Lucy V. Norling

Resolvin D2 is a potent regulator of leukocytes and controls microbial sepsis

A growing body of evidence indicates that resolution of acute inflammation is an active process. Resolvins are a new family of lipid mediators enzymatically generated within resolution networks that possess unique and specific functions to orchestrate catabasis, the phase in which disease declines. Resolvin D2 (RvD2) was originally identified in resolving exudates, yet its individual contribution in resolution remained to be elucidated. Here, we establish RvD2’s potent stereoselective actions in reducing excessive neutrophil trafficking to inflammatory loci. RvD2 decreased leukocyte–endothelial interactions in vivo by endothelial-dependent nitric oxide production, and by direct modulation of leukocyte adhesion receptor expression. In mice with microbial sepsis initiated by caecal ligation and puncture, RvD2 sharply decreased both local and systemic bacterial burden, excessive cytokine production and neutrophil recruitment, while increasing peritoneal mononuclear cells and macrophage phagocytosis. These multi-level pro-resolving actions of RvD2 translate to increased survival from sepsis induced by caecal ligation and puncture and surgery. Together, these results identify RvD2 as a potent endogenous regulator of excessive inflammatory responses that acts via multiple cellular targets to stimulate resolution and preserve immune vigilance.

Annexin 1 mediates the rapid anti-inflammatory effects of neutrophil-derived microparticles

Polymorphonuclear leukocyte (PMN)-derived microparticles display inhibitory properties on target cells as assessed in vitro; since PMNs contain abundant amounts of the endogenous anti-inflammatory protein annexin 1 (AnxA1), we tested here whether biologically active AnxA1 could be present in PMN-derived microparticles. PMN adhesion to human umbilical vein endothelial cell (HUVEC) monolayers led to the generation of microparticles that contained AnxA1, as detected by Western blotting, flow cytometry, and mass spectrometry analyses. Addition of these microparticles to recipient PMNs prior to flow over HUVEC monolayers significantly inhibited cell adhesion, an effect abrogated by a neutralizing anti-AnxA1 antibody, or an antibody raised against the AnxA1 receptor, that is termed lipoxin A(4) receptor or ALX. Intravenous delivery of human PMN-derived microparticles markedly inhibited PMN recruitment to an air pouch inflamed with IL-1beta. This anti-inflammatory effect was also dependent on endogenous AnxA1, since injection of microparticles produced from wild-type PMNs (bone marrow derived), but not from AnxA1-null PMNs, inhibited IL-1beta-induced leukocyte trafficking. In conclusion, PMN-derived microparticles contain functionally active AnxA1 that confers them anti-inflammatory properties; generation of these microparticles in the microcirculation could promote inflammatory resolution by time-dependent dampening of cell recruitment.

Cutting Edge: Humanized Nano-Proresolving Medicines Mimic Inflammation-Resolution and Enhance Wound Healing

Endogenous microparticles (MPs) were systematically profiled during the time course of self-limited inflammation. Precursors for specialized proresolving lipid mediators were identified in MPs from inflammatory exudates using liquid chromatography tandem mass spectrometry-based metabolomics. Hence, we postulated that formation of anti-inflammatory and proresolving lipid mediators could underlie beneficial effects attributed to MPs and that this process could serve as a basis for biomimicry. Using human neutrophil-derived MPs, we constructed novel nanoparticles (NPs) containing aspirin-triggered resolvin D1 or a lipoxin A4 analog. Enriched NPs dramatically reduced polymorphonuclear cell influx in murine peritonitis, shortened resolution intervals, and exhibited proresolving actions accelerating keratinocyte healing. The enriched NPs protected against inflammation in the temporomandibular joint. These findings indicate that humanized NPs, termed nano-proresolving medicines, are mimetics of endogenous resolving mechanisms, possess potent beneficial bioactions, can reduce nanotoxicity, and offer new therapeutic approaches.

Resolvin D1 Limits Polymorphonuclear Leukocyte Recruitment to Inflammatory Loci: Receptor-Dependent Actions

Objective—Resolvin D1 (RvD1) limits neutrophil recruitment during acute inflammation and is derived from omega-3 docosahexaenoic acid to promote catabasis. The contribution of its specific receptors, the lipoxin A4/Annexin-A1 receptor formyl-peptide receptor 2 (FPR2/ALX) and the orphan receptor G-protein–coupled receptor 32 (GPR32) are of considerable interest. Methods and Results—RvD1 reduced human polymorphonuclear leukocytes recruitment to endothelial cells under shear conditions as quantified using a flow chamber system. Receptor-specific antibodies blocked these anti-inflammatory actions of RvD1, with low (1 nmol/L) concentrations sensitive to GPR32 blockade, while the higher (10 nmol/L) concentration appeared FPR2/ALX-specific. Interestingly, polymorphonuclear leukocytes surface expression of FPR2/ALX but not GPR32 increased following activation with pro-inflammatory stimuli, corresponding with secretory vesicle mobilization. Lipid mediator metabololipidomics carried out with 24-hour exudates revealed that RvD1 in vivo gave a significant reduction in the levels of a number of pro-inflammatory mediators including prostaglandins and leukotriene B4. These actions of RvD1 were abolished in fpr2 null mice. Conclusion—Pro-resolving lipid mediators and their receptors, such as RvD1 and the 2 G-protein–coupled receptors, studied here regulate resolution and may provide new therapeutic strategies for diseases with a vascular inflammatory component.

The resolution of inflammation: Principles and challenges

The concept that chemokines, cytokines and pro-inflammatory mediators act in a co-ordinated fashion to drive the initiation of the inflammatory reaction is well understood. The significance of such networks acting during the resolution of inflammation however is poorly appreciated. In recent years, specific pro-resolving mediators were discovered which activate resolution pathways to return tissues to homeostasis. These mediators are diverse in nature, and include specialized lipid mediators (lipoxins, resolvins, protectins and maresins) proteins (annexin A1, galectins) and peptides, gaseous mediators including hydrogen sulphide, a purine (adenosine), as well as neuromodulator release under the control of the vagus nerve. Functionally, they can act to limit further leukocyte recruitment, induce neutrophil apoptosis and enhance efferocytosis by macrophages. They can also switch macrophages from classical to alternatively activated cells, promote the return of non-apoptotic cells to the lymphatics and help initiate tissue repair mechanisms and healing. Within this review we highlight the essential cellular aspects required for successful tissue resolution, briefly discuss the pro-resolution mediators that drive these processes and consider potential challenges faced by researchers in the quest to discover how inflammation resolves and why chronic inflammation persists.

Cutting-Edge Analysis of Extracellular Microparticles using ImageStream X Imaging Flow Cytometry

Interest in extracellular vesicle biology has exploded in the past decade, since these microstructures seem endowed with multiple roles, from blood coagulation to inter-cellular communication in pathophysiology. In order for microparticle research to evolve as a preclinical and clinical tool, accurate quantification of microparticle levels is a fundamental requirement, but their size and the complexity of sample fluids present major technical challenges. Flow cytometry is commonly used, but suffers from low sensitivity and accuracy. Use of Amnis ImageStreamX Mk II imaging flow cytometer afforded accurate analysis of calibration beads ranging from 1 μm to 20 nm; and microparticles, which could be observed and quantified in whole blood, platelet-rich and platelet-free plasma and in leukocyte supernatants. Another advantage was the minimal sample preparation and volume required. Use of this high throughput analyzer allowed simultaneous phenotypic definition of the parent cells and offspring microparticles along with real time microparticle generation kinetics. With the current paucity of reliable techniques for the analysis of microparticles, we propose that the ImageStreamX could be used effectively to advance this scientific field.

Novel insights into the inhibitory effects of Galectin‐1 on neutrophil recruitment under flow

Galectin‐1 (Gal‐1) is a β‐galactoside‐binding protein endowed with anti‐inflammatory properties. The purpose of this study was to investigate the effects of endogenous and exogenous Gal‐1 on neutrophil recruitment onto TNF‐treated endothelium. The effect of human recombinant (hr)Gal‐1 on markers of neutrophil activation (CD11b expression, P‐selectin glycoprotein ligand 1, and L‐selectin shedding) was also assessed. Gal‐1 inhibited the platelet‐activating factor‐induced increase in CD11b expression in a concentration‐dependent manner, as assessed by flow cytometry. To determine the effects of Gal‐1 on neutrophil recruitment, an in vitro flow chamber was used: Preincubation of neutrophils with hrGal‐1 significantly decreased the extent of capture, rolling, and adhesion on activated endothelial monolayers. This inhibition was shared with the endogenous protein, as knockdown of endothelial Gal‐1 using small interfering RNA resulted in a significant increase in the number of cells captured and rolling. To verify the effects of Gal‐1 in an in vivo system, intravital microscopy of Gal‐1 null mice and their wild‐type counterparts was performed. Leukocyte adhesion and emigration were increased significantly in the cremasteric circulation of Gal‐1 null mice inflamed with IL‐1β. These findings indicate that Gal‐1 functions to limit neutrophil recruitment onto a TNF‐treated endothelium, a property that may underline its inhibitory effects in acute inflammation.

Resolvin D1 Limits Polymorphonuclear Leukocyte Recruitment to Inflammatory Loci

Objective—Resolvin D1 (RvD1) limits neutrophil recruitment during acute inflammation and is derived from omega-3 docosahexaenoic acid to promote catabasis. The contribution of its specific receptors, the lipoxin A4/Annexin-A1 receptor formyl-peptide receptor 2 (FPR2/ALX) and the orphan receptor G-protein–coupled receptor 32 (GPR32) are of considerable interest. Methods and Results—RvD1 reduced human polymorphonuclear leukocytes recruitment to endothelial cells under shear conditions as quantified using a flow chamber system. Receptor-specific antibodies blocked these anti-inflammatory actions of RvD1, with low (1 nmol/L) concentrations sensitive to GPR32 blockade, while the higher (10 nmol/L) concentration appeared FPR2/ALX-specific. Interestingly, polymorphonuclear leukocytes surface expression of FPR2/ALX but not GPR32 increased following activation with pro-inflammatory stimuli, corresponding with secretory vesicle mobilization. Lipid mediator metabololipidomics carried out with 24-hour exudates revealed that RvD1 in vivo gave a significant reduction in the levels of a number of pro-inflammatory mediators including prostaglandins and leukotriene B4. These actions of RvD1 were abolished in fpr2 null mice. Conclusion—Pro-resolving lipid mediators and their receptors, such as RvD1 and the 2 G-protein–coupled receptors, studied here regulate resolution and may provide new therapeutic strategies for diseases with a vascular inflammatory component.

Inhibitory control of endothelial galectin-1 on in vitro and in vivo lymphocyte trafficking

Galectin‐1 (Gal‐1) is a β‐galactoside‐binding protein, the expression of which is increased in endothelial cells on exposure to proinflammatory stimuli. Through binding of several receptors (CD7, CD45, and CD43) Gal‐1 is known to induce apoptosis of activated T lymphocytes, an effect thought to mediate the beneficial effects it exerts in various inflammatory models. The data presented here highlights another function for Gal‐1, that of a negative regulator of T‐cell recruitment to the endothelium under both physiological and pathophysiological conditions. We have shown, using siRNA to knockdown Gal‐1 in endothelial cells, that endogenous Gal‐1 limits T‐cell capture, rolling, and adhesion to activated endothelial cells under flow. Furthermore, the reverse effect is observed when exogenous human recombinant Gal‐1 is added to activated endothelial monolayers whereby a dramatic reduction in lymphocyte recruitment is seen. These findings are corroborated by studies in Gal‐1 null mice in which homing of wild‐type (WT) T lymphocytes is significantly increased to mesenteric lymph nodes and to the inflamed paw in a model of delayed‐type hyper‐sensitivity. In conclusion, mimicking endothelial Gal‐1 actions would be a novel strategy for controlling aberrant T‐cell trafficking, hence for the development of innovative anti‐inflammatory therapeutics.—Norling, L. V., Sampaio, A. L. F., Cooper, D., Perretti, M. Inhibitory control of endothelial galectin‐1 on in vitro and in vivo lymphocyte trafficking. FASEB J. 22, 682–690 (2008)

Endogenous galectins and the control of the host inflammatory response

A new era of research is being devoted to deciphering endogenous mediators and mechanisms that are in place to resolve the inflammatory response. Accruing evidence indicates that galectins fall into this category of immunoregulatory mediators signifying their use as prospective novel anti-inflammatory agents. The focus of this review is to depict the immunoregulatory bioactivities of three members of the galectin superfamily, Galectin (Gal)-1, Gal-3 and Gal-9. Emphasis is given to the studies investigating the properties of these endogenous lectins. Gal-1, Gal-3 and Gal-9 are emerging as pertinent players in the modulation of acute and chronic inflammatory diseases, autoimmunity and cancer, and thus being increasingly recognised as molecular targets for innovative drug discovery.