Identification and Complete Stereochemical Assignments of the New Resolvin Conjugates in Tissue Regeneration in Human Tissues that Stimulate Proresolving Phagocyte Functions and Tissue Regeneration.

Resolvin conjugates in tissue regeneration (RCTRs) are new chemical signals that accelerate resolution of inflammation, infection, and tissue regeneration. Herein, using liquid chromatography-tandem mass spectrometry-based metabololipidomics, we identified RCTRs in human spleen, lymph node, bone marrow, and brain. In human spleen incubated with Staphylococcus aureus, endogenous RCTRs were increased along with conversion of deuterium-labeled docosahexaenoic acid, conferring pathway activation. Physical and biological properties of endogenous RCTRs were matched with those prepared by total organic synthesis. The complete stereochemical assignment of bioactive RCTR1 is 8R-glutathionyl-7S,17S-dihydroxy-4Z,9E,11E,13Z,15E,19Z-docosahexaenoic acid, RCTR2 is 8R-cysteinylglycinyl-7S,17S-dihydroxy-4Z,9E,11E,13Z,15E,19Z-docosahexaenoic acid, and RCTR3 is 8R-cysteinyl-7S,17S-dihydroxy-4Z,9E,11E,13Z,15E,19Z-docosahexaenoic acid. These stereochemically defined RCTRs stimulated human macrophage phagocytosis, efferocytosis, and planaria tissue generation. Proteome profiling demonstrated that RCTRs regulated both proinflammatory and anti-inflammatory cytokines with human macrophages. In microfluidic chambers, the three RCTRs limited human polymorphonuclear cell migration. In hind-limb ischemia-reperfusion-initiated organ injury, both RCTR2 and RCTR3 reduced polymorphonuclear cell infiltration into lungs. In infectious peritonitis, RCTR1 shortened the resolution intervals. Each RCTR (1 nmol/L) accelerated planaria tissue regeneration by approximately 0.5 days, with direct comparison to both maresin and protectin CTRs. Together, these results identify a new bioactive RCTR (ie, RCTR3) in human tissues and establish the complete stereochemistry and rank-order potencies of three RCTRs in vivo. Moreover, RCTR1, RCTR2, and RCTR3 each exert potent anti-inflammatory and proresolving actions with human leukocytes.

Structural elucidation and physiologic functions of specialized pro-resolving mediators and their receptors.

The acute inflammatory response is host-protective to contain foreign invaders. Many of today's pharmacopeia that block pro-inflammatory chemical mediators can cause serious unwanted side effects such as immune suppression. Uncontrolled inflammation is now considered a pathophysiologic basis associated with many widely occurring diseases such as cardiovascular disease, neurodegenerative diseases, diabetes, obesity and asthma, as well as the classic inflammatory diseases, e.g. arthritis, periodontal diseases. The inflammatory response is designated to be a self-limited process that produces a superfamily of chemical mediators that stimulate resolution of inflammatory responses. Specialized proresolving mediators (SPM) uncovered in recent years are endogenous mediators that include omega-3-derived families resolvins, protectins and maresins, as well as arachidonic acid-derived (n-6) lipoxins that stimulate and promote resolution of inflammation, clearance of microbes, reduce pain and promote tissue regeneration via novel mechanisms. Here, we review recent evidence from human and preclinical animal studies, together with the structural and functional elucidation of SPM indicating the SPM as physiologic mediators and pharmacologic agonists that stimulate resolution of inflammation and infection. These results suggest that it is time to develop immunoresolvents as agonists for testing resolution pharmacology in nutrition and health as well as in human diseases and during surgery.

Maresin conjugates in tissue regeneration biosynthesis enzymes in human macrophages.

Macrophages are central in coordinating immune responses, tissue repair, and regeneration, with different subtypes being associated with inflammation-initiating and proresolving actions. We recently identified a family of macrophage-derived proresolving and tissue regenerative molecules coined maresin conjugates in tissue regeneration (MCTR). Herein, using lipid mediator profiling we identified MCTR in human serum, lymph nodes, and plasma and investigated MCTR biosynthetic pathways in human macrophages. With human recombinant enzymes, primary cells, and enantiomerically pure compounds we found that the synthetic maresin epoxide intermediate 13S,14S-eMaR (13S,14S-epoxy- 4Z,7Z,9E,11E,16Z,19Z-docosahexaenoic acid) was converted to MCTR1 (13R-glutathionyl, 14S-hydroxy-4Z,7Z,9E,11E,13R,14S,16Z,19Z-docosahexaenoic acid) by LTC4S and GSTM4. Incubation of human macrophages with LTC4S inhibitors blocked LTC4 and increased resolvins and lipoxins. The conversion of MCTR1 to MCTR2 (13R-cysteinylglycinyl, 14S-hydroxy-4Z,7Z,9E,11E,13R,14S,16Z,19Z-docosahexaenoic acid) was catalyzed by γ-glutamyl transferase (GGT) in human macrophages. Biosynthesis of MCTR3 was mediated by dipeptidases that cleaved the cysteinyl-glycinyl bond of MCTR2 to give 13R-cysteinyl, 14S-hydroxy-4Z,7Z,9E,11E,13R,14S,16Z,19Z-docosahexaenoic acid. Of note, both GSTM4 and GGT enzymes displayed higher affinity to 13S,14S-eMaR and MCTR1 compared with their classic substrates in the cysteinyl leukotriene metabolome. Together these results establish the MCTR biosynthetic pathway and provide mechanisms in tissue repair and regeneration.

Specialized proresolving lipid mediators in patients with coronary artery disease and their potential for clot remodeling.

Inflammation in arterial walls leads to coronary artery disease (CAD). Because specialized proresolving lipid mediators (SPMs; lipoxins, resolvins, and protectins) stimulate resolution of inflammation in animal models, we tested whether n-3 fatty acids impact SPM profiles in patients with CAD and promote clot remodeling. Six patients with stable CAD were randomly assigned to either treatment with daily 3.36 g Lovaza for 1 yr or without. Targeted lipid mediator-metabololipidomics showed that both groups had absence of resolvin D1 (RvD1), RvD2, RvD3, RvD5 and resolvin E1-all of which are present in healthy patients. Those not taking Lovaza had an absence of aspirin-triggered resolvin D3 (AT-RvD3) and aspirin-triggered lipoxin B4 (AT-LXB4). Lovaza treatment restored AT-RvD3 and AT-LXB4 and gave levels of RvD6 and aspirin-triggered protectin D1 (AT-PD1) twice as high (resolvin E2 ∼5 fold) as well as lower prostaglandins. Principal component analysis indicated positive relationships for patients with CAD who were receiving Lovaza with increased AT-RvD3, RvD6, AT-PD1, and AT-LXB4 SPMs identified in Lovaza-treated patients with CAD enhanced ∼50% at 1 nM macrophage uptake of blood clots. These results indicate that patients with CAD have lower levels and/or absence of specific SPMs that were restored with Lovaza; these SPMs promote macrophage phagocytosis of blood clots. Together, they suggest that low vascular SPMs may enable progression of chronic vascular inflammation predisposing to coronary atherosclerosis and to thrombosis.-Elajami, T. K., Colas, R. A., Dalli, J., Chiang, N., Serhan, C. N., Welty, F. K. Specialized proresolving lipid mediators in patients with coronary artery disease and their potential for clot remodeling.

The resolution code of acute inflammation: Novel pro-resolving lipid mediators in resolution.

Studies into the mechanisms in resolution of self-limited inflammation and acute reperfusion injury have uncovered a new genus of pro-resolving lipid mediators coined specialized pro-resolving mediators (SPM) including lipoxins, resolvins, protectins and maresins that are each temporally produced by resolving-exudates with distinct actions for return to homeostasis. SPM evoke potent anti-inflammatory and novel pro-resolving mechanisms as well as enhance microbial clearance. While born in inflammation-resolution, SPM are conserved structures with functions discovered in microbial defense, pain, organ protection and tissue regeneration, wound healing, cancer, reproduction, and neurobiology-cognition. This review covers these SPM mechanisms and other new omega-3 PUFA pathways that open their path for functions in resolution physiology.

Proresolving actions of a new resolvin D1 analog mimetic qualifies as an immunoresolvent.

Resolution of inflammation is an active process driven by several new families of endogenous lipid mediators collectively coined specialized proresolving mediators (SPM). Here, we report a synthetic analog of resolvin D1 (RvD1) and aspirin-triggered RvD1, benzo-diacetylenic-17R-RvD1-methyl ester (BDA-RvD1), which was prepared using fewer steps than required for total organic synthesis of natural SPM. BDA-RvD1 was resistant to further metabolism by human recombinant 15-prostaglandin dehydrogenase, a major inactivation pathway for RvD1. In ischemia-reperfusion-initiated second organ injury, BDA-RvD1 intravenously (1 µg) reduced neutrophil infiltration into the lungs by 58 ± 9% and was significantly more potent than native RvD1. BDA-RvD1 at 100 ng/mouse also shortened the resolution interval, Ri, of Escherichia coli peritonitis with a similar potency as RvD1, by ~57%, from Ri 10.5 h to 4.5 h. With isolated human phagocytes, BDA-RvD1 at picomolar concentrations (10(-12) M) stimulated phagocytosis of zymosan A particles. BDA-RvD1 activated human recombinant G protein-coupled receptor 32/DRV1, an RvD1 receptor, in a dose-dependent manner. These results indicate that, both in vivo in mice and with isolated human cells, BDA-RvD1 shares defining proresolving actions of RvD1, including inhibiting leukocyte infiltration and stimulating phagocytosis. Moreover, they provide evidence for a new analog mimetic and example of an immunoresolvent, namely an agent that stimulates active resolution of inflammation, for a potential new therapeutic class.

The Regulation of Proresolving Lipid Mediator Profiles in Baboon Pneumonia by Inhaled Carbon Monoxide.

Strategies for the treatment of bacterial pneumonia beyond traditional antimicrobial therapy have been limited. The recently discovered novel genus of lipid mediators, coined "specialized proresolving mediators" (SPMs), which orchestrate clearance of recruited leukocytes and restore epithelial barrier integrity, have offered new insight into the resolution of inflammation. We performed lipid mediator (LM) metabololipidomic profiling and identification of LMs on peripheral blood leukocytes and plasma from a baboon model of Streptococcus pneumoniae pneumonia. Leukocytes and plasma were isolated from whole blood of S. pneumoniae-infected (n = 5-6 per time point) and control, uninfected baboons (n = 4 per time point) at 0, 24, 48, and 168 hours. In a subset of baboons with pneumonia (n = 3), we administered inhaled carbon monoxide (CO) at 48 hours (200-300 ppm for 60-90 min). Unstimulated leukocytes from control animals produced a proresolving LM signature with elevated resolvins and lipoxins. In contrast, serum-treated, zymosan-stimulated leukocytes and leukocytes from baboons with S. pneumoniae pneumonia produced a proinflammatory LM signature profile with elevated leukotriene B4 and prostaglandins. Plasma from baboons with S. pneumoniae pneumonia also displayed significantly reduced LM-SPM levels, including eicosapentaenoic acid-derived E-series resolvins (RvE) and lipoxins. CO inhalation increased levels of plasma RvE and lipoxins relative to preexposure levels. These results establish the leukocyte and plasma LM profiles biosynthesized during S. pneumoniae pneumonia in baboons and provide evidence for pneumonia-induced dysregulation of these proresolution programs. Moreover, these SPM profiles are partially restored with inhaled low-dose CO and SPM, which may shorten the time to pneumonia resolution.

Resolvin D3 and aspirin-triggered resolvin D3 are potent immunoresolvents.

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

Self-limited versus delayed resolution of acute inflammation: temporal regulation of pro-resolving mediators and microRNA.

Mechanisms underlying delays in resolution programs of inflammation are of interest for many diseases. Here, we addressed delayed resolution of inflammation and identified specific microRNA (miR)-metabolipidomic signatures. Delayed resolution initiated by high-dose challenges decreased miR-219-5p expression along with increased leukotriene B(4) (5-fold) and decreased (~3-fold) specialized pro-resolving mediators, e.g. protectin D1. Resolvin (Rv)E1 and RvD1 (1 nM) reduced miR-219-5p in human macrophages, not shared by RvD2 or PD1. Since mature miR-219-5p is produced from pre-miRs miR-219-1 and miR-219-2, we co-expressed in human macrophages a 5-lipoxygenase (LOX) 3'UTR-luciferase reporter vector together with either miR-219-1 or miR-219-2. Only miR-219-2 reduced luciferase activity. Apoptotic neutrophils administered into inflamed exudates in vivo increased miR-219-2-3p expression and PD1/NPD1 levels as well as decreased leukotriene B(4). These results demonstrate that delayed resolution undermines endogenous resolution programs, altering miR-219-2 expression, increasing pro-inflammatory mediators and compromising SPM production that contribute to failed catabasis and homeostasis.

Stereochemical assignment, antiinflammatory properties, and receptor for the omega-3 lipid mediator resolvin E1.

The essential fatty acid eicosapentaenoic acid (EPA) present in fish oils displays beneficial effects in a range of human disorders associated with inflammation including cardiovascular disease. Resolvin E1 (RvE1), a new bioactive oxygenated product of EPA, was identified in human plasma and prepared by total organic synthesis. Results of bioaction and physical matching studies indicate that the complete structure of RvE1 is 5S,12R,18R-trihydroxy-6Z,8E,10E,14Z,16E-EPA. At nanomolar levels, RvE1 dramatically reduced dermal inflammation, peritonitis, dendritic cell (DC) migration, and interleukin (IL) 12 production. We screened receptors and identified one, denoted earlier as ChemR23, that mediates RvE1 signal to attenuate nuclear factor-kappaB. Specific binding of RvE1 to this receptor was confirmed using synthetic [(3)H]-labeled RvE1. Treatment of DCs with small interference RNA specific for ChemR23 sharply reduced RvE1 regulation of IL-12. These results demonstrate novel counterregulatory responses in inflammation initiated via RvE1 receptor activation that provide the first evidence for EPA-derived potent endogenous agonists of antiinflammation.