Anti il 23p19

Human hepatoma cell lines HepG2, Huh-7, HepG2.215 were obtained from the China Center for Type Culture Collection (Wuhan, China). They were maintained in DMEM medium (Gibco, BRL Co. Ltd, USA) containing 10% fetal bovine serum (Gibco), 100 units/ml penicillin and 100 mg/ml streptomycin (Sigma, St Louis, MO). Cells were cultured at 37° C in a humidified atmosphere of 5% CO₂.
For analyzing HNF4α expression affected by HBV-related IL-23, HepG2.215 cells were cultured with serum-free medium for 48 h. Then supernatant was collected and mixed with DMEM in different proportions (D/S). After that, HepG2 cells were cultured with D/S or supernatant (S) with IL-23 neutralization antibody (anti-IL-23p19, R&D, Minnesota, USA) for 24 h.
Plasmid pBlue-HBV encoding the full length of HBV was preserved by our lab.

Spleen were removed from mice and gently meshed in DMEM containing 10% FBS (Life Technologies) to prepare for single cell suspensions. CD4⁺/CD25⁻ T cells were isolated by the CD4⁺CD25⁻ T cell isolation kit (Miltenyi Biotec) according to manufacturer's instruction. The purity of CD4⁺CD25⁻ populations was around 90%. CD11c⁺ DCs isolated from colitic mice receiving the different treatments were cultured for 24 hrs in the presence or absence of GTS-21 (a specific α7nAChR agonist, 100 µM) before medium was washed and co-cultured with CD4⁺CD25⁻ T cell isolated from naïve mice at ratio of 1∶3 (DCs:T cells) [25] (link) in plates coated with 10 µg/ml⁻¹ of anti-CD3 and 2 µg/ml⁻¹ of anti-CD28. In neutralization experiments, these cultures were treated with 10 µg/ml anti-IL-12p35 or anti-IL-23p19 (R&D Systems) neutralizing mAb to block the potential activities of endogenous sources of these cytokines. In a separated set, recombinant (r) IL-12p70 or rIL-23 protein (25 ng/ml⁻¹; R&D Systems) were added to the cell culture medium. Cell culture supernatants were collected at 24 hrs, and interferon-gamma (IFN-γ), IL-4 and IL-17 levels were analyzed by ELISA (R&D Systems).