Understanding the Interplay Between Inflammatory Lipid Signaling and Demyelination Process: A Lipidomics Approach Applied to 3D Human Brain Model Maria Laura Santoru (Presenter) University of Cagliari / University of Lausanne

Authors: Maria Laura Santoru (1,3), Héctor Gallart-Ayala (1), David Pamies (2,4), Tony Teav (1), Luigi Atzori (3), Marie Gabrielle Zurich (2,4) and Julijana Ivanisevic (1)
(1) Metabolomics Platform, Faculty of Biology and Medicine, University of Lausanne, Lausanne, Switzerland (2) Department of Physiology, Faculty of Biology and Medicine, University of Lausanne, Lausanne, Switzerland (3) Department of Biomedical Sciences, University of Cagliari, Cagliari, Italy (4)Swiss Center for Applied Human Toxicology (SCAHT), Switzerland

Demyelination and inflammation are two main features of multiple sclerosis. It has been proved that lipids play an important role in these two processes and to elucidate their mechanism of action in MS, a lipidomics approach was applied to iPSC-derived human 3D brain spheres system challenged by a treatment with cytokines to induce the demyelination. Combining targeted and untargeted lipidomics, we detected different classes of lipids in both spheres and their corresponding media. Among the measured lipid species, PAF and PAF related species were found to be significantly upregulated in the cytokine-treated spheres while the PUFA species and derived eicosanoids were increased in the spent media, suggesting a link between the early stage of inflammation process in MS and activated lipid signaling.

Introduction

Demyelination or myelin sheath destruction of axons and subsequent neuronal dysfunction are the main symptoms of multiple sclerosis (MS) – a chronic neurodegenerative and autoimmune disease 1-4. Initially, inflammation is transient and partial re-myelination occurs, however, this pathology progressively develops into chronic neurodegeneration associated with microglial activation and demyelination. Despite decades of intense research, the etiology of MS and molecular mechanisms implicated in myelin damage remain uncertain.

There is evidence that T cells from MS patients are clonally expanded effector memory T cells 5,6 with an increased production of pro-inflammatory cytokines (IL-17, IFN-γ, and TNF-α) 7,8. This over production of cytokines triggers the release of membrane phospholipids and sphingolipids-derived mediators, such as PAF (platelet-activating factor) and arachidonic acid that activate the immune cells and the biosynthesis of eicosanoids. These bioactive lipids act as potent enhancers of innate and adaptive immune response and are implicated in numerous inflammatory disorders 9.

Recent advances in mass spectrometry technology and bioinformatics allow broad-scale lipidomic analysis, thus offering the possibility to elucidate the biochemical mechanism(s) underlying the alterations in lipid metabolism associated with specific pathophysiological processes.

Methods

To enhance the mechanistic insights into how lipids mediate the demyelination process in MS a comprehensive and quantitative lipidomics approach was applied to a highly reproducible iPSC-derived human 3D brain spheres system16 challenged by a treatment with three different cytokines (TNF-α, IFN-γ and IL-17) to induce the demyelination process causative of MS disease progression. Control and treated brain spheres and media were collected and extracted using 80% methanol, followed by the isotope dilution and solid phase extraction (SPE) to concentrate the sample in the metabolites of interest. To prevent non-enzymatic oxidation, anti-oxidative agents were added during the extraction that was performed using argon purged solvents. Extracted spheres lysates and media were analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS) approach on a triple quadrupole instrument (QqQ) to measure 115 species of eicosanoids and 40 different species of sphingolipids. In addition, an untargeted LC-HRMS lipidomics approach using a quadrupole time-of-flight instrument (Q-TOF) was applied to expand the coverage to other lipid species (e.g. PC, PE, PAF, carnitines, etc.) also implicated in the signalling cascade of the inflammatory response. In parallel to lipidomic analysis, the extent of demyelination and inflammation was evaluated using qPCR analysis and immunostaining to determine the expression levels of genes and proteins involved in these processes.

Results

Combining both approaches, targeted and untargeted, we were able to assess several different classes of lipids, including eicosanoids, sphingolipids and phospholipids. With the targeted approach we were able to detect common PUFA precursors of eicosanoids (arachidonic acid, eicosapentaenoic acid, linoleic acid and docohexanoic acid) and 12 different eicosanoids derived from arachidonic and linoleic acid, in both spheres and their corresponding media. Moreover, with the untargeted lipidomic approach, we detected different classes of phospholipids (i.e. phosphatidylcholines, phosphatidylethanolamines), platelet activating factor (PAF) and PAF related compounds, and several carnitines. Interestingly, among the measured lipid species, PAF and PAF related species were found to be significantly upregulated in the cytokine-treated spheres. Furthermore, the PUFA were increased in the spent media, together with several eicosanoids derived from arachidonic and linoleic acid (8,9-DiHETrE, 9-KOTrE, EKODE, 5-HpETE). These results imply the production of arachidonic acid, PAF and specific eicosanoids as key lipid mediators in the early stage of inflammatory response in MS. Further analyses, in varying treatment conditions and over time, are currently in progress.

Conclusions & Discussion

These preliminary results suggest a link between the inflammation process in MS and activated lipid signaling, involving mainly the arachidonic acid derived signaling cascade. Our future studies will elucidate in more details the meaning of these changes and how they can modulate and trigger the inflammation and demyelination response in MS.

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