by Mtb. Two groups of eicosanoids are of special interest because of their opposing effects, the prostanoids that are derived by the COX pathway (34) and the lipoxins, a distinct class of LO-derived products which promote termination of inflammation (39). The identification of PGE2 and LXA4 in infected M su- pernatants was established from the diagnostic ions present in their liquid chromatography tandem mass spectrometry (LC- MS-MS) spectra (Fig. 1 A). We extended these studies by per- forming ELISA assays on H37Rv- and H37Ra-infected M . Figure 1. LXA4 and COX2 production of human M infected with H37Rv and H37Ra. (A) LC-MS-MS of endogenous PGE2 (top) and LXA4 (bottom) produced by H37Ra- (MOI 10) and H37Rv- (MOI 10) infected human M , respectively, at 24 h. The spectrum is a representative LC-MS- MS (n = 3). The prominent ions and relative intensity matched with au- thentic PGE2 and LXA4 under these LC-MS-MS conditions. (B) LXA4 production in human M infected with H37Rv and H37Ra (MOI 10:1) at 0–48 h. Differences in LXA4 concentrations in supernatants from H37Ra- and H37Rv-infected M are statistically significant (*, P < 0.002; n = 3). (C) LXA4 accumulation at 48 h in M supernatants infected with H37Ra and H37Rv (MOI 2, 5, and 10:1). The differences in LXA4 production in- duced by H37Ra and H37Rv are statistically significant at all MOIs (P < 0.01; n = 3). (D) COX2 mRNA accumulation in M infected with H37Ra or H37Rv (MOI 10:1; uninfect, uninfected). (E) Production of COX2 protein in M infected with H37Ra and H37Rv (MOI 10:1) at different time points. In all studies, n represents the number of independent experiments and the error bars represent SE. Black lines indicate that intervening lanes have been spliced out. JEM VOL. 205, November 24, 2008 A RT I C L E The production of LXA4 was measured over time in superna- tants from M infected with virulent and avirulent Mtb. Viru- lent H37Rv induce significantly higher levels of LXA4 production (1 nM; Fig. 1 B). Similar results are observed with another vir- ulent strain (Erdmann strain [not depicted]). In contrast, when M are infected with avirulent H37Ra, production of LXA4 reaches 0.4 nM at 4 and 6 h after infection but is then subse- quently down-regulated to 0.2 nM at 12 h. The amount of LXA4 production induced by H37Rv correlates with the mul- tiplicity of infection (MOI; Fig. 1 C). In contrast, the amount of LXA4 production after H37Ra infection was not increased when the MOI was raised. These findings raised the question of whether M infected with virulent Mtb negatively regulate COX2 expression. Infection of M with the virulent H37Rv induced little ac- cumulation of COX2 mRNA and COX2 protein compared with infection with the mutant Mtb strain H37Ra, suggesting that H37Rv infection inhibits COX2 expression (Fig. 1, D and E). These findings suggest that infection with virulent Mtb induces the production of LXA4 in sufficient quantities to in- hibit COX2 production, which represents an important mani- festation of bacterial virulence. The virulent Mtb strain H37Rv inhibits prostanoid production by the host M COX2, the inducible PGH synthase, is required for prostanoid synthesis (40). To study whether inhibition of COX2 expression by H37Rv had any functional consequences, we measured pros- tanoid production by Mtb-infected M . PGE2 levels of <1 nM were detected in supernatants of H37Rv-infected M during the first 48 h of infection. In contrast, levels of PGE2 were pro- duced by M infected with H37Ra, which increased over time and could be correlated with the MOI (Fig. 2, A and B). Infec- tion with H37Ra also significantly induced production of the prostanoids PGF2 (Fig. 2 C) and TXA2 (not depicted). In con- trast, the virulent Mtb strain H37Rv failed to induce significant prostanoid production (Fig. 2, A–C). To determine whether these findings were not confounded by altered bacterial uptake, survival, or replication, we measured bacterial counts. 72 h after infection, the bacterial burden was similar between M infected with H37Rv and H37Ra (unpublished data). Thus, virulent Mtb suppress COX2 expression in infected M , which leads to a global inhibition of prostanoid produc- tion. Although prostanoid synthesis is significantly inhibited by H37Rv, the amount that is produced is likely to be an impor- tant counterbalance to LXA4 because M exposed to 1 nM LXA4 are highly sensitive to PGE2 and respond to 1–2 logs lower PGE2 concentrations than M not exposed to LXA4 with a protective response against necrosis (Fig. S1, available at http://www.jem.org/cgi/content/full/jem.20080767/DC1). PGE2 suppresses mitochondrial inner membrane perturbation (MPT) and necrosis in M infected with virulent Mtb Virulent H37Rv induce rampant necrosis. In contrast, avirulent H37Ra fail to induce M necrosis (7, 8, 12). The observation 2793