Peroxisome proliferator-activated receptor delta limits the expansion of pathogenic Th cells during central nervous system autoimmunity - PubMed (original) (raw)

. 2010 Aug 2;207(8):1599-608.

doi: 10.1084/jem.20091663. Epub 2010 Jul 12.

Roopa Bhat, Daniel S Straus, Raymond A Sobel, Robert Axtell, Amanda Johnson, Kim Nguyen, Lata Mukundan, Marina Moshkova, Jason C Dugas, Ajay Chawla, Lawrence Steinman

Affiliations

Peroxisome proliferator-activated receptor delta limits the expansion of pathogenic Th cells during central nervous system autoimmunity

Shannon E Dunn et al. J Exp Med. 2010.

Abstract

Peroxisome proliferator-activated receptors (PPARs; PPAR-alpha, PPAR-delta, and PPAR-gamma) comprise a family of nuclear receptors that sense fatty acid levels and translate this information into altered gene transcription. Previously, it was reported that treatment of mice with a synthetic ligand activator of PPAR-delta, GW0742, ameliorates experimental autoimmune encephalomyelitis (EAE), indicating a possible role for this nuclear receptor in the control of central nervous system (CNS) autoimmune inflammation. We show that mice deficient in PPAR-delta (PPAR-delta(-/-)) develop a severe inflammatory response during EAE characterized by a striking accumulation of IFN-gamma(+)IL-17A(-) and IFN-gamma(+)IL-17A(+) CD4(+) cells in the spinal cord. The preferential expansion of these T helper subsets in the CNS of PPAR-delta(-/-) mice occurred as a result of a constellation of immune system aberrations that included higher CD4(+) cell proliferation, cytokine production, and T-bet expression and enhanced expression of IL-12 family cytokines by myeloid cells. We also show that the effect of PPAR-delta in inhibiting the production of IFN-gamma and IL-12 family cytokines is ligand dependent and is observed in both mouse and human immune cells. Collectively, these findings suggest that PPAR-delta serves as an important molecular brake for the control of autoimmune inflammation.

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Figures

Figure 1.

Figure 1.

PPAR-δ−/− mice develop hyperacute EAE. EAE was induced in WT or PPAR-δ−/− female (a) or male (b–e) mice (n = 8–10/group). (a and b) Mean ± SEM clinical scores of mice at various times after immunization. Results are representative of two to three experiments. (c–e) Paraffin sections of spinal cord and brain were prepared from PPAR-δ−/− and WT mice at the time of first remission and were stained with H&E and luxol fast blue. d and e show representative sections (bar, 50 µM); c shows the mean + SEM number of inflammatory foci; g shows the regional distribution of parenchymal lesions. (f) Mononuclear cells were isolated from spinal cords of WT or PPAR-δ−/− mice (n = 3/group) at 4 d after the onset of clinical signs, and then they were pooled and counted. Cells were stained with antibodies specific for cell surface markers as indicated and the percentage of each subset in the live CD45+ gate is shown. Values are mean + SEM numbers from four experiments. *, significantly different from WT.

Figure 2.

Figure 2.

PPAR-δ−/− mice exhibit higher Th cytokine production during EAE. (a and b) Mononuclear cells were isolated from spinal cords of WT or PPAR-δ−/− mice at 4 d after the onset of clinical signs, and then they were pooled (n = 3/group) and processed for intracellular cytokine staining. The number (a) and frequency (b) of CD4+ mononuclear cells that had intracellular accumulation of IFN-γ (IFN-γ+IL-17A− and IFN-γ+IL-17A+) or IL-17A (IL-17A+ IFN-γ+ and IL-17A+ IFN-γ−) are shown. (c) Representative FACs plot of IFN-γ and IL-17A staining in CD4+ gate. (d and e) Splenocytes from male age-matched WT or PPAR-δ−/− mice were harvested at 8 d after immunization and were stimulated ex vivo with MOG p35-55. Cytokines (d) were measured in culture supernatants using ELISA. Proliferation (48 h; e) was measured by [3H]-thymidine incorporation assay. Values are means + SEM of triplicate culture wells. Data in a–e are representative of three experiments. (f) Serum was collected from WT and PPAR-δ−/− mice during EAE. Titers of MOG p35-55–specific IgM and total IgG were determined by ELISA. Values are absorbance measurements of individual mice from two experiments that contained n = 5 and n = 11 mice/group. *, significantly different from WT.

Figure 3.

Figure 3.

PPAR-δ limits cytokine production by CD4+ cells. (a–c) Naive CD4+ cells were isolated from the spleens of WT or PPAR-δ−/− mice and stimulated with anti-CD3 and anti-CD28 under Th0-skewing (a, c, e, and g) or Th17-skewing (b, d, f, and h), conditions. After 4 d of culture, CD4+ cells were processed for intracellular cytokine staining. (a and b) Representative FACs plots of events in the CD4+ gate. (c and d) Cytokine levels in culture supernatants after 48 h of culture under Th0-skewing (c) or Th17-skewing (d) conditions. (e–h) mRNA levels of IFN-γ and T-bet (Th0 conditions) or IL-17A and RORγt (Th17 conditions; relative to β-actin mRNAs) were determined using real-time PCR. *, significantly different from WT. Values are means + SEM of triplicate cultures (c and d) or triplicate reactions (e–h). Data are representative of two to five experiments.

Figure 4.

Figure 4.

CD4+ T cells from PPAR-δ−/− mice are hyperresponsive. Naive CD4+ (a) or CD19+ (b) cells were isolated from the spleens of WT or PPAR-δ−/− mice and were stimulated as indicated. Proliferation of these cells was measured using a [3H]-thymidine incorporation assay. Values are means + SEM of triplicate cultures. (c and d) Splenocytes were harvested from nonimmunized WT or PPAR-δ−/− mice. The frequency of CD4+ cells that displayed a memory (CD44high and CD62Llow; c) or activated (CD69+ or CD25+; d) phenotype in each group was determined. In d, values represent mean + SEM frequencies of n = 3–4 mice/group. Data in a–d are representative of two experiments. *, significantly different from WT.

Figure 5.

Figure 5.

PPAR-δ negatively regulates expression of IL-12 family cytokines. Splenic CD11b+ cells (a and c) or peritoneal macrophages (b) were isolated from adult age-matched WT and PPAR-δ−/− mice and were cultured with 10 ng/ml LPS. (a and b) Supernatants were collected at 24 h for cytokine measurement by ELISA. (c) Cells were harvested at 0, 1, 3, or 5 h after stimulation for collection of RNA for real-time PCR measurement of p40, p35, or p19 mRNA expression (relative to β-actin). Values represent mean + SEM of triplicate cultures (a and b) or reactions (c). *, significantly different from WT. Results are representative of three experiments.

Figure 6.

Figure 6.

GW0742 suppresses IFN-γ and IL-12 family cytokines in a PPAR-δ–dependent manner. CD4+ cells (a–e) or peritoneal macrophages (f) were stimulated in the presence of 100 nM GW0742 or vehicle of equal volume. Cells were cultured in complete RPMI (a) or X-vivo 20 serum-free media (b–f). CD4+ cells were stimulated with anti-CD3 under Th0-skewing (a–d) or Th17-skewing (e) conditions, whereas peritoneal macrophages were stimulated with LPS. Cytokine production was measured after 24 or 48 h of culture by ELISA and intracellular staining was conducted after 4 d of culture as in Fig. 3. Values represent means + SEM of triplicate cultures. *, significantly different from WT. †, significantly different from vehicle counterpart. Results are representative of three experiments.

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