protective effects of PGE2 by inhibiting COX2 expression, thereby causing necrosis in Mtb-infected M . Interestingly, LXA4 on its own does not induce necrosis, indicating that the main function of LXA4 is down-regulation of prostanoid production followed by mitochondrial inner membrane de- stabilization. These results are consistent with studies demon- strating that mycobacterial burden is significantly reduced in 5-LO / mice in comparison to WT mice (38) and suggest that LXA4 negatively regulates antimycobacterial defense re- sponses. It should be noted that the concentrations of LXA4 produced are not sufficient to affect inflammatory events in other experimental systems, a feature which is almost certain to be beneficial to the Mtb pathogen. Consequently, Mtb is able to escape from the necrotic M without adverse effects and is able to infect newly recruited M . The findings in this paper argue that inhibition of PGE2 comprises a mechanism specific for virulent Mtb, a distinctly noninflammatory role for this prostanoid in the regulated re- sponse to this pathogen. Prostanoids, including PGE2, are the final product of the PGH synthases COX1 and 2. PGE2 has been extensively studied and is well known for its role in me- diating cardinal features of inflammation, including pain, vaso- dilatation essential for the control of blood flow, and leukocyte diapedesis leading to edema and fever (34). We now show that the prostanoid PGE2 protects against MPT and necrosis of M infected with virulent Mtb. Our data are consistent with recently published findings in several other experimental sys- tems that document an inhibitory role of PGE2 in cell death. This mechanism is clearly complex and can be mediated in those studies either by the EP2 receptor (57, 58) or the EP4 receptor (59). Although we definitively confirm the role of PGE2 and more precisely define its mechanistic activity by identifying EP2 as the crucial PGE2 receptor, these findings beg for further dissection of this important signaling pathway leading to increased M defense against Mtb. The mechanisms by which PGE2 stimulates induction of apoptosis of Mtb-infected M is not understood. Although PGE2 stimulation of the EP4 receptor triggers activation of PI3K and of Akt (48), blockade of mitochondrial damage by PGE2 critical for prevention of necrosis is mediated through the EP2 receptor. The EP2 subtype couples to G protein s and triggers intracellular cAMP formation. cAMP activates the PKA pathway (45), which inhibits death in several cell types (58). PKA activation was found to block cell death by phosphorylation of the proapoptotic protein Bad (49, 50, 54), causing its inactivation, and also increases the expression of Bcl-2 involving the CREB (cAMP responsive element-bind- ing) protein and inhibition of cell death (59). At present, we do not know which of these mechanisms is involved in the mitochondrial inner membrane stabilization by PGE2 and in the inhibition of LXA4 production. The innate immune system is the first line of defense against invading microbial pathogens and viruses (60). Apop- tosis characterized by intact plasma membranes, formation of apoptotic bodies and DNA fragmentation is an important component of the innate immune defense against Mtb and 2798 is the predominant death modality of the host M infected with attenuated Mtb including H37Ra and secA2 mutants (13). Apoptotic bodies are thought to provide a containment vessel for the bacilli as well as an efficient means for pathogen killing (15). Moreover, apoptotic bodies express specific cell surface receptors (61) that promote uptake and subsequent antigen presentation to DC (14). Virulent Mtb subvert apop- tosis and induce plasma membrane lysis (necrosis), leading to dissipation of the pathogens and spread of the infection. Thus, the fate of the infected M with regard to cell death modality is a critical determinant in an effective host defense response to Mtb infection. Our studies identify a novel mech- anism wherein Mtb stimulates a pathway involved in the local resolution of inflammation to subvert defense mechanisms against Mtb. To our knowledge, this is the first example of a pathway important in the resolution of inflammation that is targeted by a pathogen to counteract the innate immune sys- tem. Although it is tempting to advocate therapeutic targets of the lipoxin pathway for Mtb infection, further studies are required both to understand the mechanisms by which LXA4 production is activated via virulent Mtb and to determine the consequences of blocking the LXA4 production in the course of an infection of M with Mtb for the host. MATERIALS AND METHODS Materials. LY294002, KT5720, and propidium iodide (PI) were obtained from Sigma-Aldrich. DiOC6(3) (3,3 -dihexyloxycarbocyanine iodide), rhoda- mine-2 AM, goat anti–mouse IgG1, and anti–mouse COX1 antibodies were obtained from Invitrogen. PGE2, PGF2 , PGD2, PGI2, LTB4, TXB2, and LXA4, sheep anti–15-LO antibody, and mouse anti-COX2 antibody were ob- tained from Cayman Chemical. 15-epi-16-phenoxy-parafluoro-LXA4-methyl ester was a gift from Bayer-Schering Pharma AG. Anti– -actin mAb was ob- tained from Thermo Fisher Scientific. Mouse anti–5-LO antibody, mouse IgG1, and mouse anti–cytochrome c mAb (7H8.2C12.6H2.B4) were obtained from BD. IMDM, RPMI-1640, Opti-MEM I reduced serum medium, Oligo- fectamine, Hepes, and DTT were obtained from Invitrogen. Rabbit anti– annexin-1 polyclonal antibody, goat anti–rabbit IgG FITC conjugate, and HRP protein A were obtained from Invitrogen. Anti–phosphatidyl-serine mouse mAb (clone1H6) and rabbit IgG were obtained from Millipore. Mice. 6–10-wk-old C57BL/6 mice were obtained from Jackson Immuno- Research Laboratories. mPGES-1 / mice (N5 backcross onto the C57BL/6 background, 45) and EP1 (Ep1 / ; reference 47), EP2 / (48), EP3 / (47), and EP4 / mice (51) (n > 10 on C57BL/6 background, provided by B. Koller, University of North Carolina, Chapel Hill, NC) were bred locally. All procedures were approved by the Dana-Farber Cancer Institute Institu- tional Animal Care and Use Committee. Bacteria. The virulent Mtb strain H37Rv and the attenuated H37Ra (Amer- ican Type Culture Collection), prepared as described previously (8), were used in the in vitro experiments. The strains were grown in Middlebrook 7H9 broth (BD) with BBL Middlebrook OADC Enrichment (BD) and 0.05% Tween 80 (BD) and resuspended in 7H9 broth at 5 × 10 7 CFU/ml. Aggrega- tion was prevented by sonication for 10 s. The bacteria were allowed to settle for 10 min. Bacteria in M were quantified by lysis of the cells with 0.2% SDS in PBS. After neutralization of SDS with 50% FCS, 100 μl of cell lysates of triplicate cultures were serially diluted 10-fold, plated on 7H10 agar plates (Remel), and colonies were counted after 21 d. Alternatively, the pooled cell lysates were inoculated into triplicate Bactec 12B vials. The number of bacte- ria was determined with the Bactec model 460 TB system (BD). LIPID MEDIATORS IN MTB INFECTION | Chen et al.