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. 2017 Apr 26;12(4):e0176406.
doi: 10.1371/journal.pone.0176406. eCollection 2017.

Prostaglandin E2 produced following infection with Theiler's virus promotes the pathogenesis of demyelinating disease

Affiliations

Prostaglandin E2 produced following infection with Theiler's virus promotes the pathogenesis of demyelinating disease

Seung Jae Kim et al. PLoS One. .

Abstract

Infection of various cells with Theiler's murine encephalomyelitis virus (TMEV) activates the TLR- and melanoma differentiation-associated gene 5 (MDA5)-dependent pathways, resulting in the production of IL-1β via the activation of caspase-1 upon assembly of the node-like receptor protein 3 (NLRP3) inflammasome. The role of IL-1β in the pathogenesis of TMEV-induced demyelinating disease was previously investigated. However, the signaling effects of prostaglandin E2 (PGE2) downstream of the NLRP3 inflammasome on the immune responses to viral determinants and the pathogenesis of demyelinating disease are unknown. In this study, we investigated the levels of intermediate molecules leading to PGE2 signaling and the effects of blocking PGE2 signaling on the immune response to TMEV infection, viral persistence and the development of demyelinating disease. We demonstrate here that TMEV infection activates the NLRP3 inflammasome and PGE2 signaling much more vigorously in dendritic cells (DCs) and CD11b+ cells from susceptible SJL mice than in cells from resistant B6 mice. Inhibition of virus-induced PGE2 signaling using AH23848 resulted in decreased pathogenesis of demyelinating disease and viral loads in the central nervous system (CNS). In addition, AH23848 treatment caused the elevation of protective early IFN-γ-producing CD4+ and CD8+ T cell responses. Because the levels of IFN-β were lower in AH23848-treated mice but the level of IL-6 was similar, over-production of pathogenic IFN-β was modulated and the generation of IFN-γ-producing T cell responses was enhanced by the inhibition of PGE2 signaling. These results strongly suggest that excessive activation of the NLRP3 inflammasome and downstream PGE2 signaling contribute to the pathogenesis of TMEV-induced demyelinating disease.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Levels of NLRP3 inflammasome activation after infection with TMEV in various cells from susceptible SJL and resistant B6 mice.
Bone marrow-derived dendritic cells (BMDCs), neonatal whole brain cells, and glial cells (OD, oligodendrocytes and AS, astrocytes) derived from neonatal brains of SJL and B6 mice were compared after infection with or without TMEV for 24 h. Multiple group comparisons were done by a one-way analysis of variance (ANOVA) with Tukey-Kramer post-hoc analysis. *, P < 0.05; ***, p < 0.001. The error bars represent standard deviations (SD) of the triplicate samples.
Fig 2
Fig 2. Elevation of COX-2 and mPGES-1 levels in various cells infected with TMEV.
A. The levels of inducible COX-2 and mPGES-1, constitutive mPGES-2 and antagonistic PGDS- 2 in primary microglia, oligodendrocytes and astrocytes derived from B6 or SJL mice were assessed using qPCR after infection with TMEV (MOI = 10) for 24 h. B. The levels of Cox-2, mPGES-1 and mPGES-2 in bone marrow-derived dendritic cells (BMDCs) from resistant B6 and susceptible SJL mice were assessed using qPCR after infection with TMEV (MOI = 10) for 24 h. MOI represents the multiplicity of infection. For the cells derived from SJL mice, 2 μg of RNA and 4 μg of RNA were used for cells derived from B6 mice due to the relative levels of specific mRNAs. The error bars represent standard deviations (SD) of the triplicate samples. Multiple group comparisons were done by a one-way analysis of variance (ANOVA) with Tukey-Kramer post-hoc analysis. *, P < 0.05; **, P < 0.01; ***, p < 0.001.
Fig 3
Fig 3. Inhibition of the pathogenesis of demyelinating disease and viral loads in mice treated with a PGE2 inhibitor, AH23848.
A. TMEV was inoculated by intracerebral injection (1x106 PFU) into AH23848-treated SJL mice (n = 10) and DMSO vehicle-treated control SJL mice (n = 9) at 1, 5, and 8 dpi. The percentage of affected mice and the mean clinical scores (+/- SD) were monitored weekly. Clinical scores of individual mice were shown in S1 Table. The two-tailed p value between the control and AH23848-treated groups was very significant (p < 0.0059) based on the paired t test of the mean clinical cores (t = 4.582 with 5 degrees of freedom) between days 21 and 56 post-infection. B. Viral load levels in the CNS of AH23848-treated and control mice were assessed at 8 and 21 dpi using qPCR. The significance of the difference was assessed using two-tailed unpaired Student t test. **, P < 0.01; ***, P < 0.001.
Fig 4
Fig 4. Reduced histopathology in TMEV-infected mice treated with AH23848.
Four different sections of the brain and spinal cords of individual mice of two to three per experimental group were graded for demyelination, mononuclear infiltration, and inflammation. A representative sample is shown. Luxol Fast Blue (LFB) staining and counterstaining with H&E showed irregular vacuolation and demyelination in the white matter of the cerebellum of SJL mice (a) and reduced demyelination in PEG2 antagonist AH23848-treated SJL mice (b). Luxol Fast Blue (LFB) staining showed irregular vacuolation and demyelination in the white matter of spinal cords of SJL mice (e) and reduced demyelination in A23848-treated SJL mice (f). By H&E staining, meningitis was evident in the spinal cord of control SJL mice (g) and was not observed in A23848-treated SJL mice (h). Bielschowsky silver staining of the same areas showed the presence of irregular vacuolation and minor axonal damage and loss in SJL mice (c and i) as well as little axonal damage in the PEG2 antagonist-treated SJL mice (d and j). Original magnification, a, b, c, d = 50 μm and e, f, g, h, i, j = 100 μm.
Fig 5
Fig 5. Determination of various inflammatory cytokine levels in the CNS of TMEV-infected mice with or without AH23848 treatment.
Cytokine message levels (IFN-γ, IL-6, IFN-α and IFN-β) in the CNS were determined at 8, 14 and 21 dpi using qPCR. Relative values between the control and AH-treated mice were shown. The error bars represent standard deviations (SD) of three samples in each group. The significance of the difference was assessed using two-tailed unpaired Student t test. *, P < 0.05; **, P < 0.01; ***, p < 0.001.
Fig 6
Fig 6. Determination of IFN-γ production by CNS-infiltrating CD4+ and CD8+ T cells from TMEV-infected mice with or without AH23848 treatment at 8 dpi.
A. CNS-infiltrating mononuclear cells were restimulated for 6 h with PBS, TMEV epitope peptides or PMA/ionomycin. For CD4+ T cells, PEP represents a mixture of 1 μM VP1233-250, VP274-86, and VP324-37. For CD8+ T cells, PEP represents a mixture of 2 μM VP3159-166 and VP3173-181. The cells were then stained for CD4 or CD8 and intracellular IFN-γ. The percentages of CD4+ or CD8+ and IFN-γ+ cells are shown in the upper right corner. The data are representative of three independent experiments. B. Three separate experimental values were combined and presented as bar graphs. *, P < 0.05.
Fig 7
Fig 7. Flow cytometry profiles of CD11b+, CD4+ and CD8+ cells in the CNS of TMEV-infected control and AH23848-treated mice (n = 3) at 8 dpi.
A. The proportions of CNS-infiltrating CD4+ and CD8+ cells, CD11b+ or Gr-1+ cells in conjunction with CD45+ and FoxP3+ CD4+ cells in TMEV-infected control and AH23848-treated SJL mice were compared. B. The proportions of CD4+ and CD8+ cells, CD11b+CD45+ macrophages and activated CD45+ lymphocytes infiltrating the CNS from 3–4 experiments were analyzed using a paired Student’s t test. **, P < 0.01. C. Co-stimulatory molecules (CD80, CD86, CD40, I-As) involved in T cell activation were also slightly elevated in infiltrating macrophages from AH23848-treated mice compared to those from control mice, consistent with the elevated IFN-γ-producing T cells in the CNS. Flow cytometry data presented in A and C are representatives of three independent experiments.
Fig 8
Fig 8. Determination of CD4+ and CD8+ T cell recall responses in the periphery of TMEV-infected mice with or without AH23848 treatment at 8 and 14 dpi.
Proliferative responses to viral epitope peptides for CD4+ T cells (1 μM VP1233-250, VP274-86, and VP324-37) and those for CD8+ T cells (2 μM VP3159-166 and VP3173-181) were similar between splenic T cells from the AH23848-treated and control mice at both 8 and 14 dpi. However, the production of IFN-γ by the CD4+ and CD8+ T cells from AH23848-treated mice at 8 dpi was significantly higher than that by the T cells from the control mice. In contrast, CD4+ T cells from AH23848-treated mice at 14 dpi produced significantly lower levels of IFN-γ compared to CD4+ T cells from control mice.

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