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Sci Adv ; 5(10): eaax4895, 2019 10.
Artículo en Inglés | MEDLINE | ID: mdl-31681846


Targeting hypoxia-sensitive pathways in immune cells is of interest in treating diseases. Here, we demonstrate that physiologic hypoxia (1% O2), as encountered in bone marrow and spleen, accelerates human M2 macrophage efferocytosis of apoptotic-neutrophils and senescent erythrocytes via lipolysis-dependent biosynthesis of specialized pro-resolving mediators (SPMs), i.e. resolvins, protectins, maresins and lipoxin. SPM-production was enhanced via hypoxia in M2 macrophages interacting with neutrophils and erythrocytes enabling structural elucidation of a novel eicosapentaenoic acid (EPA)-derived resolvin, resolvin E4 (RvE4) that stimulates efferocytosis of senescent erythrocytes and more potently than aspirin in mouse hemorrhagic exudates. In hypoxia, glycolysis inhibition enhanced neutrophil RvE4-SPM biosynthesis. Human macrophage-erythrocyte co-incubations in physiologic hypoxia produced RvE4-SPM from erythrocyte stores of omega-3 fatty acids. These results indicate that hypoxic environments, including bone marrow and spleen as well as sites of inflammation, activate SPM-biosynthetic circuits that in turn stimulate resolution and clearance of senescent erythrocytes and apoptotic neutrophils.

Hipoxia/metabolismo , Metaboloma , Apoptosis , Comunicación Celular , Hipoxia de la Célula , Membrana Eritrocítica/metabolismo , Eritrocitos/metabolismo , Glucólisis , Hemorragia/patología , Humanos , Mediadores de Inflamación/metabolismo , Leucocitos/metabolismo , Macrófagos/metabolismo , Masculino , Neutrófilos
Sci Rep ; 8(1): 18050, 2018 12 21.
Artículo en Inglés | MEDLINE | ID: mdl-30575798


Specialized pro-resolving mediator(s) (SPMs) are produced from the endogenous ω-3 polyunsaturated fatty acids (PUFA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), and accelerate resolution of acute inflammation. We identified specific clusters of SPM in human plasma and serum using LC-MS/MS based lipid mediator (LM) metabololipidomics in two separate laboratories for inter-laboratory validation. The human plasma cluster consisted of resolvin (Rv)E1, RvD1, lipoxin (LX)B4, 18-HEPE, and 17-HDHA, and the human serum cluster consisted of RvE1, RvD1, AT-LXA4, 18-HEPE, and 17-HDHA. Human plasma and serum SPM clusters were increased after ω-3 supplementation (triglyceride dietary supplements or prescription ethyl esters) and low dose intravenous lipopolysaccharide (LPS) challenge. These results were corroborated by parallel determinations with the same coded samples in a second, separate laboratory using essentially identical metabololipidomic operational parameters. In these healthy subjects, two ω-3 supplementation protocols (Study A and Study B) temporally increased the SPM cluster throughout the endotoxin-challenge time course. Study A and Study B were randomized and Study B also had a crossover design with placebo and endotoxin challenge. Endotoxin challenge temporally regulated lipid mediator production in human serum, where pro-inflammatory eicosanoid (prostaglandins and thromboxane) concentrations peaked by 8 hours post-endotoxin and SPMs such as resolvins and lipoxins initially decreased by 2 h and were then elevated at 24 hours. In healthy adults given ω-3 supplementation, the plasma concentration of the SPM cluster (RvE1, RvD1, LXB4, 18-HEPE, and 17-HDHA) peaked at two hours post endotoxin challenge. These results from two separate laboratories with the same samples provide evidence for temporal production of specific pro-resolving mediators with ω-3 supplementation that together support the role of SPM in vivo in inflammation-resolution in humans.

Endotoxinas/administración & dosificación , Ácidos Grasos Omega-3/administración & dosificación , Metaboloma/efectos de los fármacos , Metabolómica/métodos , Administración Intravenosa , Adulto , Cromatografía Liquida , Suplementos Dietéticos , Ácidos Docosahexaenoicos/administración & dosificación , Relación Dosis-Respuesta a Droga , Ácido Eicosapentaenoico/administración & dosificación , Ácido Eicosapentaenoico/análogos & derivados , Endotoxinas/efectos adversos , Femenino , Voluntarios Sanos , Humanos , Inflamación/inducido químicamente , Inflamación/dietoterapia , Metabolismo de los Lípidos/efectos de los fármacos , Masculino , Proyectos de Investigación/normas , Espectrometría de Masas en Tándem , Adulto Joven
Am J Pathol ; 188(4): 950-966, 2018 04.
Artículo en Inglés | MEDLINE | ID: mdl-29571326


Resolvin conjugates in tissue regeneration (RCTRs) are new chemical signals that accelerate resolution of inflammation, infection, and tissue regeneration. Herein, using liquid chromatography-tandem mass spectrometry-based metabololipidomics, we identified RCTRs in human spleen, lymph node, bone marrow, and brain. In human spleen incubated with Staphylococcus aureus, endogenous RCTRs were increased along with conversion of deuterium-labeled docosahexaenoic acid, conferring pathway activation. Physical and biological properties of endogenous RCTRs were matched with those prepared by total organic synthesis. The complete stereochemical assignment of bioactive RCTR1 is 8R-glutathionyl-7S,17S-dihydroxy-4Z,9E,11E,13Z,15E,19Z-docosahexaenoic acid, RCTR2 is 8R-cysteinylglycinyl-7S,17S-dihydroxy-4Z,9E,11E,13Z,15E,19Z-docosahexaenoic acid, and RCTR3 is 8R-cysteinyl-7S,17S-dihydroxy-4Z,9E,11E,13Z,15E,19Z-docosahexaenoic acid. These stereochemically defined RCTRs stimulated human macrophage phagocytosis, efferocytosis, and planaria tissue generation. Proteome profiling demonstrated that RCTRs regulated both proinflammatory and anti-inflammatory cytokines with human macrophages. In microfluidic chambers, the three RCTRs limited human polymorphonuclear cell migration. In hind-limb ischemia-reperfusion-initiated organ injury, both RCTR2 and RCTR3 reduced polymorphonuclear cell infiltration into lungs. In infectious peritonitis, RCTR1 shortened the resolution intervals. Each RCTR (1 nmol/L) accelerated planaria tissue regeneration by approximately 0.5 days, with direct comparison to both maresin and protectin CTRs. Together, these results identify a new bioactive RCTR (ie, RCTR3) in human tissues and establish the complete stereochemistry and rank-order potencies of three RCTRs in vivo. Moreover, RCTR1, RCTR2, and RCTR3 each exert potent anti-inflammatory and proresolving actions with human leukocytes.

Ácidos Grasos Omega-3/química , Fagocitos/metabolismo , Regeneración/fisiología , Animales , Quimiotaxis , Infecciones por Escherichia coli/patología , Ácidos Grasos Omega-3/biosíntesis , Humanos , Inflamación/patología , Lesión Pulmonar/microbiología , Lesión Pulmonar/patología , Macrófagos/citología , Masculino , Metaboloma , Ratones , Fagocitos/citología , Fagocitosis , Planarias/fisiología , Proteoma/metabolismo , Daño por Reperfusión/microbiología , Daño por Reperfusión/patología , Bazo/metabolismo , Estereoisomerismo
J Phys Chem B ; 120(33): 8346-53, 2016 08 25.
Artículo en Inglés | MEDLINE | ID: mdl-27063350


Arachidonic acid (AA), a representative ω6-polyunsaturated fatty acid (PUFA), is a precursor of 2-series prostaglandins (PGs) that play important roles in inflammation, pain, fever, and related disorders including cardiovascular diseases. Eating fish or supplementation with the ω3-PUFAs such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) is widely assumed to be beneficial in preventing cardiovascular diseases. A proposed mechanism for a cardio-protective role of ω3-PUFAs assumes competition between AA and ω3-PUFAs for cyclooxygenases (COX), leading to reduced production of 2-series PGs. In this study, we have used a systems biology approach to integrate existing knowledge and novel high-throughput data that facilitates a quantitative understanding of the molecular mechanism of ω3- and ω6-PUFA metabolism in mammalian cells. We have developed a quantitative computational model of the competitive metabolism of AA and EPA via the COX pathway through a two-step matrix-based approach to estimate the rate constants. This model was developed by using lipidomic data sets that were experimentally obtained from EPA-supplemented ATP-stimulated RAW264.7 macrophages. The resulting model fits the experimental data well for all metabolites and demonstrates that the integrated metabolic and signaling networks and the experimental data are consistent with one another. The robustness of the model was validated through parametric sensitivity and uncertainty analysis. We also validated the model by predicting the results from other independent experiments involving AA- and DHA-supplemented ATP-stimulated RAW264.7 cells using the parameters estimated with EPA. Furthermore, we showed that the higher affinity of EPA binding to COX compared with AA was able to inhibit AA metabolism effectively. Thus, our model captures the essential features of competitive metabolism of ω3- and ω6-PUFAs.

Simulación por Computador , Ácidos Grasos Omega-3/metabolismo , Ácidos Grasos Omega-6/metabolismo , Macrófagos/metabolismo , Modelos Moleculares , Adenosina Trifosfato/metabolismo , Animales , Ácido Araquidónico/metabolismo , Línea Celular , Cinética , Ratones Endogámicos BALB C , Prostaglandina-Endoperóxido Sintasas/metabolismo , Biología de Sistemas
FASEB J ; 27(5): 1939-49, 2013 May.
Artículo en Inglés | MEDLINE | ID: mdl-23382512


Previously, we observed significant increases in spinal 12-lipoxygenase (LOX) metabolites, in particular, hepoxilins, which contribute to peripheral inflammation-induced tactile allodynia. However, the enzymatic sources of hepoxilin synthase (HXS) activity in rats remain elusive. Therefore, we overexpressed each of the 6 rat 12/15-LOX enzymes in HEK-293T cells and measured by LC-MS/MS the formation of HXB3, 12-HETE, 8-HETE, and 15-HETE from arachidonic acid (AA) at baseline and in the presence of LOX inhibitors (NDGA, AA-861, CDC, baicalein, and PD146176) vs. vehicle-treated and mock-transfected controls. We detected the following primary intrinsic activities: 12-LOX (Alox12, Alox15), 15-LOX (Alox15b), and HXS (Alox12, Alox15). Similar to human and mouse orthologs, proteins encoded by rat Alox12b and Alox12e possessed minimal 12-LOX activity with AA as substrate, while eLOX3 (encoded by Aloxe3) exhibited HXS without 12-LOX activity when coexpressed with Alox12b or supplemented with 12-HpETE. CDC potently inhibited HXS and 12-LOX activity in vitro (relative IC50s: CDC, ~0.5 and 0.8 µM, respectively) and carrageenan-evoked tactile allodynia in vivo. Notably, peripheral inflammation significantly increased spinal eLOX3; intrathecal pretreatment with either siRNA targeting Aloxe3 or an eLOX3-selective antibody attenuated the associated allodynia. These findings implicate spinal eLOX3-mediated hepoxilin synthesis in inflammatory hyperesthesia and underscore the importance of developing more selective 12-LOX/HXS inhibitors.

Araquidonato 12-Lipooxigenasa/metabolismo , Araquidonato 15-Lipooxigenasa/metabolismo , Hiperalgesia/etiología , Oxidorreductasas Intramoleculares/metabolismo , Animales , Araquidonato 12-Lipooxigenasa/efectos de los fármacos , Araquidonato 15-Lipooxigenasa/efectos de los fármacos , Células HEK293 , Humanos , Inhibidores de la Lipooxigenasa/farmacología , Masculino , Ratas
Proc Natl Acad Sci U S A ; 109(22): 8517-22, 2012 May 29.
Artículo en Inglés | MEDLINE | ID: mdl-22586114


Dietary fish oil containing ω3 fatty acids, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), elicit cardioprotective and anti-inflammatory effects through unresolved mechanisms that may involve competition and inhibition at multiple levels. Here, we report the effects of arachidonic acid (AA), EPA, and DHA supplementation on membrane incorporation, phospholipase A(2) catalyzed release, and eicosanoid production in RAW264.7 macrophages. Using a targeted lipidomics approach, we observed that Toll-like receptor 4 and purinergic receptor activation of supplemented cells leads to the release of 22-carbon fatty acids that potently inhibit cyclooxygenase pathways. This inhibition was able to shunt metabolism of AA to lipoxygenase pathways, augmenting leukotriene and other lipoxygenase mediator synthesis. In resident peritoneal macrophages, docosapentaenoic acid (DPA) was responsible for cyclooxygenase inhibition after EPA supplementation, offering fresh insights into how EPA exerts anti-inflammatory effects indirectly through elongation to 22-carbon DPA.

Ácidos Grasos Omega-3/farmacología , Macrófagos/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Receptor Toll-Like 4/metabolismo , Adenosina Trifosfato/farmacología , Animales , Ácido Araquidónico/metabolismo , Ácido Araquidónico/farmacología , Western Blotting , Línea Celular , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Células Cultivadas , Ácidos Docosahexaenoicos/metabolismo , Ácidos Docosahexaenoicos/farmacología , Eicosanoides/metabolismo , Ácidos Grasos Omega-3/metabolismo , Ácidos Grasos Insaturados/metabolismo , Ácidos Grasos Insaturados/farmacología , Cromatografía de Gases y Espectrometría de Masas , Lipopolisacáridos/farmacología , Lipooxigenasa/metabolismo , Macrófagos/citología , Macrófagos/metabolismo , Macrófagos Peritoneales/citología , Macrófagos Peritoneales/efectos de los fármacos , Macrófagos Peritoneales/metabolismo , Masculino , Lípidos de la Membrana/metabolismo , Ratones , Fosfolipasas A2/metabolismo , Prostaglandina-Endoperóxido Sintasas/metabolismo , Receptores Purinérgicos P2X7/metabolismo , Receptor Toll-Like 4/agonistas