Oxylipin Profiles as Functional Characteristics of Acute Inflammatory Responses in Astrocytes Pre-Treated with IL-4, IL-10, or LPS.

Functional phenotypes, which cells can acquire depending on the microenvironment, are currently the focus of investigations into new anti-inflammatory therapeutic approaches. Glial cells, microglia, and astrocytes are major participants in neuroinflammation, but their roles differ, as microglia are cells of mesodermal origin, while astrocytes are cells of ectodermal origin. The inflammatory phenotype of cells can be modulated by ω-6- and ω-3-polyunsaturated fatty acid-derived oxylipins, although data on changes in oxylipin profiles in different cell adaptations to pro- and anti-inflammatory stimuli are scarce. Our study aimed to compare UPLC-MS/MS-measured oxylipin profiles in various rat astrocyte adaptation states. We used cells treated for 24 h with lipopolysaccharide (LPS) for classical pro-inflammatory adaptation and with interleukin 4 (IL-4) or 10 (IL-10) for alternative anti-inflammatory adaptation, with the resulting phenotypes characterized by quantitative real-time PCR (RT-PCR). We also tested long-term, low-concentration LPS treatment (endotoxin treatment) as a model of astrocyte adaptations. The functional response of astrocytes was estimated by acute (4 h) LPS-induced cell reactivity, measured by gene expression markers and oxylipin synthesis. We discovered that, as well as gene markers, oxylipin profiles can serve as markers of pro- (A1-like) or anti-inflammatory (A2-like) adaptations. We observed predominant involvement of ω-6 polyunsaturated fatty acid (PUFA) and the cyclooxygenase branch for classical (LPS) pro-inflammatory adaptations and ω-3 PUFA and the lipoxygenase branch for alternative (IL-4) anti-inflammatory adaptations. Treatment with IL-4, but not IL-10, primes the ability of astrocytes to activate the innate immunity signaling pathways in response to LPS. Endotoxin-treated astrocytes provide an alternative anti-inflammatory adaptation, which makes cells less sensitive to acute LPS stimulation than the IL-4 induced adaptation. Taken together, the data reveal that oxylipin profiles associate with different states of polarization to generate a pro-inflammatory or anti-inflammatory phenotype. This association manifests itself both in native cells and in their responses to a pro-inflammatory stimulus.

Comparative lipidomic analysis of inflammatory mediators in the aqueous humor and tear fluid of humans and rabbits.

INTRODUCTION: Ocular inflammation is a key pathogenic factor in most blindness-causing visual disorders. It can manifest in the aqueous humor (AH) and tear fluid (TF) as alterations in polyunsaturated fatty acids (PUFAs) and their metabolites, oxylipins, lipid mediators, which are biosynthesized via enzymatic pathways involving lipoxygenase, cyclooxygenase or cytochrome P450 monooxygenase and specifically regulate inflammation and resolution pathways. OBJECTIVES: This study aimed to establish the baseline patterns of PUFAs and oxylipins in AH and TF by their comprehensive lipidomic identification and profiling in humans in the absence of ocular inflammation and comparatively analyze these compounds in the eye liquids of rabbits, the species often employed in investigative ophthalmology. METHODS: Ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was used for qualitative and quantitative characterization of lipid compounds in the analyzed samples. RESULTS: A total of 28 lipid compounds were identified, including phospholipid derivatives and PUFAs, as well as 22 oxylipins. Whereas the PUFAs included arachidonic, docosahexaenoic and eicosapentaenoic acids, the oxylipins were derived mainly from arachidonic, linoleic and α-linolenic acids. Remarkably, although the concentration of oxylipins in AH was lower compared to TF, these liquids showed pronounced similarity in their lipid profiles, which additionally exhibited noticeable interspecies concordance. CONCLUSION: The revealed correlations confirm the feasibility of rabbit models for investigating pathogenesis and trialing therapies of human eye disorders. The identified metabolite patterns suggest enzymatic mechanisms of oxylipin generation in AH and TF and might be used as a reference in ocular inflammation studies.

Mechanisms and Treatment of Light-Induced Retinal Degeneration-Associated Inflammation: Insights from Biochemical Profiling of the Aqueous Humor.

Ocular inflammation contributes to the pathogenesis of blind-causing retinal degenerative diseases, such as age-related macular degeneration (AMD) or photic maculopathy. Here, we report on inflammatory mechanisms that are associated with retinal degeneration induced by bright visible light, which were revealed while using a rabbit model. Histologically and electrophysiologically noticeable degeneration of the retina is preceded and accompanied by oxidative stress and inflammation, as evidenced by granulocyte infiltration and edema in this tissue, as well as the upregulation of total protein, pro-inflammatory cytokines, and oxidative stress markers in aqueous humor (AH). Consistently, quantitative lipidomic studies of AH elucidated increase in the concentration of arachidonic (AA) and docosahexaenoic (DHA) acids and lyso-platelet activating factor (lyso-PAF), together with pronounced oxidative and inflammatory alterations in content of lipid mediators oxylipins. These alterations include long-term elevation of prostaglandins, which are synthesized from AA via cyclooxygenase-dependent pathways, as well as a short burst of linoleic acid derivatives that can be produced by both enzymatic and non-enzymatic free radical-dependent mechanisms. The upregulation of all oxylipins is inhibited by the premedication of the eyes while using mitochondria-targeted antioxidant SkQ1, whereas the accumulation of prostaglandins and lyso-PAF can be specifically suppressed by topical treatment with cyclooxygenase inhibitor Nepafenac. Interestingly, the most prominent antioxidant and anti-inflammatory benefits and overall retinal protective effects are achieved by simultaneous administrating of both drugs indicating their synergistic action. Taken together, these findings provide a rationale for using a combination of mitochondria-targeted antioxidant and cyclooxygenase inhibitor for the treatment of inflammatory components of retinal degenerative diseases.

Cellular Model of Endotoxin Tolerance in Astrocytes: Role of Interleukin 10 and Oxylipins.

A phenomenon of endotoxin tolerance where prior exposure of cells to minute amounts of lipopolysaccharide (LPS) causes them to become refractory to a subsequent high-amount endotoxin challenge is well described for innate immune cells such as monocytes/macrophages, but it is still obscure for brain cells. We exposed primary rat cortical astrocytes to a long-term low-grade concentration of LPS, followed by stimulation with a middle-grade concentration of LPS. Inflammatory markers, i.e., pro-inflammatory cytokine TNFα, inducible enzymes COX-2 and iNOS, anti-inflammatory cytokine interleukin 10 (IL-10) detected at the mRNA and protein levels reveal similarities between astrocytes and macrophages in the model, i.e., tolerance in pro-inflammatory markers and priming in IL-10. Long-term or short-term treatment with IL-10 does not change cell sensitivity for LPS, which makes doubtful its involvement in the mechanisms of cell tolerance development. Significant changes occur in the oxylipin profiles measured by UPLC-MS/MS analysis. The priming occurs in the following compounds: 11-HETE, PGD2, PGE2, cyclopentenone prostaglandins, and TXB2. Tolerance is observed for 12-HHT, PGF2α, and 6-keto-PGF1α. As far as we know, this is the first report on changes in oxylipin profiles in the endotoxin tolerance model. The data can greatly improve the understanding of oxylipins' role in inflammatory and resolution processes in the brain and mechanisms of astrocyte involvement in neuroinflammation.

High and Low Molecular Weight Hyaluronic Acid Differentially Influences Oxylipins Synthesis in Course of Neuroinflammation.

Hyaluronic acid (HA), a major glycosaminoglycan of the extracellular matrix, has cell signaling functions that are dependent on its molecular weight. Anti-inflammatory effects for high-molecular-weight (HMW) HA and pro-inflammatory effects for low-molecular-weight (LMW) HA effects were found for various myeloid cells, including microglia. Astrocytes are cells of ectodermal origin that play a pivotal role in brain inflammation, but the link between HA with different molecular weights and an inflammatory response in these cells is not clear. We tested the effects of LMW and HMW HA in rat primary astrocytes, stimulated with Poly:IC (PIC, TLR3 agonist) and lipopolysaccharide (LPS, TLR4 agonist). Oxylipin profiles were measured by the UPLC-MS/MS analysis and metabolites HDoHEs (from docosahexaenoic acid), -HETEs, prostaglandins (from arachidonic acid), DiHOMEs and HODEs (from linoleic acid) were detected. Both, HMW and LMW HA downregulated the cyclooxygenase-mediated polyunsaturated fatty acids metabolism, LMW also reduced lipoxygenase-mediated fatty acid metabolism. Taken together, the data show that both LMW and HMW (i) influence themselves on cytokines (TNFα, IL-6, IL-10), enzymes iNOS, COX-2, and oxylipin levels in extracellular medium of cultured astrocytes, (ii) induced cellular adaptations in long-term applications, (iii) modulate TLR4- and TLR3-signaling pathways. The effects of HMW and LMW HA are predominantly revealed in TLR4- and TLR3- mediated responses, respectively.

Deuterated Arachidonic Acids Library for Regulation of Inflammation and Controlled Synthesis of Eicosanoids: An In Vitro Study.

The synthesis of signal lipids, including eicosanoids, is not fully understood, although it is key to the modulation of various inflammatory states. Recently, isotopologues of essential polyunsaturated fatty acids (PUFAs) deuterated at bis-allylic positions (D-PUFAs) have been proposed as inhibitors of non-enzymatic lipid peroxidation (LPO) in various disease models. Arachidonic acid (AA, 20:4 n-6) is the main precursor to several classes of eicosanoids, which are produced by cyclooxygenases (COX) and lipoxygenases (LOX). In this study we analyzed the relative activity of human recombinant enzymes COX-2, 5-LOX, and 15-LOX-2 using a library of arachidonic acids variably deuterated at the bis-allylic (C7, C10, and C13) positions. Kinetic parameters (KM, Vmax) and isotope effects calculated from kH/kD for seven deuterated arachidonic acid derivatives were obtained. Spectroscopic methods have shown that deuteration at the 13th position dramatically affects the kinetic parameters of COX-2 and 15-LOX-2. The activity of 5-LOX was evaluated by measuring hydroxyeicosatetraenoic acids (8-HETE and 5-HETE) using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Deuteration at the seventh and 10th positions affects the performance of the 5-LOX enzyme. A flowchart is proposed suggesting how to modulate the synthesis of selected eicosanoids using the library of deuterated isotopologues to potentially fine-tune various inflammation stages.

Sex-Mediated Differences in LPS Induced Alterations of TNFα, IL-10 Expression, and Prostaglandin Synthesis in Primary Astrocytes.

Although many neurological and psychiatric disorders reveal clear sex-dependent variations, the molecular mechanism of this process is not clear enough. Astrocytes are involved in the response of neural tissue to injury and inflammation, produce steroid hormones, and sense steroid presence. To explore the hypothesis that astrocytes may participate in sex-mediated differences of inflammatory responses, we have examined whether male and female primary rat astrocytes show different responses to lipopolysaccharide (LPS) as a toll-like receptor 4 (TLR4) agonist. Levels of mRNA and proteins of tumor necrosis factor alpha (TNFα), interleukin-10 (IL-10), and cyclooxygenase (COX)-2 were assessed using qPCR, immunoblotting, and ELISA. UPLC-MS/MS was used to detect prostaglandins (PGs). LPS stimulation resulted in different levels of cytokine production; more TNFα and less IL-10 were produced in female cells compared with male astrocytes. Although the levels of the COX-2 expression were not altered, LPS significantly induced the synthesis of PGs with notable sex-related differences. PGE2 and PGD2 were less and 6-keto-PGF1α was more upregulated in female astrocytes, and TXB2 had similar levels in cells obtained from males and females. Trilostane, an inhibitor of 3ß-Hydroxysteroid dehydrogenase (3ß-HSD), inhibited the LPS-induced TNFα production and the release of PGE2, PGD2, and 6-keto-PGF1α in female astrocytes. Thus, male and female astrocytes differentially respond to inflammatory challenges on the level of production of cytokines and steroid hormones. Sex-mediated differences in pro- and anti-inflammatory responses should be taken into consideration for the effective treatment of disorders with neuroinflammation.

Astrocytes synthesize primary and cyclopentenone prostaglandins that are negative regulators of their proliferation.

Recently, the modulation of cellular inflammatory responses via endogenous regulators became a major focus of medically relevant investigations. Prostaglandins (PGs) are attractive regulatory molecules, but their synthesis and mechanisms of action in brain cells are still unclear. Astrocytes are involved in manifestation of neuropathology and their proliferation is an important part of astrogliosis, a cellular neuroinflammatory response. The aims of our study were to measure synthesis of PGs by astrocytes, and evaluate their influence on proliferation in combination with addition of inflammatory pathway inhibitors. With UPLC-MS/MS analysis we detected primary PGs (1410 ±â€¯36 pg/mg PGE2, 344 ±â€¯24 PGD2) and cyclopentenone PGs (cyPGs) (87 ±â€¯17 15d-PGJ2, 308 ±â€¯23 PGA2) in the extracellular medium after 24-h lipopolysaccharide (LPS) stimulation of astrocytes. PGs reduced astrocytic proliferation with the following order of potencies (measured as inhibition at 20 µM): most potent 15d-PGJ2 (90%) and PGA2 (80%), > PGD2 (40%) > 15d-PGA2 (20%) > PGE2 (5%), the least potent. However, PGF2α and 2-cyclopenten-1-one, and ciglitazone and rosiglitazone (synthetic agonists of PPARγ) had no effect. Combinations of cyPGs with SC-560 or NS-398 (specific anti-inflammatory inhibitors of cyclooxygenase-1 and -2, respectively) were not effective; while GW9662 (PPARγ antagonist) or MK-741 (inhibitor of multidrug resistance protein-1, MRP1, and CysLT1 receptors) amplified the inhibitory effect of PGA2 and 15d-PGJ2. Although concentrations of individual PGs and cyPGs are low, all of them, as well as primary PGs suppress proliferation. Thus, the effects are potentially additive, and activated PGs synthesis suppresses proliferation in astrocytes.