Resolvin D4 stereoassignment and its novel actions in host protection and bacterial clearance.
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ABSTRACT:
SUBMITTER: Winkler JW
PROVIDER: S-EPMC4705531 | biostudies-other | 2016 Jan
REPOSITORIES: biostudies-other
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Project description:Local production and downstream metabolism of specialized proresolving lipid mediators (SPMs) are pivotal in regulating their biological actions during resolution of inflammation. Resolvin D4 (RvD4: 4S,5R,17S-trihydroxydocosa-6E,8E,10Z,13Z,15E,19Z hexaenoic acid) is one of the more recently elucidated SPMs with complete stereochemistry biosynthesized from docosahexaenoic acid . Here, we report a new multimilligram commercial synthesis that afforded enough material for matching, validation, and further evaluation of RvD4 functions. Using LC-MS-MS profiling, RvD4 was identified at bioactive amounts in human (1 pg/mL) and mouse bone marrow (12 pg/femur and tibia). In mouse bone marrow, ischemia increased the formation of RvD4 > 37-fold (455 pg/femur and tibia). Two separate mouse ischemic injury models were used, where RvD4 reduced second organ reperfusion lung injury > 50%, demonstrating organ protection. Structure-function relationships of RvD4 demonstrated > 40% increase in neutrophil and monocyte phagocytic function in human whole blood in comparison with 2 separate trans-containing double bond isomers that were inactive. These 2 isomers were prepared by organic synthesis: 4S,5R,17S-trihydroxydocosa-6E,8E,10E,13Z,15E,19Z-hexaenoic acid (10-trans-RvD4), a natural isomer, and 4S,5R,17S-trihydroxydocosa-6E,8E,10E,13E,15E,19Z-hexaenoic acid (10,13-trans-RvD4), a rogue isomer. Compared to leukotriene B4 , D-series resolvins (RvD1, RvD2, RvD3, RvD4, or RvD5) did not stimulate human neutrophil chemotaxis monitored via real-time microfluidics chambers. A novel 17-oxo-containing-RvD4 product of eicosanoid oxidoreductase was identified with human bone marrow cells. Comparison of 17-oxo-RvD4 to RvD4 demonstrated that with human leukocytes 17-oxo-RvD4 was inactive. Together, these provide commercial-scale synthesis that permitted a second independent validation of RvD4 complete stereochemical structure as well as evidence for RvD4 regulation in tissues and its stereoselective phagocyte responses.
| S-EPMC6136982 | biostudies-literature
Project description: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 (Ri) by ~4.5h during E. coli peritonitis after administration at peak inflammation (Tmax=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.
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Project description:Deep vein thrombosis (DVT) is a common cardiovascular disease with a major effect on quality of life, and safe and effective therapeutic measures to efficiently reduce existent thrombus burden are scarce. Using a comprehensive targeted liquid chromatography-tandem mass spectrometry-based metabololipidomics approach, we established temporal clusters of endogenously biosynthesized specialized proresolving mediators (SPMs) and proinflammatory and prothrombotic lipid mediators during DVT progression in mice. Administration of resolvin D4 (RvD4), an SPM that was enriched at the natural onset of thrombus resolution, significantly reduced thrombus burden, with significantly less neutrophil infiltration and more proresolving monocytes in the thrombus, as well as an increased number of cells in an early apoptosis state. Moreover, RvD4 promoted the biosynthesis of other D-series resolvins involved in facilitating resolution of inflammation. Neutrophils from RvD4-treated mice were less susceptible to an ionomycin-induced release of neutrophil extracellular traps (NETs), a meshwork of decondensed chromatin lined with histones and neutrophil proteins critical for DVT development. These results suggest that delivery of SPMs, specifically RvD4, modulates the severity of thrombo-inflammatory disease in vivo and improves thrombus resolution.
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Project description:Inflammation-resolution is a protective response that is mediated by specialized pro-resolving mediators (SPMs). The clearance of dead cells or efferocytosis is a critical cellular program of inflammation-resolution. Impaired efferocytosis can lead to tissue damage in prevalent human diseases, like atherosclerosis. Therefore understanding mechanisms associated with swift clearance of dead cells is of utmost clinical importance. Recently, the accumulation of necroptotic cells (NCs) was observed in human plaques and we postulated that this is due to defective clearance programs. Here we present evidence that NCs are inefficiently taken up by macrophages because they have increased surface expression of a well-known "don't eat me" signal called CD47. High levels of CD47 on NCs stimulated RhoA-pMLC signaling in macrophages that promoted "nibbling", rather than whole-cell engulfment of NCs. Anti-CD47 blocking antibodies limited RhoA-p-MLC signaling and promoted whole-cell NC engulfment. Treatment with anti-CD47 blocking antibodies to Ldlr-/- mice with established atherosclerosis decreased necrotic cores, limited the accumulation of plaque NCs and increased lesional SPMs, including Resolvin D1 (RvD1) compared with IgG controls. Mechanistically, RvD1 promoted whole-cell engulfment of NCs by decreasing RhoA signaling and activating CDC42. RvD1 specifically targeted NCs for engulfment by facilitating the release of the well-known "eat me signal" called calreticulin from macrophages in a CDC42 dependent manner. Lastly, RvD1 enhanced the clearance of NCs in advanced murine plaques. Together, these results suggest new molecules and signaling associated with the clearance of NCs, provide a new paradigm for the regulation of inflammation-resolution, and offer a potential treatment strategy for diseases where NCs underpin the pathology.
| S-EPMC7206090 | biostudies-literature
Project description:Rheumatoid arthritis (RA) is a debilitating disease characterized by persistent accumulation of leukocytes within the articular cavity and synovial tissue. Metabololipidomic profiling of arthritic joints from omega-3 supplemented mice identified elevated levels of specialized proresolving lipid mediators (SPM) including resolvin D1 (RvD1). Profiling of human RA synovial fluid revealed physiological levels of RvD1, which - once applied to human neutrophils - attenuated chemotaxis. These results prompted analyses of the antiarthritic properties of RvD1 in a model of murine inflammatory arthritis. The stable epimer 17R-RvD1 (100 ng/day) significantly attenuated arthritis severity, cachexia, hind-paw edema, and paw leukocyte infiltration and shortened the remission interval. Metabololipidomic profiling in arthritic joints revealed 17R-RvD1 significantly reduced PGE2 biosynthesis, while increasing levels of protective SPM. Molecular analyses indicated that 17R-RvD1 enhanced expression of genes associated with cartilage matrix synthesis, and direct intraarticular treatment induced chondroprotection. Joint protective actions of 17R-RvD1 were abolished in RvD1 receptor-deficient mice termed ALX/fpr2/3-/- . These investigations open new therapeutic avenues for inflammatory joint diseases, providing mechanistic substance for the benefits of omega-3 supplementation in RA.
| S-EPMC4855303 | biostudies-literature
Project description: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 A(4)/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 B(4). These actions of RvD1 were abolished in fpr2 null mice. CONCLUSIONS: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.
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Project description:Persistent activation of the innate immune system greatly influences the risk for developing metabolic complications associated with obesity. In this study, we explored the therapeutic potential of the specialized proresolving mediator (SPM) resolvin D1 (RvD1) to actively promote the resolution of inflammation in human visceral adipose tissue from obese (Ob) patients. Using liquid chromatography-tandem mass spectrometry-based metabololipidomic analysis, we identified unbalanced production of SPMs (i.e., D- and E-series resolvins, protectin D1, maresin 1, and lipoxins) with respect to inflammatory lipid mediators (i.e., leukotriene B4 and PGs) in omental adipose tissue from Ob patients. In parallel, high-throughput transcriptomic analysis revealed a unique signature in this tissue that was characterized by overactivation of the IL-10 signaling pathway. Incubation of inflamed Ob visceral adipose tissues and human macrophages with RvD1 limited excessive activation of the IL-10 pathway by reducing phosphorylation of STAT proteins. Of interest, RvD1 blocked STAT-1 and its target inflammatory genes (i.e., CXCL9), as well as persistent STAT3 activation, without affecting the IL-10 anti-inflammatory response characterized by inhibition of IL-6, IL-1?, IL-8, and TNF-?. Furthermore, RvD1 promoted resolution by enhancing expression of the IL-10 target gene heme oxygenase-1 by mechanisms dependent on p38 MAPK activity. Together, our data show that RvD1 can tailor the quantitative and qualitative responses of human inflamed adipose tissue to IL-10 and provide a mechanistic basis for the immunoresolving actions of RvD1 in this tissue. These findings may have potential therapeutic implications in obesity-related insulin resistance and other metabolic complications.
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Project description:Resolution of inflammation is an active process driven by several new families of endogenous lipid mediators collectively coined specialized proresolving mediators (SPM). Here, we report a synthetic analog of resolvin D1 (RvD1) and aspirin-triggered RvD1, benzo-diacetylenic-17R-RvD1-methyl ester (BDA-RvD1), which was prepared using fewer steps than required for total organic synthesis of natural SPM. BDA-RvD1 was resistant to further metabolism by human recombinant 15-prostaglandin dehydrogenase, a major inactivation pathway for RvD1. In ischemia-reperfusion-initiated second organ injury, BDA-RvD1 intravenously (1 ?g) reduced neutrophil infiltration into the lungs by 58 ± 9% and was significantly more potent than native RvD1. BDA-RvD1 at 100 ng/mouse also shortened the resolution interval, Ri, of Escherichia coli peritonitis with a similar potency as RvD1, by ~57%, from Ri 10.5 h to 4.5 h. With isolated human phagocytes, BDA-RvD1 at picomolar concentrations (10(-12) M) stimulated phagocytosis of zymosan A particles. BDA-RvD1 activated human recombinant G protein-coupled receptor 32/DRV1, an RvD1 receptor, in a dose-dependent manner. These results indicate that, both in vivo in mice and with isolated human cells, BDA-RvD1 shares defining proresolving actions of RvD1, including inhibiting leukocyte infiltration and stimulating phagocytosis. Moreover, they provide evidence for a new analog mimetic and example of an immunoresolvent, namely an agent that stimulates active resolution of inflammation, for a potential new therapeutic class.
| S-EPMC4421783 | biostudies-literature
Project description:Specialized proresolving mediators (SPMs) decrease NF-κB activity to prevent excessive tissue damage and promote the resolution of acute inflammation. Mechanisms for NF-κB regulation by SPMs remain to be determined. In this study, after LPS challenge, the SPMs 15-epi-lipoxin A4 (15-epi-LXA4), resolvin D1, resolvin D2, resolvin D3, and 17-epi-resolvin D1 were produced in vivo in murine lungs. In LPS-activated human bronchial epithelial cells, select SPMs increased expression of the NF-κB regulators A20 and single Ig IL-1R-related molecule (SIGIRR). Of interest, 15-epi-LXA4 induced A20 and SIGIRR in an lipoxin A4 receptor/formyl peptide receptor 2 (ALX/FPR2) receptor-dependent manner in epithelial cells and in murine pneumonia. This SPM regulated NF-κB-induced cytokines to decrease pathogen-mediated inflammation. In addition to dampening lung inflammation, surprisingly, 15-epi-LXA4 also enhanced pathogen clearance with increased antimicrobial peptide expression. Taken together, to our knowledge these results are the first to identify endogenous agonists for A20 and SIGIRR expression to regulate NF-κB activity and to establish mechanisms for NF-κB regulation by SPMs for pneumonia resolution.
| S-EPMC5906795 | biostudies-literature
Project description:Specialized proresolving mediators (SPMs) actively limit inflammation and expedite its resolution by modulating leukocyte recruitment and function. Here we profiled intramuscular lipid mediators via liquid chromatography-tandem mass spectrometry-based metabolipidomics following myofiber injury and investigated the potential role of SPMs in skeletal muscle inflammation and repair. Both proinflammatory eicosanoids and SPMs increased following myofiber damage induced by either intramuscular injection of barium chloride or synergist ablation-induced functional muscle overload. Daily systemic administration of the SPM resolvin D1 (RvD1) as an immunoresolvent limited the degree and duration of inflammation, enhanced regenerating myofiber growth, and improved recovery of muscle strength. RvD1 suppressed inflammatory cytokine expression, enhanced polymorphonuclear cell clearance, modulated the local muscle stem cell response, and polarized intramuscular macrophages to a more proregenerative subset. RvD1 had minimal direct impact on in vitro myogenesis but directly suppressed myokine production and stimulated macrophage phagocytosis, showing that SPMs can modulate both infiltrating myeloid and resident muscle cell populations. These data reveal the efficacy of immunoresolvents as a novel alternative to classical antiinflammatory interventions in the management of muscle injuries to modulate inflammation while stimulating tissue repair.
| S-EPMC7526543 | biostudies-literature