Antiinflammatory properties of a plant-derived nonsteroidal, dissociated glucocorticoid receptor modulator in experimental autoimmune encephalomyelitis

Abstract

Compound A (CpdA), a plant-derived phenyl aziridine precursor, was recently characterized as a fully dissociated nonsteroidal antiinflammatory agent, acting via activation of the glucocorticoid receptor, thereby down-modulating nuclear factor-kappaB-mediated transactivation, but not supporting glucocorticoid response element-driven gene expression. The present study demonstrates the effectiveness of CpdA in inhibiting the disease progress in experimental autoimmune encephalomyelitis (EAE), a well-characterized animal model of multiple sclerosis. CpdA treatment of mice, both early and at the peak of the disease, markedly suppressed the clinical symptoms of EAE induced by myelin oligodendrocyte glycoprotein peptide immunization. Attenuation of the clinical symptoms of EAE by CpdA was accompanied by reduced leukocyte infiltration in the spinal cord, reduced expression of inflammatory cytokines and chemokines, and reduced neuronal damage and demyelination. In vivo CpdA therapy suppressed the encephalogenicity of myelin oligodendrocyte glycoprotein peptide-specific T cells. Moreover, CpdA was able to inhibit TNF- and lipopolysaccharide-induced nuclear factor-kappaB activation in primary microglial cells in vitro, in a differential mechanistic manner as compared with dexamethasone. Finally, in EAE mice the therapeutic effect of CpdA, in contrast to that of dexamethasone, occurred in the absence of hyperinsulinemia and in the absence of a suppressive effect on the hypothalamic-pituitary-adrenal axis. Based on these results, we propose CpdA as a compound with promising antiinflammatory characteristics useful for therapeutic intervention in multiple sclerosis and other neuroinflammatory diseases.

Fig. 1.

CpdA administration ameliorates clinical symptoms and disease severity of EAE. EAE was induced in male C57BL/6 mice, and clinical symptoms were scored in PBS-, CpdA-, and DEX-treated mice after immunization with MOG peptide. A, PBS, CpdA, and DEX are administered (ip injection) every second day from the onset of acute disease (d 10 on after immunization, as indicated by the arrow). B, PBS, CpdA, and DEX are administered every second day starting from d 2 on after immunization. C, PBS and CpdA are administered every second day starting at the peak of the disease (clinical score 4). Results are displayed as mean values ± sem. *, P < 0.05 (PBS vs. CpdA), and +, P < 0.05 (PBS vs. DEX).

Fig. 2.

Reduced inflammation and demyelination in the CNS of CpdA-treated mice. PBS, CpdA, and DEX was administered every second day from the onset of acute disease (d 10 after immunization). A, Histological profiles of spinal cords from PBS-, CpdA-, and DEX-treated mice 25 d after EAE induction. Sections from the lumbar spinal cord were examined for infiltrating macrophages (MAC3, brown) and T cells (CD3, brown) by immunohistochemistry, demyelination by LFB (blue) staining, and axonal damage by APP (brown) immunohistochemistry. Scale bar, 100 μm. B, Quantification of spinal cord infiltrates, demyelination, and axonal damage from histological sections shown in panel A. Numbers of infiltrating T cells (CD3), B cells (B220), and macrophages (MAC3) in PBS-, CpdA-, and DEX-treated mice after EAE induction. Results are displayed as mean values ± sem. *, P < 0.05 (PBS vs. CpdA).

Fig. 3.

Decreased proinflammatory gene expression and impaired NF-κB DNA-binding in CNS from CpdA-treated mice. PBS and CpdA were administered every second day from the onset of acute disease (d 10 after immunization). A and B, Quantitative measurement of the indicated cytokine and chemokine mRNA expression in spinal cord from PBS- (n = 2) and CpdA-treated (n = 2) mice 10 d (= before CpdA treatment), 16 d, and 22 d after immunization, and from nonimmunized control mice (n = 2). P < 0.05 (PBS vs. CpdA) for all cytokines and chemokines tested. C, ChIP analysis combined with qPCR was used to assay recruitment of p65 to the IL-6 gene promoter. The quantity of p65 (bound fraction) detected on the IL-6 promoter is shown with a correction of the SYBR green qPCR signal for input control, as percent bound over input. The IgG control indicates the threshold levels of aspecific binding. *, P < 0.05.

Fig. 4.

Impaired peripheral immune responses in CpdA-treated mice. Splenocytes from MOG peptide-immunized control (PBS-treated) and CpdA-treated mice were cultured and stimulated with the indicated concentrations of MOG peptide. A, Splenocyte cell proliferation was assessed by measurement of [³H]thymidine incorporation by liquid scintillation counting. Results are expressed as counts per min of triplicate cultures. B, Culture supernatants were collected 48 h after MOG peptide stimulation and assayed for IL-2, IFN-γ, and IL-17 by ELISA. Results are shown as mean ± sem. imm, Immunized.

Fig. 5.

Primary microglia, but not astrocytes, show impaired IκBα phosphorylation and NF-κB nuclear translocation after CpdA treatment in vitro. A, Primary microglial cultures from C57BL/6 mice were pretreated with PBS, CpdA, or DEX for 45 min and stimulated for 20 min with TNF and stained for CD11b (red) and p65 (green). Original magnification, ×400. Images are representative of two independent experiments. B, Primary astrocyte cultures from C57BL/6 mice were pretreated with PBS, CpdA, or DEX for 45 min and stimulated for 20 min with IL-1β and stained for GFAP (red) and p65 (green). Original magnification, ×400. C, Immunoblot analysis for phosporylated IκBα expression in cytoplasmic extracts from primary microglia and astrocytes either pretreated or not pretreated with CpdA. D, Immunoblot analysis for phosporylated IκBα expression in cytoplasmic extracts from primary microglia and astrocytes either pretreated or not pretreated with DEX. E, Quantitative measurement of IL-1β mRNA expression in primary microglial cells pretreated with CpdA or DEX followed by TNF stimulation for 6 h. F, Quantitative measurement of TNF mRNA expression in primary astrocytes pretreated with CpdA or DEX followed by IL-1β stimulation for 6 h.

Fig. 6.

CpdA, in contrast to DEX, does not induce GRE-driven gene expression. A, Primary microglial cultures from C57BL/6 mice were treated with solvent (/), CpdA, or DEX for 24 h and analyzed by quantitative pPCR for GILZ, FKBP51, and MKP1. B, Primary astrocyte cultures from C57BL/6 mice were treated with solvent (/), CpdA, or DEX for 6 h and analyzed by quantitative pPCR for GILZ, FKBP51, and MKP1.

Fig. 7.

CpdA, in contrast to DEX, does not induce diabetogenic or HPA axis-suppressive side effects in vivo. EAE was induced in male C57BL/6 mice. PBS, CpdA, and DEX are administered every second day starting from d 2 onward after immunization. A, Percentage body weight loss measured during EAE. B, Serum was collected 25 d after MOG peptide stimulation and assayed for insulin by ELISA. Results are shown as mean ± sem. *, P < 0.05. C, Serum was collected 25 d after MOG peptide stimulation and assayed for corticosterone via a corticosterone immunoassay. Results are shown as mean ± sem. D, Serum was collected 25 d after MOG peptide immunization, and levels of AST and ALT enzymes were measured. Results are shown as mean ± sem.