Frontline Science: Structural insights into Resolvin D4 actions and further metabolites via a new total organic synthesis and validation

Abstract

Local production and downstream metabolism of specialized proresolving lipid mediators (SPMs) are pivotal in regulating their biological actions during resolution of inflammation. Resolvin D4 (RvD4: 4S,5R,17S-trihydroxydocosa-6E,8E,10Z,13Z,15E,19Z hexaenoic acid) is one of the more recently elucidated SPMs with complete stereochemistry biosynthesized from docosahexaenoic acid . Here, we report a new multimilligram commercial synthesis that afforded enough material for matching, validation, and further evaluation of RvD4 functions. Using LC-MS-MS profiling, RvD4 was identified at bioactive amounts in human (1 pg/mL) and mouse bone marrow (12 pg/femur and tibia). In mouse bone marrow, ischemia increased the formation of RvD4 > 37-fold (455 pg/femur and tibia). Two separate mouse ischemic injury models were used, where RvD4 reduced second organ reperfusion lung injury > 50%, demonstrating organ protection. Structure-function relationships of RvD4 demonstrated > 40% increase in neutrophil and monocyte phagocytic function in human whole blood in comparison with 2 separate trans-containing double bond isomers that were inactive. These 2 isomers were prepared by organic synthesis: 4S,5R,17S-trihydroxydocosa-6E,8E,10E,13Z,15E,19Z-hexaenoic acid (10-trans-RvD4), a natural isomer, and 4S,5R,17S-trihydroxydocosa-6E,8E,10E,13E,15E,19Z-hexaenoic acid (10,13-trans-RvD4), a rogue isomer. Compared to leukotriene B , D-series resolvins (RvD1, RvD2, RvD3, RvD4, or RvD5) did not stimulate human neutrophil chemotaxis monitored via real-time microfluidics chambers. A novel 17-oxo-containing-RvD4 product of eicosanoid oxidoreductase was identified with human bone marrow cells. Comparison of 17-oxo-RvD4 to RvD4 demonstrated that with human leukocytes 17-oxo-RvD4 was inactive. Together, these provide commercial-scale synthesis that permitted a second independent validation of RvD4 complete stereochemical structure as well as evidence for RvD4 regulation in tissues and its stereoselective phagocyte responses.

Novel mediators and mechanisms in the resolution of infectious inflammation: evidence for vagus regulation

“…In inflammatory exudates, after RvD1 and RvD2 appear in resolution they are followed by RvD3 and RvD4 biosynthesized from a 4(5)‐epoxide‐containing intermediate . Their complete stereochemical assignments are confirmed using total organic synthesis .…”

“…RvD2 carries potent organ protective actions and enhances bacterial killing and clearance [49], and RvD1 protects from PMN-mediated reperfusion organ injury [50]. In inflammatory exudates, after RvD1 and RvD2 appear in resolution they are followed by RvD3 and RvD4 biosynthesized from a 4(5)-epoxide-containing intermediate [15,[51][52][53]. Their complete stereochemical assignments are confirmed using total organic synthesis [15,[51][52][53][54].…”

“…Electrical stimulation of human vagus increased both resolvin D4 and MaR1 . Of note, resolvin D4 is found in human bone marrow and controls bacterial clearance . Vagus expresses Toll receptors , and coincubations with live E. coli increased RvD4 as well as RvD6, which was not apparent in incubations of vagus alone ex vivo or on electrical stimulation .…”

Commercial scale production of RvD4 opens the resolving door to new research

“…As with all endogenous mediators, it is critical to validate the precise biochemical structures, quantify the endogenous levels, and elucidate the biological actions of these pro-resolving mediators. In the current issue of the Journal of Leukocyte Biology , Winkler et al directly address these concerns and elucidate the endogenous production, metabolism and potent anti-inflammatory effects of Resolvin D4 (RvD4) 5 .…”

“…In the current issue of the Journal of Leukocyte Biology, Winkler et al directly address these concerns and elucidate the endogenous production, metabolism, and potent anti-inflammatory effects of Resolvin D4 (RvD4). 5 Like other D-series resolvins, RvD4 is formed by sequential enzymatic reactions in vivo ( Fig. 1); 15-LOX metabolizes DHA to 17Shydroxydocosahexaenoic acid (HDHA) and 5-LOX then generates 4S,5R,17S-trihydroxydocosahexaenoic acid (that is, RvD4).…”