Fig. 2

High cholesterol burden attenuates the pro-inflammatory phenotype displayed by PAMP-activated SIM-A9 microglial cells. Cells were cholesterol-enriched by incubation with the CHO:MCD complex for 1 h. After 4 h of recovery, cells were stimulated with LPS (10 μg/ml) plus MDP (10 μg/ml) for 16 h. a Trem2 and Clec7a mRNA expression levels analyzed by selfie qRT-PCR. Transcript copies were normalized to total genomic DNA and reported as relative levels referred to the expression in CTRL cells (n = 4 independent experiments). b CLEC7A levels quantified by flow cytometry (n = 3). Histogram plots provide representative data from 3 independent experiments. c Representative confocal immunomicrographs from 3 independent experiments showing enhanced cell surface presence of TREM2 in cholesterol-enriched cells after PAMP exposure. Nuclei were stained with DRAQ5 (blue). Images from the green channel corresponding to TREM2 immunostaining are shown in black and white. Plot represents TREM2 levels per cell, quantified as the CTCF of the green channel (n = 9–10 non-overlapping images). Scale bar: 25 μm. d Heat map depicting transcriptional changes in gene expression assayed by qRT-PCR using an innate and adaptive immune response gene array. Each probe set is represented in a blue-red row Z-score scale with red indicating high expression and blue low expression. The panel on the right shows the genes whose endotoxin-mediated changes in expression are up/down-regulated by cholesterol enrichment (n = 3 independent experiments). See Additional file 2 for array dataset. e Immunofluorescence measurement of phagocytosis. Representative confocal images of microglia incubated with fluorescent Aβ and counterstained with CellMask (cytosol/plasma membrane, red) and DRAQ5 (nuclei, blue) (n = 3 independent experiments). Scale bar, 25 μm. Plot represents Aβ phagocytosed per cell, quantified as the CTCF of the green channel (n = 12 non-overlapping images). One-way ANOVA followed by the Tukey–Kramer test was applied to calculate statistical significance (*P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001)