After 12 hours, although the increased Ptgs2 expression was maintained, it was lower than that induced by Mtb 97-1200. Associated with COX-2 induction, gene expression of the prostaglandin receptors EP-2 and EP-4 was also higher in alveolar macrophages infected with 97-1200, 6 hours after infection (Figure 3B). These

findings suggest that PLCs-expressing Mycobacterium tuberculosis subverts the eicosanoid synthesis Erismodegib supplier pathway by inhibiting COX-2, EP-2, and EP-4 expression, thereby directly influencing the generation of PGE2 and its related cellular response. Figure 3 Differential mRNA expression buy NSC23766 of COX-2 and PGE 2 /LTB 4 receptors induced by Mtb isolates 97-1200 and 97-1505. mRNA expression of (A) 5-LO, FLAP, and BLT1, and (B) COX-2, EP-2, and EP-4 in alveolar macrophages

infected for 6 and 12 h with Mtb isolates 97-1200 and 97-1505. Dotted lines show the relative expression of uninfected cells (fold change = 1). All samples were normalised by Gapdh endongenous control. ***P < 0.0001; *P < 0.05 (one-way ANOVA). Data are representative of two independent experiments (error bars, s.e.m.). Eicosanoid production is differentially induced by PLC-expressing Mycobacterium tuberculosis during alveolar macrophages PND-1186 cost infection To study whether the modulation of COX-2 and eicosanoid receptor expression by the 97-1505 Mtb has effects on the biosynthesis of these mediators, we quantified PGE2 and LTB4 production by Mtb-infected alveolar macrophages at different time points. Figure 4A shows that 12 h after infection, PGE2 production induced by 97-1505 Mtb was similar to that induced by 97-1200 Ribonucleotide reductase Mtb. However, after 24 h, 97-1505 Mtb-induced PGE2 production decreased drastically and remained lower at 48 h post-infection. Differently, 24 and 48 h after infection, LTB4 production induced by the isolate 97-1505 was higher than that induced by 97-1200 (Figure 4B). Together, our

results support the idea that PLCs-expressing Mtb are involved in decreased PGE2 production and lower EP-2/4 gene expression, impairing eicosanoid-signalling pathway in alveolar macrophages. Figure 4 LTB 4 and PGE 2 production by alveolar macrophages is differentially induced by PLC-expressing Mycobacterium tuberculosis . Cells were infected with Mtb isolates 97-1200 or 97-1505 for 2, 12, 24, and 48 hours and the eicosanoid production was assessed in the supernatants by ELISA. ***P < 0.0001; **P < 0.001 (one-way ANOVA). Data are representative of three (A) and two (B) independent experiments (error bars, s.e.m.). Cell death and subversion of PGE2 production are dependent on mycobacterial PLCs Thus far, our results showed that the Mtb isolate 97-1505 induces necrotic death in alveolar macrophages, which is associated with lower expression of COX-2 and PGE2 receptors, leading to reduced production of PGE2, compared with infection by 97-1200.