Lps e coli 0127 b8

The ApoE−/− and IL10−/− mice (>10 generations backcrossed to C57BL6, ApoE−/− stock # 002052 and IL10−/− stock # 002251) were obtained from Jackson Laboratories and maintained according to recommended animal care conditions. IL10−/− ApoE−/− double knockout mice were obtained after 3 generations of crossing the individual gene knockout mice. Mice were fed normal chow diet (NIH-31, Envigo). Sterilized mouse cages, food and water were used in experiments that involved the IL10−/− genotype as previously described [14 (link)]. For chronic hypoxia experiments, 5–6 wk old female mice in standard sized mouse cages were placed in a large hypoxia chamber (Coy Laboratory Product Inc.) set at 10% O₂ and 30–70% humidity at room temperature as previously described [15 (link), 16 (link)]. CO₂ was set at ≤0.5% and all set parameters were continuously monitored with internal and external probes through a tele-alarm service (Rees Scientific). Mice were exposed to 10% O₂ for 22–23 wk prior to aortic atherosclerotic plaque analyses. The control normoxia mice were kept long term in room air (Fig. 1) or in the chamber with identical settings as described above except that O₂ was maintained at 21% (Fig. 4). For in vivo LPS stimulation studies, ApoE−/− mice were exposed to hypoxia for 5 wk and then intraperitoneally injected with 0.1 μg of LPS (E. coli 0127:B8, Sigma) per gram of body weight.

Human monocytes were isolated from buffy coats and DCs were generated as previously described [45 (link)]. TolDCs were generated by addition of Dexamethasone (10⁻⁶M Dex) (Pharmacy, LUMC, Leiden, The Netherlands), at the start of culture. On day 6 cells were harvested, washed and stimulated with 200 ng/mL LPS (E.Coli 0127:B8 Sigma-Aldrich, Zwijndrecht), 20μg/ml Poly I:C, 10 μg/ml PGN (InvivoGen, France) or 100 ng/ml IFN-γ (Peprotech, Germany). CD40L-activation was performed with CD40L transfected L cells (L-CD40L) in a DC: L cell ratio of 5:1. Non-transfected L cells served as control cells.