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Institute
Eicosanoids are lipid mediators generated from arachidonic acid with pro- and anti-inflammatory properties. Despite these lipid mediators being known for decades, quantitative determination in biological samples is still challenging due to low abundance, instability, the existence of regio- and stereoisomers, and a wide polarity range that hampers chromatographic separation. In this study, we developed a supercritical fluid chromatography mass spectrometry (SFC-MS) platform for the quantification of relevant eicosanoids. Application of a chiral amylose-based column and modifier combination of 2-propanol/acetonitrile offered separation and sufficient resolution of 11 eicosanoids (5-, 12-, 15-HETE, PGB1, LTB4, t-LTB4, 20-OH-LTB4, PGE2, PGD2, PGF2α, TxB2) with baseline separation of isobaric analytes within 12 min. The method was validated in terms of range (78–2500 ng/mL), linearity, accuracy, precision, and recovery according to EMA guidelines. Finally, we confirmed the method’s applicability by quantifying eicosanoid levels in human primary blood cells. In conclusion, we present a validated SFC-MS method for the determination of relevant eicosanoids in biological samples with a wide range of polarity while maintaining baseline separation of isobars, which allows coupling to a single quadrupole mass detector.
Abstract
Neutrophils are the most abundant leukocytes in circulation playing a key role in acute inflammation during microbial infections. Phagocytosis, one of the crucial defence mechanisms of neutrophils against pathogens, is amplified by chemotactic leukotriene (LT)B4, which is biosynthesized via 5‐lipoxygenase (5‐LOX). However, extensive liberation of LTB4 can be destructive by over‐intensifying the inflammatory process. While enzymatic biosynthesis of LTB4 is well characterized, less is known about molecular mechanisms that activate 5‐LOX and lead to LTB4 formation during host–pathogen interactions. Here, we investigated the ability of the common opportunistic fungal pathogen Candida albicans to induce LTB4 formation in neutrophils, and elucidated pathogen‐mediated drivers and cellular processes that activate this pathway. We revealed that C. albicans‐induced LTB4 biosynthesis requires both the morphological transition from yeast cells to hyphae and the expression of hyphae‐associated genes, as exclusively viable hyphae or yeast‐locked mutant cells expressing hyphae‐associated genes stimulated 5‐LOX by [Ca2+]i mobilization and p38 MAPK activation. LTB4 biosynthesis was orchestrated by synergistic activation of dectin‐1 and Toll‐like receptor 2, and corresponding signaling via SYK and MYD88, respectively. Conclusively, we report hyphae‐specific induction of LTB4 biosynthesis in human neutrophils. This highlights an expanding role of neutrophils during inflammatory processes in the response to C. albicans infections.
Natural products comprise a rich reservoir for innovative drug leads and are a constant
source of bioactive compounds. To find pharmacological targets for new or already known
natural products using modern computer-aided methods is a current endeavor in drug discovery.
Nature’s treasures, however, could be used more effectively. Yet, reliable pipelines for the
large-scale target prediction of natural products are still rare. We developed an in silico workflow
Int. J. Mol. Sci. 2020, 21, 7102; doi:10.3390/ijms21197102 www.mdpi.com/journal/ijms
Int. J. Mol. Sci. 2020, 21, 7102 2 of 18
consisting of four independent, stand-alone target prediction tools and evaluated its performance
on dihydrochalcones (DHCs)—a well-known class of natural products. Thereby, we revealed
four previously unreported protein targets for DHCs, namely 5-lipoxygenase, cyclooxygenase-1,
17β-hydroxysteroid dehydrogenase 3, and aldo-keto reductase 1C3. Moreover, we provide a
thorough strategy on how to perform computational target predictions and guidance on using the
respective tools.
Sphingosine-1-phosphate (S1P) is a versatile signaling lipid involved in the regulation of numerous cellular processes. S1P regulates cellular proliferation, migration, and apoptosis as well as the function of immune cells. S1P is generated from sphingosine (Sph), which derives from the ceramide metabolism. In particular, high concentrations of S1P are present in the blood. This originates mainly from erythrocytes, endothelial cells (ECs), and platelets. While erythrocytes function as a storage pool for circulating S1P, platelets can rapidly generate S1P de novo, store it in large quantities, and release it when the platelet is activated. Platelets can thus provide S1P in a short time when needed or in the case of an injury with subsequent platelet activation and thereby regulate local cellular responses. In addition, platelet-dependently generated and released S1P may also influence long-term immune cell functions in various disease processes, such as inflammation-driven vascular diseases. In this review, the metabolism and release of platelet S1P are presented, and the autocrine versus paracrine functions of platelet-derived S1P and its relevance in various disease processes are discussed. New pharmacological approaches that target the auto- or paracrine effects of S1P may be therapeutically helpful in the future for pathological processes involving S1P.