Interleukin 6 (il 6)

Splenocytes from IL-21eGFP and WT mice were seeded at 8 × 10⁵ cells in 0.2 ml/well into 48 well plates and stimulated with anti-CD3 (5 μg/ml) and anti-CD28 (1 μg/ml) antibodies (BD Biosciences). The cytokines IL-6 (20 ng/ml; R & D Systems) and TGFβ (5 ng/ml; BioLegend), anti-IL-4 antibodies (10 μg/ml; BioLegend), anti-IFNγ antibodies (10 μg/ml; BioLegend), and all trans retinoic acid (RA) (10 nM; Sigma) were added at the beginning of the cultures in various combinations (IL-6 alone; IL-6+TGFβ; IL-6+TGFβ+anti-IL-4+anti-IFNγ; IL-6+TGFβ+RA). Cells were cultured for 5 days and culture samples taken for analysis daily. The expression of GFP, Foxp3, and IL-17 were determined by intracellular staining, and the levels of IL-21 and IL-17 in culture supernatants were determined by ELISA.

The human hepatocyte cell lines, Huh-7 and HepG2, were purchased from Cell Bank of Shanghai Institute of Life Sciences, Chinese Academy of Sciences (Shanghai China). Cells were cultured in high-sugar DMEM medium (Gibco, Gaithersburg, MD, USA) with 10% fetal bovine serum (FBS, Biological Industries, Cromwell, CT, USA) with 100 U/ml streptomycin and 100 U/ml penicillin (HyClone Company, Logan, UT, USA) in a 37°C humidified incubator with 5% CO₂.
For IL-6 treatment experiments, the Huh-7 cells were treated with 10 ng/ml IL-6 (Sigma-Aldrich, St. Louis, MO, USA) for 72 h. After the incubation, total RNA or protein was collected for miRNA array or Western blot detections.
The total RNA of Huh-7 cell was extracted, and the miRNA microarray analysis was performed by Guangzhou Ribobio Co., Ltd. (Guangdong Province, China). One microgram of total RNA was added into a nuclease-free RNA sample PCR tube, and the small RNAs (<300 nucleotides) were separated using the MilliporeSigma Centriplus Centrifugal Concentrators Microcon YM-100 (MilliporeSigma, Burlington, MA) and adding poly(A) to the 3′ end of the small RNA for hybridization at 37°C. After hybridization, dyes labeled with specific flash biotin were used, followed by scanning and analyzing the data by Affymetrix Gene Chip™ Command Console Software (AGCC).

As described previously, astrocytes were acquired from neonatal rats (P2–3)42 (link). The mixed cells were plated onto culture flasks which were coated with poly-L-lysine, and cultured in DMEM/F12 (Gibco) containing 10% FBS (Gibco). The medium was changed every 2–3 days, and 6–10 days later, the cultures were put on shaker (2.5 g, 37 °C, 5% CO₂) overnight to remove microglia and oligodendrocyte lineage cells. The astrocytes were harvested and plated (1 × 10⁵/cm²) onto coverslips in 12-well plates for further analysis. Cells were 24 h before treatment. For IL-6 studies, astrocytes were treated with IL-6 (Sigma) at concentrations of 0, 10, 50, 100 ng/ml for further 24 h, and the level of GFAP and CCL7 were analyzed to acquire the optimum concentration of IL-6. In further studies, the optimum concentration of IL-6 was chosen to stimulate astrocytes.
Co-cultures of pure astrocytes (1.0 × 10⁵/cm²) and BMSCs or IL-1β-BMSCs (1.0 × 10⁵/cm²) were plated onto the coverslips in 24-well plates for 24 h after which IL-6 (100 ng/ml) was added. Cells were cultured for a further 24 h and the levels of GFAP and CCL7 were analyzed. For Ab blocking studies, IL-10 blocking Ab (BioLegend, San Diego, USA) or TGF-β1 neutralizing antibody (R&D Systems, Minneapolis, MN, USA) were added to the co-culture system 0.5 h before IL-6 stimulation.