Specialized pro-resolving lipid mediators are differentially altered in peripheral blood of patients with multiple sclerosis and attenuate monocyte and blood-brain barrier dysfunction

Abstract

Chronic inflammation is a key pathological hallmark of multiple sclerosis (MS) and suggests that resolution of inflammation, orchestrated by specialized pro-resolving lipid mediators (LM), is impaired. Here, through targeted-metabololipidomics in peripheral blood of patients with MS, we revealed that each disease form was associated with distinct LM profiles that significantly correlated with disease severity. In particular, relapsing and progressive MS patients were associated with high eicosanoids levels, whereas the majority of pro-resolving LM were significantly reduced or below limits of detection and correlated with disease progression. Furthermore, we found impaired expression of several pro-resolving LM biosynthetic enzymes and receptors in blood-derived leukocytes of MS patients. Mechanistically, differentially expressed mediators like LXA₄, LXB₄, RvD1 and PD1 reduced MS-derived monocyte activation and cytokine production, and inhibited inflammation-induced blood-brain barrier dysfunction and monocyte transendothelial migration. Altogether, these findings reveal peripheral defects in the resolution pathway in MS, suggesting pro-resolving LM as novel diagnostic biomarkers and potentially safe therapeutics.

Figure 1.

Identification of lipid mediators. Lipid mediators were isolated from plasma of healthy subjects (n=15) and multiple sclerosis (MS) patients (n=38) and analyzed by liquid chromatography tandem mass spectrometry. Representative MRM illustration of arachidonic acid (AA)-derived prostaglandins, leukotrienes and lipoxins, docosahexaenoic acid (DHA)- derived resolvins and protectins, and eicosapentaenoic acid (EPA)-derived resolvins, and representative tandem mass spectrometry of AA-derived PGE₂ and LXA₄, DHA-derived RvD1, RvD5 and PD1.

Figure 2.

Multiple sclerosis (MS) patients show altered lipid mediators profiles in blood. Lipid mediators (LM) were isolated from plasma of healthy subjects (HS, n=15) and MS patients (n=38) and analyzed by liquid chromatography tandem mass spectrometry. (A) Schematic representation of the arachidonic acid (AA)-derived respective LM biosynthetic pathways and their selected values between HS and MS. (B) Schematic representation of the docosahexaenoic acid (DHA)-derived respective LM biosynthetic pathways and their selected values between HS and MS. (C) Schematic representation of the eicosapentaenoic acid (EPA)-derived respective LM biosynthetic pathways and their selected values between HS and MS. Data are presented as means pg/mL±standard error of mean. *P<0.05 or **P<0.01 compared to HS, determined by Student t-test.

Figure 3.

Correlations between Expanded Disability Status Scale (EDSS) scores of patients and lipid mediators. Correlation plots between EDSS values and levels (pg/mL) of specific lipid mediators of the AA metabolome (A), DHA metabolome (B) and EPA metabolome (C) in the entire cohort of patients with multiple sclerosis. Data were compared by Spearman’s rank correlation coefficient (P<0.05).

Figure 4.

Lipid mediators (LM) are differentially altered in multiple sclerosis (MS) patients according to clinical disease phase. LM were isolated from plasma of healthy subjects (n=15), relapsing MS (n=14), remitting MS (n=12), and progressive MS (n=12) patients and analyzed by liquid chromatography tandem mass spectrometry. (A) Principal component analysis (PCA) of the LM profile. (Top) 3D score plot. (Bottom) 3D loading plot. (B) Heat map of LM fingerprint. Pro-inflammatory LM are shown in red, anti-inflammatory/proresolving LM are shown in green and pathway intermediates in black (C-E) Selected values of LM of AA metabolome (C), DHA metabolome (D), and EPA metabolome (E) from healthy subjects, relapsing, remitting and progressive MS patients. Data are presented as means pg/mL±standard error of mean. *P<0.05, **P<0.01, ***P<0.001 determined by one-way ANOVA followed by Bonferroni’s multiple comparison test.

Figure 5.

Specialized pro-resolving mediators biosynthetic enzymes and receptors are differentially expressed in multiple sclerosis (MS) patients according to the clinical disease phase. Peripheral blood mononuclear cells (PBMC, 2x10⁶ cells) from healthy subjects (n=5), relapsing MS (n=7), remitting MS (n=5), and progressive MS (n=5) patients were quantified for their mRNA content by quantitative real-time polymerase chain reaction (qRT-PCR) of lipid mediator biosynthesizing enzymes COX-2, 5-LOX, 12-LOX and 15-LOX (A) and of SPMs receptors ALX/FPR2, GPR32/DRV1, GPR18/DRV2, ChemR23/ERV and BLT1 (B). Data are means±standard error of mean of 5-7 independent experiments. *P<0.05, **P<0.01 determined by one-way ANOVA followed by Bonferroni’s multiple comparison test.

Figure 6.

Specialized pro-resolving mediators reduce monocyte activation and cytokine production in multiple sclerosis (MS) patients. Peripheral blood mononuclear cells (PBMC, 2x10⁶ cells) from relapsing MS patients (n=5) were left untreated or pre-treated with LXA, LXB₄, RvD1 or PD1 for 30 minutes. Cells were then stimulated with Imiquimod (Toll-like receptor 7 agonist) and ssRNA40 (Toll-like receptor 8 agonist) for five hours in absence or presence of Brefeldin A, stained at the cell surface and intracellularly, and analyzed by flow cytometry by gating on CD14⁺ monocytes. (A) Surface expression of CD69 positive monocytes. Data are shown as representative flow cytometry histograms and as means of fluorescence intensity (MFI)±standard error of mean of five independent experiments. **P<0.01 compared to control cells and ^P<0.05 compared to TLR7/TLR8 agonists, determined by one-way ANOVA followed by Bonferroni’s multiple comparison test. (B) Cytofluorimetric plots and percentages of intracellular pro-inflammatory cytokine production (IL-6, IL-12, IL1-b and TNF-a) from CD14⁺ monocytes. Data are presented as means± standard error of mean (SEM) of five independent experiments. **P<0.01 and ***P<0.001 compared to control cells, ^P<0.05 and #P<0.001 compared to TLR7/TLR8 agonists, determined by one-way ANOVA followed by Bonferroni’s multiple comparison test. (C) Cytofluorimetric plots and percentages of intracellular IL-10 production from CD14⁺ monocytes. Data are presented as means±SEM of four independent experiments. **P<0.01, ^P<0.05 and #P<0.001 compared to TLR7/TLR8 agonists, determined by one-way ANOVA followed by Bonferroni’s multiple comparison test.

Figure 7.

Specialized pro-resolving mediators (SPM) improve blood brain barrier (BBB) function and reduce monocyte transmigration and activation. (A) Representative scatter plots of forward scatter versus side scatter from human brain endothelial cells (BEC) and representative overlays histogram plot gated on live cells for GPR18/DRV2, GPR32/DRV1 and ALX/FPR2 surface expression. (B) Quantification of surface expression. Data are means±standard error of mean (SEM) of four independent experiments. (C) SPM receptors mRNA content in resting or TNF-a-activated BEC. Data are means±SEM of three independent experiments. Statistical analysis was carried out using Student t-test. ***P<0.001. (D and E) The functional effect of TNF-a (5 ng/mL) in the presence or absence of LXA₄, LXB₄, RvD1 or PD1 on BBB function was assessed by measuring the trans-endothelial electrical resistance (TEER) of BEC. Confluent BEC monolayer was treated as described and TEER was measured over time. Data are shown as representative TEER curves of three independent experiments. Graphs showing the TNF-a effect at selected time-points, plotted as % TNF-a effect of control BEC±SEM of three independent experiments. Statistical analysis was carried out using Student t-test. *P<0.05, **P<0.01, ***P<0.001. (F-H) Confluent BEC were stimulated for 24 hours with TNF-α in the presence or absence of LXA₄, LXB₄, RvD1 or PD1. Human monocytes (1x10⁵ cells/well) were left untreated or treated with LXA₄, LXB₄, RvD1 or PD1 prior plated on BEC. Cells were incubated for eight hours before harvesting the transmigrated cells. (F) Percentage of monocyte transmigration evaluated by flow cytometry. Data are shown as means±SEM of three independent experiments. ***P<0.001 compared to control cells and #P<0.001 compared to TNF-a stimulated cells, determined by one-way ANOVA followed by Bonferroni’s multiple comparison test. ICAM-1 expression by flow cytometry (G) and CCL2 secretion was measured by ELISA (H). Data are means±SEM of three independent experiments. ***P<0.001 compared to control cells, ^P<0.05 and #P<0.001 compared to TNF-a stimulated cells, determined by one-way ANOVA followed by Bonferroni’s multiple comparison test.