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Clinical Trial
. 2012 May 1;188(9):4527-34.
doi: 10.4049/jimmunol.1103652. Epub 2012 Mar 26.

Resolvin E2 formation and impact in inflammation resolution

Sungwhan F Oh  1 , Maria Dona, Gabrielle Fredman, Sriram Krishnamoorthy, Daniel Irimia, Charles N Serhan
Affiliations
Clinical Trial

Resolvin E2 formation and impact in inflammation resolution

Sungwhan F Oh et al. J Immunol. .

Abstract

Acute inflammation and its resolution are essential processes for tissue protection and homeostasis. In this context, specialized proresolving mediators derived from polyunsaturated fatty acids are of interest. In this study, we report that resolvin E2 (RvE2) from eicosapentaenoic acid is endogenously produced during self-limited murine peritonitis in both the initiation and resolution phases. RvE2 (1-10 nM) carries potent leukocyte-directed actions that include: 1) regulating chemotaxis of human neutrophils; and 2) enhancing phagocytosis and anti-inflammatory cytokine production. These actions appear to be mediated by leukocyte G-protein-coupled receptors as preparation of labeled RvE2 gave direct evidence for specific binding of radiolabeled RvE2 to neutrophils (K(d) 24.7 ± 10.1 nM) and resolvin E1 activation of recombinant G-protein-coupled receptors was assessed. In addition to the murine inflammatory milieu, RvE2 was also identified in plasma from healthy human subjects. RvE2 rapidly downregulated surface expression of human leukocyte integrins in whole blood and dampened responses to platelet-activating factor. Together, these results indicate that RvE2 can stimulate host-protective actions throughout initiation and resolution in the innate inflammatory responses.

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Conflict of interest statement

Conflict of Interest Disclosure

CNS is an inventor on patents [resolvins] assigned to BWH and licensed to Resolvyx Pharmaceuticals. CNS is a scientific founder of Resolvyx Pharmaceuticals and owns equity in the company. CNS’ interests were reviewed and are managed by the Brigham and Women’s Hospital and Partners HealthCare in accordance with their conflict of interest policies.

Figures

Figure 1
Figure 1. RvE2 identification in both initiation and resolution phases of self-limited inflammatory responses
A) Differential counting (left axis-line graph, neutrophils (square) and monocytes/macrophages (diamond)) and quantitative lipidomic profiling (right axis-bar graph) of 18-HEPE (grey) and RvE2 (black). B) Representative MS/MS spectra of Resolvin E2 (left) and its precursor 18-HEPE (right). C) FACS plots of exudate cells during the time course. F4/80+ monocytes/macrophages and Gr-1+ neutrophils are indicated (for details, see Materials and Methods). Results are average ± SEM of 3–4 separate mice for each time point; spectra and FACS plots are representative of separate experiments.
Figure 2
Figure 2. RvE2 stops neutrophil chemotaxis and stimulates rapid shape change: single cell monitoring
A) Neutrophils with polarized morphology during exposure to IL-8 gradient quickly become rounded and were unable to regain their polarized morphology 5 minutes after exposure to 10 nM resolvin E2 or E1. B) Time-lapse single cell PMN monitoring. IL-8 gradient was applied at t=0, and RvE2, RvE1 or vehicle alone was added at the 15-minute time point (dotted line). Size of graphs was adjusted to compare deceleration ratio before and after adding resolvins. Results represent average ± SEM of PMN displacement in Y direction, n = 12. C) Regulation of human PMN actin polymerization with RvE2. Isolated human PMN were stimulated with LTB4 and coincubated with either vehicle or 30 nM RvE2. After 15 seconds, cells were fixed in formaldehyde and stained with phalloidin to measure actin polymerization. D) FACS plots and bar graphs: RvE2 reduces LTB4-mediated actin polymerization. Results are average of three separate donors. *p<0.05; ***p<0.005. See also Supplemental Videos S1A-D.
Figure 3
Figure 3. RvE2 enhances non-phlogistic actions of human macrophages: increases in phagocytosis and IL-10
A) RvE2 enhances human macrophage phagocytosis. Macrophages were exposed to different concentrations of RvE2 for 15 minutes (at 37°C, pH 7.45), then FITC-labeled opsonized zymosan was added and incubated for 30 minutes. Particles that were not phagocytized were washed and quenched with trypan blue. Results are shown as % enhancement of phagocytosis above vehicle treatment. B) RvE2 enhances IL-10 production by human macrophages. Macrophages were incubated with different doses of RvE2 for one hour and then exposed to LPS (100 ng/mL). Supernatants were collected 24 hours later and IL-10 level was measured. Results are n≥3 for each concentration and are representative of 4 similar datasets from individual donors. *p<0.05.
Figure 4
Figure 4. RvE2 specific binding and interaction with human leukocyte GPCRs
A) Synthesis scheme of radiolabeled 18[3H]-RvE2 by chemical/biogenic approach. B) Direct binding with isolated human PMN was carried out (see Methods) and a Scatchard plot obtained (Kd = 24.7 ± 10.1 nM and Bmax = 4.9 × 103 ± 9.5 × 102/cells). Results are the average of n=3; graphs are representative of results from three separate donors. C) RvE2 competes for LTB4 with human recombinant BLT1. BLT1-overexpressing beta-arrestin cells were incubated with 30 nM LTB4 first and then increasing concentrations of RvE1 (square) or RvE2 (circle). D) RvE2 (circle) activation of human recombinant ChemR23-overexpressing beta arrestin cells (see Methods) compared to RvE1 (squares).
Figure 5
Figure 5. Resolvin E2 in human circulation and actions in whole blood
A) RvE2 is present in human plasma from healthy subjects. MS/MS extracted ion chromatogram of m/z 333→199 and 333→115 (left) revealed one peak that matched with RvE2 synthetic and authentic standard (e.g. retention time and MS/MS spectrum, right). B) In whole blood, RvE2 regulation of CD18 in human PMN (diamond) and monocytes/macrophages (triangle) ex vivo. Vehicle or increasing concentrations of RvE2 were incubated with human whole blood (30 minutes, 37°C), fixed, RBC lysed and stained with PE-labeled CD18 antibody. Results are average +/− S.E. of four separate donors. C) RvE2 reduced PAF (50 nM)-stimulated CD18 surface expression on isolated human PMN, when exposed to RvE2 for 15 min before PAF or D) when RvE2 (30 nM) was simultaneously incubated with PAF (50 nM); see Methods. Cells were fixed and analyzed at 30 min after PAF stimulation. Results are average of n=6–8 separate donors. *p<0.05; **p<0.01; ***p<0.005 using a paired Student’s two-tailed t test.
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