Interleukin 6 (il 6)

As serum is known to induce astrocyte activation, before treatment primary mouse astrocytes were washed once with HBSS and subsequently serum-starved for a variable time (1–24 h) in Sato’s serum-free medium (Insulin 10 μg ml⁻¹, transferrin 100 μg ml⁻¹, bovine serum albumin 300 μg ml⁻¹, putrescine 16 μg ml⁻¹, thyroxine 400 ng ml⁻¹, tri-iodo-thyronine 300 ng ml⁻¹, progesterone 60 ng ml⁻¹, sodium selenite 40 ng ml⁻¹, 1 mM Glutamax in DMEM, low glucose, pyruvate, Life Technologies). After serum starvation, astrocytes were treated with either pre-clustered rat recombinant EphB1-Fc (1, 5, 10 µg ml⁻¹, R&D Systems) or IL-6 (50 ng ml⁻¹, R&D systems) in Sato’s medium. For all experiments clustered EphB1-Fc was used unless stated otherwise and was referred to as ‘EphB1’ in the text. Before treatment hiPSC-derived astrocytes were cultured for 72 h in absence of BMP4 and LIF and then treated with either human recombinant EphB1-Fc (10 µg ml⁻¹, Gentaur) or IL-6 (50 ng ml⁻¹, R&D systems). Clustering of EphB1-Fc was obtained by incubating EphB1 with a clustering antibody (goat anti-human IgG, Fc fragment specific, 1:10, Jackson ImmunoResearch) for 30 min at RT. At the appropriate time point after the stimulation, cells were further processed for immunocytochemistry, WB or RNA extraction.

Levels of human TNF-α, interleukin (IL)-1β, and IL-6 present in culture media (R and D Systems, Minneapolis, MN) and the serum concentrations of TNF-α, IL-1β (R and D Systems), IL-6 (R and D Systems), apolipoprotein (apoAI; Cusabio Biotech, Wuhan, China), and apoB100 (Cusabio Biotech Co. Ltd.) were measured by ELISA according to the manufacturer’s instructions. Plasma levels of total cholesterol (TC) and TGs were analyzed in an automated analyzer (AU5400; Beckman Coulter, Fullerton, CA). Lipoproteins were isolated by sequential ultracentrifugation from 60 μl of plasma at densities (d) of <1.006 g/ml (VLDL), 1.006 ≤ d ≤1.063 g/ml (intermediate density lipoprotein and LDL) and d >1.063 g/ml HDL) in an LE-80K Ultracentrifuge (Beckman Coulter). Cholesterol levels in lipoprotein fractions were determined by enzymatic means using a colorimetric method (AU5400; Beckman).

Isolation of cytotrophoblasts (CTBs) from term placental specimens obtained from cesarean delivery without labor was performed as described (Tang et al., 2011 (link); Guzeloglu-Kayisli et al., 2020 (link)). Briefly, after removal of decidual tissues, fragments of chorionic villi were subjected to enzymatic digestion in a Hanks’ Balanced Salt Solution (Invitrogen, Carlsbad, CA) solution containing 0.25% trypsin (Invitrogen), 0.2% DNase I (Roche, Indianapolis, IN), 25 mM HEPES, 2 mM CaCl₂ and 0.8 mM MgSO₄ in at 37°C for 90 min. Cell suspension were then separated using a discontinuous (30%, 35%, 45%, 50%) Percoll gradient (GE Healthcare, Piscataway, NJ). Cells in 35%–45% Percoll interface were collected and immune-purified using mouse anti-human CD9 (R&D Inc., Minneapolis, MN) and mouse anti-CD45 (R&D) antibodies for negative selection by removing fibroblasts and immune cells. Then, the unbound cells were collected, washed, and cultured in 6-well plates in DMEM/F12 (Gibco, Life Technologies Corporation, Grand Island, NY) containing 10% FBS (Gemini Bio Products, West Sacramento, CA). Following 24 h incubation in serum-free media, cells were treated with vehicle or recombinant human IL-11 (10 ng/ml, R&D) or IL-6 (10 ng/ml, R&D) or IL-11 + IL-6 for 6 h for microarray analysis (Monzon-Bordonaba et al., 2002 (link); Monhasery et al., 2016 (link); Strickland et al., 2017 (link))

Culture supernatants IL-1β, IL-6, IL-10, TNF-α levels were measured by IL-1β (R&D Systems, Minnneapolis, MN, USA, DY201), IL-6 (R&D Systems, DY206), IL-10 (R&D Systems, DY217B), TNF-α (R&D Systems, DY210) ELISA kit. Caspase-1, ASC, Nalp3 and IL-1β was detected by western blot with anti-caspase-1 (Cell Signaling Technology, 2225S), anti-ASC (Millipore, Billerica, MA, USA, AB3607), anti-Nalp3 (Cell Signaling Technology, 13158S), and anti-IL-1β.