GM‐CSF– and M‐CSF–primed macrophages present similar resolving but distinct inflammatory lipid mediator signatures

Abstract

M1 and M2 activated macrophages (Mfs) 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 Mf colony-stimulating factor (GM-CSF) or Mf 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 Mf phenotypes released primarily proresolving lipid mediators (prostaglandin E 2 metabolite, lipoxin A 4 , 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 Mfs produced considerably more 5-lipoxygenase products, particularly leukotriene C 4 , potentially linked to M2 functions in asthma. Prostaglandins were formed by both Mf types. In the M-CSF cells, there was also an enhanced release of arachidonic acid and activation of cytosolic phospholipase A 2 . 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 Mfs 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. FASEB J. 31, 4370-4381 (2017). www.fasebj.orgMacrophages (Mfs) orchestrate the inflammatory process from early onset to the resolution phase (1, 2). A major issue in Mf biology is to characterize functional phenotypes, currently described as a heterogeneous spectrum of activated states, from M1 to M2 (3, 4). Stimulating factors, such as cytokines and pathogens, can induce specific phenotypes: originally IFN-g + LPS activation resulted in a proinflammatory M1 state, whereas IL-4 activation induced the alternatively activated M2 state. Polarized cells express distinct markers, such as transcription factors, cytokines, and surface markers; these are the main tools to define Mf phenotypes. A number of transcripts have been reported to differ between M1 and M2 Mfs, including mRNAs for enzymes involved in eicosanoid metabolism (5).Eicosanoids originate from arachidonic acid (AA), released from membrane phospholipids by phospholipases, typically cytosolic phospholipase A 2 (cPLA 2 ) (6). Free AA is further metabolized via 3 enzymatic pathways: lipoxygenase (LO), cyclooxygenase (COX), and cyto...

Dicer up-regulation by inhibition of specific proteolysis in differentiating monocytic cells

“…To yield M1/M2 macrophages, GM-CSF primed cells were treated also with LPS (100 ng/mL) and IFNγ (20 ng/mL), and M-CSF primed cells also with IL-4 (20 ng/mL) for the final 24 h of differentiation. These treatments resulted in increased formation of IL-6 and surface markers CD86 and ICAM-1 for M1 and IL-10 and surface markers CD14 and DC-SIGN for M2, as described in our previous study (26).…”

“…Preparation and Differentiation of Monocytes. Monocytes were isolated from buffy coats of 12 healthy human donors (Karolinska Hospital Blood Bank) as described previously (25,26). Peripheral blood mononuclear cells were isolated by gradient centrifugation with Ficoll-Paque PREMIUM (GE Healthcare) and seeded at 5 × 10 6 cells per mL for 2 h, allowing monocytes to adhere to plates.…”

Cell‐free nucleic acids are present in blood products and regulate genes of innate immune response

“…In addition to angiogenesis promotion, vascular endothelial growth factor is also recognized as a proinflammatory mediator . The genes of other mediators of inflammation were up regulated in the presence of CFNA: CSF1R is a receptor for colony‐stimulating factor expressed by macrophages promoting the release of proinflammatory chemokines; Ly9 is a receptor promoting T‐cell differentiation into a proinflammatory helper‐T cell Th17 leading to increased IL‐17 secretion; DNA damage‐inducible transcript 3 positively regulates the transcription of tribbles homolog 3 and the proinflammatory cytokines IL‐1 and IL‐6; and IL‐8 has been implicated in some allergic reactions . On the other hand, the inhibitors of inflammation insulin‐like growth factor‐1 and IL1‐Ra were down regulated by CFNA.…”

Leukotriene biosynthetic enzymes as therapeutic targets

“…However, synthesis of leukotrienes can occur in response to cell stress without calcium mobilization, and in some instances biosynthesis can proceed over longer periods of time, even hours, in a cell type-and stimulus-dependent manner (27,129,151). Moreover, resting monocytes cultured with GM-CSF produce significant amounts of LXA 4 , which normally involves 5-LOX activity, without concomitant synthesis of appreciable quantities of leukotrienes (152). Apparently, the activity of 5-LOX and downstream enzymes depends on a multitude of factors leading to differential profiles of mediators in any given cellular context.…”