Resolvin D3 and aspirin-triggered resolvin D3 are potent immunoresolvents

Abstract

Resolvins are a family of n-3 lipid mediators initially identified in resolving inflammatory exudates that temper inflammatory responses to promote catabasis. Here, temporal metabololipidomics with self-limited resolving exudates revealed that resolvin (Rv) D3 has a distinct time frame from other lipid mediators, appearing late in the resolution phase. Using synthetic materials prepared by stereocontrolled total organic synthesis and metabololipidomics, we established complete stereochemistry of RvD3 and its aspirin-triggered 17R-epimer (AT-RvD3). Both synthetic resolvins potently regulated neutrophils and mediators, reducing murine peritonitis and dermal inflammation. RvD3 and AT-RvD3 displayed leukocyte-directed actions, e.g., blocking human neutrophil transmigration and enhancing macrophage phagocytosis and efferocytosis. These results position RvD3 uniquely within the inflammation-resolution time frame to vantage and contribute to the beneficial actions of aspirin and essential n-3 fatty acids.

Figure 1. Endogenous biosynthesis of RvD3 and its relation to other lipid mediators in inflammation-resolution

Mice were injected i.p. with zymosan (1mg/mouse) and lavages collected at the indicated intervals. (A) Total cell counts in the peritoneal exudates were determined by light microscopy and the number of mononuclear cells and PMN determined by flow cytometry. Lipid mediators in exudates were assessed using LC-MS-MS metabololipidomics following solid phase extraction (see Experimental Procedures). Exudate levels for (B) prostaglandins and leukotrienes; (C) D-series resolvins. Results are mean ± SEM. n=3–4 mice per time point.

Figure 2. Synthetic RvD3/AT-RvD3 stereoisomers

These isomers were prepared in enantiomerically pure form via stereocontrolled total synthesis and were fully characterized by NMR spectroscopy. (A) Structures of the isomers of RvD3/AT-RvD3 and color-coded illustration depicting the origin of R or S stereochemistry of each chiral alcohol group from stereochemically pure precursors (Winkler et al., 2013). (B) Assignment of the Z or E stereochemistry for each C=C bond using 2-dimensional NMR spectroscopy. The shown ¹H-¹H gCOSY spectrum of a solution of RvD3 in CD₃OD [9.6x10⁻³M] was acquired using a Varian VNMRS 600 MHz NMR spectrometer at 25°C on a 5mm Triple Resonance PFG ¹H and referenced to the CD₃OD and an internal standard. This spectrum depicts all of the connectivities between adjacent alkenyl hydrogens (H₅-H₁₀, H₁₃-H₁₆, H₁₉-H₂₀). The colors denote a bitmap plotting method using a rainbow palette that gives depth to the positive and negative contours. The complete identification of each H-atom using this in combination with its corresponding constants (J values) permitted the unambiguous E/Z assignment of all alkenyl hydrogens (see Results).

Figure 3. Endogenous RvD3 and AT-RvD3 from resolving inflammatory exudates match synthetic compound

(A) Endogenous RvD3 was obtained from mice injected with zymosan (1mg/mouse) and exudates collected at 4h. These were subjected to lipid mediator metabololipidomics. Selected ion chromatograms (m/z 375 – 147) depicting murine resolving exudate-derived RvD3. (B) Endogenous AT-RvD3 obtained from mice administered aspirin (500μg) and zymosan (1 mg). (C) Synthetic Isomers 1 and 2. (D) co-injection of resolving exudate endogenous RvD3 with synthetic Isomer 1 (Inset: characteristic UV-absorption spectrum) (see also Figs. S2, S3 and Table S1). (E) Co-injection of resolving exudate endogenous AT-RvD3 with synthetic Isomer 2 (Inset: characteristic UV-absorption spectrum). MS-MS spectrum for (F) endogenous RvD3 (T_R=7.4 min); (G) endogenous AT-RvD3 (T_R=7.2 min); (H) synthetic RvD3 (T_R=7.4 min); (I) synthetic AT-RvD3 (T_R=7.2min) (see also Fig. S1). Representative MRM chromatograms and MS-MS spectra (n=4).

Figure 3. Endogenous RvD3 and AT-RvD3 from resolving inflammatory exudates match synthetic compound

(A) Endogenous RvD3 was obtained from mice injected with zymosan (1mg/mouse) and exudates collected at 4h. These were subjected to lipid mediator metabololipidomics. Selected ion chromatograms (m/z 375 – 147) depicting murine resolving exudate-derived RvD3. (B) Endogenous AT-RvD3 obtained from mice administered aspirin (500μg) and zymosan (1 mg). (C) Synthetic Isomers 1 and 2. (D) co-injection of resolving exudate endogenous RvD3 with synthetic Isomer 1 (Inset: characteristic UV-absorption spectrum) (see also Figs. S2, S3 and Table S1). (E) Co-injection of resolving exudate endogenous AT-RvD3 with synthetic Isomer 2 (Inset: characteristic UV-absorption spectrum). MS-MS spectrum for (F) endogenous RvD3 (T_R=7.4 min); (G) endogenous AT-RvD3 (T_R=7.2 min); (H) synthetic RvD3 (T_R=7.4 min); (I) synthetic AT-RvD3 (T_R=7.2min) (see also Fig. S1). Representative MRM chromatograms and MS-MS spectra (n=4).

Figure 4. RvD3 is a potent anti-neutrophil and cytokine regulator

RvD3, AT-RvD3 (10 ng/mouse) or vehicle (saline containing 0.1% EtOH) was administered i.v. 10 min prior to i.p. injection of zymosan (1mg/mouse). Exudates were collected 4 h later. (A) Cells were enumerated and PMN identified using flow cytometry. (B) IL-6 and (C) IL-10 levels were measured in peritoneal exudates. Results are mean ± SEM, n = 4 mice per group. * p <0.05 and *** p <0.001 vs. zymosan plus vehicle. Dorsal skin inflammation (see Experimental Procedures): Mice were injected on Day 6 with mrTNF-α (100 ng/mouse) following administration of RvD3, AT-RvD3 (10 ng) or vehicle (saline containing 0.1% EtOH) by intrapouch injection, and at 4h lavages obtained. (D) PMN by flow cytometry. Levels of (E) MCP-1, (F) IL-6 and (G) KC. Results are mean ± SEM, n = 4 mice per group. * p <0.05; ** p <0.01 vs. TNF-α plus vehicle.

Figure 5. RvD3 and AT-RvD3 reduce local prostanoids and leukotrienes in acute inflammation

Lipid mediators in peritoneal exudates collected 4h after zymosan administration were assessed using LC-MS-MS metabololipidomics following solid phase extraction. (A) Representative multiple reaction monitoring chromatograms (MRM) of selected ion pairs for arachidonic acid derived eicosanoids. a = 6-trans-LTB₄ and b = 6- trans, 12-epi-LTB₄. (B) Representative MS-MS spectra with diagnostic ions employed for the identification of TxB₂ and PGE₂ (see Experimental Procedures). Quantification of exudate lipid mediators following (C) RvD3 and (D) AT-RvD3 administration. Results are mean ± SEM, n = 4 mice per group. * p <0.05, ** p <0.01 vs. zymosan plus vehicle.

Figure 6. RvD3 and AT-RvD3 pro-resolving actions

(A) Human neutrophils were labeled with CFDA and incubated with RvD3, AT-RvD3 or vehicle (DPBS containing 0.1% EtOH) for 15 min (37 °C) prior to assessing their transmigration across HUVEC exposed to TNF-α (10 ng/ml). Results are representative of n = 4 distinct PMN preparations. (B-D) Increased macrophage phagocytosis and efferocytosis. Murine peritoneal resident macrophages were incubated with (B,C) RvD3 (black square), AT-RvD3 (open circle) (0.1 pM – 10 nM) or (D) 1 nM of select SPM (15 min, 37°C), followed by addition of (B) FITZ-zymosan or (C,D) CDFA-labeled apoptotic PMN for 1h (see also Fig. S4). Results are mean ± SEM of n=4, d=3–4. *p<0.05, ** p <0.01, *** p <0.001 vs. vehicle; ^# p <0.05, RvD3 vs. AT-RvD3; ⁺ p <0.05, vs. RvD1.

Figure 7. RvD3 and AT-RvD3 activate human GPR32

(A-D) Ligand–receptor-dependent changes in impedance with CHO cells overexpressing human GPR32. Impedance was continuously recorded with real-time monitoring across cell monolayers using an ECIS system. Results are tracings obtained from incubations of RvD1, RvD3 or AT-RvD3 (100 nM each) with CHO-GPR32 cells (A), or in the presence of anti-GPR32 Ab or non-immune rabbit serum (B-D). Results are (A) expressed as mean of 4 or (B-D) representative of 3 separate experiments. (E-F) Human GPR32 or mock-transfected human macrophages were incubated with RvD3 or AT-RvD3 (0.1 pM – 10 nM) for 15 min, followed by addition of FITZ-zymosan (60 min, 37°C). Results are expressed as mean ± SEM; n=4 macrophage preparations. * p <0.05, macrophages (MΦ) plus GPR32 vs. MΦ plus mock (see also Fig. S5).

Figure 8. DHA Resolution Metabolome

Biosynthetic scheme for D-series Resolvins and their relation to Protectins and Maresins. The position of RvD3 is depicted and that of the aspirin-triggered RvD3 within the D-series resolvins. Note that the complete stereochemistry of RvD1, RvD2 and RvD3 are established as shown. See text for further details.