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Abstract

Resolvins of the D-series are specialized pro-resolving lipid mediators that regulate cellular response by orchestrating resolution networks involved in host responses to injury and infection. Here, endogenous resolvin D4 was identified in human tissues and found to persist late into the resolution phase of acute murine Staphylococcus aureus infections. Completion of the first total synthesis of resolvin D4 established the absolute stereochemical configuration of RvD4 confirmed by matching with endogenous RvD4 from resolving exudates in dorsal pouch S. aureus infections. In vivo, RvD4 (ng/mouse) reduced neutrophilic infiltration (~40%) and enhanced uptake of apoptotic PMN (51%) by human dermal fibroblasts at concentrations as low as 0.1 nM. These results establish the complete stereochemistry of RvD4 as 4S,5R,17S-trihydroxydocosa-6E,8E,10Z,13Z,15E,19Z-hexaenoic acid and its novel pro-resolving actions in S. aureus infections as well as its potent ability to stimulate clearance of apoptotic cells by skin fibroblasts.

RvD4 stops neutrophil infiltration in sterile peritonitis and S. aureus skin infection

With the biological activity of RvD4 confirmed, we next assessed its in vivo actions in S. aureus-mediated infection probing its action as well as temporal formation. To determine its role in bacterial killing and host protection during S. aureus infections, RvD4 was administered at 200 ng/mouse concomitantly with a self-limited bacterial inoculation (105 c.f.u) into the dorsal pouch of male FVB mice. Resolution indices were then measured. RvD4 potently reduced neutrophilic infiltration by ~40% at Tmax (12 h) and shortened the resolution interval in mice treated with RvD4 from 6 to 3 hr (). Analysis by histochemical staining of dorsal pouch lining from mice administered vehicle or RvD4 gave lower PMN levels in the cavity in mice treated with RvD4 (). Treatment with RvD4 also significantly lowered bacterial counts when compared to vehicle control (). In addition, RvD4 enhanced the phagocytosis of S. aureus by isolated mouse macrophages by ~40% at 10nM (data not shown). RvD4 also regulated inflammation-initiating eicosanoids in the inflammatory exudates including LTB4 and PGD2 by 68 and 48% respectively, demonstrating potent anti-inflammatory actions in vivo ().

RvD4 exerts pro-resolving actions with human neutrophils and macrophages

To further elucidate its host-protective actions, we sought to establish the potent anti-inflammatory and pro-resolving actions of RvD4 with isolated human leukocytes. RvD4 (0.01 nM to 10 nM) promoted bacterial phagocytosis by human macrophages (12–18%) to a similar extent as the pro-resolving mediator RvD3 (). RvD4 also promoted the phagocytosis of opsonized zymosan A at concentrations as low as 0.1–10 nM, significantly enhancing macrophage uptake by 40–60% above vehicle controls ().

RvD4 exerts pro-resolving actions with human neutrophils and macrophages

The clearance of apoptotic PMN during inflammation is a cellular hallmark in resolution. To determine RvD4’s role in the clearance of apoptotic neutrophils we assessed whether RvD4 promoted macrophage efferocytosis. RvD4 statistically significantly increased macrophage efferocytosis of apoptotic neutrophils by 40–50% at concentrations as low as 0.1–10 nM (). These results suggest that RvD4 enhances human macrophage functions in clearing cellular debris and microbial particles. Together these results provide evidence that RvD4 is pro-resolving with human phagocytes.

RvD4 exerts pro-resolving actions with human fibroblasts: Comparison with RvD1 and MaR1

Given that RvD4 displayed potent pro-resolving actions in skin infection, we investigated whether it also acts on human dermal fibroblasts. RvD4 potently promoted the clearance of apoptotic PMN by human fibroblasts () by ~59% at 1 nM. Of the SPM tested, RvD4 and RvD1 at 1 nM gave the highest increase in apoptotic cell uptake (~52 and 57%). These findings demonstrate new action of SPM such as RvD1 and MaR1 (7R,14S-dihydroxydocosa-4Z,8E,10E,12Z,16Z,19Z-hexaenoic acid) in resolution and that RvD4 displays potent actions with human dermal fibroblasts ().

Discussion

In the present report we establish the complete stereochemical assignment of RvD4 as 4S,5R,17S-trihydroxydocosa-6E,8E,10Z,13Z,15E,19Z-hexaenoic acid by matching endogenous material isolated from purulent inflammatory exudates with synthetic compound () made by total organic synthesis (). RvD4 was temporally regulated during S. aureus infection as well as remained at bioactive levels (~ pM) in the late stages of resolution (). Administration of RvD4 in vivo counter regulated inflammatory initiating eicosanoids in S. aureus infection and shortened resolution indices during infection (), concepts on which the edifice of resolution is based. Administration of RvD4 in both sterile inflammation and microbial infection gave a reduction in neutrophil recruitment in vivo ( and ). RvD4 also enhanced macrophage responses with human macrophages and enhanced fibroblast responses with human fibroblasts in efferocytosis of apoptotic PMN and sterile zymosan (). Furthermore RvD4 was found to be present in a number of tissues across species suggesting its utility through evolutionary conservation ().