modifications, or simply increases in secretory protein synthesis. The UPR alters the expression of ER chaperones to enhance the degradation of misfolded proteins. Additionally, the UPR inhibits protein synthesis to decrease the load within the ER [8]. For secretory proteins to fold properly and ensure survival, cells induce ER chaperone proteins to prevent the toxic accumulation of misfolded secretory proteins and ensure proper ER homeostasis [9]. A series of ER chaperones is involved in both the regulation of protein synthesis and the induction of cell death [10,11]. Under prolonged ER stress, the UPR initiates signaling pathways that promote apoptosis. There are three ER-localized protein sensors: IRE1a (inositol-requiring 1a), PERK (double-stranded RNA- dependent protein kinase (PKR)-like ER kinase) and ATF6 (activating transcription factor 6) [12]. One of the components of the ER stress-induced apoptotic pathway is C/EBP homologous protein (CHOP) [13]. Another major UPR target protein is glucose- regulated protein 78 (GRP78/BiP), which plays an important role in protein folding and assembly, and targets misfolded proteins for degradation. A recent study demonstrated that excess or misfolded proteins from Saccharomyces cerevisiae induce ER stress [14]. A central question of the present study is whether ER stress-induced apoptotic pathways play an important role in tuberculosis, because the ER stress pathway may be important in the overall control of cell viability [8,10]. To contest this hypothesis, we measured induction of ER stress chaperones during mycobacterial infection, characterized ER stress-mediated apoptosis, and ascertained the effect of eIF2a/CHOP pathway on the survival of intracellular mycobacteria. Results Mtb infection induces programmed cell death and caspase activation Apoptosis of macrophage plays important role in host defence against mycobacterial infections [15]. To investigate whether Mtb induced cytotoxicity was associated with the apoptotic pathway, flow cytometry was used to distinguish and quantitatively determine the percentage of dead, viable, apoptotic, and necrotic cells after Mtb infection (Fig. 1). The percentage of early apoptotic and late apoptotic cells was increased from 0.4% in unstimulated control cultures to 78.5% after Mtb infection. Since caspases are involved in ER stress-mediated cell death and are also activated by Mtb [16,17], casapase activation was examined in Raw264.7 cells after Mtb infection. Procaspase-9 is known as a substrate of caspase-12 [18], which resides in the ER. Thus, we examined the effects of Mtb stimulation on activation of both caspase-9 and caspase-12. Caspase-9 and caspase-12 were activated in macro- phages after Mtb infection and activated caspase-3 was strongly expressed at 48 h after Mtb infection (Fig. 1C). To further examine whether caspase activation is involved in CHOP expression, we cultured Raw264.7 cells infected with Mtb in the presence or absence of the broad-spectrum caspase inhibitor z- VAD-fmk (administered 1 h prior to infection). We found that z- VAD-fmk block Mtb-induced CHOP activation in macrophages, suggesting that the CHOP induction in macrophage infected with Mtb resulted primarily from capsase activation (Fig. 1D). ER sensor molecules are induced by Mtb infection The transcription factor CHOP is induced by ER stress and mediates ER stress-induced apoptosis [13]. To investigate other indicators of ER stress during Mtb infection, we examined splicing of XBP-1 indicative of IRE1 activation, BiP/GRP78, CHOP expression of both mRNA and protein levels in a time-dependent ER Stress-Mediated Apoptosis in Mtb Infections manner (Fig. 2 and 3). Expression of BiP and CHOP mRNA was gradually increased after Mtb infection and reached a maximum after 3 h. During ER stress, ER membrane-localized IRE1a is activated and the phosphorylated IRE1a catalyzes the splicing of XBP-1 mRNA [12]. The ratio of mXBP-1 splicing was markedly increased at 6 h after Mtb infection in Raw264.7 cells (Fig. 2). At the protein level, CHOP and BiP were increased by Mtb infection. The induction of CHOP was increased from 24 h and peaked at 48 h after Mtb infection (Fig. 3). BiP expression was increased a little bit at 24 h and slightly decreased at 48 h, suggesting that BiP may operate to alleviate the ER stress to prevent apoptosis but prolonged ER stress may promote apoptosis through activation of CHOP. Since eIF2a phosphorylation has been suggested to be cytoprotective during ER stress [19], we examined phosphorylation of eIF2a by blotting the same membrane using an anti-phospho-eIF2a antibody. Interestingly, eIF2a phosphorylation was decreased gradually until 24 h after Mtb infection. Thus, these data indicate that Mtb infection may affect eIF2a phosphorylation in an attempt to survive within macrophages. The eIF2a-CHOP pathway may affect survival of mycobacteria in macrophages ER stress triggers apoptosis mainly through the PERK pathway via its downstream effectors phosphorylated eIF2a (p-eIF2a) and CHOP [9]. In figure 4, we show that p-eIF2a was decreased by Mtb infection. To identify the biological roles of eIF2a during Mtb infection, we used salubrinal, a selective inhibitor of eIF-2a that seems to target the PP1/GADD34 complex [20], for the determination of its effects on CHOP expression. The expression of CHOP and p-eIF2a was increased in response to salubrinal stimulation during Mtb infection.We expected that salubrinal treatment increased p-eIF2a and CHOP expression because salubrinal blocks eIF2adephosphorylation. Interestingly, low dose of salubrinal treatment did not affect the phosphorylation status of eIF2a However, treatment with a high concentration of salubrinal (50 mM) induced phosphorylation of eIF2a (Fig. 4A). Similar expression pattern of CHOP protein was also observed. These data indicated that macrophage cells responded to Mtb infection by increasing p-eIF2a and CHOP levels, likely due to induction of the UPR. The decrease in eIF2a phosphorylation observed could result from resistance of mycobacteria to host translation inhibition. To address the significance of p-eIF2a, we assessed the effects of salubrinal on the intracellular survival of Mtb in macrophages. We focused on the time point 48 h after Mtb infection because maximum CHOP production was observed. Interestingly, intra- cellular survival of Mtb was significantly decreased at a high concentration of salubrinal in Raw264.7 cells (Fig. 4B). These data provide evidence that ER stress-mediated apoptosis affects on intracellular survival of Mtb. The enhanced eIF2a phosphoryla- tion in response to Mtb infection may elicit a proapoptotic response that is counteracted by Mtb under prolonged stress. To further investigate whether eIF2a/CHOP pathway is involved in intracellular survival of mycobacteria, we prepared CHOP siRNA to evaluate intracellular survival of Mtb. Raw264.7 cells were transfected with CHOP siRNA before Mtb infection. As shown in figure 5, transfection of CHOP siRNA resulted in Mtb infection-induced suppression, compared with cells transfected with control siRNA. As expected, intracellular survival of Mtb was increased in response to siCHOP. These data suggest that CHOP expression is important for controlling intracellular mycobacteria. PLoS ONE | www.plosone.org 2 December 2011 | Volume 6 | Issue 12 | e28531