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. Author manuscript; available in PMC: 2014 Feb 21.
Published in final edited form as: Chem Biol. 2013 Feb 21;20(2):188–201. doi: 10.1016/j.chembiol.2012.11.010

Resolvin D3 and Aspirin-Triggered Resolvin D3 Are Potent Immunoresolvents

Jesmond Dalli 1, Jeremy W Winkler 2, Romain A Colas 1, Hildur Arnardottir 1, Chien-Yee C Cheng 1, Nan Chiang 1, Nicos A Petasis 2,*, Charles N Serhan 1,*
PMCID: PMC3583372  NIHMSID: NIHMS428424  PMID: 23438748

Summary

Resolvins are a new 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 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.

Introduction

In response to stress and/or invasion, acute inflammation is host-protective (Medzhitov, 2010) and, when uncontrolled, unresolved inflammation is linked to many widely occurring diseases (Dinarello, 2010; Majno et al., 1982; Nathan and Ding, 2010; Tauber and Chernyak, 1991). The ideal outcome for an acute inflammatory response is complete resolution. In this context, in self-limited inflammatory exudates we identified potent local mediators within the resolution phase that possess novel anti-inflammatory and pro-resolving actions coined specialized pro-resolving mediators that include the resolvins, protectins and maresins (for reviews, see (Serhan, 2007; Serhan and Petasis, 2011)). Thus, within the initiation phase, chemical mediators such as prostaglandins, leukotrienes, cytokines and chemokines dictate the leukocyte influx traffic (Dinarello, 2010; Samuelsson, 2012) that is actively counter regulated by pro-resolving mediators, which orchestrate the resolution phase, catabasis and the return to homeostasis (Serhan, 2007).

The resolvins are biosynthesized from essential omega-3 fatty acids (Serhan, 2007; Serhan et al., 2002; Serhan and Petasis, 2011). The E-series resolvins, i.e., resolvin E1, resolvin E2 and resolvin E3 (Isobe et al., 2012), are produced from eicosapentaenoic acid (EPA) (reviewed in (Serhan, 2007; Serhan and Petasis, 2011)). D-series resolvins including RvD1 (Sun et al., 2007), RvD2 (Spite et al., 2009), RvD5 (Chiang et al., 2012) and their aspirin-triggered versions are biosynthesized from docosahexaenoic acid (DHA) (Serhan et al., 2002). Each resolvin possesses potent pro-resolving actions that include limiting neutrophil tissue infiltration, counter-regulation of chemokines and cytokines, reduction in pain and stimulation of macrophage-mediated actions (i.e. efferocytosis, bacterial and debris clearance). Given their unique mechanism of pro-resolving actions, resolvins demonstrate potent actions in animal disease models (reviewed in ref. Serhan and Petasis, 2011). Like many other autacoids, the actions of resolvins are stereochemically selective, reflecting their routes of biosynthesis. The classic eicosanoids, for example, each carry well established stereoselectivity in their actions (Samuelsson, 2012; Shimizu, 2009). Hence, establishing the complete stereochemical assignment for each of the separate resolvin structures is of considerable interest.

The original identification of the D-series resolvins reported the structural elucidation of several distinct bioactive structures denoted resolvin D1 through resolvin D6 in resolving murine exudates, their biosynthesis by isolated human leukocytes and potent actions in vivo in murine as well as human acute inflammation (Serhan et al., 2002). From the basic structures of the D-series resolvins it was deemed important to establish each of their complete stereochemical assignments to permit further mass spectral quantitative methods for in vivo studies as well as confirmation and extension of their roles in inflammation and active resolution. Along these lines, we determined the complete stereochemical assignments of RvD1 as 7S,8R,17S-trihydroxydocosa-4Z,9E,11E,13Z,15E,19Z-hexaenoic acid and its aspirin-triggered AT-(17R epimer)-RvD1 as 7S,8R,17R-trihydroxydocosa-4Z,9E,11E,13Z,15E,19Z-hexaenoic acid employing a stereocontrolled total organic synthesis approach that permitted confirmation of RvD1 potent anti-inflammatory and pro-resolving actions (Serhan and Petasis, 2011; Sun et al., 2007) as well as document RvD1 production by human neutrophils, blood, trout hematopoietic organs and in brain injury (Hong et al., 2007). Hence, RvD1 actions were confirmed and extended to controlling pain (Ji et al., 2011; Lima-Garcia et al., 2011; Xu et al., 2010), reducing airway inflammation (Rogerio et al., 2012), murine colitis (Bento et al., 2011) and stimulating resolution mechanisms in vivo in mice and with human macrophages in vitro (Recchiuti et al., 2011) as well as RvD1 identification in human blood (Mas et al., 2012; Psychogios et al., 2011). For resolvin D2, establishing its complete stereochemistry as 7S,16R,17S-trihydroxydocosa-4Z,8E,10Z,12E,14E,19Z-hexaenoic acid permitted the identification of resolvins’ ability to enhance the innate immune response without rendering immune suppression of the host (Spite et al., 2009). This also enabled the elucidation of the potent protective and pro-resolving actions of RvD2 in colitis (Bento et al., 2011), in reducing pain (Ji et al., 2011) and its presence in both human tissue (Mas et al., 2012) and salmon (Raatz et al., 2011).

The basic structure 4S,11,17S-trihydroxydocosa-5Z,7,9,13,15E,19Z-hexaenoic acid identified in murine exudates and with human leukocytes was denoted resolvin D3 (RvD3) (Serhan et al., 2002). By definition, resolvin D3 novel actions include potent reduction of neutrophil infiltration in vivo in both murine peritonitis and dorsal skin pouches. RvD3 also reduced human neutrophil transendothelial migration (Serhan et al., 2002), each cardinal actions of a pro-resolving mediator. Of interest, RvD3 from endogenous sources of DHA is elevated in colitis in fat-1 transgenic mice that overexpress the enzyme that increases tissue levels of n-3 essential fatty acids without feeding DHA or EPA (Hudert et al., 2006). Also, RvD3 produced from endogenous DHA is elevated in ischemic injury of the kidney (Duffield et al., 2006). Given these properties, we’ve focused on establishing RvD3’s complete stereochemistry and temporal positioning within inflammation-resolution. Here, we report the stereochemical assignments for both RvD3 and AT-RvD3 and their potent anti-inflammatory and pro-resolving actions using synthetic materials, establishing the potent actions of these new members of the D-series resolvins.

Results

RvD3 is uniquely positioned within the resolution frame of inflammation

Self-limited inflammation results in a rapid increase in infiltration of neutrophils and their eventual loss from the tissue (Fig. 1A). This is accompanied by a concomitant non-phlogistic increase in mononuclear cells. In this context, the resolution interval (Bannenberg et al., 2005) was ~11 h. Each cell population was identified using flow cytometry and light microscopy (see inset for 12 and 24 h). This system is an ideal example of the temporal relationship from initiation to resolution (Bannenberg et al., 2005). In this context, prostaglandins are rapidly produced (Fig. 1B) and, concomitant with neutrophil infiltration into the tissue, leukotriene B4 and PGE2 levels reach maximum within 4 h. LTB4 levels rapidly drop and return to essentially baseline within 12 h, whereas the levels of both PGE2 and PGD2 persist into the resolution phase, where they were shown earlier to stimulate the production of pro-resolving mediators by stimulating the transcriptional regulation of a key enzyme in this process, human 15-lipoxygenase type 1 (Levy et al., 2001).

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

Figure 1

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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.

Using this setting, which amply qualifies initiation and resolution phase, we next determined the profile of D-series resolvins within the DHA metabolome in order to temporally stage each (Fig. 1C). As anticipated, endogenous production of resolvins D1, D2 and D5 lagged behind appearance of the leukotrienes and was coincident with the resolution phase as determined from maximal PMN timepoint (Tmax), to the 50% reduction in PMN (T50), defining the resolution interval (Ri). Unexpectedly, the levels of resolvin D3 increased at 24 h post-initiation that persisted to 72 h, well into the resolution timeframe of the local inflammatory response. Thus, endogenous production of D-series resolvins gave a distinct separation between resolvins D1 and D2 accumulation early in the resolution phase and the persistence of resolvin D3, which gives a unique position within the resolution phase. Hence, we focused our attention on resolvin D3.

Synthetic RvD3 and AT-RvD3

During the initial isolation and structural characterization of RvD3 and AT-RvD3 (Serhan et al., 2002), the E/Z stereochemistry of all C=C bonds and the R/S chirality of the hydroxyl groups at C4 and C11 could not be determined since these structures were deduced and proposed using results from LC-MS-MS and GC-MS, which does not permit direct determination of stereochemical nature of these molecules. In order to fully elucidate the stereochemical assignments of these mediators, we devised a stereocontrolled synthetic approach to obtain several potential stereoisomers of these molecules (Winkler et al., 2013). The complete structures of two synthetic RvD3 isomers, the 17S-isomer 1 and the epimeric 17R-isomer 2 which were obtained in stereochemically pure form, are shown in Fig. 2A. The complete structure and stereochemistry for both isomers were unambiguously established through the stereocontrolled synthetic approach and were confirmed via spectroscopic analysis. The R/S chirality for each of the three OH groups (Fig. 2A) was ensured by starting with enantiomerically pure starting materials with the same chirality and by employing synthetic transformations that retain this chirality throughout the synthesis. The Z/E geometry for each of the C=C bonds was secured by employing highly stereocontrolled processes that produce the desired geometry in each position. Further structural confirmation and unambiguous assignment of the Z/E geometry of each C=C bond for both isomer 1 and isomer 2 were established via 2D COSY NMR spectroscopy (Fig. 2B) (see Experimental Procedures).

Figure 2. Synthetic RvD3/AT-RvD3 stereoisomers.

Figure 2

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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).

Matching of endogenous RvD3 and AT-RvD3 with synthetic materials

Since both RvD3 and its aspirin triggered epimer are produced in murine inflammatory exudates (Duffield et al., 2006; Serhan et al., 2002) it was important to establish the complete stereochemistry as well as confirm its assigned structure and actions with synthetic material prepared by stereocontrolled total organic synthesis. To this end authentic RvD3 and its aspirin-triggered form (AT-RvD3) were obtained from endogenous DHA in murine resolving exudates treated with or without aspirin and chromatographed using LC-MS-MS based metabololipidomics (Fig. 3A,B) for direct comparison with synthetic material (Fig. 3C). Authentic RvD3 from both murine exudates (Fig. 3A,B) and human macrophages (Fig. S1A) gave a sharp peak in liquid chromatography, retention time (TR) = 7.4 min, clearly separating from AT-RvD3 that produced a sharp peak TR= 7.2 min. LC-MS-MS of the synthetic isomers demonstrated that isomer 1 gave a sharp peak [TR= 7.4 min] and isomer 2 eluted at TR= 7.2 min (Fig. 3C). Co-injection with material prepared by total organic synthesis and authentic RvD3 demonstrated co-elution with isomer 1 at TR= 7.4 min (Fig. 3D). Similarly, co-injection of synthetic isomer 2 with authentic AT-RvD3 obtained from aspirin-treated inflammation demonstrated co-elution TR = 7.2 min (Fig. 3E). Moreover, the two synthetic compounds contained combined characteristic conjugated triene and diene chromophores with triplet absorbance bands at λmaxMeOH ~260, 271, 281 nm for the triene and ~236 nm band for the diene component (Fig. 3D,E insets).

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

Figure 3

Figure 3

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(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 (TR=7.4 min); (G) endogenous AT-RvD3 (TR=7.2 min); (H) synthetic RvD3 (TR=7.4 min); (I) synthetic AT-RvD3 (TR=7.2min) (see also Fig. S1). Representative MRM chromatograms and MS-MS spectra (n=4).

Of interest, although the two RvD3 epimers displayed distinct chromatographic behavior, they both demonstrated essentially identical fragmentation patterns (Fig. 3F-I). Each diastereomer gave m/z 375 = M-H, m/z 357 = M-H-H2O, m/z 339 = M-H-2H2O, m/z 313 = M-H-CO2-H2O, m/z 295 = M-H-CO2-2H2O, m/z 259 = 277-H2O, m/z 215 = 249-CO2, m/z 177 = 195-H2O, m/z 159 = 195-2H2O, m/z 147 = 165-H2O and m/z 137 = 181-CO2.

To obtain further evidence for matching of RvD3, GC-MS was performed as in the original identification and basic structural elucidation (Serhan et al., 2002). RvD3 was treated with diazomethane and subsequently converted to its corresponding trimethylsilyl derivative (Fig. S2A) and subjected to GC-MS. The retention time in GC-MS for synthetic RvD3 corresponded to a C-value of 27.9 (Fig. S2B), while the mass spectrum (Supplemental Fig. 2C and Table 1) demonstrated fragmentation and ions consistent with the proposed structure of RvD3 (Serhan et al., 2002) and matched those of the derivatized synthetic materials (see Experimental Procedures). AT-RvD3 demonstrated properties similar to RvD3 (Fig. S3) with a retention time of 20.5 min (Supplemental Fig. S3B) corresponding to a C-value of 27.9 and displaying essentially an identical fragmentation pattern to RvD3 in the GC-MS (Fig. S3C). Therefore, the complete stereochemistry of RvD3 was proved to be 4S, 11R, 17S-trihydroxydocosa-5Z, 7E, 9E, 13Z, 15E, 19Z-hexaenoic acid (isomer 1, Fig. 3A,F) and that for AT-RvD3 was 4S, 11R, 17R-trihydroxydocosa-5Z, 7E, 9E, 13Z, 15E, 19Z-hexanoic acid (isomer 2, Fig. 3B,G).

RvD3 and AT-RvD3 exert potent anti-neutrophil actions

After matching the synthetic materials with endogenously produced mediators and establishing the stereochemistry of RvD3 and AT-RvD3, we required confirmation of their potent anti-inflammatory actions. Systemic administration of RvD3 and its aspirin triggered epimer in vivo immediately prior to zymosan (1mg) challenge at doses as low as 10 ng/mouse significantly reduced the number of transmigrated neutrophils into the peritoneal cavity (~43%; Fig. 4A). Assessment of exudate cytokine and chemokine levels demonstrated a significant downregulation of the pro-inflammatory cytokine IL-6 (Fig. 4B) and an increase in levels of the pro-resolving cytokine IL-10 (Fig. 4C).

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

Figure 4

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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.

The biosynthesis and action of specialized pro-resolving mediators is regulated both temporally and spatially (Serhan and Savill, 2005), therefore we next assessed the actions of the synthetic RvD3 diastereomers in murine dorsal air-pouches. Local administration of RvD3 significantly reduced neutrophil transmigration in response to TNF-α-initiated acute inflammation (~65% reduction; Fig. 4D). Assessment of inflammatory exudate cytokine and chemokine levels demonstrated a significant reduction in MCP-1 (~70%), IL-6 (~90%) and KC (~50%) (Fig. 4E-G). Similarly, AT-RvD3 potently reduced PMN recruitment into the pouch (~75%; Fig. 4D), while also reducing exudate pro-inflammatory cytokine and chemokine levels (Fig. 4E-G). Together these results demonstrate the potent local and systemic anti-inflammatory actions of RvD3 and AT-RvD3 counter-regulating local pro-inflammatory mediator production and dampening leukocyte recruitment.

RvD3 and AT-RVD3 counter-regulate eicosanoids

Given that pro-resolving lipid mediators also regulate eicosanoid biosynthesis (Spite et al., 2009) we next assessed the ability of systemically delivered RvD3 as well as AT-RvD3 to regulate local eicosanoid production in vivo. We performed lipid mediator metabololipidomics using exudates obtained at 4 h after zymosan administration to identify potential mechanism(s) underlying the actions of systemically administered RvD3 and AT-RvD3. In these experiments, we targeted and identified the following eicosanoids: prostaglandin (PG)D2, PGE2 and thromboxane (Tx)B2 from the cyclooxygenase pathways and LTB4 from the 5-lipoxygenase (LOX) pathway (Fig. 5A). Identifications were validated using published criteria (Dalli and Serhan, 2012) as illustrated for TxB2 and PGE2 (Fig. 5B). Using multiple reaction monitoring (MRM), we quantified individual mediator levels and found that systemically delivered RvD3 significantly reduced LTB4 (~80%), PGD2 (~67%) and TxB2 (~50%) while stimulating the production of PGE2 (Fig. 5C). AT-RvD3 reduced local LTB4, PGD2 and TxB2 levels while increasing PGE2 (Fig. 5D). Together these results demonstrate that both synthetic RvD3 and AT-RvD3 possess potent anti-inflammatory actions regulating local eicosanoid levels, characteristic of specialized pro-resolving mediators.

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

Figure 5

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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.

RvD3 and AT-RvD3 exert potent pro-resolving actions

Having found that both RvD3 and AT-RvD3 exert potent anti-inflammatory and pro-resolving actions in murine systems in vivo, we next investigated their actions with human leukocytes. Exposure of human PMN to RvD3 or AT-RvD3 at concentrations as low at 10−11 M reduced transmigration ~25% (Fig. 6A). AT-RvD3 displayed greater anti-migratory properties at 1 and 10 pM (p < 0.05). We next assessed the ability of both RvD3 and its aspirin triggered form to stimulate macrophage phagocytosis, a key step in the resolution of inflammation (Serhan and Savill, 2005). Incubation of murine naive peritoneal macrophages with RvD3 or AT-RvD3 dose dependently increased macrophage phagocytosis of zymosan particles (Fig. 6B). At 10 and 100 pM, AT-RvD3 was more potent than RvD3. Together these results demonstrate that both RvD3 epimers exert potent anti-inflammatory and pro-resolving actions with leukocytes.

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

Figure 6

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(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.

Since removal of apoptotic cells and cellular debris, a process termed efferocytosis, is a cellular hallmark of tissue resolution (Serhan and Savill, 2005), we next assessed whether RvD3 and its aspirin-triggered epimer stimulated murine macrophage efferocytosis, a cardinal pro-resolving action essential for complete resolution. Synthetic RvD3 and AT-RvD3 were each found to potently stimulate macrophage efferocytosis of apoptotic human PMN (Fig. 6C and Fig. S4A). Next, we determined the rank order potencies of specialized pro-resolving mediators (SPM) and found that RvD3 and PD1 at 1 nM were the most potent in stimulating efferocytosis with both mouse bone marrow and resident macrophages, followed by MaR1, RvD2 and RvD1 (Fig. 6D and Fig. S4B). These results demonstrate and confirm the potent anti-inflammatory and pro-resolving actions defining RvD3 and AT-RvD3 with synthetic materials, human and mouse leukocytes as well as reducing levels of pro-inflammatory cytokines, i.e. MCP-1, IL-6 and KC (Fig. 4).

Activation of human GPR32

We next tested whether RvD3 activates the human RvD1 receptor (DRV1) GPR32, since we identified a specific G protein-coupled receptor (GPCR) denoted GPR32 that is activated by RvD1 (Krishnamoorthy et al., 2012; Krishnamoorthy et al., 2010) and RvD5 (Chiang et al., 2012). We employed Electrical Cell Substrate Impedance Sensing (ECIS), which monitors impedance changes upon ligand binding to receptors (Krishnamoorthy et al., 2012). In this system, RvD3 (-25.8 ± 5.0 Ω) and AT-RvD3 (−20.7 ± 3.6 Ω) at 100 nM each elicited rapid changes in impedance with CHO cells expressing recombinant human GPR32 (Fig. 7A), to a similar magnitude as RvD1 (−19.1 ± 1.8 Ω) (Krishnamoorthy et al., 2012). These changes in impedance were reduced when cells were incubated with anti-human GPR32 antibody (30 min, 37°C) prior to the addition of RvD1, RvD3 or AT-RvD3 (Fig. 7B–D).

Figure 7. RvD3 and AT-RvD3 activate human GPR32.

Figure 7

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(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).

To further examine ligand-receptor relationships, we used a beta-arrestin reporter system (Krishnamoorthy et al., 2012), which confirmed that both RvD3 and AT-RvD3 directly activated this receptor (0.1 pM-10 nM; Fig. S5A). In addition, phagocytic activity with human macrophages increased by RvD3 and AT-RvD3 proved to be dependent on GPR32, since phagocytosis was significantly enhanced with GPR32 overexpression compared to mock transfected cells (Fig. 7E-F and Fig. S5B,C). Together, these results demonstrated that RvD3 and AT-RvD3 each activated human GPR32, which contributes to their pro-resolving actions in stimulating macrophage uptake of microbial particles (Fig. 7E–F).

Discussion

Mounting evidence indicates that resolution of acute inflammation is an active process where lipid mediators play a pivotal role (Serhan, 2011). In this regard, the resolvins have emerged as uniquely positioned within the resolution phase of acute inflammation to actively counter-regulate the initiation signals as well as stimulate specific pro-resolving responses. Initiation of an inflammatory response involves the orchestrated trafficking of leukocytes from peripheral blood into the tissue. The chemical mediators involved are lipid mediators such as the eicosanoids, prostaglandins and leukotrienes (Samuelsson, 2012; Shimizu, 2009) and the pro-inflammatory cytokines and chemokines (Dinarello, 2010). The stereoselective actions are a hallmark of the lipid-derived mediators, since they evoke potent and cell type-specific actions (Coulthard et al., 2012; Samuelsson, 2012). Hence, given the potent actions of the D-series resolvins including resolvins D1 and D2, it was deemed important to establish the when-and-where, i.e. temporal relationships and actions of resolvin (Rv) D3 within inflammation-resolution.

In the present report, we establish the complete stereochemistry of RvD3 and its aspirin-triggered 17R epimer, AT-RvD3. Within the time course of self-limited local inflammatory response, RvD3 stood apart from the other D-series resolvins in that it remained elevated well into late resolution phase. The complete stereochemistry of RvD3 derived from mouse exudates as well as human leukocytes was matched to synthetic materials defined by stereocontrolled organic synthesis and NMR. The complete structure of RvD3 proved to be 4S,11R,17S-trihydroxydocosa-5Z,7E,9E,13Z,15E,19Z-hexaenoic acid, and its aspirin-triggered epimer 4S,11R,17R-trihydroxydocosa-5Z,7E,9E,13Z,15E,19Z-hexaenoic acid (Figs. 1–3). Both RvD3 and its aspirin-triggered epimer potently counter-regulate pro-inflammatory mediators as well as limit neutrophil infiltration in vivo. Translating to human cells both RvD3 and AT-RvD3 blocked human neutrophil transmigration across endothelial cells (Fig. 6), a response pivotal to regulating the size of the inflammatory exudate in situ and potential collateral tissue damage (Majno et al., 1982). Importantly, RvD3 proved to be a potent enhancer of the uptake of apoptotic neutrophils by macrophages as well as a stimulator of IL-10. Together, these results establish the structural assignments and the elucidation of the isolated bioactive RvD3 and AT-RvD3 (Serhan et al., 2002).

These actions of RvD3 are the hallmark responses defining a pro-resolving mediator and immunoresolvent (Serhan, 2007, 2011). Resolvin D3 fulfilled these defining criteria as well as proved to be among the most potent of the resolvins identified to date within inflammatory exudates. It is now widely acknowledged that pro-inflammatory mediators when produced in excess can disrupt normal resolution that can lead to chronic inflammation (Serhan and Savill, 2005) and even to an eicosanoid storm via rapid activation of the inflammasome (von Moltke et al., 2012). Active containment via enhanced phagocytosis of microbes as well as the efferocytosis of apoptotic PMN leads to the clearance of invaders and cellular debris to return to homeostasis (Chiang et al., 2012). Having established the pro-resolving bioaction, complete stereochemistry, and synthesis of RvE1, RvD1 and RvD2 as well as PD1 (reviewed in Serhan and Petasis, 2011) permitted demonstration of their potent actions by others in many diverse systems, including colitis (Bento et al., 2011), controlling pain (Ji et al., 2011) as well as regulating important processes such as macrophage polarization and promoting resolution of adipose tissue inflammation in obesity (Titos et al., 2011).

Resolvin D3 is distinguished from resolvins D1 and D2 by its late appearance in vivo, promoting pro-resolving actions with human leukocytes as well as in vivo (Figs. 4–7, 8). Given the strategic ‘late appearance’ of RvD3 during resolution of inflammation (Fig. 1), it is very likely that RvD3 is produced by macrophage sub-types known to appear late within the resolution phase of self-limited response to challenge (Stables et al., 2011). Figure 8 depicts the DHA-resolution metabolome and the position of RvD3 within the resolvin biosynthesis pathway and its relation to other families of DHA-derived bioactive mediators, i.e. protectins and maresins, and resolvin family members. These late resolution-phase macrophages display high levels of 15-LOX and hence the capacity to produce pro-resolving mediators (Dalli and Serhan, 2012; Stables et al., 2011).

Figure 8. DHA Resolution Metabolome.

Figure 8

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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.

Using metabolomic approaches, as in the present study (Fig. 1), has led to identification of previously unexplored biochemical pathways in inflammation and its resolution as well as in many other disease processes (Levy et al., 2001; Patti et al., 2012; Serhan et al., 2000). We’ve used this approach to profile the mediators and cell types within the initiation and resolution response (Fig. 1), demonstrating that there is a clear temporal dissociation between the initiation and resolution phase of self-limited acute inflammatory response, self-limited in that it resolves on its own. We also devised quantitative indices in order to evaluate substances that regulate the magnitude and duration of the inflammatory response and its subsequent resolution (Bannenberg et al., 2005). Thus, if an acute inflammatory response does not resolve, the potential for it to progress to chronic inflammation and/or abscess formation may lie in the extent of the presence of pro-inflammatory mediators or consequently the potential loss and/or delay in the accumulation of pro-resolving mediators.

In murine peritonitis, the production of both PGE2 and LTB4 anteceded the production of D-series resolvins. Importantly, PGD2 and PGE2 levels linger during the time course of resolution (Fig. 1B), and as demonstrated earlier PGE2 and PGD2 in isolated human leukocytes activate the translation of messenger RNA to 15-LOX type I, a pivotal enzyme in production of lipoxins (reviewed in Romano, 2010) as well as D-series resolvins (Fig. 1C). The finding that RvD3 persists well into the resolution phase and the return to homeostasis suggests that, in addition to counter-regulating pro-inflammatory-initiating chemical mediators such as the prostaglandins and cytokines and regulating leukocyte traffic and functions as demonstrated herein, RvD3 can also play a role in other metabolic processes relevant to homeostasis.

Resolvins regulate target cell expression of pro-inflammatory cytokines at transcriptional as well as at translational levels by interacting with specific receptors on the surface of leukocytes (Chiang et al., 2012). Pro-resolving mediators including resolvins also regulate adhesion molecules (i.e. CD11b/CD18) on leukocytes that down-regulate cell-cell interactions (Dona et al., 2008; El Kebir et al., 2012). Although not formally addressed here in the context of RvD3, the reduction in pro-inflammatory cytokines and eicosanoids (i.e. LT) by this mediator along with its aspirin-triggered form are likely to also reflect the regulation of cell-cell interactions as is the case with lipoxins, which reduce cell adhesion molecules, cell-cell interactions and transcellular biosynthesis of LM mediators (Brady and Serhan, 1992).

Aspirin, in addition to having an anti-thrombotic role and ability to block platelet thromboxane production (Samuelsson, 2012), was the lead agent used to produce nonsteroidal anti-inflammatories that could also block prostaglandin production (Vane, 1982). The well-appreciated anti-thrombotic properties of aspirin were revealed via the structural elucidation of thromboxane A2 (Samuelsson, 1982). However, the anti- inflammatory actions of low-dose aspirin (75–81 mg doses) and its anti-inflammatory capacity with anti-neutrophil activities were not appreciated until the aspirin-triggered pathways for novel anti-inflammatory mediators were uncovered (Chiang and Serhan, 2004; Morris et al., 2009). It is now apparent from evidence obtained by many investigators (for example, refs. Claria and Planaguma, 2005 and references within; Maderna and Godson, 2005; Wu et al., 2012) that aspirin can jump-start resolution by not only blocking thromboxane and certain prostaglandins, but also by triggering aspirin-triggered lipid mediators such as the aspirin-triggered lipoxins and the aspirin-triggered resolvins (Serhan et al., 2002).

Along these lines, aspirin-triggered LXA4 is produced on dermal challenge (Morris et al., 2009) and a stable aspirin-triggered LXA4 analog 15R/S-methyl-LXA4 was found effective in treating infantile eczema in a double blind two center clinical trial (Wu et al., 2012). Hence the complete structural elucidation of the previously unknown aspirin-triggered lipid mediators (Chiang and Serhan, 2004; Serhan et al., 2002) can have far-reaching implications for clinical utility. In the present experiments, the aspirin-triggered 17R form of resolvin D3 proved to be highly potent (Figs. 4–7), as is the case for other aspirin-triggered lipid mediators. The acetylation of COX-2 by aspirin imposes restrictions in size in the catalytic pocket, which blocks prostaglandin production but still lead to the oxidation of polyunsaturated fats such as arachidonic acid and DHA in vascular endothelial cells and mucosal epithelial cells. In the case of DHA, 17R-hydroxy-docosahexaenoic acid (17-HDHA) is produced. Albeit at low enzyme catalytic turn over levels, the 17R-HDHA precursor is produced by endothelial cells (Serhan et al., 2000; Serhan et al., 2002) that have an extensive biomass in humans in that the endothelium lines vessels from head to toe in all organs, making this a relevant site to produce 17R-HDHA in vivo (Serhan et al., 2002) as is the case for COX-2 in mucosal epithelia that also has an extensive biomass and surface area lining the barrier function of many organs (Louis et al., 2005; Serhan et al., 2002).

In the clinical scenario in the U.S.A., aspirin is commonly administered at one of three doses, 81, 325 or 650 mg daily (Chiang et al., 2004; Furst and Hillson, 2001). The lower dose of 81 mg daily is the anti-thrombotic dose and gives both reduction in peripheral blood TXB2 levels and a concomitant increase in AT-15-epi-LXA4 in healthy volunteers (Chiang et al., 2004). Biosynthesis of AT-15-epi-LXA4 following low-dose aspirin administration is activated on dermal challenge in human subjects and regulates neutrophil influx, demonstrating the anti-inflammatory actions of low-dose aspirin (Morris et al., 2009). This protective action of low-dose aspirin is a function of both age and gender in that men older than 50 display reduced capacity to produce AT-lipid mediators (Chiang et al., 2006; Morris et al., 2010). Since RvD3 appears late in the resolution phase in murine exudates (Fig. 1), the biosynthesis of its aspirin-triggered epimer would also be expected to occur late into the resolution phase at the site of inflammation within tissues. This along with the findings that the aspirin-triggered epimer 17R-resolvin D1 is characteristically more resistant to enzymatic inactivation (Sun et al., 2007) likely leads to elevated local levels of RvD3 and AT-RvD3. Hence, AT-RvD3 may also provide an attractive biomarker of aspirin therapy in humans.

Studies addressing structure activity relationships of pro-resolving mediators demonstrate that R and S chirality at carbons 7 and 8 from the carboxylic acid end of DHA, i.e., RvD1, and carbons 5 and 6 in AA, i.e. LXA4, are critical in dictating the ability of these resolvins and lipoxins to engage their respective receptors (Serhan and Petasis, 2011), for example, 7S,8R in RvD1. These receptors appear to be more flexible with respect to the chirality of the hydroxyl group at carbon 17 for the resolvins and carbon 15 for the lipoxins, whereby mediators with either R or S stereochemistry at these positions are able to bind and activate their cognate GPCR to a similar extent (Chiang et al., 2000; Krishnamoorthy et al., 2012). This is also the case for RvD3 and its aspirin-triggered form, which we find here to bind and activate DRV1/GPR32 to a similar extent to each other as well as RvD1 (Fig. 7).

It is noteworthy that 17-HDHA is present in blood of healthy individuals (Mas et al., 2012; Psychogios et al., 2011) and is bioactive in animal disease models (Bento et al., 2011; Lima-Garcia et al., 2011) likely as a result of its local transformation by leukocytes to D-series resolvins (Serhan et al., 2002). In addition, the 17R aspirin-triggered lipid mediators such as aspirin-triggered RvD1 remain elevated in tissues longer because they are less susceptible to local inactivation via dehydrogenation (Serhan, 2007). In this regard, aspirin-triggered RvD3 also proved to be a potent mediator in blocking neutrophil transmigration as well as enhancing pro-resolving responses (Fig. 6) such as other aspirin-triggered members of the DHA metabolome (Serhan and Petasis, 2011). Regulating neutrophil responses can prevent collateral tissue damage and unlike other agents that are anti-inflammatory, the resolvins and particularly D-series resolvins do this without immune suppression, as D-series resolvins, i.e. resolvin D2 and resolvin D5, enhance bacterial clearance in sepsis (Spite et al., 2009) and infections (Chiang et al., 2012).

Recently, lymph nodes were found to produce 17-HDHA, where it enhances antibody production, and the D-series resolvins and metabolome are also present in other lymphoid tissues of the mouse such as spleen (Ramon et al., 2012). Hence, it is likely that in addition to the role of specialized pro-resolving mediators in bringing acute inflammation to homeostasis, preventing a potential for chronicity, these mediators may also play a role in acquired immunity. Although the levels in mice of n-3 essential fatty acids (e.g. EPA, DHA) and n-6 (e.g. arachidonic acid) (Weldon and Whelan, 2011) are different from those in human tissues (De Caterina, 2011), there is still a prevailing notion that n-3 fatty acids such as DHA have important actions in maintaining human health and preventing disease (Calder, 2012) including cardiovascular diseases (De Caterina, 2011). With the complete stereochemistry of RvD3 assigned in the present studies, namely its double-bond geometry, chirality of its alcohols as well as anti-inflammatory and pro-resolving actions, they now permit gauging RvD3’s role in the DHA metabolome of bioactive molecules.

Significance

The results of the present report establish the stereochemistry of RvD3 and its aspirin-triggered form produced by human macrophages and in murine inflammatory exudates during inflammation-resolution. These results also place the endogenous accumulation of RvD3 within the late phase of the acute inflammatory response. RvD3 and its aspirin-triggered form both displayed potent anti-inflammatory (i.e. regulating PMN tissue infiltration and pro-inflammatory mediator production) and pro-resolving actions (i.e. stimulating macrophage efferocytosis and phagocytosis). The present findings provide new opportunities to evaluate the role of the DHA resolution metabolome (Serhan, 2007) that can also be evoked with aspirin treatment. These novel mediators facilitate the transition from acute inflammation to homeostasis without immunosuppression.

Experimental Procedures

NMR of Synthetic RvD3

The following chemical shifts and coupling constants were recorded (for more details, see Fig. 2): H5 (5.42 ppm), H6 (6.08 ppm, J=4.4, 11.2 Hz), H7 (6.59 ppm, J=11.4, 14.7 Hz), H8 (6.24 ppm, J=10.7, 14.7 Hz), H9 (6.33 ppm, J=10.7, 15.1 Hz), H10 (5.75 ppm, J=15.1, 6.6 Hz), H13 (5.47 ppm), H14 (6.08 ppm, J=4.4, 11.2 Hz), H15 (6.52 ppm, J=11.1, 15.3 Hz), H16 (5.70 ppm, J=6.5, 15.1 Hz), H19 (5.44 ppm), H20 (5.37 ppm).

Acute Inflammation

Male FVB mice (6- to 8-weeks-old) purchased from Charles River Laboratories were fed ad libitum Laboratory Rodent Diet 20 - 5058 (Lab Diet, Purina Mills). All animal experimental procedures were approved by the Standing Committee on Animals of Harvard Medical School (protocol no. 02570) and complied with institutional and U.S. National Institutes of Health (NIH) guidelines. Peritonitis: Zymosan (1mg/ml; Sigma-Aldrich) was injected intraperitoneally (i.p.) 10 minutes after intravenous (i.v.) administration of RvD3, AT-RvD3 (10 ng) or vehicle (0.1% EtOH in 100 μL saline). Peritoneal lavages were collected 4–72 h after zymosan administration. Leukocyte numbers and differential counts were assessed using Turks solution and flow cytometry analysis as detailed below. Cytokine and chemokine levels were assessed in cell-free supernatants by multiplex ELISA. Lavages were also placed in 2 volumes of methanol and subjected to lipid mediator metabololipidomics. Dorsal skin pouch: 2.5 ml of sterile air were injected subcutaneously on days 0 and 3. On day 6, mouse recombinant TNF-α (100 ng/mouse; R&D Systems) was administered into the airpouch in combination with either RvD3 or AT-RvD3 (10 ng/mouse). Four hours later pouches were lavaged with DPBS−/−, leukocytes were enumerated and differential count conducted; cytokine/chemokine and lipid mediator levels were assessed as above.

Flow Cytometry

Murine peritoneal and airpouch exudate cells were suspended in FACS buffer (5% BSA in DPBS), incubated with Fc block (15 min, 4°C; BD Pharmingen) then rat anti-mouse (from eBioscience) CD11b-PerCP/Cy5.5 (Clone:M1/70) and Ly6G-FITC (Clone 1A8) (30 min, 4°C) or appropriate isotype controls. Staining was assessed using FACSDiva CantoII (BD Biosciences) and analyzed using FlowJo (Tree Star Inc.).

Leukocyte Functional Responses

Macrophage phagocytosis and efferocytosis

To obtain apoptotic PMN, human PMN were isolated by density-gradient Ficoll-Histopaque from human peripheral blood. Blood was obtained from healthy human volunteers giving informed consent under protocol # 1999-P-001297 approved by the Partners Human Research Committee. PMN were labeled with carboxyfluorescein diacetate-succinimidyl ester (CFDA; 10μM, 30 min, 37°C) and cultured overnight (5x106 cells/ml in DPBS+/+). Mouse resident peritoneal macrophages (MFΦ) were plated onto 96-well plates (Costar) at 5 x104 cells/well and incubated with SPM (15 min, 37°C) followed by phagocytosis of fluorescent labeled apoptotic PMN or FITC zymosan as described (Dalli and Serhan, 2012). Fluorescent-labeled PMN were then added at a 3:1 ratio (PMN to macrophages). Human macrophages were prepared from peripheral blood mononuclear cells (Dalli and Serhan, 2012). Macrophages (3 × 106 cells in a 10 cm petri dish) were transfected with pcDNA3 or with expression vector for human GPR32 (5 μg) using Jet-Pei transfection reagent following manufacturer’s instructions (Polyplus-transfection SA). After transfection (48 h), cells were plated onto 96-well plates (50,000 cells/well), and phagocytosis carried out 24 h after re-plating. Expression of GPR32 was verified by flow cytometry using a polyclonal rabbit anti-human GPR32 antibody (GeneTex, GTX108119). Phagocytosis was then assessed; vide supra. PMN transendothelial migration was performed as in (Serhan et al., 2002).

Lipid Mediator Metabololipidomics

All samples for LC-MS-MS-based lipidomics were subject to solid-phase extraction as in (Dalli and Serhan, 2012). Prior to sample extraction, d4-LTB4 and d4-PGE2, representing each region in the chromatographic analysis (500 pg each), were added to facilitate quantification. Extracted samples were analyzed by a LC-UV-MS-MS system, QTrap 5500 (ABSciex) equipped with an Agilent HP1100 binary pump and diode-array detector. An Agilent Eclipse Plus C18 column (50 mm × 4.6 mm × 1.8 μm) was used with a gradient of methanol/water/acetic acid of 55:45:0.01 (v/v/v) to 100:0:0.01 at 0.5 mL/min flow rate. To monitor and quantify the levels of targeted lipid mediators (LMs), we used multiple reaction monitoring (MRM) with signature ion fragments for each molecule (6 diagnostic ions and calibration curves) (Dalli and Serhan, 2012). GC-MS was carried out as in (Serhan et al., 2002).

GPCR-Beta-Arrestin System and Ligand-Receptor Interactions

Ligand-receptor interactions were monitored using the Beta Arrestin PathHunter system (Discoverx) and carried out essentially as in (Krishnamoorthy et al., 2012) with CHO cells stably overexpressing recombinant human GPR32 (hGPR32: DRV1) receptors tagged with pro-link label of beta-galactosidase and beta-arrestin linked to the EA fragment of beta-galactosidase. Briefly, cells were plated in 96-well plates (20,000 cells/well) 24 h prior to initiating experiments. Test compounds were incubated with cells (60 min, 37 °C) and receptor activation was determined by measuring chemiluminescence using the PathHunter detection kit (Discoverx).

Ligand selectivity using Electrical Cell-Substrate Impedance Sensing System (ECIS)

Ligand-receptor interactions were monitored by measuring impedance across cultured CHO-hGPR32 cell monolayers using an ECIS (Applied Biophysics) and carried out (Krishnamoorthy et al., 2012). For antibody incubations, anti-GPR32 Ab or rabbit serum was incubated with cells in the ECIS chambers at 1:50 dilutions for 30 min before addition of compounds.

Statistics

All data were expressed as means ± SEM. Differences between groups were compared using Student's t test (2 groups) or 1-way ANOVA (multiple groups) followed by post hoc Bonferroni test. The criterion for statistical significance was P<0.05.

Supplementary Material

01

Highlights.

  • RvD3 accumulates in late inflammation-resolution.

  • Aspirin triggers biosynthesis of RvD3 17R-epimer (AT-RvD3) that is pro-resolving.

  • Complete stereochemistry of RvD3 and AT-RvD3 are established.

  • RvD3 and AT-RvD3 govern leukocyte functions and local inflammatory responses.

Acknowledgments

The authors thank Mary Halm Small for expert assistance in manuscript preparation and Dr. James A. Lederer (Brigham and Women’s Hospital) for multiplex ELISA. This study was supported by National Institutes of Health grant P01GM095467.

Footnotes

Disclosure

CNS is an inventor on patents [resolvins] assigned to BWH and licensed to Resolvyx Pharmaceuticals. CNS is a scientific founder of Resolvyx Pharmaceuticals and owns equity in the company. CNS’ interests were reviewed and are managed by the Brigham and Women’s Hospital and Partners HealthCare in accordance with their conflict of interest policies. NAP is an inventor on patents assigned to the University of Southern California licensed for clinical development and retains stock in Resolvyx Pharmaceuticals.

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