Lipid Mediators in the Resolution of Inflammation

Mounting of the acute inflammatory response is crucial for host defense and pivotal to the development of chronic inflammation, fibrosis, or abscess formation versus the protective response and the need of the host tissues to return to homeostasis. Within self-limited acute inflammatory exudates, novel families of lipid mediators are identified, named resolvins (Rv), protectins, and maresins, which actively stimulate cardinal signs of resolution, namely, cessation of leukocytic infiltration, counterregulation of proinflammatory mediators, and the uptake of apoptotic neutrophils and cellular debris. The biosynthesis of these resolution-phase mediators in sensu stricto is initiated during lipid-mediator class switching, in which the classic initiators of acute inflammation, prostaglandins and leukotrienes (LTs), switch to produce specialized proresolving mediators (SPMs). In this work, we review recent evidence on the structure and functional roles of these novel lipid mediators of resolution. Together, these show that leukocyte trafficking and temporal spatial signals govern the resolution of self-limited inflammation and stimulate homeostasis.Resolution of an acute inflammatory response is the ideal outcome of this protective host response with return of the tissue to homeostasis (Majno and Joris 2004; Serhan et al. 2010). Lipid mediators are widely appreciated for their important roles in initiating the leukocyte traffic required in host defense (Cotran et al. 1999). These include the classic eicosanoids, prostaglandins (PGs) and leukotrienes (LTs) (Samuelsson et al. 1987; Samuelsson 2012), that stimulate blood flow changes, edema, and neutrophil influx to tissues (Flower 2006). Novel resolution-phase mediators that possess potent proresolving actions were identified and named resolvins, protectins, and maresins. Further studies established that these three families as well as lipoxins function together with their aspirin-triggered (AT) forms (collectively termed specialized proresolving mediators [SPMs]) and are biosynthesized during active resolution (Serhan 2004; Serhan and Chiang 2013). The complete stereochemistry of each of the main SPMs is established and their potent actions confirmed via total organic synthesis (Serhan and Petasis 2011). Given increased availability of certain SPMs, a body of literature emerged that expands their potent proresolving and anti-inflammatory actions and functions originally identified for the SPMs. In this work, we review and update the roles and actions of the SPMs, focusing on recent results with resolvins, protectins, and maresins, in active resolution mechanisms.Professor Rod Flower of the William Harvey Research Institute, University of London once recited the quotation from Juvenal, a Roman poet, to introduce these new concepts and findings: Quis custodiet ipsos custodes? Who will guard the guards themselves? Hence, this quote is apropos to begin this article focusing on novel chemical mediators of resolution. The guards, the innate immune system phagocytes, certainly require direction (Serhan 2004; Perretti and D''Acquisto 2009) in the form of chemoattractants and chemical signals to appropriately control their function(s) and permit clearance of microbes and cellular debris without tissue injury; the cardinal signs of resolution.     

Characterization of antimicrobial compounds from a common fern, Pteris biaurita

Methanol extract was prepared from the fronds of Pteris biaurita and partial purification was done by solvent partitioning with diethyl ether and ethyl acetate, followed by hydrolysis and further partitioning with ethyl acetate. The three fractions, thus obtained were bioassayed separately against five test fungi--Curvularia lunata, Fomes lamaoensis, Poria hypobrumea, Fuasrium oxysporum and a bacterium--Bacillus pumilus, by spore germination, radial growth and agar cup techniques. Results revealed that ethyl acetate fraction (III) contained the active principle. TLC plate bioassay of the active fraction revealed inhibition zone at an Rf of 0.5-0.65. Silica gel from this region was scraped, eluted in methanol and subjected to UV-spectrophotometric analysis. An absorption maxima of 278 nm was recorded. HPLC analysis of TLC-eluate revealed a single peak with retention time of 8.1 min. GC-MS analysis revealed six major peaks in the retention time range of 7.2-10.9 min. Comparison with GC-MS libraries revealed that the extracts may contain a mixture of eicosenes and heptadecanes.     

Annexin A1 regulates neutrophil clearance by macrophages in the mouse bone marrow

Under homeostatic conditions, a proportion of senescent CXCR4(hi) neutrophils home from the circulation back to the bone marrow, where they are phagocytosed by bone marrow macrophages. In this study, we have identified an unexpected role for the anti-inflammatory molecule annexin A1 (AnxA1) as a critical regulator of this process. We first observed that AnxA1(-/-) mice have significantly increased neutrophil numbers in their bone marrow while having normal levels of GM and G colony-forming units, monocytes, and macrophages. Although AnxA1(-/-) mice have more neutrophils in the bone marrow, a greater proportion of these cells are senescent, as determined by their higher levels of CXCR4 expression and annexin V binding. Consequently, bone marrow neutrophils from AnxA1(-/-) mice exhibit a reduced migratory capacity in vitro. Studies conducted in vitro also show that expression of AnxA1 is required for bone marrow macrophages, but not peritoneal macrophages, to phagocytose apoptotic neutrophils. Moreover, in vivo experiments indicate a defect in clearance of wild-type neutrophils in the bone marrow of AnxA1(-/-) mice. Thus, we conclude that expression of AnxA1 by resident macrophages is a critical determinant for neutrophil clearance in the bone marrow.     

Resolvin d1 and resolvin d2 govern local inflammatory tone in obese fat

The unprecedented increase in the prevalence of obesity and obesity-related disorders is causally linked to a chronic state of low-grade inflammation in adipose tissue. Timely resolution of inflammation and return of this tissue to homeostasis are key to reducing obesity-induced metabolic dysfunctions. In this study, with inflamed adipose, we investigated the biosynthesis, conversion, and actions of Resolvins D1 (RvD1, 7S,8R,17S-trihydroxy-4Z,9E,11E,13Z,15E,19Z-docosahexaenoic acid) and D2 (RvD2, 7S,16R,17S-trihydroxy-4Z,8E,10Z,12E,14E,19Z-docosahexaenoic acid), potent anti-inflammatory and proresolving lipid mediators (LMs), and their ability to regulate monocyte interactions with adipocytes. Lipid mediator-metabololipidomics identified RvD1 and RvD2 from endogenous sources in human and mouse adipose tissues. We also identified proresolving receptors (i.e., ALX/FPR2, ChemR23, and GPR32) in these tissues. Compared with lean tissue, obese adipose showed a deficit of these endogenous anti-inflammatory signals. With inflamed obese adipose tissue, RvD1 and RvD2 each rescued impaired expression and secretion of adiponectin in a time- and concentration-dependent manner as well as decreasing proinflammatory adipokine production including leptin, TNF-α, IL-6, and IL-1β. RvD1 and RvD2 each reduced MCP-1 and leukotriene B(4)-stimulated monocyte adhesion to adipocytes and their transadipose migration. Adipose tissue rapidly converted both resolvins (Rvs) to novel oxo-Rvs. RvD2 was enzymatically converted to 7-oxo-RvD2 as its major metabolic route that retained adipose-directed RvD2 actions. These results indicate, in adipose, D-series Rvs (RvD1 and RvD2) are potent proresolving mediators that counteract both local adipokine production and monocyte accumulation in obesity-induced adipose inflammation.     

Maresin Biosynthesis and Identification of Maresin 2, a New Anti-Inflammatory and Pro-Resolving Mediator from Human Macrophages

Maresins are a new family of anti-inflammatory and pro-resolving lipid mediators biosynthesized from docosahexaenoic acid (DHA) by macrophages. Here we identified a novel pro-resolving product, 13R,14S-dihydroxy-docosahexaenoic acid (13R,14S-diHDHA), produced by human macrophages. PCR mapping of 12-lipoxygenase (12-LOX) mRNA sequence in human macrophages and platelet showed that they are identical. This human 12-LOX mRNA and enzyme are expressed in monocyte-derived cell lineage, and enzyme expression levels increase with maturation to macrophages or dendritic cells. Recombinant human 12-LOX gave essentially equivalent catalytic efficiency (k_cat/K_M) with arachidonic acid (AA) and DHA as substrates. Lipid mediator metabololipidomics demonstrated that human macrophages produce a novel bioactive product 13,14-dihydroxy-docosahexaenoic acid in addition to maresin-1, 7R,14S-dihydroxy-4Z,8E,10E,12Z,16Z,19Z-docosahexaenoic acid (MaR1). Co-incubations with human recombinant 12-LOX and soluble epoxide hydrolase (sEH) demonstrated that biosynthesis of 13,14-dihydroxy-docosahexaenoic acid (13,14-diHDHA) involves the 13S,14S-epoxy-maresin intermediate produced from DHA by 12-LOX, followed by conversion via soluble epoxide hydrolase (sEH). This new 13,14-diHDHA displayed potent anti-inflammatory and pro-resolving actions, and at 1 ng reduced neutrophil infiltration in mouse peritonitis by ∼40% and at 10 pM enhanced human macrophage phagocytosis of zymosan by ∼90%. However, MaR1 proved more potent than the 13R,14S-diHDHA at enhancing efferocytosis with human macrophages. Taken together, the present findings demonstrate that macrophages produced a novel bioactive product identified in the maresin metabolome as 13R,14S-dihydroxy-docosahexaenoic acid, from DHA via conversion by human 12-LOX followed by sEH. Given its potent bioactions, we coined 13R,14S-diHDHA maresin 2 (MaR2).     

Lipid and lipid mediator profiling of human synovial fluid in rheumatoid arthritis patients by means of LC–MS/MS

Human synovial fluid (SF) provides nutrition and lubrication to the articular cartilage. Particularly in arthritic diseases, SF is extensively accumulating in the synovial junction. During the last decade lipids have attracted considerable attention as their role in the development and resolution of diseases became increasingly recognized. Here, we describe a capillary LC–MS/MS screening platform that was used for the untargeted screening of lipids present in human SF of rheumatoid arthritis (RA) patients. Using this platform we give a detailed overview of the lipids and lipid‐derived mediators present in the SF of RA patients. Almost 70 different lipid components from distinct lipid classes were identified and quantification was achieved for the lysophosphatidylcholine and phosphatidylcholine species. In addition, we describe a targeted LC–MS/MS lipid mediator metabolomics strategy for the detection, identification and quantification of maresin 1, lipoxin A₄ and resolvin D5 in SF from RA patients. Additionally, we present the identification of 5S,12S-diHETE as a major marker of lipoxygenase pathway interactions in the investigated SF samples. These results are the first to provide a comprehensive approach to the identification and profiling of lipids and lipid mediators present in SF and to describe the presence of key anti-inflammatory and pro-resolving lipid mediators identified in SF from RA patients.     

Heterogeneity in Neutrophil Microparticles Reveals Distinct Proteome and Functional Properties

Altered plasma neutrophil microparticle levels have recently been implicated in a number of vascular and inflammatory diseases, yet our understanding of their actions is very limited. Herein, we investigate the proteome of neutrophil microparticles in order to shed light on their biological actions. Stimulation of human neutrophils, either in suspension or adherent to an endothelial monolayer, led to the production of microparticles containing >400 distinct proteins with only 223 being shared by the two subsets. For instance, postadherent microparticles were enriched in alpha-2 macroglobulin and ceruloplasmin, whereas microparticles produced by neutrophils in suspension were abundant in heat shock 70 kDa protein 1. Annexin A1 and lactotransferrin were expressed in both microparticle subsets. We next determined relative abundance of these proteins in three types of human microparticle samples: healthy volunteer plasma, plasma of septic patients and skin blister exudates finding that these proteins were differentially expressed on neutrophil microparticles from these samples reflecting in part the expression profiles we found in vitro. Functional assessment of the neutrophil microparticles subsets demonstrated that in response to direct stimulation neutrophil microparticles produced reactive oxygen species and leukotriene B₄ as well as locomoted toward a chemotactic gradient. Finally, we investigated the actions of the two neutrophil microparticles subsets described herein on target cell responses. Microarray analysis with human primary endothelial cells incubated with either microparticle subset revealed a discrete modulation of endothelial cell gene expression profile. These findings demonstrate that neutrophil microparticles are heterogenous and can deliver packaged information propagating the activation status of the parent cell, potentially exerting novel and fundamental roles both under homeostatic and disease conditions.The emerging notion that cells can communicate by packaged information represents a major shift in our understanding of cell-to-cell interaction in complex settings including inflammation (1). Packaging of mediators (irrespective of their chemical nature) in structures that can be transported through the vascular and lymphatic systems might avoid their rapid dilution and removal by biological fluids and allow the target cell or tissue to receive a biologically relevant amount of a given molecule. As an example, TNF-α produced by mast cells in the mouse paw can reach the lymph nodes unmodified, wrapped up in small structures or vesicles (2). In this respect, the last few years have witnessed augmented understanding in microparticle function.Described over 50 years ago (reviewed in (3, 4), microparticles are heterogeneous in nature with their size varying between 0.2 and 1.0 μm, and are characterized by an outer membrane composed of a phospholipid bilayer and cell surface proteins. The mechanism of microparticle production is not fully understood, though it may follow processes not dissimilar from those observed in apoptosis, involving membrane detachment from the anchoring cytoskeleton and loss of membrane symmetry, which leads to exposure of negatively charged phospholipids (5–7). Proteins found on the outer leaflet of the microparticle cell membrane are believed to reflect both the origin and activation status of the parental cell (8, 9); for instance, microparticles from neutrophils express CD66b and CD62L (10, 11). We have recently identified the selective expression of the potent anti-inflammatory and proresolving protein Annexin A1 (ANXA1) on the surface of microparticles generated from neutrophils adherent to endothelial monolayers, when compared with those prepared from quiescent neutrophils (12). Microparticle production is not restricted to one subset of cells and using cell specific antigens the relative contribution of different cell types to the total microparticle population in a particular environment can be assessed. This has allowed for the analysis of different microparticle populations (the focus being by and large platelet- and endothelial-derived microparticles) in a number of pathologies in the quest to identify robust biomarkers for disease and treatment (13–15). With regard to inflammatory diseases, examples would include plasma samples in sepsis (16), psoriatic arthritis (17), and scleroderma (18). However, the vast majority of these studies have only determined microparticle expression patterns with respect to the cell type of origin, without addressing the possibility that microparticle composition—even when generated from the same leukocyte subset—might differ in relation to disease status and/or mode of cell activation. Of note recent work has also demonstrated that the production of neutrophil microparticles during self-limited inflammation is temporally regulated suggesting that these microparticles are important in orchestrating inflammation-resolution (1).Recent work has established that microparticles can elicit a variety of biological processes ranging from angiogenesis to anti-inflammation; so that it is very unlikely they can continue to be considered “cell debris,” as initially postulated. The following are some examples, relevant to the present study. Ingestion of platelet microparticles alters the phenotype of macrophages, leading to the false identification of endothelial cell progenitor cells in culture (19). Likewise, sonic hedgehog can be transferred, via microparticles, to dysfunctional endothelial cells, restoring the activity of nitric oxide synthase with downstream production of nitric oxide (20). Microparticles can carry functionally active receptor proteins to target cells (21, 22). Finally, in vivo generation of microparticles has been observed within the inflamed microcirculation. Real time analysis of leukocyte recruitment has visualized microparticle release from leukocytes squeezing through an endothelial barrier, providing evidence for their formation in vivo together with potential functional relevance in relation to cell migration (23).On stimulation, neutrophils produce microparticles with rapid and nongenomic anti-inflammatory properties, in vitro and in vivo, reliant on their expression of ANXA1 (12). Whereas these findings are consistent with those obtained by Gasser and colleagues (24) who described inhibitory properties of neutrophil microparticles, other studies have suggested that the same cell type can produce microparticles that elicit activating properties, for instance upon incubation with endothelial cells or monocytes for longer time-points (25, 26). Thus, to gain further insight into the potential mechanisms involved in mediating such distinct effects, we deemed it important to determine the total proteome of neutrophil microparticles. Having established that different stimulation conditions yield microparticle populations with distinct protein profiles, we corroborated our observations in two distinct clinical scenarios, characterizing neutrophil microparticles from skin blister exudates and plasma samples from sepsis patients using a select group of proteins identified in our proteomic profile. We also established that the two microparticles subsets differentially modulate endothelial cell gene expression profile and thereby function, as determined by connectivity map analysis.     

Gene expression signature-based approach identifies a pro-resolving mechanism of action for histone deacetylase inhibitors

Despite several therapies being currently available to treat inflammatory diseases, new drugs to treat chronic conditions with less side effects and lower production costs are still needed. An innovative approach to drug discovery, the Connectivity Map (CMap), shows how integrating genome-wide gene expression data of drugs and diseases can accelerate this process. Comparison of genome-wide gene expression data generated with annexin A1 (AnxA1) with the CMap revealed significant alignment with gene profiles elicited by histone deacetylase inhibitors (HDACIs), what made us to hypothesize that AnxA1 might mediate the anti-inflammatory actions of HDACIs. Addition of HDACIs (valproic acid, sodium butyrate and thricostatin A) to mouse macrophages caused externalization of AnxA1 with concomitant inhibition of cytokine gene expression and release, events that occurred independently as this inhibition was retained in AnxA1 null macrophages. In contrast, novel AnxA1-mediated functions for HDACIs could be unveiled, including promotion of neutrophil apoptosis and macrophage phagocytosis, both steps crucial for effective resolution of inflammation. In a model of acute resolving inflammation, administration of valproic acid and sodium butyrate to mice at the peak of disease accelerated resolution processes in wild type, but much more modestly in AnxA1 null mice. Deeper analyses revealed a role for endogenous AnxA1 in the induction of neutrophil death in vivo by HDACIs. In summary, interrogation of the CMap revealed an unexpected association between HDACIs and AnxA1 that translated in mechanistic findings with particular impact on the processes that regulate the resolution of inflammation. We propose non-genomic modulation of AnxA1 in immune cells as a novel mechanism of action for HDACIs, which may underlie their reported efficacy in models of chronic inflammatory pathologies.     

Evidence for an anti-inflammatory loop centered on polymorphonuclear leukocyte formyl peptide receptor 2/lipoxin A4 receptor and operative in the inflamed microvasculature

The importance of proresolving mediators in the overall context of the resolution of acute inflammation is well recognized, although little is known about whether these anti-inflammatory and proresolving molecules act in concert. In this article, we focused on lipoxin A(4) (LXA(4)) and annexin A1 (AnxA1) because these two very different mediators converge on a single receptor, formyl peptide receptor type 2 (FPR2/ALX). Addition of LXA(4) to human polymorphonuclear leukocytes (PMNs) provoked a concentration- and time-dependent mobilization of AnxA1 onto the plasma membrane, as determined by Western blotting and flow cytometry analyses. This property was shared by another FPR2/ALX agonist, antiflammin-2, and partly by fMLF or peptide Ac2-26 (an AnxA1 derivative that can activate all three members of the human FPR family). An FPR2/ALX antagonist blocked AnxA1 mobilization activated by LXA(4) and antiflammin-2. Analysis of PMN degranulation patterns and phospho-AnxA1 status suggested a model in which the two FPR2/ALX agonists mobilize the cytosolic (and not the granular) pool of AnxA1 through an intermediate phosphorylation step. Intravital microscopy investigations of the inflamed mesenteric microvasculature of wild-type and AnxA1(-/-) mice revealed that LXA(4) provoked leukocyte detachment from the postcapillary venule endothelium in the former (>50% within 10 min; p < 0.05), but not the latter genotype (～15%; NS). Furthermore, recruitment of Gr1(+) cells into dorsal air-pouches, inflamed with IL-1β, was significantly attenuated by LXA(4) in wild-type, but not AnxA1(-/-), mice. Collectively, these data prompt us to propose the existence of an endogenous network in anti-inflammation centered on PMN AnxA1 and activated by selective FPR2/ALX agonists.     

Identification and Actions of a Novel Third Maresin Conjugate in Tissue Regeneration: MCTR3

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.