Nan Chiang

Resolving inflammation: dual anti-inflammatory and pro-resolution lipid mediators

Active resolution of acute inflammation is a previously unrecognized interface between innate and adaptive immunity. Once thought to be a passive process, the resolution of inflammation is now shown to involve active biochemical programmes that enable inflamed tissues to return to homeostasis. This Review presents new cellular and molecular mechanisms for the resolution of inflammation, revealing key roles for eicosanoids, such as lipoxins, and recently discovered families of endogenous chemical mediators, termed resolvins and protectins. These mediators have anti-inflammatory and pro-resolution properties, thereby protecting organs from collateral damage, stimulating the clearance of inflammatory debris and promoting mucosal antimicrobial defence.

Resolvin E1 and protectin D1 activate inflammation-resolution programmes

Resolution of acute inflammation is an active process essential for appropriate host responses, tissue protection and the return to homeostasis. During resolution, specific omega-3 polyunsaturated fatty-acid-derived mediators are generated within resolving exudates, including resolvin E1 (RvE1) and protectin D1 (PD1). It is thus important to pinpoint specific actions of RvE1 and PD1 in regulating tissue resolution. Here we report that RvE1 and PD1 in nanogram quantities promote phagocyte removal during acute inflammation by regulating leukocyte infiltration, increasing macrophage ingestion of apoptotic polymorphonuclear neutrophils in vivo and in vitro, and enhancing the appearance of phagocytes carrying engulfed zymosan in lymph nodes and spleen. In this tissue terrain, inhibition of either cyclooxygenase or lipoxygenases—pivotal enzymes in the temporal generation of both pro-inflammatory and pro-resolving mediators—caused a ‘resolution deficit’ that was rescued by RvE1, PD1 or aspirin-triggered lipoxin A4 analogue. Also, new resolution routes were identified that involve phagocytes traversing perinodal adipose tissues and non-apoptotic polymorphonuclear neutrophils carrying engulfed zymosan to lymph nodes. Together, these results identify new active components for postexudate resolution traffic, and demonstrate that RvE1 and PD1 are potent agonists for resolution of inflamed tissues.

Novel docosanoids Inhibit brain ischemia-reperfusion-mediated leukocyte infiltration and pro-inflammatory gene expression

Ischemic stroke triggers lipid peroxidation and neuronal injury. Docosahexaenoic acid released from membrane phospholipids during brain ischemia is a major source of lipid peroxides. Leukocyte infiltration and pro-inflammatory gene expression also contribute to stroke damage. In this study using lipidomic analysis, we have identified stereospecific messengers from docosahexaenoate-oxygenation pathways in a mouse stroke model. Aspirin, widely used to prevent cerebrovascular disease, activates an additional pathway, which includes the 17R-resolvins. The newly discovered brain messenger 10,17S-docosatriene potently inhibited leukocyte infiltration, NFκB, and cyclooxygenase-2 induction in experimental stroke and elicited neuroprotection. In addition, in neural cells in culture, this lipid messenger also inhibited both interleukin 1-β-induced NFκB activation and cyclooxygenase-2 expression. Thus, the specific novel bioactive docosanoids generated in vivo counteract leukocyte-mediated injury as well as pro-inflammatory gene induction. These results challenge the view that docosahexaenoate only participates in brain damage and demonstrate that this fatty acid is also the endogenous precursor to a neuroprotective signaling response to ischemia-reperfusion.

Molecular Circuits of Resolution: Formation and Actions of Resolvins and Protectins

The cellular events underlying the resolution of acute inflammation are not known in molecular terms. To identify anti-inflammatory and proresolving circuits, we investigated the temporal and differential changes in self-resolving murine exudates using mass spectrometry-based proteomics and lipidomics. Key resolution components were defined as resolution indices including Ψmax, the maximal neutrophil numbers that are present during the inflammatory response; Tmax, the time when Ψmax occurs; and the resolution interval (Ri) from Tmax to T50 when neutrophil numbers reach half Ψmax. The onset of resolution was at ∼12 h with proteomic analysis showing both haptoglobin and S100A9 levels were maximal and other exudate proteins were dynamically regulated. Eicosanoids and polyunsaturated fatty acids first appeared within 4 h. Interestingly, the docosahexaenoic acid-derived anti-inflammatory lipid mediator 10,17S-docosatriene was generated during the Ri. Administration of aspirin-triggered lipoxin A4 analog, resolvin E1, or 10,17S-docosatriene each either activated and/or accelerated resolution. For example, aspirin-triggered lipoxin A4 analog reduced Ψmax, resolvin E1 decreased both Ψmax and Tmax, whereas 10,17S-docosatriene reduced Ψmax, Tmax, and shortened Ri. Also, aspirin-triggered lipoxin A4 analog markedly inhibited proinflammatory cytokines and chemokines at 4 h (20–50% inhibition), whereas resolvin E1 and 10,17S-docosatriene’s inhibitory actions were maximal at 12 h (30–80% inhibition). Moreover, aspirin-triggered lipoxin A4 analog evoked release of the antiphlogistic cytokine TGF-β. These results characterize the first molecular resolution circuits and their major components activated by specific novel lipid mediators (i.e., resolvin E1 and 10,17S-docosatriene) to promote resolution.

Resolvin D1 binds human phagocytes with evidence for proresolving receptors

Endogenous mechanisms that act in the resolution of acute inflammation are essential for host defense and the return to homeostasis. Resolvin D1 (RvD1), biosynthesized during resolution, displays potent and stereoselective anti-inflammatory actions, such as limiting neutrophil infiltration and proresolving actions. Here, we demonstrate that RvD1 actions on human polymorphonuclear leukocytes (PMNs) are pertussis toxin sensitive, decrease actin polymerization, and block LTB4-regulated adhesion molecules (β2 integrins). Synthetic [3H]-RvD1 was prepared, which revealed specific RvD1 recognition sites on human leukocytes. Screening systems to identify receptors for RvD1 gave two candidates—ALX, a lipoxin A4 receptor, and GPR32, an orphan—that were confirmed using a β-arrestin-based ligand receptor system. Nuclear receptors including retinoid X receptor-α and peroxisome proliferator-activated receptor-α, -δ, -γ were not activated by either resolvin E1 or RvD1 at bioactive nanomolar concentrations. RvD1 enhanced macrophage phagocytosis of zymosan and apoptotic PMNs, which increased with overexpression of human ALX and GPR32 and decreased with selective knockdown of these G-protein-coupled receptors. Also, ALX and GPR32 surface expression in human monocytes was up-regulated by zymosan and granulocyte-monocyte–colony-stimulating factor. These results indicate that RvD1 specifically interacts with both ALX and GPR32 on phagocytes and suggest that each plays a role in resolving acute inflammation.

Resolvin E1 selectively interacts with leukotriene B4 receptor BLT1 and ChemR23 to regulate inflammation

Resolvin E1 (RvE1) is a potent anti-inflammatory and proresolving mediator derived from omega-3 eicosapentaenoic acid generated during the resolution phase of inflammation. RvE1 possesses a unique structure and counterregulatory actions that stop human polymorphonuclear leukocyte (PMN) transendothelial migration and PMN infiltration in several murine inflammatory models. To examine the mechanism(s) underlying anti-inflammatory actions on PMNs, we prepared [3H]RvE1 and characterized its interactions with human PMN. Results with membrane fractions of human PMN demonstrated specific binding with a Kd of 48.3 nM. [3H]RvE1 specific binding to human PMN was displaced by leukotriene B4 (LTB4) and LTB4 receptor 1 (BLT1) antagonist U-75302, but not by chemerin peptide, a ligand specific for another RvE1 receptor ChemR23. Recombinant human BLT1 gave specific binding with [3H]RvE1 with a Kd of 45 nM. RvE1 selectively inhibited adenylate cyclase with BLT1, but not with BLT2. In human PBMC, RvE1 partially induced calcium mobilization, and blocked subsequent stimulation by LTB4. RvE1 also attenuated LTB4-induced NF-κB activation in BLT1-transfected cells. In vivo anti-inflammatory actions of RvE1 were sharply reduced in BLT1 knockout mice when given at low doses (100 ng i.v.) in peritonitis. In contrast, RvE1 at higher doses (1.0 μg i.v.) significantly reduced PMN infiltration in a BLT1-independent manner. These results indicate that RvE1 binds to BLT1 as a partial agonist, potentially serving as a local damper of BLT1 signals on leukocytes along with other receptors (e.g., ChemR23-mediated counterregulatory actions) to mediate the resolution of inflammation.

The Lipoxin Receptor ALX: Potent Ligand-Specific and Stereoselective Actions in Vivo

Lipoxins (LXs) and aspirin-triggered LX (ATL) are trihydroxytetraene-containing eicosanoids generated from arachidonic acid that are distinct in structure, formation, and function from the many other proinflammatory lipid-derived mediators. These endogenous eicosanoids have now emerged as founding members of the first class of lipid/chemical mediators involved in the resolution of the inflammatory response. Lipoxin A4 (LXA4), ATL, and their metabolic stable analogs elicit cellular responses and regulate leukocyte trafficking in vivo by activating the specific receptor, ALX. ALX was the first receptor cloned and identified as a G protein-coupled receptor (GPCR) for lipoxygenase-derived eicosanoids with demonstrated cell type-specific signaling pathways. ALX at the level of DNA has sequence homology to the N-formylpeptide receptor and as an orphan GPCR was initially referred to as the N-formylpeptide receptor-like 1. Although LXA4 is the endogenous potent ligand for ALX activation, a number of peptides can also activate this receptor to stimulate calcium mobilization and chemotaxis in vitro. In contrast with LXA4, the counterparts of many of these peptides in vivo remain to be established. The purpose of this review is to highlight the molecular characterization of the ALX receptor and provide an overview of the ALX-LXA4 axis responsible for anti-inflammatory and proresolving signals in vivo. The information in this review provides further support for the initial nomenclature proposition for this GPCR as ALX.

Endogenous lipid- and peptide-derived anti-inflammatory pathways generated with glucocorticoid and aspirin treatment activate the lipoxin A4 receptor.

Aspirin (ASA) and dexamethasone (DEX) are widely used anti-inflammatory agents yet their mechanism(s) for blocking polymorphonuclear neutrophil (PMN) accumulation at sites of inflammation remains unclear. Here, we report that inhibition of PMN infiltration by ASA and DEX is a property shared by aspirin-triggered lipoxins (ATL) and the glucocorticoid-induced annexin 1 (ANXA1)-derived peptides that are both generated in vivo and act at the lipoxin A4 receptor (ALXR/FPRL1) to halt PMN diapedesis. These structurally diverse ligands specifically interact directly with recombinant human ALXR demonstrated by specific radioligand binding and function as well as immunoprecipitation of PMN receptors. In addition, the combination of both ATL and ANXA1-derived peptides limited PMN infiltration and reduced production of inflammatory mediators (that is, prostaglandins and chemokines) in vivo. Together, these results indicate functional redundancies in endogenous lipid and peptide anti-inflammatory circuits that are spatially and temporally separate, where both ATL and specific ANXA1-derived peptides act in concert at ALXR to downregulate PMN recruitment to inflammatory loci.

Infection regulates pro-resolving mediators that lower antibiotic requirements.

Underlying mechanisms for how bacterial infections contribute to active resolution of acute inflammation are unknown. Here, we performed exudate leukocyte trafficking and mediator-metabololipidomics of murine peritoneal Escherichia coli infections with temporal identification of pro-inflammatory (prostaglandins and leukotrienes) and specialized pro-resolving mediators (SPMs). In self-resolving E. coli exudates (105 colony forming units, c.f.u.), the dominant SPMs identified were resolvin (Rv) D5 and protectin D1 (PD1), which at 12 h were at significantly greater levels than in exudates from higher titre E. coli (107 c.f.u.)-challenged mice. Germ-free mice had endogenous RvD1 and PD1 levels higher than in conventional mice. RvD1 and RvD5 (nanograms per mouse) each reduced bacterial titres in blood and exudates, E. coli-induced hypothermia and increased survival, demonstrating the first actions of RvD5. With human polymorphonuclear neutrophils and macrophages, RvD1, RvD5 and PD1 each directly enhanced phagocytosis of E. coli, and RvD5 counter-regulated a panel of pro-inflammatory genes, including NF-κB and TNF-α. RvD5 activated the RvD1 receptor, GPR32, to enhance phagocytosis. With self-limited E. coli infections, RvD1 and the antibiotic ciprofloxacin accelerated resolution, each shortening resolution intervals (Ri). Host-directed RvD1 actions enhanced ciprofloxacin’s therapeutic actions. In 107 c.f.u. E. coli infections, SPMs (RvD1, RvD5, PD1) together with ciprofloxacin also heightened host antimicrobial responses. In skin infections, SPMs enhanced vancomycin clearance of Staphylococcus aureus. These results demonstrate that specific SPMs are temporally and differentially regulated during infections and that they are anti-phlogistic, enhance containment and lower antibiotic requirements for bacterial clearance.

Endogenous pro-resolving and anti-inflammatory lipid mediators: a new pharmacologic genus.

Complete resolution of an acute inflammatory response and its return to homeostasis are essential for healthy tissues. Here, we overview ongoing efforts to characterize cellular and molecular mechanisms that govern the resolution of self‐limited inflammation. Systematic temporal analyses of evolving inflammatory exudates using mediator lipidomics‐informatics, proteomics, and cellular trafficking with murine resolving exudates demonstrate novel endogenous pathways of local‐acting mediators that share both anti‐inflammatory and pro‐resolving properties. In murine systems, resolving‐exudate leukocytes switch their phenotype to actively generate new families of mediators from major omega‐3 fatty acids EPA and DHA termed resolvins and protectins. Recent advances on their biosynthesis and actions are reviewed with a focus on the E‐series resolvins (RvE1, RvE2), D series resolvins (RvD1, RvD2) and the protectins including neuroprotectin D1/protectin D1 (NPD1/PD1) as well as their aspirin‐triggered epimeric forms. Members of each new family demonstrate potent stereo‐specific actions, joining the lipoxins as endogenous local signals that govern resolution and endogenous anti‐inflammation mechanisms. In addition to their origins and roles in resolution biology in the immune system, recent findings indicate that these new mediator families also display potent protective actions in lung, kidney, and eye as well as enhance microbial clearance. Thus, these endogenous agonists of resolution pathways constitute a novel genus of chemical mediators that possess pro‐resolving, anti‐inflammatory, and antifibrotic as well as host‐directed antimicrobial actions. These may be useful in the design of new therapeutics and treatments for diseases with the underlying trait of uncontrolled inflammation and redox organ stress.