Gas6 Induces Myelination through Anti-Inflammatory IL-10 and TGF-β Upregulation in White Matter and Glia

Abstract

The Gas6-TAM (Tyro3, Axl, Mer) ligand-receptor system is believed to promote central nervous system (CNS) (re)myelination and glial cell development. An additional important function of Gas6-TAM signalling appears to be the regulation of immunity and inflammation, which remains to be fully elucidated in the CNS. Here, we characterised the expression of TAM receptors and ligands in individual CNS glial cell types, observing high expression of Gas6 and the TAM receptors, Mer and Axl, in microglia, and high expression of Tyro3 in astrocytes. We also investigated the effect of Gas6 on the inflammatory cytokine response in the optic nerve and in mixed glial cell cultures from wildtype and single TAM receptor knockout mice. In wildtype and Mer-deficient cultures, Gas6 significantly stimulated the expression of the anti-inflammatory/pro-repair cytokines interleukin 10 (IL-10) and transforming growth factor β (TGF-β), whereas this effect was absent in either Tyro3 or Axl knockout cultures. Furthermore, Gas6 caused upregulation of myelin basic protein (MBP) expression in optic nerves, which was blocked by a neutralising antibody against IL-10. In conclusion, our data show that microglia are both a major source of Gas6 as well as an effector of Gas6 action in the CNS through the upregulation of anti-inflammatory and pro-repair mediators. Furthermore, the presence of both Axl and Tyro3 receptors appears to be necessary for these effects of Gas6. In addition, IL-10, alongside suppressing inflammation and immunity, mediates the pro-myelinating mechanism of Gas6 action in the optic nerve. Therefore, Gas6 may present an attractive target for novel therapeutic interventions for demyelinating as well as neuroinflammatory disorders of the CNS.

Keywords: Gas6; IL-10; TAM receptor; TGF-β; astrocytes; microglia; myelination; optic nerve; vitamin K; white matter.

Figure 1

Expression of TAM (Tyro3, Axl, Mer) receptors and Gas6 in pure primary cultures of microglia, astrocytes and oligodendrocyte precursor cells (OPCs). Quantitative RT-PCR analysis of mRNA expression of the genes for Tyro3, Axl, Mer and the ligand Gas6 in extracts from mouse primary glial cell cultures. Values represent mean ± SEM (n = 3 experiments from different mice for all samples).

Figure 2

Effect of Gas6 on interleukin 10 (IL-10) gene expression in cultured mouse optic nerve. qRT-PCR was performed on extracts from optic nerves using specific il10 primers/probe, using Gapdh as reference gene. Gas6 caused the upregulation of the il10 gene only in wildtype and Mer knockout optic nerves. Values represent mean ± SEM (n = 4 experiments for wildtype (* p < 0.05 vs. control); n = 3 for Tyro3^−/− and Axl^−/−; n = 2 for Mer^−/−). The ‘Control’ bar represents a normalised value based on every experiment for each mouse tissue experiment having its own untreated sample.

Figure 3

Effect of Gas6 on IL-10 and IL-10 receptor (IL-10R) expression in primary mixed glial cell cultures at gene and protein levels. (A) qRT-PCR analysis was performed on extracts from wildtype cultures treated with control, Gas6 and lipopolysaccharide (LPS). Values represent mean ± SEM (n = 7 for IL-10, n = 6 for IL-10R). (B) Mouse IL-10 ELISA was performed on the medium of wildtype mixed glial cultures under the same experimental treatments as in (A). Values represent mean ± SEM (n = 6 experiments); # p = 0.08 vs. control. (C) qRT-PCR analysis on extracts from mock-, Gas6 and LPS-treated TAM single-receptor knockout mixed glial cell cultures. Values represent mean ± SEM (n = 3); * p < 0.05 vs. control.

Figure 4

Effect of Gas6 on transforming growth factor β (TGF-β) gene expression in mixed glial cell cultures, measured by qRT-PCR analysis. TGF-β expression in wildtype and single-TAM-knockout primary mixed glial cell cultures, following mock, Gas6 or LPS treatment. Values represent mean ± SEM (n = 5 experiments for wildtype, n = 3 experiments for TAM knockouts); * p < 0.05 and # p = 0.08 vs control.

Figure 5

Effect of Gas6 on myelination through myelin basic protein (MBP) expression in cultured optic nerves, analysed by western blotting. Cultured optic nerves were incubated with agents for 3 days. The graphs show the band densitometric quantification of MBP protein levels relative to β-actin protein in each sample. (A) Wildtype optic nerves in culture were incubated with Gas6 in the absence or presence of anti-IL-10 antibody. Lanes correspond to the following: (1) Mock, (2) Gas6, (3) Gas6+anti-IL-10, (4) anti-IL-10. Values represent mean ± SEM (n = 4 experiments for all the treatments except for the anti-IL-10 group; n = 3 experiments); ** p < 0.01, # p = 0.052 vs. Gas6; no significant difference between Mock and the anti-IL-10 treatments; analysis of variance followed by Dunnet’s multiple comparison. (B) Effects of Gas6 on MBP protein expression in TAM single-receptor knockout mouse optic nerve cultures. Lanes correspond to the following: (1) Tyro3^−/− mock-treated, (2) Tyro3^−/− Gas6-treated, (3) Axl^−/− mock-treated, (4) Axl^−/− Gas6-treated, (5) Mer^−/− mock-treated, (6) Mer^−/− Gas6-treated. Values represent mean ± SEM (n = 3 experiments); * p < 0.05.

Figure 6

Schematic diagram of two possible mechanisms through which Gas6 exerts its effect on glial cells through IL-10 and TGF-β induction. (A) Gas6 indirectly upregulates IL-10 via TGF-β. In this pathway, Gas6 activates astrocytes to release TGF-β which then acts on microglia to stimulate IL-10 production and contributes to the enhancement of myelination. (B) In addition, Gas6 could activate IL-10 in microglia via Axl prior to TGF-β upregulation, which then could enhance myelination, as well as stimulate TGF-β in astrocytes. This pathway could occur in the different cell types in parallel; in this case, TGF-β is indirectly activated via IL-10.