LXA4 by Mtb is a potential mechanism that prevents PGE2 production and leads to cell necrosis. The prostanoid receptor EP2 is involved in protection of the mitochondrial inner membrane of Mtb-infected M by PGE2 The action of PGE2 on the M is mediated by four distinct G protein–coupled E prostanoid (EP) receptors, referred to as EP1, EP2, EP3, and EP4 (43), and mice deficient in the four PGE2 receptors have been generated (44–47). All four receptors are expressed by human M (Fig. 5 A). The EP1 receptor is functionally connected to Gq, a G protein that increases Ca2+i. The EP2 and EP4 receptors activate G pro- teins that activate adenylate cyclase leading to increased cAMP production. The EP3 receptor inhibits adenylate cy- clase via Gi and reduces cAMP levels. To confirm the cen- tral role of PGE2 and to more precisely determine its action via specific EP receptors in protection of the mitochondrial inner membrane from MPT, we determined the effect of exogenous PGE2 on cationic dye release in WT M and M from homozygote EP1, EP2, EP3, and EP4 / mice infected with H37Ra and H37Rv. M from WT, EP1, EP3, and EP4 / mice responded to increasing exogenous PGE2 concentrations with enhanced inhibition of cationic dye release induced by H37Rv. Cationic dye release from the mitochondria in EP2 / M infected with H37Rv could not be inhibited even by high concentrations of PGE2, indicating that EP2 is the major PGE2 receptor responsible for inhibition of mitochondrial cationic dye release. Identi- cal results were obtained when the cells were infected with H37Ra (Fig. 5 B). Stimulation of EP2 and EP4 receptors triggers activation of adenylate cyclase (48). EP2 is associated with activation of protein kinase A (PKA) (49–51). In contrast, signaling through EP4 mainly stimulates phosphatidylinositol-3 kinase (PI3K)–mediated processes (49, 52). To confirm that the inhibition of mitochondrial cationic dye release is modu- lated by stimulation of EP2, we examined the effect of the specific PKA inhibitor KT5720 (53) on PGE2-dependent inhibition of mitochondrial cationic dye release. We also confirmed lack of involvement of the EP4-stimulated PI3K pathway by testing the effect of the PI3K inhibitor LY294002 on mitochondrial cationic dye release (54). KT5720, at concentrations previously shown to protect cells from apop- tosis (10 7 M [reference 53]), abrogated the protective ef- fect of PGE2 on M (Fig. 5 C). In agreement with the postulated hypothesis that H37Ra is unable to induce ne- crosis in infected M because of induction of PGE2 produc- tion, addition of KT5720 to H37Ra-infected M significantly augmented M necrosis (Fig. 5 C). In contrast, LY294002 had no effect (Fig. 5 D). KT5720 and LY294002 alone had no effect on mitochondrial cationic dye release induced by H37Rv. These results are consistent with a mechanism wherein EP2 activation of PKA, rather than EP4 activation of PI3K, me- diates the protective effect of PGE2 on mitochondria of Mtb- infected M . 2796 Pulmonary Mtb infection in mice is controlled by PGE2 The findings presented in the previous section indicate that infection with virulent Mtb leads to the induction of LXA4 and suppression of PGE2, which results in the necrotic death of infected M . We therefore performed in vitro experi- ments with M from WT and PGES / mice. Fig. 6 A shows that after 4 d of H37Rv infection, the bacterial growth was comparable between WT and PGES / M . However, 7 d after infection the bacterial burden was significantly higher in PGES / M than in WT M . These data sug- gested that PGE2 plays a major regulatory role in controlling bacterial growth in M and predicted that an alteration in the balance between PGE2 and LXA4 might change the in vivo outcome of infection. Indeed, 5-LO / mice are more Figure 5. EP2 mediates PGE2-dependent protection against cat- ionic mitochondrial dye release. Data are presented as means ± SE. (A) EP1, EP2, EP3, and EP4 are constitutively expressed in human M . Their expression is not increased by either H37Ra or H37Rv infection. (B) EP2 / M fail to respond to PGE2 by down-regulating DiCO6(3) re- lease from the mitochondria infected with H37Rv (top) or with H37Ra (bottom), indicating that EP2 mediates the protective function of PGE2. M from EP1, EP3, and EP4 / mice were equally responsive to 1 μM PGE2 (*, statistically significant; P < 0.01; n = 5). (C) The cAMP-dependent PKA inhibitor KT5720 abrogates inhibition of mitochondrial cationic dye release by PGE2 (black columns; P < 0.01; n = 3). Addition of KT5720 to H37Ra-infected (MOI 10:1) M enhanced M necrosis (gray columns; *, P < 0.01; n = 3). (D) The PI3K inhibitor LY294002 does not abrogate inhibi- tion of mitochondrial cationic dye release by PGE2 (not significant; n = 3). In all studies, n represents the number of independent experiments. LIPID MEDIATORS IN MTB INFECTION | Chen et al.