Reduced Angiopoietin-Like 4 Expression in Multiple Sclerosis Lesions Facilitates Lipid Uptake by Phagocytes via Modulation of Lipoprotein-Lipase Activity

Figure 1. ANGPTL4 expression is strikingly reduced in active MS lesions. (A) Active white matter lesions are characterized by loss of PLP and the presence of HLA-DR positive cells containing PLP-positive fragments (insert). Inactive white matter lesions display a loss of PLP immunoreactivity and absence of HLA-DR positive cells. Active white matter lesions display a marked decreased expression of ANGPTL4 compared to normal appearing white matter and inactive lesions. ANGPTL4 is predominantly localized to astrocytes (insert) (scale bar = 500 μm). (B) Double immunofluorescence labeling shows co-localization of ANGPTL4 (green) with GFAP-immunoreactive astrocytes (red) (scale bar = 5 μm). (C) Quantification of fluorescence intensity of ANGPTL4 in NAWM, active lesions and inactive lesions shows downregulation of ANGPTL4 in active MS lesions (One-way ANOVA, N = 6 for NAWM, 5 for active lesion and 4 for inactive lesion). *p < 0.05.

Figure 2. LPL is expressed on iba1 positive cells in active MS lesion. (A) Active white matter lesion is characterized by loss of PLP. Active lesions showed enhanced LPL immunoreactivity in microglia/macrophages (insert) (scale bar = 50 μm). (B) Double immunofluorescent labeling shows presence of lipoprotein-lipase (green) positive Iba1 (red) cells in MS lesions (scale bar = 10 μm).

Figure 3. Astrocytic ANGPTL4 expression is not influenced by myelin, but is by crosstalk with macrophages. (A) Astrocytes express ANGTPL4 at similar levels under normal conditions compared to 24 h treatment with myelin (Mann Whitney, N=8 for control and 11 for myelin treatment). Expression of ANGPTL4 is significantly reduced in astrocytes cultures in the presence of alternatively activated macrophages (Student's t-test, N = 7) (B). **p < 0.01.

Figure 4. ANGPTL4 inhibits uptake of myelin derived lipids but not myelin phagocytosis. (A) Increased Oil-Red-O staining of monocyte derived macrophages incubated for 4 hr with myelin, compared to control and to treatment of myelin together with recombinant Angptl4 or Orlistat. (scale bar = 50 μm) (B) Quantification of Oil-Red-O staining. (One-way ANOVA, N = 5) (C) ANGPTL4 inhibits macrophage LPL activity as determined by LPL activity assay. (Mann Whitney, N = 4) (D,E) ANGPTL4 treatment did not influence phagocytosis of myelin as determined by flow cytometry (One-way ANOVA, N = 5). *p < 0.05,**p < 0.01, ****p ≤ 0.0001.