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. 2011 Aug 26;18(8):976-87.
doi: 10.1016/j.chembiol.2011.06.008.

Novel proresolving aspirin-triggered DHA pathway

Charles N Serhan  1 , Gabrielle Fredman, Rong Yang, Sergey Karamnov, Ludmila S Belayev, Nicolas G Bazan, Min Zhu, Jeremy W Winkler, Nicos A Petasis
Affiliations

Novel proresolving aspirin-triggered DHA pathway

Charles N Serhan et al. Chem Biol. .

Abstract

Endogenous mechanisms in the resolution of acute inflammation are of interest because excessive inflammation underlies many pathologic abnormalities. We report an aspirin-triggered DHA metabolome that biosynthesizes a potent product in inflammatory exudates and human leukocytes, namely aspirin-triggered Neuroprotectin D1/Protectin D1 [AT-(NPD1/PD1)]. The complete stereochemistry of AT-(NPD1/PD1) proved to be 10R,17R-dihydroxydocosa-4Z,7Z,11E,13E,15Z,19Z-hexaenoic acid. The chirality of hydroxyl groups and geometry of the conjugated triene system essential for bioactivity were established by matching biological materials with stereochemically pure isomers prepared by organic synthesis. AT-(NPD1/PD1) reduced neutrophil (PMN) recruitment in murine peritonitis in a dose-dependent fashion whereby neither a Δ(15)-trans-isomer nor DHA was effective. With human cells, AT-(NPD1/PD1) decreased transendothelial PMN migration as well as enhanced efferocytosis of apoptotic human PMN by macrophages. These results indicate that AT-(NPD1/PD1) is a potent anti-inflammatory proresolving molecule.

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Figures

Figure 1
Figure 1. Synthetic protectins prepared for lipidomics and matching
Neuroprotectin D1/protectin D1, aspirin-triggered neuroprotectin D1/protectin D1 and related isomers.
Figure 2
Figure 2. Strategy for the total organic synthesis of isomerically pure protectin isomers and their stereochemical assignment by NMR spectroscopy
The total synthesis of AT- (NPD1/PD1) (Panel A) started with the epoxide opening of the R-glycidol derivative 1 by metallated derivatives of alkynes 2 and 3 leading after several steps to the synthesis of key intermediates 4 and 5, which were subjected cross-coupling to form the acetylenic precursor 6, what after selective hydrogenation and ester hydrolysis afforded AT-(NPD1/PD1). The NMR assignments of the olefinic region of three AT-(NPD1/PD1) stereoisomers, are shown in Panels B, C and D, show the anticipated chemical shifts and coupling constants for each isomer.
Figure 3
Figure 3. LC-MS-MS lipidomics and properties of synthetic AT-(NPD1/PD1), NPD1/PD1,and their isomers
MRM chromatogram (m/z 359 > 153) co-injection of synthetic AT- (NPD1/PD1), 10S,17R-AT-(NPD1/PD1) isomer and NPD1/PD1. AT-(NPD1/PD1) and NPD1/PD1 separate in this LC system with retention times of 8.2 and 12.0 min. Representative tandem mass and UV spectra of synthetic AT-(NPD1/PD1) (B), Δ15-trans-NPD1/PD1 (C) and NPD1/PD1 (D).
Figure 4
Figure 4. LC-MS-MS matching: comparisons for biologic and synthetic AT-(NPD1/PD1)
(A) Representative MRM analysis (m/z 359 > 153) of incubations of cells from zymosan A (1mg/mouse) induced peritonitis with exudate taken at 24 h. Cells (50 × 106/ml) were treated with aspirin (2mM, 20 min, 37ºC), and then incubated with DHA (5 μg/mL) and A23187 (5 μM) for 20 min at 37ºC. Panels B (methyl formate fraction obtained from mouse peritonitis exudates) and C are representative MRM analyses (m/z 375 > 153) of synthetic AT-(NPD1/PD1) and coinjection of synthetic AT-(NPD1/PD1). Panels D and E are representative online UV (insets) and tandem mass spectra of exudate AT-(NPD1/PD1) from mouse peritonitis exudates (Panel D) and synthetic AT-(NPD1/PD1) (Panel E). Panel F, representative tandem mass spectrum of AT- (NPD1/PD1) obtained from incubations of isolated human PMN. Human PMN (50 × 106/ml) were incubated with TNFα (100ng/mL, 4h, 37ºC) followed by aspirin (2 mM), DHA (5 μg/ml), and A23187 (5 μM) for 20 min at 37ºC. (n=3)
Figure 5
Figure 5. AT-(NPD1/PD1) reduces leukocyte infiltration in peritonitis: direct comparison to NPD1/PD1
Panels A-D: Peritonitis was initiated by peritoneal injection of 500 ng TNFαalone, TNFαplus synthetic AT-(NPD1/PD1) (0.01 – 10.0 ng) or NPD1/PD1 (0.01 – 10 ng). Peritoneal lavages were obtained at 4 h and leukocytes were enumerated (see Experimental Procedures). (A) Total leukocyte numbers, (B) dose-response: percent reduction of total leukocytes, (C) total PMN numbers and (D) dose-response: percent reduction of PMN infiltration. Representative flow cytometry dot plots (C, inset). TNFα, gray bars; AT- (NPD1/PD1), black bars; NPD1/PD1, white bars. Values are mean + SEM of n=7. Panels E-H: AT-(NPD1/PD1) dose response and direct comparison to DHA. Zymosan A alone (100 μg/mouse), zymosan A plus AT-(NPD1/PD1) (0.01 – 100 ng/mouse) or zymosan A plus DHA (1 and 10 ng/mouse) were injected (i.p.) and lavages were obtained at 4 h. (E and G) Total exudate leukocyte (PMN, monocyte, lymphocyte) numbers, (F and H) total PMN. Panels E and F insets: percent reduction in total leukocytes and PMN. Zymosan A, gray bars; AT-(NPD1/PD1), black bars; DHA, white bars; Δ15-trans-AT-(NPD1/PD1), striped bars. All values mean + SEM of n=3. *p<0.05, **p<0.01, ***p<0.001 versus vehicle; Xp< 0.05, XXp<0.01 AT-(NPD1/PD1) versus NPD1/PD1. Using one-way and two-way ANOVA indicated that AT-NPD1 and NPD1/PD1 were not significantly different from each other, P > 0.05.
Figure 6
Figure 6. AT-(NPD1/PD1) limits human PMN transmigration across human endothelial cells and enhances human macrophage efferocytosis of apoptotic human PMNs
Panel A: Neutrophils (106 cells per monolayer) were exposed to vehicle containing buffer or indicated concentrations of NPD1/PD1 (square), AT-(NPD1/PD1) (triangle), Δ15-trans-AT-NPD1/AT-PD1 (diamond) or DHA (circle) (15 min, pH 7.45, PBS-/-, 37ºC). Transmigration was initiated with addition of 10-8 M LTB4 (90 min, 37ºC). Results are mean ±SEM obtained from 3-5 separate PMN donors, each point in triplicate. Panel B: HUVECs were plated (0.2×106/well) in an ECIS chamber for 24 h. After 24 h, PMN were isolated from human donors and incubated with AT- (NPD1/PD1) or NPD1/PD1 (1 nM) for 15 min, 37ºC. LTB4 (10nM) and PMN (106 cells) were then added to the HUVECS. Impedance changes were monitored using Applied Biophysics ECIS software. (B) Representative real time tracing of impedance changes. Results are mean ± S.E.M. of n=3 separate donors and HUVEC preparations, *p<0.05. Using a two-way ANOVA, NPD1/PD1 was not significantly different from AT-NPD1/PD1 in reducing PMN transendothelial migration, P > 0.05. Panels C and D: Human macrophages (105 per well) were exposed to vehicle containing buffer or indicated concentrations of AT-(NPD1/PD1) (black triangles) or NPD1/PD1 (black squares) (15 min, pH 7.45, PBS -/-, 37ºC). Uptake was initiated with addition of CFDA-labeled human apoptotic PMNs (3×105/well, 60 min, 37ºC). Increase in efferocytosis was determined by monitoring total fluorescence from PMNs; mean ± SEM, *p < 0.05 vs. vehicle, n = 3 healthy subjects.
Figure 7
Figure 7. Proposed biosynthesis scheme for aspirin-triggered protectins and AT- (NPD1/PD1)
The stereochemistry and anti-inflammatory-proresolving bioactions of the members of this pathway are determined (see text for details). The stereochemistry of the epoxide intermediate is shown in tentative configuration.
See this image and copyright information in PMC

Comment in

  • Aspirin-triggered metabolites of EFAs.
    Makriyannis A, Nikas SP. Makriyannis A, et al. Chem Biol. 2011 Oct 28;18(10):1208-9. doi: 10.1016/j.chembiol.2011.10.005. Chem Biol. 2011. PMID: 22035788 Free PMC article.

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