RETRACTED: Oral Administration of Myelin Oligodendrocyte Glycoprotein Attenuates Experimental Autoimmune Encephalomyelitis through Induction of Th2/Treg Cells and Suppression of Th1/Th17 Immune Responses

Abstract

Multiple Sclerosis (MS) is a demyelinating autoimmune disorder of the central nervous system (CNS). Experimental autoimmune encephalomyelitis (EAE) has been widely used to determine the pathogenesis of the disease and evaluate new treatment strategies for MS. Therefore, we investigated the efficacy of oral administration of a Myelin Oligodendrocyte Glycoprotein (MOG) in the treatment of EAE. Female C57BL/6 mice were utilized in three groups (Control group, received PBS orally; prevention group, oral administration of MOG_35-55 two weeks before EAE induction; treatment group, oral administration of MOG_35-55 after EAE induction). MOG administration, both as prevention and treatment, significantly controlled clinical score, weight loss, CNS inflammation, and demyelination, mainly through the modulation of T cell proliferation, and reduction in pro-inflammatory cytokines and transcription factors, including TNF-α, IFN-γ, IL-17, T-bet, and ROR-γt. MOG administration, both as prevention and treatment, also induced anti-inflammatory cytokines and transcription factors, including IL-4, TGF-β, GATA-3, and Foxp3. The results showed that oral administration of MOG, both as prevention and treatment, could efficiently control EAE development. Immunomodulatory mechanisms include the induction of Th2 and Treg cells and the suppression of pro-inflammatory Th1 and Th17 cells.

Keywords: Immunomodulatory mechanisms; Multiple Sclerosis; Myelin Oligodendrocyte Glycoprotein; experimental autoimmune encephalomyelitis.

Figure 1

Oral administration of MOG inhibited the development of EAE in C57BL/6 mice. Prevention group received 250 μg of MOG_35–55 orally two weeks before EAE induction until 14 days after EAE induction, every day, and treatment group received MOG_35–55 orally after EAE induction until 14 days post-immunization. Mice were monitored for signs of EAE, and the results for all mice, were presented as (a) mean clinical score, and (b) body weight. Results were expressed as mean ± SEM. ** p < 0.01, *** p < 0.001, compared with control group. Mice were divided into three groups: 1. Control group (CTRL, n = 9); 2. Prevention group with MOG (n = 9); 3. Treatment group with MOG (n = 9).

Figure 2

MOG administration ameliorates CNS inflammation and demyelination. Histopathological evaluation of CNS from all the mice was performed. Brain sections from each group were collected on day 25 post-immunization, fixed in paraformaldehyde, and embedded in paraffin. Five µm sections from different regions of the brain from each of the groups were stained with (a) Hematoxylin and eosin, to enumerate infiltrating leukocytes, and (b) Luxol fast blue to assess demyelination. Scale bars: 100 μm. (c) CNS inflammatory foci and infiltrating inflammatory cells were quantified. Pathological scores, including inflammation and demyelination, were analyzed and shown as mean scores of pathological inflammation or demyelination ± SEM. *** p < 0.001, when compared with control group. Mice were divided into three groups: 1. Control group (CTRL, n = 9); 2. Prevention group with MOG (n = 9); 3. Treatment group with MOG (n = 9).

Figure 3

Oral administration of MOG suppresses T-cell proliferation. Splenocytes and lymph nodes were harvested on day 25 post-immunization and cultured in PHA (20 μg/mL), as a positive control, or with MOG (20 μg/mL) for 72 h on 96-well plates. Proliferation responses were tested using a Cell Proliferation ELISA, BrdU (colorimetric) kit (Roche Applied Science, Indianapolis, USA). The proliferation assay was conducted in triplicates. Data presented as mean optical density ± SEM. *** p < 0.001, when compared with control group. Mice were divided into three groups: 1. Control group (CTRL, n = 9); 2. Prevention group with MOG (n = 9); 3. Treatment group with MOG (n = 9).

Figure 4

Oral administration of MOG suppressed pro-inflammatory cytokines production and enhanced anti-inflammatory cytokines production in splenocytes and lymph nodes from EAE mice. Splenocytes and lymph nodes from immunized mice from all groups were isolated on day 25 post-immunization and re-stimulated with MOG35–55 (20 μg/mL) for 72 h. Cell culture supernatants were collected and indicated cytokine levels were measured by ELISA. Cytokine assays were conducted in duplicate wells. (a) Anti-inflammatory cytokines IL-4, IL-10, and TGF-β; (b) pro-inflammatory cytokines IFN-γ, TNF-α, and IL-17 released by splenocytes in culture, ±SEM. Results from lymph nodes were similar to splenocytes. ** p < 0.01, *** p < 0.001, when compared with control group. Mice were divided into three groups: 1. Control group (CTRL, n = 9); 2. Prevention group with MOG (n = 9); 3. Treatment group with MOG (n = 9).

Figure 5

Gene expression of cytokines and transcription factors in CNS. On day 25 post-immunization, brains and spinal cords were collected, and mRNA levels of cytokines and transcription factors were assessed by real-time quantitative PCR. The assay was run in triplicates, fold change expression of genes was determined, and then compared to the control group. (a) Th1- and Th17-related cytokines and transcription factors IFN-γ, T-bet, IL-17, and ROR-γt; (b) Th2- and Treg-related cytokines and transcription factors IL-4, GATA3, TGF- β, and FoxP3. Results were expressed as fold change normalized to the control group. ** p < 0.01, *** p < 0.001. Mice were divided into three groups: 1. Control group (CTRL, n = 9); 2. Prevention group with MOG (n = 9); 3. Treatment group with MOG (n = 9).