Novel n-3 immunoresolvents: structures and actions

Abstract

Resolution of inflammation is now held to be an active process where autacoids promote homeostasis. Using functional-metabololipidomics and in vivo systems, herein we report that endogenous n-3 docosapentaenoic (DPA) acid is converted during inflammation-resolution in mice and by human leukocytes to novel n-3 products congenerous to D-series resolvins (Rv), protectins (PD) and maresins (MaR), termed specialized pro-resolving mediators (SPM). The new n-3 DPA structures include 7,8,17-trihydroxy-9,11,13,15E,19Z-docosapentaenoic acid (RvD1(n-3 DPA)), 7,14-dihydroxy-8,10,12,16Z,19Z-docosapentaenoic acid (MaR1(n-3 DPA)) and related bioactive products. Each n-3 DPA-SPM displayed protective actions from second organ injury and reduced systemic inflammation in ischemia-reperfusion. The n-3 DPA-SPM, including RvD1(n-3 DPA) and MaR1(n-3 DPA), each exerted potent leukocyte directed actions in vivo. With human leukocytes each n-3 DPA-SPM reduced neutrophil chemotaxis, adhesion and enhanced macrophage phagocytosis. Together, these findings demonstrate that n-3 DPA is converted to novel immunoresolvents with actions comparable to resolvins and are likely produced in humans when n-3 DPA is elevated.

Figure 1. n-3 DPA-derived products display potent anti-inflammatory and tissue protective actions in vivo that are comparable to RvD1.

(a) Structures of DHA and n-3 DPA. (b) Ischemia was induced by applying tourniquets to the hind limb of 6-8-week-old male FvB mice. After 1 h, tourniquets were removed and reperfusion ensued for 3 h. 10 min prior to reperfusion, vehicle (saline containing 0.1% EtOH), RvD1 (500 ng) or a mixture of n-3 DPA-derived products (see Methods for details) were administered intravenously. At the end of reperfusion, lungs were collected; (c) tissue histology by H&E staining (x200) and (d) MPO levels were assessed. (e) Blood was collected, incubated with rat anti-mouse Ly6G and rat anti-mouse CD41 antibodies and neutrophil leukocyte aggregates were assessed by flow cytometry. (f) Plasma prostanoid and (g) leukotriene levels were assessed by lipid mediator metabololipidomics. Results c are representative n = 4. Results d–e are mean ± SEM. n = 4. * P < 0.05, ** P < 0.01 vs. vehicle mice.

Figure 2. n-3 DPA levels increase in acute inflammation and is converted to novel products in vivo.

Mice were subjected to ischemia reperfusion injury (see Methods for details) at 2 h of reperfusion, blood was collected via cardiac puncture and plasma was obtained by centrifugation, products were extracted and monohydroxy n-3 DPA levels were assessed by lipid mediator metabololipidomics. (a) Plasma polyunsaturated fatty acid levels; (b) Representative chromatographs obtained by Multiple Reaction Monitoring (MRM) of the parent ion (Q₁) m/z 345 and a diagnostic daughter ion (Q₃) m/z 327. (c) Monohydroxy-containing levels in plasma of sham mice and mice subjected to I/R. Results for a and c are mean ± SEM. n = 4. Results for b are representative of n = 4. *P < 0.05; **P < 0.01; ***P < 0.01 vs. sham mice.

Figure 3. Identification of novel endogenous n-3 DPA pro-resolving mediators.

Mice were subjected to ischemia reperfusion injury (see Methods and Fig. 2 for details). Two h into reperfusion, blood was collected and lipid mediators identified by lipid mediator metabololipidomics. (a) Representative chromatographs obtained by Multiple Reaction Monitoring of the parent ion (Q₁) and a diagnostic daughter ion (Q₃) in the MS-MS of n3-DPA resolvins, protectins and maresins. Representative MS-MS spectra used for identification of (b) RvD1_{n-3 DPA}, (c) RvD5_{n-3 DPA}, and (d) PD1_{n-3 DPA}. Results are representative of n = 4.

Figure 4. Self-limited inflammation: Endogenous formation of novel immunoresolvents from n-3 DPA.

Mice were treated with 0.1 mg zymosan i.p.; after the indicated time intervals peritoneal exudates were collected. (a) Exudate leukocyte counts obtained by light microscopy and flow cytometry. Exudate levels for (b) prostaglandin (PG) E₂ and leukotriene (LT) B₄; (c) resolvins, (d) protectins and (e) maresins were measured by lipid mediator metabololipidomics. Results are mean ± SEM. n = 4 mice per time point.

Figure 5. n-3 DPA immunoresolvents display potent anti-inflammatory actions in vivo.

The indicated n-3 DPA-derived product mixtures were administered by intravenous injection 10 min prior to the intraperitoneal administration of zymosan (0.1 mg, 500 μl PBS) to 6–8-week-old male FvB mice. At 4 h, peritoneal exudates were collected and the (a) number of infiltrated neutrophils was assessed by light microscopy and flow cytometry. Exudate levels for the pro-inflammatory mediators (b) IL6 and (c) MCP-1 were determined by cytokine array. The ratio of RvD1_{n-3 DPA} to RvD2_{n-3 DPA} was ~3:1 (A), the ratio of MaR1_{n-3 DPA} to MaR2_{n-3 DPA} was ~4:1 (B); the ratio of RvD5_{n-3 DPA} to PD1_{n-3 DPA} was ~9:1 (C). Results are mean ± SEM. n = 4. * P < 0.05, ** P < 0.01 vs. zymosan mice.

Figure 6. Human neutrophils produce novel n-3 DPA-derived immunoresolvents.

Human neutrophils were prepared from peripheral blood (see Methods for details), suspended in DPBS (80 × 10⁶/ml) and incubated with serum treated zymosan (0.1 mg) and n-3 DPA (1 μM, 30 min, 37°C, pH 7.45); incubations were stopped with ice-cold methanol and products assessed by lipid mediator metabololipidomics. (a) Representative chromatographs obtained by Multiple Reaction Monitoring of the parent ion (Q₁) and a diagnostic daughter ion (Q₃) in the MS/MS. Representative MS/MS spectra used for identification of (b) RvD1_{n-3 DPA} and (c) RvD5_{n-3 DPA}. Results are representative of n = 4.

Figure 7. Reduction in human neutrophil chemotaxis, neutrophil-endothelia cell adhesion and stimulation of macrophage phagocytosis by n-3 DPA-derived immunoresolvents.

(a) Left panel: micrographs depict PKH26-labeled neutrophils adherent to WGA-Alexafluor® 488-labeled HUVEC stimulated with TNF-α (10 ng/ml, 4 h, 37°C, 0.1% FCS) with or without n-3 DPA resolvins (1 nM, 15 min, 37°C, pH7.45; ×40 magnification). Right panel: Fluorescently labeled human neutrophils were incubated with vehicle (0.1% EtOH in PBS) or n-3 DPA products. These were then added to TNF-α-stimulated HUVEC and incubated for 30 min (37°C), non-adherent cells were washed and extent of neutrophil adhesion assessed using a SpectraMax M3 Plate reader. (b) Neutrophils were incubated with vehicle (0.1% EtOH in PBS) or n-3 DPA products (1 nM, 15 min, 37°C, pH7.45) prior to loading on ChemoTx chambers and assessing chemotaxis towards IL-8 (100 ng/ml, 90 min, 37°C, pH7.45). (c) Macrophages were incubated with vehicle (0.1% EtOH in PBS) or n-3 DPA products (1 nM, 15 min, 37°C, pH7.45) prior to addition of fluorescently labeled zymosan (1:10 macrophages to zymosan). After 60 min (37°C, pH7.45), the incubation was stopped, extracellular fluorescence quenched using trypan blue and phagocytosis assessed using a SpectraMax M3 Plate reader. The ratio of RvD1_{n-3 DPA} to RvD2_{n-3 DPA} (A) was ~3:1; the ratio of RvD5_{n-3 DPA} to PD1_{n-3 DPA} (B) was ~9:1; the ratio of PD1_{n-3 DPA} to PD2_{n-3 DPA} (C) was ~1:5; the ratio of MaR1_{n-3 DPA} to MaR2_{n-3 DPA} (D) was ~4:1. Results are mean ± SEM. n = 4 independent neutrophil and macrophage preparations (*P < 0.05; **P < 0.05 vs. vehicle incubated cells). Bar = 50 μM.

Figure 8. Biosynthetic schemes proposed for novel n-3 docosapentaenoic acid products and their actions.

At the site of injury, n-3 DPA is converted to (a) 17-HpDPA that undergoes further conversion by lipoxygenation to the n-3 DPA resolvins. 17-HpDPA is also a substrate for enzymatic conversion to an epoxide intermediate that is next enzymatically hydrolyzed to the n-3 DPA protectin structures. (b) n-3DPA is also converted to 14-lipoxygenation to yield 14-HpDPA that is further converted to an epoxide intermediate and then enzymatically hydrolyzed to MaR1_{n-3 DPA} and/or MaR2_{n-3 DPA}. 14-HpDPA can also undergo a second oxygenation at the omega −1 position to yield the MaR3_{n-3 DPA}. Note that each product is depicted in the 17S and 14S configuration based on the results obtained from chiral lipidomics that indicated S as the predominate form of each but may also carry 17R as well as 14R chirality from lipoxygenase reactions as these lesser components (see Supplementary Fig. 2 and text for details). The complete stereochemistries of these novel mediators remain to be established and are depicted in their likely configuration based on biogenic synthesis (see Supplementary Figures 3–5 for retention times, UV and MS-MS for each of these mediators).