PD_{n-3 DPA} biosynthesis and actions in human macrophages

K. Pistorius¹, P. R. Souza¹, K. G. Primdahl², R. A. Colas¹, M. Aursnes², T. V. Hansen², J. Dalli¹. ¹Lipid Mediator Unit, William Harvey Research Institute, London, United Kingdom, ²Department of Pharmaceutical Chemistry, School of Pharmacy, University of Oslo, Oslo, Norway.

Introduction: Macrophages are central in orchestrating the clearance of apoptotic cells and cellular debris during inflammation, with the mechanism(s) regulating this process remaining of interest. Herein, using lipid mediator profiling we investigated the n-3 docosapentanoic acid (DPA)-derived protectin (PD_{n-3 DPA}) biosynthetic pathway, and the role of these mediators in regulating both human macrophage phenotype and efferocytosis.

Method: Human monocytes were incubated with 15-lipoxygenase (LOX) inhibitor (10μM), 15-LOX inhibitor + 16S,17S-epoxy-7Z,10Z,12E,14E,19Z-DPA (16S,17S-ePD_{n-3 DPA}) (1nM), 15-LOX inhibitor + PD1_{n-3 DPA} (1nM), or vehicle (37^oC, RPMI). On day 7, cells were collected, expression of lineage markers was assessed using flow cytometry, efferocytosis investigated using fluorescently labelled apoptotic cells, and lipid mediator profiles were identified using liquid chromatography tandem mass spectrometry (1). Monocytes or 0.2 μM hr15-LOX type 1 were incubated with 10μM n-3 DPA for 3 min and quenched using acidified methanol. Total organic synthesis was used to synthesis 16S,17S-ePD_{n-3 DPA}, which was incubated with macrophages or inactivated macrophages (incubated at 100^oC), followed by the addition of E. coli (2.5x10⁸ CFU/mL) for 15 min at 37°C, or with and an epoxide hydrolase inhibitor (AUDA; 25μM) for 20 min (RT). Lipid mediators were identified and quantified using lipid mediator profiling.

Results: Inhibiting human 15-LOX, the initiating enzyme in the PD_{n-3 DPA} biosynthetic pathway, significantly reduced several macrophage lineage markers, including CD206 and CD163 (n=7), and macrophage efferocytosis (n=6; p<0.05). Evidence for the formation of an allyllic epoxide in the PD_{n-3 DPA} biosynthetic pathway in human macrophages (n=4), a reaction catalysed by h15-LOX (n=3) was obtained using acid methanol trapping. Human macrophages incubated with enantiomerically pure 16S,17S-epoxy-PD_{n-3 DPA} yielded PD1_{n-3 DPA} and PD2_{n-3 DPA} (n=4). The reaction was catalysed by macrophage epoxide hydrolases, since incubation of cells at 100^oC significantly reduced the conversion of this intermediate to the bioactive products (n=4; p<0.05). Incubations of human macrophages with either 16S,17S-epoxy-PD_{n-3 DPA} or PD1_{n-3 DPA} restored the expression of several of the lineage markers downregulated by 15-LOX inhibition, including CD206 and CD64 (n=7; p<0.05), and rescued their efferocytosis ability (n=6; p<0.05).

Conclusion: Taken together these results establish the PD_{n-3 DPA} biosynthetic pathway in human macrophages and its role in regulating macrophage resolution responses.

References:

(1) Dalli, J. et al (2013). Sci Rep 3: 1940