of microsomal prostaglandin E synthase (mPGES) / M de- ficient of PGE2 production with H37Ra should mimic the vir- ulent status in H37Rv infection and stimulate necrosis. As anticipated, PGES / M (42) are unable to produce PGE2 after infection with avirulent H37Ra and show a concomitant phenotypic switch to susceptibility to necrosis after infection (Fig. 3 D). These results demonstrate that PGES activity in- hibits Mtb-induced necrosis. Addition of exogenous PGE2 to H37Ra-infected PGES / M restored the avirulent pheno- type with striking inhibition of necrosis and confirms that PGE2 is a central mediator in cell death regulation of Mtb-infected M (Fig. 3 D). To ascertain that induction of human and mouse M necro- sis by Mtb is caused by related mechanisms, we determined the amount of necrosis after infection with the same MOIs of H37Ra or H37Rv. The results show that both human and mouse M have a similar necrosis response after infection (Fig. S2, available at http://www.jem.org/cgi/content/full/jem.20080767/DC1). Defining the existence of distinct mechanisms that regulate necrosis and apoptosis, and having observed a PGE2-dependent modulation in the context of Mtb infection, we next investigated whether PGE2 has a direct role in the regulation of M apopto- sis. In this study, we assessed PGE2-stimulated cytochrome c release from the mitochondrial intermembrane space (19), a correlate of apoptosis. Interestingly, we find that PGE2 does not modulate cytochrome c release in H37Ra or H37Rv-infected M (Fig. 3 E), nor does it cause cytochrome c release in absence of infection (not depicted). Thus, PGE2 plays no role in the in- duction of apoptosis. Necrosis is positively regulated by LXA4 through inhibition of prostanoid synthesis leading to mitochondrial inner membrane perturbation In the mouse model of Mtb infection, LXA4 production cor- relates with reduced resistance against Mtb (38). Our data re- veal a relationship between LXA4 production and M necrosis after virulent Mtb infection in vitro. These observations col- lectively suggest a functional contribution for LXA4 in the in- duction of necrosis. We reasoned that if LXA4 production is a mechanism by which virulent H37Rv promote necrosis in in- fected M , addition of LXA4 to M infected with nonvirulent H37Ra should confer a virulent phenotype with regard to Mtb-induced necrosis. Indeed, LXA4 significantly enhanced cationic dye release from the mitochondria, a correlate of ne- crosis, in H37Ra-infected M (Fig. 4 A, left). Importantly, LXA4 administration in the absence of Mtb infection has no effect on M necrosis (Fig. 4 A, left). Moreover, addition of LXA4 to H37Ra-infected PGES / M , which are unable to synthesize PGE2, does not increase cationic dye release from the mitochondria (Fig. 4 A, right). These results suggest that in Mtb-infected M , LXA4 acts by down-regulating PGE2 syn- thesis to induce necrosis. To investigate whether induction of necrosis by LXA4 is based on an inhibitory effect of LXA4 on PGE2 synthesis, we determined whether LXA4 blocks synthesis of COX2, thus inhibiting generation of PGE2. Indeed, 10 9 M of stable LXA4 JEM VOL. 205, November 24, 2008 A RT I C L E analogue added to H37Ra-infected M dramatically inhib- ited both COX2 protein and PGE2 production induced by H37Ra (Fig. 4 B). Silencing of the 5-LO gene (Fig. 4 C, left) in M infected with the virulent H37Rv inhibited H37Rv- induced LXA4 production (Fig. 4 C, right) and reduced ne- crosis (Fig. 4 D), demonstrating that 5-LO activity is required for LXA4 production by H37Rv-infected M . Finally, in- hibition of LXA4 synthesis in H37Rv-infected M by 5-LO gene silencing led to PGE2 production (Fig. 4 E). These re- sults not only provide additional proof that LXA4 suppresses PGE2 production but demonstrate that the induction of Figure 4. Effect of LXA4 on Mtb-infected M . (A, left) addition of 10 9 M LXA4 to H37Ra-infected human M (MOI 10:1) enhances mito- chondrial cationic dye release (*, statistically significant difference; n = 3; P = 0.04). (A, right) LXA4 by itself is ineffective. Addition of 10 9 and 10 10 M LXA4 to PGES / mouse M infected with H37Ra (MOI 5:1) does not affect mitochondrial cationic dye release (not significant; n = 3). (B, left) Production of COX2 protein by H37Ra (MOI 10:1)-infected M is inhibited by the addition of 10 9 M LXA4. Western analysis of M extracts. The actions of LXA4 were mimicked by its metabolic stable analogue (not depicted). (B, right) PGE2 production by M (*, P < 0.001; n = 3). Black lines indicate that intervening lanes have been spliced out. (C and D) Tar- geted silencing (t) of the 5-LO gene (C, 70% inhibition at t 50) abro- gates production of LXA4 by H37Rv-infected M compared with nontargeted (nt) silencing (D, left, 50 nM siRNA; P = 0.001; n = 3) and reduces mitochondrial cationic dye release after H37Rv infection (D, right; nt, nontargeted; P < 0.04; n = 3). Black lines indicate that intervening lanes have been spliced out. (E) Targeted silencing of the 5-LO gene re- constitutes production of PGE2 after infection of M with H37Rv, indicat- ing that block of PGE2 production is caused by the action of LXA4 (P < 0.003; n = 3). Data are presented as means ± SE. In all studies, n repre- sents the number of independent experiments (*, statistically significant). 2795