Original Research Article
Oxylipin profiling in endothelial cells in vitro – Effects of DHA and hydrocortisone upon an inflammatory challenge

https://doi.org/10.1016/j.prostaglandins.2019.106352Get rights and content

Highlights

  • Oxylipin profiles were measured in an in vitro model of endothelial inflammation.

  • TNFα/IL-1β triggered the COX pathway and the release of pro-inflammatory oxylipins.

  • DHA inhibited inflammatory pathways while turning on the pro-resolution program.

  • Hydrocortisone blunted the release of both inflammatory and resolution oxylipins.

Abstract

Omega-3 poly-unsaturated fatty acids have been shown to have beneficial effects on several inflammatory-driven endpoints such as cardiovascular diseases. The anti-inflammatory effects of docosahexaenoic acid (DHA) are largely mediated through various oxylipins. Yet, mechanistic insights are limited. Here, we measured 53 oxylipins using LC-MS/MS in an in vitro model of endothelial cell inflammation, and compared the changes induced by DHA to hydrocortisone, a well-established anti-inflammatory drug. DHA modified several oxylipins derived from different precursors such as DHA, AA, LA and EPA. In response to a TNFα and IL-1-β challenge, DHA clearly reduced many COX-derived pro-inflammatory oxylipins, yet to a minor extent when compared to hydrocortisone. DHA also upregulated metabolites from the CYP and LOX pathways as opposed to hydrocortisone. Thus, DHA reduced pro-inflammation and enhanced pro-resolution, while hydrocortisone blunted both the pro- and anti-inflammatory pathways. Our results may fuel further research on the mitigation of corticosteroids adverse side-effects.

Introduction

Omega-3 poly-unsaturated fatty acids (ω-3-PUFAs) such as docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) are potent modulators of inflammation in vivo. Epidemiological and intervention studies have shown beneficial effects of ω-3-PUFAs consumption (either as individual fatty acids or in the form of ω-3-PUFAs-rich oils) on a number of inflammatory-driven endpoints such as cardiovascular health [1], rheumatoid arthritis [2] or cancer [3], [4]. The mode of action of dietary ω-3-PUFAs, although still not fully characterized, has been shown so far to rely on 2 main pillars: inhibition of established inflammatory pathways [5] [6], and activation of the inflammation resolution pathways [7]. These effects are for a large part mediated by the oxygenated downstream metabolites of ω−3-PUFAs, which belong to the so-called oxylipins family.

Oxylipins are a class of biologically active lipids derived from ω−3- and ω−6-PUFAs via the cyclooxygenase (COX), lipoxygenase (LOX) and cytochrome P450 (CYP) pathways. Their role as major soluble mediator of cell-cell communication is being increasingly recognized and a large number of these compounds have been well characterized. Eicosanoids, the ω-6 arachidonic acid (AA)-derived oxylipins such as prostaglandins (PG), leukotrienes (LT) and thromboxanes (TBX) are long known to mediate the cardinal features of tissue inflammation during the acute early phase and contribute to orchestrate the subsequent immune responses [8].

More recently, the range of activities mediated by lipid oxidation products has broadened with the discovery that another class of oxylipins, coined specialized pro-resolving mediators (SPM) holds a key role in the active return to homeostasis during the later stage of the inflammatory reaction, the so-called resolution phase. SPMs are downstream metabolites of DHA and EPA, the best characterized SPM families being the resolvins and maresins, as well as the lipoxins which are AA-derived [9]. Their effects have been reported in numerous in vitro and in vivo models where they have been shown to down-regulate intracellular inflammatory responses, blunt the recruitment of neutrophils to the site of inflammation while attracting monocytes that will promote the return to normal tissue structure and function via non-phlogistic phagocytosis of apoptotic activated neutrophils [10].

Next to these well-characterized lipids, the family of oxylipins also comprises a number of intermediate bioactive molecules that are synthetized along the PUFAs metabolic pathways. Many of these molecules are still poorly characterized and lack a clear biological function. Over the last decade, research on oxylipins has been accelerated by the advancements of analytical detection methods [11,12]. Lipidomic platforms using state-of-the-art LC-MS/MS and LC-HR-MS systems are now available to simultaneously measure numerous oxylipin species in small volumes of biological samples. These platforms have shown their great potential of monitoring oxylipin profiles as biomarkers for a wide range of conditions such as malaria [13], influenza [14], dyslipidemia [15], post-surgical inflammation [16,17] and have been used to link pharmaceutical or dietary interventions to changes in bioactive lipids at the whole body level [18,19]. Since the in vivo effects of ω-3-PUFAs are suspected to be mediated for a large part by their oxylipin metabolites, several groups have investigated the plasma oxylipin changes in response to dietary supplementation with ω-3 rich oils or individual ω-3-FA [[20], [21], [22], [23]]. These effects could be linked to various clinical functional outcomes such as improved vascular function [24] or reduced pro-inflammatory mediator production from ex vivo-stimulated blood [25].

Although these studies provide key insights in the biology of oxylipins at the whole body level they do not provide sufficient mechanistic understanding at the cellular level, which, however, is crucial to unravel the cross-regulation between the different cellular actors and enzymatic pathways involved in oxylipin synthesis. Mechanistic insights can be obtained by applying oxylipin profiling to in vitro systems and measuring full oxylipin profiles in supernatants of cells exposed to various inflammatory stimuli. Although reported before, articles mentioning this approach are scarce and limited in their scope regarding cell type and lipid metabolic pathway [[26], [27], [28], [29]].

Here, we studied the modulation of oxylipin production in response to DHA in a single-cell culture system relevant to vascular function. We used an in vitro model of endothelial cell inflammation and measured 53 oxylipins in the supernatant in response to TNFα and IL-1β, a challenge characteristic of sterile inflammation [30]. To assess anti-inflammatory effects upon this challenge, we used hydrocortisone, a potent anti-inflammatory drug as positive control. We compared this anti-inflammatory response with the effects of increasing doses of DHA. We report clear differences in the oxylipin profiles that suggest a fundamental difference between the pro-resolution and anti-inflammation mechanisms of action from the perspective of lipid metabolism.

Section snippets

Reagents

Recombinant human cytokines, cell culture-grade hydrocortisone solution (50 μM) and DHA (purity ≥ 98%) were purchased from Sigma-Aldrich (Zwijndrecht, The Netherlands) and stored at −20 °C as 100 mM stock solution in ethanol. For all experiments, the DHA stock solution was first mixed with a carrier protein (fatty acid free human serum albumin (Sigma) solubilised in PBS) at a 3:1 M ratio. Dulbecco’s modified Eagle medium (DMEM), heat-inactivated foetal bovine serum (HI-FBS) and antibiotics for

Results

All results, presented as relative response ratio with standard deviation and statistical significance level are compiled in the Table 1. The Fig. 1 was derived from these absolute concentrations, showing the general effects of the various pre-treatments on the oxylipin output (lipid precursor and metabolic pathways utilisation as well as total oxylipin output). In addition, the fold changes calculated from these data were used to produce the heatmaps in Fig. 2. In brief, the general effects

Discussion

We report here for the first time the profiles of oxylipins secreted from an endothelial cell line in an in vitro model of sterile inflammation. The EA.hy926 cell line produced 53 oxylipins that could be detected with our lipidomics platform [16]. In response to a TNFα/IL-1β challenge, the COX pathway was triggered to produce AA-derived oxylipins, in particular a set of known pro-inflammatory prostaglandins and thromboxane, a key feature of TNFα/IL-1β signalling. All these challenge-induced

Funding

This research was financed by The Netherlands Metabolomics Centre which is part of The Netherlands Genomics Initiative/Netherlands Organization for Scientific Research.

Contributors

AM, KS and PO performed the experiments, DJ performed the statistical analysis, AM wrote the manuscript, RV and DJ coordinated the study and all authors reviewed the manuscript before submission. All authors have approved the final manuscript.

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  • Cited by (1)

    1

    Current address: Janssen R&D, Beerse, Belgium.

    2

    Current address: IMcoMET B.V., Rotterdam, The Netherlands.

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