Omega-3 polyunsaturated fatty acids protect against inflammation through production of LOX and CYP450 lipid mediators: relevance for major depression and for human hippocampal neurogenesis

Abstract

Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) can exert antidepressant, anti-inflammatory and neuroprotective properties, but the exact molecular mechanism underlying their effects is still not fully understood. We conducted both in vitro and clinical investigations to test which EPA or DHA metabolites are involved in these anti-inflammatory, neuroprotective and antidepressant effects. In vitro, we used the human hippocampal progenitor cell line HPC0A07/03C, and pre-treated cells with either EPA or DHA, followed by interleukin 1beta (IL1β), IL6 and interferon-alpha (IFN-α). Both EPA and DHA prevented the reduction in neurogenesis and the increase in apoptosis induced by these cytokines; moreover, these effects were mediated by the lipoxygenase (LOX) and cytochrome P450 (CYP450) EPA/DHA metabolites, 5-hydroxyeicosapentaenoic acid (HEPE), 4-hydroxydocosahexaenoic acid (HDHA), 18-HEPE, 20-HDHA, 17(18)-epoxyeicosatetraenoic acid (EpETE) and 19(20)-epoxydocosapentaenoic acid (EpDPA), detected here for the first time in human hippocampal neurones using mass spectrometry lipidomics of the supernatant. In fact, like EPA/DHA, co-treatment with these metabolites prevented cytokines-induced reduction in neurogenesis and apoptosis. Moreover, co-treatment with 17(18)-EpETE and 19(20)-EpDPA and the soluble epoxide hydroxylase (sEH) inhibitor, TPPU (which prevents their conversion into dihydroxyeicosatetraenoic acid (DiHETE)/ dihydroxydocosapentaenoic acid (DiHDPA) metabolites) further enhanced their neurogenic and anti-apoptotic effects. Interestingly, these findings were replicated in a sample of n = 22 patients with a DSM-IV Major Depressive Disorder, randomly assigned to treatment with either EPA (3.0 g/day) or DHA (1.4 g/day) for 12 weeks, with exactly the same LOX and CYP450 lipid metabolites increased in the plasma of these patients following treatment with their precursor, EPA or DHA, and some evidence that higher levels of these metabolites were correlated with less severe depressive symptoms. Overall, our study provides the first evidence for the relevance of LOX- and CYP450-derived EPA/DHA bioactive lipid metabolites as neuroprotective molecular targets for human hippocampal neurogenesis and depression, and highlights the importance of sEH inhibitors as potential therapeutic strategy for patients suffering from depressive symptoms.

Fig. 1. Enzymatic synthesis pathways of ω-3 PUFAs and timeline of experiments with ω-3 PUFAs and derived lipid mediators.

a ω-3 PUFAs are metabolised by COX, LOX and CYP450 enzymes. COX and LOX enzymes convert ω-3 PUFAs into prostanoids, hydroxy fatty acids and leukotrienes, while CYP450 monooxygenases convert ω-3 PUFAs into epoxy and hydroxy fatty acids. Epoxy fatty acids are then metabolised via the sEH enzyme to the corresponding dihydroxy metabolites. b HPC0A07/03C cells were plated for 24 h in proliferating media and then pre-treated with either EPA or DHA for 48h in differentiating media. After 48 h, EPA or DHA were removed and treatment with cytokines IL1β, IL6 or IFN-α was added for additional 6 or 48 h. At the end of the 6h period, supernatant was collected, and metabolites were analysed. Whereas, at the end of the 48 h period, cells were fixed and stained for markers of neurogenesis (DCX and Map2) and apoptosis (CC3). c In another experiment, cells were pre-treated with EPA or DHA and an inhibitor of COX-2 inhibitor (CAS 416901-58-1), LOX enzymes (NDGA) or CYP450 enzymes (SKF525A) for 48 h, followed by 48h treatment with cytokines (IL1β, IL6 or IFN-α) and again the inhibitors (COX-2, LOX or CYP450 inhibitor). Cells were then fixed and stained for markers of neurogenesis (DCX and Map2) and apoptosis (CC3). d Cells were plated for 24h in proliferating media, and then differentiated for 48 h in vehicle condition without any treatment. Media was then changed and treatment with cytokines (IL1β, IL6 or IFN-α) and EPA- or DHA-derived metabolites (5-HEPE, 18-HEPE, 4HDHA or 20-HDHA) were added for additional 6h or 48h. At the end of the 6h period mRNA was extracted for gene expression of STAT1, NF-kB and AQP4. Whereas, at the end of the 48 h period, cells were fixed and stained with DCX, Map2 and CC3, and supernatant was collected for cytokines analysis. e Cells were plated for 24h in proliferating media, and then differentiated for 48 h in vehicle condition without any treatment. Media was then changed and treatment with cytokines (IL1β, IL6 or IFN-α) and EPA- or DHA-derived metabolites (17(18)-EpETE, 19(20)-EpDPA, 17(18)-DiHETE, 19(20)-DiHDPA), with or without TPPU, were added for additional 6 or 48 h. At the end of the 6h period mRNA was extracted for gene expression of STAT1, NF-kB and AQP4. Whereas, at the end of the 48 h period, cells were fixed and stained with DCX, Map2 and CC3, and supernatant was collected for cytokines analysis. Legend: EPA, eicosapentaenoic acid; DHA, docosahexaenoic acid; COX-2, cyclooxygenase-2; LOX, lipoxygenase; CYP450, cytochrome P450; HEPE, hydroxyeicosapentaenoic acid; HDHA, hydroxydocosahexaenoic acid; EpETE, epoxydocosapentaenoic acid; EpDPA, epoxydocosapentaenoic acid; DiHETE, dihydroxyeicosatetraenoic acid; DiHDPA, dihydroxydocosapentaenoic acid; sEH, soluble epoxide hydroxylase; IL1β, interleukin 1beta; IL6, interleukin 6; IFN-α, interferon-alpha; DCX, doublecortin; Map2, microtubule-associated protein 2; CC3, caspase 3; STAT1, activator of transcription 1; NF-kB, nuclear factor-κB; AQP4, aquaporin 4.

Fig. 2. Pre-treatment with EPA or DHA prevents cytokines-induced reduction in neurogenesis and increase in apoptosis, and stimulates the production of EPA- and DHA-derived LOX and CYP450 lipid mediators.

a–i Pre-treatment of cells with either EPA or DHA followed by IL1β, IL6 or IFN-α prevented the reduction in neurogenesis (DCX+ and Map2+ cells) and/or increase in apoptosis (CC3+ cells) induced by the cytokines alone. Two-way ANOVA with Bonferroni’s post hoc test was performed. Data are shown as mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, compared with vehicle treatment or as indicated. j Heatmap showing metabolites production in supernatant of cells pre-treated for 48 h during differentiation with either EPA or DHA, followed by 6 h treatment with cytokines (IL1β, IL6 or IFN-α). Legend: LA, linoleic acid; DGLA, dihomo-γ-linolenic acid; AA, arachidonic acid; EPA, eicosapentaenoic acid; DHA, docosahexaenoic acid; COX-2, cyclooxygenase-2; LOX, lipoxygenase; CYP450, cytochrome P450; HODE, hydroxyoctadecadienoic acid; EpOME, epoxyoctadecenoic acid; DiHOME, dihydroxyoctadecenoic acid; HETrE, hydroxyeicosatrienoic acid; HETE, hydroxyeicosatetraenoic acid; DHET, dihydroxyeicosatrienoic acid; HEPE, hydroxyeicosapentaenoic acid; EpETE, epoxyeicosatetraenoic acid; DiHETE, dihydroxyeicosatetraenoic acid; HDHA, hydroxydocosahexaenoic acid; EpDPA, epoxydocosapentaenoic acid; DiHDPA, dihydroxydocosapentaenoic acid.

Fig. 3. Treatment with higher concentrations of LOX and CYP450 lipid mediators prevents cytokines-induced reduction in neurogenesis and increase in apoptosis.

a–i Co-treatment of cells with higher concentrations of 5-HEPE, 18-HEPE, 4-HDHA or 20-HDHA and IL1β, IL6 or IFN-α partially prevented decrease in DCX+ and Map+cells, and increase in CC3+cells caused by treatment with cytokines alone. j–r Co-treatment of cells with higher concentrations of 17(18)-EpETE or 19(20)-EpDPA and IL1β, IL6 or IFN-α prevented decrease in DCX+ and Map+cells, and increase in CC3+cells caused by treatment with cytokines alone, and this effect was enhanced by treatment with the sEH inhibitor TPPU. One-way ANOVA with Bonferroni’s post hoc test was performed. Data are shown as mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001, compared with vehicle treatment or as indicated.

Fig. 4. Treatment with CYP450 lipid mediators and sEH inhibitor fully prevents cytokines-induced production of inflammatory cytokines.

a–l Co-treatment with 5-HEPE, 18-HEPE, 4-HDHA, 20-HDHA, 17(18)-EpETE or 19(20)-EpDPA and either IL1β, IL6 or IFN-α prevented the increase in the production of the same cytokines (IL1β, IL6 or IFN-α), induced by treatment with IL1β, IL6 or IFN-α alone. Moreover, co-treatment with 17(18)-EpETE or 19(20)-EpDPA, IL1β, IL6 or IFN-α, and the sHE inhibitor TPPU further enhanced the effect of 17(18)-EpETE and 19(20)-EpDPA. One-way ANOVA with Bonferroni’s post hoc test was performed. Data are shown as mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, compared with vehicle treatment or as indicated.

Fig. 4. Treatment with CYP450 lipid mediators and sEH inhibitor fully prevents cytokines-induced production of inflammatory cytokines.

a–l Co-treatment with 5-HEPE, 18-HEPE, 4-HDHA, 20-HDHA, 17(18)-EpETE or 19(20)-EpDPA and either IL1β, IL6 or IFN-α prevented the increase in the production of the same cytokines (IL1β, IL6 or IFN-α), induced by treatment with IL1β, IL6 or IFN-α alone. Moreover, co-treatment with 17(18)-EpETE or 19(20)-EpDPA, IL1β, IL6 or IFN-α, and the sHE inhibitor TPPU further enhanced the effect of 17(18)-EpETE and 19(20)-EpDPA. One-way ANOVA with Bonferroni’s post hoc test was performed. Data are shown as mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, compared with vehicle treatment or as indicated.

Fig. 5. Increased levels of LOX and CYP450 lipid mediators in depressed patients receiving ω-3 PUFAs intervention.

a Demographic and clinical characteristics of the depressed patients by treatment groups. Chi-square χ² test (for categorical variables) or Mann Whitney U (for continuous values). Continuous data are shown as mean, categorical data are presented as n (%). b, c Differences in HAM-D scores before and after treatment with either EPA or DHA. d–f 5-, 8-, 9-, 11-, 12-, 15-, 18-HEPE and 8(9)-, 11(12)-, 14(15)-, 17(18)-EpETE, -DiHETE metabolites levels before and after treatment with either EPA. g–i 4-, 7-, 8-, 10-, 11-, 13-, 14-, 20-HDHA, 10(11)-, 13(14)-, 16(17)-, 19(20)-EpDPA, -DiHDPA metabolites levels before and after treatment with either DHA. Wilcoxon’s t test, with Bonferroni’s post hoc test was performed. Data are shown as mean ± SEM; *p < 0.05, **p < 0.01, ****p < 0.0001, compared as indicated. Lower limit of detection was reported for 11(12)-, 14(15), 17(18)-EpETE, 8(9)-DiHETE, 16(17), 19(20)-EpDPA.