Release of interleukin-10 and neurotrophic factors in the choroid plexus: possible inductors of neurogenesis following copolymer-1 immunization after cerebral ischemia

Abstract

Copolymer-1 (Cop-1) is a peptide with immunomodulatory properties, approved by the Food and Drug Administration of United States in the treatment of multiple sclerosis. Cop-1 has been shown to exert neuroprotective effects and induce neurogenesis in cerebral ischemia models. Nevertheless, the mechanism involved in the neurogenic action of this compound remains unknown. The choroid plexus (CP) is a network of cells that constitute the interphase between the immune and central nervous systems, with the ability to mediate neurogenesis through the release of cytokines and growth factors. Therefore, the CP could play a role in Cop-1-induced neurogenesis. In order to determine the participation of the CP in the induction of neurogenesis after Cop-1 immunization, we evaluated the gene expression of various growth factors (brain-derived neurotrophic factor, insulin-like growth factor 1, neurotrophin-3) and cytokines (tumor necrosis factor alpha, interferon-gamma, interleukin-4 (IL-4), IL-10 and IL-17), in the CP at 14 days after ischemia. Furthermore, we analyzed the correlation between the expression of these genes and neurogenesis. Our results showed that Cop-1 was capable of stimulating an upregulation in the expression of the genes encoding for brain-derived neurotrophic factor, insulin-like growth factor 1, neurotrophin-3 and IL-10 in the CP, which correlated with an increase in neurogenesis in the subventricular and subgranular zone. As well, we observed a downregulation of IL-17 gene expression. This study demonstrates the effect of Cop-1 on the expression of growth factors and IL-10 in the CP, in the same way, presents a possible mechanism involved in the neurogenic effect of Cop-1.

Keywords: Cop-1; Copaxone; choroid plexus; focal cerebral ischemia; glatiramer acetate; growth factors; immunomodulation; protective autoimmunity; stroke; tMCAo.

Figure 1

Effect of Cop-1 on neurological deficit and neurogenesis in tMCAo rats. (A) Neurological deficit of rats subjected to tMCAo (*P < 0.05 for Control or CFA vs. SS + Cop-1; **P = 0.01 for Control or CFA vs. CFA + Cop-1 on day 7; *P < 0.05 for Control or CFA vs. SS + Cop-1; **P < 0.01 for Control or CFA vs. CFA + Cop-1 on day 14 post ischemia. (B, C) Neurogenesis in the SVZ (B) and SGZ (C) at 14 days after ischemia. Quantification of BrdU⁺/DCX⁺ cells. (A–C) Each bar represents mean ± SEM of 5 rats. *P < 0.05, **P < 0.01, ***P < 0.0001. Kruskal Wallis followed by Dunn's multiple comparison test was used. Each test was performed in triplicate. tMCAo: Transient middle cerebral artery occlusion; CFA: complete Freund's adjuvant; SS: saline solution; SVZ: subventricular zone; SGZ: subgranular zone; BrdU⁺/DCX⁺: neuroblasts.

Figure 2

Correlation between neurogenesis and neurological deficit. (A, B) Correlation between neurogenesis (number of BrdU⁺/DCX⁺ cells) at the SVZ and neurological deficit (scores) in rats in the CFA + Cop-1 group (Spearman correlation coefficient r = –0.86, P < 0.05; A) and Cop-1 + SS group (r = –0.70, P < 0.05; B). (C, D) Correlation between neurogenesis (number of BrdU⁺/DCX⁺ cells) in the SGZ and neurological deficit (scores) in the Cop-1 + CFA group (r = –0.57, P = 0.17; C) and SS + Cop-1 group (r = –0.35, P = 0.25; D). CFA: Complete Freund's adjuvant; SS: saline solution; SVZ: subventricular zone; SGZ: subgranular zone; BrdU⁺/DCX⁺: neuroblasts.

Figure 3

Representative microphotographs of the immunolocalization of BrdU⁺/DCX⁺ cells in the SVZ and SGZ. (A) BrdU⁺/DCX⁺ in the SVZ. In the upper part, BrdU⁺ cells (green) are distinguished on the left and DCX⁺ cells (red) on the right side. Double-label (BrdU⁺/DCX⁺) is presented on the lower part in yellow. The arrows indicate the position of some neuroblasts. On the right side, a representative microphotograph of BrdU⁺/DCX⁺ cells from each group is observed. A higher number of double-labeled cells are appreciated for the groups treated with Cop-1. (B) BrdU⁺/DCX⁺ in the SGZ. BrdU⁺ cells in the SGZ are presented in the upper left side, DCX⁺ cells in the right side and double labeling of BrdU⁺/DCX⁺ in the lower part. Arrows indicate some neuroblasts. On the right side, the images indicate BrdU⁺/DCX⁺ cells in all groups, a higher number of neuroblasts is observed in the groups treated with Cop-1. Cop-1: Copolymer-1; CFA: complete Freund's adjuvant. SS: Saline solution. SVZ: subventricular zone; SGZ: subgranular zone; BrdU⁺: positive cells for 5-bromo-2-bromodeoxyuridine; DCX⁺: positive cells for doublecortin; BrdU⁺/DCX⁺: neuroblasts.

Figure 4

Effect of Cop-1 on gene expression of pro-inflammatory cytokines in the CP (quantitative PCR). Relative expression of the genes encoding for INF-γ (A), TNF-α (B), IL-1β (C), and IL-17 (D) at 14 days after tMCAo. Bars represents the mean ± SEM of 5 rats from each group. *P < 0.05, vs. control. Kruskal Wallis followed by Dunn's multiple comparison test was used. This experiment was performed in triplicate. Cop-1: Copolymer-1; TNF-α: tumor necrosis factor-α; IL-1β: interleukin-1β; INF-γ: interferon-γ; CFA: complete Freund's adjuvant; SS: saline solution; CP: choroid plexus; A.U.: arbitary unit; PCR: polymerase chain reaction.

Figure 5

Effect of Cop-1 on gene expression of anti-inflammatory cytokines in the CP (quantitative PCR). Relative expression of interleukin-4 (IL-4) (A) and IL-10 genes (B) 14 days after tMCAo. Bars represent the mean ± SEM of 5 rats from each group. *P < 0.05, vs. CFA and control groups. Kruskal Wallis followed by Dunn's multiple comparison test was used. This experiment was performed in triplicate. Cop-1: Copolymer-1; CFA: complete Freund's adjuvant; SS: saline solution; CP: choroid plexus; A.U.: arbitary unit; PCR: polymerase chain reaction.

Figure 6

Effect of Cop-1 on the expression of genes encoding for growth factors in the CP (quantitative PCR). Relative gene expression of BDNF (A), NT-3 (B), and IGF-1 (C) at 14 days after tMCAo. Bars represent mean ± SEM of 5 rats from each group. *P < 0.05, **P < 0.001, ***P < 0.0001. Kruskal Wallis followed by Dunn's multiple comparison test was used. This experiment was performed in triplicate. Cop-1: Copolymer-1; CFA: complete Freund's adjuvant; SS: saline solution; CP: choroid plexus; BDNF: brain-derived neurotrophic factor; IGF-1: insulin-like growth factor type 1; NT-3: neurotrophin-3; PCR: polymerase chain reaction; A.U.: arbitary unit.

Figure 7

Correlation of IL-10 or growth factors with neurogenesis in the SVZ of CFA plus Cop-1-treated rats. (A) IL-10 (spearman correlation coefficient r = 0.9, P < 0.01). (B) IGF-1 (r = 0.78, P < 0.01). (C) BDNF (r = 0.9, P = 0.04). (D) NT-3 (r = 0.9; P = 0.04). CFA: Complete Freund's adjuvant; SS: saline solution; SVZ: subventricular zone; BrdU⁺/DCX⁺: neuroblasts; Cop-1: copolymer-1; IL: interleukin; BDNF: brain-derived neurotrophic factor; IGF-1: insulin-like growth factor type 1; NT-3: neurotrophin-3; A.U.: arbitary unit; A.U.: arbitary unit.

Figure 8

Correlation of IL-10 or growth factors with neurogenesis in the SGZ of CFA plus Cop-1-treated rats. (A) IL-10 (Spearman correlation coefficient r = 0.9, P =0.008). (B) IGF-1 (r = 0.7, P < 0.05). (C) BDNF (r = 0.9, P = 0.04). (D) NT-3 (r = 0.9, P = 0.04). CFA: Complete Freund's adjuvant; SS: saline solution; SGZ: subgranular zone; BrdU⁺/DCX⁺: neuroblasts; Cop-1: copolymer-1; IL: interleukin; BDNF: brain-derived neurotrophic factor; IGF-1: insulin-like growth factor type 1; NT-3: neurotrophin-3; A.U.: arbitary unit.