Novel proresolving and tissue-regenerative resolvin and protectin sulfido-conjugated pathways

Jesmond Dalli; Sesquile Ramon; Paul C Norris; Romain A Colas; Charles N Serhan

doi:10.1096/fj.14-268441

. 2015 Feb 20;29(5):2120–2136. doi: 10.1096/fj.14-268441

Novel proresolving and tissue-regenerative resolvin and protectin sulfido-conjugated pathways

Jesmond Dalli ^1,¹, Sesquile Ramon ^1,¹, Paul C Norris ¹, Romain A Colas ¹, Charles N Serhan ^1,²

PMCID: PMC4415017 PMID: 25713027

Abstract

Local mediators orchestrate the host response to both sterile and infectious challenge and resolution. Recent evidence demonstrates that maresin sulfido-conjugates actively resolve acute inflammation and promote tissue regeneration. In this report, we investigated self-limited infectious exudates for novel bioactive chemical signals in tissue regeneration and resolution. By use of spleens from Escherichia coli infected mice, self-resolving infectious exudates, human spleens, and blood from patients with sepsis, we identified 2 new families of potent molecules. Characterization of their physical properties and isotope tracking demonstrated that the bioactive structures contained a docosahexaenoate backbone and sulfido-conjugated triene or tetraene double-bond systems. Activated human phagocytes converted 17-hydro(peroxy)-4Z,7Z,10Z,13Z,15E,19Z-docosahexaenoic acid to these bioactive molecules. Regeneration of injured planaria was accelerated with nanomolar amounts of 16-glutathionyl, 17-hydroxy-4Z,7Z,10,12,14,19Z-docosahexaenoic acid and 16-cysteinylglycinyl, 17-hydroxy-4Z,7Z,10,12,14,19Z-docosahexaenoic acid (Protectin sulfido-conjugates) or 8-glutathionyl, 7,17-dihydroxy-4Z,9,11,13Z,15E,19Z-docosahexaenoic acid and 8-cysteinylglycinyl, 7,17-dihydroxy-4Z,9,11,13Z,15E,19Z-docosahexaenoic acid (Resolvin sulfido-conjugates). Each protectin and resolvin sulfido-conjugate dose dependently (0.1–10 nM) stimulated human macrophage bacterial phagocytosis, phagolysosomal acidification, and efferocytosis. Together, these results identify 2 novel pathways and provide evidence for structural elucidation of new resolution moduli. These resolvin and protectin conjugates identified in mice and human infected tissues control host responses promoting catabasis.—Dalli, J., Ramon, S., Norris, P. C., Colas, R. A., Serhan, C. N. Novel proresolving and tissue-regenerative resolvin and protectin sulfido-conjugated pathways.

Keywords: inflammation, resolution, leukocytes, infection

Acute inflammation is a protective response mounted by the host following injury and/or infection (1–3). When this response becomes deregulated, it leads to unabated leukocyte activation and chronic inflammation. The acute response is coordinated by autacoids that include the arachidonic acid-derived prostaglandins (PGs), some of which (i.e., PGE₂ and PGD₂) regulate edema formation and leukotriene (LT) B₄ that mediate leukocyte recruitment to the site (1, 4, 5). Excessive production of these bioactive mediators is thought to be the cause of many chronic inflammatory conditions. Thus, an extensive effort was undertaken in recent decades to inhibit production of proinflammatory mediators (3–6) in conditions where their excessive production was associated with the underlying pathology. Although in the short term this approach yielded some clinical benefit in select conditions, in the long term, it was found to lead to immune suppression (1).

It is now appreciated that resolution of inflammation is an active cellular and biochemical process orchestrated by local acting mediators that include gaseous molecules such as hydrogen sulfide (7) and essential fatty acid (EFA)-derived signals, the latter constituting a new genus of specialized proresolving mediators [SPMs; recently reviewed in (8)]. This novel genus of mediators includes the arachidonic acid-derived lipoxins, the eicosapentaenoic acid-derived E-series resolvins, and the docosahexaenoic acid (DHA)-derived D-series resolvins (RvD), protectins (PD), and maresins (8–10). These counterregulate proinflammatory mediator production, including PGs, LTs, and select cytokines (8, 10). They also stimulate leukocyte responses including bacterial phagocytosis and efferocytosis of apoptotic cells, key processes in the clearance of infections and return to homeostasis, without apparent immune suppression (1, 8). In addition to these actions that are shared by all proresolving mediators, each SPM displays characteristic roles. For example, resolvin D1 (7S,8R,17S-trihydroxy-docosa-4Z,9E,11E,13Z,15E,19Z-hexaenoic acid; RvD1), resolvin D2 (7S,16R,17S-trihydroxy-docosa-4Z,8E,10Z,12E,14E,19Z-hexaenoic acid; RvD2), and resolvin D5 (7S,17S-dihydroxy-docosa-4Z,8E,10Z,13Z,15E,19Z-hexaenoic acid; RvD5) enhance clearance of bacterial infections (8, 11), and resolvin E1 (5S,12R,18R-trihydroxy-eicosa-6Z,8E,10E,14Z,16E-pentaenoic acid; RvE1) promotes clearance of viral infections (12) and neutrophil apoptosis (13). In addition, maresin 1 (7R,14S-dihydroxy-docosa-4Z,8E,10E,12Z,16Z,19Z-hexaenoic acid; MaR1) and RvE1 promote tissue regeneration (8, 14).

We recently identified a new family of sulfido-conjugated mediators, produced during resolution of Escherichia coli infection, that promote wound repair and tissue regeneration (14). Because they share biosynthetic pathway components with the maresins, given that their production was initiated by oxygenation at carbon 14, these mediators were coined maresin conjugate in tissue regeneration (MCTR). In the present report, we assessed whether additional novel sulfido-containing molecules are found in self-resolving infections and whether they carry biologic actions. With mouse and human spleens, self-resolving infectious exudates, human phagocytes, and human sepsis plasma, we identified 17-series sulfido-conjugated pathways, namely protectin sulfido-conjugates and resolvin sulfido-conjugates. These molecules were found to carry potent anti-inflammatory, tissue-regenerative, and proresolving actions with planaria in vivo and human cells in vitro.

MATERIALS AND METHODS

Cell isolations

Human polymorphonuclear neutrophils (PMNs) were isolated from peripheral blood as in (15). In brief, whole blood was collected from healthy volunteers according to Partners Human Research Committee Protocol (1999P001297). Red blood cells were lysed with hypotonic buffer. PMNs were isolated using Ficoll-Histopaque 1077-1 (Sigma-Aldrich, St. Louis, MO, USA) density gradient and resuspended in Dulbecco’s PBS.

Human macrophages were obtained from peripheral blood mononuclear cells isolated from leukopacks, procured from Children’s Hospital Blood Bank (Boston, MA, USA). Monocytes were cultured for 7 d in RPMI 1640 medium (Life Technologies, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (Invitrogen, Grand Island, NY, USA), 2 mM l-glutamine (Lonza, Basel, Switzerland) penicillin-streptomycin (Lonza), and granulocyte macrophage-stimulating factor (10 ng/ml; R&D Systems, Minneapolis, MN, USA) (15).

Macrophage bacterial phagocytosis, efferocytosis, and phagolysosomal acidification

Human macrophages were plated in 96-well plates (5 × 10⁴ cells per well) for 24 h, and phagocytosis or efferocytosis was assessed as in (14). In brief, apoptotic PMNs were obtained by culturing cells overnight in PBS^−/− (5 × 10⁶ cells/ml). Apoptotic human PMNs were labeled with bisbenzimide H33342 trihydrochloride (Sigma-Aldrich). Human macrophages were incubated with 0.1–10 nM concentration of test products, and then labeled apoptotic PMNs were cocultured with macrophages for 40 min [37°C (pH 7.45)]. Fluorescence was read on a SpectraMax M3 plate reader (Molecular Devices, Sunnyvale, CA, USA), and results were analyzed using SoftMax Pro (Molecular Devices).

Macrophage phagocytosis was assessed using fluorescently labeled E. coli (serotype O6:K2:H1) with BacLight Green Bacterial Stain (Life Technologies, Eugene, OR, USA). E. coli [2.5 × 10⁶ colony-forming units (CFU)/well] were then added to macrophages previously plated in 96-well plates (40 min at 37°C) and incubated with 0.1–10 nM concentration of test compounds [15 min at 37°C (pH 7.45)]. Fluorescence was measured on a SpectraMax M3 plate reader, and results were analyzed using SoftMax Pro.

Macrophage phagolysosomal acidification was assessed by incubating macrophages with pHrodo dye (Invitrogen) following the manufacturer’s instructions. Subsequently, cells were incubated with 1 nM of test compounds (15 min at 37°C), E. coli (2.5 × 10⁶ CFU/well) were added, and fluorescence was assessed after 60 min (37°C) using a BZ9000 microscope equipped with a ×20 objective (Keyence, Itasca, IL, USA) and a fluorescence plate reader.

Microbially induced mouse peritonitis

FVB mice, 6–8-week-old, purchased from Charles River Laboratories (Wilmington, MA, USA) were fed ad libitum Laboratory Rodent Diet 20-5058 (Lab Diet; Purina Mills, St. Louis, MO, USA). Mouse experimental procedures were approved by the Standing Committee on Animals of Harvard Medical School (Protocol 02570) and complied with institutional and U.S. National Institutes of Health (NIH) guidelines. E. coli (serotype O6:K2:H1) was cultured in Luria-Bertani broth and harvested at midlog phase (OD_{600 nm} ≈ 0.5 absorbance units; 5 × 10⁸ CFU/ml). Mice were given an intraperitoneal injection containing E. coli (5 × 10⁴ CFU/mouse). Four hours later, mice were administered 15 × 10⁶ apoptotic PMNs or saline via intraperitoneal injection, and 8 h later, peritoneal exudates were collected as described in (11). Cellular composition was determined by differential leukocyte count and flow cytometry. For flow cytometry, cells were labeled with fluorescently conjugated antibodies against mouse surface CD11b (clone M1/70; eBioscience, San Diego, CA, USA), F4/80 (clone BM8; eBioscience), Ly-6G (clone RB6-8C; eBioscience), and intracellular stained with anti-E. coli antibody (clone GTX408556; GeneTex, Irvine, CA, USA). Bacterial clearance was measured by culturing exudates on plates containing Luria-Bertani agar overnight at 37°C.

Sulfido-conjugate biosynthesis and liquid chromatography tandem mass spectrometry identification

Cell incubations, self-limited infectious exudates, mouse spleens, deidentified human spleens (purchased from Cooperative Human Tissue Network, Philadelphia, PA, USA), and deidentified human plasma from patients diagnosed with sepsis (purchased from Dx Biosamples, San Diego, CA, USA) were placed in 2 volumes of methanol. For lipid mediator (LM) profiling, 500 pg deuterium-labeled internal standards d₈-5S-HETE and d₅-LTC₄ was added to facilitate quantification and assessment of sample recovery. Samples were then held at −20°C for 45 min to allow for protein precipitation and were centrifuged (1200 g at 4°C for 10 min). Products were extracted using solid-phase extraction as described (14) and eluted using methanol. Eluted isolates were then brought to dryness under nitrogen and suspended in methanol:water (50:50) for LM metabololipidomics. For LM metabololipidomics of sulfido-conjugates, the liquid chromatography tandem mass spectrometry (LC-MS-MS) system was operated as described (14) with minor modifications. A Shimadzu LC-20AD HPLC (Tokyo, Japan) and a Shimadzu SIL-20AC autoinjector paired with a QTrap 5500 (AB Sciex, Framingham, MA, USA) were used. A Poroshell 120 EC-C18 column (100 mm × 4.6 mm × 2.7 μm; Agilent Technologies, Santa Clara, CA, USA) was kept in a column oven maintained at 50°C (ThermaSphere model TS-130; Phenomenex, Torrance, CA, USA), and LMs were eluted with a mobile phase consisting of methanol:water:acetic acid at 55:45:0.1 (vol:vol:vol) that was isocratic for 1 min, ramped to 70:30:0.1 (vol:vol:vol) over 5 min, then to 80:20:0.1 (vol:vol:vol) for 2 min, then isocratic 80:20:0.1 (vol:vol:vol) for the next 3 min, and ramped to 98:2:0.1 (vol:vol:vol) over 3 min. This was subsequently maintained at 98:2:0.1 (vol:vol:vol) for 3 min, and the flow rate was maintained at 0.60 ml/min. The QTrap 5500 was operated in positive ionization mode using scheduled multiple reaction monitoring (MRM) coupled with information-dependent acquisition and enhanced product ion scan.

Human macrophage cell line (KG1A, 1 × 10⁷ cells/ml; American Type Culture Collection, Manassas, VA, USA) was suspended in PBS^+/+ incubated with 17S-hydro(peroxy)-4Z,7Z,10Z,13Z,15E,19Z-docosahexaenoic acid (17-HpD; 30 μM) and E. coli [1 × 10⁸ CFU/ml at 37°C (pH 7.45) for 30 min]. The 17-HpD was produced by incubation of DHA with soybean lipoxygenase and isolated as in (10). There were 2 volumes of methanol then added, and products were extracted using C18 columns as outlined above. In select experiments, mouse spleens were incubated with d₅-DHA [10 µM (pH 7.45) for 30 min, PBS^+/+] and norepinephrine (10 µM). Incubations were stopped with 2 volumes of ice-cold methanol and products extracted as above.

Products used for biologic evaluation and structure elucidation were obtained as detailed above and isolated using online UV-RP-HPLC (1100 Series; Agilent Technologies) and a Poroshell 120 EC-C18 column (100 mm × 4.6 mm × 2.7 μm) with the 2 mobile phases consisting of solvent A [methanol:acetonitrile (35:65 vol:vol)] and solvent B (water containing 8.3 M acetic acid and buffered to pH 5.7 with ammonium hydroxide). Solvent A was maintained at 10% for 0.3 min, then ramped to 30% over 0.2 min. This was maintained for 1.5 min and then ramped to 50% over 0.1 min, and the flow rate was reduced from 0.6 to 0.2 ml/min. Solvent B was ramped to 53% and the flow rate increased to 0.3 ml/min over the subsequent 38 min. This was then ramped to 100% over the next 8 min and the flow rate increased to 0.6 ml/min, which was maintained for 5 min. In select experiments, isolated products were incubated with freshly prepared diazomethane in diethyl ether for 30 min at room temperature. Samples were then brought to dryness, and products were assessed by LC-MS-MS using MRM of the following ion pairs: 492 > 135, 508 > 135, 565 > 193, 549 > 193, 692 > 336, and 708 > 336.

In select experiments, human macrophages (∼4.5 × 10⁷ cells/ml) were incubated with DHA (10.5 µM) or 17-HpD [10 µM at 37°C (pH 7.45)] and E. coli (1:50) for 30 min at 37°C. Incubations were stopped with 2 volumes of ice-cold methanol, products were extracted, and levels assessed by LC-MS-MS. Macrophages were also incubated with or without Acivicin [2.5 mM at 37°C, PBS (pH 7.45)], a γ-glutamyl transferase (GGT) agent that inhibits LT D₄ formation (16), before addition of 17-HpD (10 µM) and E. coli (1:50; 60 min), and products were taken to LC-MS-MS.

Planaria regeneration

Planaria (Dugesia japonica) were kept in water (Poland Spring; Nestlé Waters North America, Stamford, CT, USA) at 18°C. All animals were starved for at least 7 d before the experiments. Tissue regeneration was assessed as described previously (10). In brief, planaria were subjected to head resection postoccularly (surgical injury). The posterior portions of the planaria were then placed in spring water containing 0.01% EtOH, 16-glutathionyl, 17-hydroxyl-4Z,7Z,10,12,14,19Z-docosahexaenoic acid plus 16-cysteinylglycinyl, 17-hydroxyl-4Z,7Z,10,12,14,19Z-docosahexaenoic acid (50 nM; each) or 8-glutathionyl, 7,17-dihydroxyl-4Z,9,11,13Z,15E,19Z-docosahexaenoic acid plus 8-cysteinylglycinyl, 7,17-dihydroxyl-4Z,9,11,13Z,15E,19Z-docosahexaenoic acid (50 nM; each). The extent of tissue regeneration during a 6-d period was determined using captured images of the regenerating blastemas at regular intervals (24 h). These images were analyzed using ImageJ software (NIH, Bethesda, MD, USA). A tissue regeneration index (TRI) was used that took into consideration the size of the regenerated tissue total area (A) and the postocular width (W), where TRI = A/W (10).

Statistical analysis

All results are expressed as the mean ± sem. Differences between groups were compared using 1-way ANOVA (multiple groups) followed by post hoc Bonferroni test, or 2-way ANOVA (multiple groups, multiple time points) followed by post hoc Bonferroni or Tukey tests. The criterion for statistical significance was P ≤ 0.05.

RESULTS

Identification of novel sulfido-conjugates during self-resolving E. coli infections in mice and with human spleens

To investigate the production of novel molecules during self-limited acute inflammation, we initiated peritonitis in mice with a self-limited E. coli inoculum (11). Given that the spleen is key in regulating immune responses during infections and is rich in LMs (17), we profiled spleens from E. coli-inoculated mice during the onset of the inflammatory response and its resolution. LC-MS-MS-based LM profiling targeting molecules containing a DHA backbone and carrying a glutathionyl, cysteinylglycinyl, or a cysteinyl group gave 2 distinct peaks: the first eluting with a retention time (T_R) of 9.7 min (I), and the second peak (II) at T_R 10.4 min (Fig. 1A).

Figure 1. — In *E. coli*-infected mouse spleens, identification of novel sulfido-conjugates. Mice were inoculated with *E. coli* (1 × 10⁵ CFU/mouse) and spleens harvested. Products were extracted using C18 solid-phase columns and investigated by LC-MS-MS (see Materials and Methods). A) MRM chromatograms for the identified sulfido-conjugated products. B and C) MS-MS spectra employed for the identification of (B) 16-glutathionyl, 17-hydroxy-4Z,7Z,10,12,14,19Z-docosahexaenoic acid (I) and (C) 16-cysteinyl, 17-hydroxy-4Z,7Z,10,12,14,19Z-docosahexaenoic acid (II). D) Quantification of sulfido-conjugated mediators in mouse spleen during self-resolving infections (16-GS,17-OH,C₂₂H₃₀O₂ = 16-glutathionyl, 17-hydroxy-4Z,7Z,10,12,14,19Z-docosahexaenoic acid; 16-CY,17-OH,C₂₂H₃₀O₂ = 16-cysteinyl, 17-hydroxy-4Z,7Z,10,12,14,19Z-docosahexaenoic acid). Results for (A–C) are representative of n = 9 mice. Results for (D) are the mean ± sem (n = 3 mice per time interval).

Assessment of the mass spectrometry (MS) spectrum for product beneath peak I gave a parent ion (M+H) with mass-to-charge ratio (m/z) of 650 and a daughter ion with m/z of 308, suggesting that this contained a DHA backbone carrying a hydroxy and a glutathionyl group (14). The MS-MS fragmentation pattern for this molecule gave ions with an m/z of 565 and 548, consistent with the hydroxy group being carried at carbon 17 (Fig. 1B) of the 22-carbon backbone. In addition, ions with m/z 275, 231, and 213 were consistent with the glutathione group carried at carbon 16 of the DHA backbone, thus indicating that this molecule was 16-glutathionyl, 17-hydroxy-4Z,7Z,10,12,14,19Z-docosahexaenoic acid. The MS spectrum of signal II gave a parent ion with m/z of 464 and a daughter ion with m/z 343 and 121 that are consistent with a molecule that contains a 22-carbon DHA backbone, a hydroxy group, and a cysteinyl group. The MS-MS spectrum for this molecule gave ions with m/z 365, consistent with an alcohol group at carbon 17, and ions with m/z 275, 231, and 213, consistent with a cysteinyl group carried at carbon 16, thus indicating that this molecule was 16-cysteinyl, 17-hydroxy-4Z,7Z,10,12,14,19Z-docosahexaenoic acid (Fig. 1C). We next assessed the levels for each of these molecules during the course of infection. Using MRM, we found that their levels increased during the course of the inflammatory response, with highest levels found at the 24 h interval (Fig. 1D). Of note, cysteinyl LT levels were substantially lower in spleens from mice with peritonitis, during the resolution phase, than the new DHA-derived sulfido-conjugates (Fig. 1D). These results suggest that the novel sulfido-conjugated molecules may regulate leukocyte responses during the resolution of inflammation.

We next investigated the production of sulfido-conjugates at the site of inflammation. LC-MS-MS-based LM metabololipidomics of self-limited inflammatory exudates from E. coli-inoculated mice gave 3 peaks (Fig. 2A). Peak I gave a T_R of 8.0 min, a parent ion in the MS with m/z 666, and a daughter ion with m/z 308. These were consistent with a DHA backbone carrying 2 hydroxy and a glutathionyl group (Fig. 2A, B). Assessment of the MS-MS spectrum for this molecule gave ions with m/z 537 and 203, indicating that the hydroxy groups were carried at carbon positions 7 and 17, whereas ions with m/z 247, 217, 211, 199, and 185 were consistent with the glutathionyl group at carbon position 8. Thus, the structure was assigned as 8-glutathionyl, 7,17-dihydroxy-4Z,9,11,13Z,15E,19Z-docosahexaenoic acid.

Figure 2. — *E. coli*-infected mouse exudates gave novel sulfido-conjugates. Mice were inoculated with *E. coli* (1 × 10⁵ CFU/mouse) and exudates collected 12 or 24 h later. Products were extracted using C18 solid-phase columns and investigated by LC-MS-MS (see Materials and Methods). A) MRM chromatograms for the identified sulfido-conjugated products. B and C) MS-MS spectra employed for the identification of (B) 8-glutathionyl, 7,17-dihydroxy-4Z,9,11,13Z,15E,19Z-docosahexaenoic acid and (C)16-glutathionyl, 17-hydroxy-4Z,7Z,10,12,14,19Z-docosahexaenoic acid. D) Quantification of sulfido-conjugated mediators in mouse exudates during self-resolving infections (16-GS,17-OH,C₂₂H₃₀O₂ = 16-glutathionyl, 17-hydroxy-4Z,7Z,10,12,14,19Z-docosahexaenoic acid; 8-GS, 7,17-diOH,C₂₂H₂₉O₂ = 8-glutathionyl, 7,17-dihydroxy-4Z,9,11,13Z,15E,19Z-docosahexaenoic acid; 16-CYGL,17-OH,C₂₂H₃₀O₂ = 16-cysteinylglycinyl, 17-hydroxy-4Z,7Z,10,12,14,19Z-docosahexaenoic acid). Results for (A–C) are representative of n = 18 mice. Results for (D) are the mean ± sem (n = 6 mice per interval). Lavage volume = 5 ml each mouse.

Peak II gave a T_R of 9.3 min with a parent ion in MS of m/z 521 and daughter ion with m/z 179; this was consistent with a DHA backbone carrying 1 hydroxy and a cysteinylglycinyl. The MS-MS spectrum for this molecule gave ions with m/z 442, consistent with the hydroxy group being carried on carbon 17. Ions with m/z 275, 231, and 213 were consistent with a cysteinylglycinyl group carried at carbon 16; therefore, the structure of this product was assigned as 16-cysteinylglycinyl, 17-hydroxy-4Z,7Z,10,12,14,19Z-docosahexaenoic acid. Peak III gave essentially the same retention and MS-MS fragmentation at that found for 16-glutathionyl, 17-hydroxy-4Z,7Z,10,12,14,19Z-docosahexaenoic acid (Fig. 2A, C). Temporal regulation of these molecules demonstrates that during the course of inflammation in infectious inflammatory exudates, levels of 8-glutathionyl, 7,17-dihydroxy-4Z,9,11,13Z,15E,19Z-docosahexaenoic acid reached a maximum during the initial phase of the inflammatory response, whereas the levels of 16-cysteinylglycinyl, 17-hydroxy-4Z,7Z,10,12,14,19Z-docosahexaenoic acid and 16-glutathionyl, 17-hydroxy-4Z,7Z,10,12,14,19Z-docosahexaenoic acid were higher in exudates obtained 24 h after E. coli inoculation (Fig. 2D), which is within the resolution phase. These results demonstrate the presence of novel sulfido-conjugated DHA-derived products in infectious inflammatory exudates and their temporal regulation during self-limited inflammation.

Having identified sulfido-conjugated molecules with mouse spleens and infectious exudates, for human translation, we next sought evidence for these products with human spleens (see Supplemental Table S1). LC-MS-MS analysis gave 3 distinct peaks with T_R 8.0, 9.7, and 10.4 min (Fig. 3A). The parent ion for the product with T_R 8.0 min gave an m/z of 666 and ions in the MS-MS spectrum with an m/z of 359 that are consistent with a DHA backbone carrying 2 hydroxy groups (Fig. 3B). In addition, the ions with m/z 537, 199, and 185 are consistent with alcohols at carbons 7 and 17 for the product under peak I. Whereas ions in the MS-MS from peak I with m/z 520 and 217 are consistent with a glutathionyl group on carbon 8, thus, the structure of the molecule was assigned as 8-glutathionyl, 7,17-dihydroxy-4Z,9,11,13Z,15E,19Z-docosahexaenoic acid (Fig. 3B). The molecule with T_R 9.7 min gave an MS-MS spectrum that was essentially the same as that of 16-glutathionyl, 17-hydroxy-4Z,7Z,10,12,14,19Z-docosahexaenoic acid (Fig. 3C) and that with T_R 10.4 min gave an MS-MS spectrum consistent with 16-cysteinyl, 17-hydroxy-4Z,7Z,10,12,14,19Z-docosahexaenoic acid (Fig. 3D).

Figure 3. — Human spleen identification of novel sulfido-conjugates. Products from human spleens were extracted using C18 solid-phase columns and investigated by LC-MS-MS. A) MRM chromatograms for the identified sulfido-conjugated products. B–D) MS-MS spectra employed for the identification of (B) 8-glutathionyl, 7,17-dihydroxy-4Z,9,11,13Z,15E,19Z-docosahexaenoic acid, (C) 16-glutathionyl,17-hydroxy-4Z,7Z,10,12,14,19Z-docosahexaenoic acid, and (D) 16-cysteinyl,17-hydroxy-4Z,7Z,10,12,14,19Z-docosahexaenoic acid. Results are representative of n = 3 human spleens.

Identification of sulfido-conjugated molecules with human leukocytes

Human macrophages

Given the presence of a 17-hydroxy group in each of the molecules identified with mouse and human tissues (Figs. 1, 2, and 3), we assessed whether the 17-HpD was precursor in the biosynthesis of these novel molecules. Incubation of macrophages with serum-treated zymosan and 17-HpD gave 8-glutathionyl, 7,17-dihydroxy-4Z,9,11,13Z,15E,19Z-docosahexaenoic acid (peak II), 16-cysteinylglycinyl, 17-dihydroxy-4Z,7Z,10,12,14,19Z-docosahexaenoic acid (peak III), 16-glutathionyl, 17-hydroxy-4Z,7Z,10,12,14,19Z-docosahexaenoic acid (peak IV), and 16-cysteinyl, 17-hydroxy-4Z,7Z,10,12,14,19Z-docosahexaenoic acid (peak V; Fig. 4A, B and Supplemental Fig. S1). The parent ion for the product with T_R 7.2 min (peak I) gave an m/z of 537 and ions in the MS-MS spectrum with an m/z of 359 that are consistent with a DHA backbone carrying 2 hydroxy groups (Supplemental Fig. S1). In addition, ions with m/z 438, 393, and 143 are consistent with 2 alcohol groups at carbons 7 and 17 for the product beneath peak I. Whereas ions in the MS-MS from peak I with m/z 247, 217, 203, 199, 185, and 179 are consistent with a cysteinylglycine group on carbon 8, thus, the structure of the molecule was assigned as 8-cysteinylglycinyl, 7,17-dihydroxy-4Z,9,11,13Z,15E,19Z-docosahexaenoic acid (Supplemental Fig. S1).

Figure 4. — 17 lipoxygenation of DHA is precursor to novel sulfido-conjugates with human phagocytes. Human macrophages and neutrophils were incubated 30 min at 37°C with 1 μg 17-HpD plus 100 ng zymosan. A) Representative MRM chromatograms for each of the identified 17-series sulfido-conjugates in human macrophages (left panel) and PMN (right panel). B) Representative MS-MS spectra used for the identification of 8-glutathionyl, 7,17-dihydroxy-4Z,9,11,13Z,15E,19Z-docosahexaenoic acid (left panel) and 16-glutathionyl, 17-hydroxy-4Z,7Z,10,12,14,19Z-docosahexaenoic acid (right panel). C) Quantification of identified sulfido-conjugates. 8-GS, 7,17-diOH-C₂₂H₂₉O₂ = 8-glutathionyl, 7,17-dihydroxy-4Z,9,11,13Z,15E,19Z-docosahexaenoic acid; 8-CYGL, 7,17-diOH-C₂₂H₂₉O₂ = 8-cysteinylglycinyl, 7,17-dihydroxy-4Z,9,11,13Z,15E,19Z-docosahexaenoic acid; 16-GS,17-OH,C₂₂H₃₀O₂ = 16-glutathionyl, 17-hydroxy-4Z,7Z,10,12,14,19Z-docosahexaenoic acid; 16-CYGL,17-OH,C₂₂H₃₀O₂ = 16-cysteinylglycinyl, 17-hydroxy-4Z,7Z,10,12,14,19Z-docosahexaenoic acid; 16-CY,17-OH,C₂₂H₃₀O₂ = 16-cysteinyl, 17-hydroxy-4Z,7Z,10,12,14,19Z-docosahexaenoic acid. Results for (A) and (B) are representative of n = 3 healthy donors for each cell type. Results for (C) are the mean (n = 3 healthy donors for each cell type).

Human PMN

Incubation of human neutrophils with serum-treated zymosan and 17-HpD also gave 5 distinct peaks with T_Rs and MS-MS fragmentation spectra (Fig. 4A, B, right panel) that were essentially the same as those found with human macrophages (Fig. 4A, left panel). Assessment of levels for each of the identified molecules in these incubations using MRM demonstrated that 16-glutathionyl, 17-hydroxy-4Z,7Z,10,12,14,19Z-docosahexaenoic acid and 8-cysteinylglycinyl, 7,17-dihydroxy-4Z,9,11,13Z,15E,19Z-docosahexaenoic acid were the more abundant molecules obtained from human neutrophils (Fig. 4C). Whereas, 16-cysteinylglycinyl, 17-hydroxy-4Z,7Z,10,12,14,19Z-docosahexaenoic acid and 8-glutathionyl, 7,17-dihydroxy-4Z,9,11,13Z,15E,19Z-docosahexaenoic acid were the more abundant molecules identified with human macrophages. These results demonstrate that activated human PMNs and macrophages can convert 17-HpD to novel 17-series sulfido-conjugated products with each cell type giving characteristic sulfido-conjugated product profiles.

Physical properties of novel sulfido-conjugated products

To obtain further evidence for the proposed structures, we next investigated deuterium incorporation from d₅-DHA into each of the identified molecules. This gave the expected 5 Da shift in the parent ion of each of these products, where, for example, the m/z of 16-glutathionyl, 17-hydroxy-4Z,7Z,10,12,14,19Z-docosahexaenoic acid increased from 650 to 655, as did the m/z of diagnostic ions including that of the ion resulting from a carbon 16–17 break that increased from m/z 98 to 103 (Table 1) (14). The proposed structures were also investigated following diazomethane to obtain trimethyl and dimethyl derivatives of the parent molecules. Incubation of 8-glutathionyl, 7,17-dihydroxy-4Z,9,11,13Z,15E,19Z-docosahexaenoic acid with diazomethane led to an increase in the parent ion mass from m/z 666 to 708, indicating the addition of 3 methyl groups. In addition, characteristic ions including that resulting from a 7–8 carbon break increased from m/z 143 to 157. Similar results were also obtained for 16-cysteinylglycinyl, 17-hydroxy-4Z,7Z,10,12,14,19Z-docosahexaenoic acid, 16-cysteinyl, 17-hydroxy-4Z,7Z,10,12,14,19Z-docosahexaenoic acid, 8-glutathionyl, 7,17-dihydroxy-4Z,9,11,13Z,15E,19Z-docosahexaenoic acid, and 8-cysteinylglycinyl, 7,17-dihydroxy-4Z,9,11,13Z,15E,19Z-docosahexaenoic acid (Table 1).

TABLE 1.

Biologic systems and physical properties of deuterium-labeled, di- and trimethyl ester derivatives of 17-series sulfido-conjugates

Product	LC T_R (min)	LC-MS-MS prominent ions	d₅-derivative prominent ions	Methyl-derivative prominent ions	UV λ_max (nm)^a	Tissue source
PCTR1	9.7	650 (M+H), 632, 614, 593, 575, 565, 548, 521, 503, 418, 400, 343, 325, 308, 275, 257, 239, 231, 213, 179, 162, 144, 129, 98	655 (M+H), 637, 619, 580, 526, 508, 423, 405, 348, 330, 308, 275, 257, 245, 239, 231, 227, 213, 179, 162, 144, 129, 103	692 (M+H), 674, 660, 656, 621, 549, 357 339, 336, 289, 259, 245, 193, 176	280	Human macrophages
						Human PMN
						Human spleen
						Mouse infectious exudate
						Mouse spleen
PCTR2	9.3	521 (M+H), 442, 343, 325, 275, 239, 231, 213, 201, 179, 162, 131	526 (M+H), 508, 482, 348, 330, 275, 257	549 (M+H), 531, 505, 478, 357, 289, 241, 193, 176	280	Human macrophages
						Human PMN
						Mouse infectious exudate
PCTR3	10.4	464 (M+H), 446, 428, 365, 347, 343, 325, 275, 257, 239, 231, 227, 213, 121	469 (M+H), 451, 348, 275, 257, 245, 239, 231, 227, 213	492 (M+H), 474, 320, 289, 253, 245, 227, 135	280	Human macrophages
						Human PMN
						Human spleen
						Human sepsis plasma
						Mouse spleen
RCTR1	8.0	666 (M+H), 648, 591, 537, 519, 416, 341, 323, 308, 247, 217, 211, 203, 199, 185, 179, 162, 143	671 (M+H), 653, 635, 578, 542, 524, 506, 439, 421, 403, 364, 346, 328, 308, 252, 234, 222, 216, 208, 204, 179, 143	708 (M+H), 690, 672, 610, 580, 565, 529, 355, 336, 217, 193, 185, 157	308	Human macrophages Human PMN
						Human spleen
						Mouse infectious exudate
RCTR2	7.2	537 (M+H), 519, 501, 438, 393, 341, 247, 217, 211, 203, 199, 185, 179, 162, 143	542 (M+H), 524, 506, 469, 328, 252, 234, 222, 207, 190, 179	565 (M+H), 547, 529, 448, 373, 337, 211, 193, 185	308	Human macrophages
RCTR2	7.2		542 (M+H), 524, 506, 469, 328, 252, 234, 222, 207, 190, 179	565 (M+H), 547, 529, 448, 373, 337, 211, 193, 185	308	Mouse infectious exudate
RCTR3	8.5	480, 462, 444, 412, 382, 359, 341, 323, 247, 217, 211, 199, 185, 121	^b	^b	^b	Human macrophages

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^aλ_max was determined using online UV-RP-HPLC (1100 Series) and a Poroshell 120 EC-C18 column (100 mm × 4.6 mm × 2.7 μm) with the mobile phase consisting of methanol:water 50:50 (vol:vol). ^bNot determined.

Further evidence for each of the deduced structures was obtained by investigating the UV chromophore for each identified molecule. Here, we found that the UV chromophore for 8-glutathionyl, 7,17-dihydroxy-4Z,9,11,13Z,15E,19Z-docosahexaenoic acid gave triplet bands of absorption with λ_max^{methanol/water} 308 nm. Similar results were obtained with 8-cysteinylglycinyl, 7,17-dihydroxy-4Z,9,11,13Z,15E,19Z-docosahexaenoic acid (see Table 1). Assessment of the UV chromophores for 16-glutathionyl, 17-hydroxy-4Z,7Z,10,12,14,19Z-docosahexaenoic acid, 16-cysteinylglycinyl, 17-hydroxy-4Z,7Z,10,12,14,19Z-docosahexaenoic acid and 16-cysteinyl, 17-hydroxy-4Z,7Z,10,12,14,19Z-docosahexaenoic acid gave a triplet band of absorption with λ_max^{methanol/water} 280 nm (Table 1).

Product-precursor relationships

To obtain further evidence for the biosynthetic pathways of these sulfido-conjugated mediators, we investigated the product-precursor relationships for DHA with the novel molecules and human macrophages. DHA was rapidly converted to 16-glutathionyl, 17-hydroxy-4Z,7Z,10,12,14,19Z-docosahexaenoic acid that reached a maximum at 15 min (Fig. 5). The levels of 16-cysteinylglycinyl, 17-hydroxy-4Z,7Z,10,12,14,19Z-docosahexaenoic acid gradually increased, reaching a maximum at ∼45 min. Levels for this product were maintained in these incubations over the subsequent 45 min. 16-cysteinyl, 17-hydroxy-4Z,7Z,10,12,14,19Z-docosahexaenoic acid also gradually increased over the course of the incubation, reaching a maximum at the 90 min interval (Fig. 5A).

Figure 5. — Product-precursor relationships for the novel sulfido-conjugates. Human macrophages (4.5 × 10⁷ cells/ml) were incubated with (A) DHA [37°C (pH 7.45)], (B and C) 17-HpD [37°C (pH 7.45)], and *E. coli* (1.5 × 10⁸ CFU), and product levels were assessed using LC-MS-MS (see Materials and Methods for details). D–F) Human macrophages were incubated with or without GGT inhibitor [Acivicin; 2.5 mM at 37°C (pH 7.45) for 30 min] then (D) DHA or (E and F) 17-HpD [37°C (pH 7.45)] and *E. coli* (1.5 × 10⁸ CFU). Incubations were quenched; precursor and product levels were assessed by LC-MS-MS. Results are the mean for n = 3 macrophage preparations.*P < 0.05 vs. vehicle macrophages.

Incubation of activated human macrophages with 17-HpD gave similar product-precursor relationships (Fig. 5B). In these incubations, we also found a rapid increase in 8-glutathionyl, 7,17-dihydroxy-4Z,9,11,13Z,15E,19Z-docosahexaenoic acid that reached an apparent maximum at ∼15 min. Levels for 8-cysteinylglycinyl, 7,17-dihydroxy-4Z,9,11,13Z,15E,19Z-docosahexaenoic acid and 8-cysteinyl, 7,17-dihydroxy-4Z,9,11,13Z,15E,19Z-docosahexaenoic acid reached a maximum at later intervals (Fig. 5C). Of note, incubations with human macrophages, E. coli, and a GGT inhibitor (Acivicin), which inhibits LTD₄ formation (16), led to an increase in 16-glutathionyl, 17-hydroxy-4Z,7Z,10,12,14,19Z-docosahexaenoic acid in these incubations and a decrease in 16-cysteinyl, 17-hydroxy-4Z,7Z,10,12,14,19Z-docosahexaenoic acid (Fig. 5D, E). We also found a statistically significant increase in 8-glutathionyl, 7,17-dihydroxy-4Z,9,11,13Z,15E,19Z-docosahexaenoic acid and a decrease in 8-cysteinyl, 7,17-dihydroxy-4Z,9,11,13Z,15E,19Z-docosahexaenoic acid levels in activated macrophages incubated with Acivicin (Fig. 5F). These results provide evidence for product-precursor relationships in the biosynthesis of these sulfido-conjugates (vide infra).

Apoptotic neutrophils produce endogenous 17-series sulfido-conjugates and stimulate the phagocytosis and clearance of bacteria in vivo

We next investigated the endogenous production of the novel sulfido-conjugates by apoptotic PMNs because they play key roles in signaling resolution during acute inflammation (8). Using LC-MS-MS-based metabololipidomics and matching T_Rs as well as MS-MS fragmentation spectra (see Table 1), we identified in human apoptotic PMNs incubations 8-cysteinylglycinyl, 7,17-dihydroxy-4Z,9,11,13Z,15E,19Z-docosahexaenoic acid (peak I), 16-glutathionyl, 17-hydroxy-4Z,7Z,10,12,14,19Z-docosahexaenoic acid (peak II), and 16-cysteinylglycinyl, 17-hydroxy-4Z,7Z,10,12,14,19Z-docosahexaenoic acid (peak III; Fig. 6A, B). MRM quantification of the identified sulfido-conjugated products demonstrated that 8-cysteinylglycinyl, 7,17-dihydroxy-4Z,9,11,13Z,15E,19Z-docosahexaenoic acid was the more abundant product produced by human apoptotic PMNs (Fig. 6C). In addition, administration of apoptotic neutrophils to mice 4 h after E. coli inoculation led to a significant increase in macrophage bacterial phagocytosis (Fig. 6D) and clearance in vivo (Fig. 6E).

Figure 6. — Apoptotic human PMNs produce novel sulfido-conjugates from endogenous DHA and promote *E. coli* clearance during infection. Apoptotic neutrophil (see Materials and Methods for details) products were obtained and profiled by LC-MS-MS metabololipidomics. A) MRM chromatograms for identified products. B) Representative MS-MS spectrum employed for the identification of 16-cysteinylglycinyl, 17-hydroxy-4Z,7Z,10,12,14,19Z-docosahexaenoic acid. Results are representative of n = 6 apoptotic PMN preparations. C) Quantification of identified sulfido-conjugates in apoptotic PMN preparations. 8-CYGL, 7,17-diOH-C₂₂H₂₉O₂ = 8-cysteinylglycinyl, 7,17-dihydroxy-4Z,9,11,13Z,15E,19Z-docosahexaenoic acid;_;16-GS,17-OH,C₂₂H₃₀O₂ = 16-glutathionyl, 17-hydroxy-4Z,7Z,10,12,14,19Z-docosahexaenoic acid; 16-CYGL,17-OH,C₂₂H₃₀O₂ = 16-cysteinylglycinyl, 17-hydroxy-4Z,7Z,10,12,14,19Z-docosahexaenoic acid. Results are the mean for n = 6 cell preparations. D and E) FVB mice were infected intraperitoneally with *E. coli* (1 × 10⁵ CFU/mouse). After 4 h, mice were administered saline (*E. coli*) or apoptotic PMN (apop PMN; 15 × 10⁶ cells per mouse; i.p.). Exudates were then collected 12 h postinfection. D) Exudate macrophage phagocytosis of *E. coli* was assessed by flow cytometry. E) Bacterial titers present in the peritoneum were measured. Results for (A) and (B) are representative of n = 3 neutrophil preparations. Results for (C) are the mean ± sem (n = 3 mice per group). **P ≤ 0.01 and ***P ≤ 0.001 vs. *E. coli* mice.

Novel 17-series sulfido-conjugates accelerate tissue regeneration

We next sought evidence for the biologic actions carried by the novel products. Given their temporal biosynthesis during resolution of self-limited inflammation (Figs. 1 and 2) and production by human macrophages, we assessed the ability of these products to stimulate tissue regeneration. Here, we used a planaria tissue regeneration model (Fig. 7A). Planaria undergo both restorative and physiologic regeneration via evolutionarily conserved pathways making it an ideal system to test the tissue-regenerative actions of vertebrate-derived molecules (14, 18). Head resection gave a TRI_max (maximum tissue regeneration) at 6 d and T₅₀ (the interval at which 50% regeneration occurred) ∼4 d (Fig. 7A, B). Incubation of planaria with 16-glutathionyl, 17-hydroxy-4Z,7Z,10,12,14,19Z-docosahexaenoic acid and 16-cysteinylglycinyl, 17-hydroxy-4Z,7Z,10,12,14,19Z-docosahexaenoic acid gave an acceleration in tissue regeneration and a decrease in T₅₀ to ∼3 d. Incubation of injured planaria with 8-glutathionyl, 7,17-dihydroxy-4Z,9,11,13Z,15E,19Z-docosahexaenoic acid and 8-cysteinylglycinyl, 7,17-dihydroxy-4Z,9,11,13Z,15E,19Z-docosahexaenoic acid (Fig. 7C) also led to an acceleration in tissue regeneration with a reduction in T₅₀ from ∼4.5 to ∼3.4 d.

Figure 7. — Novel sulfido-conjugates accelerate tissue regeneration in planaria. A–C) Planaria were surgically injured, then kept in water containing (B) 16-glutathionyl, 17-hydroxy-4Z,7Z,10,12,14,19Z-docosahexaenoic acid plus 16-cysteinylglycinyl, 17-hydroxy-4Z,7Z,10,12,14,19Z-docosahexaenoic acid (50 nM; each), (C) 8-glutathionyl, 7,17-dihydroxy-4Z,9,11,13Z,15E,19Z-docosahexaenoic acid plus 8-cysteinylglycinyl, 7,17-dihydroxy-4Z,9,11,13Z,15E,19Z-docosahexaenoic acid (50 nM; each), or vehicle (water containing 0.01% EtOH), and tissue regeneration was assessed (see Materials and Methods for details). TRI₅₀, time to 50% regeneration. Results are the mean ± sem (n = 4 planaria per group). *P ≤ 0.05 and **P ≤ 0.01 vs. respective surgical injury group.

Novel sulfido-conjugated products are both anti-inflammatory and proresolving

Given the formation of these products in vivo during self-resolving infections, we next investigated actions of the new products on leukocytes at stimulating bacterial clearance. Incubation of 16-glutathionyl, 17-hydroxy-4Z,7Z,10,12,14,19Z-docosahexaenoic acid led to a dose-dependent increase in human macrophage phagocytosis of E. coli, actions that were comparable to those obtained with the proresolving mediator Protectin (D1) (10R,17S-dihydroxy-docosa-4Z,7Z,11E,13E,15Z,19Z-hexaenoic acid; PD1), also known as neuroprotectin D1 (Fig. 8A).

Figure 8. — 17-series sulfido-conjugates display potent anti-inflammatory and proresolving actions with human macrophages. A) Human macrophages (5 × 10⁴ cells per well) were incubated with the indicated concentrations of novel sulfido-conjugated mediators [15 min at 37°C (pH 7.45)] before the addition of fluorescently labeled bacteria (2.5 × 10⁶ cells per well for 40 min at 37°C). Nonphagocytosed cells were washed, extracellular fluorescence was quenched, and phagocytosis assessed using a fluorescence plate reader. B) Human macrophages (5 × 10⁴ cells per well) were incubated with a pH-sensitive fluorophore (30 min at 37°C), then incubated with 10 nM sulfido-conjugated mediators [15 min at 37°C (pH 7.45)] prior to addition of bacteria (2.5 × 10⁶ cells per well for 60 min at 37°C), and assessment of fluorescence was performed using a BZ9000 microscope equipped with a ×20 objective. C) Human macrophages (5 × 10⁴ cells per well) were incubated with the indicated concentrations of the novel sulfido-conjugated products for 10 min at 37°C. Fluorescently labeled apoptotic PMNs (1.5 × 10⁵ cells per well) were then added (40 min at 37°C), and efferocytosis was measured using a fluorescent plate reader. Results for (B) are representative of n = 3 macrophage preparations. Results for (A) and (C) are the mean ± sem (n = 3 macrophage preparations). *P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001 vs. vehicle incubated macrophages.

Phagolysosomal acidification is a critical step in the disposal of phagocytosed bacteria (19), so we next investigated whether 16-glutathionyl,17-hydroxy-4Z,7Z,10,12,14,19Z-docosahexaenoic acid promoted this process. Incubation of macrophages with 16-glutathionyl, 17-hydroxy-4Z,7Z,10,12,14,19Z-docosahexaenoic acid in the presence of bacteria led to a dose-dependent increase in macrophage phagolysosomal acidification (Fig. 8B and n = 3; P < 0.05).

Macrophage clearance of apoptotic cells and cellular debris is a key step in the resolution of inflammation (1, 8). We next investigated whether 16-glutathionyl, 17-hydroxy-4Z,7Z,10,12,14,19Z-docosahexaenoic acid promoted macrophage clearance of apoptotic PMNs. Incubation of macrophages with this product led to a dose-dependent increase in the ability of human macrophages to uptake apoptotic human PMNs (Fig. 8C). Incubation of macrophages with 16-cysteinylglycinyl, 17-hydroxy-4Z,7Z,10,12,14,19Z-docosahexaenoic acid, 16-cysteinyl, 17-hydroxy-4Z,7Z,10,12,14,19Z-docosahexaenoic acid, 8-glutathionyl, 7,17-dihydroxy-4Z,9,11,13Z,15E,19Z-docosahexaenoic acid and 8-cysteinylglycinyl, 7,17-dihydroxy-4Z,9,11,13Z,15E,19Z-docosahexaenoic acid also led to a dose-dependent increase in human macrophage phagocytosis of E. coli, phagolysosomal acidification, and efferocytosis (Fig. 8). Taken together, these results indicate that the novel sulfido-containing products possess potent tissue-regenerative and proresolving actions.

For human translation, we profiled plasma from patients diagnosed with sepsis (see Supplemental Table S1 and Table 2) and compared levels of the new sulfido-conjugated molecules to those of MCTRs (14) as well as proinflammatory cysteinyl LTs (4, 9). LC-MS-MS-based LM metabololipidomics gave 16-cysteinyl, 17-hydroxy-4Z,7Z,10,12,14,19Z-docosahexaenoic acid, 13-glutathionyl, 14-hydroxy-4Z,7Z,9,11,16Z,19Z-docosahexaenoic acid (MCTR1), 13-cysteinylglycinyl, 14-hydroxy-4Z,7Z,9,11,16Z,19Z-docosahexaenoic acid (MCTR2), 13-cysteinyl,14-hydroxy-4Z,7Z,9,11,16Z,19Z-docosahexaenoic acid (MCTR3), and LTE₄ (Table 2). MRM quantification demonstrated that MCTR3 was the more abundant sulfido-conjugated mediator identified in these plasma samples and was at levels comparable with those of LTE₄ (Table 2).

TABLE 2.

SPM sulfido-conjugate levels in human and mouse tissues

Product	24 h Mouse infected spleens (pg/mg)	24 h Resolving mouse exudates (pg/lavage)	Human autopsy spleen (pg/100 mg)	Human sepsis plasma (pg/ml)
PCTR1	19.4 ± 4.8	3.9 ± 2.6	83.1 ± 24.5	^a
PCTR2	^a	3.4 ± 2.8	^a	^a
PCTR3	23.3 ± 4.4	^a	124.5 ± 51.7	1.5 ± 0.8
RCTR1	^a	5.8 ± 2.7	36.0 ± 11.2	^a
RCTR2	^a	^a	^a	^a
RCTR3	^a	^a	^a	^a
MCTR1	10.0 ± 4.1	1.8 ± 0.7	13.6 ± 5.7	3.1 ± 0.9
MCTR2	22.2 ± 17.5	2.4 ± 1.1	22.9 ± 6.3	^a
MCTR3	53.0 ± 14.9	13.4 ± 3.7	33.1 ± 6.2	8.7 ± 2.5
LTC₄	43.3 ± 15.4	8.3 ± 2.2	240.7 ± 73.5	^a
LTD₄	^a	4.9 ± 1.3	49.3 ± 26.7	^a
LTE₄	^a	^a	^a	12.9 ± 2.9
	n = 3 mice	n = 6 mice	n = 3 patients	n = 10 patients

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Samples were extracted using solid-phase extraction columns with an automated extractor, and each was eluted with hexane, methyl formate, and methanol. The methanol fractions were then taken from LC-MS-MS. Products were identified (see Materials and Methods) and quantified using MRM and calibration curves with an r² of 0.99. Results are the mean ± sem. Lavage volume = 5 ml each mouse. ^aBelow limits, ∼1 pg.

DISCUSSION

In the present report, we identified novel 17-series sulfido-containing molecules that were proresolving and tissue regenerative. These molecules were identified with human spleens and activated phagocytes as well as with mouse spleens and inflammatory exudates during E. coli peritonitis. They were produced via 17-lipoxygenation of DHA that was subsequently converted to monohydroxy or dihydroxy peptide conjugates. Incubation of these products with surgically injured planaria gave accelerated tissue regeneration. They also stimulated macrophage efferocytosis of apoptotic PMNs and phagocytosis of bacteria. Together, these results establish the structures of new 17-series sulfido-conjugates, their actions in tissue regeneration, and resolution of infections.

During infections, the host mounts a cellular response to contain and clear the invading pathogens (20). At the site of infection, neutrophils are the first responders, where they phagocytose and clear bacteria. Neutrophils also produce mediators in the resolution phase, including resolvins and protectins, that regulate macrophage responses (8, 20). Herein, we identified with both healthy human neutrophils and apoptotic cells a novel family of sulfido-conjugated products (Figs. 4 and 6). Their production was temporally regulated, possibly reflecting the temporal shift in leukocyte populations at the site of infections (11, 15). Human macrophages and PMNs were found to convert 17-HpD to sulfido-conjugates. Of note, specific product levels were found to differ between human apoptotic PMNs, healthy PMNs and macrophages, suggesting that the biosynthesis of these mediators is distinctly regulated in human leukocyte subtypes and during distinct stages of the inflammatory response (Figs. 4 and 6).

The spleen plays a critical role in host response to both sterile and infectious challenges (8, 20). The 17-series sulfido-conjugates were identified during the course of self-limited infections in both mouse spleens and infectious exudates (Figs. 1 and 2), suggesting a role for these products in regulating host responses to the invading pathogen at both the site of infections and in lymphoid tissues. These sulfido-conjugated molecules were also identified with human spleens (Fig. 3), suggesting that findings made with mice may also be relevant to humans.

LT-modifying agents have utility in select clinical conditions where the biology of the cysteinyl LTs, such as their regulation of smooth muscle contraction and amplification of the inflammatory response, is important (4, 21). In Fig. 1, we found that levels of 17-series sulfido-conjugates were higher than those of arachidonic acid-derived cysteinyl LTs, indicating that their biosynthesis is distinctly regulated from those of the arachidonic acid-derived LTC₄, LTD₄, and LTE₄. This is in line with published findings demonstrating selective mobilization and utilization of DHA to produce proresolving mediators, namely RvD1, PD1, and 17-HD, that display potent host-protective actions in lymphoid organs (22, 23).

During self-limited inflammation, proresolving mediators ensure the activation of mechanisms that are key in preventing exuberant host responses, the clearance of offending pathogens, and the return to homeostasis (8). Accumulation of apoptotic cells at the site of inflammation can result in the release of intracellular material and amplification of the inflammatory response as the apoptotic cells progress to necrosis, a process also observed in trauma patients (20, 24). Thus, clearance of spent cells and other cellular debris is key in the resolution of inflammation and catabasis. The molecules identified herein were found to potently and dose-dependently promote human macrophage clearance of apoptotic cells (Fig. 8). In addition to clearing apoptotic cells, macrophages are also important in clearing invading pathogens. By comparison to PD1 and RvD2, the sulfido-conjugated products, at picomolar-to-nanomolar concentrations, increased uptake of E. coli in a dose-dependent manner. The actions of proresolving mediators on macrophages are characteristically bell-shaped dose responses (11, 14, 25). The results obtained herein with the sulfido-conjugated mediators also gave dose-dependent actions with features similar to those of a bell-shaped dose response (Fig. 8). Of note, maximal activity for increased E. coli phagocytosis by human macrophages was obtained at lower concentrations than with apoptotic cells. This suggests that these mediators may activate different intracellular pathways when promoting the phagocytosis of bacteria versus that of apoptotic cells. These sulfido-conjugated mediators were found to also increase phagolysosomal acidification during bacterial phagocytosis, which is a fundamental process in bacterial killing and clearance (20). In line with these findings, administration of human apoptotic neutrophils, found to carry the new sulfido-conjugated molecules, promoted macrophage phagocytosis and clearance of bacteria during E. coli infections in vivo (Fig. 6). Identification of these novel products in apoptotic cells at levels commensurate with their biologic actions (Fig. 8) suggests that they may exert a protective role when apoptotic PMNs are at sites of infections (Fig. 4).

Reestablishment of barrier function and repair of damaged tissues are critical in ensuring the return to homeostasis following both sterile and/or infectious injury (20). Planaria have emerged as a useful system to assess organ and tissue regeneration because these pathways are conserved throughout evolution (18). We have recently identified a number of mediators that promote tissue regeneration in planaria (8, 14). MaR1 and RvE1 were the first mediators identified to promote tissue regeneration in these organisms (8). In a search to uncover mechanisms in tissue regeneration, we recently identified a new family of mediators, coined MCTR. MCTRs are produced by regenerating planaria as well as in human milk and resolving infectious exudates, and promote tissue regeneration (14). Acceleration of tissue regeneration in planaria was also obtained with 16-glutathionyl, 17-hydroxy-4Z,7Z,10,12,14,19Z-docosahexaenoic acid and 16-cysteinylglycinyl, 17-hydroxy-4Z,7Z,10,12,14,19Z-docosahexaenoic acid or with 8-glutathionyl, 7,17-dihydroxy-4Z,9,11,13Z,15E,19Z-docosahexaenoic acid and 8-cysteinylglycinyl, 7,17-dihydroxy-4Z,9,11,13Z,15E,19Z-docosahexaenoic acid. These findings indicate that these sulfido-conjugated mediators may be shared signals in controlling the complex processes involved in resolution of inflammation, tissue regeneration, and the return to homeostasis. These results also highlight the primordial origins of local acting mediators, including resolvins, protectins, and MCTRs. Indeed, these ω-3 mediators have now been identified in a number of species ranging from planaria, to the Peruvian anchovies, salmon, mice, and humans (8, 14, 26).

LM biosynthesis involves stereospecific enzymatic conversion of precursor fatty acids (1). DHA is precursor to 17-series sulfido-conjugates because incubation of deuterium-labeled DHA with mouse spleens gave products with the expected 5 Da shift in their m/z ratio (Table 1). Human macrophages and PMNs incubated with DHA also gave 17-series sulfido-conjugates (n = 3), and assessment of apoptotic PMN product profiles demonstrated that these cells converted endogenous DHA to these molecules.

The biosynthetic pathways for the novel sulfido-conjugated products are proposed in Fig. 9. This takes into account results from the present analysis with the mechanisms proposed for the biosynthesis of MCTR (14), protectins, and D-series resolvins (10), and the arachidonate pathway including cysteinyl LTs (27, 28). In this proposed scheme, DHA is first converted via 17-lipoxygenation to 17-HpD. This intermediate can undergo a second lipoxygenation at the carbon 7 position yielding 7S,17S-dihydro(peroxy)-4Z,8E,10Z,13Z,15E,19Z-docosahexaenoic acid. This product can then be enzymatically converted to an allylic epoxide that is in turn enzymatically converted to 8-glutathionyl, 7,17-dihydroxy-4Z,9,11,13Z,15E,19Z-docosahexaenoic acid. The addition of glutathione to an allylic epoxide is governed by glutathione S-transferase enzymes in the biosynthesis of MCTR (14) and cysteinyl LTs (4, 6). This 8-glutathionyl, 7,17-dihydroxy-4Z,9,11,13Z,15E,19Z-docosahexaenoic acid can then be converted by GGTs to 8-cysteinylglycinyl, 7,17-dihydroxy-4Z,9,11,13Z,15E,19Z-docosahexaenoic acid and subsequently to 8-cysteinyl, 7,17-dihydroxy-4Z,9,11,13Z,15E,19Z-docosahexaenoic acid.

Figure 9. — Proposed biosynthesis of sulfido-protectins and sulfido-resolvins. Structures and pathways are depicted in likely stereochemistry based on biosynthetic evidence (see main text for details). Their stereochemistries as shown are tentative. PCTR1, 16-glutathionyl, 17-hydroxy-4Z,7Z,10,12,14,19Z-docosahexaenoic acid; PCTR2, 16-cysteinylglycinyl, 17-hydroxy-4Z,7Z,10,12,14,19Z-docosahexaenoic acid; PCTR3, 16-cysteinyl, 17-hydroxy-4Z,7Z,10,12,14,19Z-docosahexaenoic acid; RCTR1, 8-glutathionyl, 7,17-hydroxy-4Z,9,11,13Z,15E,19Z-docosahexaenoic acid; RCTR2, 8-cysteinylglycinyl, 7,17-hydroxy-4Z,9,11,13Z,15E,19Z-docosahexaenoic acid; RCTR3, 8-cysteinyl, 7,17-hydroxy-4Z,9,11,13Z,15E,19Z-docosahexaenoic acid.

The 17-HpD is also enzymatically converted to a 16(17)-epoxide intermediate (29) then to 16-glutathionyl, 17-hydroxy-4Z,7Z,10,12,14,19Z-docosahexaenoic acid. GGT enzymes then convert this product to 16-cysteinylglycinyl, 17-hydroxy-4Z,7Z,10,12,14,19Z-docosahexaenoic acid and then to 16-cysteinyl, 17-hydroxy-4Z,7Z,10,12,14,19Z-docosahexaenoic acid. The interrelationships between products carrying glutathionyl, cysteinylglycinyl, and cysteinyl in the proposed biosynthetic pathways are also supported by product-precursor relationships obtained with DHA, 17-HpD, a GGT inhibitor, and human macrophages (Fig. 5). Of note, because mammalian lipoxygenases insert molecular oxygen predominantly in the S-stereochemistry, we propose that the stereochemistry for the hydroxy groups at the 7 and 17 positions is retained in the biosynthesis of the novel products. It is conceivable that the R-containing diastereomers of these products may also be of biologic relevance in the resolution of inflammation and tissue regeneration. In addition, given that glutathione S-transferase enzymes in the biosynthesis of cysteinyl LTs stereoselectively add glutathione to the allylic epoxide in an R configuration in the carbon 6 position of arachidonate (6, 30), it is likely that the stereochemistry at carbon 8 and 16 in the new pathways (Fig. 9) is retained in the R configuration.

We coin the tetraene-containing molecules as resolvin conjugate in tissue regeneration (RCTR; Fig. 9) because they display potent tissue-regenerative actions and share their biosynthetic pathway, structural features, as well as proresolving actions with the D-series resolvins (8, 10). We coin the triene-containing molecules as protectin conjugate in tissue regeneration (PCTR) because they share biosynthetic pathway, structural features, and biologic actions with the DHA-derived protectins as well as display potent tissue-regenerative actions. Together, these results demonstrate that ω-3 EFAs can be converted via subsequent lipoxygenase activity to an allylic epoxide intermediate. This intermediate is in turn converted to potent bioactive signaling molecules via either enzymatic hydrolysis (8) or conjugation.

In summation, using key bioactions in resolution and tissue regeneration as well as LC-MS-MS-based LM metabololipidomics, we carried out structure elucidation of 2 novel bioactive families of 17-series sulfido-conjugates. These products displayed host-protective, proresolving, and tissue-regenerative actions in both vertebrates and invertebrates as repairers. Specifically, the new peptide-containing molecules promoted phagocytosis of bacteria and efferocytosis of apoptotic cells by macrophages, were present in human sepsis, and accelerated tissue regeneration in planaria. In 1930, EFA deficiencies were shown to result in uncontrolled infections and death (31). Elucidation of these mediators and pathways as well as the determination of their potent bioactions (Fig. 9) provide mechanistic evidence for the host-protective actions for the precursor DHA. In addition, these findings expand the scope and biologic roles for peptide LMs and afford signals in the regulation of host responses to infections and tissue injury.

Supplementary Material

Supplemental Data

supp_29_5_2120__index.html^{(902B, html)}

Acknowledgments

The authors thank Mary Halm Small for expert assistance in manuscript preparation and Drs. Michael Levin and Junji Mokmura (Tufts University, Medford, MA, USA) for providing Dugesia japonica seed colonies and helpful discussions. This work was supported by the U.S. National Institutes of Health (Grant P01GM095467) and a Mérieux Research Grant from the Institut Mérieux (Lyon, France).

Glossary

17-HpD: 17S-hydro(peroxy)-4Z,7Z,10Z,13Z,15E,19Z-docosahexaenoic acid
17-HD: 17S-hydroxy-4Z,7Z,10Z,13Z,15E,19Z-docosahexaenoic acid
CFU: colony-forming unit
DHA: 4Z, 7Z,10Z,13Z, 16Z,19Z-docosahexaenoic acid
EFA: essential fatty acid
GGT: γ-glutamyl transferase
HpD: hydro(peroxy)-docosahexaenoic acid
LC-MS-MS: liquid chromatography tandem mass spectrometry
LM: lipid mediator
LT: leukotriene
MaR1: Maresin 1 (7R,14S-dihydroxy-docosa-4Z,8E,10E,12Z,16Z,19Z-hexaenoic acid)
MCTR: maresin conjugate in tissue regeneration
MCTR1: 13-glutathionyl,14-hydroxy-docosahexaenoic acid
MCTR3: 13-cysteinyl,14-hydroxy-docosahexaenoic acid
MRM: multiple reaction monitoring
MS: mass spectrometry
m/z: mass-to-charge ratio
PCTR: protectin conjugate in tissue regeneration
PD1: Protectin (D1) (10R, 17S-dihydroxy-docosa-4Z,7Z,11E,13E,15Z,19Z-hexaenoic acid)
PG: prostaglandin
PMN: polymorphonuclear neutrophil
RCTR: resolvin conjugate in tissue regeneration
RvD1: resolvin D1 (7S,8R,17S-trihydroxy-docosa-4Z,9E,11E,13Z,15E,19Z-hexaenoic acid)
RvD2: resolvin D2 (7S,16R,17S-trihydroxy-docosa-4Z,8E,10Z,12E,14E,19Z-hexaenoic acid)
RvE1: resolvin E1 (5S,12R,18R-trihydroxy-eicosa-6Z,8E,10E,14Z,16E-pentaenoic acid)
SPM: specialized proresolving mediator
T₅₀: time to 50% regeneration
T_R: retention time
TRI: tissue regeneration index
TRI_max: maximum tissue regeneration

Footnotes

This article includes supplemental data. Please visit http://www.fasebj.org to obtain this information.

REFERENCES

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Associated Data

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Supplementary Materials

Supplemental Data

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supp_fj.14-268441_Supplemental_Figure1.pdf^{(90.4KB, pdf)}

supp_fj.14-268441_Supplemental_Table1.docx^{(45.5KB, docx)}

[B1] 1.Buckley C. D., Gilroy D. W., Serhan C. N. (2014) Proresolving lipid mediators and mechanisms in the resolution of acute inflammation. Immunity 40, 315–327 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B2] 2.Avicenna, adapted by L. Bakhtiar (1999) The Canon of Medicine (al-Qanun fi'l-tibb), G. B. I. W., Incorporated, Chicago [Google Scholar]

[B3] 3.Eltzschig H. K., Sitkovsky M. V., Robson S. C. (2012) Purinergic signaling during inflammation. N. Engl. J. Med. 367, 2322–2333 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B4] 4.Samuelsson B. (2012) Role of basic science in the development of new medicines: examples from the eicosanoid field. J. Biol. Chem. 287, 10070–10080 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B5] 5.Nathan C. (2012) Fresh approaches to anti-infective therapies. Sci. Transl. Med. 4, 140sr2. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B6] 6.Martinez Molina D., Wetterholm A., Kohl A., McCarthy A. A., Niegowski D., Ohlson E., Hammarberg T., Eshaghi S., Haeggström J. Z., Nordlund P. (2007) Structural basis for synthesis of inflammatory mediators by human leukotriene C4 synthase. Nature 448, 613–616 [DOI] [PubMed] [Google Scholar]

[B7] 7.Flannigan K. L., Agbor T. A., Blackler R. W., Kim J. J., Khan W. I., Verdu E. F., Ferraz J. G., Wallace J. L. (2014) Impaired hydrogen sulfide synthesis and IL-10 signaling underlie hyperhomocysteinemia-associated exacerbation of colitis. Proc. Natl. Acad. Sci. USA 111, 13559–13564 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B8] 8.Serhan C. N. (2014) Pro-resolving lipid mediators are leads for resolution physiology. Nature 510, 92–101 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B9] 9.Samuelsson B., Dahlén S. E., Lindgren J. A., Rouzer C. A., Serhan C. N. (1987) Leukotrienes and lipoxins: structures, biosynthesis, and biological effects. Science 237, 1171–1176 [DOI] [PubMed] [Google Scholar]

[B10] 10.Serhan C. N., Hong S., Gronert K., Colgan S. P., Devchand P. R., Mirick G., Moussignac R. L. (2002) Resolvins: a family of bioactive products of omega-3 fatty acid transformation circuits initiated by aspirin treatment that counter proinflammation signals. J. Exp. Med. 196, 1025–1037 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B11] 11.Chiang N., Fredman G., Bäckhed F., Oh S. F., Vickery T., Schmidt B. A., Serhan C. N. (2012) Infection regulates pro-resolving mediators that lower antibiotic requirements. Nature 484, 524–528 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B12] 12.Rajasagi N. K., Reddy P. B., Suryawanshi A., Mulik S., Gjorstrup P., Rouse B. T. (2011) Controlling herpes simplex virus-induced ocular inflammatory lesions with the lipid-derived mediator resolvin E1. J. Immunol. 186, 1735–1746 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B13] 13.El Kebir D., Gjorstrup P., Filep J. G. (2012) Resolvin E1 promotes phagocytosis-induced neutrophil apoptosis and accelerates resolution of pulmonary inflammation. Proc. Natl. Acad. Sci. USA 109, 14983–14988 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B14] 14.Dalli J., Chiang N., Serhan C. N. (2014) Identification of 14-series sulfido-conjugated mediators that promote resolution of infection and organ protection. Proc. Natl. Acad. Sci. USA 111, E4753–E4761 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B15] 15.Dalli J., Serhan C. N. (2012) Specific lipid mediator signatures of human phagocytes: microparticles stimulate macrophage efferocytosis and pro-resolving mediators. Blood 120, e60–e72 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B16] 16.Orning L., Bernström K., Hammarström S. (1981) Formation of leukotrienes E3, E4 and E5 in rat basophilic leukemia cells. Eur. J. Biochem. 120, 41–45 [DOI] [PubMed] [Google Scholar]

[B17] 17.McCauley L. K., Dalli J., Koh A. J., Chiang N., Serhan C. N. (2014) Cutting edge: Parathyroid hormone facilitates macrophage efferocytosis in bone marrow via proresolving mediators resolvin D1 and resolvin D2. J. Immunol. 193, 26–29 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B18] 18.Sánchez Alvarado A. (2006) Planarian regeneration: its end is its beginning. Cell 124, 241–245 [DOI] [PubMed] [Google Scholar]

[B19] 19.Russell D. G., Gordon S. (2009) Phagocyte-Pathogen Interactions: Macrophages and the Host Response to Infection, ASM Press, Washington, D.C. [Google Scholar]

[B20] 20.Majno G., Joris I. (2004) Cells, Tissues, and Disease: Principles of General Pathology, Oxford University Press, Bethesda, MD [Google Scholar]

[B21] 21.Funk C. D. (2005) Leukotriene modifiers as potential therapeutics for cardiovascular disease. Nat. Rev. Drug Discov. 4, 664–672 [DOI] [PubMed] [Google Scholar]

[B22] 22.Miki Y., Yamamoto K., Taketomi Y., Sato H., Shimo K., Kobayashi T., Ishikawa Y., Ishii T., Nakanishi H., Ikeda K., Taguchi R., Kabashima K., Arita M., Arai H., Lambeau G., Bollinger J. M., Hara S., Gelb M. H., Murakami M. (2013) Lymphoid tissue phospholipase A2 group IID resolves contact hypersensitivity by driving antiinflammatory lipid mediators. J. Exp. Med. 210, 1217–1234 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B23] 23.Taketomi Y., Ueno N., Kojima T., Sato H., Murase R., Yamamoto K., Tanaka S., Sakanaka M., Nakamura M., Nishito Y., Kawana M., Kambe N., Ikeda K., Taguchi R., Nakamizo S., Kabashima K., Gelb M. H., Arita M., Yokomizo T., Nakamura M., Watanabe K., Hirai H., Nakamura M., Okayama Y., Ra C., Aritake K., Urade Y., Morimoto K., Sugimoto Y., Shimizu T., Narumiya S., Hara S., Murakami M. (2013) Mast cell maturation is driven via a group III phospholipase A2-prostaglandin D2-DP1 receptor paracrine axis. Nat. Immunol. 14, 554–563 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B24] 24.Zhang Q., Raoof M., Chen Y., Sumi Y., Sursal T., Junger W., Brohi K., Itagaki K., Hauser C. J. (2010) Circulating mitochondrial DAMPs cause inflammatory responses to injury. Nature 464, 104–107 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B25] 25.Serhan C. N., Dalli J., Karamnov S., Choi A., Park C. K., Xu Z. Z., Ji R. R., Zhu M., Petasis N. A. (2012) Macrophage proresolving mediator maresin 1 stimulates tissue regeneration and controls pain. FASEB J. 26, 1755–1765 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B26] 26.Oh S. F., Vickery T. W., Serhan C. N. (2011) Chiral lipidomics of E-series resolvins: aspirin and the biosynthesis of novel mediators. Biochim. Biophys. Acta 1811, 737–747 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B27] 27.Hammarström S., Samuelsson B., Clark D. A., Goto G., Marfat A., Mioskowski C., Corey E. J. (1980) Stereochemistry of leukotriene C-1. Biochem. Biophys. Res. Commun. 92, 946–953 [DOI] [PubMed] [Google Scholar]

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[B29] 29.Hong S., Gronert K., Devchand P. R., Moussignac R. L., Serhan C. N. (2003) Novel docosatrienes and 17S-resolvins generated from docosahexaenoic acid in murine brain, human blood, and glial cells. Autacoids in anti-inflammation. J. Biol. Chem. 278, 14677–14687 [DOI] [PubMed] [Google Scholar]

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PERMALINK

Novel proresolving and tissue-regenerative resolvin and protectin sulfido-conjugated pathways

Jesmond Dalli

Sesquile Ramon

Paul C Norris

Romain A Colas

Charles N Serhan

Abstract

MATERIALS AND METHODS

Cell isolations

Macrophage bacterial phagocytosis, efferocytosis, and phagolysosomal acidification

Microbially induced mouse peritonitis

Sulfido-conjugate biosynthesis and liquid chromatography tandem mass spectrometry identification

Planaria regeneration

Statistical analysis

RESULTS

Identification of novel sulfido-conjugates during self-resolving E. coli infections in mice and with human spleens

Figure 1.

Figure 2.

Figure 3.

Identification of sulfido-conjugated molecules with human leukocytes

Human macrophages

Figure 4.

Human PMN

Physical properties of novel sulfido-conjugated products

TABLE 1.

Product-precursor relationships

Figure 5.

Apoptotic neutrophils produce endogenous 17-series sulfido-conjugates and stimulate the phagocytosis and clearance of bacteria in vivo

Figure 6.

Novel 17-series sulfido-conjugates accelerate tissue regeneration

Figure 7.

Novel sulfido-conjugated products are both anti-inflammatory and proresolving

Figure 8.

TABLE 2.

DISCUSSION

Figure 9.

Supplementary Material

Acknowledgments

Glossary

Footnotes

REFERENCES

Associated Data

Supplementary Materials

ACTIONS

PERMALINK

RESOURCES

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