Principal component analysis (PCA) was performed at each time point of euthanasia on the 90 available biomarkers. At each time point, the effect of LPS treatment was evaluated comparing Control vs. LPS groups and the effect of supplementations was evaluated comparing non-supplemented vs. supplemented LPS groups. For each analysis, we will only describe the number of components that explain at least 50% of the total variance.

30 Min post-LPS: multivariate analysisLPS-treated mice did not significantly differ from control mice after 30 min of exposureFig. 1

Multivariate analysis of control and LPS groups at 30 min: individual map of PCA

The first 3 components from the PCA analysis explained 54.2% of the total variance (first component, ‘dim 1’: 26.6%; second component, ‘dim 2’: 16.1%; third component, ‘dim 3’: 11.5%) (Fig. 1). No significant differences between LPS and Control group were revealed on these dimensions meaning that the profiles between Control and LPS groups cannot be distinguished.

Supplementations increased inflammatory and antioxidant responses in 30 min LPS-treated animalsFig. 2

Multivariate analysis of supplemented and non-supplemented LPS groups at 30 min: individual map of PCA

The first 4 components derived from the PCA analysis explained 53.9% of the total variance (first component, ‘dim 1’: 27.2%; second component, ‘dim 2’: 10.4%; third component, ‘dim 3’: 9%; fourth component, ‘dim 4’: 7.3%) (Fig. 2). Table 2 shows the correlations between biomarkers and components. PE supplemented group was significantly different from the other groups on dim 2, for which it showed positive scores (p = 0.001). Specifically, dim 2 revealed a positive correlation with proinflammatory markers CCL2, TNFα, IL-1β, NFκB, CXCL10, CXCL9, microglial markers Clec7a, SOCS3, CD11c, CD206, ApoE and antioxidant markers GPx1 and Catalase. PE + om-3 supplemented group was significantly different from the other groups on dim 4, for which it showed negative scores (p = 0.001). Dim 4 showed a positive correlation with antioxidant marker GSS and C16-2OH, a hydroxy fatty acid and a negative correlation with PGD2, LTB4, PGE2, derived from AA and 10-HODE, derived from LA. No distinction between the supplemented groups were found on dimensions 1 and 3.

Table 2 Multivariate analysis of supplemented and non-supplemented LPS groups at 30 min2 H post-LPS: multivariate analysisLPS modulated the oxylipin profile and increased proinflammatory and antioxidant markers after 2 h of exposureFig. 3

Multivariate analysis of control and LPS groups at 2 h: individual map of PCA

The first 4 components from the PCA analysis explained 56.9% of the total variance (first component, ‘dim 1’: 25.6%; second component, ‘dim 2’: 13.7%; third component, ‘dim 3’: 10%; fourth component, ‘dim 4’: 7.6%) (Fig. 3). Control and LPS groups significantly differed from each other on dim 1 and dim 2. In fact, LPS group exhibited negative scores on dim 1 and positive scores on dim 2 whereas it was the opposite for Control group (dim 1: p < 0.001 and dim 2: p = 0.016). Table 3 shows the correlation between biomarkers and components. Dim 1 was positively correlated with various LA- and AA-derived oxylipins and hydroxy fatty acids, as well as 12-LOX, an oxylipin biosynthesis enzyme and Clec7a, a microglial marker. Dim 1 was also negatively correlated with PGE2 and LTB4, derived from AA, COX-2, involved in oxylipin biosynthesis, microglial marker CD86 and proinflammatory cytokines and chemokines TNFα, IL-1β, CCL2, IL-6 (both in spleen and hippocampus), CXCL10 and CXCL9. Dim 2 revealed only positive correlation with oxylipins derived from AA, DHA and LA, pro-inflammatory factors NFκB, IL-6 (both in spleen and hippocampus), TNFα and IL-1β, microglial marker SOCS3, antioxidant markers Sod2, Catalase, GPx1, Sod1 and NRF2, and 15-LOX, an oxylipin biosynthesis enzyme. No significant differences between LPS group and Control group were found on dimensions 3 and 4.

Table 3 Multivariate analysis of control and LPS groups at 2 hPE + om-3 supplementation modulated oxylipin composition in 2 h LPS-treated animalsFig. 4

Multivariate analysis of supplemented and non-supplemented LPS groups at 2 h: individual map of PCA

The first 5 components derived from the PCA analysis accounted for 55.1% of the total variance, with the breakdown as follows: the first component (dim 1) contributing 23.7%, the second component (dim 2) 10.9%, the third component (dim 3) 8%, the fourth component (dim 4) 6.7% and the fifth component (dim 5) 5.8% (Fig. 4). PE + om-3 group was significantly different from LPS group on dim 4, for which it showed positive scores (p < 0.001) whereas LPS group showed negative scores (p < 0.001). Table 4 shows the correlation between biomarkers and components. Dim 4 was positively correlated with LxA4, derived from AA and C10-3OH, a hydroxy fatty acid, and negatively correlated with LTB4, also derived from AA. No distinction between the LPS groups were found on dimensions 1, 2, 3 and 5.

Table 4 Multivariate analysis of supplemented and non-supplemented LPS groups at 2 h6 H post-LPS: multivariate analysisLPS increased the proinflammatory response, decreased neuroprotection and modulated oxylipin composition after 6 h of exposureFig. 5

Multivariate analysis of control and LPS groups at 6 h: individual map of PCA

The first 2 components from the PCA analysis explained 54.2% of the total variance (first component, ‘dim 1’: 36.9%; second component, ‘dim 2’: 17.3%) (Fig. 5). Control group was significantly different from LPS group on dim 1 and dim 2. In fact, LPS group exhibited negative scores on dim 1 and positive scores on dim 2 whereas it was the opposite for Control group (dim 1: p = 0.039 and dim 2: p < 0.001). Table 5 shows the correlation between biomarkers and components. Dim 1 was positively correlated with various LA-, AA- and DHA-derived oxylipins and hydroxy fatty acids as well as neurotrophic factor BDNF and negatively correlated with LTB4, derived from AA, and IDO1. Dim 2 was positively correlated with PGE2 and PGD2, derived from AA, 3 hydroxy fatty acids, proinflammatory markers IL-1β, NFκB, IL-6 (both in spleen and hippocampus), CCL2, TNFα, IDO1, TLR4, CXCL9, CXCL10 and iNOS, microglial markers SOCS3, Arg1, CD86, CD11b and ApoE, oxidative markers NRF2, RAGE and GSS, oxylipin biosynthesis enzymes 15-LOX, 5-LOX, COX-2, and apoptosis marker Bax. Dim 2 was negatively correlated with microglial markers Trem2 and Glut5, oxylipin biosynthesis enzyme 12-LOX and neurogenesis marker DCX.

Table 5 Multivariate analysis of control and LPS groups at 6 hPE + om-3 supplementation induced a change in oxylipin profile in 6 h LPS-treated animalsFig. 6

Multivariate analysis of supplemented and non-supplemented LPS groups at 6 h: individual map of PCA

The first 5 components derived from the PCA analysis explained 53.8% of the total variance. Specifically, the first component (dim 1) contributed 29.4%, the second component (dim 2) 10.2%, the third component (dim 3) 8.2% and the fourth component (dim 4) 6.2% (Fig. 6). Table 6 shows the correlation between biomarkers and components. LPS group showed negative scores on dim 1 (p = 0.001) whereas PE + om-3 supplemented group showed positive scores (p = 0.040). Dim 1 was positively correlated with oxylipins derived from LA, AA, EPA and DHA and hydroxy fatty acids as well as microglial markers Tspo and ApoE, neuroprotective receptor TrkA and antioxidant markers GPx1, Sod2 and Sod1. PE + om-3 group was significantly different from the other groups on dim 3, for which it revealed negative scores (p < 0.001). Dim 3 was positively correlated with PGD2, derived from AA, microglial markers CD68, Glut5, CD11b and ApoE and C18-3OH. No distinctions of supplemented groups were found on dim 2 and 4.

Table 6 Multivariate analysis of supplemented and non-supplemented LPS groups at 6 hOxylipin analysisFig. 7

Heatmap representation of oxylipin concentrations in LPS groups. Red indicates higher concentration and blue indicates lower concentration (n = 9–10/group)

As the effect of supplementations seemed to be driven by a change in oxylipin profile, we investigated the impact of supplementations on the oxylipin concentrations derived from LA, AA, EPA and DHA at each time point. The generated heatmap demonstrated that gene regulation and oxylipin production were dependent on the time point and the group considered (Fig. 7). Interestingly, heatmap clearly highlighted the underexpression of oxylipins at 30 min post-LPS in the PE + om-3 group compared with the other groups and the overexpression of oxylipins at 6 h in the supplemented groups compared with the LPS and control groups.

After 30 min of exposure (Table 7), supplementations affected the amount of prostaglandins derived from AA: 8-iso-PGA2 (F(3,35) = 3.457, p = 0.027), PGD2 (F(3,35) = 3.463, p = 0.027), PGE2 (H(3) = 13.030, p = 0.005), PGF2α (H(3) = 13.870, p = 0.003), 15dPGJ2 (F(3,35) = 6.589, p = 0.001) and that of 18-HEPE (F(3,35) = 29.160, p < 0.001), derived from EPA. Indeed, PGF2α concentration was significantly higher in PE group than in LPS group (p = 0.006). PE + om-3 supplementation significantly increased PGD2, PGE2, PGF2α and decreased 15dPGJ2 compared to LPS group (PGD2: p = 0.049, PGE2: p = 0.040, PGF2α: p = 0.010, 15dPGJ2: p = 0.040). Moreover, PE + om-3 supplementation significantly increased PGD2 (p = 0.031) and PGE2 (p = 0.011) compared to om-3 group and decreased 8-iso-PGA2 compared to PE supplementation (p = 0.015) and 15dPGJ2 compared to all other groups (p = 0.001 vs. PE, p = 0.006 vs. om-3). Finally, om-3 and PE + om-3 supplementations significantly increased 18-HEPE compared to LPS and PE groups (p < 0.001 for all).

Table 7 Effect of supplementations on oxylipins concentrations in hippocampus 30 min post-LPS treatment

After 2 h of exposure (Table 8), supplementations modulated the concentrations of LA-derived 9-oxo-ODE (F(3,35) = 6.195, p = 0.002) and 13-oxo-ODE (H(3) = 12.110, p = 0.007), as well as AA-derived 8,9-EET (F(3,35) = 4.129, p = 0.013), 5-HETE (F(3,34) = 6.513, p = 0.001), 8-HETE (F(3,35) = 5.740, p = 0.003), 12-HETE (H(3) = 11.560, p = 0.009), 15-HETE (F(3,35) = 4.026, p = 0.015), 5-oxo-ETE (F(3,35) = 8.448, p < 0.001), LTB4 (F(3,28) = 3.715, p = 0.023), PGD2 (F(3,35) = 3.380, p = 0.029) and PGE2 (H(3) = 8.090., p = 0.044). 18-HEPE (F(3,35) = 23.99, p < 0.001) and 14-HDHA (F(3,35) = 3.021, p = 0.043), derived respectively from EPA and DHA, were also affected. In fact, PE supplementation significantly decreased 14-HDHA (p = 0.038), 9-oxo-ODE (p = 0.016), 5-oxo-ETE (p = 0.002), 5-HETE (p = 0.003), 8-HETE (p = 0.009), 15-HETE (p = 0.040) and LTB4 (p = 0.033) compared to LPS group. Om-3 supplementation significantly decreased 5-oxo-ETE compared to LPS group (p = 0.052). PE + om-3 supplementation significantly decreased 9-oxo-ODE (p = 0.001), 13-oxo-ODE (p = 0.005), 8,9-EET (p = 0.008), 5-oxo-ETE (p < 0.001) 5-HETE (p = 0.003), 8-HETE (p = 0.004), 12-HETE (0.006), 15-HETE (p = 0.016) and LTB4 (p = 0.053) and tented to increase PGD2 (p = 0.061) compared to LPS group. Moreover, PE supplementation significantly increased PGE2 and PE + om-3 supplementation significantly increased PGD2 compared to om-3 group (p = 0.049 and p = 0.034, respectively). Finally, om-3 and PE + om-3 supplementations significantly increased 18-HEPE compared to LPS and PE groups (p < 0.001 for all).

Table 8 Effect of supplementations on oxylipins concentrations in hippocampus 2 h post-LPS treatment

After 6 h of exposure (Table 9), supplementations significantly impacted the concentration of most oxylipins derived from LA: 9-HODE (F(3,36) = 5.133, p = 0.005), 13-HODE (F(3,36) = 4.213, p = 0.012), 9,10-DiHOME (F(3,36) = 4.511, p = 0.009), 12,13-DiHOME (F(3,36) = 9.159, p < 0.001), 9-oxo-ODE (F(3,36) = 6.698, p = 0.001), 13-oxo-ODE (F(3,36) = 5.257, p = 0.004), 9,10,13-TriHOME (F(3,36) = 4.767, p = 0.007), 9,12,13-TriHOME (H(3) = 15.570, p = 0.001). Most AA-derived oxylipins were also modulated by the supplementations: 5,6-EET (F(3,36) = 3.368, p = 0.029), 8,9-EET (F(3,36) = 10.74, p < 0.001), 11,12-EET (F(3,35) = 6.812, p = 0.001), 14,15-EET (H(3) = 12.73, p = 0.005), 5-HETE (F(3,36) = 6.736, p = 0.001), 8-HETE (F(3,36) = 6.363, p = 0.001), 15-HETE (F(3,36) = 4.274, p = 0.011), 5-oxo-ETE (F(3,36) = 9.646, p < 0.001), LTB4 (F(3,26) = 4.729, p = 0.009), LxA4 (H(3) = 11.20, p = 0.011), 8-iso-PGA2 (H(3) = 21.67, p < 0.001), PGD2 (F(3,36) = 6.050, p = 0.002), PGF2α (F(3,36) = 2.877, p = 0.049), 15dPGJ2 (F(3,36) = 10.21, p < 0.001), TXB2 (F(3,36) = 7.746, p < 0.001). The same results were found for EPA-derived 18-HEPE (H(3) = 27.41, p < 0.001) and DHA-derived 14-HDHA (F(3,36) = 5.365, p = 0.004) and 17-HDHA (F(3,36) = 6.470, p = 0.001). Indeed, compared to LPS group, all supplementations significantly increased 9,10-DiHOME (trend at p = 0.090 vs. PE, p = 0.007 vs. om-3, p = 0.048 vs. PE + om-3), 12,13-DiHOME (p = 0.003 vs. PE, p < 0.001 vs. om-3 and vs. PE + om-3), 9-oxo-ODE (p = 0.040 vs. PE, p = 0.012 vs. om-3, p < 0.001 vs. PE + om-3), 9,10,13-TriHOME (p = 0.028 vs. PE, p = 0.007 vs. om-3, trend at p = 0.065 vs. PE + om-3), 15dPGJ2 (p = 0.002 vs. PE, p = 0.012 vs. om-3, p < 0.001 vs. PE + om-3), TXB2 (p = 0.004 vs. PE, p < 0.001 vs. om-3, p = 0.005 vs. PE + om-3) and 8,9-EET (p = 0.050 vs. PE, p = 0.010 vs. om-3, p < 0.001 vs. PE + om-3). Compared to LPS group, PE supplementation induced a higher level of 9,12,13-TriHOME (p = 0.011), 5-HETE (p = 0.010), LTB4 (p = 0.006), LxA4 (p = 0.014) and 8-iso-PGA2 (p < 0.001). Om-3 supplementation increased the level of 9-HODE (p = 0.055), 13-HODE (p = 0.046), 13-oxo-ODE (p = 0.028), 9,12,13-TriHOME (p = 0.002), LxA4 (p = 0.040), 8-iso-PGA2 (p = 0.017) and tended to increase 11,12-EET (p = 0.060), 14,15-EET (p = 0.091) and PGF2α (p = 0.055) levels compared to LPS group. PE + om-3 supplementation increased the level of 9-HODE (p = 0.003), 13-HODE (p = 0.010), 13-oxo-ODE (p = 0.003), 11,12-EET (p < 0.001), 14,15-EET (p = 0.003), 5-HETE (p < 0.001), 8-HETE (p < 0.001), 15-HETE (p = 0.006), 8-iso-PGA2 (p < 0.001), 14-HDHA (p = 0.004) and 17-HDHA (p < 0.001) and decreased that of PGD2 (p = 0.005), compared to LPS group. Moreover, PE + om-3 supplementation increased the concentrations of 8,9-EET, 14-HDHA and 17-HDHA compared to PE group (p = 0.028, p = 0.030, p = 0.050, respectively) and decreased that of PGD2 compared to om-3 group (p = 0.004). In addition, PE + om-3 supplementation increased the concentrations of 5-oxo-ETE compared to the other groups (p < 0.001 vs. LPS, p = 0.012 vs. PE, p = 0.017 vs. om-3). Finally, 18-HEPE was also significantly higher in om-3 and PE + om-3 groups than in LPS and PE groups (p < 0.001 vs. LPS, om-3 vs. PE p = 0.006, PE + om-3 vs. PE p = 0.003).

Table 9 Effect of supplementations on oxylipins concentrations in hippocampus 6 h post-LPS treatmentFatty acid analysisFig. 8

Effect of LPS and supplementations on fatty acid concentrations in hippocampus. (A) Total SFAs concentration. (B) Total MUFAs concentration. (CC) Total omega-6 + omega-3 concentration. (*p < 0.05, **p < 0.01, ****p < 0.0001 vs. LPS by 2-WAY ANOVA and Dunnett’s post-hoc test, n = 9–10/group). Data are presented as mean ± SEM

We then analyzed the hippocampal composition of fatty acids that are precursors of oxylipin compounds (Fig. 8A-C). Time of exposure to LPS and treatment significantly impacted the proportions of total SFAs (time: F(2,128) = 3.522, p = 0.032; treatment: F(4,128) = 8.757, p < 0.001) and total omega-6 + omega-3 (om6 + om-3) (F(2,128) = 13.51, p < 0.001; treatment: F(4,128) = 19.97, p < 0.001). At each time point, the omega-6 and omega-3 proportions followed the same evolution as total om-6 + om-3 (data not shown). Moreover, an interaction between time and treatment was observed for the proportions of total SFAs (F(8,128) = 3.931, p < 0.001), total MUFAs (F(8,128) = 2.984, p = 0.004) and total om-6 + om-3 (F(8,128) = 9.801, p < 0.001). Indeed, compared to control group, LPS significantly decreased the proportion of total SFAs (p < 0.001) and increased that of total om-6 + om-3 (p < 0.001), 30 min after exposure but not later. Concerning the effect of the supplementations, after 30 min of exposure, all of them significantly increased total SFAs proportion (p < 0.001 for all) and decreased total om-6 + om-3 proportions compared to LPS (p < 0.001 for all). After 2 h of exposure, no effects of the supplementations were observed. After 6 h of exposure, all the supplementation significantly increased total MUFAs (PE: p = 0.011, om-3: p = 0.021, PE + om-3: p = 0.007) and decreased total om-6 + om-3 proportion (p < 0.001 for all) compared to LPS group.