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Resolving mediators are altered in multiple sclerosis
CNS by crossing the inflamed and disrupted BBB. Therefore, we addressed the question as to whether the differentially expressed SPM were able to counteract inflammation-induced BBB dysfunction, by using human brain endothelial cells (BEC) as a BBB model. We first assessed if these cells responded to SPM by looking at the expression pattern of specific SPM receptors. We found that BEC mainly express ALX/FPR2 and to a lesser extent GPR18/DVR2 and GPR32/DVR1 (Figure 7A and B). Interestingly, such marked ALX/FPR2 expression was even more evident when BEC were stimulated with TNF-a, inasmuch as inflamed cells underwent a signifi- cant upregulation of ALX/FPR2 mRNA, while they only showed a slight increase in GPR18/DVR2 and GPR32/DVR1 expression (Figure 7C). Next, we assessed whether LXA4, LXB4, RvD1 and PD1 were capable of counteracting inflammation-induced BBB dysfunction by measuring TEER in real-time. We found that these SPM were able to rescue the TNF-a-mediated decrease in TEER in a time-dependent manner, at both a 10 nM (Figure 7D and E) and 100 nM concentration (Online Supplementary Figure S7), with LXB4 starting to have a sig- nificant effect as early as at 24 hours (h) (at 10 nM but not at 100 nM) and with all SPM significantly rescuing TEER after 72 h (at both concentrations). Of note, no dose- dependency in SPM potency in rescuing TEER was observed, except for 100 nM LXA4 that showed a signifi- cant impact at 48 h. In view of these results, we next sought to investigate the SPM effect on monocyte transendothelial migration by using this human BBB model. Treatment with LXA4, LXB4, RvD1 or PD1 signifi- cantly inhibited the migration of monocytes across BEC (Figure 7F) and this action was associated with a signifi- cant reduction in the expression of endothelial adhesion molecule ICAM-1 and chemokine CCL2 (Figure 7G and H), thereby accounting for a potent anti-inflammatory action of the pro-resolving LM in preventing inflamma- tion-induced BBB dysfunction.
Discussion
This study provides an unprecedented comprehensive overview of the LM signature in plasma from MS patients with different clinical forms of the disease compared to healthy controls. Targeted LM metabololipidomics using LC-MS-MS with subsequent analyses revealed that relaps- ing MS patients display most of the AA-derived prostaglandins (i.e. PGD2 and PGE2) as well as of DHA- derived SPM like PD1 and RvD1 (and partly PDX), which were all reduced in remitting MS patients. In addition, progressive MS patients not only were characterized by the co-presence of all pro-inflammatory mediators (all of them even in higher levels than relapsing patients) but also on the appearance of some AA-derived or DHA-derived SPM, such as LXA4 and LXB4, RvD5 and PDX. Of note, the levels of PD1 and RvD1 were significantly reduced or even undetected along disease progression.
In line with the general concepts that bioactive LM undergo temporal and spatial production during inflam- mation, that SPM appear at the peak of acute inflamma- tion in order to later reduce inflammation by activating endogenous resolution programs,7,10 and that chronic inflammation may result from failed resolution mecha- nisms,12,13 our results display that during the acute phase of
the disease (relapsing form) there is an imbalance between pro-inflammatory and pro-resolving LM, in favor of the former, and an insufficient or lack of expression of many key SPM (including E-resolvins, maresins and the rest of D-resolvins), which may in turn affect the outcome of remission and thereby, in theory, even lead to disease pro- gression, although further studies are needed to fully clar- ify this. Indeed, over 80% of individuals with MS initially develop a clinical pattern with periodic relapses that reflect acute inflammation in the CNS and myelin disrup- tion as well as induced activation of innate immune cells and pro-inflammatory mediators in peripheral blood, fol- lowed by continuous remissions during which self- remyelination occurs and symptoms decrease or tem- porarily disappear.6,25 Repeated relapses and remissions lead to less and less effective remyelination, appearance of scar-like plaques (scleroses) and thus after 10-20 years, patients might evolve into a progressive form of the dis- ease, characterized by an irreversible disruption of periph- eral and central immune tolerance, neurodegeneration, and permanent cortical and subcortical gray matter atro- phy.26 More than a dozen disease-modifying (and mostly anti-inflammatory) agents are available to reduce the fre- quency of transient episodes of neurological disability and limit the accumulation of CNS lesions, but these systemi- cally applied agents not only are exclusive for RR-MS patients and not for progressive patients, but also result in severe side-effects, and none of these prevents or reverses the neurological deterioration.27 Therefore, we set out to investigate whether impairments of endogenous process- es to resolve inflammation correlate with MS progression to ultimately provide tools to either slow down inflamma- tory activation and simultaneously promote neuroprotec- tion or prevent disease progression. The primary objective of our study was to identify pro-inflammatory and pro- resolving LM and validate their structures in peripheral blood of MS patients and healthy controls, and secondly, to correlate levels with clinical outcomes. To this aim, tar- geted metabololipidomics allowed us to reveal the full spectrum of LM in plasma samples of healthy donors and of MS patients with different clinical disease forms. We found that acute MS patients are able to produce only very few SPM (i.e. lipoxins, RvD1 and PD1). This is sug- gestive of a defective resolution program during MS that could not only result in a partial recovery, but could also eventually increase the probability of evolving into the progressive form, as substantiated by an inverse correla- tion of such SPM with clinical severity. Our observed pres- ence of high levels of few SPM (LXA4, LXB4, RvD5 and PDX) in progressive MS, which instead positively corre- late with clinical severity, is indicative of a last, but inef- fective, attempt of the body to respond to an even higher inflammatory status, where all pro-inflammatory LM are consistently produced in high amounts and are also asso- ciated to disease severity. This is particularly relevant for LXA4 and LXB4 that are metabolically derived from arachi- donic acid, and that are the actual initiators of the meta- bolic switch from the omega-6 pro-inflammatory eicosanoids to the omega-3 SPM. Indeed, MS patients attempt to induce a compensatory boost of these two lipoxins in order to promote the subsequent production of all SPM, which is reflected only in an increased production of RvD5 and PDX (whose potency is much lower than its stereoisomer PD1) and not by an induction of all other SPM. Of note, typical SPM that are usually produced later
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