Neutrophils reside in the bone marrow (BM), ready for deployment to sites of injury/infection, initiating inflammation and its resolution. Here, we report that distal infections signal to the BM via resolvins to regulate granulopoiesis and BM neutrophil deployment. Emergency granulopoiesis during peritonitis evoked changes in BM resolvin D1 (RvD1) and BM RvD4. We found that leukotriene B4 stimulates neutrophil deployment. RvD1 and RvD4 each limited neutrophilic infiltration to infections, and differently regulated BM myeloid populations: RvD1 increased reparative monocytes, and RvD4 regulated granulocytes. RvD4 disengaged emergency granulopoiesis, prevented excess BM neutrophil deployment, and acted on granulocyte progenitors. RvD4 also stimulated exudate neutrophil, monocyte, and macrophage phagocytosis, and enhanced bacterial clearance. This mediator accelerated both neutrophil apoptosis and clearance by macrophages, thus expediting the resolution phase of inflammation. RvD4 stimulated phosphorylation of ERK1/2 and STAT3 in human BM-aspirate–derived granulocytes. RvD4 in the 1 to 100 nM range stimulated whole-blood neutrophil phagocytosis of Escherichia coli. RvD4 increased BM macrophage efferocytosis of neutrophils. Together, these results demonstrate the novel functions of resolvins in granulopoiesis and neutrophil deployment, contributing to the resolution of infectious inflammation.

While the acute inflammatory response to harmful stimuli is protective, unrestrained neutrophil swarming drives collateral tissue damage and inflammation. Biosynthesized from omega-3 essential polyunsaturated fatty acids, resolvins are a family of signaling molecules produced by immune cells within the resolution phase to orchestrate return to homeostasis. Understanding the mechanisms that govern biosynthesis of these potent molecules gives insight into stimulating endogenous resolution and offers fresh opportunities for preventing and treating excessive inflammation. In this report, using materials prepared by total synthesis and liquid chromatography and tandem mass spectrometry-based matching studies, we established the role of 7,8(S,S)-epoxytetraene intermediate in the biosynthesis of resolvin D1, resolvin D2, and the resolvin conjugate in tissue regeneration (RCTR1) by human phagocytes. We demonstrated that this 7,8(S,S)-epoxy-containing intermediate is directly converted to resolvin D2 by human M2-like macrophages and to resolvin D1 and RCTR1 by human macrophages, neutrophils, and peripheral blood mononuclear cells. In addition, both human recombinant soluble epoxide hydrolase (sEH) and the glutathione S-transferase leukotriene C₄synthase (LTC₄S) each catalyze conversion of this epoxide to resolvin D1 and RCTR1, respectively. MS³ion-trap scans and isotope incorporation of¹⁸O from H₂¹⁸O with sEH indicated that the oxygen atom at C-8 in resolvin D1 is derived from water. Results from molecular docking simulations with biosynthetic precursor 17S-hydroperoxy-4,7,10,13,19-cis-15-trans-docosahexaenoic acid and the epoxy intermediate were consistent with 5-lipoxygenase production of resolvin D1. Together, these results give direct evidence for the role of resolvin 7,8(S,S)-epoxytetraene intermediate in the endogenous formation of resolution-phase mediators resolvin D1, resolvin D2, and RCTR1 by human phagocytes.

Background: Specialized pro-resolving mediators (SPMs) promote resolution of inflammation, clear infections and stimulate tissue regeneration. These include resolvins, protectins, and maresins. During self-resolving acute inflammation, SPMs are produced and have key functions activating endogenous resolution response for returning to homeostasis. Herein, we addressed whether infections initiated with ongoing inflammation alter resolution programs, and if low-dose repetitive SPM regimen re-programs the resolution response. Methods: Inflammation was initiated with zymosan (1 mg/mouse) followed by E. coli (10⁵ CFU/mouse) infections carried out in murine peritonitis, and exudates collected at 4-72 h. Leukocytes were enumerated using light microscopy, percentages of PMN, monocytes and macrophages were determined using flow cytometry, and resolution indices calculated. Lipid mediators and SPM profiles were established using mass spectrometry-based metabololipidomics. Repetitive dosing with a SPM panel consisting of RvD1, RvD2, RvD5, MaR1 and RvE2 (0.1 ng/mouse each, i.p.) was given to mice, followed by zymosan challenge. Leukocyte composition, resolution indices and RNA-sequencing were carried out for the repetitive SPM treatments. Results: E. coli infections initiated acute inflammation-resolution programs with temporal SPM production in the infectious exudates. Zymosan-induced inflammation prior to E. coli peritonitis shifted exudate resolution indices and delayed E. coli clearance. Lipid mediator metabololipidomics demonstrated that E. coli infection with ongoing zymosan-induced inflammation shifted the time course of exudate SPMs, activating a SPM cluster that included RvD1, RvD5 and MaR1 during the initiation phase of infectious inflammation (0-4 h); RvD5 and MaR1 were present also in the resolution phase (24-48 h). To emulate daily SPM regimens used in humans, a repetitive subthreshold dosing of the SPM panel RvD1, RvD2, RvD5, MaR1 and RvE2 each at 0.1 ng per mouse was administered. This low-dose SPM regimen accelerated exudate PMN clearance following zymosan-induced inflammation, and shortened the resolution interval by > 70%. These low-dose SPMs regulated genes and pathways related to immune response, chemokine clearance and tissue repair, as demonstrated by using RNA-sequencing. Conclusions: Infections encountered during ongoing inflammation in mice reset the resolution mechanisms of inflammation via SPM clusters. Low-dose SPMs activate innate immune responses and pathways towards the resolution response that can be reprogrammed.

Coumadin (rac-warfarin) is the most commonly used anticoagulant in the world; however, its clinical use is often challenging because of its narrow therapeutic range and interindividual variations in response. A critical contributor to the uncertainty is variability in warfarin metabolism, which includes mostly oxidative but also reductive pathways. Reduction of each warfarin enantiomer yields two warfarin alcohol isomers, and the corresponding four alcohols retain varying levels of anticoagulant activity. Studies on the kinetics of warfarin reduction have often lacked resolution of parent-drug enantiomers and have suffered from coelution of pairs of alcohol metabolites; thus, those studies have not established the importance of individual stereospecific reductive pathways. We report the first steady-state analysis of R- and S-warfarin reduction in vitro by pooled human liver cytosol. As determined by authentic standards, the major metabolites were 9R,11S-warfarin alcohol for R-warfarin and 9S,11S-warfarin alcohol for S-warfarin. R-warfarin (V_max 150 pmol/mg per minute, K_m 0.67 mM) was reduced more efficiently than S-warfarin (V_max 27 pmol/mg per minute, K_m 1.7 mM). Based on inhibitor phenotyping, carbonyl reductase-1 dominated R-and S-warfarin reduction, followed by aldo-keto reductase-1C3 and then other members of that family. Overall, the carbonyl at position 11 undergoes stereospecific reduction by multiple enzymes to form the S alcohol for both drug enantiomers, yet R-warfarin undergoes reduction preferentially. This knowledge will aid in assessing the relative importance of reductive pathways for R- and S-warfarin and factors influencing levels of pharmacologically active parent drugs and metabolites, thus impacting patient dose responses.

Neutrophils have vital proinflammatory protective functions, but gene expression changes in neutrophils found in inflamed tissues suggest additional proresolving effects. We identified a neutrophil subset with a distinct phenotype and function that emerges in mouse lungs during resolution of injury. These resolution-phase neutrophils increased expression of Siglec-F (sialic acid–binding Ig-like lectin F), Alox15 (12/15-lipoxygenase), and Csf1 ( colony-stimulating factor 1 ). Siglec-F ⁺ neutrophils promoted macrophage differentiation and produced specialized proresolving mediators that accelerated injury resolution. Neutrophil depletion hindered lung epithelial catabatic responses, whereas adoptive transfer of Siglec-F ⁺ neutrophils accelerated restitution of lung epithelial cells. Transforming growth factor–β (TGF-β) and granulocyte-macrophage colony-stimulating factor (GM-CSF), acting via activator protein-1 (AP-1)/Jun, promoted expression of Siglec-F in mouse neutrophils and ALOX15 in mouse and human neutrophils. In patients with respiratory failure, ALOX15 ⁺ neutrophils were present in the bronchoalveolar lavage samples, and their frequency correlated with improved oxygenation. Thus, Siglec-F ⁺ ALOX15 ⁺ proresolving neutrophils contribute to tissue injury responses.