Lipopolysaccharides (lps)

Four populations of cells derived from the macrophage protocol described above were stimulated with LPS (i.e. non-adherent cells from the EB myeloid differentiation phase -Mac Day0-, cells at the end of the Macrophage differentiation phase incubated for 7 days with M-CSF (100 ng/ml) -Mac Day7 M-CSF-, with GM-CSF (50 ng/ml) -Mac Day7 GM-CSF- and with GM-CSF (10 ng/ml) + IL-34 (100 ng/ml) -Mac Day7 GM-CSF IL-34-). For each sample, LPS (Sigma Aldrich) was added to the media in two wells for a final concentration of 2.5 ng/ml while a third well was kept as control. After 2 h, Brefeldin A (Sigma Aldrich) was added to reach 5ug/ml to all wells and cells were incubated for an additional 4 h (total 6 h of stimulation with LPS). Media was collected for control and LPS wells for cytokine analysis using V-PLEX Proinflammatory Panel 1 Human Kit (Mesoscale) according to manufacturing instructions. In parallel, cells were collected using 10 mg/ml Lidocaine (Sigma Aldrich) 2 mM EDTA (ThermoFisher) solution for 5 min at 37 °C, the two wells of LPS stimulation per population were combined. Single-cell RNAseq analysis was performed aiming at 3000 cells per condition as described in ‘10× Genomics Chromium GEMs sample preparation and sequencing’.

At the end of the 12-week feeding period, lipoprotein fractions, including very low-density lipoprotein (VLDL), low-density lipoprotein (LDL) and high-density lipoprotein (HDL), were separated by fast protein liquid chromatography (FPLC) from pooled plasma (n = 12/group), using an Akta Pure instrument equipped with two Superose 6 columns (GE Healthcare, Chicago, IL, USA). Triglycerides, phospholipids, total cholesterol, and free cholesterol were measured in plasma and FPLC fractions, using enzymatic test kits from FUJIFILM Wako Chemicals (Richmond, VA, USA). After 12 weeks on the diets, mice received an I.P. injection of 1 mg/kg of lipopolysaccharide (LPS) (Sigma-Aldrich, St. Louis, MO, USA). Retro-orbital bleeding was performed at 4 h post-LPS injection, and the plasma concentrations of cytokines, including interleukin-1β (IL1β), interleukin-6 (IL6), interleukin-17a (IL17a), interleukin-10 (IL10), interleukin-12/23 (IL12/23), monocyte chemoattractant protein-1 (MCP1), and transforming growth factor- β1 (TGFβ1) were measured using a customized multiplex assay system (Uplex, Mesoscale, Gaithersburg, MD, USA).