells were assessed by binding of Annexin V-FITC according to the manufacturer’s instructions (BD Pharmingen, San Diego, CA). Binding of Annexin V-FITC and PI was analyzed by BD FACSCanto II flow cytometer (BD Biosciences) with FlowJo 7.6 software (Tree Star Inc). Gene silencing using small interfering RNA Silencing of CHOP was achieved by the small interfering RNA (siRNA) technique. The siRNA (200 nM) for mouse CHOP mRNA target sequences (Bioneer Corporation, South Korea) and negative control siRNAs were purchased from Santa Cruz biotechnology, Inc. (Santa Cruz Biotechnology, Santa Cruz, CA). The siRNA oligonucleotides were transfected with into cultured Raw264.7 cells using Lipofectamine 2000 (Invitrogen, CA, USA) according to the manufacturer’s instructions. After 5 h PLoS ONE | www.plosone.org 9 December 2011 | Volume 6 | Issue 12 | e28531