that infection of M with H37Rv induces LXA4 and inhibits COX2 and prostanoid production, whereas H37Ra infection fails to induce LXA4 but stimulates COX2 and prostanoid production, led to the hypothesis that H37Rv-induced LXA4 production may foster M necrosis via inhibition of PGE2 production. Conversely, H37Ra infection may stimulate PGE2- mediated inhibition of LXA4 and prevent M necrosis. By determining the release of the cationic dye DiCO6(3) from mitochondria, a correlate of M necrosis (41), we first inves- tigated whether prostanoids block induction of H37Rv-in- duced necrosis. Indeed, increasing micromolar concentrations of PGE2 significantly decreased cationic dye release from the mitochondria of human M infected with virulent H37Rv (MOI 10) after 24 h (Fig. 3, A–C). These results were dupli- cated in experiments with mouse M (unpublished data). Addition of exogenous PGE2 also inhibited necrosis measured by 7-AAD staining of H37Rv-infected M . Specifically, addition of 1 μM PGE2 to H37Rv-infected M decreased the number of 7-AAD–positive M after 48 h from 29 ± 1 to 17 ± 1% after infection with H37Rv (background 2.2 ± 0.5%; MOI 10:1; P < 0.01; n = 3). Other prostanoids, such as PGD2, PGF2 , PGI2, and TXA2, and leukotriene LTB4, had little or no effect on prevention of necrosis (Fig. 3 C). We next confirmed the role of PGE2 in protection against Mtb infection-induced necrosis using a loss-of-function ap- proach. In this study, we reasoned that Mtb-induced PGE2 pro- duction is a default pathway for the innate immune response, which may be interrupted in H37Rv infection. Thus, infection Figure 2. Prostanoid production of M infected with Mtb. (A) PGE2 production of human M 0–48 h after infection with H37Ra and H37Rv (MOI 10:1). Differences in PGE2 concentrations in supernatants from H37Ra- and H37Rv-infected M are statistically significant (*, P < 0.002; n = 3). (B) PGE2 production by M infected with H37Ra and H37Rv (MOI 2:1–10:1). The differences in PGE2 production induced by H37Ra in com- parison to H37Rv are statistically significant at all MOIs (*, P < 0.01; n = 3). (C) Quantification of PGE2, PGF2 , and PGD2 in 48-h supernatants from human M (5 × 106 /ml) infected with H37Ra and H37Rv (MOI 10:1; *, P < 0.002; n = 4). Data are presented as means ± SE. In all studies, n repre- sents the number of independent experiments. 2794 Figure 3. Effect of exogenous prostanoids produced by Mtb-in- fected M on H37Rv-induced mitochondrial cationic dye release. (A) PGE2 blocks DiOC6(3) release from mitochondria of human M in- fected with H37Rv (MOI 5:1; 48 h). A fluorescence-activated cell sorter diagram is shown. (B) Down-regulation of cationic dye release by increasing concentrations of PGE2 (2–10 μM) is statistically significant at all PGE2 concentrations (P < 0.01; n = 6). (C) Effect of 1 μM of various prostanoids on H37Rv-induced mitochondrial DiOC6(3) release of M . Only PGE2 inhibition of cationic dye release from the mitochondria is statistically significant (*, P < 0.007; n = 3). PGF2 has borderline activity. The concentration of LTB4 is 0.1 μM. (D) H37Ra (MOI 5:1)-induced DiOC6(3) release from mito- chondria of WT and PGES / mouse M infected in the absence and presence of 1 μM PGE2. At 48 h, cationic dye release from the mitochon- dria was measured (*, P < 0.006; n = 5). (E) 1 μM PGE2 does not alter H37Ra- and H37Rv-induced cytochrome c release from the mitochondria (not significant; n = 3). Data are presented as means ± SE. In all studies, n represents the number of independent experiments. LIPID MEDIATORS IN MTB INFECTION | Chen et al.