Proresolving Mediators Resolvin D1 Research Articles

Generation of resolving memory neutrophils through pharmacological training with 4-PBA or genetic deletion of TRAM

Neutrophils are the dominant leukocytes in circulation and the first responders to infection and inflammatory cues. While the roles of neutrophils in driving inflammation have been widely recognized, the contribution of neutrophils in facilitating inflammation resolution is under-studied. Here, through single-cell RNA sequencing analysis, we identified a subpopulation of neutrophils exhibiting pro-resolving characteristics with greater Cd200r and Cd86 expression at the resting state. We further discovered that 4-PBA, a peroxisomal stress-reducing agent, can potently train neutrophils into the resolving state with enhanced expression of CD200R, CD86, as well as soluble pro-resolving mediators Resolvin D1 and SerpinB1. Resolving neutrophils trained by 4-PBA manifest enhanced phagocytosis and bacterial-killing functions. Mechanistically, the generation of resolving neutrophils is mediated by the PPARγ/LMO4/STAT3 signaling circuit modulated by TLR4 adaptor molecule TRAM. We further demonstrated that genetic deletion of TRAM renders the constitutive expansion of resolving neutrophils, with an enhanced signaling circuitry of PPARγ/LMO4/STAT3. These findings may have profound implications for the effective training of resolving neutrophils with therapeutic potential in the treatment of both acute infection as well as chronic inflammatory diseases.

Inflammation resolution circuits are uncoupled in acute sepsis and correlate with clinical severity.

Sepsis is a critical illness characterized by dysregulated inflammatory responses lacking counter-regulation. Specialized proresolving mediators are agonists for antiinflammation and for promoting resolution, and they are protective in preclinical sepsis models. Here, in human sepsis, we mapped resolution circuits for the specialized proresolving mediators resolvin D1 and resolvin D2 in peripheral blood neutrophils and monocytes, their regulation of leukocyte activation and function ex vivo, and their relationships to measures of clinical severity. Neutrophils and monocytes were isolated from healthy subjects and patients with sepsis by inertial microfluidics and resolvin D1 and resolvin D2 receptor expression determined by flow cytometry. The impact of these resolvins on leukocyte activation was determined by isodielectric separation and leukocyte function by stimulated phagolysosome formation. Leukocyte proresolving receptor expression was significantly higher in sepsis. In nanomolar concentrations, resolvin D1 and resolvin D2 partially reversed sepsis-induced changes in leukocyte activation and function. Principal component analyses of leukocyte resolvin receptor expression and responses differentiated sepsis from health and were associated with measures of sepsis severity. These findings indicate that resolvin D1 and resolvin D2 signaling for antiinflammation and resolution are uncoupled from leukocyte activation in early sepsis and suggest that indicators of diminished resolution signaling correlate with clinical disease severity.

Metabolipidomic profiling reveals an age-related deficiency of skeletal muscle pro-resolving mediators that contributes to maladaptive tissue remodeling.

Specialized pro‐resolving mediators actively limit inflammation and support tissue regeneration, but their role in age‐related muscle dysfunction has not been explored. We profiled the mediator lipidome of aging muscle via liquid chromatography‐tandem mass spectrometry and tested whether treatment with the pro‐resolving mediator resolvin D1 (RvD1) could rejuvenate the regenerative ability of aged muscle. Aged mice displayed chronic muscle inflammation and this was associated with a basal deficiency of pro‐resolving mediators 8‐oxo‐RvD1, resolvin E3, and maresin 1, as well as many anti‐inflammatory cytochrome P450‐derived lipid epoxides. Following muscle injury, young and aged mice produced similar amounts of most pro‐inflammatory eicosanoid metabolites of cyclooxygenase (e.g., prostaglandin E2) and 12‐lipoxygenase (e.g., 12‐hydroxy‐eicosatetraenoic acid), but aged mice produced fewer markers of pro‐resolving mediators including the lipoxins (15‐hydroxy‐eicosatetraenoic acid), D‐resolvins/protectins (17‐hydroxy‐docosahexaenoic acid), E‐resolvins (18‐hydroxy‐eicosapentaenoic acid), and maresins (14‐hydroxy‐docosahexaenoic acid). Similar absences of downstream pro‐resolving mediators including lipoxin A4, resolvin D6, protectin D1/DX, and maresin 1 in aged muscle were associated with greater inflammation, impaired myofiber regeneration, and delayed recovery of strength. Daily intraperitoneal injection of RvD1 had minimal impact on intramuscular leukocyte infiltration and myofiber regeneration but suppressed inflammatory cytokine expression, limited fibrosis, and improved recovery of muscle function. We conclude that aging results in deficient local biosynthesis of specialized pro‐resolving mediators in muscle and that immunoresolvents may be attractive novel therapeutics for the treatment of muscular injuries and associated pain in the elderly, due to positive effects on recovery of muscle function without the negative side effects on tissue regeneration of non‐steroidal anti‐inflammatory drugs.

Role of the Specialized Pro-resolving Mediator Resolvin D1 in Hashimoto's Thyroiditis.

Resolvins are produced by the catabolism of polyunsaturated fatty acids (PUFAs) and play vital roles in inflammation resolution. Resolvins have been associated with autoimmune disorders. This study aimed to measure the level of Resolvin D1 (RVD1) in the serum of Hashimoto's thyroiditis (HT) patients and healthy controls (HCs) and to further analyse its correlation with thyroid autoantibodies and inflammatory factors. Sixty-three participants were recruited, namely, 30 untreated HT patients and 33 sex- and age-matched HCs. Serum RVD1 and inflammatory chemokine (MCP-1 and IP-10) levels were measured by ELISA according to the manufacturer's protocol. Serum total T3 (TT3), TT4, free T3 (FT3), FT4, thyroglobulin antibody (TgAb), thyroid peroxidase antibody (TPOAb) and thyroid-stimulating hormone (TSH) levels were measured using an electrochemiluminescence immunoassay. Thyroid homeostasis parameters, including the thyroid secretory capacity (SPINA-GT), the total deiodinase activity (SPINA-GD), Jostel's TSH index (TSHI) and the thyrotroph thyroid hormone sensitivity index (TTSI), were calculated. Serum RVD1 levels in HT patients (134.76, 85.35-201.36 pg/mL) were significantly lower than those in HCs (187.64, 131.01-326.85 pg/mL) (P=0.004). As the TPOAb level increased, the RVD1 level showed a decreasing trend (P for trend=0.002). Both multinomial and ordinal logistics analyses revealed that serum RVD1 levels were negatively correlated with TPOAb levels in the adjusted models. Moreover, RVD1 showed a negative correlation with the inflammatory chemokine IP-1 0 (r=-0.276, P=0.034), TSHI (r=-0.269, P=0.036) and TTSI (r=-0.277, P=0.031). Thyroid autoimmunity may be associated with low levels of RVD1. Decreased RVD1 levels indicate impaired resolution of inflammation in HT patients.

The Effects of Omega-3 Supplementation and Doxorubicin-Based Chemotherapy on the Murine Brain Lipidome

Abstract Objectives To determine how omega-3 (n3-FA) supplementation may confer protection against lipid modifications following doxorubicin-based chemotherapy (DOX). Methods Ovariectomized C57BL/6 mice consumed a diet with 0% or 2% kcal supplemental EPA + DHA for 4 weeks, followed by two injections of either DOX (9 mg/kg) + cyclophosphamide (90 mg/kg), or vehicle. In study 1, animals were sacrificed at 4, 7, and 14 days after the last injection (n = 120) and in study 2, at 10 days after the last injection (n = 40). Whole brain from study 1 were analyzed by targeted methods (UHPLC-MS/MS), to quantify specialized pro-resolving mediators resolvin D1 (RvD1), resolvin D2 (RvD2), resolvin D3 (RvD3), resolvin D5 (RvD5), resolvin E1 (RvE1), maresin (MaR1), and protectin (PD1). In study 2, lipidomics analyses were performed on hippocampus to determine changes in the lipidome after n3-FA supplementation and chemotherapy injection. Results Study 1 results: RvD1 was present in all samples, but no significant differences in concentration were observed regardless of treatment or dietary group. RvD3, PD1 and MaR1 were detected in a subset of samples. Study 2 results: EPA + DHA (2%) supplementation favorably altered lipids associated with cognitive function (i.e., PE (P-16:0/20:5), PE (P-18:0/22:6, with adjusted p-value equal to 0.003 and 0.04 respectively), which have been previously negatively correlated with Alzheimer's and Parkinson's disease. Chemotherapy treatment increases omega-9 fatty acids (i.e., nervonic, gadoleic and mead acid) previously positively correlated with diseases of cognitive decline (e.g., Alzheimer's, Parkinson's). No chemo*n3-FA interaction was observed (p-value > 0.05). Conclusions N3-FA supplementation favorably altered lipids associated with cognitive function. DOX increased lipids associated with diseases of cognitive decline. Future investigations will determine if the same biomarkers of n-3 FA consumption and chemotherapy are observed in human breast cancer patients. Funding Sources This research was supported by a Foods for Health Discovery Themes Initiative SEEDS grant, NIH R01CA189947, NIH Award Number Grant P30 CA016058, OSU, and OSUCCC.

Role of the Specialized Proresolving Mediator Resolvin D1 in Systemic Lupus Erythematosus: Preliminary Results.

Objective Systemic lupus erythematosus (SLE) is an autoimmune systemic disease and its pathogenesis has not yet been completely clarified. Patients with SLE show a deranged lipid metabolism, which can contribute to the immunopathogenesis of the disease and to the accelerated atherosclerosis. Resolvin D1 (RvD1), a product of the metabolism of the omega-3 polyunsaturated fatty acid docosahexaenoic acid (DHA), acts as a specialized proresolving mediator which can contribute in restoring the homeostasis in inflamed tissues. The aim of the present pilot study is to evaluate plasma levels of RvD1 in patients with SLE and healthy subjects, investigating its potential role as a biomarker of SLE and assessing its relationship with disease activity and laboratory parameters. Methods Thirty patients with SLE and thirty age- and sex-matched healthy subjects (HSs) have been consecutively recruited at Campus Bio-Medico University Hospital. RvD1 plasma levels were measured by ELISA according to the manufacturer's protocol (Cayman Chemical Co.). RvD1 levels were compared using Mann–Whitney test. Discriminatory ability for SLE has been evaluated by the area under the ROC curve. Results Lower levels of RvD1, 45.6 (35.5–57.4) pg/ml, in patients with SLE have been found compared to HSs, 65.1 (39.43–87.95) pg/ml (p = 0.0043). The area under the ROC curve (AUC) for RvD1 was 0.71 (95% CI: 0.578–0.82) and the threshold value of RvD1 for the classification of SLE was <58.4 pg/ml, sensitivity 80% (95% CI: 61.4–92.3), and specificity 63.3% (95% CI: 43.9–80.1), likelihood ratio 2.2 (95% CI: 1.3–3.6). Conclusions The present preliminary study allows hypothesizing a dysregulation of RvD1 in patients with SLE, confirming the emerging role of bioactive lipids in this disease.

Trypanosoma cruzi Produces the Specialized Proresolving Mediators Resolvin D1, Resolvin D5, and Resolvin E2.

Trypanosoma cruzi is a protozoan parasite that causes Chagas disease (CD). CD is a persistent, lifelong infection affecting many organs, most notably the heart, where it may result in acute myocarditis and chronic cardiomyopathy. The pathological features include myocardial inflammation and fibrosis. In the Brazil strain-infected CD-1 mouse, which recapitulates many of the features of human infection, we found increased plasma levels of resolvin D1 (RvD1), a specialized proresolving mediator of inflammation, during both the acute and chronic phases of infection (>100 days postinfection) as determined by enzyme-linked immunosorbent assay (ELISA). Additionally, ELISA on lysates of trypomastigotes of both strains Tulahuen and Brazil revealed elevated levels of RvD1 compared with lysates of cultured epimastigotes of T. cruzi, tachyzoites of Toxoplasma gondii, trypomastigotes of Trypanosoma brucei, cultured L6E9 myoblasts, and culture medium containing no cells. Lysates of T. cruzi-infected myoblasts also displayed increased levels of RvD1. Lipid mediator metabolomics confirmed that the trypomastigotes of T. cruzi produced RvD1, RvD5, and RvE2, which have been demonstrated to modulate the host response to bacterial infections. Plasma RvD1 levels may be both host and parasite derived. Since T. cruzi synthesizes specialized proresolving mediators of inflammation, as well as proinflammatory eicosanoids, such as thromboxane A2, one may speculate that by using these lipid mediators to modulate its microenvironment, the parasite is able to survive.

Macrophage-Derived Protein S Facilitates Apoptotic Polymorphonuclear Cell Clearance by Resolution Phase Macrophages and Supports Their Reprogramming.

The complete resolution of inflammation requires the uptake of apoptotic polymorphonuclear cells (PMN) by local macrophages (efferocytosis) and the consequent reprogramming of the engulfing phagocytes to reparative and pro-resolving phenotypes. The tyrosine kinase receptors TYRO3, AXL, and MERTK (collectively named TAM) are fundamental mediators in regulating inflammatory responses and efferocytosis. Protein S (PROS1) is a ligand for all TAM receptors that mediates various aspects of their activity. However, the involvement of PROS1 in the resolution of inflammation is incompletely understood. Here, we report the upregulation of Pros1 in macrophages during the resolution of inflammation. Selective knockout of Pros1 in the myeloid lineage significantly downregulated macrophage pro-resolving properties. Hence, Pros1-deficient macrophages engulfed fewer apoptotic PMN remnants in vivo, and exogenous PROS1 rescued impaired efferocytosis ex vivo. Moreover, Pros1-deficient peritoneal macrophages secreted higher levels of the pro-inflammatory mediators TNFα and CCL3, while they secreted lower levels of the reparative/anti-inflammatory IL-10 following exposure to lipopolysaccharide in comparison to their WT counterparts. Moreover, Pros1-deficient macrophages expressed less of the anti-inflammatory/pro-resolving enzymes arginase-1 and 12/15-lipoxygenase and produced less of the specialized pro-resolving mediator resolvin D1. Altogether, our results suggest that macrophage-derived PROS1 is an important effector molecule in regulating the efferocytosis, maturation, and reprogramming of resolution phase macrophages, and imply that PROS1 could provide a new therapeutic target for inflammatory and fibrotic disorders.

Pro-resolving lipid mediator Resolvin D1 serves as a marker of lung disease in cystic fibrosis.

BackgroundCystic fibrosis (CF) is an autosomal recessive genetic disorder that affects multiple organs, including the lungs, pancreas, liver and intestine. Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) locus lead to defective proteins and reduced Cl- secretion and Na+ hyperabsorption in the affected organs. In addition, patients suffering from CF display chronic inflammation that contributes to the pathogenesis of CF. Recent work suggests that CF patients have a reduced capacity to biosynthesize specialized pro-resolving lipid mediators (SPMs), which contributes to the development and duration of the unwanted inflammation. Alterations in the metabolism of arachidonic acid (AA) and docosahexaenoic acid (DHA) to specialized pro-resolving mediators (SPMs), like lipoxins (LXs), maresins (MaRs), protectins (PDs) and resolvins (Rvs), may play a major role on clinical impact of airway inflammation in CF.MethodsIn this study, our aims were to detect and quantitate Resolvin D1 (RvD1) in sputum and plasma from patients with CF and compare levels of RvD1 with biomarkers of inflammation and lung function. We studied 27 CF patients aged 6 to 55 years (median 16 years) in a prospective approach.ResultsDHA can be found in the plasma of our CF patients in the milligram range and is decreased in comparison to a healthy control group. The DHA-derived pro-resolving mediator Resolvin D1 (RvD1) was also present in the plasma (286.4 ± 50 pg/ mL, mean ± SEM) and sputum (30.0 ± 2.6 pg/ mL, mean ± SEM) samples from our patients with CF and showed a positive correlation with sputum inflammatory markers. The plasma concentrations of RvD1 were ten times higher than sputum concentrations. Interestingly, sputum RvD1/ IL-8 levels showed a positive correlation with FEV1 (rs = 0.3962, p< 0.05).ConclusionsSPMs, like RvD1, are well known to down-regulate inflammatory pathways. Our study shows that the bioactive lipid mediator RvD1, derived from DHA, was present in sputum and plasma of CF patients and may serve as a representative peripheral biomarker of the lung resolution program for CF patients.

Human Sepsis Eicosanoid and Proresolving Lipid Mediator Temporal Profiles: Correlations With Survival and Clinical Outcomes.

To identify and measure recently described chemical mediators, termed specialized pro-resolving mediators that actively regulate the resolution of acute-inflammation, and correlate measurements with clinical outcomes. Herein, deidentified plasma was collected from sepsis patients (n = 22 subjects) within 48 hours of admission to the ICU and on days 3 and 7 thereafter and subjected to lipid mediator profiling. Brigham and Women's Hospital Medical Intensive Care Unit. Patients in the medical ICU with sepsis. In all patients, we identified more than 30 bioactive mediators and pathway markers in peripheral blood using established criteria for arachidonic acid, eicosapentaenoic acid, and docosahexaenoic acid metabolomes. These included inflammation initiating mediators leukotriene B4 and prostaglandin E2 and pro-resolving mediators resolvin D1, resolvin D2, and protectin D1. In sepsis nonsurvivors, we found significantly higher inflammation-initiating mediators including prostaglandin F2α and leukotriene B4 and pro-resolving mediators, including resolvin E1, resolvin D5, and 17R-protectin D1 than was observed in surviving sepsis subjects. This signature was present at ICU admission and persisted for 7 days. Further analysis revealed increased respiratory failure in nonsurvivors. Higher inflammation-initiating mediators (including prostaglandin F2α) and select proresolving pathways were associated with the development of acute respiratory distress syndrome, whereas other traditional clinical indices were not predictive of acute respiratory distress syndrome development. These results provide peripheral blood lipid mediator profiles in sepsis that correlate with survival and acute respiratory distress syndrome development, thus suggesting plausible novel biomarkers and biologic targets for critical illness.

Resolvin D1 Reduces Emphysema and Chronic Inflammation

Chronic obstructive pulmonary disease is characterized, in part, by chronic inflammation that persists even after smoking cessation, suggesting that a failure to resolve inflammation plays an important role in the pathogenesis of the disease. It is widely recognized that the resolution of inflammation is an active process, governed by specialized proresolving lipid mediators, including lipoxins, resolvins, maresins, and protectins. Here, we report that proresolving signaling and metabolic pathways are disrupted in lung tissue from patients with chronic obstructive pulmonary disease, suggesting that supplementation with proresolving lipid mediators might reduce the development of emphysema by controlling chronic inflammation. Groups of mice were exposed long-term to cigarette smoke and treated with the proresolving mediator resolvin D1. Resolvin D1 was associated with a reduced development of cigarette smoke-induced emphysema and airspace enlargement, with concurrent reductions in inflammation, oxidative stress, and cell death. Interestingly, resolvin D1 did not promote the differentiation of M2 macrophages and did not promote tissue fibrosis. Taken together, our results suggest that cigarette smoking disrupts endogenous proresolving pathways and that supplementation with specialized proresolving lipid mediators is an important therapeutic strategy in chronic lung disease, especially if endogenous specialized proresolving lipid mediator signaling is impaired.

Cutting edge: Parathyroid hormone facilitates macrophage efferocytosis in bone marrow via proresolving mediators resolvin D1 and resolvin D2.

Bone marrow macrophages stimulate skeletal wound repair and osteoblastic bone formation by poorly defined mechanisms. Specialized proresolving mediators of inflammation drive macrophage efferocytosis (phagocytosis of apoptotic cells) and resolution, but little is known regarding this process in the bone marrow. In this study, metabololipidomic profiling via liquid chromatography mass spectrometry revealed higher levels of specialized proresolving mediators in the bone marrow relative to the spleen. The endocrine and bone anabolic agent parathyroid hormone increased specialized proresolving mediator levels, including resolvins (Rvs), in bone marrow. Human and murine primary macrophages efferocytosed apoptotic osteoblasts in vitro, and RvD1 and RvD2 (10 pM-10 nM) enhanced this process. These findings support a unique profile of specialized lipid mediators in bone marrow that contribute to a feedback system for resolution of inflammation and maintenance of skeletal homeostasis.