Figure 5. Effect of siCHOP on intracellular survival of Mtb H37Rv. (A) Representative images of Raw264.7 cells after transfection. Raw264.7 cells were transfected with siRNA (siControl, or siCHOP) for 5 h and then infected with Mtb H37Rv for 3 h. At 48 h after Mtb H37Rv infection, Western blot analysis was performed using antibodies directed against CHOP, BiP and b-actin. The experiments were repeated three times. (B) Quantification of intracellular survival of Mtb H37Rv in Raw264.7 cells pretreated with siRNA as described above. The cells were collected at 48 h postinfection with Mtb H37Rv and bacteria number was determined by CFU counting. The statistical significance (**P,0.01) of observed differences between siCHOP treated and untreated groups following infection with Mtb H37Rv were verified by two-tailed t-test. Data represent the mean 6 SEM of values obtained in two independent experiments performed in triplicate. doi:10.1371/journal.pone.0028531.g005 increased CHOP expression by Mtb infection was gradually decreased by ROS scavenger, NAC (Fig. 6E). These results suggest that ROS produced by Mtb infection may directly disturb ER function to activate CHOP expression even if NO production was different between live Mtb and heat-killed Mtb infected cells. ER Stress-Mediated Apoptosis in Mtb Infections Discussion Macrophages infected with mycobacteria may undergo apop- tosis to remove intracellular bacilli, whereas virulent mycobacteria prevent macrophage apoptosis to survive in cells [24]. It is important to understand the mechanisms of apoptosis regulation in the pathogenesis of tuberculosis. Recent studies suggest that ER stress sensors are increased in granulomas in response to mycobacterial infection [25]. One of the components of the ER stress-mediated apoptosis pathway is CHOP [13]. However, there is not enough evidence to explain the role of ER stress in macrophages in tuberculosis. Macrophage apoptosis induced by a high MOI-infection with virulent Mtb does not require TNF-a, caspases, or Toll signals [26,27] but culminates in macrophage necrosis. The high MOI could promote extracellular spread of infection and formation of necrotic lesions in tuberculosis, while a low MOI infection leads to classical apoptosis [26,27]. Here, we show that mycobacteria- infected macrophages undergo apoptosis at a low MOI, which is related to caspase activation. Induction of the ER stress-signaling pathway by Mtb infection has been confirmed by subsequent data showing that mRNA and protein levels of BiP and CHOP increase (Fig. 2, 3). BiP and CHOP are used as UPR markers for ER stress under pathological conditions [12]. Our results successfully showed upregulation of BiP and CHOP by Mtb infection (Fig. 3). BiP has been considered as a chaperone molecule that plays a key role in maintaining cell viability against various stressors [12,28]. In contrast, CHOP is involved in ER stress- mediated apoptosis. Together, these data suggest that the earlier induced BiP could be upregulated for cell survival and later CHOP expression might be upregulated for apoptosis by a direct response to Mtb infection and releasing factors from Mtb-infected cells. In the present study, we provided evidence that ER stress was induced by Mtb infection. We analyzed XBP-1 expression in Mtb infected macrophages because XBP-1 plays an important role in the regulation of innate immune response for host defense [29,30]. We have shown that XBP-1 splicing is induced by Mtb infection (Fig. 1). Toll-like receptors (TLRs) play important roles in controlling Mtb intracellular replication and elimination [31]. Additionally, myco- bacterial cell wall components can activate cells in a TLR- dependent manner [32]. The finding that macrophages under ER stress are hyper-responsive to TLR stimulation in an XBP-1- dependent manner supports our data that XBP-1 splicing is induced during Mtb infection [30]. Moreover, some candidate mRNA levels of regulated IRE1-dependent decay (RIDD) pathway (Heparan-a- glucosaminide N-acetyltransferase; HgNat, Biogenesis of lysosome- related organelles complex-1, subunit 1; Blos1, and Scavenger receptor class A, member 3; Scara3) were decreased after Mtb infection (data not shown), indicating that activated IRE1 during Mtb infection might degrade not only XBP-1 mRNA but also target mRNAs of RIDD pathway in response to ER stress. Thus, we suggest that TLR signaling may activate IRE1a and induce XBP-1 splicing during Mtb infection [32]. ER stress responses mediate the transient attenuation of mRNA translation by increasing eIF2a phosphorylation. Translation initiation factor eIF2a plays a key role in the regulation of protein synthesis in the ER [19]. Our results show that phosphorylation of eIF2a is decreased by Mtb infection (Fig. 3) and increased p-eIF2a by salubrinal treatment is correlated with CHOP induction (Fig. 4). Interestingly, increased p-eIF2a by salubrinal seems to reduce the intracellular survival after Mtb infection (Fig. 4). The phosphor- ylation of eIF2a at early time points post-infection was decreased after Mtb infection could be due to the loss of important functions PLoS ONE | www.plosone.org 7 December 2011 | Volume 6 | Issue 12 | e28531