*4.1. Biosynthesis of MaRs*

MaRs (from macrophage mediator in resolving inflammation) are a fourth family of DHA-derived SPMs [79]. In macrophages, MaR-1 biosynthesis is initiated by 12-LOX from DHA, producing 14S-hydroperoxydocosa-4Z,7Z,10Z,12E,16Z,19Z-hexaenoic acid—the hydroperoxy intermediate—which undergoes further conversion via enzymatic 13(14)-epoxidation. This epoxide intermediate is hydrolyzed enzymatically via an acid-catalyzed nucleophilic attack by water at carbon 7, resulting in the introduction of a hydroxyl group at that position and a double bond rearrangemen<sup>t</sup> to form the stereochemistry of bioactive MaR1, which has potent pro-resolution properties. The 13S, 14S-epoxy-MaR intermediate is also the precursor of MaR-2 (13R, 14S-dihydroxy-4Z,7Z,9E,11Z,16Z,19Z-DHA). This product of DHA biosynthesis by 12-LOX produces the 14S-hydroperoxide that is converted to the 13S, 14S-epoxy-MaR and finally converted by a soluble epoxide hydrolase into MaR-2 (Figure 3A) [49].

**Figure 3.** Synthesis and function of maresins (MaRs) and macrophages. (**A**) MaRs and MaR conjugates in tissue regeneration (MCTRs) biosynthesis. Human macrophage 12-LOX converts DHA to the 13S,14S-epoxy-maresin intermediate and hydrolase or soluble epoxide hydrolase is converted to MaR1 and MaR2, respectively. The MCTR biosynthetic pathway is initiated by lipoxygenation of 14S-HpDHA, converted by lipoxygenase activity to the 13S,14S-epoxy-maresin intermediate. MCTR1 is catalyzed by glutathione s-transferase mu4 (GSTM4) and/or leukotriene C4 synthase (LTC4S). MCTR1 is converted by gamma-glutamyl transferase (GGT) to MCTR2, which then acts as a substrate for conversion by dipeptidase (DPEP) to MCTR3. (**B**) M1 and M2 polarization of macrophages. Bone marrow-derived macrophages differentiate into mononuclear cells and gradually become mature macrophages that can be released into circulation. IFN-γ, TNF-<sup>α</sup>, and LPS stimulate macrophages into M1, IL-4 and IL-13 into M2a, IC and TLR into M2b, and IL-10 into M2c; A2AR agonist stimulates them into M2d. M1 macrophages induce a proinflammatory response, whereas M2 macrophages induce an anti-inflammatory response. M1 macrophages can also differentiate into M2 macrophages through local cues. The M1 phenotype is proinflammatory, phagocytic, and bactericidal, while the M2 macrophages act to switch off inflammation. IFN-γ: interferon gamma; TNF-α: tumor necrosis factor alpha; LPS: lipopolysaccharides; IC: immune complexes; TLR: toll-like receptor; A2AR: adenosine A2A receptor; IL: interleukin; IL-1R: IL-1 receptor.

#### *4.2. Biosynthesis of MaR Conjugates in Tissue Regeneration (MCTRs)*

Macrophages also produce a family of bioactive peptide-conjugated mediators called MCTRs [80]. MCTR compounds are produced from the 13(S), 14S-epoxide MaR intermediate during MaR biosynthesis. This epoxide intermediate is enzymatically converted to an MCTR. DHA is converted by 12-LOX into 13,14-epoxy-maresin (an intermediate of MaR-1 and MaR-2) that can be directly conjugated at C13 to glutathione by LTC4 synthase, yielding MaR conjugated in tissue regeneration 1(MCTR 1). MCTR 1 (13R-glutathionyl, 14S-hydroxy-4Z,7Z,9E,11E,13R,14S,16Z,19Z-DHA)—the first cysteinyl-SPM to be identified—is synthesized in the presence of leukotriene C4 (LTC4) synthase and γ-glutamyltransferase-μ<sup>4</sup> in human macrophages. γ-Glutamyl transferase is involved in the conversion of MCTR-1 to MCTR2 (13R-cysteinylglycinyl, 14S-hydroxy-4Z,7Z,9E,11E,13R,14S,16Z,19Z-DHA) and MCTR-3 (13R-cysteinyl,14S-hydroxy-4Z,7Z,9E,11E,13R,14S,16Z,19Z-DHA) (Figure 3A) [34,39].

#### *4.3. Function of MaRs and MCTRs*

13S, 14S-epoxy-DHA(eMaR) stimulates the conversion of the M1 macrophage phenotype to M2 and blocks LTA4 hydrolase [81]. MaR1 possesses potent pro-resolving, antinociceptive, tissue regenerative, antiaggregant, and vasculoprotective functions. Recently, MaR-2 was reported to have powerful bioregulatory effects. Similarly, MCTRs act as tissue protective and regenerative agents, with anti-inflammatory and pro-resolving properties [25]. Thus, MaRs and MCTRs are regulated during acute self-limited infectious-inflammation and possess many attributes that contribute to host defense, tissue regeneration, organ protection, and pain modulation [25,34,39,49,72].
