M1 and M2 activated macrophages (Mϕs) have different roles in inflammation. Because pathogens may first encounter resting cells, we investigated lipid mediator profiles prior to full activation. Human monocytes were differentiated with granulocyte Mϕ colony-stimulating factor (GM-CSF) or Mϕ colony-stimulating factor (M-CSF), which are known to prime toward M1 or M2 phenotypes, respectively. Lipid mediators released during resting conditions and produced in response to bacterial stimuli (LPS/N-formylmethionyl-leucyl-phenylalanine or peptidoglycan) were quantified by liquid chromatography-mass spectrometry. In resting conditions, both Mϕ phenotypes released primarily proresolving lipid mediators (prostaglandin E2 metabolite, lipoxin A4, and 18-hydroxyeicosapentaenoic acid). A striking shift toward proinflammatory eicosanoids was observed when the same cells were exposed (30 min) to bacterial stimuli: M-CSF Mϕs produced considerably more 5-lipoxygenase products, particularly leukotriene C4, potentially linked to M2 functions in asthma. Prostaglandins were formed by both Mϕ types. In the M-CSF cells, there was also an enhanced release of arachidonic acid and activation of cytosolic phospholipase A2 However, GM-CSF cells expressed higher levels of 5-lipoxygenase and 5-lipoxygenase-activating protein, and in ionophore incubations these cells also produced the highest levels of 5-hydroxyeicosatetraenoic acid. In summary, GM-CSF and M-CSF Mϕs displayed similar proresolving lipid mediator formation in resting conditions but shifted toward different proinflammatory eicosanoids upon bacterial stimuli. This demonstrates that preference for specific eicosanoid pathways is primed by CSFs before full M1/M2 activation.-Lukic, A., Larssen, P., Fauland, A., Samuelsson, B., Wheelock, C. E., Gabrielsson, S., Radmark, O. GM-CSF- and M-CSF-primed macrophages present similar resolving but distinct inflammatory lipid mediator signatures.