Resolvins: a family of bioactive products of omega-3 fatty acid transformation circuits initiated by aspirin treatment that counter proinflammation signals

Abstract

Aspirin (ASA) is unique among current therapies because it acetylates cyclooxygenase (COX)-2 enabling the biosynthesis of R-containing precursors of endogenous antiinflammatory mediators. Here, we report that lipidomic analysis of exudates obtained in the resolution phase from mice treated with ASA and docosahexaenoic acid (DHA) (C22:6) produce a novel family of bioactive 17R-hydroxy-containing di- and tri-hydroxy-docosanoids termed resolvins. Murine brain treated with aspirin produced endogenous 17R-hydroxydocosahexaenoic acid as did human microglial cells. Human COX-2 converted DHA to 13-hydroxy-DHA that switched with ASA to 17R-HDHA that also proved a major route in hypoxic endothelial cells. Human neutrophils transformed COX-2-ASA-derived 17R-hydroxy-DHA into two sets of novel di- and trihydroxy products; one initiated via oxygenation at carbon 7 and the other at carbon 4. These compounds inhibited (IC(50) approximately 50 pM) microglial cell cytokine expression and in vivo dermal inflammation and peritonitis at ng doses, reducing 40-80% leukocytic exudates. These results indicate that exudates, vascular, leukocytes and neural cells treated with aspirin convert DHA to novel 17R-hydroxy series of docosanoids that are potent regulators. These biosynthetic pathways utilize omega-3 DHA and EPA during multicellular events in resolution to produce a family of protective compounds, i.e., resolvins, that enhance proresolution status.

Figure 2.

Novel ASA triggered HDHA products generated by human recombinant COX-2 ASA. 17R-HDHA. Human recombinant COX-2 treated in the presence and absence of 2 mM ASA was incubated with DHA (10 μM, 30 min, 37°C). Incubations were stopped with 2 ml cold methanol, extracted and taken for LC-MS-MS analyses. Results are representative of incubations from more than eight separate experiments, some with 1-¹⁴C-labeled DHA. (Top) LC-MS-MS chromatogram of m/z 343 showing the presence of mono-HDHA. (Bottom) MS-MS spectrum of (left) 13-HDHA without ASA treatment and (right) 17R-HDHA with ASA treatment.

Figure 1.

Inflammatory exudates from mice treated with ASA generate novel compounds: LC-MS-MS-based lipodomic analysis. (A) TNF-α–induced leukocyte exudates from dorsal air pouches. Samples were collected at 6 h from FVB mice given ASA and DHA (Materials and Methods). Selected ion chromatogram (m/z 343) showing the production of 17R-HDHA, 7S-HDHA, and 4S-HDHA. Using the diene UV chromophores for quantitation, 7-HDHA was ∼15% of the exudate materials and was identified using a SIM trace for m/z 141 with ms/ms 343. In some exudates 17S-HDHA was also present from LO-dependent routes (see text). MS-MS for B: 17R-HDHA (m/z 343); C: 7S,17R-diHDHA (m/z 359); and D: 4,11,17R-triHDHA (m/z 375). See text for diagnostic ions. Results are representative of n = 7.

Figure 1.

Inflammatory exudates from mice treated with ASA generate novel compounds: LC-MS-MS-based lipodomic analysis. (A) TNF-α–induced leukocyte exudates from dorsal air pouches. Samples were collected at 6 h from FVB mice given ASA and DHA (Materials and Methods). Selected ion chromatogram (m/z 343) showing the production of 17R-HDHA, 7S-HDHA, and 4S-HDHA. Using the diene UV chromophores for quantitation, 7-HDHA was ∼15% of the exudate materials and was identified using a SIM trace for m/z 141 with ms/ms 343. In some exudates 17S-HDHA was also present from LO-dependent routes (see text). MS-MS for B: 17R-HDHA (m/z 343); C: 7S,17R-diHDHA (m/z 359); and D: 4,11,17R-triHDHA (m/z 375). See text for diagnostic ions. Results are representative of n = 7.

Figure 1.

Inflammatory exudates from mice treated with ASA generate novel compounds: LC-MS-MS-based lipodomic analysis. (A) TNF-α–induced leukocyte exudates from dorsal air pouches. Samples were collected at 6 h from FVB mice given ASA and DHA (Materials and Methods). Selected ion chromatogram (m/z 343) showing the production of 17R-HDHA, 7S-HDHA, and 4S-HDHA. Using the diene UV chromophores for quantitation, 7-HDHA was ∼15% of the exudate materials and was identified using a SIM trace for m/z 141 with ms/ms 343. In some exudates 17S-HDHA was also present from LO-dependent routes (see text). MS-MS for B: 17R-HDHA (m/z 343); C: 7S,17R-diHDHA (m/z 359); and D: 4,11,17R-triHDHA (m/z 375). See text for diagnostic ions. Results are representative of n = 7.

Figure 1.

Inflammatory exudates from mice treated with ASA generate novel compounds: LC-MS-MS-based lipodomic analysis. (A) TNF-α–induced leukocyte exudates from dorsal air pouches. Samples were collected at 6 h from FVB mice given ASA and DHA (Materials and Methods). Selected ion chromatogram (m/z 343) showing the production of 17R-HDHA, 7S-HDHA, and 4S-HDHA. Using the diene UV chromophores for quantitation, 7-HDHA was ∼15% of the exudate materials and was identified using a SIM trace for m/z 141 with ms/ms 343. In some exudates 17S-HDHA was also present from LO-dependent routes (see text). MS-MS for B: 17R-HDHA (m/z 343); C: 7S,17R-diHDHA (m/z 359); and D: 4,11,17R-triHDHA (m/z 375). See text for diagnostic ions. Results are representative of n = 7.

Figure 3.

Endogenous 17R-HDHA from brain and human microglial cells treated with aspirin. (A) LC-MS-MS chromatogram obtained from brain for relative abundance at m/z 327 for DHA and m/z 343 for the monohydroxy product. (B) MS-MS spectrum of brain 17R-HDHA (m/z 343). Murine brain samples were incubated with ASA (45 min, 37°C). Results are representative of n = 6 mice treated with ASA versus five mice without ASA. (C) Human microglial cells (HMG) treated with ASA; MS-MS spectrum of HMG 17R-HDHA. 10 × 10⁶ cells were exposed to 50 ng/ml TNF-α and incubated (24 h, 37°C). Cells were treated with ASA (500 μM, 30 min, 37°C) followed by addition of ionophore A23187 (5 μM, 25–30 min). Incubations were stopped with MeOH, extracted and analyzed by tandem UV, LC-MS-MS (Fig. 3 inset shows UV-chromatogram plotted at 235 nm absorbance) (n = 4, d = 20). Both 17R-HDHA and DHA were identified on the basis of individual retention times, parent ions, and daughter ions obtained.

Figure 3.

Endogenous 17R-HDHA from brain and human microglial cells treated with aspirin. (A) LC-MS-MS chromatogram obtained from brain for relative abundance at m/z 327 for DHA and m/z 343 for the monohydroxy product. (B) MS-MS spectrum of brain 17R-HDHA (m/z 343). Murine brain samples were incubated with ASA (45 min, 37°C). Results are representative of n = 6 mice treated with ASA versus five mice without ASA. (C) Human microglial cells (HMG) treated with ASA; MS-MS spectrum of HMG 17R-HDHA. 10 × 10⁶ cells were exposed to 50 ng/ml TNF-α and incubated (24 h, 37°C). Cells were treated with ASA (500 μM, 30 min, 37°C) followed by addition of ionophore A23187 (5 μM, 25–30 min). Incubations were stopped with MeOH, extracted and analyzed by tandem UV, LC-MS-MS (Fig. 3 inset shows UV-chromatogram plotted at 235 nm absorbance) (n = 4, d = 20). Both 17R-HDHA and DHA were identified on the basis of individual retention times, parent ions, and daughter ions obtained.

Figure 4.

Hypoxic HUVECs treated with ASA generate 17R-HDHA. HUVECs were exposed to TNF-α and IL-1β (both 1 ng/ml) and placed in a hypoxia chamber (3 h). The cells were treated with ASA (500 μM, 30 min) followed by DHA (20 μg/10⁶ cells/10 ml plate) and A23187 (2 μM, 60 min). (A) LC-MS-MS chromatogram of ion m/z 343 shows the presence of 17R-HDHA. (B) MS-MS spectrum (RT 21.2 min) of 17R-HDHA identified by retention time, parent ions, and daughter ions and matched with properties and authentic NMR qualified standard.

Figure 5.

Bioimpact properties of omega-3-derived resolvins. (A) Human glioma cells: inhibition of TNF-stimulated IL-1β transcripts. DBTRG-05MG cells 10⁶/ml were stimulated with 50 ng/ml of human recombinant TNF-α for 16 h to induce expression of IL-1β transcripts. Concentration dependence with COX-2 products: 17-HDHA (▪), 13-HDHA (•), and di-/tri-HDHA (□). The IC₅₀ for both compounds is ∼50 pM. (insets) Results are representative of RT-PCR gels of MG cells exposed to 100 nM of 13-HDHA or 17-HDHA and graphed after normalization of the IL-1β transcripts using GAPDH. n = 2. (B) Influence of eicosanoids and docosanoids on fMLP-induced neutrophil migration across microvascular endothelial monolayers. Neutrophils (10⁶ cells per monolayer) were exposed to vehicle containing buffer, or indicated concentrations of aspirin-triggered LXA₄ analogue (black diamonds) 5S,12,18R-triHEPE (black squares), 17R-HDHA (black circles) or 13-HDHA (black triangles) for 15 min at 37°C. Neutrophils were then layered on HMVEC monolayers and stimulated to transmigrate by a 10⁻⁸ M fMLP gradient for 1 h at 37°C. Transmigration was assessed by quantitation of the neutrophil marker myeloperoxidase. Results are presented as mean ± SEM number of PMNs (n = 8–12 monolayers per condition). (C) Reduction of PMN in murine peritonitis and skin pouch. Compounds (100 ng in 120 μl sterile saline) were injected by intravenous bolus injection into the mouse tail vein and followed by 1 ml zymosan A (1 mg/ml) into the peritoneum. Peritoneal lavages were collected (2 h) and cell types were enumerated. Air pouch–compounds (dissolved in 500 μl of PBS without Ca²⁺ or Mg²⁺) injected into the air pouch via intrapouch injection or via intravenous administration (in 120 μl sterile saline) followed by intrapouch injection of TNF-α. 4 h later air pouch lavages were collected and cells were enumerated and differentiated. Compounds were prepared by biogenic synthesis or isolated from in vivo exudates. The ratio of 7,17R-diHDHA to 4,17R-diHDHA was ∼8:1; the ratio of 4,11,17R-triHDHA and 7,16,17R-triHDHA was ∼2:1; and the ratio of di- to triHDHA was ∼1:1.3. Exudate transfers to a native mouse (see text). ATL denotes 15-epi-16-para(fluoro)-phenoxy-LXA₄ (administered at 100 ng/mouse). Values represent mean ± SEM from 3–4 different mice; *P < 0.05 when infiltrated PMN is compared with vehicle control.

Figure 5.

Bioimpact properties of omega-3-derived resolvins. (A) Human glioma cells: inhibition of TNF-stimulated IL-1β transcripts. DBTRG-05MG cells 10⁶/ml were stimulated with 50 ng/ml of human recombinant TNF-α for 16 h to induce expression of IL-1β transcripts. Concentration dependence with COX-2 products: 17-HDHA (▪), 13-HDHA (•), and di-/tri-HDHA (□). The IC₅₀ for both compounds is ∼50 pM. (insets) Results are representative of RT-PCR gels of MG cells exposed to 100 nM of 13-HDHA or 17-HDHA and graphed after normalization of the IL-1β transcripts using GAPDH. n = 2. (B) Influence of eicosanoids and docosanoids on fMLP-induced neutrophil migration across microvascular endothelial monolayers. Neutrophils (10⁶ cells per monolayer) were exposed to vehicle containing buffer, or indicated concentrations of aspirin-triggered LXA₄ analogue (black diamonds) 5S,12,18R-triHEPE (black squares), 17R-HDHA (black circles) or 13-HDHA (black triangles) for 15 min at 37°C. Neutrophils were then layered on HMVEC monolayers and stimulated to transmigrate by a 10⁻⁸ M fMLP gradient for 1 h at 37°C. Transmigration was assessed by quantitation of the neutrophil marker myeloperoxidase. Results are presented as mean ± SEM number of PMNs (n = 8–12 monolayers per condition). (C) Reduction of PMN in murine peritonitis and skin pouch. Compounds (100 ng in 120 μl sterile saline) were injected by intravenous bolus injection into the mouse tail vein and followed by 1 ml zymosan A (1 mg/ml) into the peritoneum. Peritoneal lavages were collected (2 h) and cell types were enumerated. Air pouch–compounds (dissolved in 500 μl of PBS without Ca²⁺ or Mg²⁺) injected into the air pouch via intrapouch injection or via intravenous administration (in 120 μl sterile saline) followed by intrapouch injection of TNF-α. 4 h later air pouch lavages were collected and cells were enumerated and differentiated. Compounds were prepared by biogenic synthesis or isolated from in vivo exudates. The ratio of 7,17R-diHDHA to 4,17R-diHDHA was ∼8:1; the ratio of 4,11,17R-triHDHA and 7,16,17R-triHDHA was ∼2:1; and the ratio of di- to triHDHA was ∼1:1.3. Exudate transfers to a native mouse (see text). ATL denotes 15-epi-16-para(fluoro)-phenoxy-LXA₄ (administered at 100 ng/mouse). Values represent mean ± SEM from 3–4 different mice; *P < 0.05 when infiltrated PMN is compared with vehicle control.

Figure 5.

Bioimpact properties of omega-3-derived resolvins. (A) Human glioma cells: inhibition of TNF-stimulated IL-1β transcripts. DBTRG-05MG cells 10⁶/ml were stimulated with 50 ng/ml of human recombinant TNF-α for 16 h to induce expression of IL-1β transcripts. Concentration dependence with COX-2 products: 17-HDHA (▪), 13-HDHA (•), and di-/tri-HDHA (□). The IC₅₀ for both compounds is ∼50 pM. (insets) Results are representative of RT-PCR gels of MG cells exposed to 100 nM of 13-HDHA or 17-HDHA and graphed after normalization of the IL-1β transcripts using GAPDH. n = 2. (B) Influence of eicosanoids and docosanoids on fMLP-induced neutrophil migration across microvascular endothelial monolayers. Neutrophils (10⁶ cells per monolayer) were exposed to vehicle containing buffer, or indicated concentrations of aspirin-triggered LXA₄ analogue (black diamonds) 5S,12,18R-triHEPE (black squares), 17R-HDHA (black circles) or 13-HDHA (black triangles) for 15 min at 37°C. Neutrophils were then layered on HMVEC monolayers and stimulated to transmigrate by a 10⁻⁸ M fMLP gradient for 1 h at 37°C. Transmigration was assessed by quantitation of the neutrophil marker myeloperoxidase. Results are presented as mean ± SEM number of PMNs (n = 8–12 monolayers per condition). (C) Reduction of PMN in murine peritonitis and skin pouch. Compounds (100 ng in 120 μl sterile saline) were injected by intravenous bolus injection into the mouse tail vein and followed by 1 ml zymosan A (1 mg/ml) into the peritoneum. Peritoneal lavages were collected (2 h) and cell types were enumerated. Air pouch–compounds (dissolved in 500 μl of PBS without Ca²⁺ or Mg²⁺) injected into the air pouch via intrapouch injection or via intravenous administration (in 120 μl sterile saline) followed by intrapouch injection of TNF-α. 4 h later air pouch lavages were collected and cells were enumerated and differentiated. Compounds were prepared by biogenic synthesis or isolated from in vivo exudates. The ratio of 7,17R-diHDHA to 4,17R-diHDHA was ∼8:1; the ratio of 4,11,17R-triHDHA and 7,16,17R-triHDHA was ∼2:1; and the ratio of di- to triHDHA was ∼1:1.3. Exudate transfers to a native mouse (see text). ATL denotes 15-epi-16-para(fluoro)-phenoxy-LXA₄ (administered at 100 ng/mouse). Values represent mean ± SEM from 3–4 different mice; *P < 0.05 when infiltrated PMN is compared with vehicle control.

Figure 6.

Resolvin production by human PMNs exposed to microbial zymosan: novel 17R di- and triHDHA. Human PMNs (50 × 10⁶ cells/ml) incubated with zymosan A (100 ng/ml) and 17R-HDHA (5 μg/ml, 40 min, 37°C). Results are representative of n = 4.

Figure 7.

Inflammatory exudate produces 17R-containing di- and tri-hydroxy tetraenes and triene-containing compounds: LC-MS-MS. See Fig. 1 for details. Exudates were obtained and analyzed by procedures essentially identical to those described in Fig. 1. (A) m/z were plotted at 375 (top), 359 (middle), and 343 (bottom). (B) UV absorbance was plotted at 300 nm to mark tetraene-containing chromatophores. (C) MS-MS of 7S,8,17R-triHDHA.

Figure 8.

Biosynthetic scheme proposed for resolvins: aspirin-triggered omega-3-derived products. Acetylation of COX-2 by ASA treatment generates novel 17R-H(p)DHA from DHA that is reduced to its corresponding alcohol and converted via sequential actions of a leukocyte 5–LO and leads to formation of both dihydroxy- and trihydroxy-containing docosanoids that retain their 17R configuration. Pathways are denoted in blue for omega oxidation products that are likely to be in vivo markers of enzymatic inactivation. The resolvin pathways appear to be maximally induced during the “spontaneous resolution” phase of inflammation and compounds are activated to dampen PMN infiltration, which reduces exudate PMN numbers to promote proresolution of inflammatory exudates (resolvins from EPA, the 18R-HEPE series, are denoted in green) that leads to potent inhibitors of PMN recruitment in vitro and in vivo (see pathway, right, text, and reference 2). The complete stereochemistries of the new di- and tri-hydroxy–containing compounds remain to be established and are depicted here in their likely configuration based on biogenic total synthesis. See Table II and text for further details.