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G. Kooij et al.
in the inflammatory process, and that appear also during chronic stages, namely RvD3 and RvD4, are undetected in all MS phases, further suggesting that, although progres- sive patients endeavor in a last attempt to boost a lipoxin- mediated metabolic switch towards SPM, this is not fol- lowed by actual SPM production.
Our findings are in line with the only other study that analyzed a few of such LM in MS, whereby RvD1 and PD1 were induced in highly active MS patients.28 However, this study, which was performed on cere- brospinal fluid samples, not only analyzed a smaller cohort of patients and did not take into consideration healthy subjects, but was able to detect only one-third of the LM that have been measured here. Furthermore, and most importantly, our metabololipidomics analysis was performed on three clinically distinct MS forms, which included not only MS with active relapse phases, but also patients with clear signs of remission or progression, allowing us to have a complete overview of a vast array of LM and observe how they vary along disease phases and during progression.
The recent evidence that several chronic inflammatory diseases are associated with altered SPM metabolism also supports our findings. Indeed, decreased production of lipoxins and resolvins (especially RvD1) have been linked to the pathogenesis of chronic obstructive pulmonary dis- ease,29 type-2 diabetes and obesity,30,31 inflammatory bowel disease,32,33 and rheumatoid arthritis.34 In addition, an imbalance between pro-inflammatory leukotrienes and pro-resolving SPM was observed in atherosclerosis.35 Of note, the notion that also neuroinflammatory and neu- rodegenerative diseases might be linked to a dysfunctional resolution of inflammation has very recently been put for- ward, and an impaired pro-resolution pathway, involving both specific SPM (i.e. LXA4 and RvD1) and their receptors was found in post-mortem brain tissues of patients with Alzheimer disease,36,37 where clinical trials with DHA show a reduced peripheral inflammation associated with increases in specific SPM.38 Accordingly, our observed sig- nificant and progressive reduction of DHA during MS, reaching very low levels in progressive patients, once again support a defect in producing its SPM derivatives. In line with this, Holmann et al. described deficiencies in polyunsaturated fatty acid (PUFA) and subsequent replace- ment by non-essential fatty acids in MS.39 Along these lines, untargeted metabolomics analysis of plasma sam- ples derived from mice with experimental autoimmune encephalomyelitis (EAE), the most commonly used ani- mal model for MS, revealed similar profound alterations in the omega-3 and omega-6 PUFA pathways, with several metabolites of PUFA being significantly lower in EAE mice including RvD1.40 Importantly, RvD1 supplementation ameliorated clinical signs of EAE, illustrating in vivo effica- cy of SP during neuro-inflammation.40 Epidemiological studies suggest that in particular omega-3 PUFA supple- mentation is linked with improved clinical outcomes in patients with MS.27,41,42 However, although the levels of AA and DHA could be restored by supplementation in MS patients, the efficacy of PUFA supplementation remains to be established.
Next, we further investigated the profile of peripheral blood leukocytes, and our analysis revealed distinctive expressions patterns of SPM biosynthetic enzymes and receptors in each clinical form of MS. Relapsing MS patients showed increased expression of COX-2 and 5-
LOX as well as of all five identified receptors (ALX/FPR2, GPR2/DRV1, GPR18/DRV2, ChemR23/ERV and BLT1). Interestingly, 12-LOX, which is responsible for maresins production, was consistently lower in all MS phases com- pared to healthy donors. Furthermore, the expression of SPM enzymes and receptors decreased along disease pro- gression, with the exception of 15-LOX (that remained constant in all MS phases) and of ALX/FPR2, GPR32/DRV1, and ChemR23/ERV that further increased only during remission, thus suggesting their possible involvement in promoting pro-resolution programs and neuroprotection, but subsequently dropped in progressive MS patients.
It is worth mentioning that the levels of SPM observed in the different disease phases might also be a conse- quence of a differential utilization and/or degradation, as well as a different expression, of their target receptors. Indeed, progressive patients bear the lowest amount of all SPM target receptors, yet they continue to express high levels of the proinflammatory BLT1 receptor, whose action is only blocked by E-series resolvins that are never to be found in all MS phases.
Although many types of leukocytes are involved in dis- ease progression, activated monocytes are believed to be one of the first to arrive to the brain and initiate inflamma- tion.43 In MS, the majority of monocytes display a classical inflammatory phenotype and are hyperactive.44 Here we found that monocytes isolated from RR-MS patients not only displayed a more activated and pro-inflammatory status (since their expression of CD69 and cytokines were, indeed, much higher than monocytes of healthy subjects), but also that specific SPM significantly inhibited such inflammatory responses in both healthy monocytes and those of RR-MS patients. However, the ability of SPM to modulate the inflammatory response of these peripheral cells was more evident in cells of healthy subjects, sug- gesting that, despite expressing comparable levels of pro- resolving receptors, MS patient-derived monocytes are less susceptible to SPM. These findings confirm and extend earlier reports in which such SPM were shown to reduce the inflammatory profile of human monocytes upon a pro-inflammatory stimulus.45-47 Of note, such SPM- induced effect is of crucial importance in preventing prim- ing and activation of autoreactive T cells (especially Th1 and Th17 cells) and natural killer cells, whose pathogenic- ity are strictly dependent on monocyte-derived cytokines.
Of note, the onset of MS starts when activated and autoreactive peripheral immune cells cross the BBB and start to damage myelin. In this process, BBB endothelial cells are key regulators of the neuroinflammatory response, inasmuch as when inflamed they lead to BBB disruption, upregulation of several adhesion molecules and production of chemokines, ultimately favoring leuko- cyte transmigration and subsequent MS lesion develop- ment.48 However, BBB endothelial cells also play an impor- tant role during the resolution phase of inflammation via the secretion of pro- and anti-inflammatory mediators that co-ordinate both leukocyte traffic and barrier func- tion.49 In this context, despite the fact that a great number of studies have shown the anti-inflammatory and pro- resolving effect of SPM on various cell types of the immune system, their potential impact on inflamed BBB has never been reported. Our results show for the first time, not only that BBB endothelial cells express several pro-resolving receptors, which are increased upon inflam-
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haematologica | 2020; 105(8)