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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 (R i ) by ~4.5h during E. coli peritonitis after administration at peak inflammation (T max =12h) 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., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Proinflammatory eicosanoids (prostaglandins and leukotrienes) and specialized pro-resolving mediators (SPM) are temporally regulated during infections. Here we show that human macrophage phenotypes biosynthesize unique lipid mediator signatures when exposed to pathogenic bacteria. E. coli and S. aureus each stimulate predominantly proinflammatory 5-lipoxygenase (LOX) and cyclooxygenase pathways (i.e., leukotriene B 4 and prostaglandin E 2 ) in M1 macrophages. These pathogens stimulate M2 macrophages to produce SPMs including resolvin D2 (RvD2), RvD5, and maresin-1. E. coli activates M2 macrophages to translocate 5-LOX and 15-LOX-1 to different subcellular locales in a Ca 2+ -dependent manner. Neither attenuated nor non-pathogenic E. coli mobilize Ca 2+ or activate LOXs, rather these bacteria stimulate prostaglandin production. RvD5 is more potent than leukotriene B 4 at enhancing macrophage phagocytosis. These results indicate that M1 and M2 macrophages respond to pathogenic bacteria differently, producing either leukotrienes or resolvins that further distinguish inflammatory or pro-resolving phenotypes.

Arachidonic acid (AA), a representative ω6-polyunsaturated fatty acid (PUFA), is a precursor of 2-series prostaglandins (PGs) that play important roles in inflammation, pain, fever, and related disorders including cardiovascular diseases. Eating fish or supplementation with the ω3-PUFAs such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) is widely assumed to be beneficial in preventing cardiovascular diseases. A proposed mechanism for a cardio-protective role of ω3-PUFAs assumes competition between AA and ω3-PUFAs for cyclooxygenases (COX), leading to reduced production of 2-series PGs. In this study, we have used a systems biology approach to integrate existing knowledge and novel high-throughput data that facilitates a quantitative understanding of the molecular mechanism of ω3- and ω6-PUFA metabolism in mammalian cells. We have developed a quantitative computational model of the competitive metabolism of AA and EPA via the COX pathway through a two-step matrix-based approach to estimate the rate constants. This model was developed by using lipidomic data sets that were experimentally obtained from EPA-supplemented ATP-stimulated RAW264.7 macrophages. The resulting model fits the experimental data well for all metabolites and demonstrates that the integrated metabolic and signaling networks and the experimental data are consistent with one another. The robustness of the model was validated through parametric sensitivity and uncertainty analysis. We also validated the model by predicting the results from other independent experiments involving AA- and DHA-supplemented ATP-stimulated RAW264.7 cells using the parameters estimated with EPA. Furthermore, we showed that the higher affinity of EPA binding to COX compared with AA was able to inhibit AA metabolism effectively. Thus, our model captures the essential features of competitive metabolism of ω3- and ω6-PUFAs.

Initiation and resolution of inflammation are considered to be tightly connected processes. Lipoxins (LX) are proresolution lipid mediators that inhibit phlogistic neutrophil recruitment and promote wound-healing macrophage recruitment in humans via potent and specific signaling through the LXA4 receptor (ALX). One model of lipoxin biosynthesis involves sequential metabolism of arachidonic acid by two cell types expressing a combined transcellular metabolon. It is currently unclear how lipoxins are efficiently formed from precursors or if they are directly generated after receptor-mediated inflammatory commitment. Here, we provide evidence for a pathway by which lipoxins are generated in macrophages as a consequence of sequential activation of toll-like receptor 4 (TLR4), a receptor for endotoxin, and P2X7, a purinergic receptor for extracellular ATP. Initial activation of TLR4 results in accumulation of the cyclooxygenase-2-derived lipoxin precursor 15-hydroxyeicosatetraenoic acid (15-HETE) in esterified form within membrane phospholipids, which can be enhanced by aspirin (ASA) treatment. Subsequent activation of P2X7 results in efficient hydrolysis of 15-HETE from membrane phospholipids by group IVA cytosolic phospholipase A2, and its conversion to bioactive lipoxins by 5-lipoxygenase. Our results demonstrate how a single immune cell can store a proresolving lipid precursor and then release it for bioactive maturation and secretion, conceptually similar to the production and inflammasome-dependent maturation of the proinflammatory IL-1 family cytokines. These findings provide evidence for receptor-specific and combinatorial control of pro- and anti-inflammatory eicosanoid biosynthesis, and potential avenues to modulate inflammatory indices without inhibiting downstream eicosanoid pathways.

Macrophages are central to essential physiological processes including the regulation of innate and adaptive immunity, but they are also central to a number of inflammatory disease states. These immune cells also possess remarkable plasticity and display various shades of functionalities based on changes in the surrounding molecular environment. Macrophage biology has defined various phenotypes and roles in inflammation based primarily on cytokine and chemokine profiles of cells in different activation states. Importantly, macrophages are elite producers of eicosanoids and other related lipid mediators during inflammation, but specific roles of these molecules have not generally been incorporated into the larger context of macrophage biology. In this review, we discuss the current classification of macrophage types and their roles in inflammation and disease, along with the practical challenges of studying biologically relevant phenotypes ex vivo. Using the latest advances in eicosanoid lipidomics, we highlight several key studies from our laboratory that provide a comprehensive understanding of how eicosanoid metabolism differs between macrophage phenotypes, along with how this metabolism is altered by changes in membrane fatty acid distribution and varied durations of Toll-like receptor (TLR) priming. In conclusion, we summarize several examples of the benefit of macrophage plasticity to develop accurate cellular mechanisms of lipid metabolism, and insights from lipidomic analyses about the differences in eicosanoid pathway enzyme activity in vitro vs. in cells ex vivo. Examples of new techniques to further understand the role of macrophage eicosanoid signaling in vivo are also discussed., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Dietary fish oil containing ω3 fatty acids, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), elicit cardioprotective and anti-inflammatory effects through unresolved mechanisms that may involve competition and inhibition at multiple levels. Here, we report the effects of arachidonic acid (AA), EPA, and DHA supplementation on membrane incorporation, phospholipase A(2) catalyzed release, and eicosanoid production in RAW264.7 macrophages. Using a targeted lipidomics approach, we observed that Toll-like receptor 4 and purinergic receptor activation of supplemented cells leads to the release of 22-carbon fatty acids that potently inhibit cyclooxygenase pathways. This inhibition was able to shunt metabolism of AA to lipoxygenase pathways, augmenting leukotriene and other lipoxygenase mediator synthesis. In resident peritoneal macrophages, docosapentaenoic acid (DPA) was responsible for cyclooxygenase inhibition after EPA supplementation, offering fresh insights into how EPA exerts anti-inflammatory effects indirectly through elongation to 22-carbon DPA.

Eicosanoid metabolism differs in profile and quantity between macrophages of different tissue origin and method of elicitation, as well as between primary and immortalized macrophages after activation with inflammatory stimuli. Using a lipidomic approach, we comprehensively analyzed the eicosanoids made by murine RPMs, TGEMs, BMDM, and the macrophage-like cell line RAW after stimulation with the TLR-4-specific agonist KLA. Direct correlation among total COX metabolites, COX side-products (11-HETE, 15-HETE), COX-2 mRNA, and protein at 8 h was found when comparing each cell type. Comprehensive qPCR analysis was used to compare relative transcript levels between the terminal prostanoid synthases themselves as well as between each cell type. Levels of PGE(2), PGD(2), and TxB(2) generally correlated with enzyme transcript expression of PGES, PGDS, and TBXS, providing evidence of comparable enzyme activities. PGIS transcript was expressed only in RPM and TGEM macrophages and at an exceptionally low level, despite high metabolite production compared with other synthases. Presence of PGIS in RPM and TGEM also lowered the production of PGE(2) versus PGD(2) by approximately tenfold relative to BMDM and RAW cells, which lacked this enzyme. Our results demonstrate that delayed PG production depends on the maximal level of COX-2 expression in different macrophages after TLR-4 stimulation. Also, the same enzymes in each cell largely dictate the profile of eicosanoids produced depending on the ratios of expression between them, with the exception of PGIS, which appears to have much greater synthetic capacity and competes selectively with mPGES-1.

UnlabelledPharmacologic and global gene deletion studies demonstrate that cyclooxygenase-2 (PTGS2/COX-2) plays a critical role in DMBA/TPA-induced skin tumor induction. Although many cell types in the tumor microenvironment express COX-2, the cell types in which COX-2 expression is required for tumor promotion are not clearly established. Here, cell type-specific Cox-2 gene deletion reveals a vital role for skin epithelial cell COX-2 expression in DMBA/TPA tumor induction. In contrast, myeloid Cox-2 gene deletion has no effect on DMBA/TPA tumorigenesis. The infrequent, small tumors that develop on mice with an epithelial cell-specific Cox-2 gene deletion have decreased proliferation and increased cell differentiation properties. Blood vessel density is reduced in tumors with an epithelial cell-specific Cox-2 gene deletion, compared with littermate control tumors, suggesting a reciprocal relationship in tumor progression between COX-2-expressing tumor epithelial cells and microenvironment endothelial cells. Lipidomics analysis of skin and tumors from DMBA/TPA-treated mice suggests that the prostaglandins PGE2 and PGF2α are likely candidates for the epithelial cell COX-2-dependent eicosanoids that mediate tumor progression. This study both illustrates the value of cell type-specific gene deletions in understanding the cellular roles of signal-generating pathways in complex microenvironments and emphasizes the benefit of a systems-based lipidomic analysis approach to identify candidate lipid mediators of biologic responses.ImplicationsCox-2 gene deletion demonstrates that intrinsic COX-2 expression in initiated keratinocytes is a principal driver of skin carcinogenesis; lipidomic analysis identifies likely prostanoid effectors.

Introduction The acute inflammatory response is a protective mechanism to injury or microbial invasion and ideally is self-limited, leading to complete resolution (1). The resolution of inflammation is temporally and [...], Resolution of acute inflammation is an active process orchestrated by endogenous mediators and mechanisms pivotal in host defense and homeostasis. The macrophage mediator in resolving inflammation, maresin 1 (MaR1), is a potent immunoresolvent, stimulating resolution of acute inflammation and organ protection. Using an unbiased screening of greater than 200 GPCRs, we identified MaR1 as a stereoselective activator for human leucine-rich repeat containing G protein- coupled receptor 6 (LGR6), expressed in phagocytes. MaR1 specificity for recombinant human LGR6 activation was established using reporter cells expressing LGR6 and functional impedance sensing. MaR1-specific binding to LGR6 was confirmed using [sup.3]H-labeled MaR1. With human and mouse phagocytes, MaR1 (0.01-10 nM) enhanced phagocytosis, efferocytosis, and phosphorylation of a panel of proteins including the ERK and cAMP response element-binding protein. These MaR1 actions were significantly amplified with LGR6 overexpression and diminished by gene silencing in phagocytes. Thus, we provide evidence for MaR1 as an endogenous activator of human LGR6 and a novel role of LGR6 in stimulating MaR1's key proresolving functions of phagocytes.

Supplementary information Graph: Updated Supplementary Information Source data Graph: Source Data file Supplementary information The online version contains supplementary material available at https://doi.org/10.1038/s41467-023-41667-y. 1a Graph The Source Data file has also been added to include all biological replicates for Fig. [Extracted from the article]