ER Stress-Mediated Apoptosis in Mtb Infections Figure 6. The expression of ER stress markers in Raw264.7 cells infected with live or heat-killed Mtb H37Rv. Raw264.7 cells were infected with live or heat killed Mtb H37Rv (MOI = 1 to 10) for 3 h, and then incubated for 48 h in the presence or absence of L-NAME or NAC. (A, D, E) Immunoblot analysis was performed as described in Materials and Methods. Representative data from three independent experiments are shown. (B) Effect of Mtb H37Rv infection on nitric oxide (NO) production was assessed indirectly by Griess reaction. (C) Representative flow cytometry histograms of superoxide at 48 h after Mtb H37Rv infection. Superoxide detection was evaluated by dihydroethidium (DHE) staining using flow cytometry. Data are means 6 SEM of two independent experiments performed in triplicate. CHOP expression analysis after treatment with N-nitro-L- arginine methyl ester (L-NAME), a nitric oxide inhibitor (D) and N-acetyl-L-cysteine (NAC), a superoxide inhibitor (E). The statistical significance (*P,0.05, **P,0.01 and ***P,0.001) of observed differences between inhibitor treated and untreated groups following infection with Mtb H37Rv were verified by two-tailed t-test. doi:10.1371/journal.pone.0028531.g006 that inhibit translation by Mtb in host cells. Similarly, respiratory syncytial virus (RSV) infection attenuates eIF2a phosphorylation to survive in hosts [33] and human papillomavirus E6 protein inhibits eIF2a phosphorylation to prevent PKR-mediated apop- tosis [34]. Thus, it is possible that the p-eIF2a/CHOP pathway may control intracellular survival of Mtb and the ability to regulate the UPR in macrophages. Caspase-12 is located in the ER and is responsible for ER-stress mediated apoptosis [35]. Processed caspase-12 activates caspase-9, followed by activation of caspase-3 [36]. Our study shows that caspase-12, caspase-9, and caspase-3 are activated by Mtb infection (Fig. 3), indicating that Mtb infection affected caspase-12 activation in macrophages. Activated caspase-12 initiates the proteolytic activity of other downstream caspases, including caspase-3. The importance of caspase-12 in immune responses has been reported, including dampening parasite clearance, inhibiting the production of proinflammatory cytokines, and bacterial clearance [37,38,39]. Thus, our results suggest that caspase-12 activation induced by the ER stress plays an important role in Mtb-infected macrophages. CHOP is known as an inducer of apoptosis-favoring genes in response to ER stress. Because CHOP acts to repress Bcl-2 production and causes apoptosis, we hypothesized that through its role in ER stress-mediated apoptotic signaling, CHOP protein may cause macrophages to remove intracellular mycobacteria. Our data support an intimate link between CHOP expression and intracellular survival of Mtb from apoptosis due to ER stress responses (Fig. 5). Regulation of CHOP expression has been accepted as an approach to remove cancer cells through the induction of apoptosis [40,41]. Although our data show that suppressed CHOP expression results in the increased survival of Mtb, it has yet to be determined whether the CHOP protein is critical in regulating mycobacteria in host cells. However, to our knowledge, there is no report that CHOP suppression affects intracellular survival of mycobacteria in macrophages. Previously, we reported that ESAT-6, a protein secreted from Mtb, induced ER stress in human epithelial cells [7]. We hypothesize that immunogenic substances from Mtb or intracel- lular replication of Mtb can induce ER stress responses. In the current study, it is demonstrated that living Mtb induced CHOP expression, but heat-killed Mtb could not. Moreover, heat-killed Mtb induced stronger p-eIF2a than living Mtb, indicating living Mtb suppresses eIF2a phosphorylation. The suppressed p-eIF2a PLoS ONE | www.plosone.org 8 December 2011 | Volume 6 | Issue 12 | e28531