RESUMO
The fish oil constituent docosahexaenoic acid (DHA, 22:6 n-3) is a signaling lipid with anti-inflammatory properties. The molecular mechanisms underlying the biological effect of DHA are poorly understood. Here, we report the design, synthesis, and application of a complementary pair of bio-orthogonal, photoreactive probes based on the polyunsaturated scaffold DHA and its oxidative metabolite 17-hydroxydocosahexaenoic acid (17-HDHA). In these probes, an alkyne serves as a handle to introduce a fluorescent reporter group or a biotin-affinity tag via copper(I)-catalyzed azide-alkyne cycloaddition. This pair of chemical probes was used to map specific targets of the omega-3 signaling lipids in primary human macrophages. Prostaglandin reductase 1 (PTGR1) was identified as an interaction partner that metabolizes 17-oxo-DHA, an oxidative metabolite of 17-HDHA. 17-oxo-DHA reduced the formation of pro-inflammatory lipids 5-HETE and LTB4 in human macrophages and neutrophils. Our results demonstrate the potential of comparative photoaffinity protein profiling for the discovery of metabolic enzymes of bioactive lipids and highlight the power of chemical proteomics to uncover new biological insights.
Assuntos
Ácidos Docosa-Hexaenoicos , Ácidos Graxos Ômega-3 , Humanos , Ácidos Docosa-Hexaenoicos/metabolismo , Ácidos Docosa-Hexaenoicos/farmacologia , Azidas , Cobre/farmacologia , Biotina/farmacologia , Leucotrieno B4/farmacologia , Ácidos Graxos Ômega-3/farmacologia , Macrófagos , Óleos de Peixe/farmacologia , Anti-Inflamatórios/farmacologia , Alcinos/farmacologia , Prostaglandinas , OxirredutasesRESUMO
Lipidomics has emerged as a powerful technique to study cellular lipid metabolism. As the lipidome contains numerous isomeric and isobaric species resulting in a significant overlap between different lipid classes, cutting-edge analytical technology is necessary for a comprehensive analysis of lipid metabolism. Just recently, differential mobility spectrometry (DMS) has evolved as such a technology, helping to overcome several analytical challenges. We here set out to apply DMS and the Lipidyzer™ platform to obtain a comprehensive overview of leukocyte-related lipid metabolism in the resting and activated states. First, we tested the linearity and repeatability of the platform by using HL60 cells. We obtained good linearities for most of the thirteen analyzed lipid classes (correlation coefficient > 0.95), and good repeatability (%CV < 15). By comparing the lipidome of neutrophils (PMNs), monocytes (CD14+), and lymphocytes (CD4+), we shed light on leukocyte-specific lipid patterns as well as lipidomic changes occurring through differential stimulation. For example, at the resting state, PMNs proved to contain higher amounts of triacylglycerides compared to CD4+ and CD14+ cells. On the other hand, CD4+ and CD14+ cells contained higher levels of phospholipids and ceramides. Upon stimulation, diacylglycerides, hexosylceramides, phosphatidylcholines, phosphoethanolamines, and lysophosphoethanolamines were upregulated in CD4+ cells and PMNs, whereas CD14+ cells did not show significant changes. By exploring the fatty acid content of the significantly upregulated lipid classes, we mainly found increased concentrations of very long and polyunsaturated fatty acids. Our results indicate the usefulness of the Lipidyzer™ platform for studying cellular lipid metabolism. Its application allowed us to explore the lipidome of leukocytes. Graphical abstract.
Assuntos
Leucócitos/química , Leucócitos/metabolismo , Lipídeos/química , Linhagem Celular Tumoral , Ácidos Graxos Insaturados/química , Ácidos Graxos Insaturados/metabolismo , Humanos , Metabolismo dos Lipídeos , Lipidômica , Espectrometria de MassasRESUMO
Objective: To explore the use of lipidomics for prediction of prednisolone treatment response in patients with inflammatory hand osteoarthritis. Design: The Hand Osteoarthritis Prednisolone Efficacy (HOPE) study included patients (n â= â92) with symptomatic inflammatory hand osteoarthritis, fulfilling the ACR criteria. The present analyses comprised only patients randomized to prednisolone treatment (10 âmg daily, n â= â40). Response to prednisolone treatment was defined according to the OARSI-OMERACT responder criteria at six weeks. Baseline blood samples were obtained non-fasted. Lipid species were quantified in erythrocytes with the Lipidyzer™ platform (Sciex). Oxylipins were analyzed in plasma using an in-house LC-MS/MS platform. Elastic net regularized regression was used to predict prednisolone treatment response based on common patient characteristics alone and including the patients' lipid profile. ROC analyses with 1000 bootstrapped area under the curve (AUC) was used to determine the discriminatory accuracy of the models. Results: Among included patients, 78% fulfilled the OARSI-OMERACT responder criteria. From the general patient characteristics, elastic net selected baseline hand function as only predictor of treatment response, with an AUC of 0.78 (0.56; 0.97). Addition of lipidomics resulted in an AUC of 0.92 (0.78; 0.99) and 0.85 (0.65; 0.98) for inclusion of the Lipidyzer™ platform and oxylipin platform, respectively. Conclusion: Our results suggest that the patients' lipid profile may improve the discriminative accuracy of the prediction of prednisolone treatment response in patients with inflammatory hand osteoarthritis compared to prediction by commonly measured patient characteristics alone. Hence, lipidomics may be a promising field for biomarker discovery for prediction of anti-inflammatory treatment response.
RESUMO
Inflammation is a tightly regulated process. During the past decade it has become clear that the resolution of inflammation is an active process and its dysregulation can contribute to chronic inflammation. Several cells and soluble mediators, including lipid mediators, regulate the course of inflammation and its resolution. It is, however, unclear which signals and cells are involved in initiating the resolution process. Macrophages are tissue resident cells and key players in regulating tissue inflammation through secretion of soluble mediators, including lipids. We hypothesize that persistent inflammatory stimuli can initiate resolution pathways in macrophages. In this study, we detected 21 lipids in LPS-stimulated human monocyte-derived macrophages by liquid chromatography coupled to tandem mass spectrometry. Cyclooxygenase-derived Prostaglandins were observed in the first six hours of stimulation. Interestingly, a switch towards 15-lipoxygenase products, such as the pro-resolving lipid precursors 15-HEPE and 17-HDHA was observed after 24â¯h. The RNA and protein expression of cyclooxygenase and 15-lipoxygenase were in line with this trend. Treatment with 17-HDHA increased IL-10 production of monocyte-derived macrophages and decreased LTB4 production by neutrophils, indicating the anti-inflammatory property of this lipid. These data reveal that monocyte-derived macrophages contribute to the resolution of inflammation in time by the production of pro-resolving lipids after an initial inflammatory stimulus.
Assuntos
Metabolismo dos Lipídeos , Macrófagos/metabolismo , Células Cultivadas , Humanos , Inflamação/metabolismo , Lipopolissacarídeos/farmacologia , Neutrófilos/metabolismo , Transdução de Sinais , Receptores Toll-LikeRESUMO
It is essential to measure lipid biomarkers with a high reproducibility to prevent biased results. We compared the lipid composition and inter-day reproducibility of lipid measurements in plasma and erythrocytes. Samples from 42 individuals (77% women, mean age 65 years, mean body mass index (BMI) 27 kg/m2), obtained non-fasted at baseline and after 6 weeks, were used for quantification of up to 1000 lipid species across 13 lipid classes with the Lipidyzer platform. Intraclass correlation coefficients (ICCs) were calculated to investigate the variability of lipid concentrations between timepoints. The ICC distribution of lipids in plasma and erythrocytes were compared using Wilcoxon tests. After data processing, the analyses included 630 lipids in plasma and 286 in erythrocytes. From these, 230 lipids overlapped between sample types. In plasma, 78% of lipid measurements were reproduced well to excellently, compared to 37% in erythrocytes. The ICC score distribution in plasma (median ICC 0.69) was significantly better than in erythrocytes (median ICC 0.51) (p-value < 0.001). At the class level, reproducibility in plasma was superior for triacylglycerols and cholesteryl esters while ceramides, diacylglycerols, (lyso)phosphatidylethanolamines, and sphingomyelins showed better reproducibility in erythrocytes. Although in plasma overall reproducibility was superior, differences at individual and class levels may favor the use of erythrocytes.