Specificity of eicosanoid production depends on the TLR‐4‐stimulated macrophage phenotype

Comparing different TLR‐4‐activated macrophages reveals metabolite quantity dependence on COX‐2 level and higher PGIS activity relative to other prostanoid syntheses that outcompetes with mPGES‐1. Eicosanoid metabolism differs in profile and quantity between macrophages of different tissue origin and method of elicitation, as well as between primary and immortalized macrophages after activation with inflammatory stimuli. Using a lipidomic approach, we comprehensively analyzed the eicosanoids made by murine RPMs, TGEMs, BMDM, and the macrophage‐like cell line RAW after stimulation with the TLR‐4‐specific agonist KLA. Direct correlation among total COX metabolites, COX side‐products (11‐HETE, 15‐HETE), COX‐2 mRNA, and protein at 8 h was found when comparing each cell type. Comprehensive qPCR analysis was used to compare relative transcript levels between the terminal prostanoid synthases themselves as well as between each cell type. Levels of PGE2, PGD2, and TxB2generally correlated with enzyme transcript expression of PGES, PGDS, and TBXS, providing evidence of comparable enzyme activities. PGIS transcript was expressed only in RPM and TGEM macrophages and at an exceptionally low level, despite high metabolite production compared with other synthases. Presence of PGIS in RPM and TGEM also lowered the production of PGE2versus PGD2by approximately tenfold relative to BMDM and RAW cells, which lacked this enzyme. Our results demonstrate that delayed PG production depends on the maximal level of COX‐2 expression in different macrophages after TLR‐4 stimulation. Also, the same enzymes in each cell largely dictate the profile of eicosanoids produced depending on the ratios of expression between them, with the exception of PGIS, which appears to have much greater synthetic capacity and competes selectively with mPGES‐1.