Myelin-derived lipids modulate macrophage activity by liver X receptor activation

Abstract

Multiple sclerosis is a chronic, inflammatory, demyelinating disease of the central nervous system in which macrophages and microglia play a central role. Foamy macrophages and microglia, containing degenerated myelin, are abundantly found in active multiple sclerosis lesions. Recent studies have described an altered macrophage phenotype after myelin internalization. However, it is unclear by which mechanisms myelin affects the phenotype of macrophages and how this phenotype can influence lesion progression. Here we demonstrate, by using genome wide gene expression analysis, that myelin-phagocytosing macrophages have an enhanced expression of genes involved in migration, phagocytosis and inflammation. Interestingly, myelin internalization also induced the expression of genes involved in liver-X-receptor signaling and cholesterol efflux. In vitro validation shows that myelin-phagocytosing macrophages indeed have an increased capacity to dispose intracellular cholesterol. In addition, myelin suppresses the secretion of the pro-inflammatory mediator IL-6 by macrophages, which was mediated by activation of liver-X-receptor β. Our data show that myelin modulates the phenotype of macrophages by nuclear receptor activation, which may subsequently affect lesion progression in demyelinating diseases such as multiple sclerosis.

Figure 1. Quantitative PCR validation.

Comparison of fold changes between IFNγ/IL1β-stimulated untreated (n = 5) and myelin treated macrophages (n = 5). Relative quantification of gene expression (SCD1/2, ABCA1/G1 and RXRα/β/γ) was accomplished by using the comparative C_t method. Data were normalized to the most stable reference genes, determined by Genorm (18S and PGK1).

Figure 2. Mye-macrophages have an increased capacity to transfer intracellular cholesterol towards HDL.

Macrophages were loaded for 48 hours with 1,2- [3H] cholesterol after which cells were treated with myelin for 24 hours or left untreated. HDL was used as cholesterol acceptor. The relative cholesterol efflux is defined as the amount of transported cholesterol in culture medium of mye-macrophages divided by values in control macrophage cultures. Data represent the mean of four independent experiments.

Figure 3. Myelin and T0901317 affect the expression of LXR response genes in a similar manner.

(a–c) Comparison of fold changes of LXR response genes between untreated (dotted line) and myelin- or T0901317-treated macrophages. Macrophages were treated for 24 and 48 hours with 100 µg/ml myelin or 10 µM T0901317 after which expression of ApoE and ABCA1/G1 was determined. Relative quantification of gene expression was accomplished by using the comparative C_t method. Data were normalized to the most stable reference genes, determined by Genorm (18S and PGK-1). Data represent the mean of four independent experiments. (d) Comparison of fold changes of ABCA1 between untreated (dotted line) and myelin treated wild-type, LXRα-, LXRβ- and LXRαβ-deficient mouse macrophages. Macrophages were treated 48 hours with 100 µg/ml myelin. Data were normalized to the most stable reference genes, determined by Genorm (CycA and HMBS). Data represent the mean of four independent experiments. Mye; Myelin: T09; T0901317.

Figure 4. Myelin alters the macrophage phenotype by activating the LXRβ isoform.

(a–d) Relative NO and IL-6 concentration in supernatants of IFNγ/IL-1β or LPS stimulated myelin- or T0901317-treated macrophages. The relative NO and IL-6 production is defined as the production of NO/IL-6 in experimental cultures divided by values in stimulated control cultures (dotted line). Data represent the mean of four independent experiments. (e, f) Relative NO and IL-6 concentration in supernatants of LPS stimulated myelin treated wild-type, LXRα-, LXRβ- and LXRαβ-deficient mouse macrophages. Macrophages were treated for 24 hours with myelin prior to stimulation with LPS. Data represent the mean of four independent experiments. Mye; Myelin: T09; T0901317.