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Cyclooxygenase-2 generates anti-inflammatory mediators from omega-3 fatty acids

Abstract

Electrophilic fatty acids are generated during inflammation by non-enzymatic reactions and can modulate inflammatory responses. We used a new mass spectrometry–based electrophile capture strategy to reveal the formation of electrophilic oxo-derivatives (EFOX) from the omega-3 fatty acids docosahexaenoic acid (DHA) and docosapentaenoic acid (DPA). These EFOX were generated by a cyclooxygenase-2 (COX-2)-catalyzed mechanism in activated macrophages. Modulation of COX-2 activity by aspirin increased the rate of EFOX production and their intracellular levels. Owing to their electrophilic nature, EFOX adducted to cysteine and histidine residues of proteins and activated Nrf2-dependent anti-oxidant gene expression. We confirmed the anti-inflammatory nature of DHA- and DPA-derived EFOX by showing that they can act as peroxisome proliferator-activated receptor-γ (PPARγ) agonists and inhibit pro-inflammatory cytokine and nitric oxide production, all within biological concentration ranges. These data support the idea that EFOX are signaling mediators that transduce the beneficial clinical effects of omega-3 fatty acids, COX-2 and aspirin.

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Figure 1: EFOX are produced during macrophage activation.
Figure 2: EFOX-D5 is an α,β-unsaturated oxo-derivative of DPA (2).
Figure 3: EFOX-D5 formation depends on COX-2 activity.
Figure 4: EFOX form adducts with proteins and GSH after activation of RAW264.7.
Figure 5: 17-EFOX-D6 (8) and 17-EFOX-D5 (7) modulate anti-oxidant and inflammatory responses.
Figure 6: EFOX are anti-inflammatory signaling mediators.

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Acknowledgements

This work was supported by National Institutes of Health grants HL58115 and HL64937, the American Diabetes Association (F.J.S.), and Fondazione Ri.MED, Italy (C.C.). We thank J. Ruzicka and A. Hilderbrand at Thermo Fisher Scientific for assistance with accurate mass measurements of EFOX using an LTQ OrbitrapVelos instrument.

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A.L.G., C.C., B.A.F. and F.J.S. designed experiments and prepared the manuscript. A.L.G. performed the search, discovery, and characterization of EFOX and PPARγ-reporter experiments. C.C. performed in vitro COX-2-based synthesis of EFOX, COX-2 siRNA and cell signaling experiments. G.B. performed proteomic analysis. M.P.C., T.K.R. and V.R. assisted with or performed animal-based studies. S.R.W. assisted with chemical derivatization reactions for EFOX characterization.

Corresponding authors

Correspondence to Bruce A Freeman or Francisco J Schopfer.

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Competing interests

B.A.F. acknowledges financial interest in Complexa, Inc.

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Supplementary Methods, Supplementary Figures 1–22 and Supplementary Table 1 (PDF 4255 kb)

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Groeger, A., Cipollina, C., Cole, M. et al. Cyclooxygenase-2 generates anti-inflammatory mediators from omega-3 fatty acids. Nat Chem Biol 6, 433–441 (2010). https://doi.org/10.1038/nchembio.367

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