Targeted lipidomics reveals mPGES-1-PGE2 as a therapeutic target for multiple sclerosis

Abstract

The arachidonic acid (AA) cascade produces eicosanoids, such as prostaglandins (PGs), that regulate physiological and pathological functions. Although various nonsteroidal anti-inflammatory drugs have been developed, blocking upstream components (cyclooxygenase-1 and -2) of the AA cascade leads to severe side effects, including gastrointestinal ulcers and cardiovascular events, respectively, due to the complexity of the AA cascade. Here, using an AA cascade-targeted lipidomics approach, we report that microsomal PGE synthase 1 (mPGES-1) plays a key role in experimental autoimmune encephalomyelitis (EAE). Eicosanoids (mainly PGD(2)) are produced constitutively in the spinal cord of naive mice. However, in EAE lesions, the PGE(2) pathway is favored and the PGD(2), PGI(2), and 5-lipoxygenase pathways are attenuated. Furthermore, mPGES-1(-/-) mice showed less severe symptoms of EAE and lower production of IL-17 and IFN-gamma than mPGES-1(+/+) mice. Expression of PGE(2) receptors (EP1, EP2, and EP4) was elevated in EAE lesions and correlated with clinical symptoms. Immunohistochemistry on central nervous systems of EAE mice and multiple sclerosis (MS) patients revealed overt expression of mPGES-1 protein in microglia/macrophages. Thus, the mPGES-1-PGE(2)-EPs axis of the AA cascade may exacerbate EAE pathology. Our findings have important implications for the design of therapies for MS.

Fig. 1.

AA cascade-targeted transcriptomics analysis. (A) C57BL/6 female mice were immunized with the MOG_35–55 peptide. Mice were monitored and weighed daily. Data are the mean clinical score and body weight ± SEM of eight animals. (B) Expression of AA cascade related transcripts was estimated using the comparative C_T method with the SCs of naive mice and EAE mice in the induction, acute, and chronic phases (n = 6, 5, 6, and 5 animals, respectively). The relative abundance of mRNA levels in EAE mice compared with naive mice is shown. Data represent means ± SEM. #, P < 0.001, **, P < 0.01, *, P < 0.05 compared with naive mice using the Kruskal-Wallis test with Dunn's post-hoc test.

Fig. 2.

AA cascade-targeted metabolomics analysis. (A) Eicosanoid levels in the SCs of naive mice and EAE mice in the induction, acute, and chronic phases (n = 8–10 animals) were determined. Data represent means ± SEM. #, P < 0.001, **, P < 0.01, *, P < 0.05 compared with naive mice using the Kruskal-Wallis test with Dunn's post-hoc test. (B) The means of eicosanoid levels were normalized with those of naive mice for each eicosanoid. Then, a cluster analysis was performed by the Ward method. The normalized eicosanoid levels were divided into seven levels according to the indicated color scale. (C) Correlations of COX/5-LO in naive mice (light blue) and EAE mice in the induction (pink), acute (red), and chronic (orange) phases are shown (n = 8–10 animals). Each data point represents the results from a single animal. Data were analyzed statistically by Pearson's correlation.

Fig. 3.

Suppression of EAE pathology and T_H1/T_H17 responses in the absence of mPGES-1. (A) SCs of EAE mice in the acute phase were stained with anti-mPGES-1 (green), F4/80 (red), and CD4 (red) Abs. (Scale bar, 50 μm.) These pictures are representative of two different experiments. (B) PGE₂ levels were measured in the SCs of mPGES-1^{+/+, +/−} and mPGES-1^−/− mice on day 26 (n = 13 and 7 animals, respectively). *, P < 0.05 versus mPGES-1^{+/+, +/−} mice by Mann–Whitney U test. Data represent means ± SEM. (C) mPGES-1^+/+ (filled circles) and mPGES-1^−/− (open circles) mice were immunized with the MOG_35–55 peptide and clinical scores were assessed daily for 26 days. Data represent means ± SEM from two independent experiments with a total of 14 and 9 animals for mPGES-1^+/+ and mPGES-1^−/− mice, respectively. *, P < 0.0001 versus mPGES-1^+/+ mice determined by two-way repeated measures ANOVA. (D) LN cells from EAE-induced mPGES-1^+/+ and mPGES-1^−/− mice on day 11 were stimulated in vitro with various concentrations of MOG_35–55 peptide, and the proliferative responses were measured (n = 5 animals). Data represent means ± SEM. (E) Supernatants of LN cell cultures were used to measure the concentrations of IFN-γ, IL-17, TNF-α, and IL-6 (n = 5 animals). Data represent means ± SEM. *, P < 0.05, **, P < 0.01 versus mPGES-1^+/+ mice by two-tailed Student's t test. (F) Two days after activation, cells were restimulated with PMA/ionomycin and subjected to intracellular cytokine staining for IFN-γ and IL-17 (n = 5 animals). Each data point represents the results from a single animal and the horizontal bars designate the mean values of individual groups. *, P < 0.05, by two-tailed Student's t test.

Fig. 4.

mPGES-1 expression in infiltrated macrophages of MS lesions. Brain tissues of MS patients' autopsied materials displaying periventricular demyelinating lesions were stained with anti-mPGES-1. (Scale bar, 50 μm A, B, D, and E.) The high magnification images of A and D are shown in B and E, respectively. (Scale bar, 10 μm.) The antigen-peptide blocks the signals of anti-mPGES-1 Ab staining. (Scale bar, 10 μm C and F.) Both MS #1 (subacute stage, A–C) and MS #2 (acute stage, D–F) showed extensive infiltration of macrophages characterized by foamy appearance. The mPGES-1 is colocalized with CD68-immunoreactive macrophage in the MS lesions. (Scale bar, 10 μm G–Q.)

Fig. 5.

Conceptual model for the role of AA cascade in EAE pathology. PGE₂ is produced by mPGES-1 in activated microglia (MG), infiltrating macrophages (MΦ) and dendritic cells (DC), and then activates these cells in an autocrine/paracrine manner through EP2/EP4. T cells differentiate into T_H17 cells by stimulation with IL-6 and IL-23 secreted from activated MG/MΦ/DC. T_H1 differentiation and expansion of T_H17 cells are accelerated by the direct effects of PGE₂ on EP1 and EP2/EP4.