Specialized pro-resolving mediators in vascular inflammation and atherosclerotic cardiovascular disease.

Timely resolution of the acute inflammatory response (or inflammation resolution) is an active, highly coordinated process that is essential to optimal health. Inflammation resolution is regulated by specific endogenous signalling molecules that function as 'stop signals' to terminate the inflammatory response when it is no longer needed; to actively promote healing, regeneration and tissue repair; and to limit pain. Specialized pro-resolving mediators are a superfamily of signalling molecules that initiate anti-inflammatory and pro-resolving actions. Without an effective and timely resolution response, inflammation can become chronic, a pathological state that is associated with many widely occurring human diseases, including atherosclerotic cardiovascular disease. Uncovering the mechanisms of inflammation resolution failure in cardiovascular diseases and identifying useful biomarkers for non-resolving inflammation are unmet needs. In this Review, we discuss the accumulating evidence that supports the role of non-resolving inflammation in atherosclerosis and the use of specialized pro-resolving mediators as therapeutic tools for the treatment of atherosclerotic cardiovascular disease. We highlight open questions about therapeutic strategies and mechanisms of disease to provide a framework for future studies on the prevention and treatment of atherosclerosis.

The effect of vitamin D and omega-3 fatty acid supplementation on pain prevalence and severity in older adults: a large-scale ancillary study of the VITamin D and OmegA-3 triaL (VITAL).

ABSTRACT: A diet supplemented with vitamin D and marine omega-3 fatty acids may prevent and treat painful disorders by promoting the resolution of inflammation. However, large, randomized placebo-controlled trials evaluating the effects of supplementation with omega-3 fatty acids and vitamin D on the presence and severity of pain are lacking. VITamin D and OmegA-3 triaL-Pain (VITAL-Pain) is an ancillary study to the VITAL trial, a large randomized, double-blind, placebo-controlled trial of vitamin D (2000 IU/day) and omega-3 supplementation (1 g/day) over 5.3 years of median follow-up, among 25,871 older men and women. We assessed pain among those reaching the end of the VITAL intervention phase using questions from the 2012 National Health Interview Survey. We used ordinal logistic regression to test the effect of vitamin D and omega-3 fatty acids on the odds ratio (OR) and 95% confidence interval [CI] of reporting higher pain prevalence or severity. Overall, 19,611 participants provided complete pain information at the end of the VITAL trial. The ORs for higher pain prevalence or severity for vitamin D and omega-3 supplementation vs placebo were 0.99 ([CI] 0.94-1.05) and 0.99 ([CI] 0.94-1.04), respectively. There was no interaction between the 2 active treatments. Dietary supplementation with commonly used moderate doses of vitamin D or omega-3 fatty acids over a median of 5.3 years did not result in a lower prevalence or severity of pain in middle-aged and older U.S. adults.

Glucocorticoids regulate lipid mediator networks by reciprocal modulation of 15-lipoxygenase isoforms affecting inflammation resolution.

Glucocorticoids (GC) are potent anti-inflammatory agents, broadly used to treat acute and chronic inflammatory diseases, e.g., critically ill COVID-19 patients or patients with chronic inflammatory bowel diseases. GC not only limit inflammation but also promote its resolution although the underlying mechanisms are obscure. Here, we reveal reciprocal regulation of 15-lipoxygenase (LOX) isoform expression in human monocyte/macrophage lineages by GC with respective consequences for the biosynthesis of specialized proresolving mediators (SPM) and their 15-LOX-derived monohydroxylated precursors (mono-15-OH). Dexamethasone robustly up-regulated pre-mRNA, mRNA, and protein levels of ALOX15B/15-LOX-2 in blood monocyte-derived macrophage (MDM) phenotypes, causing elevated SPM and mono-15-OH production in inflammatory cell types. In sharp contrast, dexamethasone blocked ALOX15/15-LOX-1 expression and impaired SPM formation in proresolving M2-MDM. These dexamethasone actions were mimicked by prednisolone and hydrocortisone but not by progesterone, and they were counteracted by the GC receptor (GR) antagonist RU486. Chromatin immunoprecipitation (ChIP) assays revealed robust GR recruitment to a putative enhancer region within intron 3 of the ALOX15B gene but not to the transcription start site. Knockdown of 15-LOX-2 in M1-MDM abolished GC-induced SPM formation and mono-15-OH production. Finally, ALOX15B/15-LOX-2 upregulation was evident in human monocytes from patients with GC-treated COVID-19 or patients with IBD. Our findings may explain the proresolving GC actions and offer opportunities for optimizing GC pharmacotherapy and proresolving mediator production.

Eosinophil Phenotypes Are Functionally Regulated by Resolvin D2 during Allergic Lung Inflammation.

Eosinophils (Eos) reside in multiple organs during homeostasis and respond rapidly to an inflammatory challenge. Although Eos share chemical staining properties, they also demonstrate phenotypic and functional plasticity that is not fully understood. Here, we used a murine model of allergic lung inflammation to characterize Eos subsets and determine their spatiotemporal and functional regulation during inflammation and its resolution in response to resolvin D2 (RvD2), a potent specialized proresolving mediator. Two Eos subsets were identified by CD101 expression with distinct anatomic localization and transcriptional signatures at baseline and during inflammation. CD101low Eos were predominantly located in a lung vascular niche and responded to allergen challenge by moving into the lung interstitium. CD101high Eos were predominantly located in bronchoalveolar lavage (BAL) and extravascular lung, only present during inflammation, and had transcriptional evidence for cell activation. RvD2 reduced total Eos numbers and changed their phenotype and activation by at least two distinct mechanisms: decreasing interleukin 5-dependent recruitment of CD101low Eos and decreasing conversion of CD101low Eos to CD101high Eos. Collectively, these findings indicate that Eos are a heterogeneous pool of cells with distinct activation states and spatiotemporal regulation during resolution of inflammation and that RvD2 is a potent proresolving mediator for Eos recruitment and activation.

Anti-Inflammatory and Pro-Resolving Actions of the N-Terminal Peptides Ac2-26, Ac2-12, and Ac9-25 of Annexin A1 on Conjunctival Goblet Cell Function.

Annexin A1 (AnxA1) is the primary mediator of the anti-inflammatory actions of glucocorticoids. AnxA1 functions as a pro-resolving mediator in cultured rat conjunctival goblet cells to ensure tissue homeostasis through stimulation of intracellular [Ca2+] ([Ca2+]i) and mucin secretion. AnxA1 has several N-terminal peptides with anti-inflammatory properties of their own, including Ac2-26, Ac2-12, and Ac9-25. The increase in [Ca2+]i caused by AnxA1 and its N-terminal peptides in goblet cells was measured to determine the formyl peptide receptors used by the compounds and the action of the peptides on histamine stimulation. Changes in [Ca2+]i were determined by using a fluorescent Ca2+ indicator. AnxA1 and its peptides each activated formyl peptide receptors in goblet cells. AnxA1 and Ac2-26 at 10-12 mol/L and Ac2-12 at 10-9 mol/L inhibited the histamine-stimulated increase in [Ca2+]i, as did resolvin D1 and lipoxin A4 at 10-12 mol/L, whereas Ac9-25 did not. AnxA1 and Ac2-26 counter-regulated the H1 receptor through the p42/p44 mitogen-activated protein kinase/extracellular regulated kinase 1/2, ß-adrenergic receptor kinase, and protein kinase C pathways, whereas Ac2-12 counter-regulated only through ß-adrenergic receptor kinase. In conclusion, current data show that the N-terminal peptides Ac2-26 and Ac2-12, but not Ac9-25, share multiple functions with the full-length AnxA1 in goblet cells, including inhibition of histamine-stimulated increase in [Ca2+]i and counter-regulation of the H1 receptor. These actions suggest a potential pharmaceutical application of the AnxA1 N-terminal peptides Ac2-26 and Ac2-12 in homeostasis and ocular inflammatory diseases.

ALX/FPR2 Activation by Stereoisomers of D1 Resolvins Elucidating with Molecular Dynamics Simulation.

Chronic inflammation contributes to several diseases, but its resolution is driven by specialized pro-resolving mediators (SPM) such as resolvin D1 (RvD1) and its epimer aspirin-triggered resolvin D1 (AT-RvD1), both biosynthesized from ω-3 fatty docosahexaenoic acid (DHA). RvD1 and AT-RvD1 have anti-inflammatory and pro-resolution potentials, and their effects could be mediated by formyl peptide receptor type 2 receptor ALX/FPR2, a G-protein-coupled receptor (GPCR). In this work, we performed 44 µs of molecular dynamics simulations with two complexes: FPR2@AT-RvD1 and FPR2@RvD1. Our results show the following: (i) in the AT-RvD1 simulations, the ALX/FPR2 receptor remained in the active state in 62% of the frames, while in the RVD1 simulations, the receptor remained in the active state in 74% of the frames; (ii) two residues, R201 and R205, of ALX/FPR2 appear, establishing interactions with both resolvins in all simulations (22 in total); (iii) RvD1 hydrogen bonds with R201 and R205 presented higher frequency than AT-RvD1; and (iv) residues R201 and R205 are the two receptor hotspots, demonstrated by the binding free calculations. Such results show that the ALX/FPR2 receptor remained in the active state for longer in the FPR2@RvD1 simulations than in the FPR2@AT-RvD1 simulations.

Resolvin D1 prevents injurious neutrophil swarming in transplanted lungs.

Neutrophils are the primary cell type involved in lung ischemia-reperfusion injury (IRI), which remains a frequent and morbid complication after organ transplantation. Endogenous lipid mediators that become activated during acute inflammation-resolution have gained increasing recognition for their protective role(s) in promoting the restoration of homeostasis, but their influence on early immune responses following transplantation remains to be uncovered. Resolvin D1, 7S,8R,17S-trihydroxy-4Z,9E,11E,13Z,15E,19Z-docosahexaenoic acid (RvD1), is a potent stereoselective mediator that exhibits proresolving and anti-inflammatory actions in the setting of tissue injury. Here, using metabololipidomics, we demonstrate that endogenous proresolving mediators including RvD1 are increased in human and murine lung grafts immediately following transplantation. In mouse grafts, we observe lipid mediator class switching early after reperfusion. We use intravital two-photon microscopy to reveal that RvD1 treatment significantly limits early neutrophil infiltration and swarming, thereby ameliorating early graft dysfunction in transplanted syngeneic lungs subjected to severe IRI. Through integrated analysis of single-cell RNA sequencing data of donor and recipient immune cells from lung grafts, we identify transcriptomic changes induced by RvD1. These results support a role for RvD1 as a potent modality for preventing early neutrophil-mediated tissue damage after lung IRI that may be therapeutic in the clinics.

Infectious neutrophil deployment is regulated by resolvin D4.

Neutrophils reside in the bone marrow (BM), ready for deployment to sites of injury/infection, initiating inflammation and its resolution. Here, we report that distal infections signal to the BM via resolvins to regulate granulopoiesis and BM neutrophil deployment. Emergency granulopoiesis during peritonitis evoked changes in BM resolvin D1 (RvD1) and BM RvD4. We found that leukotriene B4 stimulates neutrophil deployment. RvD1 and RvD4 each limited neutrophilic infiltration to infections, and differently regulated BM myeloid populations: RvD1 increased reparative monocytes, and RvD4 regulated granulocytes. RvD4 disengaged emergency granulopoiesis, prevented excess BM neutrophil deployment, and acted on granulocyte progenitors. RvD4 also stimulated exudate neutrophil, monocyte, and macrophage phagocytosis, and enhanced bacterial clearance. This mediator accelerated both neutrophil apoptosis and clearance by macrophages, thus expediting the resolution phase of inflammation. RvD4 stimulated phosphorylation of ERK1/2 and STAT3 in human BM-aspirate-derived granulocytes. RvD4 in the 1 to 100 nM range stimulated whole-blood neutrophil phagocytosis of Escherichia coli. RvD4 increased BM macrophage efferocytosis of neutrophils. Together, these results demonstrate the novel functions of resolvins in granulopoiesis and neutrophil deployment, contributing to the resolution of infectious inflammation.

Stereochemistry and functions of the new cysteinyl-resolvin, 4S,5R-RCTR1, in efferocytosis and erythrophagocytosis of human senescent erythrocytes.

Specialized pro-resolving lipid mediators play key functions in the resolution of the acute inflammatory response. Herein, we elucidate the stereochemical structure of the new 4S,5R-RCTR1, a cysteinyl-resolvin, recently uncovered in human leukocytes incubated with a 4S,5S-epoxy-resolvin intermediate, using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and ultra-violet (UV) spectrophotometry. With this approach, the physical properties of the new mediator prepared by total organic synthesis were matched to enzymatically produced biogenic material. In addition, we confirmed the potent biological actions of 4S,5R-RCTR1 with human M2-like macrophage phagocytosis of live bacteria, efferocytosis of apoptotic neutrophils, and erythrophagocytosis of senescent human red blood cells in a concentration-dependent manner from 0.1 to 10 nM. Taken together, these results establish the complete stereochemistry of 4S,5R-RCTR1 as 5R-glutathionyl-4S,17S-dihydroxy-6E,8E,10Z,13Z,15E,19Z-docosahexaenoic acid and give evidence of its novel bioactivities in human phagocyte responses. Moreover, they confirm and extend the stereoselective functions of the 4S,5R-RCTR1 with isolated human phagocytes of interest in the resolution of inflammation.

Resolution medicine in cancer, infection, pain and inflammation: are we on track to address the next Pandemic?

Uncontrolled inflammation giving rise to excessive tissue inflammation can lead to chronic inflammation that enhances tissue destruction, amplifying many chronic human pathologies. Normally the acute inflammatory response is protective and should be self-limited returning tissues to functional homeostasis with endogenous programmed resolution via leukocyte vasculature cell-cell interactions and crosstalk that biosynthesize pro-resolving mediators. When failed resolution takes place, as with the use of NSAIDs, tissues undergo chronic inflammation and fibrosis. Herein, we discuss these mechanisms and the role of specialized proresolving mediators, the resolvins, protectins and maresins produced from essential omega-3 fatty acids EPA and DHA, and their contributions via their cognate cell surface receptors, to the resolution response. Harnessing these pathways and their cellular mechanisms can help in providing new therapeutic approaches to many human diseases, infections, organ protection and trauma via resolution medicine to enhance the body's own resilience to challenge.

Resolvins and cysteinyl-containing pro-resolving mediators activate resolution of infectious inflammation and tissue regeneration.

This review is a synopsis of the main points from the opening presentation by the authors in the Resolution of Inflammation session at the 8th European Workshop on Lipid Mediators held at the Karolinska Institute, Stockholm, Sweden, June 29th, 2022. Specialized pro-resolving mediators (SPM) promote tissue regeneration, control infections and resolution of inflammation. These include resolvins, protectins, maresins and the newly identified conjugates in tissue regeneration (CTRs). We reported mechanisms of CTRs in activating primordial regeneration pathways in planaria using RNA-sequencing. Also, the 4S,5S-epoxy-resolvin intermediate in the biosynthesis of resolvin D3 and resolvin D4 was prepared by total organic synthesis. Human neutrophils convert this to resolvin D3 and resolvin D4, while human M2 macrophages transformed this labile epoxide intermediate to resolvin D4 and a novel cysteinyl-resolvin that is a potent isomer of RCTR1. The novel cysteinyl-resolvin significantly accelerates tissue regeneration with planaria and inhibits human granuloma formation.

Novel proresolving lipid mediator mimetic 3-oxa-PD1n-3 docosapentaenoic acid reduces acute and chronic itch by modulating excitatory and inhibitory synaptic transmission and astroglial secretion of lipocalin-2 in mice.

ABSTRACT: Specialized proresolving mediators (SPMs) have demonstrated potent analgesic actions in animal models of pathological pain. The actions of SPMs in acute and chronic itch are currently unknown. Recently, n-3 docosapentaenoic acid (DPA) was found to be a substrate for the biosynthesis of several novel families of SPMs and 3-oxa-PD1 n-3 DPA (3-oxa-PD1) is an oxidation-resistant metabolic stable analogue of the n-3 DPA-derived protectin D1 (PD1). In this article, we demonstrate that 3-oxa-PD1 effectively reduces both acute and chronic itch in mouse models. Intrathecal injection of 3-oxa-PD1 (100 ng) reduced acute itch induced by histamine, chloroquine, or morphine. Furthermore, intrathecal 3-oxa-PD1 effectively reduced chronic itch, induced by cutaneous T-cell lymphoma (CTCL), allergic contact dermatitis with dinitrofluorobenzene, and psoriasis by imiquimod. Intratumoral injection of 3-oxa-PD1 also suppressed CTCL-induced chronic itch. Strikingly, the antipruritic effect lasted for several weeks after 1-week intrathecal 3-oxa-PD1 treatment. Whole-cell recordings revealed significant increase in excitatory postsynaptic currents in spinal dorsal horn (SDH) neurons of CTCL mice, but this increase was blocked by 3-oxa-PD1. 3-oxa-PD1 further increased inhibitory postsynaptic currents in SDH neurons of CTCL mice. Cutaneous T-cell lymphoma increased the spinal levels of lipocalin-2 (LCN2), an itch mediator produced by astrocytes. 3-oxa-PD1 suppressed LCN2 production in CTCL mice and LCN2 secretion in astrocytes. Finally, CTCL-induced anxiety was alleviated by intrathecal 3-oxa-PD1. Our findings suggest that 3-oxa-PD1 potently inhibits acute and chronic itch through the regulation of excitatory or inhibitory synaptic transmission and astroglial LCN2 production. Therefore, stable SPM analogs such as 3-oxa-PD1 could be useful to treat pruritus associated with different skin injuries.

Stereocontrolled total synthesis of Resolvin D4 and 17(R)-Resolvin D4.

The total synthesis of Resolvin D4 and its 17(R)-hydroxy-epimer is reported. These lipid-based natural products are biosynthesized from docosahexaenoic acid (DHA, C22:6) during the body's rapid cellular and chemical response to injurious stimuli and are part of a large class of bioactive molecules that resolve inflammation. Our convergent synthesis employed a chiral pool strategy starting from glycidol derivatives and D-erythrose to introduce stereogenic centers. A copper(I)-mediated cross coupling between propargyl bromide and terminal acetylenic precursors yielded core structures of late-stage key intermediates. A simultaneous Lindlar reduction of the skipped diynyl moiety followed by silyl group cleavage securely completed the synthesis. The synthetic availability of these molecules helped further elucidate their stereoselective biofunctions.

Protectins: Their biosynthesis, metabolism and structure-functions.

Several lipoxygenase enzymes and cyclooxygenase-2 stereoselectively convert the polyunsaturated fatty acids arachidonic acid, eicosapentaenoic acid, docosahexaenoic acid, and n-3 docosapentaenoic acid into numerous oxygenated products. Biosynthetic pathway studies have shown, during the resolution phase of acute inflammation, that distinct families of endogenous products are formed. These products were named specialized pro-resolving mediators, given their specialized functions in the inflammation-resolution circuit, enhancing the return of inflamed and injured tissue to homeostasis. The lipoxins, resolvins, protectins and maresins, together with the sulfido-conjugates of the resolvins, protectins and maresins, constitute the four individual families of these local mediators. When administrated in vivo in a wide range of human disease models, the specialized pro-resolving mediators display potent bioactions. The detailed and individual biosynthetic steps constituting the biochemical pathways, the metabolism, recent reports on structure-function studies and pharmacodynamic data of the protectins, are presented herein. Emphasis is on the structure-function results on the recent members of the sulfido conjugated protectins and further metabolism of protectin D1. Moreover, the members of the individual families of specialized pro-resolving mediators and their biosynthetic precursor are presented. Today 43 specialized pro-resolving mediators possessing pro-resolution and anti-inflammatory bioactions are reported and confirmed, constituting a basis for resolution pharmacology. This emerging biomedical field provides a new approach for drug discovery, that is also discussed.