Epoxygenases regulate inflammatory resolution: a role for macrophage phagocytosis?

Jonas Bystrom1, Derek Gilroy2, Matthew Edin3, Darryl Zeldin3, David Bishop-Bailey1. 1William Harvey Research institute, Barts School of Medicine, Queen Mary University of London, EC1M 6BQ, London, United Kingdom, 2Centre for Pharmacology, Department of Medicine, UCL, WC1 6JJ, London, United Kingdom, 3NIEHS, NIH, 27709, Reseach Triangle Park, United States.

Epoxygenases are CYP450 enzymes which epoxygenate arachidonic acid producing biologically active epoxyeicosatrienoic acids (EET’s). We have found two classes of these enzymes CYP2J and CYP2C by RT-PCR in monocytes and macrophages. To assess the role of these epoxygenases during the inflammatory response we used a murine model of zymosan-induced peritonitis. C57B/6 mice were injected with 1 mg zymosan i.p. This induces an acute inflammation (6h) and a longer monocyte driven resolution phase (12-72h). During the development of the inflammatory resolution we inhibited epoxygenases by injection of the epoxygenase inhibitor SKF525A (30 mg/kg; i.p.) or PBS at 24 and 36 hr. Mice were sacrificed at 48 hr, and the peritoneal cavity washed out with 2 ml of sterile PBS-citrate. Peritoneal cells were analysed by flowcytometry and RT-PCR while the exudates were analysed by mass-spectrometry for lipids content. The mouse epoxygenase ortholog CYP2J6 was detected in inflammatory cells by RT-PCR. Further, the stable metabolite of 11,12-EET, 11,12-dihydroxyeicosatrienoic acid was found present in the inflamed peritoneum at 40 pm/mL while SKF525A treatment reduced the levels to 30 pm/mL, p<0.05. Strikingly, SKF525A induced a 20 - fold increase of Ly-6C inflammatory monocytes in the peritoneum compared to vehicle (vehicle 0.51 +/- 0.2, SKF treated 11.1 +/- 4.8 million cells/mL, p = 0.002). Ly-C6 positive monocytes were isolated by magnetic separation and were found to be bigger in size (analysis by flowcytometry), expressing CCR2, and low levels of CX3CR1 by RT-PCR. Moreover the SKF524A treatment induced the yeast cell wall receptor Dectin-1 mRNA expression (1.7 fold increase, p = 0.04). In vitro, 24 hr treatment with 100 nM PMA was used to differentiate THP-1 cells to macrophage phenotype. Differentiated cells were treated with or without 10 uM SKF for a further 12 hr, and subsequently fed fluoroscein-labelled zymosan for an additional 3 hr. Phagocytosis was assessed at 494 nm and by fluorescent microscopy. SKF treatment was found to induce zymosan uptake, p<0.0001. Student’s T-test was used to determine whether differences in this study were statistically significant.

In conclusion, using an animal model and cell line we have shown that during infectious assault epoxygenases in monocytes/macrophages regulates inflammatory resolution and pathogen phagocytosis. Epoxygenase products may therefore represent novel mediators for inflammation control by directly regulating monocyte/macrophage functions.