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

Journal: Nature communications

Article Title: Comparative therapeutic efficacy of remdesivir and combination lopinavir, ritonavir, and interferon beta against MERS-CoV.

doi: 10.1038/s41467-019-13940-6

Figure Lengend Snippet: Fig. 5 Therapeutic RDV reduces replication and pathology. Percent starting weight of 10–12-week-old female Ces1c−/−hDPP4 mice infected with 5E + 04 pfu MERS M35C4 and treated with a subcutaneous vehicle for RDV (N = 13) or remdesivir (RDV, 25 mg/kg, N = 14) BID beginning 1 dpi or b vehicle for LPV/RTV-IFNb (N = 15), LPV/RTV-IFNb low (N = 16) or LPV/RTV-IFNb high (N = 16) beginning 1 dpi. Oral vehicle or lopinavir/ritonavir (160/40 mg/kg) was administered orally once daily. IFNb low (1x human equivalent dose of 1.6 MIU/kg) and high (25x human equivalent dose of 40 MIU/kg) or PBS vehicle were administered via subcutaneous injection every other day. Asterisks indicate statistical differences by two-way ANOVA with Tukey’s multiple comparison test. c Lung hemorrhage 6 dpi for all animals in a, b scored on a scale of 0–4, where 0 is a normal pink healthy lung and 4 is a diffusely discolored dark red lung. d MERS-CoV lung titer 6 dpi in mice as described in a, b. Asterisks indicate statistical significance (N group described in a and b, P < 0.05) by one-way ANOVA with Kruskal–Wallis test for (c, d). Data for a–d are compiled from two independent experiments. For the box and whisker plots, the boxes encompass the 25th to 75th percentile, the line is at the median, while the whiskers represent the range. e Representative photomicrographs of MERS-CoV antigen (brown) and hematoxylin stained nuclei (blue) in mouse lung tissue sections from 6 dpi. The black bar is 100 µM.

Article Snippet: Recombinant mouse IFNb protein was purchased from R&D Systems (8234-MB/CF, 1.2 × 109 IU/mg calibrated against Murine IFN-beta WHO International Standard) for the in vivo studies and reconstituted in PBS.

Techniques: Infection, Injection, Comparison, Whisker Assay, Staining