Maresin 1 biosynthesis during platelet-neutrophil interactions is organ-protective

Abstract

Unregulated acute inflammation can lead to collateral tissue injury in vital organs, such as the lung during the acute respiratory distress syndrome. In response to tissue injury, circulating platelet-neutrophil aggregates form to augment neutrophil tissue entry. These early cellular events in acute inflammation are pivotal to timely resolution by mechanisms that remain to be elucidated. Here, we identified a previously undescribed biosynthetic route during human platelet-neutrophil interactions for the proresolving mediator maresin 1 (MaR1; 7R,14S-dihydroxy-docosa-4Z,8E,10E,12Z,16Z,19Z-hexaenoic acid). Docosahexaenoic acid was converted by platelet 12-lipoxygenase to 13S,14S-epoxy-maresin, which was further transformed by neutrophils to MaR1. In a murine model of acute respiratory distress syndrome, lipid mediator metabololipidomics uncovered MaR1 generation in vivo in a temporally regulated manner. Early MaR1 production was dependent on platelet-neutrophil interactions, and intravascular MaR1 was organ-protective, leading to decreased lung neutrophils, edema, tissue hypoxia, and prophlogistic mediators. Together, these findings identify a transcellular route for intravascular maresin 1 biosynthesis via platelet-neutrophil interactions that regulates the extent of lung inflammation.

Keywords: inflammation; lung; maresin; platelet; resolution.

Fig. 1.

MaR1 is produced by N-PAs via transcellular biosynthesis. (A–C) MaR1 production by isolated human peripheral blood platelets incubated with d₅-DHA before incubation with or without neutrophils in the presence of PAF or thrombin. Levels of d₅-MaR1 were assessed by liquid chromatography (LC)–MS/MS metabololipidomics. (A) Representative multiple reaction monitoring (MRM) chromatograms (m/z = 364–250) and (B) MS/MS spectrum from PAF-treated samples used for the identification of d₅-MaR1. (C) d₅-MaR1 levels in incubations with different neutrophil-to-platelet ratios. Results for A and B are representative of n = 3 healthy donor blood isolations. Results for C are mean ± SEM for n = 3 healthy donor blood isolations. *P < 0.05. (D–F) Human peripheral blood neutrophils were isolated by density centrifugation. Cells were suspended at 1 × 10⁸ in PBS with 10 mg/mL BSA, then incubated with (D) 10 µM 14HpDHA or (E) 10 µM 13S,14S-eMaR and 0.1 mg of serum-treated zymosan (15 min, 37 °C, pH 7.45). (F) Representative MS/MS spectrum used for the identification of MaR1 (7R,14S-dihydroxydocosa-4Z,8E,10E,12Z,16Z,19Z-hexaenoic acid). (G) Levels of MaR1 in fresh human whole blood pretreated with anti-human P-selectin or isotype control antibody after treatment with PAF (500 nM). Values represent the mean ± SEM for n = 7 healthy volunteers. *P < 0.05.

Fig. 2.

MaR1 is produced during acid-induced ALI in a temporally regulated manner. Lipid mediator metabololipidomics was performed to identify lung MaR1 and its biosynthetic pathway markers 7-epi-Δ12-trans-MaR1 and Δ12-trans-MaR1. (A) MRM ion chromatograms, where Q1 is the parent ion and Q3 a characteristic daughter ion (m/z = 359–221), present in lung homogenates obtained 24 h after intratracheal HCl. (B) Levels of MaR1 in lung homogenates from injured mice were determined at baseline, 2, 24, 48, and 72 h after intratracheal HCl administration. (C) Levels of MaR1 and nonenzymatic hydrolysis products 2 h after HCl in neutrophil-depleted mice (anti-Ly6G) and mice pretreated with an isotype control antibody. (D) Levels of MaR1 2 h after HCl in mice administered anti-mouse P-selectin or isotype control antibody. (E) Levels of MaR1 and nonenzymatic hydrolysis products 24 h after HCl in neutrophil-depleted mice (anti-Ly6G) and mice pretreated with an isotype control antibody. Values represent the mean ± SEM for n ≥ 4 mice. *P < 0.05.

Fig. 3.

MaR1 reduces lung inflammation and restores pulmonary capillary endothelial permeability. (A) Hematoxylin and eosin stain of right and left lungs from mice treated with MaR1 or vehicle. Results are representative from n = 3. (Scale bar, 50 µm.) (B) Endothelial barrier integrity determined by measurement of Evans blue dye in perfused whole lung in mice exposed to MaR1 or vehicle. (C) Tissue hypoxia determined by pimonidazole (Hypoxyprobe) immunostaining in injured left lung sections obtained from mice treated with MaR1 or vehicle. Pimonidazole staining appears brown. (D) Prophlogistic mediator levels in BALF and plasma (CysLTs) were analyzed by cytokine bead array or ELISA (CysLTs). In all experiments, tissues and samples were harvested 24 h after intratracheal HCl, and MaR1 was administered i.v 1 h after HCl. All values represent the mean ± SEM for n ≥ 5 mice from two independent experiments. *P < 0.05.

Fig. 4.

Intravascular MaR1 reduces neutrophil recruitment by decreasing N-PAs. (A) BALF cell counts after administration of MaR1 or vehicle. (B) Lung interstitial neutrophils (circled in flow cytometry dot plots) determined by differential immunostaining of intravascular and whole lung neutrophils. (C) Plasma TxB₂ levels 2 h after intratracheal HCl from mice given MaR1 or vehicle determined by ELISA. (D) N-PAs determined by flow cytometry of murine whole blood 24 h after intratracheal HCl administration and treatment with MaR1 or vehicle. (E) Whole blood MaR1 levels, (F) airway neutrophil counts, and (G) whole blood N-PA 24 h after intratracheal HCl in mice transfused with platelets (500 × 10⁶ platelets, intravenously) pretreated with baicalein or vehicle. Values represent the mean ± SEM for n ≥ 5 mice from two independent experiments. *P < 0.05. (H) Human N-PA in whole blood after incubation with PAF or LTB₄ and pretreatment with MaR1 or vehicle determined by flow cytometry. (I) Platelet and neutrophil activation after incubation with PAF or LTB₄ and coincubation with MaR1 or vehicle quantified by flow cytometry analysis of P-selectin and CD11b surface expression, respectively. Values represent the mean ± SEM for n ≥ 5 blood donors. *P < 0.05 by paired t test.

Fig. 5.

Proposed (platelet–neutrophil) and established (macrophage) biosynthetic routes for MaR1. (A) During neutrophil–platelet interactions, platelet 12-lipoxyenase converts DHA to 14S-hydroperoxy-DHA and 13S,14S-epoxy-maresin, which is then converted by an epoxide hydrolase in neutrophils to MaR1. (B) Macrophages express both 12-lipoxygenase and the hydrolase required to conduct MaR1 biosynthesis.