First thought to be byproducts of platelet activation carrying no significant biological activity, microvesicles are now increasingly appreciated to regulate critical aspects of the host immune response. Since their first description in platelets by Wolf in 1967 (46), the production of these microstructures has been described in many cell types including leukocytes, muscle cells and endothelial cells (47). They also carry distinct functions in both the initiation and resolution of acute inflammation [reviewed in Ref. (48)]. Microvesicles carry a wide range of molecular cargos including miRNA that are implicated in the regulation of hematopoiesis (49) as well as in protection during ischemia-reperfusion-mediated kidney injury (50). The pro-inflammatory cytokine IL1-β is also carried by microvesicles and its loading into these structures is thought to be one of the mechanisms by which this cytokine, which does not possess a secretion motif, is released from cells (51). Morphogens, such as Sonic Hedgehog, are also part of the cargo carried by subsets of these microvesicles, where microvesicles enriched in Sonic Hedgehog were found to promote angiogenesis and thus may play a role in tumor growth (52).

Recently, we found that neutrophil-derived microvesicles also display anti-inflammatory actions (53), carry precursors for the biosynthesis of pro-resolving mediators (29, 54), and display potent pro-resolving and host protective actions (54, 55). Since evolving self-limited inflammatory exudates produce functional microvesicles that also signal to stimulate resolution of inflammation in mice (54), we investigated whether these microvesicles also regulated macrophage efferocytosis. Indeed these microvesicles dose dependently regulated macrophage efferocytosis. Assessment LM-SPM concentrations in these microstructures demonstrated that neutrophil microvesicles, in addition to carrying precursors for the production of the pro-resolving mediators, also carried bioactive mediators including Protectin (PD) 1 (29).

Using a LM profiling approach, we found that microvesicles regulate macrophage LM-SPM profiles. Incubation of macrophages with neutrophil-derived microvesicles increased the biosynthesis of lipoxygenase- and cyclooxygenase-derived LM. These increased the biosynthesis of DHA-derived D-series resolvins, EPA-derived E-series resolvins, and the AA-derived lipoxins and prostanoids (29). Among the SPM that are upregulated, we observed significant increases in RvD5, MaR1, PD1, RvE2, and RvE1. Of interest, incubation of macrophages with G-protein inhibitors (pertussis toxin and cholera toxin) reduced SPM biosynthesis without altering prostanoid levels. Together, these results demonstrated that microparticles selectively stimulate macrophage SPM production in a G-protein-coupled receptor-dependent manner (29).

Having observed increases in SPM levels and efferocyotosis when macrophages were incubated with neutrophil-derived microvesicles and that these microstructures carried elevated concentrations of several SPM precursors, the question arose whether microvesicles contributed to macrophage SPM biosynthesis by donating specific precursors. The potential contribution of microvesicles to transcellular biosynthesis was assessed during macrophage efferocytosis (29). For this purpose, we employed precursurs labeled with deuterium, which can be distinguished from endogenous precursors, to investigate the contribution of essential fatty acids derived from neutrophil-derived microvesicles. During efferocytosis, microvesicles were found to contribute to the production of d₅-RvD2 and d₅-RvD5 from the D-series resolvins and d₅-PD1 from the protectin family (Figure 1). These results demonstrated that during macrophage efferocytosis transcellular biosynthesis contributes to LM production where both microvesicles contribute substrate utilized in the SPM production.

Recent studies suggest that the process of efferocytosis reprograms macrophage responses altering cytokine production (7). Our studies demonstrate that the regulation of functional responses also extends to the production of both pro-resolving and pro-inflammatory LM profiles. Efferocytosis of apoptotic PMN by macrophages increases SPM biosynthesis, primarily, RvD1, RvD2, and LXB₄. SPM biosynthesis during efferocytosis was further upregulated by microvesicles, increases that correlate with an enhancement in the uptake of apoptotic neutrophils by macrophages (29). Of note, addition of microvesicles to macrophages further upregulated macrophage biosynthesis of RvD2, LXB₄, and RvE2, while reducing PGF_2α and TXB₂ (29).

Apoptotic cells were also found to supply precursors to macrophages for the biosynthesis of both eicosanoids and SPM. This contribution was determined using a similar approach to that used for microvesicles where apoptotic neutrophils were enriched in deuterium-labeled (d) precursors. These studies demonstrate that substrates obtained from apoptotic neutrophils were also utilized for the production of pro-resolving mediators, whereas d₅-PD1 and d₅-RvE2 were produced when the substrate was obtained from either microvesicles or apoptotic cells d₅-MaR1 and d₈-LXB₄ were only identified in incubations with labeled apoptotic cells (29) (Figure 1). These results suggest that the origin of the substrate, and potentially its subcellular localization, may influence its contribution to specific mediator families.

Because apoptotic neutrophils stimulate LM biosynthesis in macrophages, we investigated whether this finding held for different macrophage subtypes. Assessment of LM biosynthesis after apoptotic cell efferocytosis demonstrated that the uptake of apoptotic neutrophils by classically activated macrophages upregulated the SPM production and reduced prostanoid biosynthesis. Of note, incubation of apoptotic neutrophils with M2 macrophages reduced overall LM production, including SPMs (29). These results indicated that the regulation of SPM profiles by apoptotic cells is also cell type dependent and may reflect the distinct biological actions of diverse cell types within the initiation-resolution spectrum.

SPM exert their biological actions via activating receptors of the G-protein-coupled receptor superfamily. RvD1, AT-RvD1, RvD3, AT-RvD3, LXA₄, and AT-LXA₄ activate the lipoxin receptor ALX/FPR2; in humans, these mediators also activate the orphan receptor DRV1/GPR32. RvD5 was also recently shown to activate DRV1/GPR32, while RvE1 binds to and activates ERV1/ChemR23; and DRV2/GPR18 mediates the biological actions of RvD2 (9, 23, 31, 39). Interested readers are referred to Ref. (9) for a detailed review on the biology of SPM receptors. In addition to displaying agonist actions to specific GPCR, RvE1, MaR1, and the MaR1 further metabolite 22-OH-MAR1 are also partial agonists/antagonists to LTB₄ receptor BLT1.

Activation of the pro-resolving receptors by their cognate mediators occurs in a stereospecific manner with even minor changes to their structure resulting in a significant loss in their ability to bind and activate these receptors. Recent studies suggest that the expression of SPM receptors differs between macrophage subsets thus suggesting that pro-resolving mediators differentially regulate the biological actions of distinct macrophage subsets. Murine peritoneal macrophages express the murine homologs of the ALX/FPR2, DRV2/GPR18, and ERV1/ChemR23 that mediate the biologic actions of their cognate SPM in regulating pro-inflammatory cytokine production, upregulating bacterial clearance and efferocytosis of apoptotic cells (9). In humans, expression of ERV1/Cherm23 was recently suggested to be restricted to macrophages obtained under conditions leading to a classically activated phenotype (42).

Studies using LM metabolomics and self-resolving exudates uncovered a new family pro-resolving mediators produced by macrophages, these mediators were coined macrophage mediators in resolving inflammation (maresins) (56). In the biosynthesis of maresins, DHA is lipoxygenated at carbon 14 yielding 14S-hydro(peroxy)-docosa-4Z,7Z,10Z,12E,16Z,19Z-hexaenoic acid (14-HpDHA) that is further converted to an allylic epoxide, reactions carried out by macrophage 12-lipoxgenase (30, 57). Using stereocontrolled total organic synthesis, enantiomerically pure 13S,14S-epoxy-docosa-4Z,7Z,9E,11E,16Z,19Z-hexaenoic acid (13S,14S-eMaR) was prepared and its stereochemistry confirmed by nuclear magnetic resonance spectroscopy. In macrophages, this intermediate is enzymatically converted to the pro-resolving mediator MaR1 (Figure 2). The stereochemistry of MaR1 was recently established as 7R,14S-dihydroxydocosa-4Z,8E,10E,12Z,16Z,19Z-hexaenoic acid, using a matching approach with material obtained from biological systems and total organic synthesis (11). The biological actions of MaR1 were also confirmed using synthetic material that included regulating leukocyte responses including limiting neutrophil infiltration in murine peritonitis (ng/mouse range) as well as enhancing human macrophage uptake of apoptotic neutrophils (11).

The biological actions of MaR1 extend beyond the regulation of cellular trafficking, where studies using planaria demonstrate that this mediator accelerates tissue regeneration following surgical injury (11). Upon injury, planaria also produce MaR1 from deuterium-labeled (d₅)-DHA in a lipoxygenase-dependent manner, since an inhibitor to this class of enzymes reduced MaR1 formation and also the ability of planaria to regenerate damaged tissues (11). Planaria are simple organisms capable of rapid regeneration, the process where in mammalian tissue macrophages play a central role. Factors involved in planaria tissue regeneration remained to be identified (58). Hence, we investigated whether MaR1 displays properties in controlling tissue regeneration. To this end, the anterior portions of planaria were surgically removed and the animals given MaR1. This accelerated tissue regeneration with the appearance of head regeneration was evident as early as 3 days post surgery. MaR1 addition at concentrations as low as 1 nM enhanced head regeneration as early as day 3 post injury (11).

MaR1 also possess potent anti-nociceptive actions, dose dependently inhibiting transient receptor potential cation channel subfamily V member 1 (TRPV1) currents in neurons, blocking capsaicin (CAP, 100 nM)-induced inward currents (IC₅₀ = 0.49 ± 0.02) and reducing both inflammatory and chemotherapy-induced neuropathic pain in mice (11, 59, 60).

Of interest, 13S,14S-eMaR, the biosynthetic intermediate in the MaR1 metabolome, is also bioactive. Indeed this intermediate inhibits LTB₄ formation by human leukotriene A₄ hydrolase (LTA₄H) ~40% (p < 0.05) to a similar extent as LTA₄ (~50%, p < 0.05). Furthermore, LTA₄H was not involved in converting 13S,14S-eMaR to MaR1 pointing to the involvement of a yet unidentified epoxide hydrolase in catalyzing this biosynthetic step. 13S,14S-eMaR also reduced (~60%; p < 0.05) arachidonic acid conversion by human 12-LOX and promotes macrophage phenotype switch toward an M2 profile with similar potency as MaR1 (30). Incubation of M1 macrophages with either 13S,14S-eMaR (10 nM) or MaR1 (10 nM) led to significant reductions in the M1 lineage markers CD54 and CD80 expression and a concomitant upregulation of the M2 lineage markers CD163 and CD206 (30). We also investigated the conversion of 13S,14S-eMaR to MaR1 by different human macrophage subtypes. 13S,14S-eMaR (2 µM) with M2 macrophages gave higher MaR1 levels then when the 13S,14S-eMaR was incubated with M1 macrophages. These results suggest that the MaR1 metabolome is central in regulating macrophage function and mediating the biological actions of these phagocytes (30).

Given the roles that macrophages play in the orchestrating wound healing, we questioned whether during the later stages of resolution these cells produce a distinct group of chemical signals that initiate tissue repair and regeneration. Using a systematic approach, we uncovered a group of peptide-lipid conjugated molecules that in addition to carrying the defining SPM pro-resolving actions, including the ability to regulate leukocyte trafficking and counter-regulate pro-inflammatory mediator production, also promote tissue repair and regeneration (8). The human macrophage 12-lipoxygenase is the initiating enzyme in the formation of these new signaling molecules, converting docosahexaenoic acid to 14S-HpDHA and then to 13S,14S-eMaR. Given that the initial biosynthetic steps are shared with MaR1, and carried a carbon 14 position alcohol, these bioactive molecules were coined as MCTR. The intermediate epoxide is converted to 13R-glutathionyl,14S-hydroxy-4Z,7Z,9E,11E,13R,14S,16Z,19Z-docosahexaenoic acid (MCTR1), a step that in human macrophages is catalyzed by Glutathione S-transferase MU 4 (GSTM4) and leukotriene C₄ synthase (LTC₄S) (61) (Figure 2). Of note, the two enzymes are shared with the cysteinyl LT pathway where they also catalyze the conversion of LTA₄ to leukotriene C₄ (LTC₄). Each of these enzymes displayed different affinities to the two substrates, where LTC₄S displayed a higher affinity to LTA₄, whereas GSTM4 displayed a higher affinity toward 13S,14S-eMAR. These findings suggest that in addition to substrate availability, the relative expression of the two enzymes in one cell type may determine the balance between the inflammation-, contraction-, and stress-initiating LTC₄ (7) in contrast with the tissue-regenerative pathway of MCTRs. MCTR1 is precursor to 13R-cysteinylglycinyl,14S-hydroxy-4Z,7Z,9E,11E,13R,14S,16Z,19Z-docosahexaenoic acid (MCTR2), a conversion catalyzed by gamma-glutamyl transferase (Figure 2). This mediator in addition to carrying pro-resolving and tissue regenerative actions is also precursor to the third member of the MCTR family of mediators, 13R-cysteinyl,14S-hydroxy-4Z,7Z,9E,11E,13R,14S,16Z,19Z-docosahexaenoic acid (MCTR3), a reaction that is catalyzed by dipeptidases (61) (Figure 2). Of note, these enzymes are also shared with the cysteinyl LT biosynthetic pathway suggesting that processes regulating substrate availability may be critical in determining the macrophage LM phenotype and therefore function.

The production and biological actions of members of the maresin family are evolutionary conserved with identification in planaria mouse infectious exudates, human breast milk, spleen, and plasma from sepsis patients (Figure 2). Using deuterium-labeled docosahexaenoic acid, we found that MaR1 was produced by planaria following surgical injury (11). MCTR1 and MCTR2 were also identified in regenerating planaria and incubation of these mediators with planaria accelerated tissue regeneration (8). This increase in the rate of regeneration was associated with an early upregulation of a number of genes that in these animals are associated with head-to-tail differentiation suggesting that these molecules form part of the tissue regeneration process engaged following injury in planaria. This is further supported by the finding that planaria express genes that are homologous to MCTR biosynthetic enzymes including GSTM4, which are upregulated in regenerating tissues (8). Inhibition of these enzymes using both genetic approaches and small molecule inhibitors reduced MCTR levels as well as the ability of planaria to regenerate. In mice, MCTRs were also found to regulate tissue repair and regeneration in lung tissue where administration of these mediators during ischemia-reperfusion-mediated injury protected the lung from leukocyte-mediated damage and upregulated the expression of molecules that are associated with cell proliferation and tissue repair in the lung (8).

Therefore, SPM emerge both as potent regulators of macrophage responses of interest during the resolution phase of acute inflammatory responses and effectors in macrophage-mediated responses. Since the means and methodologies to identify these mediators have only recently become widely available, a number of aspects of their biology in humans have only just started to be explored such as the production of these pro-resolving mediators in human tissues (62–64). In addition, a number of questions still remain to be addressed such as the when and where these mediators are produced in human tissues in health and disease and the relevance of resolution processes that may fail giving rise to human disease. In this context, we recently found that following either a systemic insult or local inflammatory stimulus LM biosynthesis is differentially regulated between males and females. In these studies, vaccination with typhoid vaccine lead to peripheral blood leukocyte activation and endothelia dysfunction in males but not in females. This was associated with an increase peripheral blood E-series resolvin and a downregulation in the levels of LTB₄ in females (65). Local challenge using chantaridin led to a rapid resolution of the inflammatory response in females that was associated with an increased exudate RvD and decreased LTB₄ concentrations. Differences in tissue SPM concentrations were also recently reported in patients with inflammatory arthritis where a correlation between synovial RvE2 concentrations and pain was observed, with higher concentrations of RvE2 in these inflammatory exudates correlating with lower pain in arthritic patients (66). Thus, these results underscore the role of SPM in controlling tissue inflammation in humans and the utility of measuring these mediators as a potential diagnostic tool in patient stratification.

Since resolution of inflammation is a fundamental process in all human tissues and that phagocytes and specifically macrophages play a central role in orchestrating this response as well as tissue repair and regeneration, immunoresolvents (such as resolvins, protectins, and maresins) may provide a novel therapeutic approach for diseases characterized by uncontrolled inflammation and failed resolution. In this context, recent findings demonstrate that enriching microvesicles with SPM may represent a novel therapeutic approach to control chronic inflammation and promote tissue regeneration. In minipigs, administration of a single dose of a novel pro-resolving nanomedicine, produced by enriching microvesicles in a lipoxin analog, markedly reduced periodontal disease, and promoted bone regeneration (67). These results, together with many other recent studies, emphasize that using agonists to reprogram the immune cells rather then inhibiting the inflammatory response may represent a novel paradigm to controlling inflammation without compromising the host immune response.