NR4A1 agonist cytosporone B attenuates neuroinflammation in a mouse model of multiple sclerosis

Abstract

Nuclear receptor subfamily 4 group A1 (NR4A1) is an orphan nuclear receptor, which is expressed in the majority of cells. NR4A1 expression in peripheral blood mononuclear cells is low during the preclinical stage of multiple sclerosis. Knockout of the Nr4a1 gene in mice can aggravate the symptoms of experimental autoimmune encephalomyelitis (EAE), which is an animal model of multiple sclerosis. In this study, we intragastrically administered the NR4A1 agonist cytosporone B (Csn-B) to mice after inducing EAE. After treatment with Csn-B, the clinical symptoms in the EAE mice were substantially attenuated compared with that in PBS-treated control mice. The percentages of CD4⁺ T cells and F4/80⁺ cells in the central nervous system were decreased. In addition, interferon-γ and interleukin-17 production by proinflammatory Th1/Th17 cells in the central nervous system and interferon-γ levels in splenocytes were decreased after Csn-B treatment. These findings suggest that the NR4A1 agonist Csn-B can alleviate nerve injury after EAE induction, and, therefore, may be useful as a potential treatment for multiple sclerosis.

Keywords: NR4A1; NR4A1 agonist; Th1; Th17; cytosporone B (Csn-B); experimental autoimmune encephalomyelitis; macrophages; microglia; multiple sclerosis; treatment.

Figure 1

Experimental flow chart. Csn-B: Cytosporone B; EAE: experimental autoimmune encephalomyelitis; PBS: phosphate buffer saline.

Figure 2

Th1 and Th17 cell differentiation is decreased in vitro after treatment of naïve CD4⁺ T cells with cytosporone B (Csn-B). Naïve CD4⁺ T cells were separated by microbeads and differentiation was induced to generate T helper (Th)1 or Th17 cells. The cells were treated with 5 or 10 µg/mL Csn-B, and PBS was used in the control group. Intracellular levels of interferon (IFN)-γ (for Th1) and interleukin (IL)-17 (for Th17) in differentiated CD4⁺ T cells were determined using flow cytometry based on the gated populations (left), and the percentages of IFN-γ⁺ and IL-17⁺ cells within the CD4⁺ T cell population were analyzed (right). Data are expressed as means ± SEM. **P < 0.01, ****P < 0.001 (one-way analysis of variance followed by Bonferroni post hoc test). Csn-B: cytosporone B; IFN-γ: interferon-γ; IL-17: interleukin-17; PBS: phosphate buffer saline.

Figure 3

The NR4A1 agonist cytosporone B (Csn-B) can suppress the development of experimental autoimmune encephalomyelitis symptoms in vivo. After induction of experimental autoimmune encephalomyelitis with myelin oligodendrocyte glycoprotein peptide 35–55 and pertussis toxin, 2.5, 10, or 20 mg/kg Csn-B or PBS was administered to the mice once per day from day 10 to 21 post-immunization (the arrow represents the time of administration). The clinical function score showed no significant difference between the mice treated with 2.5 mg/kg/d Csn-B and the PBS control group, whereas the clinical function scores for the 10 and 20 mg/kg/d Csn-B groups were significantly lower than the scores for mice treated with PBS. Data are presented as means ± SEM (n = 5/group). ****P < 0.001 (one-way analysis of variance followed by Bonferroni post hoc test). Csn-B: cytosporone B; MOG35–55: myelin oligodendrocyte glycoprotein-35–55; PBS: phosphate buffer saline.

Figure 4

Cytosporone B (Csn-B) attenuates clinical symptoms and reduces demyelination and inflammatory cell infiltration in the central nervous system. After induction of experimental autoimmune encephalomyelitis (EAE) with myelin oligodendrocyte glycoprotein peptide 35–55 and pertussis toxin, 10 mg/kg Csn-B or PBS was administered to the mice once per day from day 14 to 21 post-immunization. L3 spinal cords were harvested on day 21. (A) Clinical symptom scores. The arrow represents the time of Csn-B or PBS administration. (B) Luxol fast blue (LFB) staining shows demyelination, and hematoxylin-eosin (HE) staining shows inflammation (left panel). PBS-treated EAE mice demonstrated more severe demyelination and greater inflammatory cell infiltration (arrows) compared with that observed in the Csn-B-treated EAE mice. The pathological scores for inflammation and demyelination are presented in the bar graphs (right panel). Scale bars: 200 µm. (C) Flow cytometry was used to detect CD4⁺ T and F4/80⁺ cells in the central nervous system of PBS-treated (left plot) and Csn-B-treated (right plot) based on the gated populations. The black boxes represent the distribution and percentage of F4/80⁺ cells, and the oval boxes represent the distribution and percentage of CD4⁺ cells. Absolute numbers of CD4⁺ T and F4/80⁺ cells are presented in the bar graphs (right panel). Data are presented as means ± SEM (n = 4 per group). **P < 0.01, ****P < 0.001 (Student’s t-test). Csn-B: Cytosporone B; HE: hematoxylin-eosin; LFB: Luxol fast blue; PBS: phosphate buffer saline.

Figure 5

Macrophages/microglia display an enhanced anti-inflammatory cell phenotype after cytosporone B (Csn-B) treatment. After induction of experimental autoimmune encephalomyelitis, 10 mg/kg Csn-B or PBS was administered once per day from day 14 to 21 post-immunization. The mice were sacrificed on day 21. Infiltrating cells in the central nervous system (CNS) and splenocytes were isolated and stained with the pan-macrophage marker F4/80; M1 markers Ia, CD86, CD40, and IL-12; and the M2 markers CD206 and IL-4. The cells were analyzed using flow cytometry. Expression of these markers in the CNS and spleens was presented as the mean fluorescence intensity (MFI) (left), and the quantitative results are shown in the bar graphs (right). Data are expressed as means ± SEM. ***P < 0.005, ****P < 0.001 (Student’s t-test). Csn-B: Cytosporone B; IA: I region associated antigen; IL: interleukin; MOG35–55: myelin oligodendrocyte glycoprotein-35–55; PBS: phosphate buffer saline.

Figure 6

Cytosporone B (Csn-B) decreases T helper (Th)1 and Th17 cell responses in vivo. After induction of experimental autoimmune encephalomyelitis, 10 mg/kg Csn-B or PBS was administered once per day from day 14 to 21 post-immunization. The mice were sacrificed on day 21. Infiltrating cells in the central nervous system (CNS) and splenocytes were isolated. (A) Upper: CD4⁺ T cells were gated within the CNS cell or splenocyte populations and analyzed by flow cytometry. The black boxes represent the distribution and percentages of IFN-γ⁺ and IL-17⁺ cells. Lower: Percentages of IFN-γ⁺ and IL-17⁺ cells. (B) Splenocytes were cultured in medium containing the myelin oligodendrocyte glycoprotein peptide 35–55 (25 µg/mL) for 48 and 72 hours. Supernatants were collected and cytokine levels were analyzed by enzyme-linked immunosorbent assays. Data are presented as means ± SEM (n = 4 per group). *P < 0.05, ***P < 0.005, ****P < 0.001 (Student’s t-test). Csn-B: Cytosporone B; IL-17: interleukin-17; INF-γ: interferon-γ; MOG35–55: myelin oligodendrocyte glycoprotein-35-55; PBS: phosphate buffer saline.