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-gamma (PPAR gamma) 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.

Figure 1. EFOXs are produced during macrophage activation

a, MRM scans following the neutral loss of 78 reveal EFOXs adducted with BME in cell extracts from activated (upper) and non-activated (N/A, lower) RAW 264.7 cells. b, EFOX levels in PMA-differentiated THP-1 cells 8 h post activation. c, EFOX-D₅ levels in RAW264.7 cells activated with the indicated compounds at the following concentrations: LPS (0.5 μg/ml), Kdo₂ (0.5 μg/ml), IFNγ (200 U/ml), PMA (3.24 μM), fMLP (1 μM). Data are expressed as mean ± S.D. (n=4), where * = significantly different (p<0.01) from “PMA + IFNγ + LPS,” and # = significant difference (p<0.01) between “LPS” and “Kdo₂ + IFNγ” (one-way ANOVA, post-hoc Tukey’s test). d, EFOX-D₅ levels in RAW264.7 cells were quantified at the indicated time points post activation. ND indicates not detectable.

Figure 2. EFOX-D₅ is an α,β-unsaturated oxo-derivative of DPA

a, Characteristic BME-EFOX fragmentation pattern derived from the EPI of BME-EFOX-D₅. b, EFOX-D₅ levels in activated RAW264.7 cells grown for 3 days in medium supplemented with the indicated FA. ND indicates not detectable. c, Diagram of NaBH₄ reaction. d, MRM scans monitoring for the m/z transitions 343.2/299.2 (13-EFOX-D₅/loss of CO₂; upper and middle panel) and 345.2/327.2 (hydroxy-DPA/loss of H₂O; lower panel) in RAW264.7 cell lysates purified for EFOX-D₅ ± NaBH₄. e, MS/MS fragmentation of EFOX-D₅ purified from activated RAW 264.7 cells and reduced with NaBH₄.

Figure 3. EFOX-D₅ formation is dependent on COX-2 activity

a, b, EFOX-D₅ levels in RAW264.7 activated and treated with the indicated inhibitors at the following concentrations: genistein (25 μM), MAFP (25 μM), MK886 (500 nM), ETYA (25 μM), OKA (50 nM), ASA (200 μM), indomethacin (25 μM), ibuprofen (100 μM), diclofenac (1 μM) and NS-398 (4 μM). ND indicates not detectable. Data are expressed as mean ± S.D. (n=4), where * = significantly different (p<0.01) from “Kdo₂ + IFNγ” (one-way ANOVA, post-hoc Tukey’s test). c, Temporal formation of hydroxy-precursors (MRM 345/327) of EFOX-D₅ synthesized in vitro by purified ovine COX-2 + DPA ± ASA. d, e, Chromatographic profiles (left panels) and spectra (right panels) of the two isomers formed by COX-2 ± ASA. f, BME-adducted 17-EFOX-D₅ standard (MRM 421.2/343.2) and EFOX-D₅ BME-adducts from activated RAW264.7 cells ± ASA. g–i, Production of EFOX-D₅ and OH-DPA by RAW264.7 cell lysates supplemented with OH-DPA, DPA, or vehicle alone. Full and empty symbols indicate activated and non-activated cell lysates respectively.

Figure 4. EFOXs form adducts with proteins and GSH following activation of RAW264.7

a, Cell lysates from activated RAW264.7 cells were split into two groups: treatment with BME followed by protein precipitation with acetonitrile (“Total”) and protein precipitation followed by BME treatment (“Free + small molecule adducted”). b, Chemical structure and fragmentation pattern of GS-13-EFOX-D₅. c, Chromatographic profiles and positive ion mass spectra of GSH adducts of 13-EFOX-D₅ and 17-EFOX-D₅ derived from standards (upper panels), cell medium (middle panel) and cell pellet (lower panel), N/A profiles correspond to non-activated cell samples. Grey chromatograms represent GS-17-EFOX-D₅ (upper panel left) and GS-13-EFOX-D₅ (upper panel right) standards. Fragments 345.3 and 523.3 were monitored in cell media and cell pellet samples respectively.

Figure 5. 17-EFOX-D₆ and 17-EFOX-D₅ modulate anti-oxidant and inflammatory responses

a, b, RAW264.7 cells treated with 17-EFOX-D₆ and 17-EFOX-D₅ were harvested at 1 h for nuclear Nrf2 (a) and 18 h for of HO-1 and NQO-1 (b). c, d, RAW264.7 cells pretreated for 6 h with 17-EFOX-D₆ and 17-EFOX-D₅ were harvested 12 h post activation and IL-6, MCP-1, IL-10 levels, and nitrite levels were measured in the cell media and normalized to protein. d, iNOS and COX-2 levels were detected in cell lysates. e, PPARγ beta-lactamase reporter assays for Rosiglitazone, 17-EFOX-D₆, 17-EFOX-D₅, 15d-PGJ₂, 17-hydroxyDHA, DPA, and DHA.