Pro-Resolving Maresins in Inflammation: New Pathway and Mechanisms With Oral Pathogens

Objectives The aim of this thesis was to study the biosynthesis of Maresin 1 (MaR1) in human macrophage and to investigate the potential defect of this pathway in patients with localized aggressive periodontitis as well as the role of MaR1 in regulating phagocyte functions and leukocyte-platelet aggregation using LAP as a disease model. Methods Human recombinant enzyme (12-lipoxygenase and soluble epoxide hydrolyse) and synthetic intermediates were used to investigate the maresin pathway by LC/MS/MS-based lipid mediator (LM) metabololipidomics. Human macrophage 12-LOX sequence was crosschecked with platelet-type 12-LOX. Enzyme expression levels in human myeloid mononuclear lineage were determined with flow cytometry. Peripheral blood was collected from LAP patients and age/gender-matched healthy controls (HC). Neutrophils and monocytes were isolated, and macrophages were obtained from monocytes with GM-CSF incubation. LAP and HC macrophage 12-LOX expression levels were monitored by flow cytometry and endogenous maresin metabolite levels were investigated using LM metabololipidomics. Periodontal pathogens including Porphyromonas gingivalis (P. g) and Aggregatibacter actinomycetemcotimans (A. a) were incubated with neutrophils and macrophages to investigate phagocyte functions. Phagocytosis and intracellular anti-microbial reactive oxygen species (ROS) production were determined using fluorescence plate reader. Bacterial killing by macrophages and in whole blood was evaluated by assessing residual colony forming units following incubation with bacteria. Results We demonstrated that human macrophage 12-LOX is identical with platelet-type 12-LOX and is upregulated from monocyte to different mononuclear lineage. 12-LOX converts DHA to 14S-HpDHA, which is a key intermediate in MaR1 biosynthesis. In addition, soluble epoxide hydrolyze (sEH) is responsible for the further production of novel anti-inflammatory and pro-resolving Maresin 2 (MaR2; 13, 14S, di-HDHA). Since 12-LOX is the key-initiating enzyme, investigation begin with macrophages from LAP patients, which have lower 12-LOX expression (~30% lower) and reduced MaR1 (87.8±50 vs. 239.1±32 pg/ 106 cells) levels compared to macrophages from healthy controls (HC). Functionally, LAP macrophages gave impaired phagocytosis (~40% lower) and killing of periodontal pathogens including Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans. In addition, neutrophils from LAP patients also displayed diminished kinetics (~30% slower) and reduced maximal phagocytosis (~20% lower) of these pathogens. Exogenous MaR1 rescued the impairment with LAP phagocytes enhancing phagocytosis (31-65% increase, 1nM), intracellular anti-microbial reactive oxygen species production (26-71% increase, 1nM) and bacterial killing of these periodontal pathogens in vitro. Additionally, MaR1 retained biological action and enhanced bacterial killing in the whole blood ex vivo from HC and LAP patients (22-37% reduction of bacterial titers, 10nM). MaR1 counter-regulated leukocyte-platelet aggregation against two stimulus (platelet activating factor (PAF) and P. gingivalis) and regulated the expression levels of surface adhesion molecules (CD18 and P-selectin). Patients with LAP had elevated leukocyte-platelet aggregation (40% higher compared to HC) in the whole blood that could be reduced by MaR1 in a dose-dependent manner (40-60% reduction of the excess aggregates, 1-100nM). Conclusions These results indicate that 12-LOX is the key enzyme in maresin biosynthetic pathway. LAP macrophages, had lower 12-LOX expression levels and dysregulated maresin biosynthesis. Reduced phagocyte functions were established with both LAP macrophages and neutrophils; exogenous MaR1 rescued this impairment with LAP patients and also enhanced the functions of HC phagocytes. Additionally, MaR1 enhanced bacterial killing and reduced elevated leukocyte-platelet aggregates in LAP whole blood. Together these results suggest that therapeutics targeting maresin pathway may have clinical application treating LAP and oral diseases associated with infection and uncontrolled inflammation