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Protectins, Resolvins and Maresins - Specialized
Pro-Resolving Mediators

Structural formula of docosahexaenoic acidThe oxygenated metabolites or oxylipins derived from eicosapentaenoic acid (20:5(n-3) or EPA), docosapentaenoic acid (22:5(n-3)) and especially docosahexaenoic acid (22:6(n-3) or DHA) and termed the (neuro)protectins, resolvins and maresins have potent anti-inflammatory and immunoregulatory actions at concentrations in the nanomolar and picomolar range. They are produced by dioxygen-dependent oxidation from these omega-3 essential fatty acids, which are the focus of considerable interest among nutritionists because of the perceived beneficial effects for the health of consumers. Although the mechanisms by which such effects are exerted are not entirely clear, it seems likely that their oxygenated metabolites play a significant part. Simple lipoxygenase and cytochrome P450 metabolites of EPA and DHA, which have related biological properties, are discussed elsewhere on this site for reasons of practical convenience.

The most important of the protectins and related oxylipins are derived from DHA and are dihydroxylated E,E,Z-docosatrienes, i.e. acyclic lipoxygenase metabolites containing a conjugated E,E,Z‑triene unit flanked by two secondary allylic alcohols. However, the nomenclature can be confusing as it has changed as the science has developed. The trivial name 'neuroprotectin' was coined by Professor Charles N. Serhan and colleagues as they were first found in neuronal tissue, though the term 'protectin' was later preferred when it was realized that they occur in many more animal tissues. Subsequently, oxylipins with similar structural features but formed by different enzymatic routes were characterized and termed 'maresins'. Other oxygenated derivatives of DHA and EPA were named 'resolvins' or 'resolution-phase interaction products', because these compounds were first encountered in resolving inflammatory exudates (pus). As this range of lipid mediators has expanded with continuing research, they have been collectively termed ‘specialized pro-resolving mediators’ or ‘SPMs’. Oxylipins derived from EPA are designated as SPMs of the E series, while those formed from the precursor DHA are denoted as either SPMs of the D series. The lipoxins, derived from arachidonic acid, are usually considered to be SPMs also.

A pattern has emerged in which acute inflammation is initiated in animal tissues by prostaglandins and leukotrienes, before there is a switch in metabolism to the production of SPMs. Both initiation and resolution of acute inflammation are required to maintain healthy tissues and SPMs are key factors, as is discussed below.

It should be recognized that establishing the precise stereochemistry of the double bonds and hydroxyl groups in these compounds, which is essential for their biological functions, has been a heroic task involving total synthesis of large numbers of possible isomers for structural comparison with those formed naturally.

1.  Protectins (Neuroprotectins)

In studies of metabolite formation from DHA in brain tissue in response to aspirin treatment, it was shown that new docosanoids were produced that were initially termed ‘neuroprotectins’. Like the leukotrienes, there are three double bonds in conjugation, hence the term ‘docosatriene’ is sometimes employed, although there are six double bonds in total. As it is now recognized that the formation and actions of these docosanoids are not restricted to neuronal tissue, the simpler term ‘protectins’ is preferable. For example, protectin D1 is present in murine inflammatory exudates and lung, in peripheral human blood and exhaled breath condensates, and in a wide range of cell types. The biosynthetic pathway to neuroprotectin NPD1 or protectin D1, as established in murine brain tissue, and human leukocytes and lymphocytes, is illustrated.

Protectin biosynthesis

The 15-lipoxygenase product 17S-hydroperoxy-DHA is converted first to a 16(17)-epoxide and then to the 10,17-dihydroxy-docosatriene (10R,17S-dihydroxy-docosa-4Z,7Z,11E,13E,15Z,19Z-hexaenoic acid), denoted as 10R,17S-DT or PD1 (or NPD1). Each step in the biosynthetic sequence is under precise stereochemical control by enzymes, and the structures of these DHA-derived mediators are highly conserved from fish to humans. Synthesis of PD1 is induced as a response to oxidative stress and/or activation of neurotrophins, and this highly stereospecific structure is essential for biological activity.

Further oxygenation can occur, and 17S-hydroperoxy-DHA can react with 5-lipoxygenase to form 10S,17S-dihydroxy-docosatriene in a double di-oxygenation reaction, as illustrated, together with some of the 7,17-isomer. However, the intermediate 16,17-epoxy-docosatriene can also undergo non-enzymatic hydrolysis to yield 10R/S,17S-dihydroxy- and 16R/S,17S-dihydroxy-docosatrienes.

Both 22:5(n-3) and 22:5(n-6) fatty acids are also good substrates for 15-lipoxygenase. Thus, 22:5(n-3) is first subjected to 17-lipoxygenation and then via an epoxy intermediate to either 10,17-dihydroxy-7Z,11,13,15,19Z-docosapentaenoic acid (designated PD1n-3 DPA) or 16,17-dihydroxy-7Z,10,13,14,19Z-docosapentaenoic acid (PD2n-3 DPA). 22:5(n-6) gives 17S-hydroxy-22:5(n-6) and 10,17S-dihydroxy-22:5(n-6) as the main products. An omega-hydroxylated analogue of PD1 (22-OH-PD1) has also been detected in animal tissues. All of these are potent anti-inflammatory agents.

Unfortunately, an isomer generated from DHA by the action of plant 15-lipoxygenase in a double di-oxygenation reaction was misidentified as PD1 and was made available commercially under that name. This isomer (now designated ‘PDX’) differs in the geometry of double bonds in the conjugated triene, which is E,Z,E for PDX and E,E,Z for PD1, and also in the configuration of carbon 10, which is S in PDX and R in PD1.

Formula of protectin PDX

It has different properties from authentic PD1, and its use has lead to some erroneous reports in the literature. On the other hand, PDX is now known to be a minor endogenous metabolite in murine peritonitis, and it has been found to have useful biological properties in its own right. For example, oxylipins with the E,Z,E-conjugated triene motif and collectively named 'poxytrins' (PUFA oxygenated trienes) may have antithrombotic potential.

2.  Aspirin-Triggered Protectins

An important additional route to protectin formation produces the 17R-epimer, i.e. 10R,17R-dihydroxy-docosa-4Z,7Z,11E,13E,15Z,19Z-hexaenoic acid, and requires the intervention of aspirin (acetylsalicylic acid), the mode of action of which was described in our web page on prostaglandins. In brief, aspirin blocks the catalytic site of COX-1 by acetylating it irreversibly, but it can only partially block that of COX-2. The acetylated COX-2 retains lipoxygenase-like activity similar to that of 15-lipoxygenase, but with the oxygen insertion in the R- rather than S-configuration as is the case with lipoxygenases; the initial oxygenation reaction is not catalysed by unacetylated COX-2 or COX-1. With arachidonate as substrate, the 15R-HETE is converted to lipoxins, the first specific lipid mediators known to initiate resolution of inflammation. In effect, low-dose aspirin jump starts the resolution phase. Note that unlike aspirin, most non-steroidal anti-inflammatory drugs inhibit cyclooxygenases reversibly and can delay complete resolution.

Structure of aspirin-triggered protectin D1

The production of 'aspirin-triggered' resolvins (see below) and then of protectins were first identified in resolving exudates and brain in mice and is now well established for human cells. Once more, the anti-inflammatory effects of the 10R,17R-isomer are dependent on the precise stereochemistry, as the 10S,17R-dihydroxy-docosatriene is essentially inactive in vivo.

3.  Resolvins

(R)-Resolvins (aspirin-triggered): Resolvins are produced both from EPA and DHA, and the trans-cellular mechanism is illustrated below for those derived from EPA. Biosynthesis of the 18R (illustrated) and 18S resolvins of the E series (name derived from EPA) has elements in common with the synthesis of the epi-lipoxins, leukotrienes and of course the protectins.

Biosynthesis of the 18R-resolvins

In vascular endothelial cells derived from blood vessels during inflammation, the cyclooxygenase enzyme COX-2 that has been acetylated by aspirin introduces an 18R-hydroperoxy group into the EPA molecule (see previous section). The product is reduced to the corresponding hydroxy compound and transferred to a neighbouring leukocyte before a 5S-hydroperoxy group is introduced into the molecule by the action of 5-lipoxygenase, as in the biosynthesis of the leukotrienes. A further reduction step produces 15S,18R-dihydroxy-EPE or resolvin E2 (RvE2). Alternatively, the 5S-hydroperoxy,18R-hydroxy-EPE intermediate is converted to a 5,6-epoxy fatty acid in polymorphonuclear neutrophils in humans and eventually to 5S,12R,18R-trihydroxy-6Z,8E,10E,14Z,16E-eicosapentaenoic acid or resolvin E1 (RvE1) by an enzyme required for the biosynthesis of leukotrienes in leukocytes, i.e. leukotriene A4 hydrolase (LTA4H). There is also a resolvin E3 (RvE3), 17,18-dihydroxy-5Z,8Z,11Z,13E,15E-EPE, synthesised by eosinophils via the 12/15-lipoxygenase pathway.

DHA is converted to 17R-resolvins by a similar aspirin-triggered COX-2 mechanism to the previous (in the absence of aspirin, COX-2 in human microvascular endothelial cells converts DHA to 13S-hydroxy-DHA). Thence, enzymatic epoxidation generates either 7S,(8)-epoxy or 4S,(5)-epoxy intermediates, which are acted upon by 5-lipoxygenase to yield the resolvins. The former produces the aspirin-triggered resolvins D1 and D2 illustrated ('D' nomenclature derived from DHA), while the latter produces the aspirin-triggered resolvins D3 and D4 with all containing a 17R-hydroxyl group. AT-RvD1 is 7S,8R,17R-trihydroxy-docosa-4Z,9E,119E,13Z,159E,19Z-hexaenoic acid.

Formulae of aspirin-triggered resolvins D1 and D2

(S)-Resolvins: The highly specific stereochemistry of resolvin E1 is required for activation of a ligand-specific receptor and thence for its biological activity down to picomolar concentrations. However, epimeric 18S-resolvins are also produced in vivo by related biosynthetic pathways, with the first step catalysed by 15-LOX as for protectins, and these have their own distinctive biological activities. Similarly, the precursor 18(R)- and 18(S)-hydroxy-EPEs have been shown to have anti-inflammatory effects in vitro.

In an alternative reaction in the absence of aspirin in human whole blood, isolated leukocytes and glial cells, 15-lipoxygenase generates 17S-hydroxy-DHA as the initial product. This is converted to 7S-hydroperoxy,17S-hydroxy-DHA by the action of a lipoxygenase, and thence via epoxy intermediates to resolvin (RvD1 or 7S,8R,17S-trihydroxy-docosa-4Z,9E,11E,13Z,15E,19Z-hexaenoic acid) and epimeric resolvin D2 (RvD2 or 7S,16R,17S-trihydroxy-docosa-4Z,8E,10Z,12E,14E,19Z-hexaenoic acid), i.e. all contain a 17S-hydroxyl group.

A further lipoxygenase-generated intermediate from 17S-hydroxy-DHA is transformed via an epoxide 4S,5S-epoxy-17S-hydroxy-DHA to resolvins D3 and D4. 17R- and 17S-hydroxy-DHA have anti-inflammatory properties of their own, although they have generally been viewed simply as pathway markers and have been found in blood samples. Two further resolvins designated RvD5 and RvD6 have now been characterized.

Formulae of resolvins D3 and D4

The docosapentaenoic acid (22:5(n-3) or DPA) can also be converted to resolvins, first by 17-lipoxygenation to 17-hydroperoxy-8Z,10Z,13Z,15E,19Z-docosapentaenoic acid followed by a 5-lipoxygenase-like reaction to yield three products, of which the most abundant is 7,8,17-trihydroxy-9,11,13,15E,19Z-docosapentaenoic acid (designated RvD1n-3 DPA). Four further metabolites of DPA have a hydroxyl group in position 13 and have been designated as 13-series resolvins (RvTs).

Catabolism: Resolvin E1 is eventually de-activated in tissues by oxidation via at least four distinct pathways, including conversion to 18- and 12-oxo-RvE1, for example, prior to further oxidation. Similarly, RvD1 is de-activated by oxidation to 8- or 17-oxo-RvD as the first step in catabolism.

4.  Maresins and Related Docosanoids

An alternative single oxygenation is found in human macrophages and platelets in which a mediator termed maresin 1 (7R,14S-dihydroxy-docosa-4Z,8E,10E,12Z,16Z,19Z-hexaenoic acid - ‘macrophage mediator in resolving inflammation’ or ‘MaR1’) is formed via the action of human 12-lipoxygenase. Biosynthesis is initiated in macrophages by 14-lipoxygenation of DHA, producing the hydroperoxy intermediate 14S-hydroperoxydocosa-4Z,7Z,10Z,12E,16Z,19Z-hexaenoic acid, which undergoes further conversion via enzymatic 13(14)-epoxidation followed by hydrolysis. The required enzymes have yet to be characterized.

Biosynthesis of Maresin 1

In addition, 13S,14S-epoxy-maresin (13,14-eMar) is the precursor for 13R,14S-dihydroxy-docosahexaenoic acid, designated maresin 2 (MaR2), which also displays potent anti-inflammatory and pro-resolving actions. Similar oxygenated compounds with anti-inflammatory properties are formed from 22:5(n-3) and 22:5(n-6) fatty acids with those derived from the former designated MaR2n-3 DPA and MaR3n-3 DPA. MaR1 has been found in primitive invertebrates suggesting that its structure and function have been conserved in evolution.

Formula of 14S,21S-dihydroxy-DHAMaresin-like di-oxygenated metabolites of DHA involving sequential oxidation by 12-LOX and enzymes of the cytochrome P450 family have been shown to occur in macrophages to produce docosanoids such as 14S,21S-dihydroxy-docosa-4Z,7Z,10Z,12E,16Z,19Z-hexaenoic acid and its epimers. Their synthesis is induced by wounding and they have been shown to promote the healing of wounds. Similar 14,22-dihydroxy-metabolites are synthesised by a related mechanism in leukocytes and platelets and also promote wound healing. 14S,20R-dihydroxy-DHA is produced by the action of 12/15-lipoxygenase in eosinophils and has been detected in inflammatory exudates.

5.  Sulfido-Conjugates of Specialized Pro-Resolving Mediators

Sulfido-peptide conjugated mediators with some structural similarity to the cysteinyl-leukotrienes and with novel biological properties are now known to be produced by SPMs, and they have been termed protectin conjugates in tissue regeneration (PCTR), resolvin conjugates in tissue regeneration (RCTR), and maresin conjugates in tissue regeneration (MCTR). For example, while 13S,14S-epoxy-maresin has biological activity in its own right, it is also the precursor of a family of novel mediators with some structural similarity to the cysteinyl-leukotrienes, i.e. 13-glutathionyl,14-hydroxy-docosahexaenoic (MCTR1), 13-cysteinylglycinyl,14-hydroxy-docosahexaenoic (MCTR2) and 13-cysteinyl,14-hydroxy-docosahexaenoic (MCTR3) acids. They promote the resolution of infection and organ protection.

Proposed biosynthetic route to sulfido-conjugated mediators

Similarly, sulfido-conjugates are produced by resolvins and protectins, and those of the 17-series are produced by human leukocytes and are highly abundant in lymphatic tissue. Macrophages produce many such metabolites but in particular a protectin analogue PCTR1, derived from 16S,17S-epoxy-protectin, which enhances resolution of bacterial infections.

6.  Biological Activity

The protectins, resolvins and maresins (SPMs) are distinctive lipids with highly stereospecific structures, which are endogenous local mediators with strong anti-inflammatory effects in addition to some immunoregulatory activities at picomolar to nanomolar concentrations. They are part of the molecular mechanisms that contribute to removal of inflammatory cells and restoration of tissue integrity once the need for the inflammatory response is over, i.e. they actively assist in the resolution of inflammation, once thought to be a passive process. It has become apparent that a novel aspect of the resolution process is that SPMs are able to induce changes in the phenotype of macrophages toward a pro-resolution state.

Acute inflammation in response to infection or tissue damage is usually characterized by heat, redness, swelling and pain at a simple observational level, and by oedema, accumulation of neutrophils, and then by accumulation of monocytes and macrophages at a cellular level. The latter are the first line of defense of the innate immune system to defeat pathogens and toxins. While the symptoms of chronic inflammation may not appear as serious as those of acute inflammation, the consequences can be equally dangerous without proper resolution. Leukotrienes (especially LTB4) and prostaglandins (PGE2 and PGD2) derived from arachidonic acid are important in the early stages of the inflammatory process by stimulating the migration of neutrophils to the affected tissue. Protectins, resolvins and maresins are autacoids, which are produced at the site of inflammation to help tissues to return to health by promoting resolution of inflammation through recruitment of non-inflammatory monocytes. The lipoxins, derived from an omega-6 fatty acid, are also important in the latter process. Macrophages and mast cells are then able to remove excess neutrophils together with cellular debris via draining lymph nodes, ideally without leaving remnants of the host defences or of the invading microorganisms or other inflammatory initiators.

It is evident that the presence of aspirin uniquely facilitates the resolution of inflammation. Thus, at local sites of inflammation, aspirin treatment enhances the conversion of the omega-3 fatty acids EPA and DHA to 18R- and 17R-oxygenated products, respectively, i.e. resolvins of the E and D series, which carry potent anti-inflammatory signals. So far two receptors have been identified that mediate the activities of RvD1 and RvE1, designated GPR32 and ChemR23, respectively, while resolvin RvD2 activates a cell surface G protein-coupled receptor, GPR18/DRV2.

During inflammation, polymorphonuclear neutrophils are produced which have generally beneficial effects in countering disease by removing invading pathogens by phagocytosis, but in the longer term or if malfunctioning they may eventually cause trauma and tissue damage through infiltration into tissues. The resolvins, like the lipoxins, appear to have an important role in regulating and indeed inhibiting these harmful effects. In so doing, they oppose the effects of some of the pro-inflammatory prostanoids. For example, nanomolar concentrations of resolvin E1 dramatically reduce dermal inflammation, peritonitis, dendritic cell migration and interleukin production. RvE1 blocks excessive platelet aggregation, and it also limits the effects of certain human pathogens by enhancing phagocytosis by polymorphonuclear leukocytes. Similarly, RvD2 and RvD3 have extremely potent regulatory actions on neutrophil trafficking in the picogram range in vivo by stimulating resolution and enhancing innate host defense mechanisms via a specific receptor. Thus, RvD2 interacting with DRV2 ameliorates the effects of bacterial sepsis in experimental animals. In general, RvD3 appears late in resolution suggesting that it has a special role in the last stages of the process. RvD4 has novel pro-resolving actions in Staphyllococcus aureus infections and is a powerful stimulant for the clearance of apoptotic cells by skin fibroblasts.

In relation to atherosclerotic plaques, it has been reported that the deleterious effects of leukotriene LTB4 resulting from an excessive inflammatory response are countered by the presence of specialized proresolving mediators, especially RvD1, suggesting a new therapeutic approach to promote plaque stability.

Scottish thistleProtectins appear to operate in the same way as the resolvins in brain tissue. Thus, (N)PD1 has anti-inflammatory effects and protects retinal epithelial cells from apoptosis induced by oxidative stress. Indeed, it promotes nerve regeneration. In addition, it has protective effects in animal models of stroke and of Alzheimer's disease. Amongst its activities in non-neuronal tissues, it promotes apoptosis of T cells, it has beneficial effects towards asthma in nanogram amounts, and it is reported to have therapeutic potential against virus infections. Protectins and maresins reduce neuropathic pain in experimental animals, the latter by interfering with vanilloid receptor TRPV1 in dorsal root ganglion neurons and blocking capsaicin activity.

Similarly, maresin 1 is a powerful regulator of resolution of inflammation, tissue regeneration and pain. Maresins appear to be especially important for tissue regeneration and wound healing in the later stages of resolution. In addition, the epoxy intermediate in maresin biosynthesis, 13S,14S-epoxy-maresin, affects macrophage activity and function independently by inhibiting the production of leukotrienes and other inflammatory eicosanoids from arachidonic acid.

At nanomolar concentrations, sulfido-conjugates derived from epoxy-maresin were shown to resolve E. coli infections, and in general they constitute a novel mechanism of chemical signalling that contains infections, stimulates resolution of inflammation, and promotes the restoration of function in human tissues and those of experimental animals. Similarly, protectin and resolvin sulfido-conjugates stimulated human macrophages and limited the effects of bacterial infections in a dose-dependent manner.

Although the specialized pro-resolving mediators are produced locally to terminate inflammation, they also reach the circulation and are found in human peripheral blood, suggesting that they may act as anti-inflammatory signals in tissues other than those in which they originate. Proresolving mediators together with lipoxins have been found at bioactive levels in human breast milk and in animal placenta, so it is possible that they may have regulatory functions in normal physiological development as well as in pathophysiological processes.

It is now well established that administration of lipoxins, protectins, resolvins and maresins in vivo and in vitro in animal models can aid the process of recovery from inflammation without compromising host defences by causing immune suppression. It is evident that such compounds and their metabolism have considerable potential for therapeutic intervention in acute inflammation or chronic inflammatory disease, and they have been tested in a wide range of experimental models, including peritonitis, periodontitis, colitis, arthritis, dry eye, inflammatory pain, cardiovascular disease, asthma and other lung ailments, and even cancer. A phase III clinical trial of a RvE1 analogue against dry eye syndrome is underway, and NPD1/PD1 is in clinical development for neurodegenerative diseases. Such compounds also appear to beneficial towards attack by bacteria, fungi, viruses and parasites, including such debilitating diseases as influenza and malaria. For example, protectin PDX has therapeutic potential in that it suppresses replication of the influenza virus by inhibiting the nuclear export of viral mRNA rather than by regulating the resolution of inflammation. As endogenous mediators, their use may be associated with fewer side effects than other drugs.

From a nutritional or health standpoint, it has been suggested that dietary supplements of the precursor omega-3 fatty acids, taken together with aspirin, may ameliorate the clinical symptoms of many inflammatory disorders by regulating the time course of resolution via the production of resolvins and protectins amongst other effects.

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