Interleukin 6 (il 6)

Fed hormones/metabolites levels were determined by collecting tail blood from mice that were without food for 3 h. Fasted hormones/metabolites levels were assessed in mice provided only with water ad libitum and without food for the indicated period. Time at day at which blood was collected was the same between groups. Tail vein blood was assayed for glucose levels using a standard glucometer (Nova Biomedical). Plasma was collected by centrifugation in EDTA-coated tubes (Kent Scientific) and assayed for leptin (Crystal Chem. Inc., Downers Grove, IL), insulin (Crystal Chem. Inc.), TNF-α (Biovision) and IL-6 (Biovision) levels using the indicated commercially available kits.

Anti-DNP IgE, DNP-bovine serum albumin (BSA), dexamethasone, and Evans blue were purchased from Sigma (St. Louis, MO, USA). The RBL-2H3 cell line (KCLB-22256) was purchased from the Korean Cell Line Bank (Seoul, Korea). Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum, and penicillin-streptomycin (PS) were purchased from Hyclone (Logan, UT, USA). The histamine, TNF-α, IL-6, IL-1β, and IL-4 ELISA kits were purchased from Biovision (Milpitas, CA, USA). Anti-phospho-p38 (Thr180/Tyr182), anti-phospho-ERK (L352), anti-phospho-JNK (T183/Y185), anti-p38, anti-ERK, anti-JNK, and β-actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Boehmeria nivea is an eco-friendly pesticide-free product, which was purchased from a farming association (Yeonggwang, Korea).

HeLa cells (ATCC) were maintained in low glucose Dulbecco's Modified Eagle Medium (DMEM) supplemented with Heat-inactivated fetal calf serum (10% vol/vol) (FCS, Gibco), 2mM GlutaMAX (Invitrogen), and 0.1 mM nonessential amino acids at 37°C under 5% CO₂ atmosphere. C. rodentium was cultured in Luria Broth at 37°C, 200 rpm with appropriate antibiotics for 8 h and then subculture (1/500) in DMEM with low glucose and grown overnight at 37°C without agitation in 5% CO2 incubator. After 3 h of starvation in DMEM only, cells were infected for 3 h. HeLa cells were incubated with IL-6 (Biovision, 50ng/ml) for 30 min. prior to analyzing cell extracts by Western blotting, using Hax-1 as a loading control.

Fed hormones/metabolites levels were determined by collecting tail blood from mice that were without food for 3 h. Fasted hormones/metabolites levels were assessed in mice provided only with water ad libitum and without food for the indicated period. Time at day at which blood was collected was the same between groups. Tail vein blood was assayed for glucose levels using a standard glucometer (Nova Biomedical). Plasma was collected by centrifugation (1000 xg) in EDTA-coated tubes (Kent Scientific) and assayed using the indicated commercially available kits: β-hydroxybutyrate (Sigma), insulin (Crystal Chem. Inc.), TNF-α (Biovision), IL1-β (Biovision) and IL-6 (Biovision), S100A9 (R&D) and NEFA (Wako Chemicals).

After centrifuging the cell suspension for 10 minutes at 300 xg (RT), each cell pellet was resuspended in T-cell medium (2 x 10⁶ cells/mL) and the cultures rested in a 5% CO₂ and 37°C incubator for 90 minutes. Four culture conditions were created per subject: in the presence of either GSK2794776A or GSK2794778A [17 (link)] and in Th17 driving medium [T-cell medium containing 10 ng/mL IL-1β (Cell Guidance Systems #GFH167AF), 20 ng/mL IL-6 (BioVision #6464–10), 10 ng/mL IL-23 (BioVision #6470–10), 2 ng/mL TGFβ (BioVision #6479–10), 1 ng/mL IL-2 (BioVision #6461–10), 2 μg/mL anti-IL-4 (BioLegend #500815), 2 μg/mL anti-IFNγ (BioLegend #506513)] or in T-cell maintenance medium. Isolated naïve CD4⁺ T cells were then distributed into a 6-well plate containing each of the four conditions stated above in a cell density of 1 x 10⁶ cells/mL. Simultaneously, Dynabeads Human T-Activator CD3/CD28 (Life Technologies #11131D) were prepared according to the manufacturer instructions and resuspended in T cell medium. Bead suspension was then added to each well to give a final ratio of 1 bead: 50 cells, as described by Purvis et al (2010) [18 (link)]. Cells were incubated for 6 days in a 5% CO₂ and 37°C.
Significant differences between groups were determined using unpaired t-tests. Analyses were performed using Microsoft Excel and a p-value < 0.05 was considered statistically significant.