Inflammation and its natural resolution are host-protective responses triggered by infection or injury. The resolution phase of inflammation is regulated by enzymatically produced specialized pro-resolving mediators. We recently identified a new class of peptide-conjugated specialized pro-resolving mediators that carry potent tissue regenerative actions that belong to the protectin family and are coined protectin conjugates in tissue regeneration (PCTR). Herein, with the use of microbial-induced peritonitis in mice and liquid chromatography-tandem mass spectrometryebased lipid mediator metabololipidomics, we found that PCTR1 is temporally regulated during self-resolving infection. When administered at peak of inflammation, PCTR1 enhanced macrophage recruitment and phagocytosis of Escherichia coli, decreased polymorphonuclear leukocyte infiltration, and counter-regulated inflammation-initiating lipid mediators, including prostaglandins. In addition, biologically produced PCTR1 promoted human monocyte and macrophage migration in a dose-dependent manner (0.001 to 10.0 nmol/L). We prepared PCTR1 via organic synthesis and confirmed that synthetic PCTR1 increased macrophage and monocyte migration, enhanced macrophage efferocytosis, and accelerated tissue regeneration in planaria. With human macrophage subsets, PCTR1 levels were significantly higher in M2 macrophages than in M1 phenotype, along with members of the resolvin conjugates in tissue regeneration and maresin conjugate families. In contrast, M1 macrophages gave higher levels of cysteinyl leukotrienes. Together, these results demonstrate that PCTR1 is a potent monocyte/macrophage agonist, regulating key anti-inflammatory and pro-resolving processes during bacterial infection. (Am J Pathol 2016, 186: 962e973; http://dx

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.

Maresin conjugates in tissue regeneration (MCTR) are a new family of evolutionarily conserved chemical signals that orchestrate host responses to promote tissue regeneration and resolution of infections. Herein, we identified the novel MCTR3 and established rank order potencies and matched the stereochemistries of MCTR1, MCTR2 and MCTR3 using material prepared by total organic synthesis and mediators isolated from both mouse and human systems. MCTR3 was produced from endogenous substrate by E. coli activated human macrophages and identified in sepsis patients. Each of the three synthetic MCTR dose-dependently (1–100nM) accelerated tissue regeneration in planaria by 0.6–0.9 days. When administered at the onset or peak of inflammation, each of the MCTR promoted resolution of E. coli infections in mice. They increased bacterial phagocytosis by exudate leukocytes (~15–50%), limited neutrophil infiltration (~20–50%), promoted efferocytosis (~30%) and reduced eicosanoids. MCTR1 and MCTR2 upregulated human neutrophil and macrophage phagocytic responses where MCTR3 also proved to possess potent actions. These results establish the complete stereochemistry and rank order potencies for MCTR1, MCTR2 and MCTR3 that provide novel resolution moduli in regulating host responses to clear infections and promote tissue regeneration.

Maresin 1 (MaR1) is an immunoresolvent that governs resolution of acute inflammation and its local metabolism in the context infectious-inflammation is of interest. Here, we investigated the MaR1 metabolome in infectious exudates and its bioactions in regulating leukocyte responses in the context of bacterial infection. In Escherichia coli infectious exudates, MaR1 was temporally regulated with maximal levels at 4 h (2.2 ± 0.4 pg/lavage). In these exudates we also identified two novel products and their structure elucidation gave 22-hydroxy-MaR1 and 14-oxo-MaR1. Using human primary leukocytes we found that neutrophils primarily produced 22-OH-MaR1 whereas the main macrophage product was 14-oxo-MaR1. Both 22-OH-MaR1 and 14-oxo-MaR1 incubated with human primary macrophages gave dose dependent increases in macrophage phagocytosis of ~75% at 1 pM 22-OH-MaR1 and ~25% at 1 pM 14-oxo-MaR1, while 14-oxo-MaR1 was less active than MaR1 at higher concentrations. Together these findings establish the temporal regulation of MaR1 during infectious inflammation as well as elucidate the structures and actions of two novel MaR1 further metabolites that carry bioactivities.

Resolution of infection and inflammation is governed by innate immune cells. The resolvin family of n-3 mediators produced by resolving exudates stimulates clearance of neutrophils and attenuates pro-inflammatory signals. Using metabololipidomics, endogenous resolvin D3 (RvD3) was identified in self-resolving exudates during active E. coli infection. Through a new, independent synthetic route for RvD3, we matched endogenous and synthetic RvD3 and determined that RvD3 (ng doses) potently reduced the resolution interval (Ri) by ~4.5 h during E. coli peritonitis after administration at peak inflammation (Tmax = 12 h) and increased leukocyte phagocytosis of E. coli and neutrophils as well as reduced proinflammatory cytokines, chemokines, MMP-2 and MMP-9. At pM-nM concentrations, RvD3 also enhanced human macrophage efferocytosis and bacterial phagocytosis, increased neutrophil bacterial phagocytosis and intracellular ROS generation, and reduced human platelet-PMN aggregation. These results provide additional evidence for potent RvD3 immunoresolvent actions in host defense, host protection and antimicrobial defense.

Sulforaphane (SFN), a naturally occurring isothiocyanate, has shown promising chemopreventive and anti‐cancer potential. Our preliminary studies demonstrate that SFN induces apoptosis in HL‐60 leukemia cells. Our data show a significant increase in SFN‐induced apoptotic and dead cells, as well as slowed cellular division over 72 hours. However, the mechanism by which SFN affects HL‐60 cells to induce apoptosis and block cellular division is not well understood. To this end, microarray experiments were used to compare gene expression profiles of untreated and SFN‐treated cells. Of particular interest, there was a 3.52‐fold upregulation of Bcl‐2 as well as upregulation of other Bcl‐2 related genes in the presence of SFN and a 100‐fold downregulation of Myc after treatment with SFN. The Myc gene is constitutively active in most cancers and this downregulation is of particular interest for potential anti‐cancer effects of the mechanism of SFN. Using FITC labeled monoclonal antibodies and flow cytometry, we are currently investigating SFN’s effects on protein expression of Bcl‐2 and Myc to determine the consistency or inconsistency with microarray data. Initial experiments suggest a difference in Myc expression between SFN treated and untreated HL‐60 cells while no difference was observed in Bcl‐2 levels. Subsequent experiments will utilize different doses of SFN and exposure times.