Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: ADAM17 Boosts Cholesterol Efflux and Downstream Effects of High-Density Lipoprotein on Inflammatory Pathways in Macrophages

doi: 10.1161/atvbaha.121.315145

Figure Lengend Snippet: Figure 1. Proinflammatory and anti-inflammatory responses in macrophages from APOA1Tg; Ldlr−/− mice mimic those of HDL (high-density lipoprotein). Peritoneal cells from male Ldlr−/− and APOA1Tg; Ldlr−/− control littermate mice were collected 4 days after thioglycolate injection. Macrophages were isolated from other cell types using a macrophage isolation kit and were further purified by a 1 h adhesion protocol before stimulation with LPS (lipopolysaccharide) or IFNβ (interferon β). A, Effect of LPS (10 ng/mL, 4 h) on inflammatory gene expression in macrophages isolated from Ldlr−/− and APOA1Tg; Ldlr−/− mice (n=5). B, Conditioned media were collected at indicated times from LPS-stimulated macrophages to analyze the release of TNF (tumor necrosis factor) α and CXCL1 (C-X-C motif chemokine ligand 1; n=4). C, Effect of LPS on the type 1 IFN-inducible genes Ifit2 and Mx1 analyzed in peritoneal macrophages from Ldlr−/− and APOA1Tg; Ldlr−/− mice (n=5–6). D, Effect of IFNβ on Ifit2 and Irf7 mRNA levels analyzed in peritoneal macrophages from Ldlr−/− and APOA1Tg; Ldlr−/− mice (n=5). E, Effect on STAT1 (signal transducer and activator of transcription 1) phosphorylation in IFNβ-stimulated peritoneal macrophages isolated from Ldlr−/− and APOA1Tg; Ldlr−/− mice (n=4). Phospho-STAT1 band intensity was normalized to that of total STAT1 and quantified (bar graph on the right). β-actin was used as an additional loading control. F, Representative photos and quantification (right) of lipid raft staining in thioglycolate-elicited peritoneal macrophages from Ldlr−/− and APOA1Tg; Ldlr−/− mice (n=7–13). G, BMDMs from female C57BL/6J mice were pretreated with HDL (100 μg/ mL) for 18 h. The cells were then stimulated with LPS (10 ng/mL) in the absence of HDL for 4 h, and inflammatory gene expression (Tnfa and Ifit2) was determined using quantitative polymerase chain reaction (n=4–7). H, BMDMs from male C57BL/6J mice were pretreated with HDL for 18 h. The cells were then stimulated with LPS (10 ng/mL) in the absence of HDL for 4 h to determine the phosphorylation status of p38 MAPK and STAT1 by immunoblot (n=4–5). Band intensities of p-p38 MAPK and p-STAT1 were normalized to those of total p38MAPK and STAT1, respectively, and quantified (bar graphs on the right). Data are shown as mean±SEM. Tests for normality (Shapiro-Wilk) and equal variance (Brown-Forsythe) were performed for each of the data sets. P values were determined accordingly by Kruskal-Wallis followed by Dunn multiple comparison tests (A), 1-way ANOVA followed by Tukey multiple comparison tests (C—Ifit2, D, E, H), Brown-Forsythe ANOVA followed by Dunnett multiple comparison tests (C—Mx1), 2-way ANOVA followed by Sidak multiple comparison test (B and G), or unpaired 2-tailed nonparametric Mann-Whitney test (F). Data are representative of at least 3 independent experiments performed in replicates. APOA1 indicates apolipoprotein A1.

Article Snippet: BMDMs were isolated and cultured as described previoulsy.37 After 7 days, BMDMs were treated with HDL isolated from APOA1Tg; Ldlr−/− mice in RPMI1640 medium containing 2% FBS for 18 hours, followed by washing of the cells and stimulation with ultrapure LPS, recombinant mouse IFNβ, TNFα (20 ng/mL; R&D systems; 410-MT), R848 (2 μg/mL, InvivoGen; tlrl-r848), or Poly:IC (10 μg/mL, Tocris Bioscience; 4287) for the indicated periods of time in the presence of 30% L929conditioned medium.

Techniques: Control, Injection, Isolation, Purification, Gene Expression, Phospho-proteomics, Staining, Real-time Polymerase Chain Reaction, Western Blot, Comparison, MANN-WHITNEY