Cells and culture. Mononuclear cells from leukopacks of healthy donors or from mouse spleens (7) were plated for FACS analysis at 4 × 10 5 cells/ml/ well in 12-well cluster plates (Corning) and for PI staining and transfection with small interfering RNA (siRNA) at 5 × 105 cells/ml/well in 12-well cluster plates. Human M were cultured for 7 d in IMDM with 10% human AB serum (Gemini Bio-Products) and mouse spleen M for 8–10 d in RPMI 1640 (Invitrogen) with 10% fetal bovine serum (Gemini Bio-Products), 1% Hepes, 1% penicillin/streptomycin, and 0.1% -mercaptoethanol. In vitro infection of M . CD11b+ cells were purified from thioglycollate (3%)-elicited peritoneal M harvested from WT and PGES / mice by MACS column purification. The purity of the CD11b+ M was 90%. CD11b+ M (105/well) were allowed to adhere in a 96-well culture plate for 24 h. Adherent cells were infected with virulent H37Rv (MOI 10: 1) for varying time periods. At different time points, cells were washed extensively with PBS and lysed in H20 for 5 min, and mycobacterial growth was evalu- ated by enumeration of the bacilli for 21 d by plating the cell lysates on Mid- dlebrook 7H10 agar plates and incubating at 37°C. Aerosol infection of mice. C57BL/6 and mPGES-1 / mice were in- fected with virulent Mtb (Erdman strain) via the aerosol route using a nose- only exposure unit (Intox Products) and received 100 CFU/mouse (3, 15, 16). After 5 wk, mice were killed by CO2 inhalation and the left lung was asepti- cally removed and individually homogenized in 0.9% NaCl-0.02% Tween 80 with a Mini-Bead Beater 8 (BioSpec Products, Inc.). Viable bacteria were enumerated by plating 10-fold serial dilutions of organ homogenates onto 7H11 agar plates (Remel). Colonies were counted after 3 wk of incubation at 37°C. LC-MS-MS and ELISA. PGE2, PGF2 , PGD2, and LXA4 concentrations in cell supernatants were determined using ELISA kits (Oxford Biomedical Re- search) according to the recommendations of the manufacturer. The TXA2 concentration was determined with the TXB2 express EIA kit (Cayman Chemical). 50 μl of culture media from infected M was added to wells in duplicate and absorbance was determined using a microplate reader. The lipid concentration was determined using standard curves. LXA4 and PGE2 identity were confirmed using LC-MS-MS diagnostic ions and their physical prop- erties. For LC-MS-MS, an LCQ ion trap mass spectrometer (Finnigan Corp.) with an electrospray ionization probe was used (62). Assessment of MPT in M . MPT was assessed in M by measuring re- tention of the cationic dye DiOC6(3) within the mitochondria (41). Cells were loaded with 3 nM DiOC6(3) in IMDM for 20 min at 37°C. After washing, 20 μg digitonin was added per milliliter and the M were incu- bated at 37°C for 20 min and fixed with 4% paraformaldehyde for 20 min at room temperature. Cells were dislodged with a rubber policeman, washed with PBS, and resuspended in PBS with 1% BSA. Flow cytometry was per- formed under FL-1 conditions using a BD FACSort flow cytometer (BD). Western blotting. After incubation with Mtb (MOI 10:1), cells were har- vested and lysed with SDS sample buffer (62.5 mM Tris-HCl, pH 6.8, 2% wt/vol SDS, 10% glycerol, 50 mM DTT, 0.01% wt/vol bromophenol blue). The cell lysates were sonicated for 10 s, centrifuged at 10,000 g for 10 min, and dissolved in 15% SDS by using -actin as a loading control. Mouse anti- COX2 antibody (1:1 dilution), anti– -actin (1:2,000 dilution; Thermo Fisher Scientific) antibody, mouse anti–5-LO antibody (1:250 dilution; BD) and ovine anti–15-LO antibody (1:250 dilution; Cayman Chemical) were used to detect 5-LO and 15-LO. Rabbit anti-EP1, -EP2, -EP3, and -EP4 receptor antibodies (1:200; Cayman Chemical) were used to detect EP1, EP2, EP3, and EP4 receptor protein. RT-PCR. Total RNA was extracted as specified in cells-to-cDNA II kit (Ambion). In brief, 5 × 105 mononuclear cells/ml/well in 12-well cluster plates were dislodged and lysed with 100 μl of ice-cold cell lysis II buffer and then 2 μl DNase I was added. Primer sequences used for COX2 were JEM VOL. 205, November 24, 2008 A RT I C L E TTCAAATGAGATTGTGGGAAAATTGCT and AGATCATCTCTG- CCTGAGTATCTT. Primer sequences for GAPDH were ACCACAG- TCCATGCCATCAC and TCCACCACCCTGTTGCTGTA. PI staining. Adherent mPGES / spleen M adherent were stained with 2.5 μg/ml PI in RPMI containing 10% FBS at 4°C for 10 min and washed with ice-cold PBS and H2O. Dried and mounted coverslips were examined using a fluorescence microscope and photographed with a digital camera (DFC300; Leica). siRNA transfection. The 5-LO siRNA target sequence (AAATGCCA- CAAGGATTTACCC) targeted against NM_000698 and a scrambled con- trol siRNA sequence (GCCCTCTATCGAATAAGACAA) designed with siRNA target finder (Ambion) (63) was used. The siRNAs were synthesized from DNA templates with the Silencer siRNA construction kit (Ambion) according to the manufacturer’s instructions. Cells were cultured in IMDM with 10% human AB serum, and the medium was changed 1 d before trans- fection. All siRNAs were used at a final concentration of 50 nM by diluting with Opti-MEM I reduced serum medium. To oligofectamine (1:200 dilu- tion; Invitrogen), fresh IMDM containing 30% human AB serum (Gemini Bio-Products) was added to bring the serum concentration to 10%. After 72 h of transfection at 37°C, the cells were infected with Mtb. To examine the effect of siRNA transfection, cells were harvested and analyzed using North- ern or Western blotting. Statistics. Results are expressed as mean ± SEM. The data were analyzed by using Excel Statistical Software (Microsoft) using the Student’s t test for normally distributed data with equal variances. CFU were log10 transformed and analyzed using the Mann Whitney nonparametric t test. A p-value <0.05 was considered statistically significant. Online supplemental material. Fig. S1 shows sensitization of human M with 1 nM LXA4, 1 h before infection with H37Ra (MOI 10:1) to decreas- ing concentrations of PGE2. Fig. S2 shows FACS analysis of necrotic cells (7-ADD) in human and murine M cultures infected with MOI 10:1 and 20:1 H37Rv and H37Ra for 72 h. We are grateful to Dr. Beverly Koller (University of North Carolina, Chapel Hill, NC) for providing the EP-1 / , EP-2 / , EP-3 / , EP-4 / , and PGES / mice. We are also grateful to Dr. Yan Lu (LSU center for Neuroscience, New Orleans, LA) for lipidomic analyses of LC-MS-MS results. This work was funded by the National Institutes of Health grants (AI50216 and AI072143) and the Fonds de la Recherche en Santé du Québec Postdoctoral Fellowship (to M. Divangahi). The authors have no conflicting financial interests. Submitted: 9 April 2008 Accepted: 29 September 2008 REFERENCES 1. Corbett, E.L., C.J. Watt, N. Walker, B.G. Williams, M.C. Raviglione, and C. Dye. 2003. The growing burden of tuberculosis: global trends, and interactions with the HIV epidemic. Arch. Intern. Med. 163: 1009–1021. 2. Dannenberg, A.M., and G.A. Rook. 1994. Pathogenesis of pulmonary tuberculosis: an interplay of tissue-damaging and macrophage-activating immune responses – dual mechanisms that control bacillary multiplica- tion. In Tuberculosis. Pathogenesis, Protection and Control. Bloom B R, editor. American Society for Microbiology, Washington DC. 459–484. 3. Leemans, J.C., N.P. Juffermans, S. Florquin, N. van Rooijen, M.J. Verwordeldonk, A. Verbon, S.J.H. van Deventer, T. van der Poll. 2001. 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