A New E-Series Resolvin: RvE4 Stereochemistry and Function in Efferocytosis of Inflammation-Resolution

Abstract

The resolution of the acute inflammatory response is governed by phagocytes actively clearing apoptotic cells and pathogens. Biosynthesis of the specialized pro-resolving mediators (SPMs) is pivotal in the resolution of inflammation via their roles in innate immune cells. Resolvin E4 (RvE4: 5S,15S-dihydroxy-eicosapentaenoic acid) is a newly uncovered member of the E-series resolvins biosynthesized from eicosapentaenoic acid (EPA) recently elucidated in physiologic hypoxia. This new resolvin was termed RvE4 given its ability to increase efferocytosis of apoptotic cells by macrophages. Herein, we report on the total organic synthesis of RvE4 confirming its unique structure, complete stereochemistry assignment and function. This synthetic RvE4 matched the physical properties of biogenic RvE4 material, i.e. ultra-violet (UV) absorbance, chromatographic behavior, and tandem mass spectrometry (MS²) fragmentation, as well as bioactivity. We confirmed RvE4 potent responses with human M2 macrophage efferocytosis of human apoptotic neutrophils and senescent red blood cells. Together, these results provide direct evidence for the assignment of the complete stereochemistry of RvE4 as 5S,15S-dihydroxy-6E,8Z,11Z,13E,17Z-eicosapentaenoic acid and its bioactions in human phagocyte response.

Keywords: M2 macrophages; eicosapentaenoic acid; lipid mediator; omega-3 fatty acids; phagocytes; pro-resolving; pro-resolving mediator.

Illustration Scheme 1

Proposed biosynthesis of Resolvin E4 (RvE4) by hypoxic human leukocytes. The proposed biosynthesis is initiated by lipoxygenation of EPA by 15-lipoxygenase (15-LOX) to 15S-hydroperoxy-EPE (15S-HpEPE); see (17). This intermediate can be further reduced to the corresponding hydroxyl intermediate 15S-HEPE. The resultant products are 15S-HpEPE or 15S-HEPE and can undergo a second enzymatic lipoxygenation to yield a hydroperoxyl group at the carbon C-5 position, which is reduced to form RvE4.

Figure 1

Retrosynthetic strategy for total organic synthesis and spectroscopic properties of Resolvin E4. The synthesis of RvE4 was accomplished by total organic synthesis from stereochemically pure starting building blocks and was characterized using NMR spectroscopy. (A) Synthetic precursors were prepared from enantiomerically pure commercially available starting materials and assembled using carbon-carbon bond coupling reactions between precursors C1–C7 (black), and C8–C20 (blue) to ensure absolute regio- and stereochemical control. (B) Double-bond geometry was assigned using two-dimensional ¹H-¹H NMR using a JNM ECZ-400S NMR spectrometer with a 400MHz magnet at 20.7°C on a JEOL ROYALPROBE^TM and referenced to the methanol-d₄ (CD₃OD) internal standard. The purple plot depicts positive contours of cross-peaks along the diagonal axis allowing for the full and detailed proton assignment. The enlarged region highlights the Z/E olefinic protons H₆-H₉, H₁₁-H₁₄, and H₁₇-H₁₈. This, in addition to the full ¹H-NMR spectrum of chemical shifts and coupling constants, confirmed the structure of Resolvin E4.

Figure 2

Matching physical properties of biogenic and synthetic RvE4. Screen captures of (A) Biogenic RvE4 monitored by liquid chromatography-tandem mass spectrometry (LC-MS/MS) in negative ion MRM mode for m/z 333>115. Inset: UV spectrum with ${{\lambda }_{\text{max}}}^{\text{MeOH}}=244 nm$. (B) MS/MS fragmentation spectrum with ions matching RvE4. (C) Synthetic RvE4 monitored by LC-MS/MS in negative ion MRM mode for m/z 333>115. Inset: UV spectrum with ${{\lambda }_{\text{max}}}^{\text{MeOH}}=244 nm$. (D) MS/MS fragmentation spectrum with ions matching RvE4. (E) Biogenic and synthetic RvE4 were co-injected and monitored by LC-MS/MS in negative ion MRM mode for m/z 333>115. Inset: RvE4 structure with proposed fragmentations. (F) MS/MS fragmentation spectrum with ions matching RvE4. Arrows on y-axes indicate threshold; 5% for chromatograms (A, C, E), 1% for spectrum (B, D, F).

Figure 3

RvE4 increases human M2 macrophage efferocytosis of senescent red blood cells. Human M2 macrophages were plated on a 6-well plate (2 x 10⁶ cells/well), incubated with synthetic RvE4 (10^-11M to 10^-7M), biogenic RvE4 (10^-7 M), or vehicle (0.01% ethanol vol/vol) for 15 min followed by CFSE-labeled senescent red blood cells (sRBCs) (1:50, M2:sRBCs). After 60 min of co-incubation at 37°C, plates were washed thoroughly to remove non-ingested sRBCs, and cells were fixed with DPBS containing 4% paraformaldehyde for 15 min on ice. Cells were washed with DPBS and removed from plates with a cell scraper in DPBS. M2 macrophages were then taken to flow cytometry to assess efferocytosis. (A) Flow cytometry gating strategy for M2 macrophages. (B) Representative histogram of intracellular CFSE-labeled sRBCs. (C) Dose-Response: percent increase in M2 efferocytosis of sRBCs above vehicle by synthetic RvE4 (solid blue line). EC₅₀ was estimated using non-linear regression (dashed gray line) with log (agonist) vs. response (three parameters). (D) Comparison between synthetic RvE4 (10 nM) and biogenic RvE4 (10 nM): percent increase of M2 efferocytosis of sRBCs above the vehicle (C, D). Results are the percent of efferocytosis above vehicle and expressed as mean ± SEM, n=5 (sRBCs). *p < 0.05, **p < 0.01, and ****p < 0.0001 when compared to vehicle (as control). † (p=0.73) no statistically significant differences when compared between synthetic and biogenic RvE4. Statistical analysis was carried out using one-way ANOVA with Bonferroni multiple comparisons test (C) or two-tailed Student’s t-test (D).

Figure 4

Synthetic RvE4 increases human M2 macrophage efferocytosis of apoptotic human neutrophils. Human M2 macrophages were plated on a 6-well plate (2 x 10⁶ cells/well), incubated with synthetic RvE4 (10^-11 to 10^-7M) or vehicle (0.01% ethanol) for 15 min followed by CFSE-labeled apoptotic neutrophils (1:5 M2: apoptotic neutrophils). After 60 min of co-incubation at 37°C, plates were washed thoroughly to remove non-ingested neutrophils. M2 macrophages were fixed with DPBS containing 4% paraformaldehyde for 15 min on ice. Cells were washed with DPBS and removed from plates with a cell scraper in DPBS. Cells were then taken to flow cytometry to assess efferocytosis. (A) Flow cytometry gating strategy for M2 macrophages. (B) Representative histogram of intracellular CFSE-labeled apoptotic neutrophils. (C) Dose-Response: percent increases in M2 macrophage efferocytosis of apoptotic PMN above vehicle by synthetic RvE4 (solid black line). EC₅₀ was estimated using non-linear regression (dashed gray line) with log (agonist) vs. response (three parameters). Results are represented as mean ± SEM. n=3 healthy human donors. ***p < 0.001 and ****p < 0.0001 compared to vehicle (as control). Statistical analysis was carried out using one-way ANOVA with Bonferroni multiple comparisons test.

Figure 5

Biosynthetic scheme for Resolvin E4 and E-series resolvins. Eicosapentaenoic acid (EPA) is converted to 18R-HEPE via acetylation of COX-2 by aspirin (12) or P450 (13), which is subsequently lipoxygenated by 5-LOX to produce the intermediate 5S-hydroperoxy-18R-HEPE. 5S-hydroperoxy-18R-HEPE can be either reduced to RvE2 (5S,18R-dihydroxy-6E,8Z,11Z,14Z,16E-eicosapentaenoic acid) (14) or to 5S,6S-epoxy-18R-HEPE by enzymatic epoxidation by 5-LOX confirmed by trapping experiments. 5S,6S-epoxy-18R-HEPE can be further hydrolyzed to produce RvE1 (5S,12R,18R-trihydroxy-6Z,8E,10E,14Z,16E-eicosapentaenoic acid) (11, 12). EPA can also be lipoxygenated by 15-LOX to produce RvE3 (17R,18R-dihydroxy-5Z,8Z,11Z,13E,15E-eicosapentaenoic acid) (15). Stereochemistry and proposed biosynthetic route of RvE4 and confirmed actions (❖ as demonstrated in the present paper) adding to the family of E-series resolvins derived from EPA.