Low-Density Lipoprotein Receptor Deficiency Attenuates Neuroinflammation through the Induction of Apolipoprotein E

Abstract

Objective: We aimed to determine the role of the low-density lipoprotein receptor (LDLr) in neuroinflammation by inducing experimental autoimmune encephalomyelitis (EAE) in ldlr knock out mice.

Methods: MOG_35-55 induced EAE in male and female ldlr^-/- mice was assessed clinically and histopathologically. Expression of inflammatory mediators and apolipoprotein E (apoE) was investigated by qPCR. Changes in protein levels of apoE and tumor necrosis factor alpha (TNFα) were validated by western blot and ELISA, respectively.

Results: Ldlr^-/--attenuated EAE disease severity in female, but not in male, EAE mice marked by a reduced proinflammatory cytokine production in the central nervous system of female ldlr^-/- mice. Macrophages from female ldlr^-/- mice showed a similar decrease in proinflammatory mediators, an impaired capacity to phagocytose myelin and enhanced secretion of the anti-inflammatory apoE. Interestingly, apoE/ldlr double knock out abrogated the beneficial effect of ldlr depletion in EAE.

Conclusion: Collectively, we show that ldlr^-/- reduces EAE disease severity in female but not in male EAE mice, and that this can be explained by increased levels of apoE in female ldlr^-/- mice. Although the reason for the observed sexual dimorphism remains unclear, our findings show that LDLr and associated apoE levels are involved in neuroinflammatory processes.

Keywords: apolipoprotein E; experimental autoimmune encephalomyelitis; low-density lipoprotein receptor; multiple sclerosis; neuroinflammation.

Figure 1

Ldlr^−/− reduces experimental autoimmune encephalomyelitis (EAE) severity in female but not in male mice. Daily EAE score of female [p < 0.0079; wild-type (WT) vs ldlr^−/− by two-way ANOVA] (n = 19–20, pooled from three independent experiments) (A) and male (p < 0.2221; WT vs ldlr^−/− by two-way ANOVA) (n = 5) (B) mice. The mean score [t(31) = 3.050; p < 0.0048] (C), the maximum score [t(37) = 2.349; p < 0.0244] (D), and the day of onset of the disease [t(29) = 1.848; p < 0.0748] (E).

Figure 2

Ldlr deficiency has no significant influence on macrophage and T cell infiltration into the central nervous system. Immunohistological sections of spinal cord tissue from wild-type (WT) and ldlr^−/− mice at day 18 (A,B) and day 33 (C,D) post immunization. Sections were stained against F4/80 for macrophages (A,C) and CD3 for T cells (B,D). At least six sections per mouse were quantified (n = 5). Representative images can be found in the supplementary material section S1/2.

Figure 3

Ldlr deficiency has no influence on T cell proliferation. Stimulation index (SI) of T cells isolated from lymph nodes (LN) of wild-type (WT) and LDLR deficient mice (n = 3). Cells were collected on day 9 post immunization and proliferation was analyzed by means of a thymidine assay. Unstimulated cells an SI of 1 (A). Gene expression of transcription factors (T-bet, GATA3, RORγt, and Foxp3) in T cells isolated from LN (B) and spleen (C) from WT and ldlr^−/− female mice on day 9 post immunization (n = 4).

Figure 4

Relative expression of proinflammatory genes in the spinal cord of ldlr^−/− mice. Expression of Tnfα [t(10) = 3.668; p < 0.0044], IL-6 (MWU = 0; 5; 7; p < 0.0026) and iNOS [t(10) = 2.588; p < 0.0271] for male and female ldlr^−/− mice at experimental autoimmune encephalomyelitis (EAE) peak (n = 5–7) (A). Expression of Tnfα [t(13) = 2.467; p < 0.0284], IL-6 [t(12) = 2.553; p < 0.0254], and iNOS [t(13) = 1.313; p < 0.2118] for wild-type (WT) and ldlr^−/− female mice at EAE peak (n = 5–7) (B). Expression of Tnfα [t(7) = 1.106; p < 0.3054], IL-6 [t(7) = 0.7745; p < 0.4641], and iNOS [t(7) = 1.370; p < 0.2129] for WT and ldlr^−/− male mice at EAE peak (n = 5–7) (C). Data were normalized to the most stable reference genes, determined by Genorm (Ywhaz and Pgk1).

Figure 5

Ldlr deficiency influences the production of inflammatory mediators by macrophages. TNFα production [t(17) = 3.444; p < 0.0032] (n = 6) (A), NO production [t(22) = 3.368; p < 0.0029] (n = 6) (B), phagocytosis (mean fluorescence) [t(28) = 3.638; p < 0.0012] (n = 6) (C), lrp1 expression (MWU = 2; 5; 8; p < 0.0063) (n = 5–8) (D), Tnfα expression [t(9) = 3.630; p < 0.0055] (E), iNOS expression [t(9) = 2.020; p < 0.0742] (F), IL-12 expression (MWU = 0; 4; 6; p < 0.0095) (G), IL-6 expression [t(9) = 2.391; p < 0.0405] (H), and Arg1 expression [t(8) = 5.846; p < 0.0004] (I) in peritoneal macrophages isolated from wild-type (WT) and ldlr^−/− female mice (n = 6).

Figure 6

Apolipoprotein E (ApoE) expression by peritoneal macrophages. Comparison of apoE gene expression between male and female ldlr^−/− mice (MWU = 1; 6; 6; p < 0.0476) (n = 6) (A), wild-type (WT) and ldlr^−/− female mice (MWU = 1; 6; 6; p < 0.0044) (n = 6) (B), and WT and ldlr^−/− male mice (n = 6) (C). Western blot analysis of cell culture medium from LPS-stimulated peritoneal macrophages, stained for apoE (n = 3) (D).

Figure 7

Lack of apolipoprotein E (apoE) abrogates the reduced neuroinflammatory response in female ldlr^−/− mice. Daily clinical EAE score of female WT and apoE^−/− and ldlr^−/− mice (n = 6) (A). ELISA to measure TNFα production (n = 6) (B), myelin phagocytosis assay (n = 6) (C), Griess assay to measure NO production [t(15) = 5.091; p < 0.0001] (n = 6) (D), for WT and apoE^−/− and ldlr^−/− female mice. ApoE^−/− female mice show a similar EAE disease score compared to WT female mice (n = 9–11) (E).