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Resolving mediators are altered in multiple sclerosis
mary progressive MS). Recent studies suggest that chronic inflammation and autoimmunity could be a consequence of failure to resolve inflammation, and this resolution of inflammation is mediated by newly discovered metabo- lites termed specialized pro-resolving lipid mediators (SPM),7 temporally and spatially synthesized from ω-3 polyunsaturated fatty acids [eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA)].7,8 During the process of resolution of inflammation, the very same cells recruited to the inflammatory milieu and that produce inflammato- ry mediators (mainly innate immune cells) undergo a tem- poral lipid mediator (LM) class switch, whereby they stop producing classical eicosanoids (prostaglandins, leukotrienes, thromboxanes) from ω-6 arachidonic acid and start to biosynthesize SPM mainly from ω-3 EPA and DHA,7,8 through the stereoselective and concerted action of the same enzymes engaged in classical eicosanoids pro- duction, namely cyclo-oxygenase (COX) COX-2, lipoxy- genases (LOX) LOX-5, LOX-12 and LOX-15, as well as cytochrome P450 and several pathway specific epoxide hydrolases.7 These LM are potent and extinguish the eicosanoid-induced inflammation by activating local reso- lution programs,7-9 also by directly modulating oxidative stress10 and T-cell responses,11 via five separate G protein- coupled receptors (e.g. ALX/FPR2, GPR32/DRV1, ChemR23/ERV, BLT1 and GPR18/DRV2),9 without evok- ing unwanted side effects as opposed to the immunosup- pressive agents that are currently most used as disease- modifying treatments. Despite the increase in data sug- gesting that SPM metabolism and functions are differen- tially altered in several chronic peripheral and brain inflammatory diseases,7,10,12,13 research on these LM in MS and how they contribute to disease is of high interest. Given this, we aimed to determine whether MS patients at different phases of disease, compared to healthy sub- jects, displayed different levels and abilities to endoge- nously synthesize and respond to ω-6- and ω-3-derived pro-inflammatory (eicosanoids) and pro-resolving LM (lipoxins, resolvins, protectins and maresins) by means of targeted lipid metabololipidomics in human plasma and by evaluating the expression of their enzymes and key tar- get receptors in peripheral blood mononuclear cells (PBMC). Finally, we investigated whether specific SPM could modulate the inflammatory response of MS patient- derived monocytes and whether they could attenuate inflammation-induced BBB dysfunction as well as mono- cyte-transendothelial migration, which all represent key pathological hallmarks of MS pathogenesis.14
Methods
Multiple sclerosis patients
Peripheral blood was collected from two different cohorts. The first cohort was admitted to the neurological clinic of the University Hospital Tor Vergata, Rome, (14 females and 6 males, mean age 34.51±3.35 years) and the second cohort to the San Camillo Hospital of Rome (13 females and 5 males, mean age 38.24±2.76 years). Both cohorts were diagnosed as suffering from relapsing-remitting MS (RR-MS, n=26) or primary progressive (P-MS, n=12). Fifteen age-matched healthy subjects (HS, n=15) were used as controls. See Table 1 and Online Supplementary Methods for diagnostic details. All subjects gave their written informed consent to the study which was approved by the ethics committees of Tor Vergata Hospital and of San Camillo Hospital, Rome.
Liquid chromatography-tandem mass spectrometry-based metabololipidomics and analysis
Total lipids were extracted from plasma samples with solid phase C18 cartridges. Liquid chromatography-tandem mass spec- trometry (LC-MS-MS) was used to perform absolute quantifica- tions of all LM.15,16 Lipidomics data were analyzed by principal component analysis using SIMCA 13.0.3 software (MKS Data Analytics Solution Umea, Sweden) and by volcano plots using MetaboAnalyst (http://www.metaboanalyst.ca).
Human leukocyte and brain endothelial cell treatments
Freshly isolated PBMC from HS or MS patients were left untreated or pretreated with SPM and then stimulated with Imiquimod and ssRNA40 for five hours in presence of brefeldin A.17-19 Human brain endothelial cell line hCMEC/D3 cells were grown and treated with TNF-a in the presence or absence of SPM.20
Flow cytometry
Peripheral blood mononuclear cells were assayed for surface immunophenotype (CD14, CD16, CD69) and intracellular cytokine production (TNF-a, IL-1b, IL-6 and IL-12) by multiple flu- orochrome-conjugated antibody staining. hCMEC/D3 were assayed for anti-ICAM-1 (REK-1) and SPM receptors (GPR32, ALX/FPR2 and GPR18) through primary specific antibodies fol- lowed by fluorochrome-conjugated secondary antibodies.11,17
Real-time quantitative polymerase chain reaction
Total RNA was extracted from PBMC and hCMEC/D3, and retro-transcribed to cDNA. Specific probes for SPM receptors and SPM biosynthetic enzymes were used to assess relative mRNA
Table 1. Demographic data of patients with multiple sclerosis (MS) and control subjects.
N. of subjects
Mean age
Female/male
Disease duration* (years) MeanEDSS Corticosteroids# (yes/no) DMT# (yes/no)
N. of Gd+ T2 brain MRI lesions (%)
Healthy
n=14
0 0 0
Relapsing MS
n=14
0/0
0/0 10-20 (65%)
Remitting MS
n=12
0/0
0/0 10-20 (45%)
Progressive MS
n=12
0/0
0/0 >20 (50%)
36.12±1.77 9/6
-
36.82±2.91 10/4 3.6
37.67±2.23 9/3
4.2
38.82±2.54 8/4
6.1
- <3(1.5–3) <3(1–1.5) >3(4–6)
*Diseasedurationwasdefinedasthetimefromdiseaseonsettothetimeofsampling(inyears).#Attimeofsampling. EDSS:ExpandedDisabilityStatusScalescores;DMT:dis- ease modifying treatments; Gd: gadolinium; MRI: magnetic reasonance imaging.
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