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G. Kooij et al.
lowed a similar trend, being generally increased in both relapsing MS and progressive MS patients compared to healthy subjects (with PGD2 and PGE2 being also signifi- cant for relapsing MS and LXA4 and LXB4 for progressive MS), while showing reduced levels in remitting MS patients compared to clinically active forms. For all of these LM, progressive patients consistently displayed higher levels compared to relapsing patients (Figure 4B and C). Of note, levels of their precursor AA moved in the opposite direction (Online Supplementary Figure S2A), sug- gesting an active metabolic conversion into such LM asso- ciated to disease phase. Other key metabolites of AA, including HETE, PGF2a and LTB4, appeared with specific trends of expression, with 5-HETE being mostly found in progressive MS and 12-HETE in remitting MS, while 15-HETE and PGF2a steadily increased along disease forms (Online Supplementary Figure S2A). Levels of aspirin-trig- gered (AT) lipoxins were detected only for AT-LXA4, which was particularly present in progressive patients (Table 2 and Figure 4B). As for the DHA metabolome, pro- resolving LM RvD5 and PDX were slightly increased in relapsing MS and significantly increased in progressive MS, with remitting MS showing similar levels compared to healthy subjects (Figure 4C). Furthermore, RvD1 and PD1 were increased in relapsing MS, with the latter being also significant and showing a reduction in both remitting and progressive patients, while the former being reduced in remitting MS and undetectable in progressive MS (Figure 4C). Of interest, the levels of their precursor DHA, although showing an initial, yet not significant, increase in relapsing MS, were progressively reduced along disease forms (Online Supplementary Figure S2B). Other pathway makers and metabolites of DHA were significantly increased, especially in remitting (14-HDHA and 17- HDHA) or progressive (17-HDHA and 4-HDHA) patients (Online Supplementary Figure S2B). As for EPA metabolome, levels of 15-HEPE and 18-HEPE were reduced in relapsing and progressive MS, returning back to control levels in remitting MS, showing a similar trend as their precursor EPA (Figure 4D and Online Supplementary Figure S2C). However, 12-HEPE was particularly high in both relapsing and remitting MS (Figure 4E). Specific LM fingerprints are also shown by volcano plots when comparing every two groups against each other, with SPM like RvD1 and PD1 being reduced along disease progression and others like LXB4 and pro-inflammatory prostaglandins being pro- duced during the active phases of disease, especially in progressive patients (Online Supplementary Figure S3).
Of note, both cohorts of healthy donors and MS patients displayed an almost identical LM profile (Online Supplementary Figure S4), with pro-inflammatory AA- derived prostanoids being induced in relapsing and pro- gressive patients and reduced during remission, whereas lipoxins being slightly induced during the relapsing phase and even more during the progressive phase. As for the DHA- and EPA-derived SPM, both cohorts showed an induction of RvD1 and PD1 during the relapsing phase, which were both reduced or even undetected along dis- ease progression, and showed a significant induction of RvD5 in progressive patients. The only SPM that was dis- cordant was PDX, being significantly induced during the relapsing phase in the first cohort of MS patients, whereas in the second cohort, PDX was markedly induced in pro- gressive patients (Online Supplementary Figure S4).
Having observed that each clinical form of MS is char-
acterized by differential profiles in the levels of SPM, we next sought to evaluate whether this was associated to contradistinctive capacities to produce them and/or to respond to them. Thus, we further characterized the dif- ferent forms of MS by investigating the mRNA expres- sion of the main SPM biosynthetic enzymes and their known receptors in peripheral blood leukocytes. While COX-2 and 5-LOX were particularly induced in relapsing MS to be then strongly reduced in both remitting and pro- gressive patients, 15-LOX was consistently found in all MS forms (Figure 5A). On the contrary, 12-LOX expres- sion was antithetical, inasmuch as its levels were substan- tially reduced in relapsing and remitting MS and started to recover in progressive MS patients (Figure 5A). As for SPM receptor expression, ALX/FPR2, DRV1 and ERV all displayed a similar pattern, with their expression levels being induced in relapsing MS, reaching their highest expression in remitting MS, and then exhibiting a reduc- tion in progressive MS (Figure 5B). In addition, while BLT1 receptor expression was induced only in the active phases of the disease, DRV2 was strongly induced in relapsing MS and markedly reduced in both remitting and progressive patients, to expression levels much lower than healthy subjects (Figure 5B).
Specific specialized pro-resolving mediators attenuate monocyte inflammatory responses in multiple sclerosis patients
The observed differences of MS patients in producing distinct SPM profiles prompted us to examine whether peripheral blood leukocytes of MS patients were respon- sive to the immunomodulatory activity of disease-affect- ed SPM. Accordingly, we tested the ability of SPM that showed an initial induction in relapsing MS patients and are subsequently diminished along disease progression (RvD1 and PD1), and SPM that showed a higher expres- sion in relapsing MS patients and no induction in remit- ting MS (LXA4, LXB4), to evaluate their potential to affect the activation and cytokine production of activated monocytes obtained from RRMS patients, i.e. in the dis- ease phase where such mediators were initially increased and then decreased. To do so, we analyzed the expression of activation markers and inflammatory cytokines in SPM-treated monocytes that were then challenged with two different viral Toll-like receptors (TLR) agonists: TLR7 and TLR8 (see Online Supplementary Figure S5A for gating strategy). As expected, the simultaneous stimula- tion of monocytes of relapsing MS patients with selective agonists of viral Toll-like receptors (TLR) 7 and TLR8 induced a strong upregulation of the activation marker CD69 on their cell surface compared to resting mono- cytes (Figure 6A). Treatment of activated monocytes with LXA4, LXB4, RvD1 or PD1 caused a significant reduction in CD69 surface expression (Figure 6A), indicating an overall ability of these SPM to attenuate the general acti- vation of myeloid cells. More specifically, all these tested SPM were equally able to significantly reduce the intra- cellular production of several pro-inflammatory cytokines. Indeed, the high levels of TNF-a, IL-1b, IL-6 and IL-12 production from activated monocytes of relaps- ing MS patients were equivalently and significantly reduced by LXA4, LXB4, RvD1 and PD1, although RvD1 and PD1 showed a higher vigor in reducing IL-6 produc- tion (Figure 6B and Online Supplementary Figure S5B). The immunomodulatory activity of these SPM was even
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